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Sample records for calcineurin-inhibitor-free de-novo immunosuppression

  1. Renal impairment after liver transplantation - a pilot trial of calcineurin inhibitor-free vs. calcineurin inhibitor sparing immunosuppression in patients with mildly impaired renal function after liver transplantation

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    Gerhardt T

    2009-05-01

    Full Text Available Abstract Objectives Chronic kidney disease is frequent in patients after orthotopic liver transplantation (OLT and has impact on survival. Patients receiving calcineurin inhibitors (CNI are at increased risk to develop impaired renal function. Early CNI reduction and concomitant use of mycophenolat mofetil (MMF has been shown to improve renal function. Methods The aim of this trial was to compare dose-reduced CNI/MMF versus CNI-free MMF/prednisone-based treatment in stable patients after OLT with respect to glomerular filtration rate (GFR. 21 patients [GFR 44.9 ± 9.9 mL/min/1.73 m2 measured by 99m-Tc-DTPA-clearance, serum creatinine (SCr 1.5 ± 0.42 mg/dL] were randomized either to exchange CNI for 10 mg prednisone (group 1; n = 8 or to receive CNI at 25% of the initial dose (group 2; n = 13 each in combination with 1000 mg MMF b.i.d. Results At month 12 mean SCr (-0.3 ± 0.4 mg/dL, p = 0.031 and GFR improved (8.6 ± 13.1 mL/min/1.73 m2, p = 0.015 in group 2 but remained unchanged in group 1. Main side effects were gastroinstestinal symptoms (14.3% and infections (4.8%. Two biopsy proven, steroid-responsive rejections occurred. In group 1 mean diastolic blood pressure (BP increased by 11 ± 22 mmHg (p = 0.03. Conclusions Reduced dose CNI in combination with MMF but not CNI-free-immunosuppression leads to improvement of GFR in patients with moderately elevated SCr levels after OLT. Addition of steroids resulted in increased diastolic blood pressure presumably counterbalancing the benefits of CNI withdrawal on renal function.

  2. Are calcineurin inhibitors-free regimens ready for prime time?

    Science.gov (United States)

    Vincenti, Flavio

    2012-11-01

    The goal of research in transplant therapeutics is to achieve safe and effective immunosuppression strategies that allow durable engraftment free of toxicities. The calcineurin inhibitors (CNIs) regimens, because of their inherent toxicities (including nephrotoxicity), have been unable to meet these promises. Over the past decade acute cellular rejection decreased dramatically with a concomitant robust increase in 1-year graft survival; however, long-term graft outcome showed only modest improvement. This is due in part to the toxicities of the immunosuppressive drugs. The quest for a toxicity-free-CNI-free regimen has been both intense and frustrating. A turning point in CNIs-free therapy may have occurred with the recent approval of belatacept, which represents a new paradigm in immunosuppression: biological therapy for chronic immunosuppression devoid of the usual toxicities associated with the CNIs. Belatacept, a fusion receptor protein, blocks costimulation signals necessary for the activation of T cells. Although costimulation blockade has not been shown to induce tolerance, it can provide safe and effective immunosuppression without renal or cardiovascular toxicities. The approval of belatacept in both the United States and Europe for use in renal transplantation will finally push CNI-free regimens into prime time. Novel biologics such as ASKP1240 (a human anti-CD40 monoclonal antibody) and one small molecule, tofacitinib, may advance further the use of CNI-free regimens in organ transplantation.

  3. De novo glomerular diseases after renal transplantation.

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    Ponticelli, Claudio; Moroni, Gabriella; Glassock, Richard J

    2014-08-07

    Glomerular diseases developing in the kidney allograft are more often recurrences of the original disease affecting the native kidneys. However, in an undefined number of cases de novo, glomerular diseases unrelated to the original disease in the native kidneys can develop in the transplanted kidney. The clinical presentation and histologic features of de novo diseases are often similar to those features observed in patients with primary or secondary GN in the native kidneys. However, in transplanted kidneys, the glomerular, vascular, and tubulointerstitial changes are often intertwined with structural abnormalities already present at the time of transplant or caused by antibody- or cell-mediated allograft rejection, immunosuppressive drugs, or superimposed infection (most often of a viral nature). The pathophysiology of de novo glomerular diseases is quite variable. In rare cases of de novo minimal change disease, circulating factors increasing the glomerular permeability likely participate. Maladaptive hemodynamic changes and tissue fibrosis caused by calcineurin inhibitors or other factors may be involved in the pathogenesis of de novo FSGS. The exposure of cryptic podocyte antigens may favor the development of de novo membranous nephropathy. Many cases of de novo membranoproliferative GN are related to hepatitis C virus infection. Patients with Alport syndrome lacking antigenic epitopes in their glomerular basement membrane may develop antibodies against these glomerular basement membrane antigens expressed in the transplanted kidney. Infection may cause acute GN to have a heterogeneous clinical presentation and outcome. De novo pauci-immune GN in renal transplant is rare. Preexisting or acquired intolerance to glucose may, in the long term, cause diabetic nephropathy. The prognosis of de novo diseases depends on the type of GN, the severity of lesions caused by the alloimmune response, or the efficacy of immunosuppressive therapy. In most cases, the management

  4. Immunosuppression in the elderly renal allograft recipient

    DEFF Research Database (Denmark)

    Montero, Nuria; Pérez-Sáez, María José; Pascual, Julio

    2016-01-01

    avoidance using mycophenolate mofetil (MMF). Observational cohort studies looked at different antibody induction strategies, calcineurin-inhibitors based maintenance immunosuppression, calcineurin-inhibitor-free sirolimus-based therapy and use of MMF versus azathioprine. Treatment with interleukin-2...... receptor antibody induction, calcineurin-inhibitor minimization with MMF and steroid minimization is advisable in the low immunologic risk elderly recipient, considering the increased risk of toxicities, infection and malignancies. In the high immunologic risk elderly recipient, taking into account...... the morbid consequences of acute rejection in the elderly, observational studies support antibody induction with depletive antibodies, calcineurin-inhibitor, MMF and steroids; calcineurin-inhibitor-minimization is not recommended. CONCLUSIONS: There is very limited evidence for the benefits and harms...

  5. De Novo Glutamine Synthesis

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    Qiao He MD

    2016-04-01

    Full Text Available Purpose: The aim of this study was to investigate the role of de novo glutamine (Gln synthesis in the proliferation of C6 glioma cells and its detection with 13N-ammonia. Methods: Chronic Gln-deprived C6 glioma (0.06C6 cells were established. The proliferation rates of C6 and 0.06C6 cells were measured under the conditions of Gln deprivation along with or without the addition of ammonia or glutamine synthetase (GS inhibitor. 13N-ammonia uptake was assessed in C6 cells by gamma counting and in rats with C6 and 0.06C6 xenografts by micro–positron emission tomography (PET scanning. The expression of GS in C6 cells and xenografts was assessed by Western blotting and immunohistochemistry, respectively. Results: The Gln-deprived C6 cells showed decreased proliferation ability but had a significant increase in GS expression. Furthermore, we found that low concentration of ammonia was sufficient to maintain the proliferation of Gln-deprived C6 cells, and 13N-ammonia uptake in C6 cells showed Gln-dependent decrease, whereas inhibition of GS markedly reduced the proliferation of C6 cells as well as the uptake of 13N-ammoina. Additionally, microPET/computed tomography exhibited that subcutaneous 0.06C6 xenografts had higher 13N-ammonia uptake and GS expression in contrast to C6 xenografts. Conclusion: De novo Gln synthesis through ammonia–glutamate reaction plays an important role in the proliferation of C6 cells. 13N-ammonia can be a potential metabolic PET tracer for Gln-dependent tumors.

  6. De novo trisomy 16p

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    Juan, J.L.C.; Cigudosa, J.C.; Gomez, A.O. [Univ. of La Laguna, Tenerife, Canary Islands (Spain)] [and others

    1997-01-20

    We report on a patient with psychomotor retardation and a pattern of malformations comprising single umbilical artery, craniofacial anomalies, severe truncal hypotonia, and lower-limb hyporreflexia. G-banding cytogenetics demonstrated a 16p+ chromosome. Parental chromosomes were normal. The use of fluorescent in situ hybridization (FISH) showed that this extra material derived from chromosome 16. High-resolution G-banding demonstrated a duplicated segment on the 16p arm, confirming our suspicion of a de novo tandem duplication; hence, the cytogenetic diagnosis was given as 46,XY,dir dup(16)(p11.2{r_arrow}p12). 9 refs., 3 figs.

  7. De novo hepatitis B infection after liver transplantation: source of disease, incidence, and impact.

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    Fabia, R; Levy, M F; Crippin, J; Tillery, W; Netto, G J; Aguanno, J; Dysert, P; Goldstein, R M; Husberg, B S; Gonwa, T A; Klintmalm, G B

    1998-03-01

    New-onset hepatitis B (de novo B) after liver transplantation (OLTX) is an emerging concern. The goals of our study were to determine the incidence and pattern of this infection, to attempt determination of risk factors and the role of immunosuppression, and to review its morbidity/mortality. Over a 10-year period, 1078 OLTX were performed in 956 patients at our institution. Eight hundred twenty-six patients had proven negative hepatitis B surface antigen (HBsAg) before transplantation. Among these, 14 patients (1.7%), 8 women and 6 men, ages 21-59 years (median, 42 years), developed positive HBsAg after transplantation and were defined as de novo B. In 10 of 14 patients (71%), positive HBsAg was revealed during routine annual visits, whereas 4 patients had titer verification prompted by illness. Blood product use (cryoprecipitate, fresh-frozen plasma, platelets, and packed red blood cells) during the transplant hospitalization was similar between groups. Pretransplant hepatitis C infection was more prevalent among the 14 patients with de-novo B (7 of 14, 50% v 129 of 812, 16%; P infected patients is 24 (5-51) months. Twelve of these 14 de novo B patients were not clinically ill, with normal or near-normal transaminase levels. One of 14 has died from complications related to hepatic artery revascularization, and another is well after repeat OLTX for biliary strictures. Half of these de novo B patients remain free from viral antigens in their transplanted liver tissue. The high percentage of positive hepatitis C patients who acquire de novo B may indicate a link between these two viral infections and potential risk factor for de novo B. The origins of this infection are most likely multifactorial, needing further study. De novo B after liver transplantation is preliminarily associated with little clinical morbidity and mortality.

  8. De novo Transcriptome Analysis in Perennial Ryegrass

    DEFF Research Database (Denmark)

    Farrell, Jacqueline Danielle; Byrne, Stephen; Asp, Torben

    selection will be the availability of a reference genome, and efforts are underway within our group to deliver this. An important step in de novo assembly will be defining the gene set, and the availability of transcriptome sequencing data will greatly aid gene prediction and validation, and the development...... of functional markers for improved ryegrass breeding. Therefore, the goal of this study is to analyze a de novo assembly of the perennial ryegrass transcriptome from the same inbred genotype being used for de novo genome assembly. Furthermore, we also conducted de novo transcriptome assembly with other......Perennial ryegrass (Lolium perenne L.) is an important grass species for both forage and amenity purposes for temperate regions worldwide. It is envisaged that breeding efforts may be enhanced with the assistance of new breeding technologies such as genomic selection. A major step towards genomic...

  9. Challenging immunosuppression treatment in lung transplant recipients with kidney failure.

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    Högerle, Benjamin A; Kohli, Neeraj; Habibi-Parker, Kirsty; Lyster, Haifa; Reed, Anna; Carby, Martin; Zeriouh, Mohamed; Weymann, Alexander; Simon, André R; Sabashnikov, Anton; Popov, Aron-Frederik; Soresi, Simona

    2016-03-01

    Kidney failure after lung transplantation is a risk factor for chronic kidney disease. Calcineurin inhibitors are immunosuppressants which play a major role in terms of postoperative kidney failure after lung transplantation. We report our preliminary experience with the anti-interleukin-2 monoclonal antibody Basiliximab utilized as a "calcineurin inhibitor-free window" in the setting of early postoperative kidney failure after lung transplantation. Between 2012 and 2015 nine lung transplant patients who developed kidney failure for more than 14 days were included. Basiliximab was administrated in three doses (Day 0, 4, and 20) whilst Tacrolimus was discontinued or reduced to maintain a serum level between 2 and 4 ng/mL. Baseline glomerular filtration rate pre transplant was normal for all patients. Seven patients completely recovered from kidney failure (67%, mean eGFR pre and post Basiliximab: 42.3 mL/min/1.73 m(2) and 69 mL/min/1.73 m(2)) and were switched back on Tacrolimus. Only one of these patients still needs ongoing renal replacement therapy. Two patients showed no recovery from kidney failure and did not survive. Basiliximab might be a safe and feasible therapeutical option in patients which are affected by calcineurin inhibitor-related kidney failure in the early post lung transplant period. Further studies are necessary to confirm our preliminary results.

  10. Safety, tolerability, and efficacy of everolimus in de novo liver transplant recipients : 12-and 36-month results

    NARCIS (Netherlands)

    Levy, Gary; Schmidli, Heinz; Punch, Jeffrey; Tuttle-Newhall, Elizabeth; Mayer, David; Neuhaus, Peter; Samuel, Didier; Nashan, Bjorn; Klempnauer, Juergen; Langnas, Alan; Calmus, Yvon; Rogiers, Xavier; Abecassis, Michael; Freeman, Richard; Sloof, Maarten; Roberts, John

    2006-01-01

    Everolimus is a macrolide immunosuppressive agent with known consistent absorption. In this double-blind study, we examined the safety and tolerability of everolimus vs. placebo in de novo liver transplant recipients. One hundred and nineteen liver allograft recipients were randomized to 1 of 4 grou

  11. Proteomics-grade de novo sequencing approach

    DEFF Research Database (Denmark)

    Savitski, Mikhail M; Nielsen, Michael L; Kjeldsen, Frank

    2005-01-01

    known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach...... is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65...

  12. De novo mutations in epileptic encephalopathies

    NARCIS (Netherlands)

    Allen, Andrew S.; Berkovic, Samuel F.; Cossette, Patrick; Delanty, Norman; Dlugos, Dennis; Eichler, Evan E.; Epstein, Michael P.; Glauser, Tracy; Goldstein, David B.; Han, Yujun; Heinzen, Erin L.; Hitomi, Yuki; Howell, Katherine B.; Johnson, Michael R.; Kuzniecky, Ruben; Lowenstein, Daniel H.; Lu, Yi-Fan; Madou, Maura R. Z.; Marson, Anthony G.; Mefford, Heather C.; Nieh, Sahar Esmaeeli; O'Brien, Terence J.; Ottman, Ruth; Petrovski, Slave; Poduri, Annapurna; Ruzzo, Elizabeth K.; Scheffer, Ingrid E.; Sherr, Elliott H.; Yuskaitis, Christopher J.; Abou-Khalil, Bassel; Alldredge, Brian K.; Bautista, Jocelyn F.; Berkovic, Samuel F.; Boro, Alex; Cascino, Gregory D.; Consalvo, Damian; Crumrine, Patricia; Devinsky, Orrin; Dlugos, Dennis; Epstein, Michael P.; Fiol, Miguel; Fountain, Nathan B.; French, Jacqueline; Friedman, Daniel; Geller, Eric B.; Glauser, Tracy; Glynn, Simon; Haut, Sheryl R.; Hayward, Jean; Helmers, Sandra L.; Joshi, Sucheta; Kanner, Andres; Kirsch, Heidi E.; Knowlton, Robert C.; Kossoff, Erich; Kuperman, Rachel; Kuzniecky, Ruben; Lowenstein, Daniel H.; McGuire, Shannon M.; Motika, Paul V.; Novotny, Edward J.; Ottman, Ruth; Paolicchi, Juliann M.; Parent, Jack M.; Park, Kristen; Poduri, Annapurna; Scheffer, Ingrid E.; Shellhaas, Renee A.; Sherr, Elliott H.; Shih, Jerry J.; Singh, Rani; Sirven, Joseph; Smith, Michael C.; Sullivan, Joseph; Thio, Liu Lin; Venkat, Anu; Vining, Eileen P. G.; Von Allmen, Gretchen K.; Weisenberg, Judith L.; Widdess-Walsh, Peter; Winawer, Melodie R.

    2013-01-01

    Epileptic encephalopathies are a devastating group of severe childhood epilepsy disorders for which the cause is often unknown(1). Here we report a screen for de novo mutations in patients with two classical epileptic encephalopathies: infantile spasms (n = 149) and Lennox-Gastaut syndrome (n = 115)

  13. Development of de novo major involvement during follow-up in Behçet's syndrome.

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    Talarico, Rosaria; Cantarini, Luca; d'Ascanio, Anna; Figus, Michele; Favati, Benedetta; Baldini, Chiara; Tani, Chiara; Neri, R; Bombardieri, Stefano; Mosca, Marta

    2016-01-01

    The primary aim of the study was to evaluate the incidence of de novo major involvement during follow-up in a cohort of patients with Behçet's syndrome (BS); the secondary aim was to analyse the epidemiological profile and the long-term outcome of those patients who developed new major involvement. Among our cohort of 120 BS patients, we evaluated all subjects who had no major organ involvement during the early years of their disease; specifically, at disease onset, the 52% of the cohort presented a prevalent mucocutaneous involvement. The primary outcomes were represented by the following: Hatemi et al. (Rheum Dis Clin North Am 39(2):245-61, 2013) the incidence of de novo major involvement during the follow-up and Hatemi et al. (Clin Exp Rheumatol 32(4 Suppl 84):S112-22, 2014) the use of immunosuppressive drugs during the follow-up. We have defined the development of de novo major involvement during the follow-up as the occurrence of severe ocular, vascular or CNS involvement after a latency period from the diagnosis of at least 3 years. Among 62 patients characterized by a mild onset of disease, we observed that after at least 3 years from the diagnosis, 21 BS patients (34%) still developed serious morbidities. Specifically, three patients developed ocular involvement, nine patients developed neurological involvement and nine patients presented vascular involvement. Comparing main epidemiological and clinical findings of the two groups, we observed that patients who developed de novo major involvement were more frequently males and younger; furthermore, 95% of these patients were characterized by a young onset of disease (p < 0.001). Being free of major organ complication in the first years of BS is not necessary a sign of a favourable outcome. Globally, the development of de novo major involvement during the coursfce of BS suggests that a tight control is strongly recommended during the course of the disease.

  14. Incidence, risk factors and outcomes of de novo malignancies post liver transplantation

    Institute of Scientific and Technical Information of China (English)

    Pavan; Kedar; Mukthinuthalapati; Raghavender; Gotur; Marwan; GhabrilMarwan; Ghabril

    2016-01-01

    Liver transplantation(LT) is associated with a 2 to7 fold higher, age and gender adjusted, risk of de novo malignancy. The overall incidence of de novo malignancy post LT ranges from 2.2% to 26%, and 5 and 10 years incidence rates are estimated at 10% to 14.6% and 20% to 32%, respectively. The main risk factors for de novo malignancy include immunosuppression with impaired immunosurveillance, and a number of patient factors which include; age, latent oncogenic viral infections, tobacco and alcohol use history, and underlying liver disease. The most common cancers after LT are non-melanoma skin cancers, accounting for approximately 37% of de novo malignancies, with a noted increase in the ratio of squamous to basal cell cancers. While these types of skin cancer do not impact patient survival, post-transplant lymphoproliferative disorders and solid organ cancer, accounting for 25% and 48% of malignancies, are associated with increased mortality. Patients developing these types of cancer are diagnosed at more advanced stages, and their cancers behave more aggressively compared with the general population. Patients undergoing LT for primary sclerosing cholangitis(particularly with inflammatory bowel disease) and alcoholic liver disease have high rates of malignancies compared with patients undergoing LT for other indications. These populations are at particular risk for gastrointestinal and aerodigestive cancers respectively. Counseling smoking cessation, skin protection from sun exposure and routine clinical follow-up are the current approach in practice. There are no standardized surveillance protocol, but available data suggests that regimented surveillance strategies are needed and capable of yielding cancer diagnosis at earlier stages with better resulting survival. Evidence-based strategies are needed to guide optimal surveillance and safe minimization of immunosuppression.

  15. De novo psychogenic seizures after epilepsy surgery: case report

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    MONTENEGRO MARIA AUGUSTA

    2000-01-01

    Full Text Available The occurrence of de novo psychogenic seizures after epilepsy surgery is rare, and is estimated in 1.8% to 3.6%. Seizures after epilepsy surgery should be carefully evaluated, and de novo psychogenic seizures should be considered especially when there is a change in the ictal semiology. We report a patient with de novo psychogenic seizures after anterior temporal lobe removal for refractory temporal lobe epilepsy. Once psychogenic seizures were diagnosed and psychiatric treatment was started, seizures stopped.

  16. Evolution and function of de novo originated genes.

    Science.gov (United States)

    Wu, Dong-Dong; Zhang, Ya-Ping

    2013-05-01

    De novo origination has recently been appreciated to be an important mechanism contributing to the origin of genes. Evidence indicates that de novo originated genes can evolve important and even essential functions rapidly. We present an "adaptation following neutrality" process to explain the evolution of essential function of new genes. How new de novo originated genes become involved in pathways and interact with other old genes, and the exact functions of these new genes, however, remains largely undocumented. Examinations of the function of de novo origin and the function of noncoding RNA genes should become more frequent and appreciated in the future studies.

  17. Donor-transmitted, donor-derived, and de novo cancer after liver transplant.

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    Chapman, Jeremy R; Lynch, Stephen V

    2014-03-01

    Cancer is the third most common cause of death (after cardiovascular disease and infection) for patients who have a functioning kidney allograft. Kidney and liver transplant recipients have similar cancer risks because of immunosuppression but different risks because of differences in primary diseases that cause renal and hepatic failure and the inherent behavior of cancers in the liver. There are 4 types of cancer that may develop in liver allograft recipients: (1) recurrent cancer, (2) donor-transmitted cancer, (3) donor-derived cancer, and (4) de novo cancer. Identification of potential donor cancer transmission may occur at postmortem examination of a deceased donor or when a probable donor-transmitted cancer is identified in another recipient. Donor-transmitted cancer after liver transplant is rare in Australia, the United Kingdom, and the United States. Aging of the donor pool may increase the risk of subclinical cancer in donors. Liver transplant recipients have a greater risk of de novo cancer than the general population, and risk factors for de novo cancer in liver transplant recipients include primary sclerosing cholangitis, alcoholic liver disease, smoking, and increased age. Liver transplant recipients may benefit from cancer screening because they have a high risk, are clearly identifiable, and are under continuous medical supervision.

  18. De novo autoimmune hepatitis after liver transplantation.

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    Lohse, Ansgar W; Weiler-Norman, Christina; Burdelski, Martin

    2007-10-01

    The Kings College group was the first to describe a clinical syndrome similar to autoimmune hepatitis in children and young adults transplanted for non-immune mediated liver diseases. They coined the term "de novo autoimmune hepatitis". Several other liver transplant centres confirmed this observation. Even though the condition is uncommon, patients with de novo AIH are now seen in most of the major transplant centres. The disease is usually characterized by features of acute hepatitis in otherwise stable transplant recipients. The most characteristic laboratory hallmark is a marked hypergammaglobulinaemia. Autoantibodies are common, mostly ANA. We described also a case of LKM1-positivity in a patients transplanted for Wilson's disease, however this patients did not develop clinical or histological features of AIH. Development of SLA/LP-autoantibodies is also not described. Therefore, serologically de novo AIH appears to correspond to type 1 AIH. Like classical AIH patients respond promptly to treatment with increased doses of prednisolone and azathioprine, while the calcineurin inhibitors cyclosporine or tacrolimus areof very limited value - which is not surprising, as almost all patients develop de novo AIH while receiving these drugs. Despite the good response to treatment, most patients remain a clinical challenge as complete stable remissions are uncommon and flares, relapses and chronic disease activity can often occur. Pathogenetically this syndrome is intriguing. It is not clear, if the immune response is directed against allo-antigens, neo-antigens in the liver, or self-antigens, possibly shared by donor and host cells. It is very likely that the inflammatory milieu due to alloreactive cells in the transplanted organ contribute to the disease process. Either leading to aberrant antigen presentation, or providing co-stimulatory signals leading to the breaking of self-tolerance. The development of this disease in the presence of treatment with calcineurin

  19. De novo computer-aided design of novel antiviral agents.

    Science.gov (United States)

    Massarotti, Alberto; Coluccia, Antonio; Sorba, Giovanni; Silvestri, Romano; Brancale, Andrea

    2012-01-01

    Computer-aided drug design techniques have become an integral part of the drug discovery process. In particular, de novo methodologies can be useful to identify putative ligands for a specific target relying only on the structural information of the target itself. Here we discuss the basic de novo approaches available and their application in antiviral drug design.:

  20. Frequency and Complexity of De Novo Structural Mutation in Autism

    Science.gov (United States)

    Brandler, William M.; Antaki, Danny; Gujral, Madhusudan; Noor, Amina; Rosanio, Gabriel; Chapman, Timothy R.; Barrera, Daniel J.; Lin, Guan Ning; Malhotra, Dheeraj; Watts, Amanda C.; Wong, Lawrence C.; Estabillo, Jasper A.; Gadomski, Therese E.; Hong, Oanh; Fajardo, Karin V. Fuentes; Bhandari, Abhishek; Owen, Renius; Baughn, Michael; Yuan, Jeffrey; Solomon, Terry; Moyzis, Alexandra G.; Maile, Michelle S.; Sanders, Stephan J.; Reiner, Gail E.; Vaux, Keith K.; Strom, Charles M.; Zhang, Kang; Muotri, Alysson R.; Akshoomoff, Natacha; Leal, Suzanne M.; Pierce, Karen; Courchesne, Eric; Iakoucheva, Lilia M.; Corsello, Christina; Sebat, Jonathan

    2016-01-01

    Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement. PMID:27018473

  1. Novel variation and de novo mutation rates in population-wide de novo assembled Danish trios

    DEFF Research Database (Denmark)

    Besenbacher, Søren; Liu, Siyang; Gonzalez-Izarzugaza, Jose Maria;

    2015-01-01

    sequencing 10 trios to high depth (50). We report 536k novelSNVs and 283k novel short indels from mapping approaches and develop a population-widede novo assembly approach to identify 132k novel indels larger than 10 nucleotides with lowfalse discovery rates. We identify a higher proportion of indels and SVs...... than previous effortsshowing the merits of high coverage and de novo assembly approaches. In addition, we usetrio information to identify de novo mutations and use a probabilistic method to providedirect estimates of 1.27e8 and 1.5e9 per nucleotide per generation for SNVs and indels, respectively.......Building a population-specific catalogue of single nucleotide variants (SNVs), indels andstructural variants (SVs) with frequencies, termed a national pan-genome, is critical forfurther advancing clinical and public health genetics in large cohorts. Here we report a Danishpan-genome obtained from...

  2. Necessidade de novos antibióticos

    OpenAIRE

    Brito,Monique Araujo de; Cordeiro, Benedito Carlos

    2012-01-01

    Os antibióticos são uma classe de fármacos indispensável; sem eles haveria perdas na expectativa de vida que conquistamos ao longo de décadas. Mas a potência dos antibióticos está ameaçada pela resistência bacteriana. Há uma explosão de casos reportados na literatura. De fato, o aumento da resistência bacteriana, principalmente entre patógenos potencialmente perigosos, tem levado ao aumento na necessidade de novos fármacos e novas classes de antibióticos, tanto para infecções adquiridas em ho...

  3. De Novo Kidney Regeneration with Stem Cells

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    Shinya Yokote

    2012-01-01

    Full Text Available Recent studies have reported on techniques to mobilize and activate endogenous stem-cells in injured kidneys or to introduce exogenous stem cells for tissue repair. Despite many recent advantages in renal regenerative therapy, chronic kidney disease (CKD remains a major cause of morbidity and mortality and the number of CKD patients has been increasing. When the sophisticated structure of the kidneys is totally disrupted by end stage renal disease (ESRD, traditional stem cell-based therapy is unable to completely regenerate the damaged tissue. This suggests that whole organ regeneration may be a promising therapeutic approach to alleviate patients with uncured CKD. We summarize here the potential of stem-cell-based therapy for injured tissue repair and de novo whole kidney regeneration. In addition, we describe the hurdles that must be overcome and possible applications of this approach in kidney regeneration.

  4. From immunosuppression to tolerance.

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    Adams, David H; Sanchez-Fueyo, Alberto; Samuel, Didier

    2015-04-01

    The past three decades have seen liver transplantation becoming a major therapeutic approach in the management of end-stage liver diseases. This is due to the dramatic improvement in survival after liver transplantation as a consequence of the improvement of surgical and anaesthetic techniques, of post-transplant medico-surgical management and of prevention of disease recurrence and other post-transplant complications. Improved use of post-transplant immunosuppression to prevent acute and chronic rejection is a major factor in these improved results. The liver has been shown to be more tolerogenic than other organs, and matching of donor and recipients is mainly limited to ABO blood group compatibility. However, long-term immunosuppression is required to avoid severe acute and chronic rejection and graft loss. With the current immunosuppression protocols, the risk of acute rejection requiring additional therapy is 10-40% and the risk of chronic rejection is below 5%. However, the development of histological lesions in the graft in long-term survivors suggest atypical forms of graft rejection may develop as a consequence of under-immunosuppression. The backbone of immunosuppression remains calcineurin inhibitors (CNI) mostly in association with steroids in the short-term and mycophenolate mofetil or mTOR inhibitors (everolimus). The occurrence of post-transplant complications related to the immunosuppressive therapy has led to the development of new protocols aimed at protecting renal function and preventing the development of de novo cancer and of dysmetabolic syndrome. However, there is no new class of immunosuppressive drugs in the pipeline able to replace current protocols in the near future. The aim of a full immune tolerance of the graft is rarely achieved since only 20% of selected patients can be weaned successfully off immunosuppression. In the future, immunosuppression will probably be more case oriented aiming to protect the graft from rejection and at

  5. De Novo Collapsing Glomerulopathy in a Renal Allograft Recipient

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    Kanodia K

    2008-01-01

    Full Text Available Collapsing glomerulopathy (CG, characterized histologically by segmental/global glomerular capillary collapse, podocyte hypertrophy and hypercellularity and tubulo-interstitial injury; is characterized clinically by massive proteinuria and rapid progressive renal failure. CG is known to recur in renal allograft and rarely de novo. We report de novo CG 3 years post-transplant in a patient who received renal allograft from haplo-identical type donor.

  6. Genovo: De Novo Assembly for Metagenomes

    Science.gov (United States)

    Laserson, Jonathan; Jojic, Vladimir; Koller, Daphne

    Next-generation sequencing technologies produce a large number of noisy reads from the DNA in a sample. Metagenomics and population sequencing aim to recover the genomic sequences of the species in the sample, which could be of high diversity. Methods geared towards single sequence reconstruction are not sensitive enough when applied in this setting. We introduce a generative probabilistic model of read generation from environmental samples and present Genovo, a novel de novo sequence assembler that discovers likely sequence reconstructions under the model. A Chinese restaurant process prior accounts for the unknown number of genomes in the sample. Inference is made by applying a series of hill-climbing steps iteratively until convergence. We compare the performance of Genovo to three other short read assembly programs across one synthetic dataset and eight metagenomic datasets created using the 454 platform, the largest of which has 311k reads. Genovo's reconstructions cover more bases and recover more genes than the other methods, and yield a higher assembly score.

  7. Novor: real-time peptide de novo sequencing software.

    Science.gov (United States)

    Ma, Bin

    2015-11-01

    De novo sequencing software has been widely used in proteomics to sequence new peptides from tandem mass spectrometry data. This study presents a new software tool, Novor, to greatly improve both the speed and accuracy of today's peptide de novo sequencing analyses. To improve the accuracy, Novor's scoring functions are based on two large decision trees built from a peptide spectral library with more than 300,000 spectra with machine learning. Important knowledge about peptide fragmentation is extracted automatically from the library and incorporated into the scoring functions. The decision tree model also enables efficient score calculation and contributes to the speed improvement. To further improve the speed, a two-stage algorithmic approach, namely dynamic programming and refinement, is used. The software program was also carefully optimized. On the testing datasets, Novor sequenced 7%-37% more correct residues than the state-of-the-art de novo sequencing tool, PEAKS, while being an order of magnitude faster. Novor can de novo sequence more than 300 MS/MS spectra per second on a laptop computer. The speed surpasses the acquisition speed of today's mass spectrometer and, therefore, opens a new possibility to de novo sequence in real time while the spectrometer is acquiring the spectral data. Graphical Abstract ᅟ.

  8. Novor: Real-Time Peptide de Novo Sequencing Software

    Science.gov (United States)

    Ma, Bin

    2015-11-01

    De novo sequencing software has been widely used in proteomics to sequence new peptides from tandem mass spectrometry data. This study presents a new software tool, Novor, to greatly improve both the speed and accuracy of today's peptide de novo sequencing analyses. To improve the accuracy, Novor's scoring functions are based on two large decision trees built from a peptide spectral library with more than 300,000 spectra with machine learning. Important knowledge about peptide fragmentation is extracted automatically from the library and incorporated into the scoring functions. The decision tree model also enables efficient score calculation and contributes to the speed improvement. To further improve the speed, a two-stage algorithmic approach, namely dynamic programming and refinement, is used. The software program was also carefully optimized. On the testing datasets, Novor sequenced 7%-37% more correct residues than the state-of-the-art de novo sequencing tool, PEAKS, while being an order of magnitude faster. Novor can de novo sequence more than 300 MS/MS spectra per second on a laptop computer. The speed surpasses the acquisition speed of today's mass spectrometer and, therefore, opens a new possibility to de novo sequence in real time while the spectrometer is acquiring the spectral data.

  9. Genetic variation and the de novo assembly of human genomes.

    Science.gov (United States)

    Chaisson, Mark J P; Wilson, Richard K; Eichler, Evan E

    2015-11-01

    The discovery of genetic variation and the assembly of genome sequences are both inextricably linked to advances in DNA-sequencing technology. Short-read massively parallel sequencing has revolutionized our ability to discover genetic variation but is insufficient to generate high-quality genome assemblies or resolve most structural variation. Full resolution of variation is only guaranteed by complete de novo assembly of a genome. Here, we review approaches to genome assembly, the nature of gaps or missing sequences, and biases in the assembly process. We describe the challenges of generating a complete de novo genome assembly using current technologies and the impact that being able to perfectly sequence the genome would have on understanding human disease and evolution. Finally, we summarize recent technological advances that improve both contiguity and accuracy and emphasize the importance of complete de novo assembly as opposed to read mapping as the primary means to understanding the full range of human genetic variation.

  10. Installing hydrolytic activity into a completely de novo protein framework

    Science.gov (United States)

    Burton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

  11. De novo copy number variations in cloned dogs from the same nuclear donor

    OpenAIRE

    Jung, Seung-Hyun; Yim, Seon-Hee; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Kim, Tae-Min; Kim, Jin-Soo; Lee, Byeong Chun; Chung, Yeun-Jun

    2013-01-01

    Background Somatic mosaicism of copy number variants (CNVs) in human body organs and de novo CNV event in monozygotic twins suggest that de novo CNVs can occur during mitotic recombination. These de novo CNV events are important for understanding genetic background of evolution and diverse phenotypes. In this study, we explored de novo CNV event in cloned dogs with identical genetic background. Results We analyzed CNVs in seven cloned dogs using the nuclear donor genome as reference by array-...

  12. Point mutations as a source of de novo genetic disease

    NARCIS (Netherlands)

    Ligt, J. de; Veltman, J.A.; Vissers, L.E.L.M.

    2013-01-01

    Family-based next generation sequencing (NGS) has recently pointed to an important role for de novo germline point mutations in both rare and common genetic disorders associated with reduced fitness. In this review we highlight the impact of the mutational target size on the frequency of diseases ca

  13. De novo synthesis of milk triglycerides in humans

    Science.gov (United States)

    Mammary gland (MG) de novo lipogenesis contributes significantly to milk fat in animals but little is known in humans. Objective: To test the hypothesis that the incorporation of 13C carbons from [U-13C]glucose into fatty acids (FA) and glycerol in triglycerides (TG) will be greater: 1) in milk tha...

  14. Genes from scratch--the evolutionary fate of de novo genes.

    Science.gov (United States)

    Schlötterer, Christian

    2015-04-01

    Although considered an extremely unlikely event, many genes emerge from previously noncoding genomic regions. This review covers the entire life cycle of such de novo genes. Two competing hypotheses about the process of de novo gene birth are discussed as well as the high death rate of de novo genes. Despite the high death rate, some de novo genes are retained and remain functional, even in distantly related species, through their integration into gene networks. Further studies combining gene expression with ribosome profiling in multiple populations across different species will be instrumental for an improved understanding of the evolutionary processes operating on de novo genes.

  15. Monitoring of nuclear factor of activated T-cell-regulated gene expression in de novo and long-term liver transplant recipients treated with cyclosporine a.

    Science.gov (United States)

    Herden, Uta; Kromminga, Arno; Hagel, Christine; Hartleb, Jürgen; Nashan, Björn; Sterneck, Martina; Fischer, Lutz

    2011-04-01

    Pharmacodynamic drug monitoring might allow an improved use of immunosuppressive medication in transplant recipients. We assessed whether drug concentrations reflect the effect of cyclosporine (CsA) on expression of nuclear factor of activated T-cells-regulated cytokines. CsA drug concentrations and expression of interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor in stimulated blood lymphocytes were determined predose (C0) and 2 hours after (C2) CsA intake in 20 de novo (less than 3 months) and 20 long-term (3 months to 10 years) liver transplant patients. The residual cytokine expression at C2 relative to C0 was calculated. Mean CsA C0 and C2 concentrations were 236 and 776 μg/L in de novo and 100 and 573 μg/L in long-term liver transplant patients, respectively. Two hours after CsA intake, the residual cytokine expression for all cytokines was comparable in both groups (de novo patients mean 16%; long-term patients mean 17%). CsA C2 concentrations showed a significant (P < 0.01) correlation with the residual cytokine expression of interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor in both de novo and long-term patients, whereas CsA C0 concentrations did not. The data suggest that CsA C2 concentrations, but not C0 concentrations, reflect the effect of CsA on downregulation of cytokine expression in both de novo and long-term liver transplant patients.

  16. Does Papillary Carcinoma of Thyroglossal Duct Cyst Develop De Novo?

    Directory of Open Access Journals (Sweden)

    Tekin Baglam

    2015-01-01

    Full Text Available Backround. Thyroglossal duct cyst (TDC is a developmental abnormality of the thyroid gland. Due to embryological remnants of thyroid tissue located in the TDC, the same malignant tumors that develop in the thyroid gland can also develop in the TDC. Methods. We present the unique case of a 39-year-old female with simultaneous de novo papillary carcinoma in a TDC and the thyroid gland. Results. With the suspicion of simultaneous papillary carcinoma in the TDC and the thyroid gland, Sistrunk procedure with total thyroidectomy and central neck exploration was performed. Conclusion. The clinician should have a high index of suspicion upon encountering papillary carcinoma of the TDC to differentiate de novo papillary carcinoma in the TDC from those originating from the thyroid gland, because papillary carcinoma in TDC may originate from an occult thyroid papillary carcinoma.

  17. Does Papillary Carcinoma of Thyroglossal Duct Cyst Develop De Novo?

    Science.gov (United States)

    Baglam, Tekin; Binnetoglu, Adem; Yumusakhuylu, Ali Cemal; Demir, Berat; Askan, Gokce; Sari, Murat

    2015-01-01

    Backround. Thyroglossal duct cyst (TDC) is a developmental abnormality of the thyroid gland. Due to embryological remnants of thyroid tissue located in the TDC, the same malignant tumors that develop in the thyroid gland can also develop in the TDC. Methods. We present the unique case of a 39-year-old female with simultaneous de novo papillary carcinoma in a TDC and the thyroid gland. Results. With the suspicion of simultaneous papillary carcinoma in the TDC and the thyroid gland, Sistrunk procedure with total thyroidectomy and central neck exploration was performed. Conclusion. The clinician should have a high index of suspicion upon encountering papillary carcinoma of the TDC to differentiate de novo papillary carcinoma in the TDC from those originating from the thyroid gland, because papillary carcinoma in TDC may originate from an occult thyroid papillary carcinoma.

  18. De novo mutations in ataxin-2 gene and ALS risk.

    Directory of Open Access Journals (Sweden)

    José Miguel Laffita-Mesa

    Full Text Available Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2 is the genetic cause of spinocerebellar ataxia type 2 (SCA2. Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS. Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.

  19. Revisiting de novo drug design: receptor based pharmacophore screening.

    Science.gov (United States)

    Amaravadhi, Harikishore; Baek, Kwanghee; Yoon, Ho Sup

    2014-01-01

    De novo drug design methods such as receptor or protein based pharmacophore modeling present a unique opportunity to generate novel ligands by employing the potential binding sites even when no explicit ligand information is known for a particular target. Recent developments in molecular modeling programs have enhanced the ability of early programs such as LUDI or Pocket that not only identify the key interactions or hot spots at the suspected binding site, but also and convert these hot spots into three-dimensional search queries and virtual screening of the property filtered synthetic libraries. Together with molecular docking studies and consensus scoring schemes they would enrich the lead identification processes. In this review, we discuss the ligand and receptor based de novo drug design approaches with selected examples.

  20. Reasons for treating secondary AML as de novo AML

    DEFF Research Database (Denmark)

    Ostgård, Lene Sofie Granfeldt; Kjeldsen, Eigil; Holm, Mette Skov;

    2010-01-01

    ) in 77 cases (49%), CMPD (chronic myeloproliferative disorder) in 43 cases (27%) and was therapy-related AML (t-AML) in 37 cases (24%). Median age at diagnosis of AML was 69 yr in secondary cases when compared to 66 yr in de novo cases (P = 0.006). In univariate analyses, secondary AML was associated...... with an inferior complete remission (CR) rate (P = 0.008) and poorer overall survival (OS, P = 0.003) whereas in complete remitters, disease-free survival (DFS) of secondary cases was equal to that of de novo cases. Interestingly, in all further analyses of CR-rates, OS and DFS, when correcting for the influence...

  1. From de novo mutations to personalized therapeutic interventions in autism.

    Science.gov (United States)

    Brandler, William M; Sebat, Jonathan

    2015-01-01

    The high heritability, early age at onset, and reproductive disadvantages of autism spectrum disorders (ASDs) are consistent with an etiology composed of dominant-acting de novo (spontaneous) mutations. Mutation detection by microarray analysis and DNA sequencing has confirmed that de novo copy-number variants or point mutations in protein-coding regions of genes contribute to risk, and some of the underlying causal variants and genes have been identified. As our understanding of autism genes develops, the spectrum of autism is breaking up into quanta of many different genetic disorders. Given the diversity of etiologies and underlying biochemical pathways, personalized therapy for ASDs is logical, and clinical genetic testing is a prerequisite.

  2. Novel variation and de novo mutation rates in population-wide de novo assembled Danish trios

    DEFF Research Database (Denmark)

    Besenbacher, Søren; Liu, Siyang; Gonzalez-Izarzugaza, Jose Maria;

    2015-01-01

    than previous effortsshowing the merits of high coverage and de novo assembly approaches. In addition, we usetrio information to identify de novo mutations and use a probabilistic method to providedirect estimates of 1.27e8 and 1.5e9 per nucleotide per generation for SNVs and indels, respectively....

  3. De Novo Mutations in Moderate or Severe Intellectual Disability

    OpenAIRE

    Hamdan, Fadi F; Myriam Srour; Jose-Mario Capo-Chichi; Hussein Daoud; Christina Nassif; Lysanne Patry; Christine Massicotte; Amirthagowri Ambalavanan; Dan Spiegelman; Ousmane Diallo; Edouard Henrion; Alexandre Dionne-Laporte; Anne Fougerat; Pshezhetsky, Alexey V; Sunita Venkateswaran

    2014-01-01

    Genetics is believed to have an important role in intellectual disability (ID). Recent studies have emphasized the involvement of de novo mutations (DNMs) in ID but the extent to which they contribute to its pathogenesis and the identity of the corresponding genes remain largely unknown. Here, we report a screen for DNMs in subjects with moderate or severe ID. We sequenced the exomes of 41 probands and their parents, and confirmed 81 DNMs affecting the coding sequence or consensus splice site...

  4. Glucose-dependent de Novo Lipogenesis in B Lymphocytes

    Science.gov (United States)

    Dufort, Fay J.; Gumina, Maria R.; Ta, Nathan L.; Tao, Yongzhen; Heyse, Shannon A.; Scott, David A.; Richardson, Adam D.; Seyfried, Thomas N.; Chiles, Thomas C.

    2014-01-01

    Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation. PMID:24469453

  5. Effective de novo assembly of fish genome using haploid larvae.

    Science.gov (United States)

    Iwasaki, Yuki; Nishiki, Issei; Nakamura, Yoji; Yasuike, Motoshige; Kai, Wataru; Nomura, Kazuharu; Yoshida, Kazunori; Nomura, Yousuke; Fujiwara, Atushi; Kobayashi, Takanori; Ototake, Mitsuru

    2016-02-01

    Recent improvements in next-generation sequencing technology have made it possible to do whole genome sequencing, on even non-model eukaryote species with no available reference genomes. However, de novo assembly of diploid genomes is still a big challenge because of allelic variation. The aim of this study was to determine the feasibility of utilizing the genome of haploid fish larvae for de novo assembly of whole-genome sequences. We compared the efficiency of assembly using the haploid genome of yellowtail (Seriola quinqueradiata) with that using the diploid genome obtained from the dam. De novo assembly from the haploid and the diploid sequence reads (100 million reads per each datasets) generated by the Ion Proton sequencer (200 bp) was done under two different assembly algorithms, namely overlap-layout-consensus (OLC) and de Bruijn graph (DBG). This revealed that the assembly of the haploid genome significantly reduced (approximately 22% for OLC, 9% for DBG) the total number of contigs (with longer average and N50 contig lengths) when compared to the diploid genome assembly. The haploid assembly also improved the quality of the scaffolds by reducing the number of regions with unassigned nucleotides (Ns) (total length of Ns; 45,331,916 bp for haploids and 67,724,360 bp for diploids) in OLC-based assemblies. It appears clear that the haploid genome assembly is better because the allelic variation in the diploid genome disrupts the extension of contigs during the assembly process. Our results indicate that utilizing the genome of haploid larvae leads to a significant improvement in the de novo assembly process, thus providing a novel strategy for the construction of reference genomes from non-model diploid organisms such as fish.

  6. Immunosuppressive strategies and management

    Institute of Scientific and Technical Information of China (English)

    Shi-hui PAN

    2008-01-01

    have failed to adequately control chronic rejection in most of solid organ transplantation except liver transplantation.Eady post-transplant complications aye generally related to the operation,the severity of pre-operative illness,immunologic status,and the quality of the donor organ.Careful recipient and donor selection is paramount to minimize severity of disease and medical comorbidities.These early complications include allograft dysfunction,cardiovascular and hemodynamic instability,and immunosuppressive drug-induced adverse effects.Acute infection remains a common and serious early complication despite new and effective drug therapies,placing the responsibility on the clinician for early recognition and treatment.Emerging resistant bacteria and fungi require early and aggressive intervention.Unlike infection,early aUograft rejection is usually limited and manageable with the newer immunosuppressive agents.However,it must be distinguished from other causes of allograft dysfunction(ie.recurrent hepatitis C,ealcineurin induced nephrotoxicity,or infection).Recently approved Cylex@immune cell function assay allows clinicians to tailor and individualize immunosuppression to prevent organ rejection while minimizing infection and complications.Improved patient and allograft survival has enabled transplant recipients to reach milestones and return to productive lives provided they are compliant. It was also challenged the clinician to manage the long-term complications of immunosuppression therapy, adverse drug interaction, recurrent diseases and chronic allograft failure. Long-term immunosuppressive therapy places transplant recipients at risk for renal insufficiency, cardiovascular and metabolic diseases, de novo malignancies, and psychosocial challenges. The management of viral hepatitis C re-infection, chronic allograft nephropathy, vasculopathy, and obliterative bronchiolitis is currently the greatest challenges facing the transplant specialist. The management of

  7. De novo assembly of highly diverse viral populations

    Directory of Open Access Journals (Sweden)

    Yang Xiao

    2012-09-01

    Full Text Available Abstract Background Extensive genetic diversity in viral populations within infected hosts and the divergence of variants from existing reference genomes impede the analysis of deep viral sequencing data. A de novo population consensus assembly is valuable both as a single linear representation of the population and as a backbone on which intra-host variants can be accurately mapped. The availability of consensus assemblies and robustly mapped variants are crucial to the genetic study of viral disease progression, transmission dynamics, and viral evolution. Existing de novo assembly techniques fail to robustly assemble ultra-deep sequence data from genetically heterogeneous populations such as viruses into full-length genomes due to the presence of extensive genetic variability, contaminants, and variable sequence coverage. Results We present VICUNA, a de novo assembly algorithm suitable for generating consensus assemblies from genetically heterogeneous populations. We demonstrate its effectiveness on Dengue, Human Immunodeficiency and West Nile viral populations, representing a range of intra-host diversity. Compared to state-of-the-art assemblers designed for haploid or diploid systems, VICUNA recovers full-length consensus and captures insertion/deletion polymorphisms in diverse samples. Final assemblies maintain a high base calling accuracy. VICUNA program is publicly available at: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/ viral-genomics-analysis-software. Conclusions We developed VICUNA, a publicly available software tool, that enables consensus assembly of ultra-deep sequence derived from diverse viral populations. While VICUNA was developed for the analysis of viral populations, its application to other heterogeneous sequence data sets such as metagenomic or tumor cell population samples may prove beneficial in these fields of research.

  8. De Novo Assembly of Highly Substituted Morpholines and Piperazines

    Science.gov (United States)

    2017-01-01

    The morpholine and piperazine with their remarkable physical and biochemical properties are popular heterocycles in organic and medicinal chemistry used in rational property design. However, in the majority of cases these rings are added to an existing molecule in a building block approach thus limiting their substitution pattern and diversity. Here we introduce a versatile de novo synthesis of the morpholine and piperazine rings using multicomponent reaction chemistry. The large scale amenable building blocks can be further substituted at up to four positions, making this a very versatile scaffold synthesis strategy. Our methods thus fulfill the increasing demand for novel building block design and nontraditional scaffolds which previously were not accessible PMID:28102692

  9. [Three cases of de novo multiple myeloma after kidney transplantation].

    Science.gov (United States)

    Nieto-Ríos, John Fredy; Zuluaga, Mónica; Serna, Lina María; Aristizábal, Arbey; Ocampo-Kohn, Catalina; Gálvez, Kenny Mauricio; Flórez, Adriana Alejandra; Zuluaga, Gustavo

    2016-12-01

    Light chain-associated kidney compromise is frequent in patients with monoclonal gammopathies; it affects the glomeruli or the tubules, and its most common cause is multiple myeloma. It may develop after a kidney transplant due to recurrence of a preexisting multiple myeloma or it can be a de novo disease manifesting as graft dysfunction and proteinuria. A kidney biopsy is always necessary to confirm the diagnosis.We describe three cases of kidney graft dysfunction due to multiple myeloma in patients without presence of the disease before the transplant.

  10. Relapse of HCV Genotype 1b Infection After Sofosbuvir/Ledipasvir Treatment Presenting as De Novo Cryoglobulinemic Vasculitis

    Science.gov (United States)

    Moreno, Alan D.; Joseph, Nora; Kim, George; Fimmel, Claus J.

    2017-01-01

    Relapse of hepatitis C virus (HCV) genotype 1 infection after combination therapy with sofosbuvir and ledipasvir is unusual. We report a treatment-naïve, non-cirrhotic patient in whom the relapse of genotype 1b HCV infection was accompanied by de novo cryoglobulinemic vasculitis and glomerulonephritis, requiring hemodialysis for acute renal failure. Sequence analysis revealed several resistance-associated variants in the HCV NS5a gene but not in NS3/4A. The patient’s vasculitis was successfully treated with immunosuppression and plasmapheresis, followed by retreatment of HCV with a combination of sofosbuvir, simeprevir, and ribavirin. The patient achieved sustained virological response, recovered his renal function, and remains in remission from cryoglobulinemia. PMID:28184378

  11. Cyclosporine Sparing Effect of Enteric-Coated Mycophenolate Sodium in De Novo Kidney Transplantation

    Science.gov (United States)

    Lee, Su Hyung; Oh, Chang-Kwon; Kim, Myoung Soo; Kim, Sung Joo; Ha, Jongwon

    2017-01-01

    Purpose The increased tolerability of enteric-coated mycophenolate sodium (EC-MPS), compared to mycophenolate mofetil, among kidney transplant recipients has the potential to facilitate cyclosporine (CsA) minimization. Therefore, a prospective trial to determine the optimum EC-MPS dose in CsA-based immunosuppression regimens is necessary. Materials and Methods A comparative, parallel, randomized, open-label study was performed for 140 patients from four centers to compare the efficacy and tolerability of low dose CsA with standard dose EC-MPS (the investigational group) versus standard dose CsA with low dose EC-MPS (the control group) for six months in de novo kidney transplant recipients. Graft function, the incidence of efficacy failure [biopsy-confirmed acute rejection (BCAR), death, graft loss, loss to follow-up], and adverse events were compared. Results The mean estimated glomerular filtration rate (eGFR) of the investigational group at six months post-transplantation was non-inferior to that of the control group (confidence interval between 57.3 mL/min/1.73m2 and 67.4 mL/min/1.73 m2, p0.05) in the incidence of discontinuations and serious adverse events (SAE) between the groups. Conclusion CsA minimization using a standard dose of EC-MPS kept the incidence of acute rejection and additional risks as low as conventional immunosuppression and provided therapeutic equivalence in terms of renal graft function and safety issues. PMID:27873516

  12. Post-zygotic Point Mutations Are an Underrecognized Source of De Novo Genomic Variation

    NARCIS (Netherlands)

    Acuna-Hidalgo, R.; Bo, T.; Kwint, M.P.; Vorst, M. van de; Pinelli, M.; Veltman, J.A.; Hoischen, A.; Vissers, L.E.L.M.; Gilissen, C.

    2015-01-01

    De novo mutations are recognized both as an important source of genetic variation and as a prominent cause of sporadic disease in humans. Mutations identified as de novo are generally assumed to have occurred during gametogenesis and, consequently, to be present as germline events in an individual.

  13. 76 FR 61103 - Draft Guidance for Industry and Food and Drug Administration Staff; De Novo Classification...

    Science.gov (United States)

    2011-10-03

    ... Food, Drug, and Cosmetic Act (FD&C Act), also known as the de novo classification process. FDA is... HUMAN SERVICES Food and Drug Administration Draft Guidance for Industry and Food and Drug Administration Staff; De Novo Classification Process (Evaluation of Automatic Class III Designation);...

  14. A simple method suitable to study de novo root organogenesis

    Directory of Open Access Journals (Sweden)

    Xiaodong eChen

    2014-05-01

    Full Text Available De novo root organogenesis is the process in which adventitious roots regenerate from detached or wounded plant tissues or organs. In tissue culture, appropriate types and concentrations of plant hormones in the medium are critical for inducing adventitious roots. However, in natural conditions, regeneration from detached organs is likely to rely on endogenous hormones. To investigate the actions of endogenous hormones and the molecular mechanisms guiding de novo root organogenesis, we developed a simple method to imitate natural conditions for adventitious root formation by culturing Arabidopsis thaliana leaf explants on B5 medium without additive hormones. Here we show that the ability of the leaf explants to regenerate roots depends on the age of the leaf and on certain nutrients in the medium. Based on these observations, we provide examples of how this method can be used in different situations, and how it can be optimized. This simple method could be used to investigate the effects of various physiological and molecular changes on the regeneration of adventitious roots. It is also useful for tracing cell lineage during the regeneration process by differential interference contrast observation of -glucuronidase staining, and by live imaging of proteins labeled with fluorescent tags.

  15. De novo malignant solitary fibrous tumor of the kidney

    Directory of Open Access Journals (Sweden)

    Chen Siu-Chung

    2011-10-01

    Full Text Available Abstract The kidney is a relatively infrequent site for solitary fibrous tumor (SFT. Among the previously reported cases, only two cases of malignant renal SFT developing via dedifferentiation from a pre-existing benign SFT have been reported. Here we reported a case of de novo malignant renal SFT clinically diagnosed as renal cell carcinoma in a 50-year-old woman. The tumor was circumscribed but unencapsulated and showed obvious hemorrhagic necrosis. Microscopically, the tumor was composed of patternless sheets of alternating hypercellular and hypocellular areas of spindle cells displaying mild to moderate nuclear atypia, frequent mitoses up to 8 per 10 high power fields, and a 20% Ki-67 proliferative index. Immunohistochemical studies revealed reactivity for CD34, CD99 and vimentin, with no staining for all other markers, confirming the diagnosis of SFT. No areas of dedifferentiation were seen after extensive sampling. Based on the pathologic and immunohistochemical features, a diagnosis of de novo malignant renal SFT was warranted. Our report expands the spectrum of malignant progression in renal SFTs. Even though this patient has been disease-free for 30 months, long-term follow-up is still mandatory.

  16. De novo malignant solitary fibrous tumor of the kidney.

    Science.gov (United States)

    Hsieh, Tsan-Yu; ChangChien, Yi-Che; Chen, Wen-Hsiang; Chen, Siu-Chung; Chang, Liang-Che; Hwang, Cheng-Cheng; Chein, Hui-Ping; Chen, Jim-Ray

    2011-10-05

    The kidney is a relatively infrequent site for solitary fibrous tumor (SFT). Among the previously reported cases, only two cases of malignant renal SFT developing via dedifferentiation from a pre-existing benign SFT have been reported. Here we reported a case of de novo malignant renal SFT clinically diagnosed as renal cell carcinoma in a 50-year-old woman. The tumor was circumscribed but unencapsulated and showed obvious hemorrhagic necrosis. Microscopically, the tumor was composed of patternless sheets of alternating hypercellular and hypocellular areas of spindle cells displaying mild to moderate nuclear atypia, frequent mitoses up to 8 per 10 high power fields, and a 20% Ki-67 proliferative index. Immunohistochemical studies revealed reactivity for CD34, CD99 and vimentin, with no staining for all other markers, confirming the diagnosis of SFT. No areas of dedifferentiation were seen after extensive sampling. Based on the pathologic and immunohistochemical features, a diagnosis of de novo malignant renal SFT was warranted. Our report expands the spectrum of malignant progression in renal SFTs. Even though this patient has been disease-free for 30 months, long-term follow-up is still mandatory.

  17. De novo Evolution of a Small Choroidal Melanoma

    Science.gov (United States)

    Aleksidze, Nino; Medina, Carlos A.; Singh, Arun D.

    2015-01-01

    Aim To report the evolution of a de novo choroidal melanoma. Method This is a case report of a 22-year-old white male patient who has been undergoing periodic examination for a choroidal ‘freckle’ since 10 years of age. Results In 2007, a fundus photograph of the left eye showed a nondescript area of choroidal hyperpigmentation temporal to the fovea. Progressive growth was observed and, by 2012, the lesion had become well circumscribed and raised. One year later, a 4.5 × 4.5 × 1.5 mm, dome-shaped, pigmented, choroidal mass with subretinal fluid and orange pigmentation was evident. The lesion was classified as a small choroidal melanoma. Six months after plaque radiotherapy, tumor regression with total resolution of the subretinal fluid was documented. Conclusion The distinction between small choroidal melanoma and choroidal nevus is not absolute; therefore, some choroidal melanomas may initially be mislabeled as choroidal nevi because of their small size until continued growth identifies them to be small choroidal melanomas. In our case, the documented growth of the choroidal lesion on each consecutive visit and its high-risk features strongly suggest that it had been a melanoma from the beginning. To our knowledge, this is only the second documented case of a de novo evolution of a small choroidal melanoma. PMID:27231689

  18. Molecular characterization of de novo secondary trisomy 13

    Energy Technology Data Exchange (ETDEWEB)

    Shaffer, L.G.; McCaskill, C.; Han, Jin-Yeong [Baylor College of Medicine, Houston, TX (United States); Choo, K.H.A. [Murdoch Institute, Melbourne (Australia); Cutillo, D.M.; Donnenfeld, A.E. [Pennyslvania Hospital, PA (United States); Weiss, L.; Van Dyke, D.L. [Henry Ford Hospital, Detroit, MI (United States)

    1994-11-01

    Unbalanced Robertsonian translocations are a significant cause of mental retardation and fetal wastage. The majority of homologous rearrangements of chromosome 21 in Down syndrome have been shown to be isochromosomes. Aside from chromosome 21, very little is known about other acrocentric homologous rearrangements. In this study, four cases of de novo secondary trisomy 13 are presented. FISH using alpha-satellite sequences, rDNA, and a pTRI-6 satellite I sequence specific to the short arm of chromosome 13 showed all four rearrangements to be dicentric an apparently devoid of ribosomal genes. Three of four rearrangements retained the pTRI-6 satellite I sequence. Case 1 was the exception, showing a deletion of this sequence in the rearrangement, although both parental chromosomes 13 had strong positive hybridization signals. Eleven microsatellite markers from chromosome 13 were also used to characterize the rearrangements. Of the four possible outcomes, one maternal Robertsonian translocation, two paternal isochromosomes, and one maternal isochromosomes were observed. A double recombination was observed in the maternally derived rob(13q13q). No recombination events were detected in any isochromosome. The parental origins and molecular chromosomal structure of these cases are compared with previous studies of de novo acrocentric rearrangements. 20 refs., 3 figs., 2 tabs.

  19. Origins of De Novo Genes in Human and Chimpanzee.

    Directory of Open Access Journals (Sweden)

    Jorge Ruiz-Orera

    2015-12-01

    Full Text Available The birth of new genes is an important motor of evolutionary innovation. Whereas many new genes arise by gene duplication, others originate at genomic regions that did not contain any genes or gene copies. Some of these newly expressed genes may acquire coding or non-coding functions and be preserved by natural selection. However, it is yet unclear which is the prevalence and underlying mechanisms of de novo gene emergence. In order to obtain a comprehensive view of this process, we have performed in-depth sequencing of the transcriptomes of four mammalian species--human, chimpanzee, macaque, and mouse--and subsequently compared the assembled transcripts and the corresponding syntenic genomic regions. This has resulted in the identification of over five thousand new multiexonic transcriptional events in human and/or chimpanzee that are not observed in the rest of species. Using comparative genomics, we show that the expression of these transcripts is associated with the gain of regulatory motifs upstream of the transcription start site (TSS and of U1 snRNP sites downstream of the TSS. In general, these transcripts show little evidence of purifying selection, suggesting that many of them are not functional. However, we find signatures of selection in a subset of de novo genes which have evidence of protein translation. Taken together, the data support a model in which frequently-occurring new transcriptional events in the genome provide the raw material for the evolution of new proteins.

  20. Origins of De Novo Genes in Human and Chimpanzee.

    Science.gov (United States)

    Ruiz-Orera, Jorge; Hernandez-Rodriguez, Jessica; Chiva, Cristina; Sabidó, Eduard; Kondova, Ivanela; Bontrop, Ronald; Marqués-Bonet, Tomàs; Albà, M Mar

    2015-12-01

    The birth of new genes is an important motor of evolutionary innovation. Whereas many new genes arise by gene duplication, others originate at genomic regions that did not contain any genes or gene copies. Some of these newly expressed genes may acquire coding or non-coding functions and be preserved by natural selection. However, it is yet unclear which is the prevalence and underlying mechanisms of de novo gene emergence. In order to obtain a comprehensive view of this process, we have performed in-depth sequencing of the transcriptomes of four mammalian species--human, chimpanzee, macaque, and mouse--and subsequently compared the assembled transcripts and the corresponding syntenic genomic regions. This has resulted in the identification of over five thousand new multiexonic transcriptional events in human and/or chimpanzee that are not observed in the rest of species. Using comparative genomics, we show that the expression of these transcripts is associated with the gain of regulatory motifs upstream of the transcription start site (TSS) and of U1 snRNP sites downstream of the TSS. In general, these transcripts show little evidence of purifying selection, suggesting that many of them are not functional. However, we find signatures of selection in a subset of de novo genes which have evidence of protein translation. Taken together, the data support a model in which frequently-occurring new transcriptional events in the genome provide the raw material for the evolution of new proteins.

  1. De Novo duplication in Charcot-Marie-Tooth Type 1A

    Energy Technology Data Exchange (ETDEWEB)

    Mandich, P.; Bellone, E.; Ajmar, F. [and others

    1996-09-01

    We read with interest the paper on {open_quotes}Prevalence and Origin of De Novo Duplications in Charcot-Marie-Tooth Disease Type 1A: First Report of a De Novo Duplication with a Maternal Origin,{close_quotes}. They reported their experience with 10 sporadic cases of Charcot-Marie-Tooth type 1A (CMT1A) in which it was demonstrated that the disease had arisen as the result of a de novo duplication. They analyzed the de novo-duplication families by using microsatellite markers and identified the parental origin of the duplication in eight cases. In one family the duplication was of maternal origin, whereas in the remaining seven cases it was of paternal origin. The authors concluded that their report was the first evidence of a de novo duplication of maternal origin, suggesting that this is not a phenomenon associated solely with male meiosis. 7 refs.

  2. Interpreting the role of de novo protein-coding mutations in neuropsychiatric disease.

    Science.gov (United States)

    Gratten, Jacob; Visscher, Peter M; Mowry, Bryan J; Wray, Naomi R

    2013-03-01

    Pedigree, linkage and association studies are consistent with heritable variation for complex disease due to the segregation of genetic factors in families and in the population. In contrast, de novo mutations make only minor contributions to heritability estimates for complex traits. Nonetheless, some de novo variants are known to be important in disease etiology. The identification of risk-conferring de novo variants will contribute to the discovery of etiologically relevant genes and pathways and may help in genetic counseling. There is considerable interest in the role of such mutations in complex neuropsychiatric disease, largely driven by new genotyping and sequencing technologies. An important role for large de novo copy number variations has been established. Recently, whole-exome sequencing has been used to extend the investigation of de novo variation to point mutations in protein-coding regions. Here, we consider several challenges for the interpretation of such mutations in the context of their role in neuropsychiatric disease.

  3. DeNovoID: a web-based tool for identifying peptides from sequence and mass tags deduced from de novo peptide sequencing by mass spectroscopy.

    Science.gov (United States)

    Halligan, Brian D; Ruotti, Victor; Twigger, Simon N; Greene, Andrew S

    2005-07-01

    One of the core activities of high-throughput proteomics is the identification of peptides from mass spectra. Some peptides can be identified using spectral matching programs like Sequest or Mascot, but many spectra do not produce high quality database matches. De novo peptide sequencing is an approach to determine partial peptide sequences for some of the unidentified spectra. A drawback of de novo peptide sequencing is that it produces a series of ordered and disordered sequence tags and mass tags rather than a complete, non-degenerate peptide amino acid sequence. This incomplete data is difficult to use in conventional search programs such as BLAST or FASTA. DeNovoID is a program that has been specifically designed to use degenerate amino acid sequence and mass data derived from MS experiments to search a peptide database. Since the algorithm employed depends on the amino acid composition of the peptide and not its sequence, DeNovoID does not have to consider all possible sequences, but rather a smaller number of compositions consistent with a spectrum. DeNovoID also uses a geometric indexing scheme that reduces the number of calculations required to determine the best peptide match in the database. DeNovoID is available at http://proteomics.mcw.edu/denovoid.

  4. Post-steroid neuropsychiatric manifestations are significantly more frequent in SLE compared with other systemic autoimmune diseases and predict better prognosis compared with de novo neuropsychiatric SLE.

    Science.gov (United States)

    Shimizu, Yuka; Yasuda, Shinsuke; Kako, Yuki; Nakagawa, Shin; Kanda, Masatoshi; Hisada, Ryo; Ohmura, Kazumasa; Shimamura, Sanae; Shida, Haruki; Fujieda, Yuichiro; Kato, Masaru; Oku, Kenji; Bohgaki, Toshiyuki; Horita, Tetsuya; Kusumi, Ichiro; Atsumi, Tatsuya

    2016-08-01

    In patients with systemic lupus erythematosus (SLE), neuropsychiatric (NP) symptoms sometimes occur after administration of corticosteroids, making differential diagnosis between NPSLE and steroid-induced psychosis challenging for clinicians. The aim of this study was to clarify the characteristics of post-steroid NP disease (PSNP) in patients with SLE. Clinical courses of 146 patients with SLE and 162 with other systemic autoimmune diseases, all in the absence of NP manifestations on admission, were retrospectively analyzed. Forty-three NPSLE patients on admission (de novo NPSLE) were also investigated. All patients were consecutively recruited and treated with 40mg/day or more of prednisolone in Hokkaido University Hospital between April 2002 and March 2015. The prevalence of PSNP was strikingly higher in SLE patients than other systemic autoimmune diseases (24.7% vs. 7.4%, OR 4.09, 95% CI 2.04-8.22). As independent risk factors to develop PSNP in SLE patients, past history of mental disorder and the presence of antiphospholipid syndrome were identified using multiple logistic regression analysis. In patients with PSNP-SLE, mood disorder was significantly more frequent than in de novo NPSLE (47.2% vs. 20.9%, OR 3.38, 95% CI 1.26-9.04). Of PSNP-SLE patients, two-thirds were with one or more abnormal findings in cerebrospinal fluid, electroencephalogram, MRI or SPECT. Majority of our PSNP-SLE patients received intensified immunosuppressive treatments and experienced improvement in most cases. PSNP-SLE had better relapse-free survival than de novo NPSLE (p<0.05, log rank test). In conclusion, PSNP frequently occurred in patients with SLE and treated successfully with immunosuppressive therapy, indicating that NPSLE is likely to harbor patients with PSNP-SLE.

  5. De novo assembly of a haplotype-resolved human genome.

    Science.gov (United States)

    Cao, Hongzhi; Wu, Honglong; Luo, Ruibang; Huang, Shujia; Sun, Yuhui; Tong, Xin; Xie, Yinlong; Liu, Binghang; Yang, Hailong; Zheng, Hancheng; Li, Jian; Li, Bo; Wang, Yu; Yang, Fang; Sun, Peng; Liu, Siyang; Gao, Peng; Huang, Haodong; Sun, Jing; Chen, Dan; He, Guangzhu; Huang, Weihua; Huang, Zheng; Li, Yue; Tellier, Laurent C A M; Liu, Xiao; Feng, Qiang; Xu, Xun; Zhang, Xiuqing; Bolund, Lars; Krogh, Anders; Kristiansen, Karsten; Drmanac, Radoje; Drmanac, Snezana; Nielsen, Rasmus; Li, Songgang; Wang, Jian; Yang, Huanming; Li, Yingrui; Wong, Gane Ka-Shu; Wang, Jun

    2015-06-01

    The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

  6. Accurate de novo design of hyperstable constrained peptides

    Energy Technology Data Exchange (ETDEWEB)

    Bhardwaj, Gaurav; Mulligan, Vikram Khipple; Bahl, Christopher D.; Gilmore, Jason M.; Harvey, Peta J.; Cheneval, Olivier; Buchko, Garry W.; Pulavarti, Surya V. S. R. K.; Kaas, Quentin; Eletsky, Alexander; Huang, Po-Ssu; Johnsen, William A.; Greisen, Per Jr; Rocklin, Gabriel J.; Song, Yifan; Linsky, Thomas W.; Watkins, Andrew; Rettie, Stephen A.; Xu, Xianzhong; Carter, Lauren P.; Bonneau, Richard; Olson, James M.; Coutsias, Evangelos; Correnti, Colin E.; Szyperski, Thomas; Craik, David J.; Baker, David

    2016-09-14

    Covalently-crosslinked peptides present attractive opportunities for developing new therapeutics. Lying between small molecule and protein therapeutics in size, natural crosslinked peptides play critical roles in signaling, virulence and immunity. Engineering novel peptides with precise control over their three-dimensional structures is a significant challenge. Here we describe the development of computational methods for de novo design of conformationally-restricted peptides, and the use of these methods to design hyperstable disulfide-stabilized miniproteins, heterochiral peptides, and N-C cyclic peptides. Experimentally-determined X-ray and NMR structures for 12 of the designs are nearly identical to the computational models. The computational design methods and stable scaffolds provide the basis for a new generation of peptide-based drugs.

  7. Herpes simplex virus 1 induces de novo phospholipid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Sutter, Esther [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Oliveira, Anna Paula de; Tobler, Kurt [Electron microscopy, Institute of Virology, University of Zuerich (Switzerland); Schraner, Elisabeth M. [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Sonda, Sabrina [Institute of Parasitology, University of Zuerich (Switzerland); Kaech, Andres [Center for Microscopy and Image Analysis, University of Zuerich (Switzerland); Lucas, Miriam S. [Electron Microscopy ETH Zuerich (EMEZ), Swiss Federal Institute of Technology, Zuerich (Switzerland); Ackermann, Mathias [Electron microscopy, Institute of Virology, University of Zuerich (Switzerland); Wild, Peter, E-mail: pewild@access.uzh.ch [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland)

    2012-08-01

    Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [{sup 3}H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [{sup 3}H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and that de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.

  8. De novo transcriptome assembly of shrimp Palaemon serratus

    Directory of Open Access Journals (Sweden)

    Alejandra Perina

    2017-03-01

    Full Text Available The shrimp Palaemon serratus is a coastal decapod crustacean with a high commercial value. It is harvested for human consumption. In this study, we used Illumina sequencing technology (HiSeq 2000 to sequence, assemble and annotate the transcriptome of P. serratus. RNA was isolated from muscle of adults individuals and, from a pool of larvae. A total number of 4 cDNA libraries were constructed, using the TruSeq RNA Sample Preparation Kit v2. The raw data in this study was deposited in NCBI SRA database with study accession number of SRP090769. The obtained data were subjected to de novo transcriptome assembly using Trinity software, and coding regions were predicted by TransDecoder. We used Blastp and Sma3s to annotate the identified proteins. The transcriptome data could provide some insight into the understanding of genes involved in the larval development and metamorphosis.

  9. Nonequilibrium mechanisms underlying de novo biogenesis of Golgi cisternae

    CERN Document Server

    Sachdeva, Himani; Rao, Madan

    2016-01-01

    A central issue in cell biology is the physico-chemical basis of organelle biogenesis in intracellular trafficking pathways, its most impressive manifestation being the biogenesis of Golgi cisternae. At a basic level, such morphologically and chemically distinct compartments should arise from an interplay between the molecular transport and chemical maturation. Here, we formulate analytically tractable, minimalist models, that incorporate this interplay between transport and chemical progression in physical space, and explore the conditions for de novo biogenesis of distinct cisternae. We propose new quantitative measures that can discriminate between the various models of transport in a qualitative manner- this includes measures of the dynamics in steady state and the dynamical response to perturbations of the kind amenable to live-cell imaging.

  10. De-novo design of antimicrobial peptides for plant protection.

    Directory of Open Access Journals (Sweden)

    Benjamin Zeitler

    Full Text Available This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of "healthy" food, these peptides might serve as templates for novel antibacterial and antifungal agents.

  11. De novo mutations in moderate or severe intellectual disability.

    Science.gov (United States)

    Hamdan, Fadi F; Srour, Myriam; Capo-Chichi, Jose-Mario; Daoud, Hussein; Nassif, Christina; Patry, Lysanne; Massicotte, Christine; Ambalavanan, Amirthagowri; Spiegelman, Dan; Diallo, Ousmane; Henrion, Edouard; Dionne-Laporte, Alexandre; Fougerat, Anne; Pshezhetsky, Alexey V; Venkateswaran, Sunita; Rouleau, Guy A; Michaud, Jacques L

    2014-10-01

    Genetics is believed to have an important role in intellectual disability (ID). Recent studies have emphasized the involvement of de novo mutations (DNMs) in ID but the extent to which they contribute to its pathogenesis and the identity of the corresponding genes remain largely unknown. Here, we report a screen for DNMs in subjects with moderate or severe ID. We sequenced the exomes of 41 probands and their parents, and confirmed 81 DNMs affecting the coding sequence or consensus splice sites (1.98 DNMs/proband). We observed a significant excess of de novo single nucleotide substitutions and loss-of-function mutations in these cases compared to control subjects, suggesting that at least a subset of these variations are pathogenic. A total of 12 likely pathogenic DNMs were identified in genes previously associated with ID (ARID1B, CHD2, FOXG1, GABRB3, GATAD2B, GRIN2B, MBD5, MED13L, SETBP1, TBR1, TCF4, WDR45), resulting in a diagnostic yield of ∼29%. We also identified 12 possibly pathogenic DNMs in genes (HNRNPU, WAC, RYR2, SET, EGR1, MYH10, EIF2C1, COL4A3BP, CHMP2A, PPP1CB, VPS4A, PPP2R2B) that have not previously been causally linked to ID. Interestingly, no case was explained by inherited mutations. Protein network analysis indicated that the products of many of these known and candidate genes interact with each other or with products of other ID-associated genes further supporting their involvement in ID. We conclude that DNMs represent a major cause of moderate or severe ID.

  12. De novo mutations in moderate or severe intellectual disability.

    Directory of Open Access Journals (Sweden)

    Fadi F Hamdan

    2014-10-01

    Full Text Available Genetics is believed to have an important role in intellectual disability (ID. Recent studies have emphasized the involvement of de novo mutations (DNMs in ID but the extent to which they contribute to its pathogenesis and the identity of the corresponding genes remain largely unknown. Here, we report a screen for DNMs in subjects with moderate or severe ID. We sequenced the exomes of 41 probands and their parents, and confirmed 81 DNMs affecting the coding sequence or consensus splice sites (1.98 DNMs/proband. We observed a significant excess of de novo single nucleotide substitutions and loss-of-function mutations in these cases compared to control subjects, suggesting that at least a subset of these variations are pathogenic. A total of 12 likely pathogenic DNMs were identified in genes previously associated with ID (ARID1B, CHD2, FOXG1, GABRB3, GATAD2B, GRIN2B, MBD5, MED13L, SETBP1, TBR1, TCF4, WDR45, resulting in a diagnostic yield of ∼29%. We also identified 12 possibly pathogenic DNMs in genes (HNRNPU, WAC, RYR2, SET, EGR1, MYH10, EIF2C1, COL4A3BP, CHMP2A, PPP1CB, VPS4A, PPP2R2B that have not previously been causally linked to ID. Interestingly, no case was explained by inherited mutations. Protein network analysis indicated that the products of many of these known and candidate genes interact with each other or with products of other ID-associated genes further supporting their involvement in ID. We conclude that DNMs represent a major cause of moderate or severe ID.

  13. De novo identification of viral pathogens from cell culture hologenomes

    Directory of Open Access Journals (Sweden)

    Patowary Ashok

    2012-01-01

    Full Text Available Abstract Background Fast, specific identification and surveillance of pathogens is the cornerstone of any outbreak response system, especially in the case of emerging infectious diseases and viral epidemics. This process is generally tedious and time-consuming thus making it ineffective in traditional settings. The added complexity in these situations is the non-availability of pure isolates of pathogens as they are present as mixed genomes or hologenomes. Next-generation sequencing approaches offer an attractive solution in this scenario as it provides adequate depth of sequencing at fast and affordable costs, apart from making it possible to decipher complex interactions between genomes at a scale that was not possible before. The widespread application of next-generation sequencing in this field has been limited by the non-availability of an efficient computational pipeline to systematically analyze data to delineate pathogen genomes from mixed population of genomes or hologenomes. Findings We applied next-generation sequencing on a sample containing mixed population of genomes from an epidemic with appropriate processing and enrichment. The data was analyzed using an extensive computational pipeline involving mapping to reference genome sets and de-novo assembly. In depth analysis of the data generated revealed the presence of sequences corresponding to Japanese encephalitis virus. The genome of the virus was also independently de-novo assembled. The presence of the virus was in addition, verified using standard molecular biology techniques. Conclusions Our approach can accurately identify causative pathogens from cell culture hologenome samples containing mixed population of genomes and in principle can be applied to patient hologenome samples without any background information. This methodology could be widely applied to identify and isolate pathogen genomes and understand their genomic variability during outbreaks.

  14. The limits of de novo DNA motif discovery.

    Directory of Open Access Journals (Sweden)

    David Simcha

    Full Text Available A major challenge in molecular biology is reverse-engineering the cis-regulatory logic that plays a major role in the control of gene expression. This program includes searching through DNA sequences to identify "motifs" that serve as the binding sites for transcription factors or, more generally, are predictive of gene expression across cellular conditions. Several approaches have been proposed for de novo motif discovery-searching sequences without prior knowledge of binding sites or nucleotide patterns. However, unbiased validation is not straightforward. We consider two approaches to unbiased validation of discovered motifs: testing the statistical significance of a motif using a DNA "background" sequence model to represent the null hypothesis and measuring performance in predicting membership in gene clusters. We demonstrate that the background models typically used are "too null," resulting in overly optimistic assessments of significance, and argue that performance in predicting TF binding or expression patterns from DNA motifs should be assessed by held-out data, as in predictive learning. Applying this criterion to common motif discovery methods resulted in universally poor performance, although there is a marked improvement when motifs are statistically significant against real background sequences. Moreover, on synthetic data where "ground truth" is known, discriminative performance of all algorithms is far below the theoretical upper bound, with pronounced "over-fitting" in training. A key conclusion from this work is that the failure of de novo discovery approaches to accurately identify motifs is basically due to statistical intractability resulting from the fixed size of co-regulated gene clusters, and thus such failures do not necessarily provide evidence that unfound motifs are not active biologically. Consequently, the use of prior knowledge to enhance motif discovery is not just advantageous but necessary. An implementation of

  15. On the Origin of De Novo Genes in Arabidopsis thaliana Populations.

    Science.gov (United States)

    Li, Zi-Wen; Chen, Xi; Wu, Qiong; Hagmann, Jörg; Han, Ting-Shen; Zou, Yu-Pan; Ge, Song; Guo, Ya-Long

    2016-08-03

    De novo genes, which originate from ancestral nongenic sequences, are one of the most important sources of protein-coding genes. This origination process is crucial for the adaptation of organisms. However, how de novo genes arise and become fixed in a population or species remains largely unknown. Here, we identified 782 de novo genes from the model plant Arabidopsis thaliana and divided them into three types based on the availability of translational evidence, transcriptional evidence, and neither transcriptional nor translational evidence for their origin. Importantly, by integrating multiple types of omics data, including data from genomes, epigenomes, transcriptomes, and translatomes, we found that epigenetic modifications (DNA methylation and histone modification) play an important role in the origination process of de novo genes. Intriguingly, using the transcriptomes and methylomes from the same population of 84 accessions, we found that de novo genes that are transcribed in approximately half of the total accessions within the population are highly methylated, with lower levels of transcription than those transcribed at other frequencies within the population. We hypothesized that, during the origin of de novo gene alleles, those neutralized to low expression states via DNA methylation have relatively high probabilities of spreading and becoming fixed in a population. Our results highlight the process underlying the origin of de novo genes at the population level, as well as the importance of DNA methylation in this process.

  16. Calcineurin inhibitor-free mycophenolate mofetil/sirolimus maintenance in liver transplantation: the randomized spare-the-nephron trial.

    Science.gov (United States)

    Teperman, Lewis; Moonka, Dilip; Sebastian, Anthony; Sher, Linda; Marotta, Paul; Marsh, Christopher; Koneru, Baburao; Goss, John; Preston, Dennis; Roberts, John P

    2013-07-01

    Mycophenolate mofetil (MMF) and sirolimus (SRL) have been used for calcineurin inhibitor (CNI) minimization to reduce nephrotoxicity following liver transplantation. In this prospective, open-label, multicenter study, patients undergoing transplantation from July 2005 to June 2007 who were maintained on MMF/CNI were randomized 4 to 12 weeks after transplantation to receive MMF/SRL (n = 148) or continue MMF/CNI (n = 145) and included in the intent-to-treat population. The primary efficacy endpoints were the mean percentage change in the calculated glomerular filtration rate (GFR) and a composite of biopsy-proven acute rejection (BPAR), graft lost, death, and lost to follow-up 12 months after transplantation. Patients were followed for a median of 519 days after randomization. MMF/SRL was associated with a significantly greater renal function improvement from baseline with a mean percentage change in GFR of 19.7 ± 40.6 (versus 1.2 ± 39.9 for MMF/CNI, P = 0.0012). The composite endpoint demonstrated the noninferiority of MMF/SRL versus MMF/CNI (16.4% versus 15.4%, 90% confidence interval = -7.1% to 9.0%). The incidence of BPAR was significantly greater with MMF/SRL (12.2%) versus MMF/CNI (4.1%, P = 0.02). Graft loss (including death) occurred in 3.4% of the MMF/SRL-treated patients and in 8.3% of the MMF/CNI-treated patients (P = 0.04). Malignancy-related deaths were less frequent with MMF/SRL. Adverse events caused withdrawal for 34.2% of the MMF/SRL-treated patients and for 24.1% of the MMF/CNI-treated patients (P = 0.06). The use of MMF/SRL is an option for liver transplant recipients who can benefit from improved renal function but is associated with an increased risk of rejection (but not graft loss).

  17. De Novo Human Cardiac Myocytes for Medical Research: Promises and Challenges

    Science.gov (United States)

    Hamel, Veronique; Cheng, Kang; Liao, Shudan; Lu, Aizhu; Zheng, Yong; Chen, Yawen; Xie, Yucai

    2017-01-01

    The advent of cellular reprogramming technology has revolutionized biomedical research. De novo human cardiac myocytes can now be obtained from direct reprogramming of somatic cells (such as fibroblasts), from induced pluripotent stem cells (iPSCs, which are reprogrammed from somatic cells), and from human embryonic stem cells (hESCs). Such de novo human cardiac myocytes hold great promise for in vitro disease modeling and drug screening and in vivo cell therapy of heart disease. Here, we review the technique advancements for generating de novo human cardiac myocytes. We also discuss several challenges for the use of such cells in research and regenerative medicine, such as the immature phenotype and heterogeneity of de novo cardiac myocytes obtained with existing protocols. We focus on the recent advancements in addressing such challenges.

  18. Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery

    Science.gov (United States)

    Sen, K. Ilker; Tang, Wilfred H.; Nayak, Shruti; Kil, Yong J.; Bern, Marshall; Ozoglu, Berk; Ueberheide, Beatrix; Davis, Darryl; Becker, Christopher

    2017-01-01

    Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests.

  19. De novo histoid leprosy: A case report from a post-elimination area

    Directory of Open Access Journals (Sweden)

    Yasmeen J Bhat

    2015-01-01

    Full Text Available Histoid leprosy is an uncommon variant of lepromatous leprosy that usually follows treatment failure. Occasionally it occurs de novo without any history of previous inadequate or irregular treatment. We, hereby, report a case of de novo histoid leprosy in a 25-year-old man from the post-elimination area of Kashmir, where the prevalence rate of the disease was reported to be 0.17/10000 in March, 2013 (NLEP.

  20. De novo nonsense mutations in ASXL1 cause Bohring-Opitz syndrome

    DEFF Research Database (Denmark)

    Hoischen, Alexander; van Bon, Bregje W M; Rodríguez-Santiago, Benjamín;

    2011-01-01

    Bohring-Opitz syndrome is characterized by severe intellectual disability, distinctive facial features and multiple congenital malformations. We sequenced the exomes of three individuals with Bohring-Opitz syndrome and in each identified heterozygous de novo nonsense mutations in ASXL1, which...... is required for maintenance of both activation and silencing of Hox genes. In total, 7 out of 13 subjects with a Bohring-Opitz phenotype had de novo ASXL1 mutations, suggesting that the syndrome is genetically heterogeneous....

  1. De novo assembly and phasing of a Korean human genome.

    Science.gov (United States)

    Seo, Jeong-Sun; Rhie, Arang; Kim, Junsoo; Lee, Sangjin; Sohn, Min-Hwan; Kim, Chang-Uk; Hastie, Alex; Cao, Han; Yun, Ji-Young; Kim, Jihye; Kuk, Junho; Park, Gun Hwa; Kim, Juhyeok; Ryu, Hanna; Kim, Jongbum; Roh, Mira; Baek, Jeonghun; Hunkapiller, Michael W; Korlach, Jonas; Shin, Jong-Yeon; Kim, Changhoon

    2016-10-13

    Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of

  2. De novo ORFs in Drosophila are important to organismal fitness and evolved rapidly from previously non-coding sequences.

    Directory of Open Access Journals (Sweden)

    Josephine A Reinhardt

    Full Text Available How non-coding DNA gives rise to new protein-coding genes (de novo genes is not well understood. Recent work has revealed the origins and functions of a few de novo genes, but common principles governing the evolution or biological roles of these genes are unknown. To better define these principles, we performed a parallel analysis of the evolution and function of six putatively protein-coding de novo genes described in Drosophila melanogaster. Reconstruction of the transcriptional history of de novo genes shows that two de novo genes emerged from novel long non-coding RNAs that arose at least 5 MY prior to evolution of an open reading frame. In contrast, four other de novo genes evolved a translated open reading frame and transcription within the same evolutionary interval suggesting that nascent open reading frames (proto-ORFs, while not required, can contribute to the emergence of a new de novo gene. However, none of the genes arose from proto-ORFs that existed long before expression evolved. Sequence and structural evolution of de novo genes was rapid compared to nearby genes and the structural complexity of de novo genes steadily increases over evolutionary time. Despite the fact that these genes are transcribed at a higher level in males than females, and are most strongly expressed in testes, RNAi experiments show that most of these genes are essential in both sexes during metamorphosis. This lethality suggests that protein coding de novo genes in Drosophila quickly become functionally important.

  3. Selecting Superior De Novo Transcriptome Assemblies: Lessons Learned by Leveraging the Best Plant Genome.

    Directory of Open Access Journals (Sweden)

    Loren A Honaas

    Full Text Available Whereas de novo assemblies of RNA-Seq data are being published for a growing number of species across the tree of life, there are currently no broadly accepted methods for evaluating such assemblies. Here we present a detailed comparison of 99 transcriptome assemblies, generated with 6 de novo assemblers including CLC, Trinity, SOAP, Oases, ABySS and NextGENe. Controlled analyses of de novo assemblies for Arabidopsis thaliana and Oryza sativa transcriptomes provide new insights into the strengths and limitations of transcriptome assembly strategies. We find that the leading assemblers generate reassuringly accurate assemblies for the majority of transcripts. At the same time, we find a propensity for assemblers to fail to fully assemble highly expressed genes. Surprisingly, the instance of true chimeric assemblies is very low for all assemblers. Normalized libraries are reduced in highly abundant transcripts, but they also lack 1000s of low abundance transcripts. We conclude that the quality of de novo transcriptome assemblies is best assessed through consideration of a combination of metrics: 1 proportion of reads mapping to an assembly 2 recovery of conserved, widely expressed genes, 3 N50 length statistics, and 4 the total number of unigenes. We provide benchmark Illumina transcriptome data and introduce SCERNA, a broadly applicable modular protocol for de novo assembly improvement. Finally, our de novo assembly of the Arabidopsis leaf transcriptome revealed ~20 putative Arabidopsis genes lacking in the current annotation.

  4. De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa.

    Science.gov (United States)

    Kim, Hyun A; Shin, Ah-Young; Lee, Min-Seon; Lee, Hee-Jeong; Lee, Heung-Ryul; Ahn, Jongmoon; Nahm, Seokhyeon; Jo, Sung-Hwan; Park, Jeong Mee; Kwon, Suk-Yoon

    2016-02-01

    Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family.

  5. De novo transcriptome assembly of mangosteen (Garcinia mangostana L.) fruit.

    Science.gov (United States)

    Matra, Deden Derajat; Kozaki, Toshinori; Ishii, Kazuo; Poerwanto, Roedhy; Inoue, Eiichi

    2016-12-01

    Garcinia mangostana L. (Mangosteen), of the family Clusiaceae, is one of the economically important tropical fruits in Indonesia. In the present study, we performed de novo transcriptomic analysis of Garcinia mangostana L. through RNA-Seq technology. We obtained the raw data from 12 libraries through Ion Proton System. Clean reads of 191,735,809 were obtained from 307,634,890 raw reads. The raw data obtained in this study can be accessible in DDBJ database with accession number of DRA005014 with bioproject accession number of PRJDB5091. We obtained 268,851 transcripts as well as 155,850 unigenes, having N50 value of 555 and 433 bp, respectively. Transcript/unigene length ranged from 201 to 5916 bp. The unigenes were annotated with two main databases from NCBI and UniProtKB, respectively having annotated-sequences of 73,287 and 73,107, respectively. These transcriptomic data will be beneficial for studying transcriptome of Garcinia mangostana L.

  6. Dynamic actin controls polarity induction de novo in protoplasts.

    Science.gov (United States)

    Zaban, Beatrix; Maisch, Jan; Nick, Peter

    2013-02-01

    Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  7. Enzyme-like replication de novo in a microcontroller environment.

    Science.gov (United States)

    Tangen, Uwe

    2010-01-01

    The desire to start evolution from scratch inside a computer memory is as old as computing. Here we demonstrate how viable computer programs can be established de novo in a Precambrian environment without supplying any specific instantiation, just starting with random bit sequences. These programs are not self-replicators, but act much more like catalysts. The microcontrollers used in the end are the result of a long series of simplifications. The objective of this simplification process was to produce universal machines with a human-readable interface, allowing software and/or hardware evolution to be studied. The power of the instruction set can be modified by introducing a secondary structure-folding mechanism, which is a state machine, allowing nontrivial replication to emerge with an instruction width of only a few bits. This state-machine approach not only attenuates the problems of brittleness and encoding functionality (too few bits available for coding, and too many instructions needed); it also enables the study of hardware evolution as such. Furthermore, the instruction set is sufficiently powerful to permit external signals to be processed. This information-theoretic approach forms one vertex of a triangle alongside artificial cell research and experimental research on the creation of life. Hopefully this work helps develop an understanding of how information—in a similar sense to the account of functional information described by Hazen et al.—is created by evolution and how this information interacts with or is embedded in its physico-chemical environment.

  8. Spaced Seed Data Structures for De Novo Assembly.

    Science.gov (United States)

    Birol, Inanç; Chu, Justin; Mohamadi, Hamid; Jackman, Shaun D; Raghavan, Karthika; Vandervalk, Benjamin P; Raymond, Anthony; Warren, René L

    2015-01-01

    De novo assembly of the genome of a species is essential in the absence of a reference genome sequence. Many scalable assembly algorithms use the de Bruijn graph (DBG) paradigm to reconstruct genomes, where a table of subsequences of a certain length is derived from the reads, and their overlaps are analyzed to assemble sequences. Despite longer subsequences unlocking longer genomic features for assembly, associated increase in compute resources limits the practicability of DBG over other assembly archetypes already designed for longer reads. Here, we revisit the DBG paradigm to adapt it to the changing sequencing technology landscape and introduce three data structure designs for spaced seeds in the form of paired subsequences. These data structures address memory and run time constraints imposed by longer reads. We observe that when a fixed distance separates seed pairs, it provides increased sequence specificity with increased gap length. Further, we note that Bloom filters would be suitable to implicitly store spaced seeds and be tolerant to sequencing errors. Building on this concept, we describe a data structure for tracking the frequencies of observed spaced seeds. These data structure designs will have applications in genome, transcriptome and metagenome assemblies, and read error correction.

  9. De novo design of protein mimics of B-DNA.

    Science.gov (United States)

    Yüksel, Deniz; Bianco, Piero R; Kumar, Krishna

    2016-01-01

    Structural mimicry of DNA is utilized in nature as a strategy to evade molecular defences mounted by host organisms. One such example is the protein Ocr - the first translation product to be expressed as the bacteriophage T7 infects E. coli. The structure of Ocr reveals an intricate and deliberate arrangement of negative charges that endows it with the ability to mimic ∼24 base pair stretches of B-DNA. This uncanny resemblance to DNA enables Ocr to compete in binding the type I restriction modification (R/M) system, and neutralizes the threat of hydrolytic cleavage of viral genomic material. Here, we report the de novo design and biophysical characterization of DNA mimicking peptides, and describe the inhibitory action of the designed helical bundles on a type I R/M enzyme, EcoR124I. This work validates the use of charge patterning as a design principle for creation of protein mimics of DNA, and serves as a starting point for development of therapeutic peptide inhibitors against human pathogens that employ molecular camouflage as part of their invasion stratagem.

  10. Ultrafast de novo docking combining pharmacophores and combinatorics.

    Science.gov (United States)

    Gastreich, Marcus; Lilienthal, Markus; Briem, Hans; Claussen, Holger

    2006-12-01

    We report on a successful de novo design approach which relies on the combination of multi-million compound combinatorial docking under receptor-based pharmacophore constraints. Inspired by a rationale by A.R. Leach et al., we document on the unification of two steps into one for ligand assembly. In the original work, fragments known to bind in protein active sites were connected forming novel ligand compounds by means of generic skeleton linkers and following a combinatorial approach. In our approach, the knowledge of fragments binding to the protein has been expressed in terms of a receptor-based pharmacophore definition. The combinatorial linking step is performed in situ during docking, starting from combinatorial libraries. Three sample scenarios growing in size and complexity (combinatorial libraries of 1 million, 1.3 million, and 22.4 million compounds) have been created to illustrate the method. Docking could be accomplished between minutes and several hours depending on the outset; the results were throughout promising. Technically, a module compatibility between FlexX(C) and FlexX-Pharm has been established. The background is explained, and the crucial points from an information scientist's perspective are highlighted.

  11. MITOS: improved de novo metazoan mitochondrial genome annotation.

    Science.gov (United States)

    Bernt, Matthias; Donath, Alexander; Jühling, Frank; Externbrink, Fabian; Florentz, Catherine; Fritzsch, Guido; Pütz, Joern; Middendorf, Martin; Stadler, Peter F

    2013-11-01

    About 2000 completely sequenced mitochondrial genomes are available from the NCBI RefSeq data base together with manually curated annotations of their protein-coding genes, rRNAs, and tRNAs. This annotation information, which has accumulated over two decades, has been obtained with a diverse set of computational tools and annotation strategies. Despite all efforts of manual curation it is still plagued by misassignments of reading directions, erroneous gene names, and missing as well as false positive annotations in particular for the RNA genes. Taken together, this causes substantial problems for fully automatic pipelines that aim to use these data comprehensively for studies of animal phylogenetics and the molecular evolution of mitogenomes. The MITOS pipeline is designed to compute a consistent de novo annotation of the mitogenomic sequences. We show that the results of MITOS match RefSeq and MitoZoa in terms of annotation coverage and quality. At the same time we avoid biases, inconsistencies of nomenclature, and typos originating from manual curation strategies. The MITOS pipeline is accessible online at http://mitos.bioinf.uni-leipzig.de.

  12. Spaced Seed Data Structures for De Novo Assembly

    Directory of Open Access Journals (Sweden)

    Inanç Birol

    2015-01-01

    Full Text Available De novo assembly of the genome of a species is essential in the absence of a reference genome sequence. Many scalable assembly algorithms use the de Bruijn graph (DBG paradigm to reconstruct genomes, where a table of subsequences of a certain length is derived from the reads, and their overlaps are analyzed to assemble sequences. Despite longer subsequences unlocking longer genomic features for assembly, associated increase in compute resources limits the practicability of DBG over other assembly archetypes already designed for longer reads. Here, we revisit the DBG paradigm to adapt it to the changing sequencing technology landscape and introduce three data structure designs for spaced seeds in the form of paired subsequences. These data structures address memory and run time constraints imposed by longer reads. We observe that when a fixed distance separates seed pairs, it provides increased sequence specificity with increased gap length. Further, we note that Bloom filters would be suitable to implicitly store spaced seeds and be tolerant to sequencing errors. Building on this concept, we describe a data structure for tracking the frequencies of observed spaced seeds. These data structure designs will have applications in genome, transcriptome and metagenome assemblies, and read error correction.

  13. Integrated genomic analyses of de novo pathways underlying atypical meningiomas

    Science.gov (United States)

    Harmancı, Akdes Serin; Youngblood, Mark W.; Clark, Victoria E.; Coşkun, Süleyman; Henegariu, Octavian; Duran, Daniel; Erson-Omay, E. Zeynep; Kaulen, Leon D.; Lee, Tong Ihn; Abraham, Brian J.; Simon, Matthias; Krischek, Boris; Timmer, Marco; Goldbrunner, Roland; Omay, S. Bülent; Baranoski, Jacob; Baran, Burçin; Carrión-Grant, Geneive; Bai, Hanwen; Mishra-Gorur, Ketu; Schramm, Johannes; Moliterno, Jennifer; Vortmeyer, Alexander O.; Bilgüvar, Kaya; Yasuno, Katsuhito; Young, Richard A.; Günel, Murat

    2017-01-01

    Meningiomas are mostly benign brain tumours, with a potential for becoming atypical or malignant. On the basis of comprehensive genomic, transcriptomic and epigenomic analyses, we compared benign meningiomas to atypical ones. Here, we show that the majority of primary (de novo) atypical meningiomas display loss of NF2, which co-occurs either with genomic instability or recurrent SMARCB1 mutations. These tumours harbour increased H3K27me3 signal and a hypermethylated phenotype, mainly occupying the polycomb repressive complex 2 (PRC2) binding sites in human embryonic stem cells, thereby phenocopying a more primitive cellular state. Consistent with this observation, atypical meningiomas exhibit upregulation of EZH2, the catalytic subunit of the PRC2 complex, as well as the E2F2 and FOXM1 transcriptional networks. Importantly, these primary atypical meningiomas do not harbour TERT promoter mutations, which have been reported in atypical tumours that progressed from benign ones. Our results establish the genomic landscape of primary atypical meningiomas and potential therapeutic targets. PMID:28195122

  14. De novo transcriptome assembly of mangosteen (Garcinia mangostana L. fruit

    Directory of Open Access Journals (Sweden)

    Deden Derajat Matra

    2016-12-01

    Full Text Available Garcinia mangostana L. (Mangosteen, of the family Clusiaceae, is one of the economically important tropical fruits in Indonesia. In the present study, we performed de novo transcriptomic analysis of Garcinia mangostana L. through RNA-Seq technology. We obtained the raw data from 12 libraries through Ion Proton System. Clean reads of 191,735,809 were obtained from 307,634,890 raw reads. The raw data obtained in this study can be accessible in DDBJ database with accession number of DRA005014 with bioproject accession number of PRJDB5091. We obtained 268,851 transcripts as well as 155,850 unigenes, having N50 value of 555 and 433 bp, respectively. Transcript/unigene length ranged from 201 to 5916 bp. The unigenes were annotated with two main databases from NCBI and UniProtKB, respectively having annotated-sequences of 73,287 and 73,107, respectively. These transcriptomic data will be beneficial for studying transcriptome of Garcinia mangostana L.

  15. TBro: visualization and management of de novo transcriptomes

    Science.gov (United States)

    Ankenbrand, Markus J.; Weber, Lorenz; Becker, Dirk; Förster, Frank; Bemm, Felix

    2016-01-01

    RNA sequencing (RNA-seq) has become a powerful tool to understand molecular mechanisms and/or developmental programs. It provides a fast, reliable and cost-effective method to access sets of expressed elements in a qualitative and quantitative manner. Especially for non-model organisms and in absence of a reference genome, RNA-seq data is used to reconstruct and quantify transcriptomes at the same time. Even SNPs, InDels, and alternative splicing events are predicted directly from the data without having a reference genome at hand. A key challenge, especially for non-computational personnal, is the management of the resulting datasets, consisting of different data types and formats. Here, we present TBro, a flexible de novo transcriptome browser, tackling this challenge. TBro aggregates sequences, their annotation, expression levels as well as differential testing results. It provides an easy-to-use interface to mine the aggregated data and generate publication-ready visualizations. Additionally, it supports users with an intuitive cart system, that helps collecting and analysing biological meaningful sets of transcripts. TBro’s modular architecture allows easy extension of its functionalities in the future. Especially, the integration of new data types such as proteomic quantifications or array-based gene expression data is straightforward. Thus, TBro is a fully featured yet flexible transcriptome browser that supports approaching complex biological questions and enhances collaboration of numerous researchers. Database URL: tbro.carnivorom.com PMID:28025338

  16. De novo transcriptome of the Hemimetabolous German cockroach (Blattella germanica.

    Directory of Open Access Journals (Sweden)

    Xiaojie Zhou

    Full Text Available BACKGROUND: The German cockroach, Blattella germanica, is an important insect pest that transmits various pathogens mechanically and causes severe allergic diseases. This insect has long served as a model system for studies of insect biology, physiology and ecology. However, the lack of genome or transcriptome information heavily hinder our further understanding about the German cockroach in every aspect at a molecular level and on a genome-wide scale. To explore the transcriptome and identify unique sequences of interest, we subjected the B. germanica transcriptome to massively parallel pyrosequencing and generated the first reference transcriptome for B. germanica. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1,365,609 raw reads with an average length of 529 bp were generated via pyrosequencing the mixed cDNA library from different life stages of German cockroach including maturing oothecae, nymphs, adult females and males. The raw reads were de novo assembled to 48,800 contigs and 3,961 singletons with high-quality unique sequences. These sequences were annotated and classified functionally in terms of BLAST, GO and KEGG, and the genes putatively coding detoxification enzyme systems, insecticide targets, key components in systematic RNA interference, immunity and chemoreception pathways were identified. A total of 3,601 SSRs (Simple Sequence Repeats loci were also predicted. CONCLUSIONS/SIGNIFICANCE: The whole transcriptome pyrosequencing data from this study provides a usable genetic resource for future identification of potential functional genes involved in various biological processes.

  17. Dynamic Actin Controls Polarity Induction de novo in Protoplasts

    Institute of Scientific and Technical Information of China (English)

    Beatrix Zaban; Jan Maisch; Peter Nick

    2013-01-01

    Cell polarity and axes are central for plant morphogenesis.To study how polarity and axes are induced de novo,we investigated protoplasts of tobacco Nicotiana tabacum cv.BY-2 expressing fluorescentlytagged cytoskeletal markers.We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages.The synthesis of a new cell wall marks the transition to the first stage of regeneration,and proceeds after a long preparatory phase within a few minutes.During this preparatory phase,the nucleus migrates actively,and cytoplasmic strands remodel vigorously.We probed this system for the effect of anti-cytoskeletal compounds,inducible bundling of actin,RGD-peptides,and temperature.Suppression of actin dynamics at an early stage leads to aberrant tripolar cells,whereas suppression of microtubule dynamics produces aberrant sausagelike cells with asymmetric cell walls.We integrated these data into a model,where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis.Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments,and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

  18. De novo transcriptome assembly of a sour cherry cultivar, Schattenmorelle

    Directory of Open Access Journals (Sweden)

    Yeonhwa Jo

    2015-12-01

    Full Text Available Sour cherry (Prunus cerasus in the genus Prunus in the family Rosaceae is one of the most popular stone fruit trees worldwide. Of known sour cherry cultivars, the Schattenmorelle is a famous old sour cherry with a high amount of fruit production. The Schattenmorelle was selected before 1650 and described in the 1800s. This cultivar was named after gardens of the Chateau de Moreille in which the cultivar was initially found. In order to identify new genes and to develop genetic markers for sour cherry, we performed a transcriptome analysis of a sour cherry. We selected the cultivar Schattenmorelle, which is among commercially important cultivars in Europe and North America. We obtained 2.05 GB raw data from the Schattenmorelle (NCBI accession number: SRX1187170. De novo transcriptome assembly using Trinity identified 61,053 transcripts in which N50 was 611 bp. Next, we identified 25,585 protein coding sequences using TransDecoder. The identified proteins were blasted against NCBI's non-redundant database for annotation. Based on blast search, we taxonomically classified the obtained sequences. As a result, we provide the transcriptome of sour cherry cultivar Schattenmorelle using next generation sequencing.

  19. De novo C3 glomerulonephritis in a renal allograft.

    Science.gov (United States)

    Nahm, Ji Hae; Song, Seung Hwan; Kim, Yu Seun; Cheong, Hae-Il; Lim, Beom Jin; Kim, Beom Seok; Jeong, Hyeon Joo

    2016-01-01

    C3 glomerulonephritis (C3GN) is a recently described, rare glomerular disease characterized by predominant or sole glomerular C3 deposits. Morphologic features of C3GN are similar to those of dense deposit disease (DDD); however, ribbon-like intramembranous electron-dense deposits are absent in the former. We report a case of de novo C3GN in a renal allograft with morphologic transformation to DDD. A 6-year-old boy presented with congenital left renal agenesis and right ureteropelvic junction obstruction. The patient underwent pyeloplasty but experienced recurrent urinary tract infections. At the age of 22 years, he received a renal allograft from a living related donor. C3GN was diagnosed after 1 year of transplantation; initial histology showed minimal mesangiopathy and this progressed to mesangial proliferation and membranoproliferative features over the next 7 years. Serum creatinine levels were stabilized with anti-rejection treatments for combating repeated episodes of acute rejection; however, glomerular and tubular band-like electron-dense deposits became evident.

  20. De novo transcriptome assembly of Sorghum bicolor variety Taejin

    Directory of Open Access Journals (Sweden)

    Yeonhwa Jo

    2016-06-01

    Full Text Available Sorghum (Sorghum bicolor, also known as great millet, is one of the most popular cultivated grass species in the world. Sorghum is frequently consumed as food for humans and animals as well as used for ethanol production. In this study, we conducted de novo transcriptome assembly for sorghum variety Taejin by next-generation sequencing, obtaining 8.748 GB of raw data. The raw data in this study can be available in NCBI SRA database with accession number of SRX1715644. Using the Trinity program, we identified 222,161 transcripts from sorghum variety Taejin. We further predicted coding regions within the assembled transcripts by the TransDecoder program, resulting in a total of 148,531 proteins. We carried out BLASTP against the Swiss-Prot protein sequence database to annotate the functions of the identified proteins. To our knowledge, this is the first transcriptome data for a sorghum variety derived from Korea, and it can be usefully applied to the generation of genetic markers.

  1. De novo transcriptome assembly of Sorghum bicolor variety Taejin.

    Science.gov (United States)

    Jo, Yeonhwa; Lian, Sen; Cho, Jin Kyong; Choi, Hoseong; Kim, Sang-Min; Kim, Sun-Lim; Lee, Bong Choon; Cho, Won Kyong

    2016-06-01

    Sorghum (Sorghum bicolor), also known as great millet, is one of the most popular cultivated grass species in the world. Sorghum is frequently consumed as food for humans and animals as well as used for ethanol production. In this study, we conducted de novo transcriptome assembly for sorghum variety Taejin by next-generation sequencing, obtaining 8.748 GB of raw data. The raw data in this study can be available in NCBI SRA database with accession number of SRX1715644. Using the Trinity program, we identified 222,161 transcripts from sorghum variety Taejin. We further predicted coding regions within the assembled transcripts by the TransDecoder program, resulting in a total of 148,531 proteins. We carried out BLASTP against the Swiss-Prot protein sequence database to annotate the functions of the identified proteins. To our knowledge, this is the first transcriptome data for a sorghum variety derived from Korea, and it can be usefully applied to the generation of genetic markers.

  2. Prevalence and origin of De Novo duplications in Charcot-Marie-Tooth disease type 1A: First report of a De Novo duplication with a maternal origin

    Energy Technology Data Exchange (ETDEWEB)

    Blair, I.P.; Nash, J.; Gordon, M.J.; Nicholson, G.A. [Univ. of Sydney, New South Wales (United Kingdom)

    1996-03-01

    Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. Sporadic cases of CMT have been described since the earliest reports of the disease. The most frequent form of the disorder, CMT1A, is associated with a 1.5-Mb DNA duplication on chromosome 17p11.2, which segregates with the disease. In order to investigate the prevalence of de novo CMT1A duplications, this study examined 118 duplication-positive CMT1A families. In 10 of these families it was demonstrated that the disease had arisen as the result of a de novo mutation. By taking into account the ascertainment of families, it can be estimated that {>=}10% of autosomal dominant CMT1 families are due to de novo duplications. The CMT1A duplication is thought to be the product of unequal crossing over between parental chromosome 17 homologues during meiosis. Polymorphic markers from within the duplicated region were used to determine the parental origin of these de novo duplications in eight informative families. Seven were of paternal and one of maternal origin. This study represents the first report of a de novo duplication with a maternal origin and indicates that it is not a phenomenon associated solely with male meioses. Recombination fractions for the region duplicated in CMT1A are larger in females than in males. That suggests that oogenesis may be afforded greater protection from misalignment during synapsis, and/or that there may be lower activity of those factors or mechanisms that lead to unequal crossing over at the CMT1A locus. 41 refs., 2 figs.

  3. MRUniNovo: an efficient tool for de novo peptide sequencing utilizing the hadoop distributed computing framework.

    Science.gov (United States)

    Li, Chuang; Chen, Tao; He, Qiang; Zhu, Yunping; Li, Kenli

    2016-12-19

    Tandem mass spectrometry-based de novo peptide sequencing is a complex and time-consuming process. The current algorithms for de novo peptide sequencing cannot rapidly and thoroughly process large mass spectrometry datasets. In this paper, we propose MRUniNovo, a novel tool for parallel de novo peptide sequencing. MRUniNovo parallelizes UniNovo based on the Hadoop compute platform. Our experimental results demonstrate that MRUniNovo significantly reduces the computation time of de novo peptide sequencing without sacrificing the correctness and accuracy of the results, and thus can process very large datasets that UniNovo cannot.

  4. Further Insights into Brevetoxin Metabolism by de Novo Radiolabeling

    Directory of Open Access Journals (Sweden)

    Kevin Calabro

    2014-06-01

    Full Text Available The toxic dinoflagellate Karenia brevis, responsible for early harmful algal blooms in the Gulf of Mexico, produces many secondary metabolites, including potent neurotoxins called brevetoxins (PbTx. These compounds have been identified as toxic agents for humans, and they are also responsible for the deaths of several marine organisms. The overall biosynthesis of these highly complex metabolites has not been fully ascertained, even if there is little doubt on a polyketide origin. In addition to gaining some insights into the metabolic events involved in the biosynthesis of these compounds, feeding studies with labeled precursors helps to discriminate between the de novo biosynthesis of toxins and conversion of stored intermediates into final toxic products in the response to environmental stresses. In this context, the use of radiolabeled precursors is well suited as it allows working with the highest sensitive techniques and consequently with a minor amount of cultured dinoflagellates. We were then able to incorporate [U-14C]-acetate, the renowned precursor of the polyketide pathway, in several PbTx produced by K. brevis. The specific activities of PbTx-1, -2, -3, and -7, identified by High-Resolution Electrospray Ionization Mass Spectrometer (HRESIMS, were assessed by HPLC-UV and highly sensitive Radio-TLC counting. We demonstrated that working at close to natural concentrations of acetate is a requirement for biosynthetic studies, highlighting the importance of highly sensitive radiolabeling feeding experiments. Quantification of the specific activity of the four, targeted toxins led us to propose that PbTx-1 and PbTx-2 aldehydes originate from oxidation of the primary alcohols of PbTx-7 and PbTx-3, respectively. This approach will open the way for a better comprehension of the metabolic pathways leading to PbTx but also to a better understanding of their regulation by environmental factors.

  5. Myelodysplastic syndrome evolving from aplastic anemia treated with immunosuppressive therapy: efficacy of hematopoietic stem cell transplantation.

    Science.gov (United States)

    Kim, Sung-Yong; Le Rademacher, Jennifer; Antin, Joseph H; Anderlini, Paolo; Ayas, Mouhab; Battiwalla, Minoo; Carreras, Jeanette; Kurtzberg, Joanne; Nakamura, Ryotaro; Eapen, Mary; Deeg, H Joachim

    2014-12-01

    A proportion of patients with aplastic anemia who are treated with immunosuppressive therapy develop clonal hematologic disorders, including post-aplastic anemia myelodysplastic syndrome. Many will proceed to allogeneic hematopoietic stem cell transplantation. We identified 123 patients with post-aplastic anemia myelodysplastic syndrome who from 1991 through 2011 underwent allogeneic hematopoietic stem cell transplantation, and in a matched-pair analysis compared outcome to that in 393 patients with de novo myelodysplastic syndrome. There was no difference in overall survival. There were no significant differences with regard to 5-year probabilities of relapse, non-relapse mortality, relapse-free survival and overall survival; these were 14%, 40%, 46% and 49% for post-aplastic anemia myelodysplastic syndrome, and 20%, 33%, 47% and 49% for de novo myelodysplastic syndrome, respectively. In multivariate analysis, relapse (hazard ratio 0.71; P=0.18), non-relapse mortality (hazard ratio 1.28; P=0.18), relapse-free survival (hazard ratio 0.97; P=0.80) and overall survival (hazard ratio 1.02; P=0.88) of post-aplastic anemia myelodysplastic syndrome were similar to those of patients with de novo myelodysplastic syndrome. Cytogenetic risk was independently associated with overall survival in both groups. Thus, transplant success in patients with post-aplastic anemia myelodysplastic syndrome was similar to that in patients with de novo myelodysplastic syndrome, and cytogenetics was the only significant prognostic factor for post-aplastic anemia myelodysplastic syndrome patients.

  6. High frequencies of de novo CNVs in bipolar disorder and schizophrenia.

    LENUS (Irish Health Repository)

    Malhotra, Dheeraj

    2011-12-22

    While it is known that rare copy-number variants (CNVs) contribute to risk for some neuropsychiatric disorders, the role of CNVs in bipolar disorder is unclear. Here, we reasoned that a contribution of CNVs to mood disorders might be most evident for de novo mutations. We performed a genome-wide analysis of de novo CNVs in a cohort of 788 trios. Diagnoses of offspring included bipolar disorder (n = 185), schizophrenia (n = 177), and healthy controls (n = 426). Frequencies of de novo CNVs were significantly higher in bipolar disorder as compared with controls (OR = 4.8 [1.4,16.0], p = 0.009). De novo CNVs were particularly enriched among cases with an age at onset younger than 18 (OR = 6.3 [1.7,22.6], p = 0.006). We also confirmed a significant enrichment of de novo CNVs in schizophrenia (OR = 5.0 [1.5,16.8], p = 0.007). Our results suggest that rare spontaneous mutations are an important contributor to risk for bipolar disorder and other major neuropsychiatric diseases.

  7. Asymptomatic De Novo Inflammatory Bowel Disease Late After Liver Transplantation for Primary Sclerosing Cholangitis: A Case Report.

    Science.gov (United States)

    Åberg, F; Abdulle, A; Mäkelä, A; Nissinen, M

    2015-11-01

    Guidelines recommend colonoscopy screening for possible asymptomatic inflammatory bowel disease (IBD) in all patients diagnosed with primary sclerosing cholangitis (PSC). PSC-IBD warrants regular dysplasia-surveillance colonoscopy. However, no consensus exists regarding follow-up colonoscopy in PSC patients without IBD who remain asymptomatic. We describe a 43-year-old female who had undergone liver transplantation (LT) due to advanced PSC. Previous colonoscopies had been normal. The post-transplantation course was uneventful, with no rejections and signs of PSC recurrence. Immunosuppression was by tacrolimus monotherapy. She was asymptomatic with normal inflammation markers. A protocol colonoscopy, performed as general dysplasia surveillance 8 years post-transplantation, revealed mucopurulent-covered small superficial ulcerations and erythema diffusely distributed from the cecal to sigmoid colon with intervening normal mucosa and rectal sparing. Histologic examination showed patchy chronic colitis with crypt architectural distortion and mild-moderate inflammation activity. Infection samples were negative. Findings complied with de novo IBD, type unclassified. In conclusion, the link between PSC and clinically silent IBD may manifest after the PSC diagnosis and even several years after LT. Given the increased colorectal cancer risk associated with PSC, IBD, and LT, repeat colonoscopy might be warranted in PSC patients without IBD at initial assessment, and also after LT.

  8. Defining the maize transcriptome de novo using deep RNA-Seq

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Gross, Stephen; Choi, Cindy; Zhang, Tao; Lindquist, Erika; Wei, Chia-Lin; Wang, Zhong

    2011-06-01

    De novo assembly of the transcriptome is crucial for functional genomics studies in bioenergy research, since many of the organisms lack high quality reference genomes. In a previous study we successfully de novo assembled simple eukaryote transcriptomes exclusively from short Illumina RNA-Seq reads [1]. However, extensive alternative splicing, present in most of the higher eukaryotes, poses a significant challenge for current short read assembly processes. Furthermore, the size of next-generation datasets, often large for plant genomes, presents an informatics challenge. To tackle these challenges we present a combined experimental and informatics strategy for de novo assembly in higher eukaryotes. Using maize as a test case, preliminary results suggest our approach can resolve transcript variants and improve gene annotations.

  9. Defining the maize transcriptome de novo using deep RNA-Seq

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Gross, Stephen; Choi, Cindy; Zhang, Tao; Lindquist, Erika; Wei, Chia-Lin; Wang, Zhong

    2011-06-02

    De novo assembly of the transcriptome is crucial for functional genomics studies in bioenergy research, since many of the organisms lack high quality reference genomes. In a previous study we successfully de novo assembled simple eukaryote transcriptomes exclusively from short Illumina RNA-Seq reads [1]. However, extensive alternative splicing, present in most of the higher eukaryotes, poses a significant challenge for current short read assembly processes. Furthermore, the size of next-generation datasets, often large for plant genomes, presents an informatics challenge. To tackle these challenges we present a combined experimental and informatics strategy for de novo assembly in higher eukaryotes. Using maize as a test case, preliminary results suggest our approach can resolve transcript variants and improve gene annotations.

  10. De novo assembly of human genomes with massively parallel short read sequencing

    DEFF Research Database (Denmark)

    Li, Ruiqiang; Zhu, Hongmei; Ruan, Jue;

    2010-01-01

    genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities......Next-generation massively parallel DNA sequencing technologies provide ultrahigh throughput at a substantially lower unit data cost; however, the data are very short read length sequences, making de novo assembly extremely challenging. Here, we describe a novel method for de novo assembly of large...... for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way....

  11. Evaluating de novo sequencing in proteomics: already an accurate alternative to database-driven peptide identification?

    Science.gov (United States)

    Muth, Thilo; Renard, Bernhard Y

    2017-03-21

    While peptide identifications in mass spectrometry (MS)-based shotgun proteomics are mostly obtained using database search methods, high-resolution spectrum data from modern MS instruments nowadays offer the prospect of improving the performance of computational de novo peptide sequencing. The major benefit of de novo sequencing is that it does not require a reference database to deduce full-length or partial tag-based peptide sequences directly from experimental tandem mass spectrometry spectra. Although various algorithms have been developed for automated de novo sequencing, the prediction accuracy of proposed solutions has been rarely evaluated in independent benchmarking studies. The main objective of this work is to provide a detailed evaluation on the performance of de novo sequencing algorithms on high-resolution data. For this purpose, we processed four experimental data sets acquired from different instrument types from collision-induced dissociation and higher energy collisional dissociation (HCD) fragmentation mode using the software packages Novor, PEAKS and PepNovo. Moreover, the accuracy of these algorithms is also tested on ground truth data based on simulated spectra generated from peak intensity prediction software. We found that Novor shows the overall best performance compared with PEAKS and PepNovo with respect to the accuracy of correct full peptide, tag-based and single-residue predictions. In addition, the same tool outpaced the commercial competitor PEAKS in terms of running time speedup by factors of around 12-17. Despite around 35% prediction accuracy for complete peptide sequences on HCD data sets, taken as a whole, the evaluated algorithms perform moderately on experimental data but show a significantly better performance on simulated data (up to 84% accuracy). Further, we describe the most frequently occurring de novo sequencing errors and evaluate the influence of missing fragment ion peaks and spectral noise on the accuracy. Finally

  12. Fact or fiction: updates on how protein-coding genes might emerge de novo from previously non-coding DNA

    Science.gov (United States)

    Schmitz, Jonathan F; Bornberg-Bauer, Erich

    2017-01-01

    Over the last few years, there has been an increasing amount of evidence for the de novo emergence of protein-coding genes, i.e. out of non-coding DNA. Here, we review the current literature and summarize the state of the field. We focus specifically on open questions and challenges in the study of de novo protein-coding genes such as the identification and verification of de novo-emerged genes. The greatest obstacle to date is the lack of high-quality genomic data with very short divergence times which could help precisely pin down the location of origin of a de novo gene. We conclude that, while there is plenty of evidence from a genetics perspective, there is a lack of functional studies of bona fide de novo genes and almost no knowledge about protein structures and how they come about during the emergence of de novo protein-coding genes. We suggest that future studies should concentrate on the functional and structural characterization of de novo protein-coding genes as well as the detailed study of the emergence of functional de novo protein-coding genes.

  13. De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder

    DEFF Research Database (Denmark)

    Hamilton, P J; Campbell, N G; Sharma, S

    2013-01-01

    De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution...... at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first...

  14. A hybrid approach for de novo human genome sequence assembly and phasing.

    Science.gov (United States)

    Mostovoy, Yulia; Levy-Sakin, Michal; Lam, Jessica; Lam, Ernest T; Hastie, Alex R; Marks, Patrick; Lee, Joyce; Chu, Catherine; Lin, Chin; Džakula, Željko; Cao, Han; Schlebusch, Stephen A; Giorda, Kristina; Schnall-Levin, Michael; Wall, Jeffrey D; Kwok, Pui-Yan

    2016-07-01

    Despite tremendous progress in genome sequencing, the basic goal of producing a phased (haplotype-resolved) genome sequence with end-to-end contiguity for each chromosome at reasonable cost and effort is still unrealized. In this study, we describe an approach to performing de novo genome assembly and experimental phasing by integrating the data from Illumina short-read sequencing, 10X Genomics linked-read sequencing, and BioNano Genomics genome mapping to yield a high-quality, phased, de novo assembled human genome.

  15. Whole-Genome de novo Sequencing Of Quail And Grey Partridge

    DEFF Research Database (Denmark)

    Holm, Lars-Erik; Panitz, Frank; Burt, Dave;

    2011-01-01

    The development in sequencing methods has made it possible to perform whole genome de novo sequencing of species without large commercial interests. Within the EU-financed QUANTOMICS project (KBBE-2A-222664), we have performed de novo sequencing of quail (Coturnix coturnix) and grey partridge...... comparative studies towards the chicken genome and will aid in identifying evolutionarily conserved sequences within the Galliformes. The obtained sequences from quail and partridge represent a beginning of generating the whole genome sequence for these species. The continuation of establishing the genome...

  16. Biased exon/intron distribution of cryptic and de novo 3' splice sites.

    Science.gov (United States)

    Královicová, Jana; Christensen, Mikkel B; Vorechovský, Igor

    2005-01-01

    We compiled sequences of previously published aberrant 3' splice sites (3'ss) that were generated by mutations in human disease genes. Cryptic 3'ss, defined here as those resulting from a mutation of the 3'YAG consensus, were more frequent in exons than in introns. They clustered in approximately 20 nt region adjacent to authentic 3'ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3'ss that were induced by mutations outside the 3'YAG consensus (designated 'de novo') were in introns. The activation of intronic de novo 3'ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3'ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro-Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3'ss. Finally, AG-creating mutations in the PPT that produced aberrant 3'ss upstream of the predicted BPS in vivo shared a similar 'BPS-new AG' distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3'ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.

  17. De Novo Truncating Variants in ASXL2 Are Associated with a Unique and Recognizable Clinical Phenotype

    NARCIS (Netherlands)

    Shashi, V.; Pena, L.D.; Kim, K.; Burton, B.; Hempel, M.; Schoch, K.; Walkiewicz, M.; McLaughlin, H.M.; Cho, M; Stong, N.; Hickey, S.E.; Shuss, C.M.; Freemark, M.S.; Bellet, J.S.; Keels, M.A.; Bonner, M.J.; El-Dairi, M.; Butler, M; Kranz, P.G.; Stumpel, C.T.; Klinkenberg, S.; Oberndorff, K.; Alawi, M.; Santer, R.; Petrovski, S.; Kuismin, O.; Korpi-Heikkila, S.; Pietilainen, O.; Aarno, P.; Kurki, M.I.; Hoischen, A.; Need, A.C.; Goldstein, D.B; Kortum, F.

    2016-01-01

    The ASXL genes (ASXL1, ASXL2, and ASXL3) participate in body patterning during embryogenesis and encode proteins involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1 and ASXL3 have been respectively implicated

  18. 76 FR 68767 - Draft Guidance for Industry and Food and Drug Administration Staff; De Novo Classification...

    Science.gov (United States)

    2011-11-07

    ... entitled ``Draft Guidance for Industry and Food and Drug Administration Staff; De Novo Classification...] [FR Doc No: 2011-28766] DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2011-D-0689] Draft Guidance for Industry and Food and Drug Administration Staff; De...

  19. A recurrent de novo mutation in KCNC1 causes progressive myoclonus epilepsy

    DEFF Research Database (Denmark)

    Muona, M.; Berkovic, S. F.; Dibbens, L. M.

    2015-01-01

    Progressive myoclonus epilepsies (PMEs) are a group of rare, inherited disorders manifesting with action myoclonus, tonicclonic seizures and ataxia. We sequenced the exomes of 84 unrelated individuals with PME of unknown cause and molecularly solved 26 cases (31%). Remarkably, a recurrent de novo...

  20. De Novo Mutations in CHAMP1 Cause Intellectual Disability with Severe Speech Impairment

    NARCIS (Netherlands)

    Hempel, Maja; Cremer, Kirsten; Ockeloen, Charlotte W.; Lichtenbelt, Klaske D.; Herkert, Johanna C.; Denecke, Jonas; Haack, Tobias B.; Zink, Alexander M.; Becker, Jessica; Wohlleber, Eva; Johannsen, Jessika; Alhaddad, Bader; Pfundt, Rolph; Fuchs, Sigrid; Wieczorek, Dagmar; Strom, Tim M.; van Gassen, Koen L. I.; Kleefstra, Tjitske; Kubisch, Christian; Engels, Hartmut; Lessel, Davor

    2015-01-01

    CHAMP1 encodes a protein with a function in kinetochore-microtubule attachment and in the regulation of chromosome segregation, both of which are known to be important for neurodevelopment. By trio whole-exome sequencing, we have identified de novo deleterious mutations in CHAMP1 in five unrelated i

  1. De Novo Insertions and Deletions of Predominantly Paternal Origin Are Associated with Autism Spectrum Disorder

    Directory of Open Access Journals (Sweden)

    Shan Dong

    2014-10-01

    Full Text Available Whole-exome sequencing (WES studies have demonstrated the contribution of de novo loss-of-function single-nucleotide variants (SNVs to autism spectrum disorder (ASD. However, challenges in the reliable detection of de novo insertions and deletions (indels have limited inclusion of these variants in prior analyses. By applying a robust indel detection method to WES data from 787 ASD families (2,963 individuals, we demonstrate that de novo frameshift indels contribute to ASD risk (OR = 1.6; 95% CI = 1.0–2.7; p = 0.03, are more common in female probands (p = 0.02, are enriched among genes encoding FMRP targets (p = 6 × 10−9, and arise predominantly on the paternal chromosome (p < 0.001. On the basis of mutation rates in probands versus unaffected siblings, we conclude that de novo frameshift indels contribute to risk in approximately 3% of individuals with ASD. Finally, by observing clustering of mutations in unrelated probands, we uncover two ASD-associated genes: KMT2E (MLL5, a chromatin regulator, and RIMS1, a regulator of synaptic vesicle release.

  2. De novo insertions and deletions of predominantly paternal origin are associated with autism spectrum disorder

    Science.gov (United States)

    Dong, Shan; Walker, Michael F.; Carriero, Nicholas J.; DiCola, Michael; Willsey, A. Jeremy; Ye, Adam Y.; Waqar, Zainulabedin; Gonzalez, Luis E.; Overton, John D.; Frahm, Stephanie; Keaney, John F.; Teran, Nicole A.; Dea, Jeanselle; Mandell, Jeffrey D.; Bal, Vanessa Hus; Sullivan, Catherine A.; DiLullo, Nicholas M.; Khalil, Rehab O.; Gockley, Jake; Yuksel, Zafer; Sertel, Sinem M.; Ercan-Sencicek, A. Gulhan; Gupta, Abha R.; Mane, Shrikant M.; Sheldon, Michael; Brooks, Andrew I.; Roeder, Kathryn; Devlin, Bernie; State, Matthew W.; Wei, Liping; Sanders, Stephan J.

    2014-01-01

    SUMMARY Whole-exome sequencing (WES) studies have demonstrated the contribution of de novo loss-of-function single nucleotide variants to autism spectrum disorders (ASD). However, challenges in the reliable detection of de novo insertions and deletions (indels) have limited inclusion of these variants in prior analyses. Through the application of a robust indel detection method to WES data from 787 ASD families (2,963 individuals), we demonstrate that de novo frameshift indels contribute to ASD risk (OR=1.6; 95%CI=1.0-2.7; p=0.03), are more common in female probands (p=0.02), are enriched among genes encoding FMRP targets (p=6×10−9), and arise predominantly on the paternal chromosome (p<0.001). Based on mutation rates in probands versus unaffected siblings, de novo frameshift indels contribute to risk in approximately 3.0% of individuals with ASD. Finally, through observing clustering of mutations in unrelated probands, we report two novel ASD-associated genes: KMT2E (MLL5), a chromatin regulator, and RIMS1, a regulator of synaptic vesicle release. PMID:25284784

  3. De novo nonsense mutations in ASXL1 cause Bohring-Opitz syndrome

    DEFF Research Database (Denmark)

    Hoischen, Alexander; van Bon, Bregje W M; Rodríguez-Santiago, Benjamín;

    2011-01-01

    Bohring-Opitz syndrome is characterized by severe intellectual disability, distinctive facial features and multiple congenital malformations. We sequenced the exomes of three individuals with Bohring-Opitz syndrome and in each identified heterozygous de novo nonsense mutations in ASXL1, which...

  4. Three families with 'de novo' m.3243A > G mutation.

    Science.gov (United States)

    de Laat, Paul; Janssen, Mirian C H; Alston, Charlotte L; Taylor, Robert W; Rodenburg, Richard J T; Smeitink, Jan A M

    2016-12-01

    The m.3243A > G mutation is the most prevalent, disease-causing mitochondrial DNA (mtDNA) mutation. In a national cohort study of 48 families harbouring the m.3243A > G mutation, we identified three families in which the mutation appeared to occur sporadically within these families. In this report we describe these three families. Based on detailed mtDNA analysis of three different tissues using two different quantitative pyrosequencing assays with sensitivity to a level of 1% mutated mtDNA, we conclude that the m.3243A > G mutation has arisen de novo in each of these families. The symptomatic carriers presented with a variety of symptoms frequently observed in patients harbouring the m.3243A > G mutation. A more severe phenotype is seen in the de novo families compared to recent cohort studies, which might be due to reporting bias. The observation that de novo m.3243A > G mutations exist is of relevance for both diagnostic investigations and genetic counselling. Firstly, even where there is no significant (maternal) family history in patients with stroke-like episodes, diabetes and deafness or other unexplained organ dysfunction, the m.3243A > G mutation should be screened as a possible cause of the disease. Second, analysis of maternally-related family members is highly recommended to provide reliable counselling for these families, given that the m.3243A > G mutation may have arisen de novo.

  5. Advanced approaches for the characterization of a de novo designed antiparallel coiled coil peptide

    NARCIS (Netherlands)

    Pagel, K; Seeger, K; Seiwert, B; Villa, Alessandra; Mark, AE; Berger, S; Koksch, B

    2005-01-01

    We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modi. cation of two hydrophobic cor

  6. Monosaccharide templates for de novo designed 4-alpha-helix bundle proteins: template effects in carboproteins

    DEFF Research Database (Denmark)

    Brask, Jesper; Dideriksen, J.M.; Nielsen, John;

    2003-01-01

    De novo design and total chemical synthesis of proteins provide powerful approaches to critically test our understanding of protein folding, structure, and stability. The 4-alpha-helix bundle is a frequently studied structure in which four amphiphilic alpha-helical peptide strands form a hydropho......)) and melting points in chemical and thermal denaturation experiments....

  7. Phenotype and clinical course in a family with a new de novo Twinkle gene mutation

    DEFF Research Database (Denmark)

    Jeppesen, Tina Dysgaard; Schwartz, M.; Colding-Jorgensen, E.;

    2008-01-01

    The Twinkle gene product is important for mtDNA replication. Only a few reports have investigated the clinically effect of mutations in this gene. We describe a new de novo mutation (1110C > A) in the PEO1 gene in a mother and her two sons. The mother had progressive ophthalmoplegia, limb weakness...

  8. Drug-Eluting Balloons in the Treatment of Coronary De Novo Lesions

    DEFF Research Database (Denmark)

    Richelsen, Rasmus Kapalu Broge; Overvad, Thure Filskov; Jensen, Svend Eggert

    2016-01-01

    in coronary de novo lesions, either as part of a DEB-only strategy or in combination with another device, mainly a bare metal stent (BMS). By searching Pubmed and Embase we were able to identify 52 relevant studies, differing in design, intervention, and clinical setting, including patients with small vessel...

  9. De novo formal synthesis of (-)-virginiamycin M2 via the asymmetric hydration of dienoates.

    Science.gov (United States)

    Mortensen, Matthew S; Osbourn, Joshua M; O'Doherty, George A

    2007-08-02

    A de novo approach to the formal total synthesis of the macrolide natural product (-)-virginiamycin M2 has been achieved via a convergent approach. The absolute and relative stereochemistry of the nonpeptide portion of (-)-virginiamycin M2 was introduced by two Sharpless asymmetric dihydroxylation reactions.

  10. 12 CFR 925.14 - De novo insured depository institution applicants.

    Science.gov (United States)

    2010-01-01

    ...) Conditional approval. A de novo applicant that has not received its first formal, or, if unavailable, informal... better on its first formal, or if unavailable, informal or preliminary, CRA performance evaluation. (3... applicants. 925.14 Section 925.14 Banks and Banking FEDERAL HOUSING FINANCE BOARD FEDERAL HOME LOAN...

  11. De novo mutations from sporadic schizophrenia cases highlight important signaling genes in an independent sample.

    Science.gov (United States)

    Kranz, Thorsten M; Harroch, Sheila; Manor, Orly; Lichtenberg, Pesach; Friedlander, Yechiel; Seandel, Marco; Harkavy-Friedman, Jill; Walsh-Messinger, Julie; Dolgalev, Igor; Heguy, Adriana; Chao, Moses V; Malaspina, Dolores

    2015-08-01

    Schizophrenia is a debilitating syndrome with high heritability. Genomic studies reveal more than a hundred genetic variants, largely nonspecific and of small effect size, and not accounting for its high heritability. De novo mutations are one mechanism whereby disease related alleles may be introduced into the population, although these have not been leveraged to explore the disease in general samples. This paper describes a framework to find high impact genes for schizophrenia. This study consists of two different datasets. First, whole exome sequencing was conducted to identify disruptive de novo mutations in 14 complete parent-offspring trios with sporadic schizophrenia from Jerusalem, which identified 5 sporadic cases with de novo gene mutations in 5 different genes (PTPRG, TGM5, SLC39A13, BTK, CDKN3). Next, targeted exome capture of these genes was conducted in 48 well-characterized, unrelated, ethnically diverse schizophrenia cases, recruited and characterized by the same research team in New York (NY sample), which demonstrated extremely rare and potentially damaging variants in three of the five genes (MAFgene they carried. Functional de novo mutations in protein-interaction domains in sporadic schizophrenia can illuminate risk genes that increase the propensity to develop schizophrenia across ethnicities.

  12. Impact of modern chemotherapy on the survival of women presenting with de novo metastatic breast cancer

    Directory of Open Access Journals (Sweden)

    Pal Sumanta K

    2012-09-01

    Full Text Available Abstract Background Data that directly associate utilization of novel systemic therapies with survival trends in metastatic breast cancer (MBC are limited. In the setting of de novo MBC, large registry analyses cite positive temporal trends in survival, but the extent to which advances in systemic therapy have contributed to these gains is not clear. Methods The City of Hope Cancer Registry was used to identify a consecutive series of patients with de novo MBC who received their first line of therapy between 1985 and 2004. Comprehensive clinicopathologic and treatment-related data were collected for each patient. Univariate analyses were conducted via Cox regression to identify factors associated with improved survival. Multivariate analysis was also conducted via Cox regression and the stepwise procedure was used to identify independent predictors of survival. Results A total of 324 patients with de novo MBC were identified. After application of exclusion criteria, including the sole presence of supraclavicular node metastasis, 274 patients were retained in the analysis. The treatment-related characteristics associated with improved survival included: use of endocrine therapy (hazard ratio [HR] 0.60, 95%CI 0.47-0.77; P Conclusions The overall survival of women with de novo metastatic breast cancer has improved over the past 20 years. However, the contribution of conventional cytotoxic agents to this improvement is minimal.

  13. Enhanced de novo assembly of high throughput pyrosequencing data using whole genome mapping.

    Science.gov (United States)

    Onmus-Leone, Fatma; Hang, Jun; Clifford, Robert J; Yang, Yu; Riley, Matthew C; Kuschner, Robert A; Waterman, Paige E; Lesho, Emil P

    2013-01-01

    Despite major advances in next-generation sequencing, assembly of sequencing data, especially data from novel microorganisms or re-emerging pathogens, remains constrained by the lack of suitable reference sequences. De novo assembly is the best approach to achieve an accurate finished sequence, but multiple sequencing platforms or paired-end libraries are often required to achieve full genome coverage. In this study, we demonstrated a method to assemble complete bacterial genome sequences by integrating shotgun Roche 454 pyrosequencing with optical whole genome mapping (WGM). The whole genome restriction map (WGRM) was used as the reference to scaffold de novo assembled sequence contigs through a stepwise process. Large de novo contigs were placed in the correct order and orientation through alignment to the WGRM. De novo contigs that were not aligned to WGRM were merged into scaffolds using contig branching structure information. These extended scaffolds were then aligned to the WGRM to identify the overlaps to be eliminated and the gaps and mismatches to be resolved with unused contigs. The process was repeated until a sequence with full coverage and alignment with the whole genome map was achieved. Using this method we were able to achieved 100% WGRM coverage without a paired-end library. We assembled complete sequences for three distinct genetic components of a clinical isolate of Providencia stuartii: a bacterial chromosome, a novel bla NDM-1 plasmid, and a novel bacteriophage, without separately purifying them to homogeneity.

  14. Steroid-resistant Tolosa-Hunt syndrome with a de novo intracavernous aneurysm: A case report

    Directory of Open Access Journals (Sweden)

    Hiroshi Takasuna

    2016-01-01

    Conclusions: To our knowledge, this is the first report of a growing de novo C4 aneurysm in THS. Surgical intervention and administration of MTX should be attempted in steroid-resistant THS. Careful observation with serial MRI and MR angiography is important to manage the complicated THS.

  15. Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos

    DEFF Research Database (Denmark)

    Svarcova, Olga; Laurincik, Jozef; Avery, Birthe;

    2007-01-01

    developmental stages and disintegrated NPBs stage were found at the late 8-cell. Our immunofluorescence data indicate that RNA polymerase I, UBF, topoisomerase I and fibrillarin are transcribed de novo whereas nucleolin and nucleophosmin are maternally inherited as demonstrated by a-amanitin inhibition. However...

  16. De novo ring chromosome 3: a new case with a mild phenotype.

    Science.gov (United States)

    McKinley, M; Colley, A; Sinclair, P; Donnai, D; Andrews, T

    1991-08-01

    We report an 18 year old female with a de novo ring chromosome 3 found after investigation for short stature. Her karyotype was interpreted as 46,XX, r(3)(p26.2q29). Her phenotype is milder than previously reported cases and illustrates the mild end of the spectrum of the ring chromosome 3 phenotype.

  17. De novo mutations of KIAA2022 in females cause intellectual disability and intractable epilepsy

    DEFF Research Database (Denmark)

    de Lange, Iris M; Helbig, Katherine L; Weckhuysen, Sarah;

    2016-01-01

    BACKGROUND: Mutations in the KIAA2022 gene have been reported in male patients with X-linked intellectual disability, and related female carriers were unaffected. Here, we report 14 female patients who carry a heterozygous de novo KIAA2022 mutation and share a phenotype characterised by intellect...

  18. Clinical Implications of Phosphorylated STAT3 Expression in de novo Diffuse Large B-cell Lymphoma

    DEFF Research Database (Denmark)

    Ok, Chi Y; Chen, Jiayu; Xu-Monette, Ziju

    2014-01-01

    of phosphorylated STAT3 (pSTAT3) on prognosis are limited. EXPERIMENTAL DESIGN: We evaluated expression of pSTAT3 in de novo DLBCL using immunohistochemistry, gene expression profiling (GEP), and gene set enrichment analysis (GSEA). Results were analyzed in correlation with cell-of-origin (COO), critical lymphoma...

  19. A glance at quality score: implication for de novo transcriptome reconstruction of Illumina reads

    Directory of Open Access Journals (Sweden)

    Stanley Kimbung Mbandi

    2014-02-01

    Full Text Available Downstream analyses of short-reads from next-generation sequencing platforms are often preceded by a pre-processing step that removes uncalled and wrongly called bases. Standard approaches rely on their associated base quality scores to retain the read or a portion of it when the score is above a predefined threshold. It is difficult to differentiate sequencing error from biological variation without a reference using quality scores. The effects of quality score based trimming have not been systematically studied in de novo transcriptome assembly. Using RNA-Seq data produced from Illumina, we teased out the effects of quality score base filtering or trimming on de novo transcriptome reconstruction. We showed that assemblies produced from reads subjected to different quality score thresholds contain truncated and missing transfrags when compared to those from untrimmed reads. Our data supports the fact that de novo assembling of untrimmed data is challenging for de Bruijn graph assemblers. However, our results indicates that comparing the assemblies from untrimmed and trimmed read subsets can suggest appropriate filtering parameters and enable selection of the optimum de novo transcriptome assembly in non-model organisms.

  20. De novo genome assembly of Cercospora beticola for microsatellite marker development and validation

    Science.gov (United States)

    Cercospora leaf spot caused by Cercospora beticola is a significant threat to the production of sugar and table beet worldwide. A de novo genome assembly of C. beticola was used to develop eight polymorphic and reproducible microsatellite markers for population genetic analyses. These markers were u...

  1. Thermogravimetric desorption and de novo tests I: method development and validation.

    Science.gov (United States)

    Tsytsik, Palina; Czech, Jan; Carleer, Robert; Reggers, Guy; Buekens, Alfons

    2008-08-01

    Thermogravimetric analysis (TGA) has been combined with evolved gas analysis (EGA) with the purpose of simulating the thermal behaviour of filter dust samples under inert (desorption) and de novo test oxidising conditions. Emphasis is on studying de novo formation of dioxins, surrogates and precursors arising from filter dust derived from thermal processes, such as municipal solid waste incineration and metallurgy. A new method is tested for sampling and analysing dioxin surrogates and precursors in the TGA effluent, which are collected on sampling tubes; the adsorbed compounds are eventually desorbed and quantified by TD-GC-MS. The major sources of error and losses are considered, including potential sorbent artefacts, possible breakthrough of volatiles through sampling tubes, or eventual losses of semi-volatiles due to their incomplete desorption or re-condensation inside the TG Analyser. The method is optimised and validated for di- to hexa-chlorinated benzenes in a range of 10-1000 ppb with average recovery exceeding 85%. The results are compared with data obtained in similar studies, performed by other research groups. As a result, the method provides the means for simulating de novo synthesis of dioxins in fly-ash and facilitates reliable and easy estimation of de novo activity, comparable with results of other studies, in combination with wide flexibility of testing conditions.

  2. Identification of a de novo DYNC1H1 mutation via WES according to published guidelines

    Science.gov (United States)

    Ding, Dongxue; Chen, Zhao; Li, Kai; Long, Zhe; Ye, Wei; Tang, Zhaoli; Xia, Kun; Qiu, Rong; Tang, Beisha; Jiang, Hong

    2016-01-01

    De novo mutations that contribute to rare Mendelian diseases, including neurological disorders, have been recently identified. Whole-exome sequencing (WES) has become a powerful tool for the identification of inherited and de novo mutations in Mendelian diseases. Two important guidelines were recently published regarding the investigation of causality of sequence variant in human disease and the interpretation of novel variants identified in human genome sequences. In this study, a family with supposed movement disorders was sequenced via WES (including the proband and her unaffected parents), and a standard investigation and interpretation of the identified variants was performed according to the published guidelines. We identified a novel de novo mutation (c.2327C > T, p.P776L) in DYNC1H1 gene and confirmed that it was the causal variant. The phenotype of the affected twins included delayed motor milestones, pes cavus, lower limb weakness and atrophy, and a waddling gait. Electromyographic (EMG) recordings revealed typical signs of chronic denervation. Our study demonstrates the power of WES to discover the de novo mutations associated with a neurological disease on the whole exome scale, and guidelines to conduct WES studies and interpret of identified variants are a preferable option for the exploration of the pathogenesis of rare neurological disorders. PMID:26846447

  3. De novo autoimmune hepatitis in liver transplant: State-of-the-art review.

    Science.gov (United States)

    Vukotic, Ranka; Vitale, Giovanni; D'Errico-Grigioni, Antonia; Muratori, Luigi; Andreone, Pietro

    2016-03-14

    In the two past decades, a number of communications, case-control studies, and retrospective reports have appeared in the literature with concerns about the development of a complex set of clinical, laboratory and histological characteristics of a liver graft dysfunction that is compatible with autoimmune hepatitis. The de novo prefix was added to distinguish this entity from a pre-transplant primary autoimmune hepatitis, but the globally accepted criteria for the diagnosis of autoimmune hepatitis have been adopted in the diagnostic algorithm. Indeed, de novo autoimmune hepatitis is characterized by the typical liver necro-inflammation that is rich in plasma cells, the presence of interface hepatitis and the consequent laboratory findings of elevations in liver enzymes, increases in serum gamma globulin and the appearance of non-organ specific auto-antibodies. Still, the overall features of de novo autoimmune hepatitis appear not to be attributable to a univocal patho-physiological pathway because they can develop in the patients who have undergone liver transplantation due to different etiologies. Specifically, in subjects with hepatitis C virus recurrence, an interferon-containing antiviral treatment has been indicated as a potential inception of immune system derangement. Herein, we attempt to review the currently available knowledge about de novo liver autoimmunity and its clinical management.

  4. In vitro, long-range sequence information for de novo genome assembly via transposase contiguity.

    Science.gov (United States)

    Adey, Andrew; Kitzman, Jacob O; Burton, Joshua N; Daza, Riza; Kumar, Akash; Christiansen, Lena; Ronaghi, Mostafa; Amini, Sasan; Gunderson, Kevin L; Steemers, Frank J; Shendure, Jay

    2014-12-01

    We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to > 1 megabase. These pools are "subhaploid," in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome. The scaffolding approach described here, termed fragScaff, leverages coincidences between the content of different pools as a source of contiguity information. Specifically, CPT-seq data is mapped to a de novo genome assembly, followed by the identification of pairs of contigs or scaffolds whose ends disproportionately co-occur in the same indexed pools, consistent with true adjacency in the genome. Such candidate "joins" are used to construct a graph, which is then resolved by a minimum spanning tree. As a proof-of-concept, we apply CPT-seq and fragScaff to substantially boost the contiguity of de novo assemblies of the human, mouse, and fly genomes, increasing the scaffold N50 of de novo assemblies by eight- to 57-fold with high accuracy. We also demonstrate that fragScaff is complementary to Hi-C-based contact probability maps, providing midrange contiguity to support robust, accurate chromosome-scale de novo genome assemblies without the need for laborious in vivo cloning steps. Finally, we demonstrate CPT-seq as a means of anchoring unplaced novel human contigs to the reference genome as well as for detecting misassembled sequences.

  5. Efficient assembly of de novo human artificial chromosomes from large genomic loci

    Directory of Open Access Journals (Sweden)

    Stromberg Gregory

    2005-07-01

    Full Text Available Abstract Background Human Artificial Chromosomes (HACs are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb genomic loci have been reported to be successfully packaged into de novo HACs. Results We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100–200 kb genomic loci including therapeutically significant genes for human growth hormone (HGH, polycystic kidney disease (PKD1 and ß-globin. We report significant differences in the ability of different genomic loci to support de novo HAC formation, suggesting possible effects of cis-acting genomic elements. Finally, as a proof of principle, we have observed sustained ß-globin gene expression from HACs incorporating the entire 200 kb ß-globin genomic locus for over 90 days in the absence of selection. Conclusion Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate de novo HAC formation. These genomic loci

  6. Hominoid-specific de novo protein-coding genes originating from long non-coding RNAs.

    Directory of Open Access Journals (Sweden)

    Chen Xie

    2012-09-01

    Full Text Available Tinkering with pre-existing genes has long been known as a major way to create new genes. Recently, however, motherless protein-coding genes have been found to have emerged de novo from ancestral non-coding DNAs. How these genes originated is not well addressed to date. Here we identified 24 hominoid-specific de novo protein-coding genes with precise origination timing in vertebrate phylogeny. Strand-specific RNA-Seq analyses were performed in five rhesus macaque tissues (liver, prefrontal cortex, skeletal muscle, adipose, and testis, which were then integrated with public transcriptome data from human, chimpanzee, and rhesus macaque. On the basis of comparing the RNA expression profiles in the three species, we found that most of the hominoid-specific de novo protein-coding genes encoded polyadenylated non-coding RNAs in rhesus macaque or chimpanzee with a similar transcript structure and correlated tissue expression profile. According to the rule of parsimony, the majority of these hominoid-specific de novo protein-coding genes appear to have acquired a regulated transcript structure and expression profile before acquiring coding potential. Interestingly, although the expression profile was largely correlated, the coding genes in human often showed higher transcriptional abundance than their non-coding counterparts in rhesus macaque. The major findings we report in this manuscript are robust and insensitive to the parameters used in the identification and analysis of de novo genes. Our results suggest that at least a portion of long non-coding RNAs, especially those with active and regulated transcription, may serve as a birth pool for protein-coding genes, which are then further optimized at the transcriptional level.

  7. Chemical defense balanced by sequestration and de novo biosynthesis in a lepidopteran specialist.

    Directory of Open Access Journals (Sweden)

    Joel Fürstenberg-Hägg

    Full Text Available The evolution of sequestration (uptake and accumulation relative to de novo biosynthesis of chemical defense compounds is poorly understood, as is the interplay between these two strategies. The Burnet moth Zygaena filipendulae (Lepidoptera and its food-plant Lotus corniculatus (Fabaceae poses an exemplary case study of these questions, as Z. filipendulae belongs to the only insect family known to both de novo biosynthesize and sequester the same defense compounds directly from its food-plant. Z. filipendulae and L. corniculatus both contain the two cyanogenic glucosides linamarin and lotaustralin, which are defense compounds that can be hydrolyzed to liberate toxic hydrogen cyanide. The overall amounts and ratios of linamarin and lotaustralin in Z. filipendulae are tightly regulated, and only to a low extent reflect the ratio in the ingested food-plant. We demonstrate that Z. filipendulae adjusts the de novo biosynthesis of CNglcs by regulation at both the transcriptional and protein level depending on food plant composition. Ultimately this ensures that the larva saves energy and nitrogen while maintaining an effective defense system to fend off predators. By using in situ PCR and immunolocalization, the biosynthetic pathway was resolved to the larval fat body and integument, which infers rapid replenishment of defense compounds following an encounter with a predator. Our study supports the hypothesis that de novo biosynthesis of CNglcs in Z. filipendulae preceded the ability to sequester, and facilitated a food-plant switch to cyanogenic plants, after which sequestration could evolve. Preservation of de novo biosynthesis allows fine-tuning of the amount and composition of CNglcs in Z. filipendulae.

  8. Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy

    DEFF Research Database (Denmark)

    Fenrich, Keith K; Skelton, Nicole; MacDermid, Victoria E

    2007-01-01

    at 4-5 weeks post injury. The somata of axotomized CINs were identified by the presence of immunoreactivity for the axonal growth-associated protein-43 (GAP-43). Nearly half of the CINs had de novo axons that emerged from distal dendrites. These axons lacked immunoreactivity for the dendritic protein......Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injury......, develop de novo axons. Our goal was to determine whether spinal commissural interneurons (CINs), axotomized by 3-4-mm midsagittal transection at C3, form de novo axons from distal dendrites. All experiments were performed on adult cats. CINs in C3 were stained with extracellular injections of Neurobiotin...

  9. Proteomics of Soil and Sediment: Protein Identification by De Novo Sequencing of Mass Spectra Complements Traditional Database Searching

    Science.gov (United States)

    Miller, S.; Rizzo, A. I.; Waldbauer, J.

    2015-12-01

    Proteomics has the potential to elucidate the metabolic pathways and taxa responsible for in situ biogeochemical transformations. However, low rates of protein identification from high resolution mass spectra have been a barrier to the development of proteomics in complex environmental samples. Much of the difficulty lies in the computational challenge of linking mass spectra to their corresponding proteins. Traditional database search methods for matching peptide sequences to mass spectra are often inadequate due to the complexity of environmental proteomes and the large database search space, as we demonstrate with soil and sediment proteomes generated via a range of extraction methods. One alternative to traditional database searching is de novo sequencing, which identifies peptide sequences without the need for a database. BLAST can then be used to match de novo sequences to similar genetic sequences. Assigning confidence to putative identifications has been one hurdle for the implementation of de novo sequencing. We found that accurate de novo sequences can be screened by quality score and length. Screening criteria are verified by comparing the results of de novo sequencing and traditional database searching for well-characterized proteomes from simple biological systems. The BLAST hits of screened sequences are interrogated for taxonomic and functional information. We applied de novo sequencing to organic topsoil and marine sediment proteomes. Peak-rich proteomes, which can result from various extraction techniques, yield thousands of high-confidence protein identifications, an improvement over previous proteomic studies of soil and sediment. User-friendly software tools for de novo metaproteomics analysis have been developed. This "De Novo Analysis" Pipeline is also a faster method of data analysis than constructing a tailored sequence database for traditional database searching.

  10. Efficacy and safety of the HAA regimen as induction chemotherapy in 236 de novo acute myeloid leukemia

    Institute of Scientific and Technical Information of China (English)

    叶佩佩

    2013-01-01

    Objective To evaluate the efficacy and safety of the HAA regimen (homoharringtonine,cytarabine and aclarubicin) as induction chemotherapy in de novo acute myeloid leukemia (AML) .Methods The efficacy and safety of 236 de novo AML patients who received the HAA regimen as induction chemotherapy were retrospectively analyzed.The complete remission (CR) rate was assayed.Kaplan-Meier method was used to estimate overall survival (OS) and relapse free survival (RFS) ,and the differ-

  11. Alternative normalization methods demonstrate widespread cortical hypometabolism in untreated de novo Parkinson's disease

    DEFF Research Database (Denmark)

    Berti, Valentina; Polito, C; Borghammer, Per

    2012-01-01

    , recent studies suggested that conventional data normalization procedures may not always be valid, and demonstrated that alternative normalization strategies better allow detection of low magnitude changes. We hypothesized that these alternative normalization procedures would disclose more widespread...... metabolic alterations in de novo PD. METHODS: [18F]FDG PET scans of 26 untreated de novo PD patients (Hoehn & Yahr stage I-II) and 21 age-matched controls were compared using voxel-based analysis. Normalization was performed using gray matter (GM), white matter (WM) reference regions and Yakushev...... normalization. RESULTS: Compared to GM normalization, WM and Yakushev normalization procedures disclosed much larger cortical regions of relative hypometabolism in the PD group with extensive involvement of frontal and parieto-temporal-occipital cortices, and several subcortical structures. Furthermore...

  12. De novo mutations in the genome organizer CTCF cause intellectual disability

    DEFF Research Database (Denmark)

    Gregor, Anne; Oti, Martin; Kouwenhoven, Evelyn N

    2013-01-01

    and is involved in various chromatin regulation processes such as higher order of chromatin organization, enhancer function, and maintenance of three-dimensional chromatin structure. Transcriptome analyses in all three individuals with point mutations revealed deregulation of genes involved in signal transduction......An increasing number of genes involved in chromatin structure and epigenetic regulation has been implicated in a variety of developmental disorders, often including intellectual disability. By trio exome sequencing and subsequent mutational screening we now identified two de novo frameshift...... mutations and one de novo missense mutation in CTCF in individuals with intellectual disability, microcephaly, and growth retardation. Furthermore, an individual with a larger deletion including CTCF was identified. CTCF (CCCTC-binding factor) is one of the most important chromatin organizers in vertebrates...

  13. Top-down analysis of protein samples by de novo sequencing techniques

    Energy Technology Data Exchange (ETDEWEB)

    Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.; VanDuijn, Martijn M.; Liu, Xiaowen; Tolić, Nikola; Luider, Theo M.; Paša-Tolić, Ljiljana; Pevzner, Pavel A.

    2016-05-14

    MOTIVATION: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. RESULTS: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns.

  14. NxRepair: error correction in de novo sequence assembly using Nextera mate pairs

    Directory of Open Access Journals (Sweden)

    Rebecca R. Murphy

    2015-06-01

    Full Text Available Scaffolding errors and incorrect repeat disambiguation during de novo assembly can result in large scale misassemblies in draft genomes. Nextera mate pair sequencing data provide additional information to resolve assembly ambiguities during scaffolding. Here, we introduce NxRepair, an open source toolkit for error correction in de novo assemblies that uses Nextera mate pair libraries to identify and correct large-scale errors. We show that NxRepair can identify and correct large scaffolding errors, without use of a reference sequence, resulting in quantitative improvements in the assembly quality. NxRepair can be downloaded from GitHub or PyPI, the Python Package Index; a tutorial and user documentation are also available.

  15. De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

    DEFF Research Database (Denmark)

    Heinzen, Erin L; Swoboda, Kathryn J; Hitomi, Yuki;

    2012-01-01

    and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation...... in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without...... affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3....

  16. De novo appearance of a choroidal osteoma in an eye with previous branch retinal vein occlusion.

    Science.gov (United States)

    Adhi, Mehreen; Bryant, Juanita Sonya; Alwassia, Ahmad A; Chen, Carolyn; Duker, Jay S

    2013-01-01

    This report describes the de novo appearance of a choroidal osteoma occurring 8 years after laser photocoagulation for previous branch retinal vein occlusion (BRVO). A 62-year-old man presented with an asymptomatic yellowish orange lesion in the macula on fundus examination of his left eye during a regular follow-up visit for bilateral BRVO associated with macular edema that had previously been treated with laser photocoagulation. The lesion was observed for 1.5 years until a decrease in vision occurred. Fundus photography revealed a yellow-to-orange, well-defined lesion in the macular region. Fluorescein angiography was consistent with choroidal neovascularization (CNV). Optical coherence tomography and B-scan ultrasonography showed features consistent with choroidal osteoma. This is the first report of the de novo appearance of a choroidal osteoma occurring years after laser photocoagulation for BRVO. CNV developed secondary to the lesion, which was treated with intravitreal bevacizumab, leading to subjective and anatomic improvement.

  17. Exploiting molecular motors as nanomachines: the mechanisms of de novo and re-engineered cytoskeletal motors.

    Science.gov (United States)

    DelRosso, Nicole V; Derr, Nathan D

    2017-01-11

    Cytoskeletal molecular motors provide exciting proof that nanoscale transporters can be highly efficient, moving for microns along filamentous tracks by hydrolyzing ATP to fuel nanometer-size steps. For nanotechnology, such conversion of chemical energy into productive work serves as an enticing platform for re-purposing and re-engineering. It also provides a roadmap for successful molecular mechanisms that can be mimicked to create de novo molecular motors for nanotechnology applications. Here we focus specifically on how the mechanisms of molecular motors are being re-engineered for greater control over their transport parameters. We then discuss mechanistic work to create fully synthetic motors de novo and conclude with future directions in creating novel motor systems.

  18. Dominant transmission of de novo KIF1A motor domain variant underlying pure spastic paraplegia.

    Science.gov (United States)

    Ylikallio, Emil; Kim, Doyoun; Isohanni, Pirjo; Auranen, Mari; Kim, Eunjoon; Lönnqvist, Tuula; Tyynismaa, Henna

    2015-10-01

    Variants in family 1 kinesin (KIF1A), which encodes a kinesin axonal motor protein, have been described to cause variable neurological manifestations. Recessive missense variants have led to spastic paraplegia, and recessive truncations to sensory and autonomic neuropathy. De novo missense variants cause developmental delay or intellectual disability, cerebellar atrophy and variable spasticity. We describe a family with father-to-son transmission of de novo variant in the KIF1A motor domain, in a phenotype of pure spastic paraplegia. Structural modeling of the predicted p.(Ser69Leu) amino acid change suggested that it impairs the stable binding of ATP to the KIF1A protein. Our study reports the first dominantly inherited KIF1A variant and expands the spectrum of phenotypes caused by heterozygous KIF1A motor domain variants to include pure spastic paraplegia. We conclude that KIF1A should be considered a candidate gene for hereditary paraplegias regardless of inheritance pattern.

  19. PRIME: A Mass Spectrum Data Mining Tool for De Novo Sequencing and PTMs Identification

    Institute of Scientific and Technical Information of China (English)

    Bo Yan; You-Xing Qu; Feng-Lou Mao; Victor N. Olman; Ying Xu

    2005-01-01

    De novo sequencing is one of the most promising proteomics techniques for identification of protein posttranslation modifications (PTMs) in studying protein regulations and functions. We have developed a computer tool PRIME for identification of b and y ions in tandem mass spectra, a key challenging problem in de novo sequencing. PRIME utilizes a feature that ions of the same and different types follow different mass-difference distributions to separate b from y ions correctly. We have formulated the problem as a graph partition problem. A linear integer-programming algorithm has been implemented to solve the graph partition problem rigorously and efficiently. The performance of PRIME has been demonstrated on a large amount of simulated tandem mass spectra derived from Yeast genome and its power of detecting PTMs has been tested on 216 simulated phosphopeptides.

  20. Asystole in alternating hemiplegia with de novo ATP1A3 mutation.

    Science.gov (United States)

    Novy, Jan; McWilliams, Eric; Sisodiya, Sanjay M

    2014-01-01

    Alternating hemiplegia is a rare condition presenting with episodes of hemiplegia, epileptic seizures and, at times, dysautonomic attacks. De novo ATP1A3 (Na(+)/K(+) ATPase subunit) mutations were recently found to be the most common cause. We report a patient with alternating hemiplegia with de novo ATP1A3 mutation who experienced new-onset episodes of collapse in early adulthood unrelated to seizures. An implantable cardiac loop recorder documented episodes of asystole up to 5 s long. Subsequently a permanent pacemaker was implanted. ATP1A3 heart expression may be the explanation for the association of alternating hemiplegia and asystole episodes. Alternating hemiplegia has been associated with an increased risk of sudden death and lethal cardiac arrhythmias may be causative. Patients may need referral for appropriate cardiac investigations, especially if there is a change in symptoms. This case highlights the importance of clinical vigilance in patients with alternating hemiplegia.

  1. Natures balancing act: examining biosynthesis de novo, recycling and processing damaged vitamin B metabolites.

    Science.gov (United States)

    Colinas, Maite; Fitzpatrick, Teresa B

    2015-06-01

    Plants use B vitamin compounds as cofactors for metabolism. Biosynthesis de novo of these metabolites in plants is almost fully elucidated. However, salvaging of precursors as well as cofactor derivatives is only being unraveled. Furthermore, processing of these compounds when damaged by cellular activities to prevent deleterious effects on metabolism is emerging. Recent investigations indicate that the role of B vitamins goes beyond metabolism and are being linked with epigenetic traits, specific developmental cues, the circadian clock, as well as abiotic and biotic stress responses. More in depth investigations on the regulation of the provision of these compounds through biosynthesis de novo, salvage and transport is suggesting that plants may share the cost of this load by division of labor.

  2. T-IDBA: A de novo Iterative de Bruijn Graph Assembler for Transcriptome

    Science.gov (United States)

    Peng, Yu; Leung, Henry C. M.; Yiu, S. M.; Chin, Francis Y. L.

    RNA-seq data produced by next-generation sequencing technology is a useful tool for analyzing transcriptomes. However, existing de novo transcriptome assemblers do not fully utilize the properties of transcriptomes and may result in short contigs because of the splicing nature (shared exons) of the genes. We propose the T-IDBA algorithm to reconstruct expressed isoforms without reference genome. By using pair-end information to solve the problem of long repeats in different genes and branching in the same gene due to alternative splicing, the graph can be decomposed into small components, each corresponds to a gene. The most possible isoforms with sufficient support from the pair-end reads will be found heuristically. In practice, our de novo transcriptome assembler, T-IDBA, outperforms Abyss substantially in terms of sensitivity and precision for both simulated and real data. T-IDBA is available at http://www.cs.hku.hk/~alse/tidba/

  3. Whole-genome sequencing for comparative genomics and de novo genome assembly.

    Science.gov (United States)

    Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

    2015-01-01

    Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

  4. Augmenting transcriptome assembly by combining de novo and genome-guided tools.

    Science.gov (United States)

    Jain, Prachi; Krishnan, Neeraja M; Panda, Binay

    2013-01-01

    Researchers interested in studying and constructing transcriptomes, especially for non-model species, face the conundrum of choosing from a number of available de novo and genome-guided assemblers. None of the popular assembly tools in use today achieve requisite sensitivity, specificity or recovery of full-length transcripts on their own. Here, we present a comprehensive comparative study of the performance of various assemblers. Additionally, we present an approach to combinatorially augment transciptome assembly by using both de novo and genome-guided tools. In our study, we obtained the best recovery and most full-length transcripts with Trinity and TopHat1-Cufflinks, respectively. The sensitivity of the assembly and isoform recovery was superior, without compromising much on the specificity, when transcripts from Trinity were augmented with those from TopHat1-Cufflinks.

  5. NxRepair: error correction in de novo sequence assembly using Nextera mate pairs.

    Science.gov (United States)

    Murphy, Rebecca R; O'Connell, Jared; Cox, Anthony J; Schulz-Trieglaff, Ole

    2015-01-01

    Scaffolding errors and incorrect repeat disambiguation during de novo assembly can result in large scale misassemblies in draft genomes. Nextera mate pair sequencing data provide additional information to resolve assembly ambiguities during scaffolding. Here, we introduce NxRepair, an open source toolkit for error correction in de novo assemblies that uses Nextera mate pair libraries to identify and correct large-scale errors. We show that NxRepair can identify and correct large scaffolding errors, without use of a reference sequence, resulting in quantitative improvements in the assembly quality. NxRepair can be downloaded from GitHub or PyPI, the Python Package Index; a tutorial and user documentation are also available.

  6. Prenatal diagnosis of a de novo inversion of chromosome (2)(p21q11).

    Science.gov (United States)

    Hengstschläger, M; Mittermayer, C; Prusa, A R; Drahonsky, R; Repa, C; Deutinger, J; Bernaschek, G

    2003-08-01

    Prenatal diagnosis of "apparently balanced" chromosomal rearrangements, if not inherited from a parent, are problematic for genetic counsellors and families. Although the parents need to be informed about the increased risk of multiple congenital anomalies, the anomalies that the fetus is at risk can not be discussed unless a similar breakpoint and accompanying phenotype have been reported in the literature. In the reported case prenatal ultrasound examination revealed a massive hydrocephalus internus and IUGR. The karyotype of the fetus was inv(2)(p21q11) de novo. Postmortem examination revealed short palpebral fissures, hypertelorism, atypical nasiolabial configuration, microgenia, extended position of the fingers, atypical proximal inserted first toe, hydrocephalus internus, hypoplasia of the cerebellum and bulbi olfactorii, bilateral hypoplastic lungs, atrial septal defect II, small right ventricle, dysplasia of the pulmonary valve, hypoplastic pulmonary artery, right proximal ureterostenosis, hypoplastic gall bladder. This is the first description of a de novo inversion (2)(p21q11) in a fetus with multiple malformations.

  7. A strategy to identify de novo mutations in common disorders such as autism and schizophrenia.

    Science.gov (United States)

    Julie, Gauthier; Hamdan, Fadi F; Rouleau, Guy A

    2011-06-15

    There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness(1) and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them(2). This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new

  8. THE ROLE OF PRE-TRANSPLANT AND DE NOVO ALLOANTIBODIES IN KIDNEY TRANSPLANTATION

    Directory of Open Access Journals (Sweden)

    A. I. Sushkov

    2011-01-01

    Full Text Available Kidney transplant recipients may have pre-transplant alloantibodies or develop de novo anti-HLA and non- HLA antibodies after transplantation. Although these antibodies may be donor-specific or non-donor-specific, their presence may increase the risk for acute and chronic rejection, thereby decreasing allograft survival. The introduction of high sensitive and specific methods to detect anti-HLA antibodies, both before and after trans- plantation, will help to define transplant recipients who might be at increased risk for early or late allograft failure. Moreover, knowledge of alloantibody status before transplantation may help to guide the appropriate use of immunomodulatory agents and plasmapheresis to remove anti-HLA antibodies or downregulate their production. The review focuses on the associations between renal graft outcome and pre-transplant and de novo alloantibodies. 

  9. [Abnormality of blood coagulation indexes in patients with de novo acute leukemia and its clinical significance].

    Science.gov (United States)

    Xiao, Fang-Fang; Hu, Kai-Xun; Guo, Mei; Qiao, Jian-Hui; Sun, Qi-Yun; Ai, Hui-Sheng; Yu, Chang-Lin

    2013-04-01

    To explore hemorrhage risk and the clinical significance of abnormal change of prothrombin time (PT), activated partial thromboplastin time (APTT), plasma fibrinogen (FIB), plasma thrombin time (TT) and d-dimer (D-D) in de novo acute leukemia (except for APL), the different bleeding manifestations of 114 cases of de novo acute leukemia with different coagulation indexes were analyzed retrospectively. The correlation between these blood coagulation indexes and the possible correlative clinical characteristics were analysed, including age, sex, type of acute leukemia, initial white blood cell(WBC) and platelet(Plt) count, the proportion of blast cells in bone marrow and cytogenetic abnormality of patients at diagnosis. The results indicated that the incidence of abnormal blood coagulation was as high as 78.1% for de novo AL patients. These patients with 5 normal blood coagulation indexes may have mild bleeding manifestation, but the more abnormal indexes, the more severe bleeding. Both PT and D-D were sensitive indexes for diagnosis of level II bleeding. Incidence of abnormal blood coagulation significantly correlates with the proportion of blast cells in bone marrow (χ(2) = 4.184, OR = 1.021, P coagulation. It is concluded that the coagulation and fibrinolysis are abnormal in most patients with de novo acute leukemia. More abnormal indexes indicate more severe bleeding, and both PT and D-D are sensitive indexes for diagnosis of level II bleeding. Higher proportion of blast cells in bone marrow predicts higher incidence of abnormal blood clotting. Acute leukemia with elderly age, high white blood cell count and adverse cytogenetics do not predict severer abnormal blood clotting. Detection of PT, APTT, TT, FIB, and D-D may help to judge whether the patients are in a state of hypercoagulability or disseminated intravenous coagulation, which will provide experiment evidences for early intervention and medication.

  10. De Novo DNA Methyltransferase DNMT3b Interacts with NEDD8-modified Proteins*

    OpenAIRE

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; AMBINDER, RICHARD F; Hayward, S. Diane

    2010-01-01

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. ...

  11. First report of a de novo germline mutation in the MLH1 gene

    Institute of Scientific and Technical Information of China (English)

    Rein P Stulp; Yvonne J Vos; Bart Mol; Arend Karrenbeld; Monique de Raad; Huub JC van der Mijle; Rolf H Sijmons

    2006-01-01

    Hereditary non-polyposis colorectal carcinoma (HNPCC)is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven,or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation (in MSH2) has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient's parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.

  12. Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming

    OpenAIRE

    Burridge, Paul W.; Keller, Gordon; Gold, Joseph D.; Wu, Joseph C

    2012-01-01

    Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted method developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent ...

  13. De novo complex intra chromosomal rearrangement after ICSI: characterisation by BACs micro array-CGH

    Science.gov (United States)

    Kasakyan, Serdar; Lohmann, Laurence; Aboura, Azeddine; Quimsiyeh, Mazin; Menezo, Yves; Tachdjian, Gerard; Benkhalifa, Moncef

    2008-01-01

    Background In routine Assisted Reproductive Technology (ART) men with severe oligozoospermia or azoospermia should be informed about the risk of de novo congenital or chromosomal abnormalities in ICSI program. Also the benefits of preimplantation or prenatal genetic diagnosis practice need to be explained to the couple. Methods From a routine ICSI attempt, using ejaculated sperm from male with severe oligozoospermia and having normal karyotype, a 30 years old pregnant woman was referred to prenatal diagnosis in the 17th week for bichorionic biamniotic twin gestation. Amniocentesis was performed because of the detection of an increased foetal nuchal translucency for one of the fetus by the sonographic examination during the 12th week of gestation (WG). Chromosome and DNA studies of the fetus were realized on cultured amniocytes Results Conventional, molecular cytogenetic and microarray CGH experiments allowed us to conclude that the fetus had a de novo pericentromeric inversion associated with a duplication of the 9p22.1-p24 chromosomal region, 46,XY,invdup(9)(p22.1p24) [arrCGH 9p22.1p24 (RP11-130C19 → RP11-87O1)x3]. As containing the critical 9p22 region, our case is in coincidence with the general phenotype features of the partial trisomy 9p syndrome with major growth retardation, microcephaly and microretrognathia. Conclusion This de novo complex chromosome rearrangement illustrates the possible risk of chromosome or gene defects in ICSI program and the contribution of array-CGH for mapping rapidly de novo chromosomal imbalance. PMID:19105807

  14. Robust de novo pathway enrichment with KeyPathwayMiner 5

    DEFF Research Database (Denmark)

    Alcaraz, Nicolas; List, Markus; Dissing-Hansen, Martin

    2016-01-01

    Identifying functional modules or novel active pathways, recently termed de novo pathway enrichment, is a computational systems biology challenge that has gained much attention during the last decade. Given a large biological interaction network, KeyPathwayMiner extracts connected subnetworks...... several network perturbation techniques and over a range of perturbation degrees. In addition, users may now provide a gold-standard set to determine how enriched extracted pathways are with relevant genes compared to randomized versions of the original network....

  15. Compacting and correcting Trinity and Oases RNA-Seq de novo assemblies

    Science.gov (United States)

    Djari, Anis; Guiguen, Yann; Bobe, Julien; Klopp, Christophe

    2017-01-01

    Background De novo transcriptome assembly of short reads is now a common step in expression analysis of organisms lacking a reference genome sequence. Several software packages are available to perform this task. Even if their results are of good quality it is still possible to improve them in several ways including redundancy reduction or error correction. Trinity and Oases are two commonly used de novo transcriptome assemblers. The contig sets they produce are of good quality. Still, their compaction (number of contigs needed to represent the transcriptome) and their quality (chimera and nucleotide error rates) can be improved. Results We built a de novo RNA-Seq Assembly Pipeline (DRAP) which wraps these two assemblers (Trinity and Oases) in order to improve their results regarding the above-mentioned criteria. DRAP reduces from 1.3 to 15 fold the number of resulting contigs of the assemblies depending on the read set and the assembler used. This article presents seven assembly comparisons showing in some cases drastic improvements when using DRAP. DRAP does not significantly impair assembly quality metrics such are read realignment rate or protein reconstruction counts. Conclusion Transcriptome assembly is a challenging computational task even if good solutions are already available to end-users, these solutions can still be improved while conserving the overall representation and quality of the assembly. The de novo RNA-Seq Assembly Pipeline (DRAP) is an easy to use software package to produce compact and corrected transcript set. DRAP is free, open-source and available under GPL V3 license at http://www.sigenae.org/drap. PMID:28224052

  16. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences

    OpenAIRE

    Li, Heng

    2015-01-01

    Motivation: Single Molecule Real-Time (SMRT) sequencing technology and Oxford Nanopore technologies (ONT) produce reads over 10kbp in length, which have enabled high-quality genome assembly at an affordable cost. However, at present, long reads have an error rate as high as 10-15%. Complex and computationally intensive pipelines are required to assemble such reads. Results: We present a new mapper, minimap, and a de novo assembler, miniasm, for efficiently mapping and assembling SMRT and ONT ...

  17. De novo synthesis of D- and L-fucosamine containing disaccharides

    Directory of Open Access Journals (Sweden)

    Daniele Leonori

    2013-02-01

    Full Text Available The availability of rare monosaccharides that cannot be isolated from natural sources is currently limiting the access to the synthesis and the biological evaluation of complex bacterial cell-surface glycans. Here, we report the synthesis of D- and L-fucosamine building blocks by a de novo approach from L- and D-Garner aldehydes. These differentially protected monosaccharide building blocks were utilized to prepare disaccharides present on the surface of Pseudomonas aeruginosa bacteria.

  18. De novo complex intra chromosomal rearrangement after ICSI: characterisation by BACs micro array-CGH

    Directory of Open Access Journals (Sweden)

    Quimsiyeh Mazin

    2008-12-01

    Full Text Available Abstract Background In routine Assisted Reproductive Technology (ART men with severe oligozoospermia or azoospermia should be informed about the risk of de novo congenital or chromosomal abnormalities in ICSI program. Also the benefits of preimplantation or prenatal genetic diagnosis practice need to be explained to the couple. Methods From a routine ICSI attempt, using ejaculated sperm from male with severe oligozoospermia and having normal karyotype, a 30 years old pregnant woman was referred to prenatal diagnosis in the 17th week for bichorionic biamniotic twin gestation. Amniocentesis was performed because of the detection of an increased foetal nuchal translucency for one of the fetus by the sonographic examination during the 12th week of gestation (WG. Chromosome and DNA studies of the fetus were realized on cultured amniocytes Results Conventional, molecular cytogenetic and microarray CGH experiments allowed us to conclude that the fetus had a de novo pericentromeric inversion associated with a duplication of the 9p22.1-p24 chromosomal region, 46,XY,invdup(9(p22.1p24 [arrCGH 9p22.1p24 (RP11-130C19 → RP11-87O1x3]. As containing the critical 9p22 region, our case is in coincidence with the general phenotype features of the partial trisomy 9p syndrome with major growth retardation, microcephaly and microretrognathia. Conclusion This de novo complex chromosome rearrangement illustrates the possible risk of chromosome or gene defects in ICSI program and the contribution of array-CGH for mapping rapidly de novo chromosomal imbalance.

  19. Plasmodium falciparum is dependent on de novo myo-inositol biosynthesis for assembly of GPI glycolipids and infectivity.

    Science.gov (United States)

    Macrae, James I; Lopaticki, Sash; Maier, Alexander G; Rupasinghe, Thusitha; Nahid, Amsha; Cowman, Alan F; McConville, Malcolm J

    2014-02-01

    Intra-erythrocytic stages of the malaria parasite, Plasmodium falciparum, are thought to be dependent on de novo synthesis of phosphatidylinositol, as red blood cells (RBC) lack the capacity to synthesize this phospholipid. The myo-inositol headgroup of PI can either be synthesized de novo or scavenged from the RBC. An untargeted metabolite profiling of P. falciparum infected RBC showed that trophozoite and schizont stages accumulate high levels of myo-inositol-3-phosphate, indicating increased de novo biosynthesis of myo-inositol from glucose 6-phosphate. Metabolic labelling studies with (13) C-U-glucose in the presence and absence of exogenous inositol confirmed that de novo myo-inositol synthesis occurs in parallel with myo-inositol salvage pathways. Unexpectedly, while both endogenous and scavenged myo-inositol was used to synthesize bulk PI, only de novo-synthesized myo-inositol was incorporated into GPI glycolipids. Moreover, gene disruption studies suggested that the INO1 gene, encoding myo-inositol 3-phosphate synthase, is essential in asexual parasite stages. Together these findings suggest that P. falciparum asexual stages are critically dependent on de novo myo-inositol biosynthesis for assembly of a sub-pool of PI species and GPI biosynthesis. These findings highlight unexpected complexity in phospholipid biosynthesis in P. falciparum and a lack of redundancy in some nutrient salvage versus endogenous biosynthesis pathways.

  20. An 11bp region with stem formation potential is essential for de novo DNA methylation of the RPS element.

    Directory of Open Access Journals (Sweden)

    Matthew Gentry

    Full Text Available The initiation of DNA methylation in Arabidopsis is controlled by the RNA-directed DNA methylation (RdDM pathway that uses 24nt siRNAs to recruit de novo methyltransferase DRM2 to the target site. We previously described the REPETITIVE PETUNIA SEQUENCE (RPS fragment that acts as a hot spot for de novo methylation, for which it requires the cooperative activity of all three methyltransferases MET1, CMT3 and DRM2, but not the RdDM pathway. RPS contains two identical 11nt elements in inverted orientation, interrupted by a 18nt spacer, which resembles the features of a stemloop structure. The analysis of deletion/substitution derivatives of this region showed that deletion of one 11nt element RPS is sufficient to eliminate de novo methylation of RPS. In addition, deletion of a 10nt region directly adjacent to one of the 11nt elements, significantly reduced de novo methylation. When both 11nt regions were replaced by two 11nt elements with altered DNA sequence but unchanged inverted repeat homology, DNA methylation was not affected, indicating that de novo methylation was not targeted to a specific DNA sequence element. These data suggest that de novo DNA methylation is attracted by a secondary structure to which the two 11nt elements contribute, and that the adjacent 10nt region influences the stability of this structure. This resembles the recognition of structural features by DNA methyltransferases in animals and suggests that similar mechanisms exist in plants.

  1. Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol.

    Science.gov (United States)

    Sonntag, Frank; Kroner, Cora; Lubuta, Patrice; Peyraud, Rémi; Horst, Angelika; Buchhaupt, Markus; Schrader, Jens

    2015-11-01

    Over the last 10 to 15 years, metabolic engineering of microbes has become a versatile tool for high-level de novo synthesis of terpenoids, with the sesquiterpenoids armopha-1,4-diene, farnesene and artemisinic acid as prime examples. However, almost all cell factory approaches towards terpenoids to date have been based on sugar as the raw material, which is mainly used as a food resource and subject to high price volatilities. In this study we present de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens AM1. Expression of α-humulene synthase from Zingiber zerumbet in combination with farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae led to concentrations of up to 18 mg/L α-humulene. Introduction of a prokaryotic mevalonate pathway from Myxococcus xanthus in combination with ribosome binding site optimization of α-humulene and FPP synthases increased product concentration 3-fold. This value was additionally raised by 30% using a carotenoid synthesis deficient mutant strain. Final product concentrations of up to 1.65 g/L were obtained in methanol limited fed-batch cultivations, which is the highest titer of de novo synthesized α-humulene reported to date. This study demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol.

  2. Autophagy protects against de novo formation of the [PSI+] prion in yeast.

    Science.gov (United States)

    Speldewinde, Shaun H; Doronina, Victoria A; Grant, Chris M

    2015-12-15

    Prions are self-propagating, infectious proteins that underlie several neurodegenerative diseases. The molecular basis underlying their sporadic formation is poorly understood. We show that autophagy protects against de novo formation of [PSI(+)], which is the prion form of the yeast Sup35 translation termination factor. Autophagy is a cellular degradation system, and preventing autophagy by mutating its core components elevates the frequency of spontaneous [PSI(+)] formation. Conversely, increasing autophagic flux by treating cells with the polyamine spermidine suppresses prion formation in mutants that normally show a high frequency of de novo prion formation. Autophagy also protects against the de novo formation of another prion, namely the Rnq1/[PIN(+)] prion, which is not related in sequence to the Sup35/[PSI(+)] prion. We show that growth under anaerobic conditions in the absence of molecular oxygen abrogates Sup35 protein damage and suppresses the high frequency of [PSI(+)] formation in an autophagy mutant. Autophagy therefore normally functions to remove oxidatively damaged Sup35, which accumulates in cells grown under aerobic conditions, but in the absence of autophagy, damaged/misfolded Sup35 undergoes structural transitions favoring its conversion to the propagatable [PSI(+)] form.

  3. Germline De Novo Mutations in GNB1 Cause Severe Neurodevelopmental Disability, Hypotonia, and Seizures

    Science.gov (United States)

    Petrovski, Slavé; Küry, Sébastien; Myers, Candace T.; Anyane-Yeboa, Kwame; Cogné, Benjamin; Bialer, Martin; Xia, Fan; Hemati, Parisa; Riviello, James; Mehaffey, Michele; Besnard, Thomas; Becraft, Emily; Wadley, Alexandrea; Politi, Anya Revah; Colombo, Sophie; Zhu, Xiaolin; Ren, Zhong; Andrews, Ian; Dudding-Byth, Tracy; Schneider, Amy L.; Wallace, Geoffrey; Rosen, Aaron B.I.; Schelley, Susan; Enns, Gregory M.; Corre, Pierre; Dalton, Joline; Mercier, Sandra; Latypova, Xénia; Schmitt, Sébastien; Guzman, Edwin; Moore, Christine; Bier, Louise; Heinzen, Erin L.; Karachunski, Peter; Shur, Natasha; Grebe, Theresa; Basinger, Alice; Nguyen, Joanne M.; Bézieau, Stéphane; Wierenga, Klaas; Bernstein, Jonathan A.; Scheffer, Ingrid E.; Rosenfeld, Jill A.; Mefford, Heather C.; Isidor, Bertrand; Goldstein, David B.

    2016-01-01

    Whole-exome sequencing of 13 individuals with developmental delay commonly accompanied by abnormal muscle tone and seizures identified de novo missense mutations enriched within a sub-region of GNB1, a gene encoding the guanine nucleotide-binding protein subunit beta-1, Gβ. These 13 individuals were identified among a base of 5,855 individuals recruited for various undiagnosed genetic disorders. The probability of observing 13 or more de novo mutations by chance among 5,855 individuals is very low (p = 7.1 × 10−21), implicating GNB1 as a genome-wide-significant disease-associated gene. The majority of these 13 mutations affect known Gβ binding sites, which suggests that a likely disease mechanism is through the disruption of the protein interface required for Gα-Gβγ interaction (resulting in a constitutively active Gβγ) or through the disruption of residues relevant for interaction between Gβγ and certain downstream effectors (resulting in reduced interaction with the effectors). Strikingly, 8 of the 13 individuals recruited here for a neurodevelopmental disorder have a germline de novo GNB1 mutation that overlaps a set of five recurrent somatic tumor mutations for which recent functional studies demonstrated a gain-of-function effect due to constitutive activation of G protein downstream signaling cascades for some of the affected residues. PMID:27108799

  4. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions

    Science.gov (United States)

    Burton, Joshua N.; Adey, Andrew; Patwardhan, Rupali P.; Qiu, Ruolan; Kitzman, Jacob O.; Shendure, Jay

    2014-01-01

    Genomes assembled de novo from short reads are highly fragmented relative to the finished chromosomes of H. sapiens and key model organisms generated by the Human Genome Project. To address this, we need scalable, cost-effective methods enabling chromosome-scale contiguity. Here we show that genome-wide chromatin interaction datasets, such as those generated by Hi-C, are a rich source of long-range information for assigning, ordering and orienting genomic sequences to chromosomes, including across centromeres. To exploit this, we developed an algorithm that uses Hi-C data for ultra-long-range scaffolding of de novo genome assemblies. We demonstrate the approach by combining shotgun fragment and short jump mate-pair sequences with Hi-C data to generate chromosome-scale de novo assemblies of the human, mouse and Drosophila genomes, achieving – for human – 98% accuracy in assigning scaffolds to chromosome groups and 99% accuracy in ordering and orienting scaffolds within chromosome groups. Hi-C data can also be used to validate chromosomal translocations in cancer genomes. PMID:24185095

  5. De novo assembly of plant body plan: a step ahead of Deadpool

    Science.gov (United States)

    Kareem, Abdul; Radhakrishnan, Dhanya; Sondhi, Yash; Aiyaz, Mohammed; Roy, Merin V.; Sugimoto, Kaoru

    2016-01-01

    Abstract While in the movie Deadpool it is possible for a human to recreate an arm from scratch, in reality plants can even surpass that. Not only can they regenerate lost parts, but also the whole plant body can be reborn from a few existing cells. Despite the decades old realization that plant cells possess the ability to regenerate a complete shoot and root system, it is only now that the underlying mechanisms are being unraveled. De novo plant regeneration involves the initiation of regenerative mass, acquisition of the pluripotent state, reconstitution of stem cells and assembly of regulatory interactions. Recent studies have furthered our understanding on the making of a complete plant system in the absence of embryonic positional cues. We review the recent studies probing the molecular mechanisms of de novo plant regeneration in response to external inductive cues and our current knowledge of direct reprogramming of root to shoot and vice versa. We further discuss how de novo regeneration can be exploited to meet the demands of green culture industries and to serve as a general model to address the fundamental questions of regeneration across the plant kingdom. PMID:27800169

  6. Autism Spectrum Disorder in a Girl with a De Novo X;19 Balanced Translocation

    Directory of Open Access Journals (Sweden)

    Marcelo Razera Baruffi

    2012-01-01

    Full Text Available Balanced X-autosome translocations are rare, and female carriers are a clinically heterogeneous group of patients, with phenotypically normal women, history of recurrent miscarriage, gonadal dysfunction, X-linked disorders or congenital abnormalities, and/or developmental delay. We investigated a patient with a de novo X;19 translocation. The six-year-old girl has been evaluated due to hyperactivity, social interaction impairment, stereotypic and repetitive use of language with echolalia, failure to follow parents/caretakers orders, inconsolable outbursts, and persistent preoccupation with parts of objects. The girl has normal cognitive function. Her measurements are within normal range, and no other abnormalities were found during physical, neurological, or dysmorphological examinations. Conventional cytogenetic analysis showed a de novo balanced translocation, with the karyotype 46,X,t(X;19(p21.2;q13.4. Replication banding showed a clear preference for inactivation of the normal X chromosome. The translocation was confirmed by FISH and Spectral Karyotyping (SKY. Although abnormal phenotypes associated with de novo balanced chromosomal rearrangements may be the result of disruption of a gene at one of the breakpoints, submicroscopic deletion or duplication, or a position effect, X; autosomal translocations are associated with additional unique risk factors including X-linked disorders, functional autosomal monosomy, or functional X chromosome disomy resulting from the complex X-inactivation process.

  7. Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

    Science.gov (United States)

    Reig, Amanda J.; Pires, Marcos M.; Snyder, Rae Ana; Wu, Yibing; Jo, Hyunil; Kulp, Daniel W.; Butch, Susan E.; Calhoun, Jennifer R.; Szyperski, Thomas G.; Solomon, Edward I.; Degrado, William F.

    2012-11-01

    De novo proteins provide a unique opportunity to investigate the structure-function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the Due Ferri family. Originally created to catalyse the O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyse the selective N-hydroxylation of arylamines by remodelling the substrate access cavity and introducing a critical third His ligand to the metal-binding cavity. Additional second- and third-shell modifications were required to stabilize the His ligand in the core of the protein. These structural changes resulted in at least a 106-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of the geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

  8. De Novo Design of Metalloproteins and Metalloenzymes in a Three-Helix Bundle.

    Science.gov (United States)

    Plegaria, Jefferson S; Pecoraro, Vincent L

    2016-01-01

    For more than two decades, de novo protein design has proven to be an effective methodology for modeling native proteins. De novo design involves the construction of metal-binding sites within simple and/or unrelated α-helical peptide structures. The preparation of α3D, a single polypeptide that folds into a native-like three-helix bundle structure, has significantly expanded available de novo designed scaffolds. Devoid of a metal-binding site (MBS), we incorporated a 3Cys and 3His motif in α3D to construct a heavy metal and a transition metal center, respectively. These efforts produced excellent functional models for native metalloproteins/metalloregulatory proteins and metalloenzymes. Morever, these α3D derivatives serve as a foundation for constructing redox active sites with either the same (e.g., 4Cys) or mixed (e.g., 2HisCys) ligands, a feat that could be achieved in this preassembled framework. Here, we describe the process of constructing MBSs in α3D and our expression techniques.

  9. iAssembler: a package for de novo assembly of Roche-454/Sanger transcriptome sequences

    Directory of Open Access Journals (Sweden)

    Zheng Yi

    2011-11-01

    Full Text Available Abstract Background Expressed Sequence Tags (ESTs have played significant roles in gene discovery and gene functional analysis, especially for non-model organisms. For organisms with no full genome sequences available, ESTs are normally assembled into longer consensus sequences for further downstream analysis. However current de novo EST assembly programs often generate large number of assembly errors that will negatively affect the downstream analysis. In order to generate more accurate consensus sequences from ESTs, tools are needed to reduce or eliminate errors from de novo assemblies. Results We present iAssembler, a pipeline that can assemble large-scale ESTs into consensus sequences with significantly higher accuracy than current existing assemblers. iAssembler employs MIRA and CAP3 assemblers to generate initial assemblies, followed by identifying and correcting two common types of transcriptome assembly errors: 1 ESTs from different transcripts (mainly alternatively spliced transcripts or paralogs are incorrectly assembled into same contigs; and 2 ESTs from same transcripts fail to be assembled together. iAssembler can be used to assemble ESTs generated using the traditional Sanger method and/or the Roche-454 massive parallel pyrosequencing technology. Conclusion We compared performances of iAssembler and several other de novo EST assembly programs using both Roche-454 and Sanger EST datasets. It demonstrated that iAssembler generated significantly more accurate consensus sequences than other assembly programs.

  10. De novo hepatic steatosis drives atherogenic risk in liver transplantation recipients.

    Science.gov (United States)

    Idowu, Michael O; Chhatrala, Ravi; Siddiqui, M Bilal; Driscoll, Carolyn; Stravitz, R Todd; Sanyal, Arun J; Bhati, Chandra; Sargeant, Carol; Luketic, Velimir A; Sterling, Richard K; Contos, Melissa; Matherly, Scott; Puri, Puneet; Siddiqui, M Shadab

    2015-11-01

    Nonalcoholic fatty liver disease is associated with cardiovascular disease (CVD) in the general population. Despite a high prevalence of de novo hepatic steatosis after liver transplantation (LT), there are no data exploring the association between hepatic steatosis after LT and atherogenic risk. The aim of the study was to explore the impact of hepatic steatosis on serum atherogenic markers in liver transplantation recipients (LTRs). Biomarkers of CVD risk were compared in 89 LTRs with no known history of dyslipidemia, ischemic heart disease, or graft cirrhosis. To avoid potential confounders, LTRs on oral hypoglycemic agents, exogenous insulin, corticosteroids, or lipid-lowering therapy were excluded. Only patients for whom histological assessment was available after LT were included in the study. Thirty-five LTRs had de novo hepatic steatosis after LT, whereas 54 did not. Both cohorts were similar with regards to age, sex, ethnicity, and follow-up from LT. Additionally, the traditional lipid profile was similar between the 2 cohorts. LTRs with hepatic steatosis had higher serum concentrations of small-dense low-density lipoprotein cholesterol (sdLDL-C; 34.8 ± 16.9 versus 22.7 ± 11.2 mg/dL; P mass index, age, diabetes mellitus, time from transplant, and indication for LT. In conclusion, de novo hepatic steatosis after LT is associated with atherogenic lipoproteins and independent of traditional CVD risk factors.

  11. Building a better fragment library for de novo protein structure prediction.

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    Saulo H P de Oliveira

    Full Text Available Fragment-based approaches are the current standard for de novo protein structure prediction. These approaches rely on accurate and reliable fragment libraries to generate good structural models. In this work, we describe a novel method for structure fragment library generation and its application in fragment-based de novo protein structure prediction. The importance of correct testing procedures in assessing the quality of fragment libraries is demonstrated. In particular, the exclusion of homologs to the target from the libraries to correctly simulate a de novo protein structure prediction scenario, something which surprisingly is not always done. We demonstrate that fragments presenting different predominant predicted secondary structures should be treated differently during the fragment library generation step and that exhaustive and random search strategies should both be used. This information was used to develop a novel method, Flib. On a validation set of 41 structurally diverse proteins, Flib libraries presents both a higher precision and coverage than two of the state-of-the-art methods, NNMake and HHFrag. Flib also achieves better precision and coverage on the set of 275 protein domains used in the two previous experiments of the the Critical Assessment of Structure Prediction (CASP9 and CASP10. We compared Flib libraries against NNMake libraries in a structure prediction context. Of the 13 cases in which a correct answer was generated, Flib models were more accurate than NNMake models for 10. "Flib is available for download at: http://www.stats.ox.ac.uk/research/proteins/resources".

  12. De novo anti-HLA antibody responses after renal transplantation: detection and clinical impact.

    Science.gov (United States)

    Seveso, Michela; Bosio, Erika; Ancona, Ermanno; Cozzi, Emanuele

    2009-01-01

    Numerous retrospective and prospective studies have been conducted to determine the prevalence and significance on long-term graft survival of de novo post-transplant donor-specific antibodies (DSA), directed against both HLA and non-HLA molecules. Moreover, it has been postulated that the development of anti-HLA antibodies may precede the clinical manifestation of chronic rejection, therefore being considered a predictive marker. In this context, the detection of C4d deposition in the failing kidney in patients presenting de novo DSA supports the hypothesis that antibody production and complement deposition could be involved in the pathogenesis of graft failure. Due to the development of more sensitive meth-ods to detect alloantibodies, the number of transplanted patients which show the appearance of DSA at different times following transplantation has increased. Nevertheless, this increased sensitivity has allowed the identification of circulating donor-specific anti-HLA antibodies in many patients with otherwise good graft function. Such findings are worthy of discussion, as it has yet to be determined whether these circulating antibodies can only be considered an early marker of humoral rejection or whether they could play a protective role. The possible relevance of the post-transplant appearance of non-DSA should also be mentioned. This review will focus primarily on de novo anti-donor HLA antibody responses in kidney transplant patients and will only briefly deal with anti-non HLA and non-DSA that will be discussed elsewhere in this issue.

  13. Heterologous aggregates promote de novo prion appearance via more than one mechanism.

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    Fatih Arslan

    2015-01-01

    Full Text Available Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q/asparagine (N-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN

  14. The complex task of choosing a de novo assembly: lessons from fungal genomes.

    Science.gov (United States)

    Gallo, Juan Esteban; Muñoz, José Fernando; Misas, Elizabeth; McEwen, Juan Guillermo; Clay, Oliver Keatinge

    2014-12-01

    Selecting the values of parameters used by de novo genomic assembly programs, or choosing an optimal de novo assembly from several runs obtained with different parameters or programs, are tasks that can require complex decision-making. A key parameter that must be supplied to typical next generation sequencing (NGS) assemblers is the k-mer length, i.e., the word size that determines which de Bruijn graph the program should map out and use. The topic of assembly selection criteria was recently revisited in the Assemblathon 2 study (Bradnam et al., 2013). Although no clear message was delivered with regard to optimal k-mer lengths, it was shown with examples that it is sometimes important to decide if one is most interested in optimizing the sequences of protein-coding genes (the gene space) or in optimizing the whole genome sequence including the intergenic DNA, as what is best for one criterion may not be best for the other. In the present study, our aim was to better understand how the assembly of unicellular fungi (which are typically intermediate in size and complexity between prokaryotes and metazoan eukaryotes) can change as one varies the k-mer values over a wide range. We used two different de novo assembly programs (SOAPdenovo2 and ABySS), and simple assembly metrics that also focused on success in assembling the gene space and repetitive elements. A recent increase in Illumina read length to around 150 bp allowed us to attempt de novo assemblies with a larger range of k-mers, up to 127 bp. We applied these methods to Illumina paired-end sequencing read sets of fungal strains of Paracoccidioides brasiliensis and other species. By visualizing the results in simple plots, we were able to track the effect of changing k-mer size and assembly program, and to demonstrate how such plots can readily reveal discontinuities or other unexpected characteristics that assembly programs can present in practice, especially when they are used in a traditional molecular

  15. Incidence and Risk Factors of De novo Stress Urinary Incontinence after Pelvic Floor Reconstruction: A Nested Case-control Study

    Science.gov (United States)

    Wang, Shi-Yan; Cao, Ting-Ting; Wang, Run-Zhi; Yang, Xin; Sun, Xiu-Li; Wang, Jian-Liu

    2017-01-01

    Background: Some patients with pelvic organ prolapse may suffer from lower urinary tract symptoms (LUTS), especially stress urinary incontinence (SUI) named de novo SUI after pelvic floor reconstruction. This study aimed to investigate the incidence and risk factors of de novo SUI. Methods: This is a nested case-control study of 533 patients who underwent pelvic floor reconstruction due to pelvic organ prolapse (POP) at the Department of Gynecology in Peking University People's Hospital from January 2011 to March 2013. According to the inclusion and exclusion criteria, 401 patients were enrolled in the study with the follow-up rate of 74.8% (101 patients lost to follow-up). There were 75 patients with de novo SUI postoperatively. According to the ratio of 1:3, we ensured the number of control group (n = 225). The preoperative urinary dynamics, POP-quantification scores, and LUTS were compared between the two groups by univariate and multivariate logistic regression analyses to investigate the risk factors of de novo SUI. Results: The incidence of de novo SUI was 25% (75/300). Univariate analysis showed that the ratio of lower urinary tract obstruction (LUTO) before surgery in de novo SUI group was significantly higher than the control group (odds ratio [OR] = 2.1, 95% confidence interval [CI] [1.1–4.0], P = 0.022). The interaction test of LUTO and other factors displayed that Aa value was an interaction factor. With the increasing score of Aa, the incidence of de novo SUI become higher (OR = 2.1, 95% CI [1.0–3.7], P = 0.045). After multivariable adjustment, multiple regression analysis showed that LUTO was independently associated with a greater risk of de novo SUI after pelvic floor surgery (OR = 2.3, 95% CI [1.2–4.6], P = 0.013). Conclusions: Preoperative LUTO in patients with POP is a high-risk factor of de novo SUI, and high score of Aa-point is related to the occurrence of de novo SUI, which might be due to the outlet obstruction caused by bladder

  16. Targeting of de novo DNA methylation throughout the Oct-4 gene regulatory region in differentiating embryonic stem cells.

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    Rodoniki Athanasiadou

    Full Text Available Differentiation of embryonic stem (ES cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the approximately 2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the Oct-4 regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process.

  17. Emergence, Retention and Selection: A Trilogy of Origination for Functional De Novo Proteins from Ancestral LncRNAs in Primates.

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    Jia-Yu Chen

    2015-07-01

    Full Text Available While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.

  18. Whole Exome Sequencing for a Patient with Rubinstein-Taybi Syndrome Reveals de Novo Variants besides an Overt CREBBP Mutation

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    Hee Jeong Yoo

    2015-03-01

    Full Text Available Rubinstein-Taybi syndrome (RSTS is a rare condition with a prevalence of 1 in 125,000–720,000 births and characterized by clinical features that include facial, dental, and limb dysmorphology and growth retardation. Most cases of RSTS occur sporadically and are caused by de novo mutations. Cytogenetic or molecular abnormalities are detected in only 55% of RSTS cases. Previous genetic studies have yielded inconsistent results due to the variety of methods used for genetic analysis. The purpose of this study was to use whole exome sequencing (WES to evaluate the genetic causes of RSTS in a young girl presenting with an Autism phenotype. We used the Autism diagnostic observation schedule (ADOS and Autism diagnostic interview revised (ADI-R to confirm her diagnosis of Autism. In addition, various questionnaires were used to evaluate other psychiatric features. We used WES to analyze the DNA sequences of the patient and her parents and to search for de novo variants. The patient showed all the typical features of Autism, WES revealed a de novo frameshift mutation in CREBBP and de novo sequence variants in TNC and IGFALS genes. Mutations in the CREBBP gene have been extensively reported in RSTS patients, while potential missense mutations in TNC and IGFALS genes have not previously been associated with RSTS. The TNC and IGFALS genes are involved in central nervous system development and growth. It is possible for patients with RSTS to have additional de novo variants that could account for previously unexplained phenotypes.

  19. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

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    Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Morales, Jorge; Fukai, Yoshihisa; Suzuki, Shigeo; Takamiya, Shinzaburo; Tsubouchi, Akiko; Inoue, Syou [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Inoue, Masayuki [Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Kita, Kiyoshi [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Harada, Shigeharu [Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Tanaka, Akiko [Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan); Aoki, Takashi [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Nara, Takeshi, E-mail: tnara@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. Black-Right-Pointing-Pointer Disruption of the cpsII gene significantly reduced the growth of epimastigotes. Black-Right-Pointing-Pointer In particular, the CPSII-null mutant severely retarded intracellular growth. Black-Right-Pointing-Pointer The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.

  20. Adaptive Reprogramming of De Novo Pyrimidine Synthesis Is a Metabolic Vulnerability in Triple-Negative Breast Cancer.

    Science.gov (United States)

    Brown, Kristin K; Spinelli, Jessica B; Asara, John M; Toker, Alex

    2017-04-01

    Chemotherapy resistance is a major barrier to the treatment of triple-negative breast cancer (TNBC), and strategies to circumvent resistance are required. Using in vitro and in vivo metabolic profiling of TNBC cells, we show that an increase in the abundance of pyrimidine nucleotides occurs in response to chemotherapy exposure. Mechanistically, elevation of pyrimidine nucleotides induced by chemotherapy is dependent on increased activity of the de novo pyrimidine synthesis pathway. Pharmacologic inhibition of de novo pyrimidine synthesis sensitizes TNBC cells to genotoxic chemotherapy agents by exacerbating DNA damage. Moreover, combined treatment with doxorubicin and leflunomide, a clinically approved inhibitor of the de novo pyrimidine synthesis pathway, induces regression of TNBC xenografts. Thus, the increase in pyrimidine nucleotide levels observed following chemotherapy exposure represents a metabolic vulnerability that can be exploited to enhance the efficacy of chemotherapy for the treatment of TNBC.Significance: The prognosis for patients with TNBC with residual disease after chemotherapy is poor. We find that chemotherapy agents induce adaptive reprogramming of de novo pyrimidine synthesis and show that this response can be exploited pharmacologically, using clinically approved inhibitors of de novo pyrimidine synthesis, to sensitize TNBC cells to chemotherapy. Cancer Discov; 7(4); 391-9. ©2017 AACR.See related article by Mathur et al., p. 380This article is highlighted in the In This Issue feature, p. 339.

  1. De novo assembly and characterization of the Trichuris trichiura adult worm transcriptome using Ion Torrent sequencing.

    Science.gov (United States)

    Santos, Leonardo N; Silva, Eduardo S; Santos, André S; De Sá, Pablo H; Ramos, Rommel T; Silva, Artur; Cooper, Philip J; Barreto, Maurício L; Loureiro, Sebastião; Pinheiro, Carina S; Alcantara-Neves, Neuza M; Pacheco, Luis G C

    2016-07-01

    Infection with helminthic parasites, including the soil-transmitted helminth Trichuris trichiura (human whipworm), has been shown to modulate host immune responses and, consequently, to have an impact on the development and manifestation of chronic human inflammatory diseases. De novo derivation of helminth proteomes from sequencing of transcriptomes will provide valuable data to aid identification of parasite proteins that could be evaluated as potential immunotherapeutic molecules in near future. Herein, we characterized the transcriptome of the adult stage of the human whipworm T. trichiura, using next-generation sequencing technology and a de novo assembly strategy. Nearly 17.6 million high-quality clean reads were assembled into 6414 contiguous sequences, with an N50 of 1606bp. In total, 5673 protein-encoding sequences were confidentially identified in the T. trichiura adult worm transcriptome; of these, 1013 sequences represent potential newly discovered proteins for the species, most of which presenting orthologs already annotated in the related species T. suis. A number of transcripts representing probable novel non-coding transcripts for the species T. trichiura were also identified. Among the most abundant transcripts, we found sequences that code for proteins involved in lipid transport, such as vitellogenins, and several chitin-binding proteins. Through a cross-species expression analysis of gene orthologs shared by T. trichiura and the closely related parasites T. suis and T. muris it was possible to find twenty-six protein-encoding genes that are consistently highly expressed in the adult stages of the three helminth species. Additionally, twenty transcripts could be identified that code for proteins previously detected by mass spectrometry analysis of protein fractions of the whipworm somatic extract that present immunomodulatory activities. Five of these transcripts were amongst the most highly expressed protein-encoding sequences in the T

  2. Safe conversion to cicloral, a generic cylosporine, in both stable and De Novo renal transplant recipients

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    Kahn Delawir

    2010-01-01

    Full Text Available Several generic cyclosporine (CsA formulations have been developed over the last decade and are now widely available. In 2003 our local Health Department replaced Neoral with CicloHexal for the cost benefits, and we were compelled to convert all our renal transplant reci-pients to the generic CsA formulation. All renal transplant recipients were converted from Neoral to CicloHexal on a 1:1 dose basis in August/September 2003. Study 1 constitutes the retrospective review of all stable renal transplant patients and the CsA dose, CsA level and serum creatinine were noted. Study 2 constitutes the review of the records of de novo transplant patients inititated on CicloHexal compared to matched patients transplanted on Neoral before the conversion and the CsA dose, CsA level and serum creatinine noted (Study 2. There was no difference in the mean CsA dose, CsA level or serum creatinine at one month before conversion (on Neoral com-pared to one month after conversion (on CicloHexal in the 117 stable renal transplant recipients. Similarly, the mean CsA dose, CsA level and serum creatinine in de novo renal transplant reci-pients on Neoral (n= 26 were similar to those on CicloHexal (n= 23 at about seven and ten days postoperatively. In conclusion both stable and de novo renal transplant patients can be safely converted from Neoral to CicloHexal on a 1:1 dose basis.

  3. De novo analysis of transcriptome dynamics in the migratory locust during the development of phase traits.

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    Shuang Chen

    Full Text Available Locusts exhibit remarkable density-dependent phenotype (phase changes from the solitary to the gregarious, making them one of the most destructive agricultural pests. This phenotype polyphenism arises from a single genome and diverse transcriptomes in different conditions. Here we report a de novo transcriptome for the migratory locust and a comprehensive, representative core gene set. We carried out assembly of 21.5 Gb Illumina reads, generated 72,977 transcripts with N50 2,275 bp and identified 11,490 locust protein-coding genes. Comparative genomics analysis with eight other sequenced insects was carried out to identify the genomic divergence between hemimetabolous and holometabolous insects for the first time and 18 genes relevant to development was found. We further utilized the quantitative feature of RNA-seq to measure and compare gene expression among libraries. We first discovered how divergence in gene expression between two phases progresses as locusts develop and identified 242 transcripts as candidates for phase marker genes. Together with the detailed analysis of deep sequencing data of the 4(th instar, we discovered a phase-dependent divergence of biological investment in the molecular level. Solitary locusts have higher activity in biosynthetic pathways while gregarious locusts show higher activity in environmental interaction, in which genes and pathways associated with regulation of neurotransmitter activities, such as neurotransmitter receptors, synthetase, transporters, and GPCR signaling pathways, are strongly involved. Our study, as the largest de novo transcriptome to date, with optimization of sequencing and assembly strategy, can further facilitate the application of de novo transcriptome. The locust transcriptome enriches genetic resources for hemimetabolous insects and our understanding of the origin of insect metamorphosis. Most importantly, we identified genes and pathways that might be involved in locust development

  4. Rare de novo copy number variants in patients with congenital pulmonary atresia.

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    Li Xie

    Full Text Available BACKGROUND: Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. The aim of this study was to identify rare copy number variants in individuals with congenital pulmonary atresia (PA. METHODS AND RESULTS: Based on the hypothesis that rare structural variants encompassing key genes play an important role in heart development in PA patients, we performed high-resolution genome-wide microarrays for copy number variations (CNVs in 82 PA patient-parent trios and 189 controls with an Illumina SNP array platform. CNVs were identified in 17/82 patients (20.7%, and eight of these CNVs (9.8% are considered potentially pathogenic. Five de novo CNVs occurred at two known congenital heart disease (CHD loci (16p13.1 and 22q11.2. Two de novo CNVs that may affect folate and vitamin B12 metabolism were identified for the first time. A de novo 1-Mb deletion at 17p13.2 may represent a rare genomic disorder that involves mild intellectual disability and associated facial features. CONCLUSIONS: Rare CNVs contribute to the pathogenesis of PA (9.8%, suggesting that the causes of PA are heterogeneous and pleiotropic. Together with previous data from animal models, our results might help identify a link between CHD and folate-mediated one-carbon metabolism (FOCM. With the accumulation of high-resolution SNP array data, these previously undescribed rare CNVs may help reveal critical gene(s in CHD and may provide novel insights about CHD pathogenesis.

  5. De novo purine biosynthesis by two pathways in Burkitt lymphoma cells and in human spleen.

    Science.gov (United States)

    Reem, G H

    1972-05-01

    This study was designed to answer the question whether human lymphocytes and spleen cells were capable of de novo purine biosynthesis. Experiments were carried out in cell-free extracts prepared from human spleen, and from a cell line established from Burkitt lymphoma. Burkitt lymphoma cells and human spleen cells could synthesize the first and second intermediates of the purine biosynthetic pathway. Cell-free extracts of all cell lines studied contained the enzyme systems which catalyze the synthesis of phosphoribosyl-1-amine, the first intermediate unique to the purine biosynthetic pathway and of phosphoribosyl glycinamide, the second intermediate of this pathway. Phosphoribosyl-1-amine could be synthesized in cell-free extracts from alpha-5-phosphoribosyl-1-pyrophosphate (PRPP) and glutamine, from PRPP and ammonia, and by an alternative pathway, directly from ribose-5-phosphate and ammonia. These findings suggest that extrahepatic tissues may be an important source for the de novo synthesis of purine ribonucleotide in man. They also indicate that ammonia may play an important role in purine biosynthesis. The alternative pathway for the synthesis of phosphoribosyl-1-amine from ribose-5-phosphate and ammonia was found to be subject to inhibition by the end products of the purine synthetic pathway, particularly by adenylic acid and to a lesser degree by guanylic acid. The alternative pathway for phosphoribosyl-1-amine synthesis from ribose-5-phosphate and ammonia may contribute significantly towards the regulation of the rate of de novo purine biosynthesis in the normal state, in metabolic disorders in which purines are excessively produced and in myeloproliferative diseases.

  6. Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

    Science.gov (United States)

    Christen, Matthias; Deutsch, Samuel; Christen, Beat

    2015-08-21

    Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

  7. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads.

    Science.gov (United States)

    Kajitani, Rei; Toshimoto, Kouta; Noguchi, Hideki; Toyoda, Atsushi; Ogura, Yoshitoshi; Okuno, Miki; Yabana, Mitsuru; Harada, Masayuki; Nagayasu, Eiji; Maruyama, Haruhiko; Kohara, Yuji; Fujiyama, Asao; Hayashi, Tetsuya; Itoh, Takehiko

    2014-08-01

    Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wild-type samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity.

  8. De Novo Assembly and Characterization of the Transcriptome of Grasshopper Shirakiacris shirakii

    Directory of Open Access Journals (Sweden)

    Zhongying Qiu

    2016-07-01

    Full Text Available Background: The grasshopper Shirakiacris shirakii is an important agricultural pest and feeds mainly on gramineous plants, thereby causing economic damage to a wide range of crops. However, genomic information on this species is extremely limited thus far, and transcriptome data relevant to insecticide resistance and pest control are also not available. Methods: The transcriptome of S. shirakii was sequenced using the Illumina HiSeq platform, and we de novo assembled the transcriptome. Results: Its sequencing produced a total of 105,408,878 clean reads, and the de novo assembly revealed 74,657 unigenes with an average length of 680 bp and N50 of 1057 bp. A total of 28,173 unigenes were annotated for the NCBI non-redundant protein sequences (Nr, NCBI non-redundant nucleotide sequences (Nt, a manually-annotated and reviewed protein sequence database (Swiss-Prot, Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG databases. Based on the Nr annotation results, we manually identified 79 unigenes encoding cytochrome P450 monooxygenases (P450s, 36 unigenes encoding carboxylesterases (CarEs and 36 unigenes encoding glutathione S-transferases (GSTs in S. shirakii. Core RNAi components relevant to miroRNA, siRNA and piRNA pathways, including Pasha, Loquacious, Argonaute-1, Argonaute-2, Argonaute-3, Zucchini, Aubergine, enhanced RNAi-1 and Piwi, were expressed in S. shirakii. We also identified five unigenes that were homologous to the Sid-1 gene. In addition, the analysis of differential gene expressions revealed that a total of 19,764 unigenes were up-regulated and 4185 unigenes were down-regulated in larvae. In total, we predicted 7504 simple sequence repeats (SSRs from 74,657 unigenes. Conclusions: The comprehensive de novo transcriptomic data of S. shirakii will offer a series of valuable molecular resources for better studying insecticide resistance, RNAi and molecular marker discovery in the transcriptome.

  9. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    Science.gov (United States)

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  10. Sensitivity-Informed De Novo Programming for Many-Objective Water Portfolio Planning Under Uncertainty

    Science.gov (United States)

    Kasprzyk, J. R.; Reed, P. M.; Kirsch, B. R.; Characklis, G. W.

    2009-12-01

    Risk-based water supply management presents severe cognitive, computational, and social challenges to planning in a changing world. Decision aiding frameworks must confront the cognitive biases implicit to risk, the severe uncertainties associated with long term planning horizons, and the consequent ambiguities that shape how we define and solve water resources planning and management problems. This paper proposes and demonstrates a new interactive framework for sensitivity informed de novo programming. The theoretical focus of our many-objective de novo programming is to promote learning and evolving problem formulations to enhance risk-based decision making. We have demonstrated our proposed de novo programming framework using a case study for a single city’s water supply in the Lower Rio Grande Valley (LRGV) in Texas. Key decisions in this case study include the purchase of permanent rights to reservoir inflows and anticipatory thresholds for acquiring transfers of water through optioning and spot leases. A 10-year Monte Carlo simulation driven by historical data is used to provide performance metrics for the supply portfolios. The three major components of our methodology include Sobol globoal sensitivity analysis, many-objective evolutionary optimization and interactive tradeoff visualization. The interplay between these components allows us to evaluate alternative design metrics, their decision variable controls and the consequent system vulnerabilities. Our LRGV case study measures water supply portfolios’ efficiency, reliability, and utilization of transfers in the water supply market. The sensitivity analysis is used interactively over interannual, annual, and monthly time scales to indicate how the problem controls change as a function of the timescale of interest. These results have been used then to improve our exploration and understanding of LRGV costs, vulnerabilities, and the water portfolios’ critical reliability constraints. These results

  11. A stochastic inference of de novo CNV detection and association test in multiplex schizophrenia families

    Directory of Open Access Journals (Sweden)

    Shi-Heng eWang

    2013-09-01

    Full Text Available The copy number variation (CNV is a type of genetic variation in the genome. It is measured based on signal intensity measures and can be assessed repeatedly to reduce the uncertainty in PCR-based typing. Studies have shown that CNVs may lead to phenotypic variation and modification of disease expression. Various challenges exist, however, in the exploration of CNV-disease association. Here we construct latent variables to infer the discrete CNV values and to estimate the probability of mutations. In addition, we propose to pool rare variants to increase the statistical power and we conduct family studies to mitigate the computational burden in determining the composition of CNVs on each chromosome. The aim is to explore in a stochastic sense the association between the collapsing CNV variants and disease status with a Bayesian hierarchical model. This model assigns integers in a probabilistic sense to the quantitatively measured copy numbers, and is able to test simultaneously the association for all variants of interest in a regression framework. This integrative model can account for the uncertainty in copy number assignment and differentiate if the variation was de novo or inherited on the basis of posterior probabilities. For family studies, this model can accommodate the dependence within family members and among repeated CNV data. Moreover, the Mendelian rule can be assumed under this model and yet the genetic variation, including de novo and inherited variation, can still be included and quantified directly for each individual. Finally, simulation studies show that this model has high true positive and low false positive rates in the detection of de novo mutation.

  12. Sequence analysis of two de novo mutation alleles at the DXS10011 locus.

    Science.gov (United States)

    Tamura, Akiyoshi; Iwata, Misa; Takase, Izumi; Miyazaki, Tokiko; Matsui, Kiyoshi; Nishio, Hajime; Suzuki, Koichi

    2003-09-01

    We have detected two unusual alleles at the DXS10011 locus in two paternity trio cases. In one case, one allele of the daughter was found not to have been derived from the mother but the other allele was shared with the father. In the other case, the mother and the son shared no bands. Paternity in both cases was established using conventional polymorphic markers in addition to DNA markers (probabilities: >0.999999). Sequencing showed that the two de novo alleles of the children acquired a single unit (GAAA).

  13. De Novo Mutation in ABCC9 Causes Hypertrichosis Acromegaloid Facial Features Disorder.

    Science.gov (United States)

    Afifi, Hanan H; Abdel-Hamid, Mohamed S; Eid, Maha M; Mostafa, Inas S; Abdel-Salam, Ghada M H

    2016-01-01

    A 13-year-old Egyptian girl with generalized hypertrichosis, gingival hyperplasia, coarse facial appearance, no cardiovascular or skeletal anomalies, keloid formation, and multiple labial frenula was referred to our clinic for counseling. Molecular analysis of the ABCC9 gene showed a de novo missense mutation located in exon 27, which has been described previously with Cantu syndrome. An overlap between Cantu syndrome, acromegaloid facial syndrome, and hypertrichosis acromegaloid facial features disorder is apparent at the phenotypic and molecular levels. The patient reported here gives further evidence that these syndromes are an expression of the ABCC9-related disorders, ranging from hypertrichosis and acromegaloid facies to the severe end of Cantu syndrome.

  14. De Novo ACTA2 Mutation Causes a Novel Syndrome of Multisystemic Smooth Muscle Dysfunction

    Science.gov (United States)

    Milewicz, Dianna M.; Østergaard, John R.; Ala-Kokko, Leena M.; Khan, Nadia; Grange, Dorothy K.; Mendoza-Londono, Roberto; Bradley, Timothy J.; Olney, Ann Haskins; Adès, Lesley; Maher, Joseph F.; Guo, Dongchuan; Buja, L. Maximilian; Kim, Dong; Hyland, James C.; Regalado, Ellen S.

    2011-01-01

    Smooth muscle cells (SMCs) contract to perform many physiological functions, including regulation of blood flow and pressure in arteries, contraction of the pupils, peristalsis of the gut and voiding of the bladder. SMC lineage in these organs is characterized by cellular expression of the SMC isoform of α-actin, encoded by the ACTA2 gene. We report here on a unique and de novo mutation in ACTA2, R179H, that causes a syndrome characterized by dysfunction of SMCs throughout the body, leading to aortic and cerebrovascular disease, fixed dilated pupils, hypotonic bladder, malrotation and hypoperistalsis of the gut and pulmonary hypertension. PMID:20734336

  15. The 'de novo' DNA methyltransferase Dnmt3b compensates the Dnmt1-deficient intestinal epithelium.

    Science.gov (United States)

    Elliott, Ellen N; Sheaffer, Karyn L; Kaestner, Klaus H

    2016-01-25

    Dnmt1 is critical for immediate postnatal intestinal development, but is not required for the survival of the adult intestinal epithelium, the only rapidly dividing somatic tissue for which this has been shown. Acute Dnmt1 deletion elicits dramatic hypomethylation and genomic instability. Recovery of DNA methylation state and intestinal health is dependent on the de novo methyltransferase Dnmt3b. Ablation of both Dnmt1 and Dnmt3b in the intestinal epithelium is lethal, while deletion of either Dnmt1 or Dnmt3b has no effect on survival. These results demonstrate that Dnmt1 and Dnmt3b cooperate to maintain DNA methylation and genomic integrity in the intestinal epithelium.

  16. Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

    OpenAIRE

    Gordon, Catherine A.; Hartono, Stella R; Frédéric Chédin

    2013-01-01

    Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore l...

  17. Anterior abdominal wall leiomyoma arising de novo in a fertile women: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Je Young; Woo, Ji Young; Hong, Hye Suk; Yang, Ik; Lee, Yul; Hwang, Ji Young; Kim, Han Myun; Shin, Mi Kyung [Hallym University College of Medicine, Kangnam Sacred Heart Hospital, Seoul (Korea, Republic of)

    2016-01-15

    Abdominal wall leiomyoma arising de novo is very rare, hence the reported imaging findings of this disease are also rare. We reported the case of a 33-year-old woman who presented with an abdominal wall mass without antecedent gynecological surgeries. The initial abdominal computed tomography (CT) showed thickening of the left rectus abdominis and the loss of intervening fat between the rectus abdominis and the lateral abdominal muscles. After 8 months, the follow-up contrast-enhanced CT and ultrasonography (US) showed a lentiform-shaped mass with isodensity to the adjacent muscles. The US-guided biopsy was consistent with leiomyoma.

  18. De novo design of peptide scaffolds as novel preorganized ligands for metal-ion coordination.

    Science.gov (United States)

    Gamble, Aimee J; Peacock, Anna F A

    2014-01-01

    This chapter describes how de novo designed peptides can be used as novel preorganized ligands for metal ion coordination. The focus is on the design of peptides which are programmed to spontaneously self-assemble into α-helical coiled coils in aqueous solution, and how metal ion binding sites can be engineered onto and into these structures. In addition to describing the various design principles, some key examples are covered illustrating the success of this approach, including a more detailed example in the case study.

  19. Three cases of hypertension and renal arteriovenous fistula with a de novo fistula.

    Science.gov (United States)

    Melo, Natalia Correa Vieira; Mundim, Juliano Sacramento; Costalonga, Elerson Carlos; Lucon, Antonio Marmo; Santello, Jose Luiz; Praxedes, Jose Nery

    2009-05-01

    The Renal Arteriovenous Fistula (RAVF) is a rare and potentially reversible cause of hypertension and kidney and/or heart failure. The treatment of RAVF aims at preserving the most of the renal parenchyma and, concomitantly, eradicating the symptoms and hemodynamic effects caused by the RAVF. The present study reports three cases of RAVF, including one case of a de novo idiopathic RAVF, which presented with hypertension and kidney and/or heart failure and describes the therapeutic measures used to treat these patients as well as the outcomes.

  20. Recent Advances Towards The Discovery Of Drug-Like Peptides De Novo

    Science.gov (United States)

    Goldflam, Michael; Ullman, Christopher

    2015-12-01

    Peptides are important natural molecules that possess functions as diverse as antibiotics, toxins, venoms and hormones, for example. However, whilst these peptides have useful properties, there are many targets and pathways that are not addressed through the activities of natural peptidic compounds. In these circumstances, directed evolution techniques, such as phage display, have been developed to sample the diverse chemical and structural repertoire of small peptides for useful means. In this review, we consider recent concepts that relate peptide structure to drug-like attributes and how these are incorporated within display technologies to deliver peptides de novo with valuable pharmaceutical properties.

  1. Interplay between De Novo Biosynthesis and Sequestration of Cyanogenic Glucosides in Arthropods

    DEFF Research Database (Denmark)

    Fürstenberg-Hägg, Joel

    Cyanogenic glucosides (CNglcs) are phytoanticipins that are present in more than 2650 plant species. They play an important role in defense against generalist herbivory due to their bitter taste and their hydrolysis upon tissue disruption results in release of toxic hydrogen cyanide. Zygaena larvae....... Adult females are shown to transfer CNglcs to the eggs following emergence from the pupa, providing protection against predation at this vulnerable developmental stage. It is finally speculated that de novo biosynthesis of CNglcs enabled colonization of and subsequently sequestration from the food plant...

  2. A de novo designed monomeric, compact three helix bundle protein on a carbohydrate template

    DEFF Research Database (Denmark)

    Malik, Leila; Nygård, Jesper; Christensen, Niels Johan

    2015-01-01

    De novo design and chemical synthesis of proteins and of other artificial structures, which mimic them, is a central strategy for understanding protein folding and for accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial...... proteins, so called carboproteins. The hypothesis is that the template pre-organizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from...

  3.  De novo isolation of antibodies with pH-dependent binding properties

    OpenAIRE

    Bonvin, Pauline; Venet, Sophie; Fontaine, Gaëlle; Ravn, Ulla; Gueneau, Franck; Kosco-Vilbois, Marie; Proudfoot, Amanda; Fischer, Nicolas

    2015-01-01

    pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in hist...

  4. Multiple de novo vascular malformations in relation to diffuse venous occlusive disease: a case report

    Energy Technology Data Exchange (ETDEWEB)

    Desal, H.A. [Hopital Laennec, University of Nantes, Department of Neuroradiology, Nantes (France); Toronto Western Hospital, Department of Medical Imaging, University Health Network, Toronto, Ont. (Canada); Lee, S.K.; Kim, B.S.; TerBrugge, K.G. [Toronto Western Hospital, Department of Medical Imaging, University Health Network, Toronto, Ont. (Canada); Raoul, S.; Tymianski, M. [Toronto Western Hospital, Department of Neurosurgery, University Health Network, Toronto, Ont. (Canada)

    2005-01-01

    Brain vascular malformations are dynamic disorders. Although mostly considered to be of congenital origin, the improvement of clinical imaging and vasculogenesis knowledge has shown that they might also result from a biological dysfunction of the remodeling process after birth. Venous occlusive disease and ishemia may represent powerful revealing triggers and support the capillary venous origin of some vascular malformations. We report a unique case of the development of multiple de novo vascular malformations (transverse sinus dural fistula and posterior fossa cavernomas) following acoustic neuroma surgery. (orig.)

  5. De novo mutations in Plxnd1 and Rev3l cause mobius syndrome

    OpenAIRE

    Kayserili Karabey, Hülya; Tomas-Roca, Laura; Tsaalbi-Shtylik, Anastasia; Jansen, Jacob G.; Singh, Manvendra K.; Epstein, Jonathan A.; Altunoglu, Umut; Verzijl, Harriette; Soria, Laura; van Beusekom, Ellen; Roscioli, Tony; Iqbal, Zafar; Gilissen, Christian; Hoischen, Alexander; de Brouwer,Arjan P. M.; Erasmus, Corrie; Schubert, Dirk; Brunner, Han; Aytes, Antonio Perez; Marin, Faustino; Aroca, Pilar; Carta, Arturo; de Wind, Niels; Padberg, George W.; van Bokhoven, Hans

    2015-01-01

    ARTICLE Received 15 Nov 2014 | Accepted 17 Apr 2015 | Published 12 Jun 2015 De novo mutations in PLXND1 and REV3L cause Mo¨bius syndrome Laura Tomas-Roca1,2, Anastasia Tsaalbi-Shtylik3, Jacob G. Jansen3, Manvendra K. Singh4,5, Jonathan A. Epstein4, Umut Altunoglu6, Harriette Verzijl7, Laura Soria1, Ellen van Beusekom1, Tony Roscioli1,8, Zafar Iqbal1, Christian Gilissen1, Alexander Hoischen9, Arjan P.M. de Brouwer1, Corrie Erasmus7, Dirk Schubert10, Han Brunner1,11, Antoni...

  6. Whole-genome sequencing in autism identifies hot spots for de novo germline mutation

    DEFF Research Database (Denmark)

    Michaelson, Jacob J.; Shi, Yujian; Gujral, Madhusudan

    2012-01-01

    De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We...... of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our...

  7. The efficacy and safety of cyclosporine reduction in de novo renal allograft patients receiving sirolimus and corticosteroids: results from an open-label comparative study.

    Science.gov (United States)

    Mühlbacher, Ferdinand; Neumayer, Hans-Helmut; del Castillo, Domingo; Stefoni, Sergio; Zygmunt, Anthony J; Budde, Klemens

    2014-02-01

    This study evaluated the safety and efficacy of a sirolimus, corticosteroid, and cyclosporine reduction regimen in an open-label, 12-month trial of 420 de novo renal allograft recipients at 49 European transplant centers. One month post-transplantation, 357 patients were randomized to receive standard-dose cyclosporine (sCsA, n = 179) or reduced-dose cyclosporine (rCsA, n = 178). All patients also received sirolimus and corticosteroids. The primary end points were the rate of biopsy-confirmed acute rejection (BCAR) and renal function, as measured by serum creatinine. Baseline demographic and donor characteristics were similar between groups. BCAR rates at 12 months were not significantly different: 11.2% for rCsA patients and 16.2% for sCsA patients. Mean serum creatinine (±SEM) was significantly lower (1.75 ± 0.10 vs. 1.97 ± 0.07 mg/dl, P renal allograft recipients. Sirolimus administered with rCsA and corticosteroids provided adequate immunosuppression while reducing the potential for the nephrotoxic effects of cyclosporine. These findings may help to improve long-term renal allograft outcomes.

  8. De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly.

    Science.gov (United States)

    Wang, Won-Jing; Acehan, Devrim; Kao, Chien-Han; Jane, Wann-Neng; Uryu, Kunihiro; Tsou, Meng-Fu Bryan

    2015-11-26

    Vertebrate centrioles normally propagate through duplication, but in the absence of preexisting centrioles, de novo synthesis can occur. Consistently, centriole formation is thought to strictly rely on self-assembly, involving self-oligomerization of the centriolar protein SAS-6. Here, through reconstitution of de novo synthesis in human cells, we surprisingly found that normal looking centrioles capable of duplication and ciliation can arise in the absence of SAS-6 self-oligomerization. Moreover, whereas canonically duplicated centrioles always form correctly, de novo centrioles are prone to structural errors, even in the presence of SAS-6 self-oligomerization. These results indicate that centriole biogenesis does not strictly depend on SAS-6 self-assembly, and may require preexisting centrioles to ensure structural accuracy, fundamentally deviating from the current paradigm.

  9. Integration of Molecular Dynamics Based Predictions into the Optimization of De Novo Protein Designs: Limitations and Benefits.

    Science.gov (United States)

    Carvalho, Henrique F; Barbosa, Arménio J M; Roque, Ana C A; Iranzo, Olga; Branco, Ricardo J F

    2017-01-01

    Recent advances in de novo protein design have gained considerable insight from the intrinsic dynamics of proteins, based on the integration of molecular dynamics simulations protocols on the state-of-the-art de novo protein design protocols used nowadays. With this protocol we illustrate how to set up and run a molecular dynamics simulation followed by a functional protein dynamics analysis. New users will be introduced to some useful open-source computational tools, including the GROMACS molecular dynamics simulation software package and ProDy for protein structural dynamics analysis.

  10. Prenatal Diagnosis of a Fetus with de novo Supernumerary Ring Chromosome 16 Characterized by Array Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Pietro Cignini

    2011-09-01

    Full Text Available A fetus with de novo ring chromosome 16 is presented. At 20 weeks' gestation, ultrasound examination demonstrated bilateral clubfoot, bilateral renal pyelectasis, hypoplasia of the corpus callosum, and transposition of the great vessel. Amniocentesis was performed. Chromosome analysis identified a ring chromosome 16 [47,XY,r(16] and array comparative genomic hybridization (a-CGH demonstrated that the ring included the euchromatic portion 16p11.2. Postmortem examination confirmed prenatal findings. This is the first case of de novo ring chromosome 16 diagnosed prenatally with a new phenotypic pattern and also reinforces the importance of offering amniocentesis with a-CGH if fetal anomalies are detected.

  11. The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations.

    Science.gov (United States)

    Wang, Moye; Hu, Jie; Zhang, Zhuqing

    2016-04-26

    As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5-10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.

  12. Robust de novo pathway enrichment with KeyPathwayMiner 5

    DEFF Research Database (Denmark)

    Alcaraz, Nicolas; List, Markus; Dissing-Hansen, Martin;

    2016-01-01

    Identifying functional modules or novel active pathways, recently termed de novo pathway enrichment, is a computational systems biology challenge that has gained much attention during the last decade. Given a large biological interaction network, KeyPathwayMiner extracts connected subnetworks tha...... several network perturbation techniques and over a range of perturbation degrees. In addition, users may now provide a gold-standard set to determine how enriched extracted pathways are with relevant genes compared to randomized versions of the original network.......Identifying functional modules or novel active pathways, recently termed de novo pathway enrichment, is a computational systems biology challenge that has gained much attention during the last decade. Given a large biological interaction network, KeyPathwayMiner extracts connected subnetworks...... that are enriched for differentially active entities from a series of molecular profiles encoded as binary indicator matrices. Since interaction networks constantly evolve, an important question is how robust the extracted results are when the network is modified. We enable users to study this effect through...

  13. Adopting De Novo Programming Approach on IC Design Service Firms Resources Integration

    Directory of Open Access Journals (Sweden)

    James K. C. Chen

    2014-01-01

    Full Text Available The semiconductor industry has very important position in computer industry, ICT field, and new electronic technology developing. The IC design service is one of key factor of semiconductor industry development. There are more than 365 IC design service firms have been established around Hsinchu Science Park in Taiwan. Building an efficient planning model for IC design service firm resources integrating is very interest issue. This study aims to construct a planning model for IC design service firm implementation resources integration. This study uses the De Novo programming as an approach of criteria alternative to achieve optimal resource allocation on IC design firm. Results show the IC design service firm should conduct open innovation concept and utilizes design outsourcing obtains cost down and enhance IC design service business performance. This plan model of De Novo programming is not only for IC design service firm and also can apply to the other industrial implementation strategic alliance/integrating resource. This plan model is a universal model for the others industries field.

  14. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    Science.gov (United States)

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  15. De novo transcriptome assembly databases for the central nervous system of the medicinal leech.

    Science.gov (United States)

    Hibsh, Dror; Schori, Hadas; Efroni, Sol; Shefi, Orit

    2015-01-01

    The study of non-model organisms stands to benefit greatly from genetic and genomic data. For a better understanding of the molecular mechanisms driving neuronal development, and to characterize the entire leech Hirudo medicinalis central nervous system (CNS) transcriptome we combined Trinity for de-novo assembly and Illumina HiSeq2000 for RNA-Seq. We present a set of 73,493 de-novo assembled transcripts for the leech, reconstructed from RNA collected, at a single ganglion resolution, from the CNS. This set of transcripts greatly enriches the available data for the leech. Here, we share two databases, such that each dataset allows a different type of search for candidate homologues. The first is the raw set of assembled transcripts. This set allows a sequence-based search. A comprehensive analysis of which revealed 22,604 contigs with high e-values, aligned versus the Swiss-Prot database. This analysis enabled the production of the second database, which includes correlated sequences to annotated transcript names, with the confidence of BLAST best hit.

  16. Chromatin Targeting of de Novo DNA Methyltransferases by the PWWP Domain

    Institute of Scientific and Technical Information of China (English)

    Ying-ZiGe; Min-TiePu; HumairaGowher; Hai-PingWu; Jian-PingDing; AlbertJeltsch; Guo-LiangXu

    2005-01-01

    DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both en-zymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible forthe catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWPmediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation insatellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.

  17. Inactive DNMT3B splice variants modulate de novo DNA methylation.

    Science.gov (United States)

    Gordon, Catherine A; Hartono, Stella R; Chédin, Frédéric

    2013-01-01

    Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

  18. Inactive DNMT3B splice variants modulate de novo DNA methylation.

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    Catherine A Gordon

    Full Text Available Inactive DNA methyltransferase (DNMT 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3 deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

  19. A case of de novo duplication of 15q24-q26.3

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    Hye Ran Kim

    2011-06-01

    Full Text Available Distal duplication, or trisomy 15q, is an extremely rare chromosomal disorder characterized by prenatal and postnatal overgrowth, mental retardation, and craniofacial malformations. Additional abnormalities typically include an unusually short neck, malformations of the fingers and toes, scoliosis and skeletal malformations, genital abnormalities, particularly in affected males, and, in some cases, cardiac defects. The range and severity of symptoms and physical findings may vary from case to case, depending upon the length and location of the duplicated portion of chromosome 15q. Most reported cases of duplication of the long arm of chromosome 15 frequently have more than one segmental imbalance resulting from unbalanced translocations involving chromosome 15 and deletions in another chromosome, as well as other structural chromosomal abnormalities. We report a female newborn with a de novo duplication, 15q24- q26.3, showing intrauterine overgrowth, a narrow asymmetric face with down-slanting palpebral fissures, a large, prominent nose, and micrognathia, arachnodactyly, camptodactyly, congenital heart disease, hydronephrosis, and hydroureter. Chromosomal analysis showed a 46,XX,inv(9(p12q13,dup(15(q24q26.3. Array comparative genomic hybridization analysis revealed a gain of 42 clones on 15q24-q26.3. This case represents the only reported patient with a de novo 15q24-q26.3 duplication that did not result from an unbalanced translocation and did not have a concomitant monosomic component in Korea.

  20. De Novo Sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, Reveal a Variable Genomic Landscape

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    Benjamin J. Tully

    2015-01-01

    Full Text Available Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.

  1. De Novo characterization of a Cephalotaxus hainanensis transcriptome and genes related to paclitaxel biosynthesis.

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    Fei Qiao

    Full Text Available Cephalotaxus hainanensis, an endangered plant, is known to contain several metabolites with anti-cancer activity. Despite its clinical impact, the alkaloid metabolism of this species has remained largely uncharacterized. The potential of Cephalotaxus for metabolic engineering of medically interesting compounds has, so far, not been exploited, due to the almost complete lack of molecular information. We have therefore performed a high throughput RNA-seq analysis and assembled the transcriptome de novo. Raw reads comprising 4.3 Gbp were assembled de novo into 39,416 unique sequences (unigenes with a mean length of 1,089.8 bp and a total assembly size of 45.8 Mbp, which equals to more than 50 times the number of Cephalotaxaceae sequences currently deposited in the GenBank (as of August 2013. As proof of principle for medically interesting pathways, gene fragments related to paclitaxel biosynthesis were searched and detected. To verify their functionality, the metabolic product paclitaxel, and its precursor baccatin III, were identified in the leaves of C. hainanensis by HPLC, and shown to be induced by MeJA. This finding demonstrates exemplarily the potential of the annotated transcriptome as information resource for the biotechnological exploitation of plant secondary metabolism.

  2. On the mechanical role of de novo synthesized elastin in the urinary bladder wall.

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    Wognum, Silvia; Schmidt, David E; Sacks, Michael S

    2009-10-01

    The urinary bladder wall (UBW), which is composed of smooth muscle, collagen, and elastin, undergoes profound remodeling in response to changes in mechanical loading resulting from various pathologies. In our laboratory, we have observed the production of fibrillar elastin in the extracellular matrix (ECM), which makes the UBW a particularly attractive tissue to investigate smooth muscle tissue remodeling. In the present study, we explored the mechanical role that de novo elastin fibers play in altering UBW ECM mechanical behavior using a structural constitutive modeling approach. The mechanical behavior of the collagen fiber component of the UBW ECM was determined from the biaxial stress-stretch response of normal UBW ECM, based on bimodal fiber recruitment that was motivated by the UBW's unique collagen fiber structure. The resulting fiber ensemble model was then combined with an experimentally derived fiber angular distribution to predict the biaxial mechanical behavior of normal and the elastin-rich UBW ECM to elucidate the underlying mechanisms of elastin production. Results indicated that UBW ECM exhibited a distinct structure with highly coiled collagen fiber bundles and visible elastic fibers in the pathological situation. Elastin-rich UBW ECM had a distinct mechanical behavior with higher compliance, attributable to the indirect effect of elastin fibers contracting the collagen fiber network, resulting in a retracted unloaded reference state of the tissue. In conclusion, our results suggest that the urinary bladder responds to prolonged periods of high strain by increasing its effective compliance through the interaction between collagen and de novo synthesized elastic fibers.

  3. Downregulated stromal antigen 2 expression in de novo acute myeloid leukemia patients

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    Han, Qiaoyan; He, Xuefeng; Wu, Lili; Gao, Feng; Ye, Jinsong; Wu, Lingyu; Chen, Lu; Jiang, Xin; Sun, Miao; Chen, Suning

    2017-01-01

    The stromal antigen 2 (STAG2) gene encodes a component of the cohesin complex that participates in the regulation of sister chromatid separation during mitosis. When activated, STAG2 may act as a ‘caretaker’ tumor suppressor gene. As it is unknown whether STAG2 gene is responsible for the occurrence and associated with the prognosis of acute myeloid leukemia (AML), the present study analyzed the relative expression levels of STAG2 in 127 de novo AML patients and 17 healthy volunteers using reverse transcription-quantitative polymerase chain reaction. In addition, AML patients were divided into three risk groups using cytogenetic and molecular genetic abnormalities to define their risk status. STAG2 gene expression was found to be significantly downregulated in de novo AML patients, when compared with the healthy controls; however, the expression was not significantly different in the various gender and age subgroups. Furthermore, no significant difference between risk groups was detected in AML patients. Thus, the STAG2 gene may serve an important role in AML development, but is not associated with prognosis in AML.

  4. De Novo Assembly of the Transcriptome of Turritopsis, a Jellyfish that Repeatedly Rejuvenates.

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    Hasegawa, Yoshinori; Watanabe, Takashi; Takazawa, Masaki; Ohara, Osamu; Kubota, Shin

    2016-08-01

    In most animals, aging is an irreversible process; however the species Turritopsis sp. has been observed to undergo a rejuvenation process as many as 14 times. In the present study, we used multiplexed RNA libraries to obtain the transcriptome from four developmental stages (St) of Turritopsis sp., including (I) immature medusa, (II) dumpling, (III) dumpling with a short stolon, and (IV) polyp, which had recently rejuvenated. A total of 4.02 billion paired-end reads were assembled de novo, yielding 90,327 contigs. Our analyses revealed that significant blast hits were recovered for 74% of the assembled contigs, and 19% were successfully annotated with gene ontology (GO) terms. A BLAST search demonstrated that 32% of the contigs were most similar to Hydra vulgarissequences. Raw reads from each sample were mapped against the contigs to find St-specific genes. This represents the first comprehensive set of de novo transcriptome data for this species, which may provide clues toward a better understanding of cyclical rejuvenation in multicellular animals.

  5. Early presentation of de novo high grade glioma with epileptic seizures: electroclinical and neuroimaging findings.

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    Rossi, Rosario; Figus, Andrea; Corraine, Simona

    2010-10-01

    We report the clinical, EEG and neuroradiologic findings from three adult patients who developed new-onset seizure disorders as early clinical manifestations of de novo high grade glioma. The malignancies could not be recognised at the time of the first epileptic seizure because of minimal non-specific brain abnormalities, which showed no signs of necrosis or significant contrast enhancement on computed tomography and magnetic resonance imaging. Focal EEG abnormalities were recorded in all cases and appeared consistent with the neuroradiologic findings. The patients regained normal neurological status after the first seizure but rapidly developed space-occupying necrotic lesions. Two patients underwent surgery and received histological diagnoses of the tumours. Another patient was finally diagnosed with a malignant glioma based on the neuroradiologic picture and rapid progression of the cerebral lesion. It should be noted that in adult patients, new-onset epileptic seizures might reveal the presence of malignant gliomas at a very early stage in the tumour formation process. This report indicates that typical anatomoradiologic features of de novo high grade glioma, such as necrosis and rim-contrast enhancement, could be absent at the time of the first epileptic seizure but become clear within a short period after clinical presentation.

  6. Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines.

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    McKeague, Maureen; Wang, Yen-Hsiang; Cravens, Aaron; Win, Maung Nyan; Smolke, Christina D

    2016-11-01

    Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270μg/L, 61μg/L, and 3700μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.

  7. Novel de novo mutations in ZBTB20 in Primrose syndrome with congenital hypothyroidism.

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    Mattioli, Francesca; Piton, Amelie; Gérard, Bénédicte; Superti-Furga, Andrea; Mandel, Jean-Louis; Unger, Sheila

    2016-06-01

    The cardinal features of Primrose syndrome (MIM 259050) are dysmorphic facial features, macrocephaly, and intellectual disability, as well as large body size, height and weight, and calcified pinnae. A variety of neurological signs and symptoms have been reported including hearing loss, autism, behavioral abormalities, hypotonia, cerebral calcifications, and hypoplasia of the corpus callosum. Recently, heterozygous de novo missense mutations in ZBTB20, coding for a zing finger protein, have been identified in Primrose syndrome patients. We report a boy with intellectual disability carrying two de novo missense mutations in the last exon of ZBTB20 (Ser616Phe and Gly741Arg; both previously unreported). One of them, Ser616Phe, affects an amino acid located in one of the C2H2 zing-fingers involved in DNA-binding and close to other missense mutations already described. Reverse phenotyping showed that this patient presents with classic features of Primrose syndrome (dysmorphic facies, macrocephaly, hearing loss, hypotonia, hypoplasia of the corpus callosum) and, in addition, congenital hypothyroidism. Review of the literature reveals another Primrose syndrome patient with hypothyroidism and thus, this may represent an under recognized component that should be investigated in other patients. © 2016 Wiley Periodicals, Inc.

  8. Identification and characterization of a de novo partial trisomy 10p by comparative genomic hybridization (CGH).

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    Benzacken, B; Lapierre, J M; Siffroi, J P; Chalvon, A; Tachdjian, G

    1998-10-01

    We report the characterization of a de novo unbalanced chromosome rearrangement by comparative genomic hybridization (CGH) in a 15-day-old child with hypotonia and dysmorphia. We describe the combined use of CGH and fluorescence in situ hybridization (FISH) to identify the origin of the additional chromosomal material on the short arm of chromosome 6. Investigation with FISH revealed that the excess material was not derived from chromosome 6. Identification of unknown unbalanced aberrations that could not be identified by traditional cytogenetics procedures is possible by CGH analysis. Visual analysis of digital images from CGH-metaphase spreads revealed a predominantly green signal on the telomeric region of chromosome 10p. After quantitative digital ratio imaging of 10 CGH-metaphase spreads, a region of gain was found in the chromosome band 10p14-pter. The CGH finding was confirmed by FISH analysis, using a whole chromosome 10 paint probe. These results show the usefulness of CGH for a rapid characterization of de novo unbalanced translocation, unidentifiable by karyotype alone.

  9. De novo transcriptome assembly databases for the central nervous system of the medicinal leech

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    Hibsh, Dror; Schori, Hadas; Efroni, Sol; Shefi, Orit

    2015-01-01

    The study of non-model organisms stands to benefit greatly from genetic and genomic data. For a better understanding of the molecular mechanisms driving neuronal development, and to characterize the entire leech Hirudo medicinalis central nervous system (CNS) transcriptome we combined Trinity for de-novo assembly and Illumina HiSeq2000 for RNA-Seq. We present a set of 73,493 de-novo assembled transcripts for the leech, reconstructed from RNA collected, at a single ganglion resolution, from the CNS. This set of transcripts greatly enriches the available data for the leech. Here, we share two databases, such that each dataset allows a different type of search for candidate homologues. The first is the raw set of assembled transcripts. This set allows a sequence-based search. A comprehensive analysis of which revealed 22,604 contigs with high e-values, aligned versus the Swiss-Prot database. This analysis enabled the production of the second database, which includes correlated sequences to annotated transcript names, with the confidence of BLAST best hit. PMID:25977819

  10. De novo generation of defective interfering-like RNAs in broad bean mottle bromovirus.

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    Pogany, J; Romero, J; Huang, Q; Sgro, J Y; Shang, H; Bujarski, J J

    1995-10-01

    Broad been mottle virus (BBMV) is the only member of the bromoviruses that is known to accumulate defective-interfering (DI) RNAs (Romero et al., Virology 194, 576-584, 1993). De novo generation of DI-like RNAs was demonstrated during serial passages of BBMV in broad bean using either DI RNA-free virion RNA preparations or transcribed genomic RNA inocula. As for previously described DI RNAs, all but one of the characterized de novo generated DI-like RNAs were derived by a single in-frame deletion from the RNA2 component. The sole exception was derived by two shorter in-frame deletions from RNA2. The maintenance of an open reading frame by all DI-like RNAs suggests the importance of coding capacity and/or the shortened 2a protein in the accumulation of these RNAs during infection. The deletion junction sites were between nucleotides 1152 and 2366, suggesting that the retained regions are essential for the efficient accumulation of BBMV DI-like RNAs in planta. Short regions of sequence similarity and/or complementarity were revealed at the 5' and 3' junction borders. We speculate that these regions can facilitate DI (DI-like) RNA formation. In addition to DI-like RNAs, the full-length nucleotide sequences of RNA2 components of the Type and Morocco strains of BBMV are presented.

  11. De novo minimal change disease after ABO-incompatible kidney transplantation.

    Science.gov (United States)

    Mochizuki, Yasushi; Iwata, Takahisa; Nishikido, Masaharu; Uramatsu, Tadashi; Sakai, Hideki; Taguchi, Takashi

    2012-07-01

    We report the clinical and pathological findings of a case of de novo minimal change disease (MCD) after ABO-incompatible living kidney transplantation. A 62-yr-old man with end-stage renal disease associated with type I diabetes received ABO-incompatible kidney transplantation from his 58-yr-old wife. Although allograft function was excellent immediately after surgery, massive proteinuria (35 g/d) appeared on post-transplantation day 5. After the allograft biopsy taken on post-transplantation day 6, he was treated with 12 cycles of plasma exchange, but the nephrotic-range proteinuria showed no remission. The biopsy specimen showed no significant pathological findings on light microscopy, but electron microscopy showed diffuse effacement of podocyte foot processes. Based on the diagnosis of de novo MCD, the patient received intravenous methylprednisolone pulse therapy, followed by high-dose steroid maintenance therapy. The steroid therapy induced complete remission of nephrotic syndrome and stable allograft function immediately, which was also maintained at one yr after the transplantation.

  12. Sequencing, de novo assembly and comparative analysis of Raphanus sativus transcriptome.

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    Wu, Gang; Zhang, Libin; Yin, Yongtai; Wu, Jiangsheng; Yu, Longjiang; Zhou, Yanhong; Li, Maoteng

    2015-01-01

    Raphanus sativus is an important Brassicaceae plant and also an edible vegetable with great economic value. However, currently there is not enough transcriptome information of R. sativus tissues, which impedes further functional genomics research on R. sativus. In this study, RNA-seq technology was employed to characterize the transcriptome of leaf tissues. Approximately 70 million clean pair-end reads were obtained and used for de novo assembly by Trinity program, which generated 68,086 unigenes with an average length of 576 bp. All the unigenes were annotated against GO and KEGG databases. In the meanwhile, we merged leaf sequencing data with existing root sequencing data and obtained better de novo assembly of R. sativus using Oases program. Accordingly, potential simple sequence repeats (SSRs), transcription factors (TFs) and enzyme codes were identified in R. sativus. Additionally, we detected a total of 3563 significantly differentially expressed genes (DEGs, P = 0.05) and tissue-specific biological processes between leaf and root tissues. Furthermore, a TFs-based regulation network was constructed using Cytoscape software. Taken together, these results not only provide a comprehensive genomic resource of R. sativus but also shed light on functional genomic and proteomic research on R. sativus in the future.

  13. Nicotinamide phosphoribosyl transferase (Nampt) is required for de novo lipogenesis in tumor cells.

    Science.gov (United States)

    Bowlby, Sarah C; Thomas, Michael J; D'Agostino, Ralph B; Kridel, Steven J

    2012-01-01

    Tumor cells have increased metabolic requirements to maintain rapid growth. In particular, a highly lipogenic phenotype is a hallmark of many tumor types, including prostate. Cancer cells also have increased turnover of nicotinamide adenine dinucleotide (NAD(+)), a coenzyme involved in multiple metabolic pathways. However, a specific role for NAD(+) in tumor cell lipogenesis has yet to be described. Our studies demonstrate a novel role for the NAD(+)-biosynthetic enzyme Nicotinamide phosphoribosyltransferase (Nampt) in maintaining de novo lipogenesis in prostate cancer (PCa) cells. Inhibition of Nampt reduces fatty acid and phospholipid synthesis. In particular, short chain saturated fatty acids and the phosphatidylcholine (PC) lipids into which these fatty acids are incorporated were specifically reduced by Nampt inhibition. Nampt blockade resulted in reduced ATP levels and concomitant activation of AMP-activated protein kinase (AMPK) and phosphorylation of acetyl-CoA carboxylase (ACC). In spite of this, pharmacological inhibition of AMPK was not sufficient to fully restore fatty acid synthesis. Rather, Nampt blockade also induced protein hyperacetylation in PC-3, DU145, and LNCaP cells, which correlated with the observed decreases in lipid synthesis. Moreover, the sirtuin inhibitor Sirtinol, and the simultaneous knockdown of SIRT1 and SIRT3, phenocopied the effects of Nampt inhibition on fatty acid synthesis. Altogether, these data reveal a novel role for Nampt in the regulation of de novo lipogenesis through the modulation of sirtuin activity in PCa cells.

  14. Nicotinamide phosphoribosyl transferase (Nampt is required for de novo lipogenesis in tumor cells.

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    Sarah C Bowlby

    Full Text Available Tumor cells have increased metabolic requirements to maintain rapid growth. In particular, a highly lipogenic phenotype is a hallmark of many tumor types, including prostate. Cancer cells also have increased turnover of nicotinamide adenine dinucleotide (NAD(+, a coenzyme involved in multiple metabolic pathways. However, a specific role for NAD(+ in tumor cell lipogenesis has yet to be described. Our studies demonstrate a novel role for the NAD(+-biosynthetic enzyme Nicotinamide phosphoribosyltransferase (Nampt in maintaining de novo lipogenesis in prostate cancer (PCa cells. Inhibition of Nampt reduces fatty acid and phospholipid synthesis. In particular, short chain saturated fatty acids and the phosphatidylcholine (PC lipids into which these fatty acids are incorporated were specifically reduced by Nampt inhibition. Nampt blockade resulted in reduced ATP levels and concomitant activation of AMP-activated protein kinase (AMPK and phosphorylation of acetyl-CoA carboxylase (ACC. In spite of this, pharmacological inhibition of AMPK was not sufficient to fully restore fatty acid synthesis. Rather, Nampt blockade also induced protein hyperacetylation in PC-3, DU145, and LNCaP cells, which correlated with the observed decreases in lipid synthesis. Moreover, the sirtuin inhibitor Sirtinol, and the simultaneous knockdown of SIRT1 and SIRT3, phenocopied the effects of Nampt inhibition on fatty acid synthesis. Altogether, these data reveal a novel role for Nampt in the regulation of de novo lipogenesis through the modulation of sirtuin activity in PCa cells.

  15. Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

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    Maher, Geoffrey J; McGowan, Simon J; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O M

    2016-03-01

    De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

  16. Effects of GC bias in next-generation-sequencing data on de novo genome assembly.

    Science.gov (United States)

    Chen, Yen-Chun; Liu, Tsunglin; Yu, Chun-Hui; Chiang, Tzen-Yuh; Hwang, Chi-Chuan

    2013-01-01

    Next-generation-sequencing (NGS) has revolutionized the field of genome assembly because of its much higher data throughput and much lower cost compared with traditional Sanger sequencing. However, NGS poses new computational challenges to de novo genome assembly. Among the challenges, GC bias in NGS data is known to aggravate genome assembly. However, it is not clear to what extent GC bias affects genome assembly in general. In this work, we conduct a systematic analysis on the effects of GC bias on genome assembly. Our analyses reveal that GC bias only lowers assembly completeness when the degree of GC bias is above a threshold. At a strong GC bias, the assembly fragmentation due to GC bias can be explained by the low coverage of reads in the GC-poor or GC-rich regions of a genome. This effect is observed for all the assemblers under study. Increasing the total amount of NGS data thus rescues the assembly fragmentation because of GC bias. However, the amount of data needed for a full rescue depends on the distribution of GC contents. Both low and high coverage depths due to GC bias lower the accuracy of assembly. These pieces of information provide guidance toward a better de novo genome assembly in the presence of GC bias.

  17. De novo transcriptome assembly of two different peach cultivars grown in Korea

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    Yeonhwa Jo

    2015-12-01

    Full Text Available Peach (Prunus persica is one of the most popular stone fruits worldwide. Next generation sequencing (NGS has facilitated genome and transcriptome analyses of several stone fruit trees. In this study, we conducted de novo transcriptome analyses of two peach cultivars grown in Korea. Leaves of two cultivars, referred to as Jangtaek and Mibaek, were harvested and used for library preparation. The two prepared libraries were paired-end sequenced by the HiSeq2000 system. We obtained 8.14 GB and 9.62 GB sequence data from Jangtaek and Mibaek (NCBI accession numbers: SRS1056585 and SRS1056587, respectively. The Trinity program was used to assemble two transcriptomes de novo, resulting in 110,477 (Jangtaek and 136,196 (Mibaek transcripts. TransDecoder identified possible coding regions in assembled transcripts. The identified proteins were subjected to BLASTP search against NCBI's non-redundant database for functional annotation. This study provides transcriptome data for two peach cultivars, which might be useful for genetic marker development and comparative transcriptome analyses.

  18. Long-read sequencing and de novo assembly of a Chinese genome.

    Science.gov (United States)

    Shi, Lingling; Guo, Yunfei; Dong, Chengliang; Huddleston, John; Yang, Hui; Han, Xiaolu; Fu, Aisi; Li, Quan; Li, Na; Gong, Siyi; Lintner, Katherine E; Ding, Qiong; Wang, Zou; Hu, Jiang; Wang, Depeng; Wang, Feng; Wang, Lin; Lyon, Gholson J; Guan, Yongtao; Shen, Yufeng; Evgrafov, Oleg V; Knowles, James A; Thibaud-Nissen, Francoise; Schneider, Valerie; Yu, Chack-Yung; Zhou, Libing; Eichler, Evan E; So, Kwok-Fai; Wang, Kai

    2016-06-30

    Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93 Gb (contig N50: 8.3 Mb, scaffold N50: 22.0 Mb, including 39.3 Mb N-bases), together with 206 Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8 Mb of HX1-specific sequences, including 4.1 Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.

  19. Whole genome amplification and de novo assembly of single bacterial cells.

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    Sébastien Rodrigue

    Full Text Available BACKGROUND: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA and complete genome sequencing of individual cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA, and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. CONCLUSIONS/SIGNIFICANCE: The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.

  20. Arginine Methylation of SREBP1a via PRMT5 Promotes De Novo Lipogenesis and Tumor Growth.

    Science.gov (United States)

    Liu, Liu; Zhao, Xiaoping; Zhao, Li; Li, Jiajin; Yang, Hao; Zhu, Zongping; Liu, Jianjun; Huang, Gang

    2016-03-01

    Dysregulation of the sterol regulatory element-binding transcription factors sterol regulatory element-binding protein (SREBP) and SREBF activates de novo lipogenesis to high levels in cancer cells, a critical event in driving malignant growth. In this study, we identified an important posttranslational mechanism by which SREBP1a is regulated during metabolic reprogramming in cancer cells. Mass spectrometry revealed protein arginine methyltransferase 5 (PRMT5) as a binding partner of SREBP1a that symmetrically dimethylated it on R321, thereby promoting transcriptional activity. Furthermore, PRMT5-induced methylation prevented phosphorylation of SREBP1a on S430 by GSK3β, leading to its disassociation from Fbw7 (FBXW7) and its evasion from degradation through the ubiquitin-proteasome pathway. Consequently, methylation-stabilized SREBP1a increased de novo lipogenesis and accelerated the growth of cancer cells in vivo and in vitro. Clinically, R321 symmetric dimethylation status was associated with malignant progression of human hepatocellular carcinoma, where it served as an independent risk factor of poor prognosis. By showing how PRMT5-induced methylation of SREBP1a triggers hyperactivation of lipid biosynthesis, a key event in tumorigenesis, our findings suggest a new generalized strategy to selectively attack tumor metabolism.

  1. The Role of Adherence and Retreatment in De Novo Emergence of MDR-TB.

    Science.gov (United States)

    Cadosch, Dominique; Abel Zur Wiesch, Pia; Kouyos, Roger; Bonhoeffer, Sebastian

    2016-03-01

    Treatment failure after therapy of pulmonary tuberculosis (TB) infections is an important challenge, especially when it coincides with de novo emergence of multi-drug-resistant TB (MDR-TB). We seek to explore possible causes why MDR-TB has been found to occur much more often in patients with a history of previous treatment. We develop a mathematical model of the replication of Mycobacterium tuberculosis within a patient reflecting the compartments of macrophages, granulomas, and open cavities as well as parameterizing the effects of drugs on the pathogen dynamics in these compartments. We use this model to study the influence of patient adherence to therapy and of common retreatment regimens on treatment outcome. As expected, the simulations show that treatment success increases with increasing adherence. However, treatment occasionally fails even under perfect adherence due to interpatient variability in pharmacological parameters. The risk of generating MDR de novo is highest between 40% and 80% adherence. Importantly, our simulations highlight the double-edged effect of retreatment: On the one hand, the recommended retreatment regimen increases the overall success rate compared to re-treating with the initial regimen. On the other hand, it increases the probability to accumulate more resistant genotypes. We conclude that treatment adherence is a key factor for a positive outcome, and that screening for resistant strains is advisable after treatment failure or relapse.

  2. Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

    Science.gov (United States)

    Jo, Byung Hoon; Park, Tae Yoon; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je; Cha, Hyung Joon

    2016-07-01

    Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

  3. Occurrence and de novo biosynthesis of caffeine and theanine in seedlings of tea (Camellia sinensis).

    Science.gov (United States)

    Deng, Wei-Wei; Ashihara, Hiroshi

    2015-05-01

    Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

  4. A framework for the detection of de novo mutations in family-based sequencing data

    Science.gov (United States)

    Francioli, Laurent C; Cretu-Stancu, Mircea; Garimella, Kiran V; Fromer, Menachem; Kloosterman, Wigard P; Wijmenga, Cisca; Investigator, Principal; Swertz, Morris A; van Duijn, Cornelia M; Boomsma, Dorret I; Slagboom, PEline; van Ommen, Gertjan B; de Bakker, Paul IW; Swertz, Morris A; Francioli, Laurent C; van Dijk, Freerk; Menelaou, Androniki; Neerincx, Pieter BT; Pulit, Sara L; Deelen, Patrick; Elbers, Clara C; Francesco Palamara, Pier; Pe'er, Itsik; Abdellaoui, Abdel; Kloosterman, Wigard P; van Oven, Mannis; Vermaat, Martijn; Li, Mingkun; Laros, Jeroen FJ; Stoneking, Mark; de Knijff, Peter; Kayser, Manfred; Veldink, Jan H; van den Berg, Leonard H; Byelas, Heorhiy; den Dunnen, Johan T; Dijkstra, Martijn; Amin, Najaf; van der Velde, K Joeri; Hottenga, Jouke Jan; van Setten, Jessica; van Leeuwen, Elisabeth M; Kanterakis, Alexandros; Kattenberg, Mathijs; Karssen, Lennart C; van Schaik, Barbera DC; Bot, Jan; Nijman, Isaäc J; Renkens, Ivo; van Enckevort, David; Mei, Hailiang; Koval, Vyacheslav; Estrada, Karol; Medina-Gomez, Carolina; Ye, Kai; Lameijer, Eric-Wubbo; Moed, Matthijs H; Hehir-Kwa, Jayne Y; Handsaker, Robert E; McCarroll, Steven A; Sunyaev, Shamil R; Polak, Paz; Vuzman, Dana; Sohail, Mashaal; Hormozdiari, Fereydoun; Marschall, Tobias; Schönhuth, Alexander; Guryev, Victor; de Bakker, Paul IW; Slagboom, P Eline; Beekman, Marian B; de Craen, Anton JM; Suchiman, H Eka D; Hofman, Albert; van Duijn, Cornelia M; Oostra, Ben; Isaacs, Aaron; Amin, Najaf; Rivadeneira, Fernando; Uitterlinden, André G; Boomsma, Dorret I; Willemsen, Gonneke; Platteel, Mathieu; Pitts, Steven J; Potluri, Shobha; Sundar, Purnima; Cox, David R; Li, Qibin; Li, Yingrui; Du, Yuanping; Chen, Ruoyan; Cao, Hongzhi; Li, Ning; Cao, Sujie; Wang, Jun; Bovenberg, Jasper A; Brandsma, Margreet; Samocha, Kaitlin E; Neale, Benjamin M; Daly, Mark J; Banks, Eric; DePristo, Mark A; de Bakker, Paul IW

    2017-01-01

    Germline mutation detection from human DNA sequence data is challenging due to the rarity of such events relative to the intrinsic error rates of sequencing technologies and the uneven coverage across the genome. We developed PhaseByTransmission (PBT) to identify de novo single nucleotide variants and short insertions and deletions (indels) from sequence data collected in parent-offspring trios. We compute the joint probability of the data given the genotype likelihoods in the individual family members, the known familial relationships and a prior probability for the mutation rate. Candidate de novo mutations (DNMs) are reported along with their posterior probability, providing a systematic way to prioritize them for validation. Our tool is integrated in the Genome Analysis Toolkit and can be used together with the ReadBackedPhasing module to infer the parental origin of DNMs based on phase-informative reads. Using simulated data, we show that PBT outperforms existing tools, especially in low coverage data and on the X chromosome. We further show that PBT displays high validation rates on empirical parent-offspring sequencing data for whole-exome data from 104 trios and X-chromosome data from 249 parent-offspring families. Finally, we demonstrate an association between father's age at conception and the number of DNMs in female offspring's X chromosome, consistent with previous literature reports. PMID:27876817

  5. Exome analysis of Smith-Magenis-like syndrome cohort identifies de novo likely pathogenic variants.

    Science.gov (United States)

    Berger, Seth I; Ciccone, Carla; Simon, Karen L; Malicdan, May Christine; Vilboux, Thierry; Billington, Charles; Fischer, Roxanne; Introne, Wendy J; Gropman, Andrea; Blancato, Jan K; Mullikin, James C; Gahl, William A; Huizing, Marjan; Smith, Ann C M

    2017-02-17

    Smith-Magenis syndrome (SMS), a neurodevelopmental disorder characterized by dysmorphic features, intellectual disability (ID), and sleep disturbances, results from a 17p11.2 microdeletion or a mutation in the RAI1 gene. We performed exome sequencing on 6 patients with SMS-like phenotypes but without chromosomal abnormalities or RAI1 variants. We identified pathogenic de novo variants in two cases, a nonsense variant in IQSEC2 and a missense variant in the SAND domain of DEAF1, and candidate de novo missense variants in an additional two cases. One candidate variant was located in an alpha helix of Necdin (NDN), phased to the paternally inherited allele. NDN is maternally imprinted within the 15q11.2 Prader-Willi Syndrome (PWS) region. This can help clarify NDN's role in the PWS phenotype. No definitive pathogenic gene variants were detected in the remaining SMS-like cases, but we report our findings for future comparison. This study provides information about the inheritance pattern and recurrence risk for patients with identified variants and demonstrates clinical and genetic overlap of neurodevelopmental disorders. Identification and characterization of ID-related genes that assist in development of common developmental pathways and/or gene-networks, may inform disease mechanism and treatment strategies.

  6. Distinguishing between Selective Sweeps from Standing Variation and from a De Novo Mutation

    DEFF Research Database (Denmark)

    Peter, Benjamin M.; Huerta-Sanchez, Emilia; Nielsen, Rasmus

    2012-01-01

    An outstanding question in human genetics has been the degree to which adaptation occurs from standing genetic variation or from de novo mutations. Here, we combine several common statistics used to detect selection in an Approximate Bayesian Computation (ABC) framework, with the goal of discrimi......An outstanding question in human genetics has been the degree to which adaptation occurs from standing genetic variation or from de novo mutations. Here, we combine several common statistics used to detect selection in an Approximate Bayesian Computation (ABC) framework, with the goal...... of discriminating between models of selection and providing estimates of the age of selected alleles and the selection coefficients acting on them. We use simulations to assess the power and accuracy of our method and apply it to seven of the strongest sweeps currently known in humans. We identify two genes, ASPM...... and PSCA, that are most likely affected by selection on standing variation; and we find three genes, ADH1B, LCT, and EDAR, in which the adaptive alleles seem to have swept from a new mutation. We also confirm evidence of selection for one further gene, TRPV6. In one gene, G6PD, neither neutral models nor...

  7. De Novo Proteins with Life-Sustaining Functions Are Structurally Dynamic.

    Science.gov (United States)

    Murphy, Grant S; Greisman, Jack B; Hecht, Michael H

    2016-01-29

    Designing and producing novel proteins that fold into stable structures and provide essential biological functions are key goals in synthetic biology. In initial steps toward achieving these goals, we constructed a combinatorial library of de novo proteins designed to fold into 4-helix bundles. As described previously, screening this library for sequences that function in vivo to rescue conditionally lethal mutants of Escherichia coli (auxotrophs) yielded several de novo sequences, termed SynRescue proteins, which rescued four different E. coli auxotrophs. In an effort to understand the structural requirements necessary for auxotroph rescue, we investigated the biophysical properties of the SynRescue proteins, using both computational and experimental approaches. Results from circular dichroism, size-exclusion chromatography, and NMR demonstrate that the SynRescue proteins are α-helical and relatively stable. Surprisingly, however, they do not form well-ordered structures. Instead, they form dynamic structures that fluctuate between monomeric and dimeric states. These findings show that a well-ordered structure is not a prerequisite for life-sustaining functions, and suggests that dynamic structures may have been important in the early evolution of protein function.

  8. Comprehensive comparison of three different immunosuppressive regimens for liver transplant patients with hepatocellular carcinoma: steroid-free immunosuppression, induction immunosuppression and standard immunosuppression.

    Directory of Open Access Journals (Sweden)

    Yuan-Yuan Liu

    Full Text Available The different choices of immunosuppression (IS regimens influenced the outcomes of liver transplantation. Steroid was applied as a standard IS to prevent and treat rejections. However, steroid-related complications were increasingly prominent. This study compared the efficacy and safety of standard IS regimens with the efficacy and safety of steroid-free IS regimen and induction IS regimen in Chinese liver transplantation recipients for hepatocellular carcinoma (HCC. A total of 329 patients who underwent liver transplantation from January 2008 to December 2012 were retrospectively reviewed. Three different groups of patients received standard triple-drug IS regimen of steroid, tacrolimus (TAC and mycophenolate mofetil (MMF (triple-drug regimen group; n=57, induction-contained IS regimen of basiliximab, steroid, TAC and MMF (BS group; n=241, and induction-contained and steroid-free regimen of basiliximab, TAC and MMF (SF group; n=31, respectively. There were no significant differences in terms of patient, tumor-free and graft survival rates. The acute rejection rate and rejection time were equivalent in different groups. But compared with BS group, higher incidences of biliary complications (11.52% vs. 30.77%, p=0.013 and graft dysfunction (0.48% vs. 13.64%, p=0.003 were observed in SF group. Furthermore, compared with the two groups, incidence of pleural effusion was also higher in SF group (15.79%, 11.96% vs. 45.45%, respectively, both p<0.01. And a trend towards less proportion of De novo diabetes was revealed in SF group. Although it was found that patient, tumor-free and graft survival rates were equivalent among three IS regimens, higher incidences of complications were demonstrated in steroid-free regimen in patients for HCC. These findings suggested that steroid-free IS regimen has no clear advantages in comparison with standard IS regimens for liver transplant recipients with HCC and the postoperative complications should be treated with

  9. Teratogenesis and immunosuppressive treatment.

    Science.gov (United States)

    Danesi, R; Del Tacca, M

    2004-04-01

    Despite the potential risks to the mother and fetus caused by immunosuppressive drugs, uneventful pregnancies are now frequent among transplant recipients. Although there is no apparent increase in the type or incidence of malformations in the newborns or evidence of graft dysfunction, pregnancy-related complications, including premature termination and low birth weight, may be more frequent. To prevent graft rejection due to the increased immunologic reactivity of the transplant recipient during pregnancy, it is reasonable to wait 2 years after transplantation before conception, to have stable graft function and to be on low drug doses for maintenance immunosuppression. Among the immunosuppressive agents, corticosteroids may induce a number of treatment-related complications, including diabetes and osteoporosis; however, the incidence of fetal malformations during corticosteroid treatment is about 3.5%, a value close to that of the general population. Among immunosuppressive antibodies, no evidence of developmental toxicity has been demonstrated with basiliximab. On the contrary, some concerns have been raised about azathioprine, since its use has been associated with fetal abnormalities in animals; however, clinical data so far have indicated only a small teratogenic risk. Therefore, immunosuppressive therapy with selected drugs and antibodies does not apparently increase the risk of birth defects and may be continued in pregnancy. Finally, although breast-feeding is not recommended, because of drug transfer into maternal milk, the available clinical data do not support this limitation because of the low amount of drug absorbed by the infant and the absence of clinical toxicity in published case reports.

  10. De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes.

    Science.gov (United States)

    Kitchen, Sheila A; Crowder, Camerron M; Poole, Angela Z; Weis, Virginia M; Meyer, Eli

    2015-09-17

    Many nonmodel species exemplify important biological questions but lack the sequence resources required to study the genes and genomic regions underlying traits of interest. Reef-building corals are famously sensitive to rising seawater temperatures, motivating ongoing research into their stress responses and long-term prospects in a changing climate. A comprehensive understanding of these processes will require extending beyond the sequenced coral genome (Acropora digitifera) to encompass diverse coral species and related anthozoans. Toward that end, we have assembled and annotated reference transcriptomes to develop catalogs of gene sequences for three scleractinian corals (Fungia scutaria, Montastraea cavernosa, Seriatopora hystrix) and a temperate anemone (Anthopleura elegantissima). High-throughput sequencing of cDNA libraries produced ~20-30 million reads per sample, and de novo assembly of these reads produced ~75,000-110,000 transcripts from each sample with size distributions (mean ~1.4 kb, N50 ~2 kb), comparable to the distribution of gene models from the coral genome (mean ~1.7 kb, N50 ~2.2 kb). Each assembly includes matches for more than half the gene models from A. digitifera (54-67%) and many reasonably complete transcripts (~5300-6700) spanning nearly the entire gene (ortholog hit ratios ≥0.75). The catalogs of gene sequences developed in this study made it possible to identify hundreds to thousands of orthologs across diverse scleractinian species and related taxa. We used these sequences for phylogenetic inference, recovering known relationships and demonstrating superior performance over phylogenetic trees constructed using single mitochondrial loci. The resources developed in this study provide gene sequences and genetic markers for several anthozoan species. To enhance the utility of these resources for the research community, we developed searchable databases enabling researchers to rapidly recover sequences for genes of interest. Our

  11. De Novo Generation of a Unique Cervid Prion Strain Using Protein Misfolding Cyclic Amplification

    Science.gov (United States)

    Meyerett-Reid, Crystal; Wyckoff, A. Christy; Spraker, Terry; Pulford, Bruce; Bender, Heather

    2017-01-01

    ABSTRACT Substantial evidence supports the hypothesis that prions are misfolded, infectious, insoluble, and protease-resistant proteins (PrPRES) devoid of instructional nucleic acid that cause transmissible spongiform encephalopathies (TSEs). Protein misfolding cyclic amplification (PMCA) has provided additional evidence that PrPRes acts as a template that can convert the normal cellular prion protein (PrPC) present in uninfected normal brain homogenate (NBH) into the infectious misfolded PrPRES isoform. Human PrPC has been shown to spontaneously convert to a misfolded pathological state causing sporadic Creutzfeldt-Jakob disease (sCJD). Several investigators have reported spontaneous generation of prions by in vitro assays, including PMCA. Here we tested the rate of de novo generation of cervid prions in our laboratory using our standard PMCA protocol and NBH from transgenic mice expressing cervid PrPC (TgCerPrP mice). We generated de novo prions in rounds 4, 5, and 7 at low cumulative rates of 1.6, 5.0, and 6.7%, respectively. The prions caused infectious chronic wasting disease (CWD) upon inoculation into normal uninfected TgCerPrP mice and displayed unique biochemical characteristics compared to other cervid prion strains. We conclude that PMCA of cervid PrPC from normal brain homogenate spontaneously generated a new cervid prion strain. These data support the potential for cervids to develop sporadic CWD. IMPORTANCE CWD is the only known TSE that affects free-ranging wildlife, specifically cervids such as elk, deer, moose, caribou, and reindeer. CWD has become endemic in both free-ranging and captive herds in North America, South Korea, and, most recently, northern Europe. The prion research community continues to debate the origins of CWD. Original foci of CWD emergence in Colorado and Wyoming coincident with the sheep TSE scrapie suggest that scrapie prions may have adapted to cervids to cause CWD. However, emerging evidence supports the idea that cervid Pr

  12. [Traveling with immunosuppression].

    Science.gov (United States)

    Birkenfeld, G

    2014-03-01

    The rapidly increasing number of patients with immunosuppression is followed by their expectation to lead-as much as possible-a "normal" life, including long-distance travel. The advice and preventive measures for diseases associated with travelling depend overall on the mode of the patient's immunosuppression. This report explains the individual preventive possibilities, limits and risks for travellers with asplenia, common variable immunodeficiency, chronic inflammatory bowel and rheumatic diseases, HIV, as well as for patients having undergone solid organ or bone marrow transplantation or chemotherapy.

  13. Highly efficient de novo mutant identification in a sorghum bicolor tilling population using the ComSeq approach

    Science.gov (United States)

    Screening large populations for carriers of known or de novo rare SNPs is required both in Targeting induced local lesions IN genomes (TILLING) experiments in plants and analogously in screening human populations. We formerly suggested an approach that combines the celebrated mathematical field of c...

  14. Mandibulofacial Dysostosis in a Patient with a de novo 2;17 Translocation that Disrupts the HOXD Gene Cluster

    OpenAIRE

    2007-01-01

    Treacher Collins syndrome is the prototypical mandibulofacial dysostosis syndrome, but other mandibulofacial dysostosis syndromes have been described. We report an infant with mandibulofacial dysostosis and an apparently balanced de novo 2;17 translocation. She presented with severe lower eyelid colobomas requiring skin grafting, malar and mandibular hypoplasia, bilateral microtia with external auditory canal atreasia, dysplastic ossicles, hearing loss, bilateral choanal stenosis, cleft palat...

  15. Environmental exposure of the mouse germ line: DNA adducts in spermatozoa and formation of de novo mutations during spermatogenesis.

    Directory of Open Access Journals (Sweden)

    Ann-Karin Olsen

    Full Text Available BACKGROUND: Spermatozoal DNA damage is associated with poor sperm quality, disturbed embryonic development and early embryonic loss, and some genetic diseases originate from paternal de novo mutations. We previously reported poor repair of bulky DNA-lesions in rodent testicular cells. METHODOLOGY/PRINCIPAL FINDINGS: We studied the fate of DNA lesions in the male germ line. B[a]PDE-N(2-dG adducts were determined by liquid chromatography-tandem mass spectrometry, and de novo mutations were measured in the cII-transgene, in Big Blue mice exposed to benzo[a]pyrene (B[a]P; 3 x 50 mg/kg bw, i.p.. Spermatozoa were harvested at various time-points following exposure, to study the consequences of exposure during the different stages of spermatogenesis. B[a]PDE-N(2-dG adducts induced by exposure of spermatocytes or later stages of spermatogenesis persisted at high levels in the resulting spermatozoa. Spermatozoa originating from exposed spermatogonia did not contain DNA adducts; however de novo mutations had been induced (p = 0.029, specifically GC-TA transversions, characteristic of B[a]P mutagenesis. Moreover, a specific spectrum of spontaneous mutations was consistently observed in spermatozoa. CONCLUSIONS/SIGNIFICANCE: A temporal pattern of genotoxic consequences following exposure was identified, with an initial increase in DNA adduct levels in spermatozoa, believed to influence fertility, followed by induction of germ line de novo mutations with possible consequences for the offspring.

  16. Interstitial deletion 1p as a result of a de novo reciprocal 1p;2p translocation

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Jensen, P H

    1985-01-01

    A 5-month-old female patient with psychomotor retardation and minor dysmorphisms is described. Cytogenetic analysis using high-resolution banding technique revealed an interstitial deletion of the short arm of one chromosome 1 (p21----p22.2) resulting from a de novo translocation t(1;2)(p22;p25)....

  17. A high-throughput de novo sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Banfield Jillian F

    2010-03-01

    Full Text Available Abstract Background High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate de novo sequencing for identification of post-translational modifications and amino acid polymorphisms. Results In this study, a new de novo sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of Rhodopseudomonas palustris. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of de novo sequenced spectra and the sequencing accuracy. Conclusions Here, we improved de novo sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at http://compbio.ornl.gov/Vonode.

  18. Fatty acids in the de novo lipogenesis pathway and risk of coronary heart disease: the Cardiovascular Health Study123

    OpenAIRE

    Wu, Jason HY; Lemaitre, Rozenn N.; Imamura, Fumiaki; King, Irena B.; Song, Xiaoling; Spiegelman, Donna; Siscovick, David S.; Mozaffarian, Dariush

    2011-01-01

    Background: De novo lipogenesis (DNL) is an endogenous pathway whereby carbohydrates and proteins are converted to fatty acids. DNL could affect coronary heart disease (CHD) or sudden cardiac arrest (SCA) via generation of specific fatty acids. Whether these fatty acids are prospectively associated with SCA or other CHD events is unknown.

  19. Title A de novo synthesis citrate transporter VuMATE confers aluminum resistance in rice bean (vigna umbellata)

    Science.gov (United States)

    Al-activated organic acid anion efflux from roots is an important Al resistance mechanism in plants. We have conducted the homologous cloning and isolation of VuMATE (Vigna umbellata multidrug and toxic compound extrusion), a gene encoding a de novo citrate transporter from rice bean. Al treatment u...

  20. The role of superficial femoral artery endoluminal bypass in long de novo lesions and in-stent restenosis.

    NARCIS (Netherlands)

    Doomernik, D.E.; Golchehr, B.; Lensvelt, M.M.A.; Reijnen, M.M.P.J.

    2012-01-01

    AIM: Results of endovascular treatment for long de novo lesions of the superficial femoral artery (SFA) are limited by in-stent restenosis (ISR). Polytetrafluoroethylene (PTFE) covered stents are developed to reduce the incidence of ISR. This study was conducted to summarize available data on the us

  1. Identification and functional characterization of de novo FOXP1 variants provides novel insights into the etiology of neurodevelopmental disorder

    NARCIS (Netherlands)

    Sollis, E; Graham, S.A.; Vino, A.; Froehlich, H.; Vreeburg, M.; Dimitropoulou, D.; Gilissen, C.; Pfundt, R.; Rappold, G.A.; Brunner, H.G; Deriziotis, P.; Fisher, S.E.

    2016-01-01

    De novo disruptions of the neural transcription factor FOXP1 are a recently discovered, rare cause of sporadic intellectual disability (ID). We report three new cases of FOXP1-related disorder identified through clinical whole-exome sequencing. Detailed phenotypic assessment confirmed that global de

  2. Prognostic impact of concurrent MYC and BCL6 rearrangements and expression in de novo diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Ye, Qing; Xu-Monette, Zijun Y; Tzankov, Alexandar;

    2016-01-01

    and BCL6 in 898 patients with de novo diffuse large B-cell lymphoma treated with standard chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab). Neither BCL6 translocation alone (more frequent in activated B-cell like diffuse large B-cell lymphoma) nor in combination...

  3. Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

    DEFF Research Database (Denmark)

    Li, Yingrui; Zheng, Hancheng; Luo, Ruibang;

    2011-01-01

    Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise...

  4. Optic atrophy, cataracts, lipodystrophy/lipoatrophy, and peripheral neuropathy caused by a de novo OPA3 mutation

    Science.gov (United States)

    Bourne, Stephanie C.; Townsend, Katelin N.; Shyr, Casper; Matthews, Allison; Lear, Scott A.; Attariwala, Raj; Lehman, Anna; Wasserman, Wyeth W.; van Karnebeek, Clara; Sinclair, Graham; Vallance, Hilary; Gibson, William T.

    2017-01-01

    We describe a woman who presented with cataracts, optic atrophy, lipodystrophy/lipoatrophy, and peripheral neuropathy. Exome sequencing identified a c.235C > G p.(Leu79Val) variant in the optic atrophy 3 (OPA3) gene that was confirmed to be de novo. This report expands the severity of the phenotypic spectrum of autosomal dominant OPA3 mutations. PMID:28050599

  5. Beroepsdifferentiatie in de tandheelkunde 19 : Het effect van parodontale behandeling op de mate van de novo plaquevorming

    NARCIS (Netherlands)

    Dahan, M; Timmerman, M F; van Winkelhoff, A J; van der Velden, U

    2008-01-01

    In order to determine whether the quantity of bacteria in saliva and the degree of periodontal infection influence the speed of de novo plaque formation in periodontitis patients, 23 patients rinsed with 10 ml sterile saline. All teeth were then supragingivally and professionally cleaned, after whic

  6. Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

    Directory of Open Access Journals (Sweden)

    Madlung Johannes

    2010-05-01

    Full Text Available Abstract Background Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i. Results The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set. All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase. Conclusions Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

  7. De novo design of protein-protein interactions through modification of inter-molecular helix-helix interface residues.

    Science.gov (United States)

    Yagi, Sota; Akanuma, Satoshi; Yamagishi, Manami; Uchida, Tatsuya; Yamagishi, Akihiko

    2016-05-01

    For de novo design of protein-protein interactions (PPIs), information on the shape and chemical complementarity of their interfaces is generally required. Recent advances in computational PPI design have allowed for de novo design of protein complexes, and several successful examples have been reported. In addition, a simple and easy-to-use approach has also been reported that arranges leucines on a solvent-accessible region of an α-helix and places charged residues around the leucine patch to induce interactions between the two helical peptides. For this study, we adopted this approach to de novo design a new PPI between the helical bundle proteins sulerythrin and LARFH. A non-polar patch was created on an α-helix of LARFH around which arginine residues were introduced to retain its solubility. The strongest interaction found was for the LARFH variant cysLARFH-IV-3L3R and the sulerythrin mutant 6L6D (KD=0.16 μM). This artificial protein complex is maintained by hydrophobic and ionic interactions formed by the inter-molecular helical bundle structure. Therefore, by the simple and easy-to-use approach to create de novo interfaces on the α-helices, we successfully generated an artificial PPI. We also created a second LARFH variant with the non-polar patch surrounded by positively charged residues at each end. Upon mixing this LARFH variant with 6L6D, mesh-like fibrous nanostructures were observed by atomic force microscopy. Our method may, therefore, also be applicable to the de novo design of protein nanostructures.

  8. Organ-Specific Alterations in Fatty Acid De Novo Synthesis and Desaturation in a Rat Model of Programmed Obesity

    Directory of Open Access Journals (Sweden)

    Desai Mina

    2011-05-01

    Full Text Available Abstract Background Small for gestational age (SGA leads to increased risk of adult obesity and metabolic syndrome. Offspring exposed to 50% maternal food restriction in utero are born smaller than Controls (FR, catch-up in growth by the end of the nursing period, and become obese adults. The objective of the study was to determine stearoyl-CoA desaturase activity (SCD1 and rates of de novo fatty acid synthesis in young FR and Control offspring tissues at the end of the nursing period, as possible contributors to catch-up growth. Methods From gestational day 10 to term, dams fed ad libitum (Control or were 50% food-restricted to produce small FR pups. Control dams nursed all pups. At postnatal day 1 (p1 and p21, offspring body tissues were analyzed by GC/MS, and desaturation indices of palmitoleate/palmitate and oleate/stearate were calculated. SCD1 gene expression was determined by real-time PCR on adipose and liver. Offspring were enriched with deuterium that was given to dams in drinking water during lactation and de novo synthesis of offspring body tissues was determined at p21. Primary adipocyte cell cultures were established at p21 and exposed to U13C-glucose. Results FR offspring exhibited higher desaturation index in p1 and p21 adipose tissue, but decreased desaturation index in liver at p21. SCD1 gene expression at p21 was correspondingly increased in adipose and decreased in liver. FR subcutaneous fat demonstrated increased de novo synthesis at p21. Primary cell cultures exhibited increased de novo synthesis in FR. Conclusions Adipose tissue is the first site to exhibit increased de novo synthesis and desaturase activity in FR. Therefore, abnormal lipogenesis is already present prior to onset of obesity during the period of catch-up growth. These abnormalities may contribute to future obesity development.

  9. Impacts of soil moisture on de-novo monoterpene emissions from European beech, Holm oak, Scots pine, and Norway spruce

    Directory of Open Access Journals (Sweden)

    C. Wu

    2014-09-01

    Full Text Available Impacts of soil moisture on de-novo monoterpene (MT emissions from Holm oak, European beech, Scots pine, and Norway spruce were studied in laboratory experiments. The volumetric water content of the soil, Θ, was used as reference quantity to parameterize the dependency of MT emissions on soil moisture and to characterize the severity of the drought. When Θ dropped from 0.4 m3 m−3 to ~ 0.2 m3 m−3 slight increases of de-novo MT emissions were observed but with further progressing drought the emissions decreased to almost zero. The increases of MT emissions observed under conditions of mild drought were explainable by increases of leaf temperature due to lowered transpirational cooling. When Θ fell below certain thresholds, MT emissions decreased simultaneously with Θ and the relationship between Θ and MT emissions was approximately linear. The thresholds of Θ (0.044–0.19 m3 m−3 were determined as well other parameters required to describe the soil moisture dependence of de-novo MT emissions for application in the Model of Emissions of Gases and Aerosols from Nature, MEGAN. A factorial approach was found appropriate to describe the impacts of Θ, temperature, and light. Temperature and Θ influenced the emissions largely independent from each other, and, in a similar manner, light intensity and Θ acted independently on de-novo MT emissions. The use of Θ as reference quantity in a factorial approach was tenable in predicting constitutive de-novo MT emissions when Θ changed on a time scale of days. Only when soil moisture changed suddenly empirical parameterization with Θ as a reference was unsuccessful.

  10. Wound complications and surgical events in de novo heart transplant patients treated with everolimus

    DEFF Research Database (Denmark)

    Rashidi, Mitra; Esmaily, Sorosh; Fiane, Arnt E;

    2016-01-01

    .5 years (median) was an overall risk factor for surgical wound complications regardless of treatment group (p=0.025). There was no difference in the EVE versus CyA group with regards to other surgical events. Majority of events were in 1/3 of the patients. CONCLUSION: De novo initiation of EVE and early...... CyA withdrawal in HTx patients did not show any significant differences in wound complications or in total surgical events. Majority of complications were seen in a small number of patients....... (CNI) avoidance (SCHEDULE). The aim of the study was to compare wound complications between EVE and CNI based regimen. MATERIALS AND METHODS: A total of 115 patients (mean age 51 ± 13 years, 73% men) were randomized within five days post-HTx to low dose EVE and reduced dose Cyclosporine (CyA) followed...

  11. De novo design of caseinolytic protein proteases inhibitors based on pharmacophore and 2D molecular fingerprints.

    Science.gov (United States)

    Wu, Guanzhong; Zhang, Zhen; Chen, Hong; Lin, Kejiang

    2015-06-01

    Caseinolytic protein proteases (ClpP) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Inhibitors of ClpP can significantly attenuate the capability to produce virulence factors of the bacteria. In this work, we developed a workflow to expand the chemical space of potential ClpP inhibitors based on a set of β-lactones. In our workflow, an artificial pharmacophore model was generated based on HipHop and HYPOGEN method. A de novo compound library based on molecular fingerprints was constructed and virtually screened by the pharmacophore model. The results were further investigated by molecular docking study. The workflow successfully achieved potential ClpP inhibitors. It could be applied to design more novel potential ClpP inhibitors and provide theoretical basis for the further optimization of the hit compounds.

  12. Transcriptional regulator-mediated activation of adaptation genes triggers CRISPR de novo spacer acquisition

    DEFF Research Database (Denmark)

    Liu, Tao; Li, Yingjun; Wang, Xiaodi

    2015-01-01

    Acquisition of de novo spacer sequences confers CRISPR-Cas with a memory to defend against invading genetic elements. However, the mechanism of regulation of CRISPR spacer acquisition remains unknown. Here we examine the transcriptional regulation of the conserved spacer acquisition genes in Type I......, it was demonstrated that the transcription level of csa1, cas1, cas2 and cas4 was significantly enhanced in a csa3a-overexpression strain and, moreover, the Csa1 and Cas1 protein levels were increased in this strain. Furthermore, we demonstrated the hyperactive uptake of unique spacers within both CRISPR loci...... in the presence of the csa3a overexpression vector. The spacer acquisition process is dependent on the CCN PAM sequence and protospacer selection is random and non-directional. These results suggested a regulation mechanism of CRISPR spacer acquisition where a single transcriptional regulator senses the presence...

  13. Nonequilibrium description of de novo biogenesis and transport through Golgi-like cisternae

    Science.gov (United States)

    Sachdeva, Himani; Barma, Mustansir; Rao, Madan

    2016-12-01

    A central issue in cell biology is the physico-chemical basis of organelle biogenesis in intracellular trafficking pathways, its most impressive manifestation being the biogenesis of Golgi cisternae. At a basic level, such morphologically and chemically distinct compartments should arise from an interplay between the molecular transport and chemical maturation. Here, we formulate analytically tractable, minimalist models, that incorporate this interplay between transport and chemical progression in physical space, and explore the conditions for de novo biogenesis of distinct cisternae. We propose new quantitative measures that can discriminate between the various models of transport in a qualitative manner–this includes measures of the dynamics in steady state and the dynamical response to perturbations of the kind amenable to live-cell imaging.

  14. De novo calcification of liver and nodal metastases in prostate carcinoma.

    Science.gov (United States)

    Ghosh, P; Santosa, A C; Lin, G Y; Downs, T M

    2006-01-01

    Prostate cancer has a distinctly recognized pattern of metastases: multifocal and osteoblastic lesions involving the axial skeleton and non-calcified lymph nodes in the pelvic and lumbar aortic groups. Most adenocarcinomas are capable of producing macrocalcification. We report a case of prostate cancer with de novo calcified metastases to the liver and retroperitoneal lymph nodes mimicking the pattern usually seen in mucin-producing adenocarcinomas arising from the gastrointestinal tract. To our knowledge, this is the first such case to be reported in the literature. We propose a multifactorial mechanism that supports dystrophic calcification in this case. The knowledge of atypical presentation of metastatic disease can prevent diagnostic delay and prompt initiation of therapy.

  15. De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy

    DEFF Research Database (Denmark)

    Syrbe, Steffen; Hedrich, Ulrike B S; Riesch, Erik;

    2015-01-01

    Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium...... channel KV1.2, in six isolated patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual...... disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype...

  16. De novo PMP2 mutations in families with type 1 Charcot-Marie-Tooth disease.

    Science.gov (United States)

    Motley, William W; Palaima, Paulius; Yum, Sabrina W; Gonzalez, Michael A; Tao, Feifei; Wanschitz, Julia V; Strickland, Alleene V; Löscher, Wolfgang N; De Vriendt, Els; Koppi, Stefan; Medne, Livija; Janecke, Andreas R; Jordanova, Albena; Zuchner, Stephan; Scherer, Steven S

    2016-06-01

    We performed whole exome sequencing on a patient with Charcot-Marie-Tooth disease type 1 and identified a de novo mutation in PMP2, the gene that encodes the myelin P2 protein. This mutation (p.Ile52Thr) was passed from the proband to his one affected son, and segregates with clinical and electrophysiological evidence of demyelinating neuropathy. We then screened a cohort of 136 European probands with uncharacterized genetic cause of Charcot-Marie-Tooth disease and identified another family with Charcot-Marie-Tooth disease type 1 that has a mutation affecting an adjacent amino acid (p.Thr51Pro), which segregates with disease. Our genetic and clinical findings in these kindred demonstrate that dominant PMP2 mutations cause Charcot-Marie-Tooth disease type 1.

  17. A Truncating De Novo Point Mutation in a Young Infant with Severe Menkes Disease.

    Science.gov (United States)

    Lin, Yi-Jie; Ho, Che-Sheng; Hsu, Chyong-Hsin; Lin, Ju-Li; Chuang, Chih-Kuang; Tsai, Jen-Daw; Chiu, Nan-Chang; Lin, Hsiang-Yu; Lin, Shuan-Pei

    2017-02-01

    Menkes disease is a rare neurodegenerative disorder caused by mutations in ATP7A gene. Deficiency in copper-dependent enzymes results in the unique kinky hair appearance, neurodegeneration, developmental delay, seizures, failure to thrive and other connective tissue or organ abnormalities. Other than biochemical tests, DNA-based diagnosis is now playing an important role. More than two hundred mutations in ATP7A gene were identified. Early copper supplementation can help improve neurological symptoms, but not non-neurological problems. Further molecular studies are needed to identify additional mutation types and to understand the mechanism of pathogenesis. This may help in discovering the possible treatment measures to cure the disease. We present a case with the clinical features and biochemical findings, abnormal brain magnetic resonance imaging as well as the effects of treatment with copper-histidine. Direct sequencing of ATP7A gene revealed a de novo point mutation which resulted in an early stop codon with truncated protein.

  18. Anterior Abdominal Wall Leiomyoma Arising De Novo in a Perimenopausal Woman

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    Hamed A. Al-Wadaani

    2012-07-01

    Full Text Available Extrauterine or extraintestinal leiomyomas are extremely uncommon especially in the pre-peritoneal area or within the anterior abdominal wall muscles. These tumors have been ascribed to intraoperative seeding during resection of a fibroid or a leiomyoma of gut, to exogenous hormone replacement therapy or a major derangement of glucose and/or lipid metabolism. So far, there is no published report of de novo origin of anterior abdominal wall pure leiomyoma in the literature. The author herein reports a case of perimenopausal multiparous woman without any listing of previous gynecological surgery or hormone therapy who presented with a large pre-peritoneal intramuscular leiomyoma of the anterior abdominal wall. The patient underwent complete primary resection with amelioration of her symptoms.

  19. De novo mutations in HCN1 cause early infantile epileptic encephalopathy

    DEFF Research Database (Denmark)

    Nava, Caroline; Dalle, Carine; Rastetter, Agnès

    2014-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to cationic Ih current in neurons and regulate the excitability of neuronal networks. Studies in rat models have shown that the Hcn1 gene has a key role in epilepsy, but clinical evidence implicating HCN1 mutations...... in human epilepsy is lacking. We carried out exome sequencing for parent-offspring trios with fever-sensitive, intractable epileptic encephalopathy, leading to the discovery of two de novo missense HCN1 mutations. Screening of follow-up cohorts comprising 157 cases in total identified 4 additional amino...... acid substitutions. Patch-clamp recordings of Ih currents in cells expressing wild-type or mutant human HCN1 channels showed that the mutations had striking but divergent effects on homomeric channels. Individuals with mutations had clinical features resembling those of Dravet syndrome with progression...

  20. De Novo Truncating Variants in SON Cause Intellectual Disability, Congenital Malformations, and Failure to Thrive.

    Science.gov (United States)

    Tokita, Mari J; Braxton, Alicia A; Shao, Yunru; Lewis, Andrea M; Vincent, Marie; Küry, Sébastien; Besnard, Thomas; Isidor, Bertrand; Latypova, Xénia; Bézieau, Stéphane; Liu, Pengfei; Motter, Connie S; Melver, Catherine Ward; Robin, Nathaniel H; Infante, Elena M; McGuire, Marianne; El-Gharbawy, Areeg; Littlejohn, Rebecca O; McLean, Scott D; Bi, Weimin; Bacino, Carlos A; Lalani, Seema R; Scott, Daryl A; Eng, Christine M; Yang, Yaping; Schaaf, Christian P; Walkiewicz, Magdalena A

    2016-09-01

    SON is a key component of the spliceosomal complex and a critical mediator of constitutive and alternative splicing. Additionally, SON has been shown to influence cell-cycle progression, genomic integrity, and maintenance of pluripotency in stem cell populations. The clear functional relevance of SON in coordinating essential cellular processes and its presence in diverse human tissues suggests that intact SON might be crucial for normal growth and development. However, the phenotypic effects of deleterious germline variants in SON have not been clearly defined. Herein, we describe seven unrelated individuals with de novo variants in SON and propose that deleterious variants in SON are associated with a severe multisystem disorder characterized by developmental delay, persistent feeding difficulties, and congenital malformations, including brain anomalies.

  1. De Novo generation of molecular structures using optimization to select graphs on a given lattice

    DEFF Research Database (Denmark)

    Bywater, R.P.; Poulsen, Thomas Agersten; Røgen, Peter;

    2004-01-01

    A recurrent problem in organic chemistry is the generation of new molecular structures that conform to some predetermined set of structural constraints that are imposed in an endeavor to build certain required properties into the newly generated structure. An example of this is the pharmacophore...... model, used in medicinal chemistry to guide de novo design or selection of suitable structures from compound databases. We propose here a method that efficiently links up a selected number of required atom positions while at the same time directing the emergent molecular skeleton to avoid forbidden...... positions. The linkage process takes place on a lattice whose unit step length and overall geometry is designed to match typical architectures of organic molecules. We use an optimization method to select from the many different graphs possible. The approach is demonstrated in an example where crystal...

  2. A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.; Xu, C.; Andre, C.

    2011-06-23

    Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

  3. De novo formation of left-right asymmetry by posterior tilt of nodal cilia.

    Directory of Open Access Journals (Sweden)

    Shigenori Nonaka

    2005-08-01

    Full Text Available In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left-right (L-R asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.

  4. Paternal Age Explains a Major Portion of De Novo Germline Mutation Rate Variability in Healthy Individuals

    Science.gov (United States)

    Bourassa, Cynthia V.; Lemieux Perreault, Louis-Philippe; Legault, Marc-André; Barhdadi, Amina; Ambalavanan, Amirthagowri; Brendgen, Mara; Vitaro, Frank; Noreau, Anne; Dionne, Ginette; Tremblay, Richard E.; Dion, Patrick A.; Boivin, Michel; Dubé, Marie-Pierre; Rouleau, Guy A.

    2016-01-01

    De novo mutations (DNM) are an important source of rare variants and are increasingly being linked to the development of many diseases. Recently, the paternal age effect has been the focus of a number of studies that attempt to explain the observation that increasing paternal age increases the risk for a number of diseases. Using disease-free familial quartets we show that there is a strong positive correlation between paternal age and germline DNM in healthy subjects. We also observed that germline CNVs do not follow the same trend, suggesting a different mechanism. Finally, we observed that DNM were not evenly distributed across the genome, which adds support to the existence of DNM hotspots. PMID:27723766

  5. A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors

    Directory of Open Access Journals (Sweden)

    Donald M. Bryant

    2017-01-01

    Full Text Available Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.

  6. De novo mutation of keratin 9 gene in two Taiwanese patients with epidermolytic palmoplantar keratoderma.

    Science.gov (United States)

    Yang, Mei-Hui; Lee, Julia Yu-Yun; Lin, Jeng-Hsien; Chao, Sheau-Chiou

    2003-07-01

    Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant hereditary disorder of keratinization. Recent molecular studies have shown that EPPK is caused by mutations in keratin 9 gene (K9). We report 2 unrelated sporadic cases of EPPK in Taiwanese, confirmed by histopathology and electron microscopy. A de novo mutation with a C to T transition at the first nucleotide of codon 162 in K9 was detected in both patients, but not in their parents. The mutation is expected to result in an arginine to tryptophan substitution (R162W) in the beginning region of the alpha-helical 1A domain of K9. Mutations in this region could disrupt keratin filament assembly, leading to degeneration or cytolysis of keratinocytes. Mutations of this arginine codon (R162W, R162Q) are common in pedigrees with EPPK. Our mutation analysis suggests that codon 162 in K9 gene is an important hot spot for mutation in EPPK.

  7. HHrep: de novo protein repeat detection and the origin of TIM barrels.

    Science.gov (United States)

    Söding, Johannes; Remmert, Michael; Biegert, Andreas

    2006-07-01

    HHrep is a web server for the de novo identification of repeats in protein sequences, which is based on the pairwise comparison of profile hidden Markov models (HMMs). Its main strength is its sensitivity, allowing it to detect highly divergent repeat units in protein sequences whose repeats could as yet only be detected from their structures. Examples include sequences with beta-propellor fold, ferredoxin-like fold, double psi barrels or (betaalpha)8 (TIM) barrels. We illustrate this with proteins from four superfamilies of TIM barrels by revealing a clear 4- and 8-fold symmetry, which we detect solely from their sequences. This symmetry might be the trace of an ancient origin through duplication of a betaalphabetaalpha or betaalpha unit. HHrep can be accessed at http://hhrep.tuebingen.mpg.de.

  8. De novo MEIS2 mutation causes syndromic developmental delay with persistent gastro-esophageal reflux.

    Science.gov (United States)

    Fujita, Atsushi; Isidor, Bertrand; Piloquet, Hugues; Corre, Pierre; Okamoto, Nobuhiko; Nakashima, Mitsuko; Tsurusaki, Yoshinori; Saitsu, Hirotomo; Miyake, Noriko; Matsumoto, Naomichi

    2016-09-01

    MEIS2 aberrations are considered to be the cause of intellectual disability, cleft palate and cardiac septal defect, as MEIS2 copy number variation is often observed with these phenotypes. To our knowledge, only one nucleotide-level change-specifically, an in-frame MEIS2 deletion-has so far been reported. Here, we report a female patient with a de novo nonsense mutation (c.611C>G, p.Ser204*) in MEIS2. She showed severe intellectual disability, moderate motor/verbal developmental delay, cleft palate, cardiac septal defect, hypermetropia, severe feeding difficulties with gastro-esophageal reflux and constipation. By reviewing this patient and previous patients with MEIS2 point mutations, we found that feeding difficulty with gastro-esophageal reflux appears to be one of the core clinical features of MEIS2 haploinsufficiency, in addition to intellectual disability, cleft palate and cardiac septal defect.

  9. Transcriptional regulator-mediated activation of adaptation genes triggers CRISPR de novo spacer acquisition

    DEFF Research Database (Denmark)

    Liu, Tao; Li, Yingjun; Wang, Xiaodi;

    2015-01-01

    , it was demonstrated that the transcription level of csa1, cas1, cas2 and cas4 was significantly enhanced in a csa3a-overexpression strain and, moreover, the Csa1 and Cas1 protein levels were increased in this strain. Furthermore, we demonstrated the hyperactive uptake of unique spacers within both CRISPR loci......Acquisition of de novo spacer sequences confers CRISPR-Cas with a memory to defend against invading genetic elements. However, the mechanism of regulation of CRISPR spacer acquisition remains unknown. Here we examine the transcriptional regulation of the conserved spacer acquisition genes in Type I...... in the presence of the csa3a overexpression vector. The spacer acquisition process is dependent on the CCN PAM sequence and protospacer selection is random and non-directional. These results suggested a regulation mechanism of CRISPR spacer acquisition where a single transcriptional regulator senses the presence...

  10. Prognostic factors in de novo myelodysplastic syndrome in young and middle-aged people

    Directory of Open Access Journals (Sweden)

    Наталья Николаевна Климкович

    2015-01-01

    Full Text Available We spent multivariate analysis of clinical and laboratory parameters for the prediction of de-novo myelodysplastic syndromes (MDS patients aged 18-60 years. The results of clinical application of prognostic systems in MDS show that there is a large variability within individual risk groups, especially at low-risk MDS. So now hematologists conduct research aimed at identifying additional adverse risk MDS. This is done so that patients with low-risk MDS embodiments and unfavorable prognosis could benefit from early therapeutic intervention, and not only be clinician monitored until disease progression. We found that additional adverse risk factors for the development of MDS are the expression of CD95 in bone marrow ≤40 % and FLT3≥60 %. The expression level of CD95 in bone marrow cells≤40 % and FLT3≥60 % can be considered as a prognostic marker progression of MDS and time start specific therapy

  11. A Real-Time de novo DNA Sequencing Assembly Platform Based on an FPGA Implementation.

    Science.gov (United States)

    Hu, Yuanqi; Georgiou, Pantelis

    2016-01-01

    This paper presents an FPGA based DNA comparison platform which can be run concurrently with the sensing phase of DNA sequencing and shortens the overall time needed for de novo DNA assembly. A hybrid overlap searching algorithm is applied which is scalable and can deal with incremental detection of new bases. To handle the incomplete data set which gradually increases during sequencing time, all-against-all comparisons are broken down into successive window-against-window comparison phases and executed using a novel dynamic suffix comparison algorithm combined with a partitioned dynamic programming method. The complete system has been designed to facilitate parallel processing in hardware, which allows real-time comparison and full scalability as well as a decrease in the number of computations required. A base pair comparison rate of 51.2 G/s is achieved when implemented on an FPGA with successful DNA comparison when using data sets from real genomes.

  12. Stacks: building and genotyping Loci de novo from short-read sequences.

    Science.gov (United States)

    Catchen, Julian M; Amores, Angel; Hohenlohe, Paul; Cresko, William; Postlethwait, John H

    2011-08-01

    Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress. Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks can recover thousands of single nucleotide polymorphism (SNP) markers useful for the genetic analysis of crosses or populations. Stacks can generate markers for ultra-dense genetic linkage maps, facilitate the examination of population phylogeography, and help in reference genome assembly. We report here the algorithms implemented in Stacks and demonstrate their efficacy by constructing loci from simulated RAD-tags taken from the stickleback reference genome and by recapitulating and improving a genetic map of the zebrafish, Danio rerio.

  13. Annotation and Re-Sequencing of Genes from De Novo Transcriptome Assembly of Abies alba (Pinaceae

    Directory of Open Access Journals (Sweden)

    Anna M. Roschanski

    2013-01-01

    Full Text Available Premise of the study: We present a protocol for the annotation of transcriptome sequence data and the identification of candidate genes therein using the example of the nonmodel conifer Abies alba. Methods and Results: A normalized cDNA library was built from an A. alba seedling. The sequencing on a 454 platform yielded more than 1.5 million reads that were de novo assembled into 25 149 contigs. Two complementary approaches were applied to annotate gene fragments that code for (1 well-known proteins and (2 proteins that are potentially adaptively relevant. Primer development and testing yielded 88 amplicons that could successfully be resequenced from genomic DNA. Conclusions: The annotation workflow offers an efficient way to identify potential adaptively relevant genes from the large quantity of transcriptome sequence data. The primer set presented should be prioritized for single-nucleotide polymorphism detection in adaptively relevant genes in A. alba.

  14. De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints.

    Science.gov (United States)

    Smock, Robert G; Yadid, Itamar; Dym, Orly; Clarke, Jane; Tawfik, Dan S

    2016-01-28

    Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.

  15. Clinical assessment of hepatic de novo lipogenesis in non-alcoholic fatty liver disease.

    Science.gov (United States)

    Paglialunga, Sabina; Dehn, Clayton A

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD) is heralded as the next big global epidemic. Hepatic de novo lipogenesis (DNL), the synthesis of new fatty acids from non-lipid sources, is thought to play a pivotal role in the development of NAFLD. While there is currently no NAFLD-specific therapeutic agent available, pharmaceutical drugs aimed at reducing hepatic fat accretion may prove to be a powerful ally in the treatment and management of this disease. With a focus on NAFLD, the present review summarizes current techniques examining DNL from a clinical perspective, and describes the merits and limitations of three commonly used assays; stable-label isotope tracer studies, fatty acid indexes and indirect calorimetry as non-invasive measures of hepatic DNL. Finally, the application of DNL assessments in the pharmacological and nutraceutical treatment of NAFLD/NASH is summarized. In a clinical research setting, measures of DNL are an important marker in the development of anti-NAFLD treatments.

  16. Insular carcinoma: a distinct de novo entity among follicular carcinomas of the thyroid gland.

    Science.gov (United States)

    Pilotti, S; Collini, P; Mariani, L; Placucci, M; Bongarzone, I; Vigneri, P; Cipriani, S; Falcetta, F; Miceli, R; Pierotti, M A; Rilke, F

    1997-12-01

    We reclassified 720 nonmedullary invasive thyroid carcinomas diagnosed and treated between 1975 and 1993. Twenty-seven cases met the criteria of insular carcinoma and 29 cases those of widely invasive follicular carcinoma. Comparison of these histotypes with respect to pathologic stage and overall, relative, and visceral metastasis-free survival showed a significant association between histotype and pT and pN categories. In particular, pT4 (p AAA transversion at codon 61 of the N-RAS gene in insular carcinoma. These findings suggest that insular carcinoma represents a de novo entity distinct from widely invasive follicular carcinoma, that widely invasive follicular carcinoma has biologic characteristics more consistent with poorly differentiated than well-differentiated carcinomas, and that both insular carcinoma and widely invasive follicular carcinoma share similar molecular alterations.

  17. De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans.

    Science.gov (United States)

    García-Bayona, Leonor; Garavito, Manuel F; Lozano, Gabriel L; Vasquez, Juan J; Myers, Kevin; Fry, William E; Bernal, Adriana; Zimmermann, Barbara H; Restrepo, Silvia

    2014-03-10

    The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.

  18. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Science.gov (United States)

    Smadbeck, James; Peterson, Meghan B; Zee, Barry M; Garapaty, Shivani; Mago, Aashna; Lee, Christina; Giannis, Athanassios; Trojer, Patrick; Garcia, Benjamin A; Floudas, Christodoulos A

    2014-01-01

    Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should

  19. De Novo Transcriptome Sequencing of Oryza officinalis Wall ex Watt to Identify Disease-Resistance Genes

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    Bin He

    2015-12-01

    Full Text Available Oryza officinalis Wall ex Watt is one of the most important wild relatives of cultivated rice and exhibits high resistance to many diseases. It has been used as a source of genes for introgression into cultivated rice. However, there are limited genomic resources and little genetic information publicly reported for this species. To better understand the pathways and factors involved in disease resistance and accelerating the process of rice breeding, we carried out a de novo transcriptome sequencing of O. officinalis. In this research, 137,229 contigs were obtained ranging from 200 to 19,214 bp with an N50 of 2331 bp through de novo assembly of leaves, stems and roots in O. officinalis using an Illumina HiSeq 2000 platform. Based on sequence similarity searches against a non-redundant protein database, a total of 88,249 contigs were annotated with gene descriptions and 75,589 transcripts were further assigned to GO terms. Candidate genes for plant–pathogen interaction and plant hormones regulation pathways involved in disease-resistance were identified. Further analyses of gene expression profiles showed that the majority of genes related to disease resistance were all expressed in the three tissues. In addition, there are two kinds of rice bacterial blight-resistant genes in O. officinalis, including two Xa1 genes and three Xa26 genes. All 2 Xa1 genes showed the highest expression level in stem, whereas one of Xa26 was expressed dominantly in leaf and other 2 Xa26 genes displayed low expression level in all three tissues. This transcriptomic database provides an opportunity for identifying the genes involved in disease-resistance and will provide a basis for studying functional genomics of O. officinalis and genetic improvement of cultivated rice in the future.

  20. Characterization and analysis of a de novo transcriptome from the pygmy grasshopper Tetrix japonica.

    Science.gov (United States)

    Qiu, Zhongying; Liu, Fei; Lu, Huimeng; Huang, Yuan

    2016-06-11

    The pygmy grasshopper Tetrix japonica is a common insect distributed throughout the world, and it has the potential for use in studies of body colour polymorphism, genomics and the biology of Tetrigoidea (Insecta: Orthoptera). However, limited biological information is available for this insect. Here, we conducted a de novo transcriptome study of adult and larval T. japonica to provide a better understanding of its gene expression and develop genomic resources for future work. We sequenced and explored the characteristics of the de novo transcriptome of T. japonica using Illumina HiSeq 2000 platform. A total of 107 608 206 paired-end clean reads were assembled into 61 141 unigenes using the trinity software; the mean unigene size was 771 bp, and the N50 length was 1238 bp. A total of 29 225 unigenes were functionally annotated to the NCBI nonredundant protein sequences (Nr), NCBI nonredundant nucleotide sequences (Nt), a manually annotated and reviewed protein sequence database (Swiss-Prot), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of putative genes that are potentially involved in pigment pathways, juvenile hormone (JH) metabolism and signalling pathways were identified in the T. japonica transcriptome. Additionally, 165 769 and 156 796 putative single nucleotide polymorphisms occurred in the adult and larvae transcriptomes, respectively, and a total of 3162 simple sequence repeats were detected in this assembly. This comprehensive transcriptomic data for T. japonica will provide a usable resource for gene predictions, signalling pathway investigations and molecular marker development for this species and other pygmy grasshoppers.

  1. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

    Science.gov (United States)

    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

  2. Characterization of Liaoning cashmere goat transcriptome: sequencing, de novo assembly, functional annotation and comparative analysis.

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    Hongliang Liu

    Full Text Available BACKGROUND: Liaoning cashmere goat is a famous goat breed for cashmere wool. In order to increase the transcriptome data and accelerate genetic improvement for this breed, we performed de novo transcriptome sequencing to generate the first expressed sequence tag dataset for the Liaoning cashmere goat, using next-generation sequencing technology. RESULTS: Transcriptome sequencing of Liaoning cashmere goat on a Roche 454 platform yielded 804,601 high-quality reads. Clustering and assembly of these reads produced a non-redundant set of 117,854 unigenes, comprising 13,194 isotigs and 104,660 singletons. Based on similarity searches with known proteins, 17,356 unigenes were assigned to 6,700 GO categories, and the terms were summarized into three main GO categories and 59 sub-categories. 3,548 and 46,778 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Comparative analysis revealed that 42,254 unigenes were aligned to 17,532 different sequences in NCBI non-redundant nucleotide databases. 97,236 (82.51% unigenes were mapped to the 30 goat chromosomes. 35,551 (30.17% unigenes were matched to 11,438 reported goat protein-coding genes. The remaining non-matched unigenes were further compared with cattle and human reference genes, 67 putative new goat genes were discovered. Additionally, 2,781 potential simple sequence repeats were initially identified from all unigenes. CONCLUSION: The transcriptome of Liaoning cashmere goat was deep sequenced, de novo assembled, and annotated, providing abundant data to better understand the Liaoning cashmere goat transcriptome. The potential simple sequence repeats provide a material basis for future genetic linkage and quantitative trait loci analyses.

  3. A human-specific de novo protein-coding gene associated with human brain functions.

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    Chuan-Yun Li

    2010-03-01

    Full Text Available To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203. Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

  4. Brain metabolic correlates of dopaminergic degeneration in de novo idiopathic Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Berti, Valentina; Polito, Cristina; Vanzi, Eleonora; Cristofaro, Maria Teresa de; Pellicano, Giannantonio; Mungai, Francesco; Formiconi, Andreas Robert; Pupi, Alberto [University of Florence, Department of Clinical Pathophysiology, Florence (Italy); Ramat, Silvia; Marini, Paolo; Sorbi, Sandro [University of Florence, Department of Psychiatric and Neurological Sciences, Florence (Italy)

    2010-03-15

    The aim of the present study was to evaluate the reciprocal relationships between motor impairment, dopaminergic dysfunction, and cerebral metabolism (rCMRglc) in de novo Parkinson's disease (PD) patients. Twenty-six de novo untreated PD patients were scanned with {sup 123}I-FP-CIT SPECT and {sup 18}F-FDG PET. The dopaminergic impairment was measured with putaminal {sup 123}I-FP-CIT binding potential (BP), estimated with two different techniques: an iterative reconstruction algorithm (BP{sub OSEM}) and the least-squares (LS) method (BP{sub LS}). Statistical parametric mapping (SPM) multiple regression analyses were performed to determine the specific brain regions in which UPDRS III scores and putaminal BP values correlated with rCMRglc. The SPM results showed a negative correlation between UPDRS III and rCMRglc in premotor cortex, and a positive correlation between BP{sub OSEM} and rCMRglc in premotor and dorsolateral prefrontal cortex, not surviving at multiple comparison correction. Instead, there was a positive significant correlation between putaminal BP{sub LS} and rCMRglc in premotor, dorsolateral prefrontal, anterior prefrontal, and orbitofrontal cortex (p < 0.05, corrected for multiple comparison). Putaminal BP{sub LS} is an efficient parameter for exploring the correlations between PD severity and rCMRglc cortical changes. The correlation between dopaminergic degeneration and rCMRglc in several prefrontal regions likely represents the cortical functional correlate of the dysfunction in the motor basal ganglia-cortical circuit in PD. This finding suggests focusing on the metabolic course of these areas to follow PD progression and to analyze treatment effects. (orig.)

  5. The Rickettsia Endosymbiont of Ixodes pacificus Contains All the Genes of De Novo Folate Biosynthesis.

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    Daniel J Hunter

    Full Text Available Ticks and other arthropods often are hosts to nutrient providing bacterial endosymbionts, which contribute to their host's fitness by supplying nutrients such as vitamins and amino acids. It has been detected, in our lab, that Ixodes pacificus is host to Rickettsia species phylotype G021. This endosymbiont is predominantly present, and 100% maternally transmitted in I. pacificus. To study roles of phylotype G021 in I. pacificus, bioinformatic and molecular approaches were carried out. MUMmer genome alignments of whole genome sequence of I. scapularis, a close relative to I. pacificus, against completely sequenced genomes of R. bellii OSU85-389, R. conorii, and R. felis, identified 8,190 unique sequences that are homologous to Rickettsia sequences in the NCBI Trace Archive. MetaCyc metabolic reconstructions revealed that all folate gene orthologues (folA, folC, folE, folKP, ptpS required for de novo folate biosynthesis are present in the genome of Rickettsia buchneri in I. scapularis. To examine the metabolic capability of phylotype G021 in I. pacificus, genes of the folate biosynthesis pathway of the bacterium were PCR amplified using degenerate primers. BLAST searches identified that nucleotide sequences of the folA, folC, folE, folKP, and ptpS genes possess 98.6%, 98.8%, 98.9%, 98.5% and 99.0% identity respectively to the corresponding genes of Rickettsia buchneri. Phylogenetic tree constructions show that the folate genes of phylotype G021 and homologous genes from various Rickettsia species are monophyletic. This study has shown that all folate genes exist in the genome of Rickettsia species phylotype G021 and that this bacterium has the genetic capability for de novo folate synthesis.

  6. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

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    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

  7. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

  8. TP53 overexpression is an independent adverse prognostic factor in de novo myelodysplastic syndromes with fibrosis.

    Science.gov (United States)

    Loghavi, Sanam; Al-Ibraheemi, Alyaa; Zuo, Zhuang; Garcia-Manero, Guillermo; Yabe, Mariko; Wang, Sa A; Kantarjian, Hagop M; Yin, Cameron C; Miranda, Roberto N; Luthra, Raja; Medeiros, L Jeffrey; Bueso-Ramos, Carlos E; Khoury, Joseph D

    2015-10-01

    Bone marrow (BM) fibrosis is associated with poor prognosis in patients with de novo myelodysplastic syndromes (MDS). TP53 mutations and TP53 (p53) overexpression in MDS are also associated with poor patient outcomes. The prevalence and significance of TP53 mutations and TP53 overexpression in MDS with fibrosis are unknown. We studied 67 patients with de novo MDS demonstrating moderate to severe reticulin fibrosis (MDS-F). Expression of TP53 was evaluated in BM core biopsy specimens using dual-colour CD34/TP53 immunohistochemistry with computer-assisted image analysis. Mutation analysis was performed using next-generation sequencing, or Sanger sequencing methods. TP53 mutations were present in 47·1% of cases. TP53 mutation was significantly associated with TP53 expression (P = 0·0294). High levels of TP53 expression (3 +  in ≥10% cells) were associated with higher BM blast counts (P = 0·0149); alterations of chromosomes 5 (P = 0·0009) or 7 (P = 0·0141); complex karyotype (P = 0·0002); high- and very-high risk IPSS-R groups (P = 0·009); and TP53 mutations (P = 0·0003). High TP53 expression independently predicted shorter overall survival (OS) by multivariate analysis (P = TP53 by CD34-positive cells was associated with shorter OS and leukaemia-free survival (P = 0·0428). TP53 overexpression is a predictor of poor outcome in patients with MDS-F.

  9. De-Novo anti-HLA Antibodies After Renal Transplantation: Prevalance and Risk Factors

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    Burak SUVAK

    2011-01-01

    Full Text Available AIM: Development of de-novo anti-HLA antibodies in the post-transplant period might be the earliest finding of later chronic antibody mediated rejection. In this study, we aimed to investigate the incidence and risk factors of de-novo anti-HLA antibodies in our kidney allograft recipients. MATERIAL and METHODS: After exclusion, 91 (64M/27F patients having functional graft and negative HLA antibody before the transplantation were taken into the analysis. Anti-HLA antibodies were evaluated by the Luminex method. RESULTS: Duration of posttransplantation time was 38±31 months and the mean age was 38±10. Mean estimated glomerular filtration rate (GFR was 68±19 ml/min, and the biopsy proven acute rejection rate was 15.2 %. Anti- HLA antibody was observed in 12 patients (13.1%. When the anti-HLA antibody positive group was compared with the negative group, estimated GFR (58±26 ml/min vs. 69±18 ml/ min, (p=0.05, living donor/cadaveric donor (5/7 vs. 66/13 (p=0.004, and acute rejection (6/12 (%50 vs. 8/79 (%10.1 (p=0.002 were significantly different between the groups. Deceased donor and acute rejection were independent risk factors for development of anti-HLA antibody (p=0.008 and p= 0.004, respectively on multivariate analysis. CONCLUSION: In conclusion, anti-HLA antibody can be seen after renal transplantation even in stable patients. Acute rejection and deceased donor transplantation are the major risk factors for development of anti-HLA antibodies.

  10. The microaerophilic microbiota of de-novo paediatric inflammatory bowel disease: the BISCUIT study.

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    Richard Hansen

    Full Text Available INTRODUCTION: Children presenting for the first time with inflammatory bowel disease (IBD offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD. PATIENTS AND METHODS: 100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis. RESULTS: 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates. Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae. No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312. CONCLUSIONS: This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon. TRIAL REGISTRATION: This study is publically registered on the United Kingdom Clinical Research Network Portfolio (9633.

  11. De novo assembly of a field isolate genome reveals novel Plasmodium vivax erythrocyte invasion genes.

    Science.gov (United States)

    Hester, James; Chan, Ernest R; Menard, Didier; Mercereau-Puijalon, Odile; Barnwell, John; Zimmerman, Peter A; Serre, David

    2013-01-01

    Recent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P. vivax reference genome that was generated using DNA from the monkey-adapted strain Salvador I. Any genomic locus deleted in this strain would be lacking in the reference genome sequence and missed in previous analyses. Here, we report de novo assembly of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs, each containing more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P. vivax DNA sequences account for 3.8 million nucleotides and contain 792 predicted genes. Most of these contigs contain members of multigene families and likely originate from telomeric regions. Interestingly, we identify two contigs containing predicted protein coding genes similar to known Plasmodium red blood cell invasion proteins. One gene encodes the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P. knowlesi NBPXb. The second gene harbors all the hallmarks of a Plasmodium erythrocyte-binding protein, including conserved Duffy-binding like and C-terminus cysteine-rich domains. Phylogenetic analysis shows that this novel gene clusters separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showing that this gene is present in most P. vivax genomes and transcribed in blood-stage parasites suggest that P. vivax red blood cell invasion mechanisms may be more complex than currently understood. The strategy employed here complements previous genomic analyses and takes full advantage of next-generation sequencing data to provide a comprehensive characterization of genetic variations in this important malaria parasite. Further analyses of the novel protein coding genes discovered through de novo assembly have the potential to identify genes that influence key aspects of P

  12. Optimizing information in Next-Generation-Sequencing (NGS) reads for improving de novo genome assembly.

    Science.gov (United States)

    Liu, Tsunglin; Tsai, Cheng-Hung; Lee, Wen-Bin; Chiang, Jung-Hsien

    2013-01-01

    Next-Generation-Sequencing is advantageous because of its much higher data throughput and much lower cost compared with the traditional Sanger method. However, NGS reads are shorter than Sanger reads, making de novo genome assembly very challenging. Because genome assembly is essential for all downstream biological studies, great efforts have been made to enhance the completeness of genome assembly, which requires the presence of long reads or long distance information. To improve de novo genome assembly, we develop a computational program, ARF-PE, to increase the length of Illumina reads. ARF-PE takes as input Illumina paired-end (PE) reads and recovers the original DNA fragments from which two ends the paired reads are obtained. On the PE data of four bacteria, ARF-PE recovered >87% of the DNA fragments and achieved >98% of perfect DNA fragment recovery. Using Velvet, SOAPdenovo, Newbler, and CABOG, we evaluated the benefits of recovered DNA fragments to genome assembly. For all four bacteria, the recovered DNA fragments increased the assembly contiguity. For example, the N50 lengths of the P. brasiliensis contigs assembled by SOAPdenovo and Newbler increased from 80,524 bp to 166,573 bp and from 80,655 bp to 193,388 bp, respectively. ARF-PE also increased assembly accuracy in many cases. On the PE data of two fungi and a human chromosome, ARF-PE doubled and tripled the N50 length. However, the assembly accuracies dropped, but still remained >91%. In general, ARF-PE can increase both assembly contiguity and accuracy for bacterial genomes. For complex eukaryotic genomes, ARF-PE is promising because it raises assembly contiguity. But future error correction is needed for ARF-PE to also increase the assembly accuracy. ARF-PE is freely available at http://140.116.235.124/~tliu/arf-pe/.

  13. De novo assembly of the perennial ryegrass transcriptome using an RNA-Seq strategy.

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    Jacqueline D Farrell

    Full Text Available Perennial ryegrass is a highly heterozygous outbreeding grass species used for turf and forage production. Heterozygosity can affect de-Bruijn graph assembly making de novo transcriptome assembly of species such as perennial ryegrass challenging. Creating a reference transcriptome from a homozygous perennial ryegrass genotype can circumvent the challenge of heterozygosity. The goals of this study were to perform RNA-sequencing on multiple tissues from a highly inbred genotype to develop a reference transcriptome. This was complemented with RNA-sequencing of a highly heterozygous genotype for SNP calling.De novo transcriptome assembly of the inbred genotype created 185,833 transcripts with an average length of 830 base pairs. Within the inbred reference transcriptome 78,560 predicted open reading frames were found of which 24,434 were predicted as complete. Functional annotation found 50,890 transcripts with a BLASTp hit from the Swiss-Prot non-redundant database, 58,941 transcripts with a Pfam protein domain and 1,151 transcripts encoding putative secreted peptides. To evaluate the reference transcriptome we targeted the high-affinity K+ transporter gene family and found multiple orthologs. Using the longest unique open reading frames as the reference sequence, 64,242 single nucleotide polymorphisms were found. One thousand sixty one open reading frames from the inbred genotype contained heterozygous sites, confirming the high degree of homozygosity.Our study has developed an annotated, comprehensive transcriptome reference for perennial ryegrass that can aid in determining genetic variation, expression analysis, genome annotation, and gene mapping.

  14. Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus).

    Science.gov (United States)

    Thanh, Nguyen Minh; Jung, Hyungtaek; Lyons, Russell E; Njaci, Isaac; Yoon, Byoung-Ha; Chand, Vincent; Tuan, Nguyen Viet; Thu, Vo Thi Minh; Mather, Peter

    2015-10-01

    Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478bp and N50 length of 506bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

  15. Immunosuppression for islet transplantation.

    Directory of Open Access Journals (Sweden)

    Noguchi,Hirofumi

    2006-04-01

    Full Text Available The development by the Edmonton group of a sirolimus-based, steroid-free, low-tacrolimus regimen is a significant breakthrough that allows the rate of insulin independence after islet transplantation to increase from 13% to 80% at 1 year ; however, the rate is reduced to 50% at 3 years, attributed to prolonged tacrolimus exposure. Recently, immunosuppression agents such as cyclosporine, mycophenolate mofetil, and the novel agent FTY 720 have been used instead of tacrolimus. Lymphocytedepleting antibodies such as anti-thymocyte globulin, alemtuzumab, and hOKT3gamma 1 (ala, ala have been launched, and a costimulatory blockade of anti-CD40 monoclonal antibodies and CTLA4-Ig will be attempted in the near future. Moreover, the potential of a novel immunosuppressing peptide could now be realized using new technology called the protein transduction system. In this review, we show some of the most recent contributions to the advancement of knowledge in this field.

  16. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  17. Olefin cross metathesis based de novo synthesis of a partially protected L-amicetose and a fully protected L-cinerulose derivative

    Science.gov (United States)

    Hauke, Sylvia

    2014-01-01

    Summary Cross metathesis of a lactate derived allylic alcohol and acrolein is the entry point to a de novo synthesis of 4-benzoate protected L-amicetose and a cinerulose derivative protected at C5 and C1. PMID:24991253

  18. Olefin cross metathesis based de novo synthesis of a partially protected L-amicetose and a fully protected L-cinerulose derivative

    Directory of Open Access Journals (Sweden)

    Bernd Schmidt

    2014-05-01

    Full Text Available Cross metathesis of a lactate derived allylic alcohol and acrolein is the entry point to a de novo synthesis of 4-benzoate protected L-amicetose and a cinerulose derivative protected at C5 and C1.

  19. Discovery, genotyping and characterization of structural variation and novel sequence at single nucleotide resolution from de novo genome assemblies on a population scale

    DEFF Research Database (Denmark)

    Huang, Shujia; Rao, Junhua; Ye, Weijian;

    2015-01-01

    present a novel approach implemented in a single software package, AsmVar, to discover, genotype and characterize different forms of structural variation and novel sequence from population-scale de novo genome assemblies up to nucleotide resolution. Application of AsmVar to several human de novo genome...... assemblies captures a wide spectrum of structural variants and novel sequences present in the human population in high sensitivity and specificity. Our method provides a direct solution for investigating structural variants and novel sequences from de novo genome assemblies, facilitating the construction...... of population-scale pan-genomes. Our study also highlights the usefulness of the de novo assembly strategy for definition of genome structure....

  20. Increased co-expression of genes harboring the damaging de novo mutations in Chinese schizophrenic patients during prenatal development.

    Science.gov (United States)

    Wang, Qiang; Li, Miaoxin; Yang, Zhenxing; Hu, Xun; Wu, Hei-Man; Ni, Peiyan; Ren, Hongyan; Deng, Wei; Li, Mingli; Ma, Xiaohong; Guo, Wanjun; Zhao, Liansheng; Wang, Yingcheng; Xiang, Bo; Lei, Wei; Sham, Pak C; Li, Tao

    2015-12-15

    Schizophrenia is a heritable, heterogeneous common psychiatric disorder. In this study, we evaluated the hypothesis that de novo variants (DNVs) contribute to the pathogenesis of schizophrenia. We performed exome sequencing in Chinese patients (N = 45) with schizophrenia and their unaffected parents (N = 90). Forty genes were found to contain DNVs. These genes had enriched transcriptional co-expression profile in prenatal frontal cortex (Bonferroni corrected p genes (LRP1, MACF1, DICER1 and ABCA2) harboring the damaging de novo mutations are strongly prioritized as susceptibility genes by multiple evidences. Our findings in Chinese schizophrenic patients indicate the pathogenic role of DNVs, supporting the hypothesis that schizophrenia is a neurodevelopmental disease.

  1. A de novo germline MLH1 mutation in a Lynch syndrome patient with discordant immunohistochemical and molecular biology test results

    Institute of Scientific and Technical Information of China (English)

    Fabrice Airaud; Sébastien Küry; Isabelle Valo; Ingrid Maury; Dominique Bonneau; Olivier Ingster; Stéphane Bezieau

    2012-01-01

    We describe a patient with a Homo sapiens mutL homolog 1 (MLH1)-associated Lynch syndrome with previous diagnoses of two distinct primary cancers:a sigmoid colon cancer at the age of 39 years,and a right colon cancer at the age of 50 years.The mutation identified in his blood and buccal cells,c.1771delG,p.Asp591Ilefs*25,appears to be a de novo event,as it was not transmitted by either of his parents.This type of de novo event is rare in MLH1 as only three cases have been reported in the literature so far.Furthermore,the discordant results observed between replication error phenotyping and immunohistochemistry highlight the importance of the systematic use of both pre-screening tests in the molecular diagnosis of Lynch syndrome.

  2. A Swedish family with de novo alpha-synuclein A53T mutation: evidence for early cortical dysfunction

    DEFF Research Database (Denmark)

    Puschmann, Andreas; Ross, Owen A; Vilariño-Güell, Carles;

    2009-01-01

    A de novo alpha-synuclein A53T (p.Ala53 Th; c.209G > A) mutation has been identified in a Swedish family with autosomal dominant Parkinson's disease (PD). Two affected individuals had early-onset (before 31 and 40 years), severe levodopa-responsive PD with prominent dysphasia, dysarthria, and cog......A de novo alpha-synuclein A53T (p.Ala53 Th; c.209G > A) mutation has been identified in a Swedish family with autosomal dominant Parkinson's disease (PD). Two affected individuals had early-onset (before 31 and 40 years), severe levodopa-responsive PD with prominent dysphasia, dysarthria......, and cognitive decline. Longitudinal clinical follow-up, EEG, SPECT and CSF biomarker examinations suggested an underlying encephalopathy with cortical involvement. The mutated allele (c.209A) was present within a haplotype different from that shared among mutation carriers in the Italian (Contursi...

  3. De Novo Balanced Translocation t (7;16 (p22.1; p11.2 Associated with Autistic Disorder

    Directory of Open Access Journals (Sweden)

    Nadia Bayou

    2008-01-01

    Full Text Available The high incidence of de novo chromosomal aberrations in a population of persons with autism suggests a causal relationship between certain chromosomal aberrations and the occurrence of isolated idiopathic autism. We report on the clinical and cytogenetic findings in a male patient with autism, no physical abnormalities and a de novo balanced (7;16(p22.1;p16.2 translocation. G-banded chromosomes and fluorescent in situ hybridization (FISH were used to examine the patient's karyotype as well as his parents'. FISH with specific RP11-BAC clones mapping near 7p22.1 and 16p11.2 was used to refine the location of the breakpoints. This is, in the best of our knowledge, the first report of an individual with autism and this specific chromosomal aberration.

  4. Detection of phytochrome-like genes from Rhazya stricta (Apocynaceae) using de novo genome assembly.

    Science.gov (United States)

    Sabir, Jamal S M; Baeshen, Nabih A; Shokry, Ahmed M; Gadalla, Nour O; Edris, Sherif; Mutwakil, Mohammed H; Ramadan, Ahmed M; Atef, Ahmed; Al-Kordy, Magdy A; Abuzinadah, Osama A; El-Domyati, Fotouh M; Jansen, Robert K; Bahieldin, Ahmed

    2013-01-01

    Phytochrome-like genes in the wild plant species Rhazya stricta Decne were characterized using a de novo genome assembly of next generation sequence data. Rhazya stricta contains more than 100 alkaloids with multiple pharmacological properties, and leaf extracts have been used to cure chronic rheumatism, to treat tumors, and in the treatment of several other diseases. Phytochromes are known to be involved in the light-regulated biosynthesis of some alkaloids. Phytochromes are soluble chromoproteins that function in the absorption of red and far-red light and the transduction of intracellular signals during light-regulated plant development. De novo assembly of the nuclear genome of R. stricta recovered 45,641 contigs greater than 1000bp long, which were used in constructing a local database. Five sequences belonging to Arabidopsis thaliana phytochrome gene family (i.e., AtphyABCDE) were used to identify R. stricta contigs with phytochrome-like sequences using BLAST. This led to the identification of three contigs with phytochrome-like sequences covering AtphyA-, AtphyC- and AtphyE-like full-length genes. Annotation of the three sequences showed that each contig consists of one phytochrome-like gene with three exons and two introns. BLASTn and BLASTp results indicated that RsphyA mRNA and protein sequences had homologues in Wrightia coccinea and and Solanum tuberosum, respectively. RsphyC-like mRNA and protein sequence were homologous to Vitis vinifera and Vitis riparia. RsphyE-like mRNA coding and protein sequences were homologous to Ipomoea nil. Multiple-sequence alignment of phytochrome proteins indicated a homology with 30 sequences from 23 different species of flowering plants. Phylogenetic analysis confirmed that each R. stricta phytochrome gene is related to the same phytochrome gene of other flowering plants. It is proposed that the absence of phyB gene in R. stricta is due to RsphyA gene taking over the role of phyB.

  5. De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube

    OpenAIRE

    Domenico Iaria; Adriana Chiappetta; Innocenzo Muzzalupo

    2016-01-01

    In olive (Olea europaea L.), the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cu...

  6. Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

    Energy Technology Data Exchange (ETDEWEB)

    Haigh, Cathryn L., E-mail: chaigh@unimelb.edu.au [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); Drew, Simon C., E-mail: sdrew@unimelb.edu.au [Florey Department of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010 (Australia)

    2015-06-05

    The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights: • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation.

  7. Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance

    Directory of Open Access Journals (Sweden)

    Thieffry Denis

    2009-01-01

    Full Text Available Abstract Background In breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer. Results First, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab, demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation. Conclusion In this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not

  8. Increased co-expression of genes harboring the damaging de novo mutations in Chinese schizophrenic patients during prenatal development

    OpenAIRE

    Qiang Wang; Miaoxin Li; Zhenxing Yang; Xun Hu; Hei-Man Wu; Peiyan Ni; Hongyan Ren; Wei Deng; Mingli Li; Xiaohong Ma; Wanjun Guo; Liansheng Zhao; Yingcheng Wang; Bo Xiang; Wei Lei

    2015-01-01

    Schizophrenia is a heritable, heterogeneous common psychiatric disorder. In this study, we evaluated the hypothesis that de novo variants (DNVs) contribute to the pathogenesis of schizophrenia. We performed exome sequencing in Chinese patients (N = 45) with schizophrenia and their unaffected parents (N = 90). Forty genes were found to contain DNVs. These genes had enriched transcriptional co-expression profile in prenatal frontal cortex (Bonferroni corrected p 

  9. De novo transcriptome assembly and comprehensive expression profiling in Crocus sativus to gain insights into apocarotenoid biosynthesis

    OpenAIRE

    Mukesh Jain; Prabhakar Lal Srivastava; Mohit Verma; Rajesh Ghangal; Rohini Garg

    2016-01-01

    Saffron (Crocus sativus L.) is commonly known as world’s most expensive spice with rich source of apocarotenoids and possesses magnificent medicinal properties. To understand the molecular basis of apocarotenoid biosynthesis/accumulation, we performed transcriptome sequencing from five different tissues/organs of C. sativus using Illumina platform. After comprehensive optimization of de novo transcriptome assembly, a total of 105, 269 unique transcripts (average length of 1047 bp and N50 leng...

  10. Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

    Science.gov (United States)

    Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

    2013-01-01

    Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

  11. De novo assembly and characterization of the carrot transcriptome reveals novel genes, new markers, and genetic diversity

    Directory of Open Access Journals (Sweden)

    Matvienko Marta

    2011-08-01

    Full Text Available Abstract Background Among next generation sequence technologies, platforms such as Illumina and SOLiD produce short reads but with higher coverage and lower cost per sequenced nucleotide than 454 or Sanger. A challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. The scope of this study was to develop a de novo assembly of carrot ESTs from multiple genotypes using the Illumina platform, and to identify polymorphisms. Results A de novo assembly of transcriptome sequence from four genetic backgrounds produced 58,751 contigs and singletons. Over 50% of these assembled sequences were annotated allowing detection of transposable elements and new carrot anthocyanin genes. Presence of multiple genetic backgrounds in our assembly allowed the identification of 114 computationally polymorphic SSRs, and 20,058 SNPs at a depth of coverage of 20× or more. Polymorphisms were predominantly between inbred lines except for the cultivated x wild RIL pool which had high intra-sample polymorphism. About 90% and 88% of tested SSR and SNP primers amplified a product, of which 70% and 46%, respectively, were of the expected size. Out of verified SSR and SNP markers 84% and 82% were polymorphic. About 25% of SNPs genotyped were polymorphic in two diverse mapping populations. Conclusions This study confirmed the potential of short read platforms for de novo EST assembly and identification of genetic polymorphisms in carrot. In addition we produced the first large-scale transcriptome of carrot, a species lacking genomic resources.

  12. De novo dup(7)(q21q22.2) and cytogenetics of 7q21q34 duplications.

    Science.gov (United States)

    Rivera, H

    2013-01-01

    We report on a constitutional dup(7)(q21q22.2) and compile 25 similar mid-7q imbalances in order to sort out relevant cytogenetic aspects. The propositus was first karyotyped elsewhere at 2 years of age and found to have a de novo 7q+ chromosome. When reassessed at 22 years of age, he exhibited overt mental disability, marked speech delay, mild short stature, frontal bossing, and mild dysmorphisms. The patient's chromosomes were analyzed in metaphases from a lymphocyte culture by means of G-banding and FISH assays with a wcp 7 and two dual probes, namely ELN (7q11)/D7S2686 (7q22) and ELN (7q11)/D7S486, D7S522 (q31). G-bands revealed a 7q21q22.2 direct duplication that was confirmed by FISH: the 7q+ was entirely painted with the wcp and had two 7q22 signals but a single 7q31 signal. Thus, the patient's karyotype was 46,XY, dup(7)(q21q22.2).ish dup(7)(q21 q22.2)(wcp7+, ELN+, D7S2686++, D7S486+)dn. Among 26 interstitial duplications confined to the segment 7q21q34, 13 were contiguous de novo duplications, one was due to a de novo ins (19;7), and 12 were inherited from carriers of inter-/intrachromosomal insertions or complex rearrangements. Mean paternal and maternal ages in de novo contiguous duplications of paternal/unknown (n = 9) or maternal/unknown (n = 10) descent were 33.44 and 30.9 yr whereas median ages were 29 and 30, respectively. The patient's clinical picture confirms the mild or moderate phenotypical repercussion of mid-7q duplications; among 25 patients born alive, 24 (including six teenagers or older) were still alive when reported on.

  13. Impact of cytogenetics on outcome of de novo and therapy-related AML and MDS after allogeneic transplantation.

    Science.gov (United States)

    Armand, Philippe; Kim, Haesook T; DeAngelo, Daniel J; Ho, Vincent T; Cutler, Corey S; Stone, Richard M; Ritz, Jerome; Alyea, Edwin P; Antin, Joseph H; Soiffer, Robert J

    2007-06-01

    Cytogenetics has an important impact on the prognosis of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML) or myelodysplastic syndromes (MDS). However, it is unclear whether currently accepted cytogenetic risk groups, which were established for patients treated mostly with standard therapy, are optimally discriminating for patients undergoing HSCT. Also, the impact of cytogenetics in the growing population of patients with therapy-related disease has not been completely elucidated. In this study, we retrospectively analyzed data on 556 patients with AML or MDS transplanted at our institution. We examined, in multivariate analyses, the contribution of cytogenetics to survival, relapse, and nonrelapse mortality for the 476 patients with de novo disease. We used these results to establish an optimal cytogenetic grouping scheme. We then applied this grouping scheme to the 80 patients with therapy-related disease. Our proposed 3-group cytogenetic classification outperformed the established grouping schemes for both de novo and therapy-related disease. When classified by this new scheme, cytogenetics was the strongest prognostic factor for overall survival in our cohort, through its impact on the risk of relapse (and not on nonrelapse mortality). After accounting for cytogenetics, patients with therapy-related AML or MDS had an equivalent outcome to those with de novo disease. This study demonstrates the impact of cytogenetics on the risk of relapse and death for patients with both de novo and therapy-related disease undergoing transplantation; it also emphasizes the necessity of using cytogenetics to stratify patients entering clinical trials, and provides a system for doing so, which can be validated in a multi-institutional database.

  14. Amplicon Resequencing Identified Parental Mosaicism for Approximately 10% of "de novo" SCN1A Mutations in Children with Dravet Syndrome.

    Science.gov (United States)

    Xu, Xiaojing; Yang, Xiaoxu; Wu, Qixi; Liu, Aijie; Yang, Xiaoling; Ye, Adam Yongxin; Huang, August Yue; Li, Jiarui; Wang, Meng; Yu, Zhe; Wang, Sheng; Zhang, Zhichao; Wu, Xiru; Wei, Liping; Zhang, Yuehua

    2015-09-01

    The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered "de novo" by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low-level mosaicism in parents of families with seemingly "de novo" mutations will allow for better informed genetic counseling.

  15. Comparative study of de novo assembly and genome-guided assembly strategies for transcriptome reconstruction based on RNA-Seq.

    Science.gov (United States)

    Lu, Bingxin; Zeng, Zhenbing; Shi, Tieliu

    2013-02-01

    Transcriptome reconstruction is an important application of RNA-Seq, providing critical information for further analysis of transcriptome. Although RNA-Seq offers the potential to identify the whole picture of transcriptome, it still presents special challenges. To handle these difficulties and reconstruct transcriptome as completely as possible, current computational approaches mainly employ two strategies: de novo assembly and genome-guided assembly. In order to find the similarities and differences between them, we firstly chose five representative assemblers belonging to the two classes respectively, and then investigated and compared their algorithm features in theory and real performances in practice. We found that all the methods can be reduced to graph reduction problems, yet they have different conceptual and practical implementations, thus each assembly method has its specific advantages and disadvantages, performing worse than others in certain aspects while outperforming others in anther aspects at the same time. Finally we merged assemblies of the five assemblers and obtained a much better assembly. Additionally we evaluated an assembler using genome-guided de novo assembly approach, and achieved good performance. Based on these results, we suggest that to obtain a comprehensive set of recovered transcripts, it is better to use a combination of de novo assembly and genome-guided assembly.

  16. Immunosuppression by discodermolide.

    Science.gov (United States)

    Longley, R E; Gunasekera, S P; Faherty, D; Mclane, J; Dumont, F

    1993-11-30

    In summary, discodermolide, a novel, marine-derived compound, is a potent in vitro and in vivo immunosuppressive agent. Discodermolide blocks cellular proliferation in lymphoid and nonlymphoid cells. This blocking action is not due to cytotoxicity. Blockage of cell proliferation by discodermolide appears to occur at the G2/M interface of the cell cycle, similar to that observed with other types of antiproliferative drugs (i.e., doxorubicin). The cell cycle block appears to be reversible, as cells recover normal cycling patterns within 48 h after removal of the compound. Additional work with this compound is targeted towards determining the exact nature of discodermolide's mitotic block and is currently under way.

  17. An Accurate de novo Algorithm for Glycan Topology Determination from Mass Spectra.

    Science.gov (United States)

    Dong, Liang; Shi, Bing; Tian, Guangdong; Li, YanBo; Wang, Bing; Zhou, MengChu

    2015-01-01

    Determining the glycan topology automatically from mass spectra represents a great challenge. Existing methods fall into approximate and exact ones. The former including greedy and heuristic ones can reduce the computational complexity, but suffer from information lost in the procedure of glycan interpretation. The latter including dynamic programming and exhaustive enumeration are much slower than the former. In the past years, nearly all emerging methods adopted a tree structure to represent a glycan. They share such problems as repetitive peak counting in reconstructing a candidate structure. Besides, tree-based glycan representation methods often have to give different computational formulas for binary and ternary glycans. We propose a new directed acyclic graph structure for glycan representation. Based on it, this work develops a de novo algorithm to accurately reconstruct the tree structure iteratively from mass spectra with logical constraints and some known biosynthesis rules, by a single computational formula. The experiments on multiple complex glycans extracted from human serum show that the proposed algorithm can achieve higher accuracy to determine a glycan topology than prior methods without increasing computational burden.

  18. EvoDesign: De novo protein design based on structural and evolutionary profiles.

    Science.gov (United States)

    Mitra, Pralay; Shultis, David; Zhang, Yang

    2013-07-01

    Protein design aims to identify new protein sequences of desirable structure and biological function. Most current de novo protein design methods rely on physics-based force fields to search for low free-energy states following Anfinsen's thermodynamic hypothesis. A major obstacle of such approaches is the inaccuracy of the force field design, which cannot accurately describe the atomic interactions or distinguish correct folds. We developed a new web server, EvoDesign, to design optimal protein sequences of given scaffolds along with multiple sequence and structure-based features to assess the foldability and goodness of the designs. EvoDesign uses an evolution-profile-based Monte Carlo search with the profiles constructed from homologous structure families in the Protein Data Bank. A set of local structure features, including secondary structure, torsion angle and solvation, are predicted by single-sequence neural-network training and used to smooth the sequence motif and accommodate the physicochemical packing. The EvoDesign algorithm has been extensively tested in large-scale protein design experiments, which demonstrate enhanced foldability and structural stability of designed sequences compared with the physics-based designing methods. The EvoDesign server is freely available at http://zhanglab.ccmb.med.umich.edu/EvoDesign.

  19. Sup35 methionine oxidation is a trigger for de novo [PSI(+)] prion formation.

    Science.gov (United States)

    Grant, Chris M

    2015-01-01

    The molecular basis by which fungal and mammalian prions arise spontaneously is poorly understood. A number of different environmental stress conditions are known to increase the frequency of yeast [PSI(+)] prion formation in agreement with the idea that conditions which cause protein misfolding may promote the conversion of normally soluble proteins to their amyloid forms. A recent study from our laboratory has shown that the de novo formation of the [PSI(+)] prion is significantly increased in yeast mutants lacking key antioxidants suggesting that endogenous reactive oxygen species are sufficient to promote prion formation. Our findings strongly implicate oxidative damage of Sup35 as an important trigger for the formation of the heritable [PSI(+)] prion in yeast. This review discusses the mechanisms by which the direct oxidation of Sup35 might lead to structural transitions favoring conversion to the transmissible amyloid-like form. This is analogous to various environmental factors which have been proposed to trigger misfolding of the mammalian prion protein (PrP(C)) into the aggregated scrapie form (PrP(Sc)).

  20. Primary de novo malignant giant cell tumor of kidney: a case report

    Directory of Open Access Journals (Sweden)

    Torkian Bahman

    2004-06-01

    Full Text Available Abstract Background Osteoclast-like giant cell tumors are usually observed in osseous tissue or as tumors of tendon sheath, characterized by the presence of multinucleated giant cells and mononuclear stromal cells. It has been reported in various extraosseous sites including breast, skin, soft tissue, salivary glands, lung, pancreas, female genital tract, thyroid, larynx and heart. However, extraosseus occurrence of such giant cell tumors in the kidney is extremely rare and is usually found in combination with a conventional malignancy. De-novo primary malignant giant cell tumors of the kidney are unusual lesions and to our knowledge this is the second such case. Case Presentation We report a rare case of extraosseous primary denovo malignant giant cell tumor of the renal parenchyma in a 39-year-old Caucasian female to determine the histogenesis of this neoplasm with a detailed literature review. Conclusion Primary denovo malignant giant cell tumor of the kidney is extremely rare. The cellular origin of this tumor is favored to be a pluripotential mesenchymal stromal cell of the mononuclear/phagocytic cellular lineage. Awareness of this neoplasm is important in the pathological interpretation of unusual findings at either fine needle aspiration or frozen section of solid renal masses.

  1. De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins.

    Science.gov (United States)

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; Ambinder, Richard F; Hayward, S Diane

    2010-11-19

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line.

  2. De novo assembly ofZea nicaraguensis root transcriptome identiifed 5261full-length transcripts

    Institute of Scientific and Technical Information of China (English)

    JIANG Wei; LIU Hai-lan; WU Yuan-qi; ZHANG Su-zhi; LIU Jian; LU Yan-li; TANG Qi-lin; RONG Ting-zhao

    2016-01-01

    Zea nicaraguensis, a wild relative of cultivated maize (Zea mayssubsp. mays), is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function veriif-cation to discover waterlogging tolerance genes inZ. nicaraguensis is currently impractical, because little gene sequence information forZ. nicaraguensis is available in public databases. In this study,Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L–1 Ilumina 100-bp paired-end reads were generated.De novo assembly of the reads generated 81002 ifnal non-re-dundant contigs, from which 5261 full-length transcripts were identiifed. Among these full-length transcripts, 3169 had at least one Gene Ontology (GO) annotation, 2354 received cluster of orthologous groups (COG) terms, and 1992 were assigned a Kyoto encyclopedia of genes and genomes (KEGG) Orthology number. These sequence data represent a valuable resource for identiifcation ofZ. nicaraguensisgenes involved in waterlogging response.

  3. Delivery of basic fibroblast growth factors from heparinized decellularized adipose tissue stimulates potent de novo adipogenesis.

    Science.gov (United States)

    Lu, Qiqi; Li, Mingming; Zou, Yu; Cao, Tong

    2014-01-28

    Scaffolds based on decellularized adipose tissue (DAT) are gaining popularity in adipose tissue engineering due to their high biocompatibility and adipogenic inductive property. However, previous studies involving DAT-derived scaffolds have not fully revealed their potentials for in vivo adipose tissue construction. With the aim of developing a more efficient adipose tissue engineering technique based on DAT, in this study, we investigated the in vivo adipogenic potential of a basic fibroblast growth factor (bFGF) delivery system based on heparinized DAT (Hep-DAT). To generate this system, heparins were cross-linked to mouse DATs by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-Hydroxysuccinimide. The bFGF-binding Hep-DATs were first tested for controlled release ability in vitro and then transplanted subcutaneously. Highly vascularized adipose tissues were formed 6weeks after transplantation. Histology and gene expression analysis revealed that majority of the Hep-DAT scaffolds were infiltrated with host-derived adipose tissues that possessed similar adipogenic and inflammatory gene expression as endogenous adipose tissues. Additionally, strong de novo adipogenesis could also be induced when bFGF-binding Hep-DATs were thoroughly minced and injected subcutaneously. In conclusion, our study demonstrated that bFGF-binding Hep-DAT could be an efficient, biocompatible and injectable adipogenic system for in vivo adipose tissue engineering.

  4. De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy.

    Science.gov (United States)

    Syrbe, Steffen; Hedrich, Ulrike B S; Riesch, Erik; Djémié, Tania; Müller, Stephan; Møller, Rikke S; Maher, Bridget; Hernandez-Hernandez, Laura; Synofzik, Matthis; Caglayan, Hande S; Arslan, Mutluay; Serratosa, José M; Nothnagel, Michael; May, Patrick; Krause, Roland; Löffler, Heidrun; Detert, Katja; Dorn, Thomas; Vogt, Heinrich; Krämer, Günter; Schöls, Ludger; Mullis, Primus E; Linnankivi, Tarja; Lehesjoki, Anna-Elina; Sterbova, Katalin; Craiu, Dana C; Hoffman-Zacharska, Dorota; Korff, Christian M; Weber, Yvonne G; Steinlin, Maja; Gallati, Sabina; Bertsche, Astrid; Bernhard, Matthias K; Merkenschlager, Andreas; Kiess, Wieland; Gonzalez, Michael; Züchner, Stephan; Palotie, Aarno; Suls, Arvid; De Jonghe, Peter; Helbig, Ingo; Biskup, Saskia; Wolff, Markus; Maljevic, Snezana; Schüle, Rebecca; Sisodiya, Sanjay M; Weckhuysen, Sarah; Lerche, Holger; Lemke, Johannes R

    2015-04-01

    Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six isolated patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a new gene involved in human neurodevelopmental disorders through two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons.

  5. Identifying wrong assemblies in de novo short read primary sequence assembly contigs

    Indian Academy of Sciences (India)

    VANDNA CHAWLA; RAJNISH KUMAR; RAVI SHANKAR

    2016-09-01

    With the advent of short-reads-based genome sequencing approaches, large number of organisms are being sequencedall over the world. Most of these assemblies are done using some de novo short read assemblers and other relatedapproaches. However, the contigs produced this way are prone to wrong assembly. So far, there is a conspicuousdearth of reliable tools to identify mis-assembled contigs. Mis-assemblies could result from incorrectly deleted orwrongly arranged genomic sequences. In the present work various factors related to sequence, sequencing andassembling have been assessed for their role in causing mis-assembly by using different genome sequencing data.Finally, some mis-assembly detecting tools have been evaluated for their ability to detect the wrongly assembledprimary contigs, suggesting a lot of scope for improvement in this area. The present work also proposes a simpleunsupervised learning-based novel approach to identify mis-assemblies in the contigs which was found performingreasonably well when compared to the already existing tools to report mis-assembled contigs. It was observed that theproposed methodology may work as a complementary system to the existing tools to enhance their accuracy.

  6. Prenatal diagnosis and molecular cytogenetic analysis of a de novo isodicentric chromosome 18

    Science.gov (United States)

    Zhang, Yanliang; Dai, Yong; Ren, Jinghui; Wang, Linqian

    2010-01-01

    Isodicentric chromosome 18 [idic(18)] is rare structural aberration. We report on a prenatal case described by conventional and molecular cytogenetic analyses. The sonography at 24 weeks of gestation revealed multiple fetal anomalies; radial aplasia and ventricular septal defect were significant features. Routine karyotyping showed a derivative chromosome replacing one normal chromosome 18. The parental karyotypes were normal, indicating that the derivative chromosome was de novo. Array comparative genomic hybridization (array-CGH) revealed 18p11.21→qter duplication and 18p11.21→pter deletion for genomic DNA of the fetus. The breakpoint was located at 18p11.21 (between 12104527 bp and 12145199 bp from the telomere of 18p). Thus, the derivative chromosome was ascertained as idic(18)(qter→p11.21::p11.21→qter). Fluorescent in situ hybridization (FISH) confirmed that the derivative chromosome was idic(18). Our report describes a rare isodicentric chromosome 18 and demonstrates that array-CGH is a useful complementary tool to cytogenetic analysis for reliable identifying derivative chromosome. PMID:20864786

  7. De novo actin polymerization is required for model Hirano body formation in Dictyostelium

    Directory of Open Access Journals (Sweden)

    Yun Dong

    2016-06-01

    Full Text Available Hirano bodies are eosinophilic, actin-rich inclusions found in autopsied brains in numerous neurodegenerative diseases. The mechanism of Hirano body formation is unknown. Mass spectrometry analysis was performed to identify proteins from partially purified model Hirano bodies from Dictyostelium. This analysis identified proteins primarily belonging to ribosomes, proteasomes, mitochondria and cytoskeleton. Profilin, Arp/2/3 and WASH identified by mass spectrometry were found to colocalise with model Hirano bodies. Due to their roles in actin regulation, we selected these proteins for further investigation. Inhibition of the Arp2/3 complex by CK666 prevented formation of model Hirano bodies. Since Arp2/3 activation occurs via the WASH or WAVE complex, we next investigated how these proteins affect Hirano body formation. Whereas model Hirano bodies could form in WASH-deficient cells, they failed to form in cells lacking HSPC300, a member of the WAVE complex. We identified other proteins required for Hirano body formation that include profilin and VASP, an actin nucleation factor. In the case of VASP, both its G- and F-actin binding domains were required for model Hirano body formation. Collectively, our results indicate that de novo actin polymerization is required to form model Hirano bodies.

  8. De Novo Sequencing of Peptides from Top-Down Tandem Mass Spectra

    Energy Technology Data Exchange (ETDEWEB)

    Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.; VanDuijn, Martijn M.; Liu, Xiaowen; Tolić, Nikola; Dvorkin, Mikhail; Alexandrova, Sonya; Luider, Theo M.; Paša-Tolić, Ljiljana; Pevzner, Pavel A.

    2015-11-06

    De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technology, now gaining more and more popularity, opens new perspectives for protein analysis and characterization, implying a need in efficient algorithms for processing this kind of MS/MS data. Here we describe a method that allows to retrieve from a set of top-down MS/MS spectra long and accurate sequence fragments of the proteins contained in a sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down datasets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.

  9. De novo germinal mutations and other classes of non-traditional inheritance in humans

    Energy Technology Data Exchange (ETDEWEB)

    Mohrenweiser, H. [Lawrence Livermore National Laboratory, CA (United States)

    1994-12-31

    Genetic diseases provide a unique resource for the study of the molecular basis for biologically relevant, inherited variation. Review of variants at a series of disease loci suggest significant differences among loci in the relative frequency of classes of variants. Common mechanistic features are observed within each class of variant. The spectrum of events identified is a reflection of both the gene structure and the selective pressure necessary to generate a disease phenotype. This locus specificity has significant potential to compromise estimates of both background and induced germinal gene mutation rates. Aberrant inheritance has been the classical definition of a de novo germinal mutation. Recent studies have identified mosaicism as an alternative explanation for the non-traditional pattern of inheritance. Mosaicism is of unique concern for studies of induced mutation rates because this event would reflect exposure of grandparent(s) of the proband to genotoxic agents. This is in contrast to the {open_quotes}normal expectation{close_quotes} that induced mutations are the result of parental exposure. The observations on the frequency of mosaicism, in conjunction with the problems of incomplete ascertainment of alterations in DNA structure, increase the complexity of efforts to estimate induced germinal mutation rates in populations exposed to potentially genotoxic agents.

  10. Sporadic cardiac and skeletal myopathy caused by a de novo desmin mutation.

    Science.gov (United States)

    Park, K Y; Dalakas, M C; Semino-Mora, C; Lee, H S; Litvak, S; Takeda, K; Ferrans, V J; Goldfarb, L G

    2000-06-01

    Desmin myopathy is a familial or sporadic disorder characterized by intracytoplasmic accumulation of desmin in the muscle cells. We and others have previously identified desmin gene mutations in patients with familial myopathy, but close to 45% of the patients do not report previous family history of the disease. The present study was conducted to determine the cause of desmin myopathy in a sporadic patient presenting with symmetrical muscle weakness and atrophy combined with atrioventricular conduction block requiring a permanent pacemaker. A novel heterozygous R406W mutation in the desmin gene was identified by sequencing cDNA and genomic DNA. Expression of a construct containing the patient's mutant desmin cDNA in SW13 (vim-) cells demonstrated a high pathogenic potential of the R406W mutation. This mutation was not found in the patient's father, mother or sister by sequencing and restriction analysis. Testing with five microsatellite markers and four intragenic single nucleotide polymorphisms excluded alternative paternity. Haplotype analysis indicates that the patient's father was germ-line mosaic for the desmin mutation. We conclude that de novo mutations in the desmin gene may be the cause of sporadic forms of desmin-related cardiac and skeletal myopathy.

  11. PEP-FOLD: an online resource for de novo peptide structure prediction.

    Science.gov (United States)

    Maupetit, Julien; Derreumaux, Philippe; Tuffery, Pierre

    2009-07-01

    Rational peptide design and large-scale prediction of peptide structure from sequence remain a challenge for chemical biologists. We present PEP-FOLD, an online service, aimed at de novo modelling of 3D conformations for peptides between 9 and 25 amino acids in aqueous solution. Using a hidden Markov model-derived structural alphabet (SA) of 27 four-residue letters, PEP-FOLD first predicts the SA letter profiles from the amino acid sequence and then assembles the predicted fragments by a greedy procedure driven by a modified version of the OPEP coarse-grained force field. Starting from an amino acid sequence, PEP-FOLD performs series of 50 simulations and returns the most representative conformations identified in terms of energy and population. Using a benchmark of 25 peptides with 9-23 amino acids, and considering the reproducibility of the runs, we find that, on average, PEP-FOLD locates lowest energy conformations differing by 2.6 A Calpha root mean square deviation from the full NMR structures. PEP-FOLD can be accessed at http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD.

  12. ATR-16 due to a de novo complex rearrangement of chromosome 16.

    Science.gov (United States)

    Gallego, Marta S; Zelaya, Gabriela; Feliu, Aurora S; Rossetti, Liliana; Shaffer, Lisa G; Bailey, Kristen A; Bacino, Carlos A; Barreiro, Cristina Z

    2005-01-01

    We describe a child with ATR-16 [alpha-thalassemia (thal)/mental retardation], who was referred for genetic evaluation because of minor anomalies and developmental delay. Cytogenetic analysis demonstrated a de novo complex rearrangement of chromosome 16. Fluorescence in situ hybridization (FISH) analysis, using chromosome 16 subtelomeric probes, showed that this patient had a deletion of the distal short arm of chromosome 16 that contains the alpha-globin genes and a duplication of 16q. Analysis of the alpha-globin locus by Southern blot showed a half normal dose of the alpha-globin gene. Microsatellite marker studies revealed that the duplicated 16q region was maternal in origin. Hematological studies revealed anemia, hypochromia and occasional cells with Hb H inclusion bodies. A hematological screening for alpha-thal should be considered in patients with mild developmental delay and a suggestive phenotype of ATR-16 with microcytic hypochromic anemia and normal iron status. The stellate pattern of the iris, a new finding in our patient, may contribute to a better clinical delineation of both syndromes, ATR-16 and/or duplication of 16qter.

  13. De novo assembly and transcriptome analysis of the Mediterranean fruit fly Ceratitis capitata early embryos.

    Directory of Open Access Journals (Sweden)

    Marco Salvemini

    Full Text Available The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, belongs to the Tephritidae family, which includes a large number of other damaging pest species. The Medfly has been the first non-drosophilid fly species which has been genetically transformed paving the way for designing genetic-based pest control strategies. Furthermore, it is an experimentally tractable model, in which transient and transgene-mediated RNAi have been successfully used. We applied Illumina sequencing to total RNA preparations of 8-10 hours old embryos of C. capitata, This developmental window corresponds to the blastoderm cellularization stage. In summary, we assembled 42,614 transcripts which cluster in 26,319 unique transcripts of which 11,045 correspond to protein coding genes; we identified several hundreds of long ncRNAs; we found an enrichment of transcripts encoding RNA binding proteins among the highly expressed transcripts, such as CcTRA-2, known to be necessary to establish and, most likely, to maintain female sex of C. capitata. Our study is the first de novo assembly performed for Ceratitis capitata based on Illumina NGS technology during embryogenesis and it adds novel data to the previously published C. capitata EST databases. We expect that it will be useful for a variety of applications such as gene cloning and phylogenetic analyses, as well as to advance genetic research and biotechnological applications in the Medfly and other related Tephritidae.

  14. De novo assembly and transcriptome analysis of the Mediterranean fruit fly Ceratitis capitata early embryos.

    Science.gov (United States)

    Salvemini, Marco; Arunkumar, Kallare P; Nagaraju, Javaregowda; Sanges, Remo; Petrella, Valeria; Tomar, Archana; Zhang, Hongyu; Zheng, Weiwei; Saccone, Giuseppe

    2014-01-01

    The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, belongs to the Tephritidae family, which includes a large number of other damaging pest species. The Medfly has been the first non-drosophilid fly species which has been genetically transformed paving the way for designing genetic-based pest control strategies. Furthermore, it is an experimentally tractable model, in which transient and transgene-mediated RNAi have been successfully used. We applied Illumina sequencing to total RNA preparations of 8-10 hours old embryos of C. capitata, This developmental window corresponds to the blastoderm cellularization stage. In summary, we assembled 42,614 transcripts which cluster in 26,319 unique transcripts of which 11,045 correspond to protein coding genes; we identified several hundreds of long ncRNAs; we found an enrichment of transcripts encoding RNA binding proteins among the highly expressed transcripts, such as CcTRA-2, known to be necessary to establish and, most likely, to maintain female sex of C. capitata. Our study is the first de novo assembly performed for Ceratitis capitata based on Illumina NGS technology during embryogenesis and it adds novel data to the previously published C. capitata EST databases. We expect that it will be useful for a variety of applications such as gene cloning and phylogenetic analyses, as well as to advance genetic research and biotechnological applications in the Medfly and other related Tephritidae.

  15. First systematic experience of preimplantation genetic diagnosis for de-novo mutations.

    Science.gov (United States)

    Rechitsky, Svetlana; Pomerantseva, Ekaterina; Pakhalchuk, Tatiana; Pauling, Dana; Verlinsky, Oleg; Kuliev, Anver

    2011-04-01

    Standard preimplantation genetic diagnosis (PGD) cannot be applied for de-novo mutations (DNM), because neither origin nor relevant haplotypes are available for testing in single cells. PGD strategies were developed for 80 families with 38 genetic disorders, determined by 33 dominant, three recessive and two X-linked DNM. All three recessive mutations were of paternal origin, while of 93 dominant mutations, 40 were paternal, 46 maternal and seven detected in affected children. The development of specific PGD strategy for each couple involved DNA analysis of the parents and affected children prior to PGD, including a mutation verification, polymorphic marker evaluation, whole and single sperm testing to establish the normal and mutant haplotypes and PGD by polar body analysis and/or embryo biopsy. Overall, 151 PGD cycles were performed for 80 families, for which a specific PGD design has been established. The application of these protocols resulted in pre-selection and transfer of 219 (1.72 per cycle) DNM-free embryos in 127 (84.1%) PGD cycles, yielding 63 (49.6%) unaffected pregnancies and birth of 59 (46.5%) healthy children, confirmed to be free of DNM. The data show feasibility of PGD for DNM, which may routinely be performed with accuracy of over 99%, using the established PGD strategy.

  16. De novo assembly and transcriptome analysis of sclerotial development in Wolfiporia cocos.

    Science.gov (United States)

    Wu, Yayun; Zhu, Wenjun; Wei, Wei; Zhao, Xiaolong; Wang, Qi; Zeng, Wanyong; Zheng, Yonglian; Chen, Ping; Zhang, Shaopeng

    2016-08-22

    Wolfiporia cocos Ryvarden et Gilbertson, a well-known medicinal fungus in the Basidiomycetes, is widely distributed in East Asia. Its dried sclerotium, which is known as Fuling in China, has been used as a traditional crude drug in Chinese traditional medicine for thousand years. However, little is known about how the sclerotium is developed at the genetic level. In this study, the de novo sequencing of sclerotia of W. cocos (S1_initial stage; S2_developmental stage and S3_mature stage) was carried out by illumina HiSeq 2000 technology. 27,438 unigenes were assembled from ~30Gbp raw data, and 12,093 unigenes were significantly annotated. The analysis of expression profiles during development returned 304 differentially expressed genes (DEGs), which were clustered into four different groups according to their expression trends. Especially for the maturation stage (S3), the sclerotium exhibited a markedly different expression profile from other stages. We further showed that peroxisome, unsaturation of fatty acids and degradation pathway were respectively prevalent in S1, S2 and S3 stages as evidenced by enrichment analysis. To our knowledge, this study represents the first report of sclerotial development transcriptomics in W. cocos. The obtained results provide novel insights into the developmental biology of the sclerotia, which is helpful for future studies about cultivation and breeding of W. cocos.

  17. RepARK--de novo creation of repeat libraries from whole-genome NGS reads.

    Science.gov (United States)

    Koch, Philipp; Platzer, Matthias; Downie, Bryan R

    2014-05-01

    Generation of repeat libraries is a critical step for analysis of complex genomes. In the era of next-generation sequencing (NGS), such libraries are usually produced using a whole-genome shotgun (WGS) derived reference sequence whose completeness greatly influences the quality of derived repeat libraries. We describe here a de novo repeat assembly method--RepARK (Repetitive motif detection by Assembly of Repetitive K-mers)--which avoids potential biases by using abundant k-mers of NGS WGS reads without requiring a reference genome. For validation, repeat consensuses derived from simulated and real Drosophila melanogaster NGS WGS reads were compared to repeat libraries generated by four established methods. RepARK is orders of magnitude faster than the other methods and generates libraries that are: (i) composed almost entirely of repetitive motifs, (ii) more comprehensive and (iii) almost completely annotated by TEclass. Additionally, we show that the RepARK method is applicable to complex genomes like human and can even serve as a diagnostic tool to identify repetitive sequences contaminating NGS datasets.

  18. De Novo Identification of Regulatory Regions in Intergenic Spaces of Prokaryotic Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Chain, P; Garcia, E; Mcloughlin, K; Ovcharenko, I

    2007-02-20

    This project was begun to implement, test, and experimentally validate the results of a novel algorithm for genome-wide identification of candidate transcription-factor binding sites in prokaryotes. Most techniques used to identify regulatory regions rely on conservation between different genomes or have a predetermined sequence motif(s) to perform a genome-wide search. Therefore, such techniques cannot be used with new genome sequences, where information regarding such motifs has not yet been discovered. This project aimed to apply a de novo search algorithm to identify candidate binding-site motifs in intergenic regions of prokaryotic organisms, initially testing the available genomes of the Yersinia genus. We retrofitted existing nucleotide pattern-matching algorithms, analyzed the candidate sites identified by these algorithms as well as their target genes to screen for meaningful patterns. Using properly annotated prokaryotic genomes, this project aimed to develop a set of procedures to identify candidate intergenic sites important for gene regulation. We planned to demonstrate this in Yersinia pestis, a model biodefense, Category A Select Agent pathogen, and then follow up with experimental evidence that these regions are indeed involved in regulation. The ability to quickly characterize transcription-factor binding sites will help lead to a better understanding of how known virulence pathways are modulated in biodefense-related organisms, and will help our understanding and exploration of regulons--gene regulatory networks--and novel pathways for metabolic processes in environmental microbes.

  19. Rnnotator: an automated de novo transcriptome assembly pipeline from stranded RNA-Seq reads

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Bruno, Vincent M.; Fang, Zhide; Meng, Xiandong; Blow, Matthew; Zhang, Tao; Sherlock, Gavin; Snyder, Michael; Wang, Zhong

    2010-11-19

    Background: Comprehensive annotation and quantification of transcriptomes are outstanding problems in functional genomics. While high throughput mRNA sequencing (RNA-Seq) has emerged as a powerful tool for addressing these problems, its success is dependent upon the availability and quality of reference genome sequences, thus limiting the organisms to which it can be applied. Results: Here, we describe Rnnotator, an automated software pipeline that generates transcript models by de novo assembly of RNA-Seq data without the need for a reference genome. We have applied the Rnnotator assembly pipeline to two yeast transcriptomes and compared the results to the reference gene catalogs of these organisms. The contigs produced by Rnnotator are highly accurate (95percent) and reconstruct full-length genes for the majority of the existing gene models (54.3percent). Furthermore, our analyses revealed many novel transcribed regions that are absent from well annotated genomes, suggesting Rnnotator serves as a complementary approach to analysis based on a reference genome for comprehensive transcriptomics. Conclusions: These results demonstrate that the Rnnotator pipeline is able to reconstruct full-length transcripts in the absence of a complete reference genome.

  20. De Novo Transcriptome Analysis and Detection of Antimicrobial Peptides of the American Cockroach Periplaneta americana (Linnaeus.

    Directory of Open Access Journals (Sweden)

    In-Woo Kim

    Full Text Available Cockroaches are surrogate hosts for microbes that cause many human diseases. In spite of their generally destructive nature, cockroaches have recently been found to harbor potentially beneficial and medically useful substances such as drugs and allergens. However, genomic information for the American cockroach (Periplaneta americana is currently unavailable; therefore, transcriptome and gene expression profiling is needed as an important resource to better understand the fundamental biological mechanisms of this species, which would be particularly useful for the selection of novel antimicrobial peptides. Thus, we performed de novo transcriptome analysis of P. americana that were or were not immunized with Escherichia coli. Using an Illumina HiSeq sequencer, we generated a total of 9.5 Gb of sequences, which were assembled into 85,984 contigs and functionally annotated using Basic Local Alignment Search Tool (BLAST, Gene Ontology (GO, and Kyoto Encyclopedia of Genes and Genomes (KEGG database terms. Finally, using an in silico antimicrobial peptide prediction method, 86 antimicrobial peptide candidates were predicted from the transcriptome, and 21 of these peptides were experimentally validated for their antimicrobial activity against yeast and gram positive and -negative bacteria by a radial diffusion assay. Notably, 11 peptides showed strong antimicrobial activities against these organisms and displayed little or no cytotoxic effects in the hemolysis and cell viability assay. This work provides prerequisite baseline data for the identification and development of novel antimicrobial peptides, which is expected to provide a better understanding of the phenomenon of innate immunity in similar species.

  1. De novo synthesis of estrogen in pregnant uterus is critical for stromal decidualization and angiogenesis.

    Science.gov (United States)

    Das, Amrita; Mantena, Srinivasa Raju; Kannan, Athilakshmi; Evans, Dean B; Bagchi, Milan K; Bagchi, Indrani C

    2009-07-28

    Implantation is initiated when the embryo attaches to the uterine luminal epithelium during early pregnancy. Following this event, uterine stromal cells undergo steroid hormone-dependent transformation into morphologically and functionally distinct decidual cells in a unique process known as decidualization. An angiogenic network is also formed in the uterine stromal bed, critically supporting the early development of the embryo. The steroid-induced mechanisms that promote stromal differentiation and endothelial proliferation during decidualization are not fully understood. Although the role of ovarian progesterone as a key regulator of decidualization is well established, the requirement of ovarian estrogen (E) during this process remains unresolved. Here we show that the expression of P450 aromatase, a key enzyme that converts androgens to E, is markedly induced in mouse uterine stromal cells undergoing decidualization. The aromatase then acts in conjunction with other steroid biosynthetic enzymes present in the decidual tissue to support de novo synthesis of E. This locally produced E is able to support the advancement of the stromal differentiation program even in the absence ovarian E in an ovariectomized, progesterone-supplemented pregnant mouse model. Administration of letrozole, a specific aromatase inhibitor, to these mice blocked the stromal differentiation process. Gene expression profiling further revealed that the intrauterine E induces the expression of several stromal factors that promote neovascularization in the decidual tissue. Collectively, these studies identified the decidual uterus as a novel site of E biosynthesis and uncovered E-regulated maternal signaling pathways that critically control uterine differentiation and angiogenesis during early pregnancy.

  2. Recurrent de novo BICD2 mutation associated with arthrogryposis multiplex congenita and bilateral perisylvian polymicrogyria.

    Science.gov (United States)

    Ravenscroft, Gianina; Di Donato, Nataliya; Hahn, Gabriele; Davis, Mark R; Craven, Paul D; Poke, Gemma; Neas, Katherine R; Neuhann, Teresa M; Dobyns, William B; Laing, Nigel G

    2016-11-01

    Autosomal dominantly inherited mutations of BICD2 are associated with congenital-onset spinal muscular atrophy characterised by lower limb predominance. A few cases have also showed upper motor neuron pathology, including presenting with features resembling hereditary spastic paraplegia. The age-of-onset for the published families is usually at birth but also included cases with childhood- and adult-onset disease. In this report we described two isolated probands that presented in utero with features associated with reduced fetal movements. Both cases were diagnosed at birth with arthrogryposis multiplex congenita (AMC) and hypotonia. Other variable features included congenital fractures, hip dislocation, micrognathia, respiratory insufficiency, microcephaly and bilateral perisylvian polymicrogyria. Patient 1 is 4 years of age and stable, but shows significant motor developmental delay and delayed speech. Patient 2 passed away at 7 weeks of age. Through next generation sequencing we identified the same missense substitution in BICD2 (p.Arg694Cys) in both probands. Sanger sequencing showed that in both cases the mutation arose de novo. The in utero onset in both cases suggests that the p.Arg694Cys substitution may have a more deleterious effect on BICD2 function than previously described mutations. Our results broaden the phenotypes associated with BICD2 mutations to include AMC and cortical malformations and therefore to a similar phenotypic spectrum to that associated with its binding partner DYNC1H1.

  3. De Novo Assembly of the Pea (Pisum sativum L. Nodule Transcriptome

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    Vladimir A. Zhukov

    2015-01-01

    Full Text Available The large size and complexity of the garden pea (Pisum sativum L. genome hamper its sequencing and the discovery of pea gene resources. Although transcriptome sequencing provides extensive information about expressed genes, some tissue-specific transcripts can only be identified from particular organs under appropriate conditions. In this study, we performed RNA sequencing of polyadenylated transcripts from young pea nodules and root tips on an Illumina GAIIx system, followed by de novo transcriptome assembly using the Trinity program. We obtained more than 58,000 and 37,000 contigs from “Nodules” and “Root Tips” assemblies, respectively. The quality of the assemblies was assessed by comparison with pea expressed sequence tags and transcriptome sequencing project data available from NCBI website. The “Nodules” assembly was compared with the “Root Tips” assembly and with pea transcriptome sequencing data from projects indicating tissue specificity. As a result, approximately 13,000 nodule-specific contigs were found and annotated by alignment to known plant protein-coding sequences and by Gene Ontology searching. Of these, 581 sequences were found to possess full CDSs and could thus be considered as novel nodule-specific transcripts of pea. The information about pea nodule-specific gene sequences can be applied for gene-based markers creation, polymorphism studies, and real-time PCR.

  4. Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

    Institute of Scientific and Technical Information of China (English)

    Bin-Zhong Li; Guo-Liang Xu; Zheng Huang; Qing-Yan Cui; Xue-Hui Song; Lin Du; Albert Jeltsch; Ping Chen; Guohong Li; En Li

    2011-01-01

    Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.

  5. Trans-10, cis-12 conjugated linoleic acid decreases de novo lipid synthesis in human adipocytes

    DEFF Research Database (Denmark)

    Obsen, Thomas; Faergeman, Nils J; Chung, Soonkyu;

    2012-01-01

    Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid......]-oleic or [(14)C]-linoleic acids. When using [(14)C]-acetic acid and [(14)C]-pyruvic acid as substrates, 30 μM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3-24 h. Treatment with 30 μM 10......,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within...

  6. A de novo microdeletion involving PAFAH1B (LIS1 related to lissencephaly phenotype

    Directory of Open Access Journals (Sweden)

    Keiko Shimojima

    2015-09-01

    Full Text Available Lissencephaly is a type of the congenital malformation of the brain. Due to the impairments of neuronal migration, patients show absence of brain convolution manifesting smooth brain surfaces. One of the human genes responsible for lissencephaly is the platelet-activating factor acetylhydrolase 1b gene (PAFAH1B; also known as LIS1 located on 17p13.3. Patients with heterozygous deletion of this chromosomal region exhibit lissencephaly. Recently, we encountered a male patient who showed typical lissencephaly. Using a microarray analysis, we identified a 1.3 Mb submicroscopic deletion in 17p13.3. This deletion included PAFAH1B. Both of the parents showed no deletion in this region. Therefore, this was determined to be derived from de novo origin. After obtaining the written informed consent, skin fibroblasts were provided from this patient and disease-specific induced pluripotent stem (iPS cells were generated and used for medical research (Shimojima K, Okumura A, Hayashi M, Kondo T, Inoue H, and Yamamoto T. CHCHD2 is down-regulated in neuronal cells differentiated from iPS cells derived from patients with lissencephaly. Genomics, in press.

  7. Antilope - A Lagrangian Relaxation Approach to the de novo Peptide Sequencing Problem

    CERN Document Server

    Andreotti, Sandro; Reinert, Knut

    2011-01-01

    Peptide sequencing from mass spectrometry data is a key step in proteome research. Especially de novo sequencing, the identification of a peptide from its spectrum alone, is still a challenge even for state-of-the-art algorithmic approaches. In this paper we present Antilope, a new fast and flexible approach based on mathematical programming. It builds on the spectrum graph model and works with a variety of scoring schemes. Antilope combines Lagrangian relaxation for solving an integer linear programming formulation with an adaptation of Yen's k shortest paths algorithm. It shows a significant improvement in running time compared to mixed integer optimization and performs at the same speed like other state-of-the-art tools. We also implemented a generic probabilistic scoring scheme that can be trained automatically for a dataset of annotated spectra and is independent of the mass spectrometer type. Evaluations on benchmark data show that Antilope is competitive to the popular state-of-the-art programs PepNovo...

  8. Carbopeptides: carbohydrates as potential templates for de novo design of protein models.

    Science.gov (United States)

    Jensen, K J; Barany, G

    2000-07-01

    De novo design of proteins has evolved into a powerful approach for studying the factors governing protein folding and stability. Among the families of structures frequently studied is the 'four-helix bundle' in which four alpha-helical peptide strands, linked by loops, form a hydrophobic core. Assembly of protein models on a template has been suggested as a way to reduce the entropy of folding. Here we describe the potential use of a carbohydrate as such a template. The monosaccharide D-galactose was per-O-acylated with (Nbeta-Fmoc-betaAla)2O to give a penta-substituted derivative, which was converted to the corresponding glycosyl bromide and used for the glycosylation of 4-hydroxymethylbenzoic acid pentafluorophenyl ester (HMBA-OPfp). The beta-glycosidic carbohydrate template (Nbeta-Fmoc-3Ala)4-beta-D-Galp-(1-O)-MBA-OPfp thus obtained was coupled to a PAL-PEG-PS resin and simultaneously extended at the four arms to yield, after cleavage from the solid support, a carbopeptide with four identical peptide strands. Extension of this concept to, for example, synthesis of novel multiple antigenic peptides (MAPs) and synthesis of carbohydrate clusters can be easily envisioned. The ability to efficiently synthesize such structures sets the stage for further studies to test whether the carbohydrate templates do indeed nucleate folding.

  9. De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

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    Koopman Frank

    2012-12-01

    Full Text Available Abstract Background Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules. Result Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations ( Conclusion The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.

  10. Specific activation of human interleukin-5 depends on de novo synthesis of an AP-1 complex.

    Science.gov (United States)

    Schwenger, Gretchen T F; Kok, Chee Choy; Arthaningtyas, Estri; Thomas, Marc A; Sanderson, Colin J; Mordvinov, Viatcheslav A

    2002-12-06

    It is clear from the biology of eosinophilia that a specific regulatory mechanism must exist. Because interleukin-5 (IL5) is the key regulatory cytokine, it follows that a gene-specific control of IL5 expression must exist that differs even from closely related cytokines such as IL4. Two features of IL5 induction make it unique compared with other cytokines; first, induction by cyclic adenosine monophosphate (cAMP), which inhibits other T-cell-derived cytokines, and second, sensitivity to protein synthesis inhibitors, which have no effect on other cytokines. This study has utilized the activation of different transcription factors by different stimuli in a human T-cell line to study the role of conserved lymphokine element 0 (CLE0) in the specific induction of IL5. In unstimulated cells the ubiquitous Oct-1 binds to CLE0. Stimulation induces de novo synthesis of the AP-1 members JunD and Fra-2, which bind to CLE0. The amount of IL5 produced correlates with the production of the AP-1 complex, suggesting a key role in IL5 expression. The formation of the AP-1 complex is essential, but the rate-limiting step is the synthesis of AP-1, especially Fra-2. This provides an explanation for the sensitivity of IL5 to protein synthesis inhibitors and a mechanism for the specific induction of IL5 compared with other cytokines.

  11. De novo transcriptome assembly databases for the butterfly orchid Phalaenopsis equestris

    Science.gov (United States)

    Niu, Shan-Ce; Xu, Qing; Zhang, Guo-Qiang; Zhang, Yong-Qiang; Tsai, Wen-Chieh; Hsu, Jui-Ling; Liang, Chieh-Kai; Luo, Yi-Bo; Liu, Zhong-Jian

    2016-01-01

    Orchids are renowned for their spectacular flowers and ecological adaptations. After the sequencing of the genome of the tropical epiphytic orchid Phalaenopsis equestris, we combined Illumina HiSeq2000 for RNA-Seq and Trinity for de novo assembly to characterize the transcriptomes for 11 diverse P. equestris tissues representing the root, stem, leaf, flower buds, column, lip, petal, sepal and three developmental stages of seeds. Our aims were to contribute to a better understanding of the molecular mechanisms driving the analysed tissue characteristics and to enrich the available data for P. equestris. Here, we present three databases. The first dataset is the RNA-Seq raw reads, which can be used to execute new experiments with different analysis approaches. The other two datasets allow different types of searches for candidate homologues. The second dataset includes the sets of assembled unigenes and predicted coding sequences and proteins, enabling a sequence-based search. The third dataset consists of the annotation results of the aligned unigenes versus the Nonredundant (Nr) protein database, Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG) databases with low e-values, enabling a name-based search. PMID:27673730

  12. Optimizing k-mer size using a variant grid search to enhance de novo genome assembly

    Science.gov (United States)

    Cha, Soyeon; Bird, David McK

    2016-01-01

    Largely driven by huge reductions in per-base costs, sequencing nucleic acids has become a near-ubiquitous technique in laboratories performing biological and biomedical research. Most of the effort goes to re-sequencing, but assembly of de novogenerated, raw sequence reads into contigs that span as much of the genome as possible is central to many projects. Although truly complete coverage is not realistically attainable, maximizing the amount of sequence that can be correctly assembled into contigs contributes to coverage. Here we compare three commonly used assembly algorithms (ABySS, Velvet and SOAPdenovo2), and show that empirical optimization of k-mer values has a disproportionate influence on de novo assembly of a eukaryotic genome, the nematode parasite Meloidogynechitwoodi. Each assembler was challenged with about 40 million Iluumina II paired-end reads, and assemblies performed under a range of k-mer sizes. In each instance, the optimal k-mer was 127, although based on N50 values,ABySS was more efficient than the others. That the assembly was not spurious was established using the “Core Eukaryotic Gene Mapping Approach”, which indicated that 98.79% of the M. chitwoodi genome was accounted for by the assembly. Subsequent gene finding and annotation are consistent with this and suggest that k-mer optimization contributes to the robustness of assembly. PMID:28104957

  13. Transcriptome sequencing and de novo analysis of the copepod Calanus sinicus using 454 GS FLX.

    Directory of Open Access Journals (Sweden)

    Juan Ning

    Full Text Available BACKGROUND: Despite their species abundance and primary economic importance, genomic information about copepods is still limited. In particular, genomic resources are lacking for the copepod Calanus sinicus, which is a dominant species in the coastal waters of East Asia. In this study, we performed de novo transcriptome sequencing to produce a large number of expressed sequence tags for the copepod C. sinicus. RESULTS: Copepodid larvae and adults were used as the basic material for transcriptome sequencing. Using 454 pyrosequencing, a total of 1,470,799 reads were obtained, which were assembled into 56,809 high quality expressed sequence tags. Based on their sequence similarity to known proteins, about 14,000 different genes were identified, including members of all major conserved signaling pathways. Transcripts that were putatively involved with growth, lipid metabolism, molting, and diapause were also identified among these genes. Differentially expressed genes related to several processes were found in C. sinicus copepodid larvae and adults. We detected 284,154 single nucleotide polymorphisms (SNPs that provide a resource for gene function studies. CONCLUSION: Our data provide the most comprehensive transcriptome resource available for C. sinicus. This resource allowed us to identify genes associated with primary physiological processes and SNPs in coding regions, which facilitated the quantitative analysis of differential gene expression. These data should provide foundation for future genetic and genomic studies of this and related species.

  14. De novo genome assembly of Geosmithia morbida, the causal agent of thousand cankers disease.

    Science.gov (United States)

    Schuelke, Taruna A; Westbrook, Anthony; Broders, Kirk; Woeste, Keith; MacManes, Matthew D

    2016-01-01

    Geosmithia morbida is a filamentous ascomycete that causes thousand cankers disease in the eastern black walnut tree. This pathogen is commonly found in the western U.S.; however, recently the disease was also detected in several eastern states where the black walnut lumber industry is concentrated. G. morbida is one of two known phytopathogens within the genus Geosmithia, and it is vectored into the host tree via the walnut twig beetle. We present the first de novo draft genome of G. morbida. It is 26.5 Mbp in length and contains less than 1% repetitive elements. The genome possesses an estimated 6,273 genes, 277 of which are predicted to encode proteins with unknown functions. Approximately 31.5% of the proteins in G. morbida are homologous to proteins involved in pathogenicity, and 5.6% of the proteins contain signal peptides that indicate these proteins are secreted. Several studies have investigated the evolution of pathogenicity in pathogens of agricultural crops; forest fungal pathogens are often neglected because research efforts are focused on food crops. G. morbida is one of the few tree phytopathogens to be sequenced, assembled and annotated. The first draft genome of G. morbida serves as a valuable tool for comprehending the underlying molecular and evolutionary mechanisms behind pathogenesis within the Geosmithia genus.

  15. SparseAssembler: de novo Assembly with the Sparse de Bruijn Graph

    CERN Document Server

    Ye, Chengxi; Ma,; Cannon, Charles H; Pop, Mihai; Yu, Douglas W

    2011-01-01

    de Bruijn graph-based algorithms are one of the two most widely used approaches for de novo genome assembly. A major limitation of this approach is the large computational memory space requirement to construct the de Bruijn graph, which scales with k-mer length and total diversity (N) of unique k-mers in the genome expressed in base pairs or roughly (2k+8)N bits. This limitation is particularly important with large-scale genome analysis and for sequencing centers that simultaneously process multiple genomes. We present a sparse de Bruijn graph structure, based on which we developed SparseAssembler that greatly reduces memory space requirements. The structure also allows us to introduce a novel method for the removal of substitution errors introduced during sequencing. The sparse de Bruijn graph structure skips g intermediate k-mers, therefore reducing the theoretical memory space requirement to ~(2k/g+8)N. We have found that a practical value of g=16 consumes approximately 10% of the memory required by standa...

  16. De Novo Assembly and Characterization of Oryza officinalis Leaf Transcriptome by Using RNA-Seq

    Directory of Open Access Journals (Sweden)

    Ying Bao

    2015-01-01

    Full Text Available Although endeavors have been made to identify useful wild rice genes that can be used to improve cultivated rice, the virtual reservoir of genetic variation hidden within the wild relatives of cultivated rice is largely untapped. Here, using next-generation sequencing technology, we investigated the leaf transcriptome of a wild rice O. officinalis with CC genome. Approximately 23 million reads were produced in the species leaf transcriptome analysis and de novo assembly methods constructed 68,132 unigenes. Functional annotations for the unigenes were conducted using sequence similarity comparisons against the following databases: the nonredundant nucleotide database, the nonredundant protein database, the SWISS-PROT database, the Clusters of Orthologous Groups of proteins database, the Kyoto Encyclopedia of Genes and Genomes database, the Gene Ontology Consortium database, and the InterPro domains database. In addition, a total of 476 unigenes related to disease resistance were identified in O. officinalis, and these unigenes can serve as important genetic resources for cultivated rice breeding and quality improvement. The present study broadens our understanding of the genetic background of non-AA genomic wild rice species and it also provides a bridge to extend studies to other Oryza species with CC genomes.

  17. De Novo Assembly and Characterization of Sophora japonica Transcriptome Using RNA-seq

    Directory of Open Access Journals (Sweden)

    Liucun Zhu

    2014-01-01

    Full Text Available Sophora japonica Linn (Chinese Scholar Tree is a shrub species belonging to the subfamily Faboideae of the pea family Fabaceae. In this study, RNA sequencing of S. japonica transcriptome was performed to produce large expression datasets for functional genomic analysis. Approximate 86.1 million high-quality clean reads were generated and assembled de novo into 143010 unique transcripts and 57614 unigenes. The average length of unigenes was 901 bps with an N50 of 545 bps. Four public databases, including the NCBI nonredundant protein (NR, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG, and the Cluster of Orthologous Groups (COG, were used to annotate unigenes through NCBI BLAST procedure. A total of 27541 of 57614 unigenes (47.8% were annotated for gene descriptions, conserved protein domains, or gene ontology. Moreover, an interaction network of unigenes in S. japonica was predicted based on known protein-protein interactions of putative orthologs of well-studied plant genomes. The transcriptome data of S. japonica reported here represents first genome-scale investigation of gene expressions in Faboideae plants. We expect that our study will provide a useful resource for further studies on gene expression, genomics, functional genomics, and protein-protein interaction in S. japonica.

  18. Transcriptome sequencing and De Novo analysis of Youngia japonica using the illumina platform.

    Directory of Open Access Journals (Sweden)

    Yulan Peng

    Full Text Available Youngia japonica, a weed species distributed worldwide, has been widely used in traditional Chinese medicine. It is an ideal plant for studying the evolution of Asteraceae plants because of its short life history and abundant source. However, little is known about its evolution and genetic diversity. In this study, de novo transcriptome sequencing was conducted for the first time for the comprehensive analysis of the genetic diversity of Y. japonica. The Y. japonica transcriptome was sequenced using Illumina paired-end sequencing technology. We produced 21,847,909 high-quality reads for Y. japonica and assembled them into contigs. A total of 51,850 unigenes were identified, among which 46,087 were annotated in the NCBI non-redundant protein database and 41,752 were annotated in the Swiss-Prot database. We mapped 9,125 unigenes onto 163 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. In addition, 3,648 simple sequence repeats (SSRs were detected. Our data provide the most comprehensive transcriptome resource currently available for Y. japonica. C4 photosynthesis unigenes were found in the biological process of Y. japonica. There were 5596 unigenes related to defense response and 1344 ungienes related to signal transduction mechanisms (10.95%. These data provide insights into the genetic diversity of Y. japonica. Numerous SSRs contributed to the development of novel markers. These data may serve as a new valuable resource for genomic studies on Youngia and, more generally, Cichoraceae.

  19. Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome.

    Science.gov (United States)

    Goodwin, Sara; Gurtowski, James; Ethe-Sayers, Scott; Deshpande, Panchajanya; Schatz, Michael C; McCombie, W Richard

    2015-11-01

    Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available, and we used this for sequencing the Saccharomyces cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr specifically for Oxford Nanopore reads, because existing packages were incapable of assembling the long read lengths (5-50 kbp) at such high error rates (between ∼5% and 40% error). With this new method, we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: The contig N50 length is more than ten times greater than an Illumina-only assembly (678 kb versus 59.9 kbp) and has >99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly.

  20. ELN 2013 response status criteria: relevance for de novo imatinib chronic phase chronic myeloid leukemia patients?

    Science.gov (United States)

    Etienne, Gabriel; Dulucq, Stéphanie; Lascaux, Axelle; Schmitt, Anna; Bidet, Audrey; Fort, Marie-Pierre; Lippert, Eric; Bureau, Caroline; Adiko, Didier; Hayette, Sandrine; Reiffers, Josy; Nicolini, Franck-Emmanuel; Mahon, François-Xavier

    2015-01-01

    The response definitions proposed by the European Leukemia Net (ELN) have been recently modified. We evaluated the new criteria for de novo imatinib (400 mg/d) chronic phase chronic myeloid leukemia (CP-CML) patients. Response status according to the 2009 and 2013 criteria were determined in 180 unselected patients. Outcome of the subgroups of patients were then compared. The 180 patients were classified as optimal responders (OR2009; n = 113, 62.7%), suboptimal responders (SOR2009; n = 47, 26.1%) and failures (FAIL2009; n = 20, 11.1%) according to the 2009 ELN criteria and optimal responders (OR2013; n = 77, 42.7%), warnings (WAR2013; n = 59, 32.7%), and failures (FAIL2013; n = 44, 24.4%) according to the 2013 ELN criteria. No difference in terms of outcome was observed between OR2009 patients who became WAR2013 when compared with OR2013 patients. When compared with FAIL2009 patients, SOR2009 patients who became WAR2013 had better EFS, FFS, PFS, and OS. No difference was observed in PFS or OS in SOR2009 patients who became FAIL2013. The 2013 ELN response status criteria have improved patients classification in terms of response status. However, in our patient population this improvement is related to a better definition of failure rather than that of optimal response for CP-CML patients treated with IM frontline therapy.

  1. Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

    Science.gov (United States)

    Saha, Bodhisattwa; Sircar, Gaurab; Pandey, Naren; Gupta Bhattacharya, Swati

    2015-11-06

    Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

  2. De novo assembly of the transcriptome of an invasive snail and its multiple ecological applications.

    Science.gov (United States)

    Sun, J; Wang, M; Wang, H; Zhang, H; Zhang, X; Thiyagarajan, V; Qian, P Y; Qiu, J W

    2012-11-01

    Studying how invasive species respond to environmental stress at the molecular level can help us assess their impact and predict their range expansion. Development of markers of genetic polymorphism can help us reconstruct their invasive route. However, to conduct such studies requires the presence of substantial amount of genomic resources. This study aimed to generate and characterize genomic resources using high throughput transcriptome sequencing for Pomacea canaliculata, a nonmodel gastropod indigenous to Argentina that has invaded Asia, Hawaii and southern United States. De novo assembly of the transcriptome resulted in 128,436 unigenes with an average length of 419 bp (range: 150-8556 bp). Many of the unigenes (2439) contained transposable elements, showing the existence of a source of genetic variability in response to stressful conditions. A total of 3196 microsatellites were detected in the transcriptome; among 20 of the randomly tested microsatellites, 10 were validated to exhibit polymorphism. A total of 15,412 single-nucleotide polymorphisms (SNPs) were detected in the ORFs. LC-MS/MS analysis of the proteome of juveniles revealed 878 proteins, of which many are stress related. This study has demonstrated the great potential of high throughput DNA sequencing for rapid development of genomic resources for a nonmodel organism. Such resources can facilitate various molecular ecological studies, such as stress physiology and range expansion.

  3. De novo genic mutations among a Chinese autism spectrum disorder cohort

    Science.gov (United States)

    Wang, Tianyun; Guo, Hui; Xiong, Bo; Stessman, Holly A.F.; Wu, Huidan; Coe, Bradley P.; Turner, Tychele N.; Liu, Yanling; Zhao, Wenjing; Hoekzema, Kendra; Vives, Laura; Xia, Lu; Tang, Meina; Ou, Jianjun; Chen, Biyuan; Shen, Yidong; Xun, Guanglei; Long, Min; Lin, Janice; Kronenberg, Zev N.; Peng, Yu; Bai, Ting; Li, Honghui; Ke, Xiaoyan; Hu, Zhengmao; Zhao, Jingping; Zou, Xiaobing; Xia, Kun; Eichler, Evan E.

    2016-01-01

    Recurrent de novo (DN) and likely gene-disruptive (LGD) mutations contribute significantly to autism spectrum disorders (ASDs) but have been primarily investigated in European cohorts. Here, we sequence 189 risk genes in 1,543 Chinese ASD probands (1,045 from trios). We report an 11-fold increase in the odds of DN LGD mutations compared with expectation under an exome-wide neutral model of mutation. In aggregate, ∼4% of ASD patients carry a DN mutation in one of just 29 autism risk genes. The most prevalent gene for recurrent DN mutations is SCN2A (1.1% of patients) followed by CHD8, DSCAM, MECP2, POGZ, WDFY3 and ASH1L. We identify novel DN LGD recurrences (GIGYF2, MYT1L, CUL3, DOCK8 and ZNF292) and DN mutations in previous ASD candidates (ARHGAP32, NCOR1, PHIP, STXBP1, CDKL5 and SHANK1). Phenotypic follow-up confirms potential subtypes and highlights how large global cohorts might be leveraged to prove the pathogenic significance of individually rare mutations. PMID:27824329

  4. A Presença da Gastronomia Alemã na Hotelaria de Novo Hamburgo, RS

    Directory of Open Access Journals (Sweden)

    mary sandra ashton

    2013-06-01

    Full Text Available Esse estudo tem por objetivo investigar os elementos e características da gastronomia alemã presentes nos serviços hoteleiros de Novo Hamburgo. Tem ênfase na gastronomia, evidenciando e (re conhecendo os hábitos e costumes alimentares trazidos pela imigração germânica e sua relação com a oferta de alimentos nos hotéis da cidade, como diferencial no quesito turismo cultural. Para tanto, foram utilizados autores reconhecidos na área, para sustentação teórica. Adotou-se o método exploratório descritivo e entre os procedimentos técnicos utilizou-se revisão bibliográfica e pesquisa de campo com entrevistas. Como resultado desse estudo percebeu-se que há uma sintonia entre os costumes alimentares mantidos pelos germano-descendentes e a intenção dos gestores do setor hoteleiro, nas tentativas de manter as características e elementos presentes na gastronomia alemã, porém isso não representou diferencial para os hóspedes, sendo aos poucos substituída pelo modelo que utiliza elementos da gastronomia denominada internacional.

  5. De novo transcriptome assembly of 'Angeleno' and 'Lamoon' Japanese plum cultivars (Prunus salicina).

    Science.gov (United States)

    González, Máximo; Maldonado, Jonathan; Salazar, Erika; Silva, Herman; Carrasco, Basilio

    2016-09-01

    Japanese plum (Prunus salicina L.) is a fruit tree of the Rosaceae family, which is an economically important stone fruit around the world. Currently, Japanese plum breeding programs combine traditional breeding and plant physiology strategies with genetic and genomic analysis. In order to understand the flavonoid pathway regulation and to develop molecular markers associated to the fuit skin color (EST-SSRs), we performed a next generation sequencing based on Illumina Hiseq2000 platform. A total of 22.4 GB and 21 GB raw data were obtained from 'Lamoon' and 'Angeleno' respectively, corresponding to 85,404,726 raw reads to 'Lamoon' and 79,781,666 to 'Angeleno'. A total of 139,775,975 reads were filtered after removing low-quality reads and trimming the adapter sequences. De novo transcriptome assembly was performed using CLC Genome Workbench software and a total of 54,584 unique contigs were generated, with an N50 of 1343 base pair (bp) and a mean length of 829 bp. This work contributed with a specific Japanese plum skin transcriptome, providing two libraries of contrasting fruit skin color phenotype (yellow and red) and increasing substantially the GB of raw data available until now for this specie.

  6. De Novo Design of Skin-Penetrating Peptides for Enhanced Transdermal Delivery of Peptide Drugs.

    Science.gov (United States)

    Menegatti, Stefano; Zakrewsky, Michael; Kumar, Sunny; De Oliveira, Joshua Sanchez; Muraski, John A; Mitragotri, Samir

    2016-03-09

    Skin-penetrating peptides (SPPs) are attracting increasing attention as a non-invasive strategy for transdermal delivery of therapeutics. The identification of SPP sequences, however, currently performed by experimental screening of peptide libraries, is very laborious. Recent studies have shown that, to be effective enhancers, SPPs must possess affinity for both skin keratin and the drug of interest. We therefore developed a computational process for generating and screening virtual libraries of disulfide-cyclic peptides against keratin and cyclosporine A (CsA) to identify SPPs capable of enhancing transdermal CsA delivery. The selected sequences were experimentally tested and found to bind both CsA and keratin, as determined by mass spectrometry and affinity chromatography, and enhance transdermal permeation of CsA. Four heptameric sequences that emerged as leading candidates (ACSATLQHSCG, ACSLTVNWNCG, ACTSTGRNACG, and ACSASTNHNCG) were tested and yielded CsA permeation on par with previously identified SPP SPACE (TM) . An octameric peptide (ACNAHQARSTCG) yielded significantly higher delivery of CsA compared to heptameric SPPs. The safety profile of the selected sequences was also validated by incubation with skin keratinocytes. This method thus represents an effective procedure for the de novo design of skin-penetrating peptides for the delivery of desired therapeutic or cosmetic agents.

  7. De novo discovery of neuropeptides in the genomes of parasitic flatworms using a novel comparative approach.

    Science.gov (United States)

    Koziol, Uriel; Koziol, Miguel; Preza, Matías; Costábile, Alicia; Brehm, Klaus; Castillo, Estela

    2016-10-01

    Neuropeptide mediated signalling is an ancient mechanism found in almost all animals and has been proposed as a promising target for the development of novel drugs against helminths. However, identification of neuropeptides from genomic data is challenging, and knowledge of the neuropeptide complement of parasitic flatworms is still fragmentary. In this work, we have developed an evolution-based strategy for the de novo discovery of neuropeptide precursors, based on the detection of localised sequence conservation between possible prohormone convertase cleavage sites. The method detected known neuropeptide precursors with good precision and specificity in the models Drosophila melanogaster and Caenorhabditis elegans. Furthermore, it identified novel putative neuropeptide precursors in nematodes, including the first description of allatotropin homologues in this phylum. Our search for neuropeptide precursors in the genomes of parasitic flatworms resulted in the description of 34 conserved neuropeptide precursor families, including 13 new ones, and of hundreds of new homologues of known neuropeptide precursor families. Most neuropeptide precursor families show a wide phylogenetic distribution among parasitic flatworms and show little similarity to neuropeptide precursors of other bilaterian animals. However, we could also find orthologs of some conserved bilaterian neuropeptides including pyrokinin, crustacean cardioactive peptide, myomodulin, neuropeptide-Y, neuropeptide KY and SIF-amide. Finally, we determined the expression patterns of seven putative neuropeptide precursor genes in the protoscolex of Echinococcus multilocularis. All genes were expressed in the nervous system with different patterns, indicating a hidden complexity of peptidergic signalling in cestodes.

  8. Effects of Short Read Quality and Quantity on a de novo Vertebrate Transcriptome Assembly✰

    Science.gov (United States)

    Garcia, T.I.; Shen, Y.; Catchen, J.; Amores, A.; Schartl, M.; Postlethwait, J.; Walter, R. B.

    2011-01-01

    For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set. PMID:21651990

  9. Effects of short read quality and quantity on a de novo vertebrate transcriptome assembly.

    Science.gov (United States)

    Garcia, T I; Shen, Y; Catchen, J; Amores, A; Schartl, M; Postlethwait, J; Walter, R B

    2012-01-01

    For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set.

  10. De Novo Renal Cell Carcinoma in a Kidney Allograft 20 Years after Transplant

    Directory of Open Access Journals (Sweden)

    Masataka Banshodani

    2015-01-01

    Full Text Available Renal cell carcinoma (RCC in a kidney allograft is rare. We report the successful diagnosis and treatment of a de novo RCC in a nonfunctioning kidney transplant 20 years after engraftment. A 54-year-old man received a kidney transplant from his mother when he was 34 years old. After 10 years, chronic rejection resulted in graft failure, and the patient became hemodialysis-dependent. Intravenous contrast-enhanced computed tomography (CT for the evaluation of gastrointestinal symptoms revealed a solid 13 mm tumor in the kidney graft. The tumor was confirmed on ultrasound examination. This tumor had not been detected on a surveillance noncontrast CT scan. Needle biopsy showed that the tumor was an RCC. Allograft nephrectomy was performed. Pathological examination showed that the tumor was a Fuhrman Grade 2 RCC. XY-fluorescence hybridization analysis of the RCC showed that the tumor cells were of donor origin. One year after the surgery, the patient is alive and has no evidence of tumor recurrence. Regardless of whether a kidney transplant is functioning, it should periodically be imaged for RCC throughout the recipient’s lifetime. In our experience, ultrasonography or CT with intravenous contrast is better than CT without contrast for the detection of tumor in a nonfunctioning kidney transplant.

  11. The Role of Dietary Sugars and De novo Lipogenesis in Non-Alcoholic Fatty Liver Disease

    Directory of Open Access Journals (Sweden)

    J. Bernadette Moore

    2014-12-01

    Full Text Available Dietary sugar consumption, in particular sugar-sweetened beverages and the monosaccharide fructose, has been linked to the incidence and severity of non-alcoholic fatty liver disease (NAFLD. Intervention studies in both animals and humans have shown large doses of fructose to be particularly lipogenic. While fructose does stimulate de novo lipogenesis (DNL, stable isotope tracer studies in humans demonstrate quantitatively that the lipogenic effect of fructose is not mediated exclusively by its provision of excess substrates for DNL. The deleterious metabolic effects of high fructose loads appear to be a consequence of altered transcriptional regulatory networks impacting intracellular macronutrient metabolism and altering signaling and inflammatory processes. Uric acid generated by fructose metabolism may also contribute to or exacerbate these effects. Here we review data from human and animal intervention and stable isotope tracer studies relevant to the role of dietary sugars on NAFLD development and progression, in the context of typical sugar consumption patterns and dietary recommendations worldwide. We conclude that the use of hypercaloric, supra-physiological doses in intervention trials has been a major confounding factor and whether or not dietary sugars, including fructose, at typically consumed population levels, effect hepatic lipogenesis and NAFLD pathogenesis in humans independently of excess energy remains unresolved.

  12. De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation.

    Directory of Open Access Journals (Sweden)

    Yangkai Duan

    Full Text Available Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs and fatty acid ethyl esters (FAEEs, and is currently produced through the transesterification reaction of methanol (or ethanol and triacylglycerols (TAGs. TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.

  13. De novo design of potential RecA inhibitors using multi objective optimization.

    Science.gov (United States)

    Sengupta, Soumi; Bandyopadhyay, Sanghamitra

    2012-01-01

    De novo ligand design involves optimization of several ligand properties such as binding affinity, ligand volume, drug likeness, etc. Therefore, optimization of these properties independently and simultaneously seems appropriate. In this paper, the ligand design problem is modeled in a multiobjective using Archived MultiObjective Simulated Annealing (AMOSA) as the underlying search algorithm. The multiple objectives considered are the energy components similarity to a known inhibitor and a novel drug likeliness measure based on Lipinski's rule of five. RecA protein of Mycobacterium tuberculosis, causative agent of tuberculosis, is taken as the target for the drug design. To gauge the goodness of the results, they are compared to the outputs of LigBuilder, NEWLEAD, and Variable genetic algorithm (VGA). The same problem has also been modeled using a well-established genetic algorithm-based multiobjective optimization technique, Nondominated Sorting Genetic Algorithm-II (NSGA-II), to find the efficacy of AMOSA through comparative analysis. Results demonstrate that while some small molecules designed by the proposed approach are remarkably similar to the known inhibitors of RecA, some new ones are discovered that may be potential candidates for novel lead molecules against tuberculosis.

  14. Draft De Novo Transcriptome of the Rat Kangaroo Potorous tridactylus as a Tool for Cell Biology.

    Science.gov (United States)

    Udy, Dylan B; Voorhies, Mark; Chan, Patricia P; Lowe, Todd M; Dumont, Sophie

    2015-01-01

    The rat kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Australia. Cultured rat kangaroo kidney epithelial cells (PtK) are commonly used to study cell biological processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes-and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the rat kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft rat kangaroo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcriptome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcriptome will make rat kangaroo cells a more tractable system for linking molecular-scale function and cellular-scale dynamics.

  15. High throughput de novo RNA sequencing elucidates novel responses in Penicillium chrysogenum under microgravity.

    Science.gov (United States)

    Sathishkumar, Yesupatham; Krishnaraj, Chandran; Rajagopal, Kalyanaraman; Sen, Dwaipayan; Lee, Yang Soo

    2016-02-01

    In this study, the transcriptional alterations in Penicillium chrysogenum under simulated microgravity conditions were analyzed for the first time using an RNA-Seq method. The increasing plethora of eukaryotic microbial flora inside the spaceship demands the basic understanding of fungal biology in the absence of gravity vector. Penicillium species are second most dominant fungal contaminant in International Space Station. Penicillium chrysogenum an industrially important organism also has the potential to emerge as an opportunistic pathogen for the astronauts during the long-term space missions. But till date, the cellular mechanisms underlying the survival and adaptation of Penicillium chrysogenum to microgravity conditions are not clearly elucidated. A reference genome for Penicillium chrysogenum is not yet available in the NCBI database. Hence, we performed comparative de novo transcriptome analysis of Penicillium chrysogenum grown under microgravity versus normal gravity. In addition, the changes due to microgravity are documented at the molecular level. Increased response to the environmental stimulus, changes in the cell wall component ABC transporter/MFS transporters are noteworthy. Interestingly, sustained increase in the expression of Acyl-coenzyme A: isopenicillin N acyltransferase (Acyltransferase) under microgravity revealed the significance of gravity in the penicillin production which could be exploited industrially.

  16. Skin cancer in immunosuppressed transplant patients:Vigilance matters

    Institute of Scientific and Technical Information of China (English)

    Ozan Unlu; Emir Charles Roach; Alexis Okoh; May Olayan; Bulent Yilmaz; Didem Uzunaslan; Abdullah Shatnawei

    2015-01-01

    Liver transplantation (LT) is a widely-accepted, definitivetherapy of irreversible liver diseases including hepatitisC, alcoholic liver disease and metabolic liver disease.After transplantation, patients generally use a varietyof immunosuppressive medications for the rest of theirlives to prevent rejection of transplanted liver. Mortalityafter LT is mainly caused by recurrence of alcoholichepatitis which is mostly seen in the patients whoresume heavy drinking. On the other hand, de-novomalignancies after LT are not seldom. Skin cancers makeup 13.5% of the de-novo malignancies seen in thesepatients. Malignancies tend to affect survival earlier inthe course with a 53% risk of death at 5 years afterdiagnosis. We aimed to report a case who underwentLT secondary to alcoholic liver disease and developedsquamous cell carcinoma of the skin eighteen yearsafter transplantation. In summary, transplant recipientsare recommended to be educated on self examinationfor skin cancer; health care providers should be furthersuspicious during routine dermatological examinations ofthe transplant patients and biopsies of possible lesionsfor skin cancer is warranted even many years aftertransplantation.

  17. Immunosuppressive mechanisms in glioblastoma.

    Science.gov (United States)

    Nduom, Edjah K; Weller, Michael; Heimberger, Amy B

    2015-11-01

    Despite maximal surgical and medical therapy, the treatment of glioblastoma remains a seriously vexing problem, with median survival well under 2 years and few long-term survivors. Targeted therapy has yet to produce significant advances in treatment of these lesions in spite of advanced molecular characterization of glioblastoma and glioblastoma cancer stem cells. Recently, immunotherapy has emerged as a promising mode for some of the hardest to treat tumors, including metastatic melanoma. Although immunotherapy has been evaluated in glioblastoma in the past with limited success, better understanding of the failures of these therapies could lead to more successful treatments in the future. Furthermore, there is a persistent challenge for the use of immune therapy to treat glioblastoma secondary to the existence of redundant mechanisms of tumor-mediated immune suppression. Here we will address these mechanisms of immunosuppression in glioblastoma and therapeutic approaches.

  18. Immunosuppressive microenvironment in neuroblastoma

    Directory of Open Access Journals (Sweden)

    Vito ePistoia

    2013-06-01

    Full Text Available According to the cancer immunoediting model, the interplay between tumor cells and the host immune system is crucial for the control of tumor growth. NB is a pediatric tumor that presents with metastatic disease at diagnosis in about 50% of the cases, the majority of which have poor prognosis. In this Review article, immune escape pathways adopted by human neuroblastoma (NB cells are reviewed. These include intrinsic defects of tumor cells such impaired expression of the HLA class I related antigen processing machinery and functional alterations of the tumor microenvironment induced by NB cell-derived immunosuppressive molecules as MICA and HLA-G. Finally, examples of therapeutic interventions targeting the tumor microenvironment are discussed to emphasize the concept that successful cancer treatment may be achieved using this strategy.

  19. Cancer immunotherapy by immunosuppression

    Directory of Open Access Journals (Sweden)

    Prehn Liisa M

    2010-12-01

    Full Text Available Abstract We have previously suggested that the stimulatory effect of a weak immune reaction on tumor growth may be necessary for the growth of incipient tumors. In the present paper, we enlarge upon and extend that idea by collecting evidence in the literature bearing upon this new hypothesis that a growing cancer, whether in man or mouse, is throughout its lifespan, probably growing and progressing because of continued immune stimulation by a weak immune reaction. We also suggest that prolonged immunosuppression might interfere with progression and thus be an aid to therapy. While most of the considerable evidence that supports the hypothesis comes from observations of experimental mouse tumors, there is suggestive evidence that human tumors may behave in much the same way, and as far as we can ascertain, there is no present evidence that necessarily refutes the hypothesis.

  20. Comprehensive de novo peptide sequencing from MS/MS pairs generated through complementary collision induced dissociation and 351 nm ultraviolet photodissociation.

    Science.gov (United States)

    Horton, Andrew Pitchford; Robotham, Scott A; Cannon, Joe R; Holden, Dustin D; Marcotte, Edward M; Brodbelt, Jennifer S

    2017-02-24

    We describe a strategy for de novo peptide sequencing based on matched pairs of tandem mass spectra (MS/MS) obtained by collision induced dissociation (CID) and 351 nm ultraviolet photodissociation (UVPD). Each precursor ion is isolated twice with the mass spectrometer switching between CID and UVPD activation modes to obtain a complementary MS/MS pair. To interpret these paired spectra, we modified the UVnovo de novo sequencing software to automatically learn from and interpret fragmentation spectra, provided a representative set of training data. This machine learning procedure, using random forests, synthesizes information from one or multiple complementary spectra, such as the CID/UVPD pairs, into peptide fragmentation site predictions. In doing so, the burden of fragmentation model definition shifts from programmer to machine and opens up the model parameter space for inclusion of nonobvious features and interactions. This spectral synthesis also serves to transform distinct types of spectra into a common representation for subsequent activation-independent processing steps. Then, independent from precursor activation constraints, UVnovo's de novo sequencing procedure generates and scores sequence candidates for each precursor. We demonstrate the combined experimental and computational approach for de novo sequencing using whole cell E. coli lysate. In benchmarks on the CID/UVPD data, UVnovo assigned correct full-length sequences to 83% of the spectral pairs of doubly charged ions with high-confidence database identifications. Considering only top-ranked de novo predictions, 70% of the pairs were deciphered correctly. This de novo sequencing performance exceeds that of PEAKS and PepNovo on the CID spectra and that of UVnovo on CID or UVPD spectra alone. As presented here, the methods for paired CID/UVPD spectral acquisition and interpretation constitute a powerful workflow for high-throughput and accurate de novo peptide sequencing.

  1. From Leflunomide to Teriflunomide: Drug development and Immunosuppressive Oral Drugs in the Treatment of Multiple Sclerosis.

    Science.gov (United States)

    Aly, Lilian; Hemmer, Bernhard; Korn, Thomas

    2016-12-08

    Immunosuppressive drugs have been used in the treatment of multiple sclerosis (MS) for a long time. Today, the increased number of approved substances and the possibility of an oral availability of some immunomodulators improve the therapeutic repertory and increase patient satisfaction and compliance. Teriflunomide is indicated as first line oral disease modifying therapy (DMT) in relapsing-remitting MS (RRMS). Its immunosuppressive capacity results from an inhibition of de novo pyrimidine synthesis in rapidly proliferating lymphocytes. While Teriflunomide has been approved for the treatment of RRMS only since 2012, there is substantial therapeutic experience with its prodrug Leflunomide used in the treatment of rheumatoid arthritis (RA). In MS, a daily dose of 14 mg Teriflunomide reduces the annualized relapse rate (ARR) by more than 30% and disability progression by 30% compared to placebo while it provides a reasonable safety profile. This review presents an overview on oral immunosuppressants used in the treatment of MS. With an emphasis on Teriflunomide it summarizes discovery, mechanism of action and clinical effectiveness in phase II and III trials as well as important aspects for treating physicians.

  2. Impact of de novo donor-specific HLA antibodies detected by Luminex solid-phase assay after transplantation in a group of 88 consecutive living-donor renal transplantations.

    Science.gov (United States)

    Dieplinger, Georg; Ditt, Vanessa; Arns, Wolfgang; Huppertz, Andrea; Kisner, Tuelay; Hellmich, Martin; Bauerfeind, Ursula; Stippel, Dirk L

    2014-01-01

    De novo donor-specific HLA antibodies (DSA) after renal transplantation are known to be correlated with poor graft outcome and the development of acute and chronic rejection. Currently, data for the influence of de novo DSA in patient cohorts including only living-donor renal transplantations (LDRT) are limited. A consecutive cohort of 88 LDRT was tested for the occurrence of de novo DSA by utilizing the highly sensitive Luminex solid-phase assay for antibody detection. Data were analyzed for risk factors for de novo DSA development and correlated with acute rejection (AR) and graft function. Patients with de novo DSA [31 (35%)] showed a trend for inferior graft function [mean creatinine change (mg/dL/year) after the first year: 0.15 DSA (+) vs. 0.02 DSA (-) (P = 0.10)] and a higher rate of AR episodes, especially in case of de novo DSA of both class I and II [6 (55%), (P = 0.05)]. Antibody-mediated rejection (AMR) appeared in five patients and was significantly correlated with de novo DSA (P = 0.05). Monitoring for de novo DSA after LDRT may help to identify patients at risk of declining renal function. Especially patients with simultaneous presence of de novo DSA class I and class II are at a high risk to suffer AR episodes.

  3. Factors affecting de novo urinary retention after Holmium laser enucleation of the prostate.

    Directory of Open Access Journals (Sweden)

    Sung Han Kim

    Full Text Available OBJECTIVE: Patients can experience urinary retention (UR after Holmium laser enucleation of the prostate (HoLEP that requires bladder distension during the procedure. The aim of this retrospective study is to identify factors affecting the UR after HoLEP. MATERIALS AND METHODS: 336 patients, which underwent HoLEP for a symptomatic benign prostatic hyperplasia between July 2008 and March 2012, were included in this study. Urethral catheters were routinely removed one or two days after surgery. UR was defined as the need for an indwelling catheter placement following a failure to void after catheter removal. Demographic and clinical parameters were compared between the UR (n = 37 and the non-urinary retention (non-UR; n = 299 groups. RESULTS: The mean age of patients was 68.3 (±6.5 years and the mean operative time was 75.3 (±37.4 min. Thirty seven patients (11.0% experienced a postoperative UR. UR patients voided catheter free an average of 1.9 (±1.7 days after UR. With regard to the causes of UR, 24 (7.1% and 13 (3.9% patients experienced a blood clot-related UR and a non-clot related UR respectively. Using multivariate analysis (p0.05. CONCLUSIONS: De novo UR after HoLEP was found to be self-limited and it was not related to learning curve, patient age, diabetes, or operative time. Efficient morcellation and careful control of bleeding, which reduces clot formation, decrease the risk of UR after HoLEP.

  4. KrillDB: A de novo transcriptome database for the Antarctic krill (Euphausia superba)

    Science.gov (United States)

    Sales, Gabriele; Deagle, Bruce E.; Calura, Enrica; Martini, Paolo; Biscontin, Alberto; De Pittà, Cristiano; Kawaguchi, So; Romualdi, Chiara; Meyer, Bettina; Costa, Rodolfo; Jarman, Simon

    2017-01-01

    Antarctic krill (Euphausia superba) is a key species in the Southern Ocean with an estimated biomass between 100 and 500 million tonnes. Changes in krill population viability would have catastrophic effect on the Antarctic ecosystem. One looming threat due to elevated levels of anthropogenic atmospheric carbon dioxide (CO2) is ocean acidification (lowering of sea water pH by CO2 dissolving into the oceans). The genetics of Antarctic krill has long been of scientific interest for both for the analysis of population structure and analysis of functional genetics. However, the genetic resources available for the species are relatively modest. We have developed the most advanced genetic database on Euphausia superba, KrillDB, which includes comprehensive data sets of former and present transcriptome projects. In particular, we have built a de novo transcriptome assembly using more than 360 million Illumina sequence reads generated from larval krill including individuals subjected to different CO2 levels. The database gives access to: 1) the full list of assembled genes and transcripts; 2) their level of similarity to transcripts and proteins from other species; 3) the predicted protein domains contained within each transcript; 4) their predicted GO terms; 5) the level of expression of each transcript in the different larval stages and CO2 treatments. All references to external entities (sequences, domains, GO terms) are equipped with a link to the appropriate source database. Moreover, the software implements a full-text search engine that makes it possible to submit free-form queries. KrillDB represents the first large-scale attempt at classifying and annotating the full krill transcriptome. For this reason, we believe it will constitute a cornerstone of future approaches devoted to physiological and molecular study of this key species in the Southern Ocean food web. PMID:28187156

  5. De novo synthesis and properties of analogues of the self-assembling chlorosomal bacteriochlorophylls

    Energy Technology Data Exchange (ETDEWEB)

    Mass, Olga [North Carolina State Univ., Raleigh, NC (United States); Pandithavidana, Dinesh R. [North Carolina State Univ., Raleigh, NC (United States); Ptaszek, Marcin [North Carolina State Univ., Raleigh, NC (United States); Santiago, Koraliz [North Carolina State Univ., Raleigh, NC (United States); Springer, Joseph W. [Washington Univ., St. Louis, MO (United States); Jiao, Jieying [Univ. Of California, Riverside, CA (United States); Tang, Qun [Univ. Of California, Riverside, CA (United States); Kirmaier, Christine [Washington Univ., St. Louis, MO (United States); Bocian, David F. [Univ. Of California, Riverside, CA (United States); Holten, Dewey [Washington Univ., St. Louis, MO (United States); Lindsey, Jonathan S. [North Carolina State Univ., Raleigh, NC (United States)

    2011-01-01

    Natural photosynthetic pigments bacteriochlorophyllsc, d and e in green bacteria undergo self-assembly to create an organized antenna system known as the chlorosome, which collects photons and funnels the resulting excitation energy toward the reaction centers. Mimicry of chlorosome function is a central problem in supramolecular chemistry and artificial photosynthesis, and may have relevance for the design of photosynthesis-inspired solar cells. The main challenge in preparing artificial chlorosomes remains the synthesis of the appropriate pigment (chlorin) equipped with a set of functional groups suitable to direct the assembly and assure efficient energy transfer. Prior approaches have entailed derivatization of porphyrins or semisynthesis beginning with chlorophylls. This paper reports a third approach, the de novo synthesis of macrocycles that contain the same hydrocarbon skeleton as chlorosomal bacteriochlorophylls. The synthesis here of Zn(II) 3-(1-hydroxyethyl)-10-aryl-13¹-oxophorbines (the aryl group consists of phenyl, mesityl, or pentafluorophenyl) entails selective bromination of a 3,13-diacetyl-10-arylchlorin, palladium-catalyzed 13¹-oxophorbine formation, and selective reduction of the 3-acetyl group using BH₃·tBuNH₂. Each macrocycle contains a geminal dimethyl group in the pyrroline ring to provide stability toward adventitious dehydrogenation. A Zn(II) 7-(1-hydroxyethyl)-10-phenyl-17-oxochlorin also has been prepared. Altogether, 30 new hydroporphyrins were synthesized. The UV-Vis absorption spectra of the new chlorosomal bacteriochlorophyll mimics reveal a bathochromic shift of [similar]1800 cm-1 of the Qy band in nonpolar solvent, indicating extensive assembly in solution. The Zn(II) 3-(1-hydroxyethyl)-10-aryl-13¹-oxophorbines differ in the propensity to form assemblies based on the 10-substituent in the following order: mesityl

  6. De novo synthesis and functional study of primitive polypeptides in the prebiotic protein world

    Science.gov (United States)

    Fujishima, Kosuke; Wang, Kendrick; Ferreira, Raphael; Rothschild, Lynn

    DNA, RNA and proteins within a lipid-bound membrane are the core components of life, but the order of their appearance during the origin and evolution of life is still under debate. The widely accepted “RNA World” hypothesis states that RNA likely emerged prior to proteins and DNA since RNA can serve both replicative and catalytic roles. While biochemists have reproduced the synthesis, polymerization, and replication of nucleotides/RNA under controlled prebiotic conditions, such complex organic molecules were not present in significant amounts on the prebiotic Earth. In contrast, amino acids are naturally abundant in various prebiotic contexts such as carbonaceous chondrites and Urey-Miller type experiments, and many studies have demonstrated plausible prebiotic conditions that could condense/polymerize amino acids to give rise to short peptides. These findings support the basis of a “Protein World” hypothesis for life, however little has been done to study the functions of such primitive peptides. Here, we present a novel synthetic biology-based approach to the de novo synthesis of over billions of primitive peptides/proteins derived from a limited set of naturally abundant proteinogenic amino acids. Of these peptides, ones with divalent metal-binding capability are of particular interest and will be screened and identified. Certain divalent metals are likely present in prebiotic environments and both coordinate well with amino acids and catalyze reactions, which are difficult to achieve in organic chemistry. Furthermore, since D-chiral and non-proteinogenic amino acids are also abundant in the universe and may provide insight into the pathway by which life developed, the methods to analyze primitive peptides consisting of these amino acids will be discussed. By understanding this natural pathway, we will be able to better understand how life developed here on Earth and the probability of life arising elsewhere.

  7. Contact-inhibited chemotaxis in de novo and sprouting blood-vessel growth.

    Directory of Open Access Journals (Sweden)

    Roeland M H Merks

    Full Text Available Blood vessels form either when dispersed endothelial cells (the cells lining the inner walls of fully formed blood vessels organize into a vessel network (vasculogenesis, or by sprouting or splitting of existing blood vessels (angiogenesis. Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier-Graner-Hogeweg model (also called Cellular Potts Model simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller-Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally observed VE-cadherin-mediated contact inhibition of chemotaxis in the simulation causes randomly distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface-normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. Both mechanisms would also apply if force transmission through the extracellular matrix rather than chemical signaling mediated cell-cell interactions. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.

  8. Identification of Bone-Derived Factors Conferring De Novo Therapeutic Resistance in Metastatic Prostate Cancer.

    Science.gov (United States)

    Lee, Yu-Chen; Lin, Song-Chang; Yu, Guoyu; Cheng, Chien-Jui; Liu, Bin; Liu, Hsuan-Chen; Hawke, David H; Parikh, Nila U; Varkaris, Andreas; Corn, Paul; Logothetis, Christopher; Satcher, Robert L; Yu-Lee, Li-Yuan; Gallick, Gary E; Lin, Sue-Hwa

    2015-11-15

    Resistance to currently available targeted therapies significantly hampers the survival of patients with prostate cancer with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a "resistance niche" adjacent to prostate cancer-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed "osteocrines") found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to the newly formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic prostate cancer.

  9. Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement

    Indian Academy of Sciences (India)

    GHOLAMREZA FARNOOSH; KHOSRO KHAJEH; ALI MOHAMMAD LATIFI; HOSSEIN AGHAMOLLAEI

    2016-12-01

    The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the mostfrequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has beenshown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineeringof de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rationalmethod of protein engineering. In this study, Disulphide by Design software, homology modelling and moleculardynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge toimprove protein thermostability. The thermostability of the wild-type and three selected mutant enzymes wereevaluated by half-life, ΔG inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Dataanalysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min,respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124Cmutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results werecorrelated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsicfluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi alsoincreased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results weresubmitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing adisulphide bridge after bioinformatics design, even though this design would not be always successful.

  10. Effects of adipocyte lipoprotein lipase on de novo lipogenesis and white adipose tissue browning.

    Science.gov (United States)

    Bartelt, Alexander; Weigelt, Clara; Cherradi, M Lisa; Niemeier, Andreas; Tödter, Klaus; Heeren, Joerg; Scheja, Ludger

    2013-05-01

    Efficient storage of dietary and endogenous fatty acids is a prerequisite for a healthy adipose tissue function. Lipoprotein lipase (LPL) is the master regulator of fatty acid uptake from triglyceride-rich lipoproteins. In addition to LPL-mediated fatty acid uptake, adipocytes are able to synthesize fatty acids from non-lipid precursor, a process called de novo lipogenesis (DNL). As the physiological relevance of fatty acid uptake versus DNL for brown and white adipocyte function remains unclear, we studied the role of adipocyte LPL using adipocyte-specific LPL knockout animals (aLKO). ALKO mice displayed a profound increase in DNL-fatty acids, especially palmitoleate and myristoleate in brown adipose tissue (BAT) and white adipose tissue (WAT) depots while essential dietary fatty acids were markedly decreased. Consequently, we found increased expression in adipose tissues of genes encoding DNL enzymes (Fasn, Scd1, and Elovl6) as well as the lipogenic transcription factor carbohydrate response element binding protein-β. In a high-fat diet (HFD) study aLKO mice were characterized by reduced adiposity and improved plasma insulin and adipokines. However, neither glucose tolerance nor inflammatory markers were ameliorated in aLKO mice compared to controls. No signs of increased BAT activation or WAT browning were detected in aLKO mice either on HFD or after 1 week of β3-adrenergic stimulation using CL316,243. We conclude that despite a profound increase in DNL-derived fatty acids, proposed to be metabolically favorable, aLKO mice are not protected from metabolic disease per se. In addition, induction of DNL alone is not sufficient to promote browning of WAT. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

  11. De novo analysis of electron impact mass spectra using fragmentation trees

    Energy Technology Data Exchange (ETDEWEB)

    Hufsky, Franziska, E-mail: franziska.hufsky@uni-jena.de [Chair of Bioinformatics, Friedrich Schiller University, Ernst-Abbe-Platz 2, Jena (Germany); Max Planck Institute for Chemical Ecology, Beutenberg Campus, Jena (Germany); Rempt, Martin [Institute for Inorganic and Analytical Chemistry, Bioorganic Analytics, Friedrich Schiller University, Lessingstrasse 8, Jena (Germany); Rasche, Florian [Chair of Bioinformatics, Friedrich Schiller University, Ernst-Abbe-Platz 2, Jena (Germany); Pohnert, Georg [Institute for Inorganic and Analytical Chemistry, Bioorganic Analytics, Friedrich Schiller University, Lessingstrasse 8, Jena (Germany); Boecker, Sebastian, E-mail: sebastian.boecker@uni-jena.de [Chair of Bioinformatics, Friedrich Schiller University, Ernst-Abbe-Platz 2, Jena (Germany)

    2012-08-20

    Highlights: Black-Right-Pointing-Pointer We present a method for de novo analysis of accurate mass EI mass spectra of small molecules. Black-Right-Pointing-Pointer This method identifies the molecular ion and thus the molecular formula where the molecular ion is present in the spectrum. Black-Right-Pointing-Pointer Fragmentation trees are constructed by automated signal extraction and evaluation. Black-Right-Pointing-Pointer These trees explain relevant fragmentation reactions. Black-Right-Pointing-Pointer This method will be very helpful in the automated analysis of unknown metabolites. - Abstract: The automated fragmentation analysis of high resolution EI mass spectra based on a fragmentation tree algorithm is introduced. Fragmentation trees are constructed from EI spectra by automated signal extraction and evaluation. These trees explain relevant fragmentation reactions and assign molecular formulas to fragments. The method enables the identification of the molecular ion and the molecular formula of a metabolite if the molecular ion is present in the spectrum. These identifications are independent of existing library knowledge and, thus, support assignment and structural elucidation of unknown compounds. The method works even if the molecular ion is of very low abundance or hidden under contaminants with higher masses. We apply the algorithm to a selection of 50 derivatized and underivatized metabolites and demonstrate that in 78% of cases the molecular ion can be correctly assigned. The automatically constructed fragmentation trees correspond very well to published mechanisms and allow the assignment of specific relevant fragments and fragmentation pathways even in the most complex EI-spectra in our dataset. This method will be very helpful in the automated analysis of metabolites that are not included in common libraries and it thus has the potential to support the explorative character of metabolomics studies.

  12. Brain perfusion correlates of cognitive and nigrostriatal functions in de novo Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Nobili, Flavio; Arnaldi, Dario; Campus, Claudio; Ferrara, Michela; Brugnolo, Andrea; Dessi, Barbara; Girtler, Nicola; Rodriguez, Guido [University of Genoa, Clinical Neurophysiology, Department of Neurosciences, Ophthalmology and Genetics, Genoa (Italy); De Carli, Fabrizio [National Research Council, Institute of Molecular Bioimaging and Physiology, Genoa (Italy); Morbelli, Silvia; Sambuceti, Gianmario [University of Genoa, Nuclear Medicine, Department of Internal Medicine, Genoa (Italy); Abruzzese, Giovanni [University Hospital San. Martino, Clinical Neurology, Department of Neurosciences, Ophthalmology and Genetics, Genoa (Italy)

    2011-12-15

    Subtle cognitive impairment is recognized in the first stages of Parkinson's disease (PD), including executive, memory and visuospatial dysfunction, but its pathophysiological basis is still debated. Twenty-six consecutive, drug-naive, de novo PD patients underwent an extended neuropsychological battery, dopamine transporter (DAT) and brain perfusion single photon emission computed tomography (SPECT). We previously reported that nigrocaudate impairment correlates with executive functions, and nigroputaminal impairment with visuospatial abilities. Here perfusion SPECT was first compared between the PD group and age-matched controls (CTR). Then, perfusion SPECT was correlated with both DAT SPECT and four neuropsychological factors by means of voxel-based analysis (SPM8) with a height threshold of p < 0.005 at peak level and p < 0.05 false discovery rate-corrected at cluster level. Both perfusion and DAT SPECT images were flipped in order to have the more affected hemisphere (MAH), defined clinically, on the same side. Significant hypoperfusion was found in an occipital area of the MAH in PD patients as compared to CTR. Executive functions directly correlated with brain perfusion in bilateral posterior cingulate cortex and precuneus in the less affected hemisphere (LAH), while verbal memory directly correlated with perfusion in the precuneus, inferior parietal lobule and superior temporal gyrus in the LAH. Furthermore, positive correlation was highlighted between nigrocaudate and nigroputaminal impairment and brain perfusion in the precuneus, posterior cingulate and parahippocampal gyri of the LAH. These data support the evidence showing an early involvement of the cholinergic system in the early cognitive dysfunction and point to a more relevant role of parietal lobes and posterior cingulate in executive functions in PD. (orig.)

  13. STARD6 is expressed in steroidogenic cells of the ovary and can enhance de novo steroidogenesis.

    Science.gov (United States)

    LaVoie, Holly A; Whitfield, Nicole E; Shi, Bo; King, Steven R; Bose, Himangshu S; Hui, Yvonne Y

    2014-04-01

    STARD6 is a member of the StAR-related lipid transfer (START) domain family of proteins whose function thus far remains obscure. While it recently was shown to facilitate steroidogenesis in a cell-free setting, it has not been localized to steroidogenic cells of normal reproductive tissues. In a recent microarray study, we detected STARD6 mRNA in cultured porcine ovarian granulosa cells which are steroidogenic. In the present study, we examined regulation of STARD6 mRNA in porcine granulosa cultures, and found that it was not regulated by cyclic AMP, but it was reduced by combined knockdown of the transcription factors GATA4 and GATA6. We detected both STARD6 mRNA and protein in fresh granulosa cells and whole antral follicles and different stage corpora lutea of pig. The highest levels were discovered in the mid-luteal phase corpus luteum. Immunolocalization within ovarian tissues indicated robust STARD6 immunoreactivity in steroidogenic cells of the corpus luteum. Relatively lesser amounts of STARD6 signal were found in granulosa cells, theca cells, and oocytes. To test the ability of STARD6 to facilitate de novo steroidogenesis, non-steroidogenic COS-1 cells were co-transfected with components of the P450 cholesterol side-chain cleavage system, enabling them to make pregnenolone, and STARD6. STARD6 increased pregnenolone production by two- to three-fold over the empty vector control. In summary, STARD6 is found in the pig ovary, exhibits the strongest expression in highly steroidogenic luteal cells, and significantly enhances pregnenolone production in transfected COS cells independent of cyclic AMP treatment. Collectively, these findings indicate that STARD6 may contribute to steroidogenesis in ovarian cells, but also suggests other cellular functions that require cholesterol trafficking.

  14. Alteration of de novo glucose production contributes to fasting hypoglycaemia in Fyn deficient mice.

    Directory of Open Access Journals (Sweden)

    Yingjuan Yang

    Full Text Available Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of (13C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold and fructose-1,6-bisphosphate (7-fold levels as well as a reduction in glucose-6-phosphate (2-fold levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity.

  15. Critical review of NGS analyses for de novo genotyping multigene families.

    Science.gov (United States)

    Lighten, Jackie; van Oosterhout, Cock; Bentzen, Paul

    2014-08-01

    The genotyping of highly polymorphic multigene families across many individuals used to be a particularly challenging task because of methodological limitations associated with traditional approaches. Next-generation sequencing (NGS) can overcome most of these limitations, and it is increasingly being applied in population genetic studies of multigene families. Here, we critically review NGS bioinformatic approaches that have been used to genotype the major histocompatibility complex (MHC) immune genes, and we discuss how the significant advances made in this field are applicable to population genetic studies of gene families. Increasingly, approaches are introduced that apply thresholds of sequencing depth and sequence similarity to separate alleles from methodological artefacts. We explain why these approaches are particularly sensitive to methodological biases by violating fundamental genotyping assumptions. An alternative strategy that utilizes ultra-deep sequencing (hundreds to thousands of sequences per amplicon) to reconstruct genotypes and applies statistical methods on the sequencing depth to separate alleles from artefacts appears to be more robust. Importantly, the 'degree of change' (DOC) method avoids using arbitrary cut-off thresholds by looking for statistical boundaries between the sequencing depth for alleles and artefacts, and hence, it is entirely repeatable across studies. Although the advances made in generating NGS data are still far ahead of our ability to perform reliable processing, analysis and interpretation, the community is developing statistically rigorous protocols that will allow us to address novel questions in evolution, ecology and genetics of multigene families. Future developments in third-generation single molecule sequencing may potentially help overcome problems that still persist in de novo multigene amplicon genotyping when using current second-generation sequencing approaches.

  16. A de-novo STXBP1 gene mutation in a patient showing the Rett syndrome phenotype.

    Science.gov (United States)

    Romaniello, Romina; Saettini, Francesco; Panzeri, Elena; Arrigoni, Filippo; Bassi, Maria T; Borgatti, Renato

    2015-03-25

    This study reports on a 9-year-old girl who developed West syndrome and showed clinical features fulfilling the main revised diagnostic criteria for typical Rett syndrome (hand washing, severe cognitive impairment with absence of language, ataxic gait, progressive scoliosis and autistic features). Mutation analyses for methyl-CpG-binding protein 2 (MECP2), cyclin-dependent kinase-like 5 (CDKL5/STK9), ARX and Forkhead box G1 (FOXG1) genes were carried out, with negative results. A known de-novo c.1217G>A missense mutation in exon 14 leading to the substitution of a conserved residue, p.R406H in domain3b of the syntaxin-binding protein 1 (STXBP1) gene, was detected. The STXBP1 gene encodes the syntaxin-binding protein 1, a neuron-specific protein involved in synaptic vesicle release at both glutaminergic and GABAergic synapses. This function is also affected by MECP2 gene mutations, which are known to lead to a decrease in glutamate and GABA receptors' density. It is possible to speculate that the impairment in synaptic plasticity represents the pathogenic link between MECP2 and STXBP1 gene mutations. On reviewing the clinical features of the reported patients with the same mutation in the STXBP1 gene, it has been observed that poor eye contact, tremour, dyskinesia, head/hand stereotypies and both cognitive and motor progressive deterioration are common symptoms, although never considered as indicative of a Rett syndrome phenotype. In conclusion, the case described here suggests a relationship between the Rett syndrome and the STXBP1 gene not described so far, making the search for STXBP1 gene mutations advisable in patients with Rett syndrome and early onset of epilepsy.

  17. The de novo genome assembly and annotation of a female domestic dromedary of North African origin.

    Science.gov (United States)

    Fitak, Robert R; Mohandesan, Elmira; Corander, Jukka; Burger, Pamela A

    2016-01-01

    The single-humped dromedary (Camelus dromedarius) is the most numerous and widespread of domestic camel species and is a significant source of meat, milk, wool, transportation and sport for millions of people. Dromedaries are particularly well adapted to hot, desert conditions and harbour a variety of biological and physiological characteristics with evolutionary, economic and medical importance. To understand the genetic basis of these traits, an extensive resource of genomic variation is required. In this study, we assembled at 65× coverage, a 2.06 Gb draft genome of a female dromedary whose ancestry can be traced to an isolated population from the Canary Islands. We annotated 21,167 protein-coding genes and estimated ~33.7% of the genome to be repetitive. A comparison with the recently published draft genome of an Arabian dromedary resulted in 1.91 Gb of aligned sequence with a divergence of 0.095%. An evaluation of our genome with the reference revealed that our assembly contains more error-free bases (91.2%) and fewer scaffolding errors. We identified ~1.4 million single-nucleotide polymorphisms with a mean density of 0.71 × 10(-3) per base. An analysis of demographic history indicated that changes in effective population size corresponded with recent glacial epochs. Our de novo assembly provides a useful resource of genomic variation for future studies of the camel's adaptations to arid environments and economically important traits. Furthermore, these results suggest that draft genome assemblies constructed with only two differently sized sequencing libraries can be comparable to those sequenced using additional library sizes, highlighting that additional resources might be better placed in technologies alternative to short-read sequencing to physically anchor scaffolds to genome maps.

  18. Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides

    Directory of Open Access Journals (Sweden)

    Kaushik Naveen K

    2012-08-01

    Full Text Available Abstract Background A lack of vaccine and rampant drug resistance demands new anti-malarials. Methods In vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis. Results Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2 (X = F,ΔF (Fm, ΔFm IC50 >100 μM}, the lysine-branched,dimeric versions showed far greater potency {IC50 (μM Fd 1.5 , ΔFd 1.39}. The more helical and proteolytically stable ΔFd was studied for mechanistic details. ΔFq, a K-K2 dendrimer of ΔFm and (ΔFm2 a linear dimer of ΔFm showed IC50 (μM of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (ΔFd, >20 (ΔFm2 and 10 (ΔFq. FITC-ΔFd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that ΔFd binds DNA. Trophozoites and schizonts incubated with ΔFd (2.5 μM egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent. Conclusion Good selectivity (>35, good resistance index (1.1 and low cytotoxicity indicate the promise of ΔFd against malaria.

  19. De novo transcriptome analysis and molecular marker development of two Hemarthria species

    Directory of Open Access Journals (Sweden)

    Xiu eHuang

    2016-04-01

    Full Text Available Hemarthria R. Br. is an important genus of perennial forage grasses that is widely used in subtropical and tropical regions. Hemarthria grasses have made remarkable contributions to the development of animal husbandry and agro-ecosystem maintenance; however, there is currently a lack of comprehensive genomic data available for these species. In this study, we used Illumina high-throughput deep sequencing to characterize of two agriculturally important Hemarthria materials, H. compressa ‘Yaan’ and H. altissima ‘1110.’ Sequencing runs that used each of four normalized RNA samples from the leaves or roots of the two materials yielded more than 24 million high-quality reads. After de novo assembly, 137,142 and 77,150 unigenes were obtained for ‘Yaan’ and ‘1110’, respectively. In addition, a total of 86,731 ‘Yaan’ and 48,645 ‘1110’ unigenes were successfully annotated. After consolidating the unigenes for both materials, 42,646 high-quality SNPs were identified in 10,880 unigenes and 10,888 SSRs were identified in 8,330 unigenes. To validate the identified markers, high quality PCR primers were designed for both SNPs and SSRs. We randomly tested 16 of the SNP primers and 54 of the SSR primers and found that the majority of these primers successfully amplified the desired PCR product. In addition, high cross-species transferability (61.11%-87.04% of SSR markers was achieved for four other Poaceae species. The amount of RNA sequencing data that was generated for these two Hemarthria species greatly increases the amount of genomic information available for Hemarthria and the SSR and SNP markers identified in this study will facilitate further advancements in genetic and molecular studies of the Hemarthria genus.

  20. Biochemical characterization of GDP-L-fucose de novo synthesis pathway in fungus Mortierella alpina

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Yan [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Perepelov, Andrei V. [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation); Wang, Haiyan [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Zhang, Hao [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China); Knirel, Yuriy A. [N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow (Russian Federation); Wang, Lei [TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457 (China); Chen, Wei, E-mail: weichen@jiangnan.edu.cn [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 (China)

    2010-01-22

    Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. L-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-L-fucose (the nucleotide-activated form of L-fucose) in M. alpina. Genes encoding GDP-D-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-L-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k{sub m} values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 {sup o}C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-L-fucose biosynthetic pathway in fungi.

  1. De novo assembly and characterization of the complete chloroplast genome of radish (Raphanus sativus L.).

    Science.gov (United States)

    Jeong, Young-Min; Chung, Won-Hyung; Mun, Jeong-Hwan; Kim, Namshin; Yu, Hee-Ju

    2014-11-01

    Radish (Raphanus sativus L.) is an edible root vegetable crop that is cultivated worldwide and whose genome has been sequenced. Here we report the complete nucleotide sequence of the radish cultivar WK10039 chloroplast (cp) genome, along with a de novo assembly strategy using whole genome shotgun sequence reads obtained by next generation sequencing. The radish cp genome is 153,368 bp in length and has a typical quadripartite structure, composed of a pair of inverted repeat regions (26,217 bp each), a large single copy region (83,170 bp), and a small single copy region (17,764 bp). The radish cp genome contains 87 predicted protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Sequence analysis revealed the presence of 91 simple sequence repeats (SSRs) in the radish cp genome. Phylogenetic analysis of 62 protein-coding gene sequences from the 17 cp genomes of the Brassicaceae family suggested that the radish cp genome is most closely related to the cp genomes of Brassica rapa and Brassicanapus. Comparisons with the B. rapa and B. napus cp genomes revealed highly divergent intergenic sequences and introns that can potentially be developed as diagnostic cp markers. Synonymous and nonsynonymous substitutions of cp genes suggested that nucleotide substitutions have occurred at similar rates in most genes. The complete sequence of the radish cp genome would serve as a valuable resource for the development of new molecular markers and the study of the phylogenetic relationships of Raphanus species in the Brassicaceae family.

  2. De novo establishment of wild-type song culture in the zebra finch.

    Science.gov (United States)

    Fehér, Olga; Wang, Haibin; Saar, Sigal; Mitra, Partha P; Tchernichovski, Ofer

    2009-05-28

    Culture is typically viewed as consisting of traits inherited epigenetically, through social learning. However, cultural diversity has species-typical constraints, presumably of genetic origin. A celebrated, if contentious, example is whether a universal grammar constrains syntactic diversity in human languages. Oscine songbirds exhibit song learning and provide biologically tractable models of culture: members of a species show individual variation in song and geographically separated groups have local song dialects. Different species exhibit distinct song cultures, suggestive of genetic constraints. Without such constraints, innovations and copying errors should cause unbounded variation over multiple generations or geographical distance, contrary to observations. Here we report an experiment designed to determine whether wild-type song culture might emerge over multiple generations in an isolated colony founded by isolates, and, if so, how this might happen and what type of social environment is required. Zebra finch isolates, unexposed to singing males during development, produce song with characteristics that differ from the wild-type song found in laboratory or natural colonies. In tutoring lineages starting from isolate founders, we quantified alterations in song across tutoring generations in two social environments: tutor-pupil pairs in sound-isolated chambers and an isolated semi-natural colony. In both settings, juveniles imitated the isolate tutors but changed certain characteristics of the songs. These alterations accumulated over learning generations. Consequently, songs evolved towards the wild-type in three to four generations. Thus, species-typical song culture can appear de novo. Our study has parallels with language change and evolution. In analogy to models in quantitative genetics, we model song culture as a multigenerational phenotype partly encoded genetically in an isolate founding population, influenced by environmental variables and taking

  3. De Novo GMNN Mutations Cause Autosomal-Dominant Primordial Dwarfism Associated with Meier-Gorlin Syndrome.

    Science.gov (United States)

    Burrage, Lindsay C; Charng, Wu-Lin; Eldomery, Mohammad K; Willer, Jason R; Davis, Erica E; Lugtenberg, Dorien; Zhu, Wenmiao; Leduc, Magalie S; Akdemir, Zeynep C; Azamian, Mahshid; Zapata, Gladys; Hernandez, Patricia P; Schoots, Jeroen; de Munnik, Sonja A; Roepman, Ronald; Pearring, Jillian N; Jhangiani, Shalini; Katsanis, Nicholas; Vissers, Lisenka E L M; Brunner, Han G; Beaudet, Arthur L; Rosenfeld, Jill A; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Xia, Fan; Lalani, Seema R; Lupski, James R; Bongers, Ernie M H F; Yang, Yaping

    2015-12-03

    Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5' end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1(st) coding exon), c.16A>T (p.Lys6(∗)) and c.35_38delTCAA (p.Ile12Lysfs(∗)4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5' end of the geminin protein. All three GMNN mutations identified alter sites 5' to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS.

  4. Sequencing and de novo assembly of the transcriptome of the glassy-winged sharpshooter (Homalodisca vitripennis.

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    Raja Sekhar Nandety

    Full Text Available BACKGROUND: The glassy-winged sharpshooter Homalodisca vitripennis (Hemiptera: Cicadellidae, is a xylem-feeding leafhopper and important vector of the bacterium Xylella fastidiosa; the causal agent of Pierce's disease of grapevines. The functional complexity of the transcriptome of H. vitripennis has not been elucidated thus far. It is a necessary blueprint for an understanding of the development of H. vitripennis and for designing efficient biorational control strategies including those based on RNA interference. RESULTS: Here we elucidate and explore the transcriptome of adult H. vitripennis using high-throughput paired end deep sequencing and de novo assembly. A total of 32,803,656 paired-end reads were obtained with an average transcript length of 624 nucleotides. We assembled 32.9 Mb of the transcriptome of H. vitripennis that spanned across 47,265 loci and 52,708 transcripts. Comparison of our non-redundant database showed that 45% of the deduced proteins of H. vitripennis exhibit identity (e-value ≤1(-5 with known proteins. We assigned Gene Ontology (GO terms, Kyoto Encyclopedia of Genes and Genomes (KEGG annotations, and potential Pfam domains to each transcript isoform. In order to gain insight into the molecular basis of key regulatory genes of H. vitripennis, we characterized predicted proteins involved in the metabolism of juvenile hormone, and biogenesis of small RNAs (Dicer and Piwi sequences from the transcriptomic sequences. Analysis of transposable element sequences of H. vitripennis indicated that the genome is less expanded in comparison to many other insects with approximately 1% of the transcriptome carrying transposable elements. CONCLUSIONS: Our data significantly enhance the molecular resources available for future study and control of this economically important hemipteran. This transcriptional information not only provides a more nuanced understanding of the underlying biological and physiological mechanisms that

  5. Hybrid De Novo Genome Assembly Using MiSeq and SOLiD Short Read Data.

    Science.gov (United States)

    Ikegami, Tsutomu; Inatsugi, Toyohiro; Kojima, Isao; Umemura, Myco; Hagiwara, Hiroko; Machida, Masayuki; Asai, Kiyoshi

    2015-01-01

    A hybrid de novo assembly pipeline was constructed to utilize both MiSeq and SOLiD short read data in combination in the assembly. The short read data were converted to a standard format of the pipeline, and were supplied to the pipeline components such as ABySS and SOAPdenovo. The assembly pipeline proceeded through several stages, and either MiSeq paired-end data, SOLiD mate-paired data, or both of them could be specified as input data at each stage separately. The pipeline was examined on the filamentous fungus Aspergillus oryzae RIB40, by aligning the assembly results against the reference sequences. Using both the MiSeq and the SOLiD data in the hybrid assembly, the alignment length was improved by a factor of 3 to 8, compared with the assemblies using either one of the data types. The number of the reproduced gene cluster regions encoding secondary metabolite biosyntheses (SMB) was also improved by the hybrid assemblies. These results imply that the MiSeq data with long read length are essential to construct accurate nucleotide sequences, while the SOLiD mate-paired reads with long insertion length enhance long-range arrangements of the sequences. The pipeline was also tested on the actinomycete Streptomyces avermitilis MA-4680, whose gene is known to have high-GC content. Although the quality of the SOLiD reads was too low to perform any meaningful assemblies by themselves, the alignment length to the reference was improved by a factor of 2, compared with the assembly using only the MiSeq data.

  6. Hybrid De Novo Genome Assembly Using MiSeq and SOLiD Short Read Data.

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    Tsutomu Ikegami

    Full Text Available A hybrid de novo assembly pipeline was constructed to utilize both MiSeq and SOLiD short read data in combination in the assembly. The short read data were converted to a standard format of the pipeline, and were supplied to the pipeline components such as ABySS and SOAPdenovo. The assembly pipeline proceeded through several stages, and either MiSeq paired-end data, SOLiD mate-paired data, or both of them could be specified as input data at each stage separately. The pipeline was examined on the filamentous fungus Aspergillus oryzae RIB40, by aligning the assembly results against the reference sequences. Using both the MiSeq and the SOLiD data in the hybrid assembly, the alignment length was improved by a factor of 3 to 8, compared with the assemblies using either one of the data types. The number of the reproduced gene cluster regions encoding secondary metabolite biosyntheses (SMB was also improved by the hybrid assemblies. These results imply that the MiSeq data with long read length are essential to construct accurate nucleotide sequences, while the SOLiD mate-paired reads with long insertion length enhance long-range arrangements of the sequences. The pipeline was also tested on the actinomycete Streptomyces avermitilis MA-4680, whose gene is known to have high-GC content. Although the quality of the SOLiD reads was too low to perform any meaningful assemblies by themselves, the alignment length to the reference was improved by a factor of 2, compared with the assembly using only the MiSeq data.

  7. De novo transcriptome assembly for the tropical grass Panicum maximum Jacq.

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    Guilherme Toledo-Silva

    Full Text Available Guinea grass (Panicum maximum Jacq. is a tropical African grass often used to feed beef cattle, which is an important economic activity in Brazil. Brazil is the leader in global meat exportation because of its exclusively pasture-raised bovine herds. Guinea grass also has potential uses in bioenergy production due to its elevated biomass generation through the C4 photosynthesis pathway. We generated approximately 13 Gb of data from Illumina sequencing of P. maximum leaves. Four different genotypes were sequenced, and the combined reads were assembled de novo into 38,192 unigenes and annotated; approximately 63% of the unigenes had homology to other proteins in the NCBI non-redundant protein database. Functional classification through COG (Clusters of Orthologous Groups, GO (Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes analyses showed that the unigenes from Guinea grass leaves are involved in a wide range of biological processes and metabolic pathways, including C4 photosynthesis and lignocellulose generation, which are important for cattle grazing and bioenergy production. The most abundant transcripts were involved in carbon fixation, photosynthesis, RNA translation and heavy metal cellular homeostasis. Finally, we identified a number of potential molecular markers, including 5,035 microsatellites (SSRs and 346,456 single nucleotide polymorphisms (SNPs. To the best of our knowledge, this is the first study to characterize the complete leaf transcriptome of P. maximum using high-throughput sequencing. The biological information provided here will aid in gene expression studies and marker-assisted selection-based breeding research in tropical grasses.

  8. The De Novo Transcriptome and Its Functional Annotation in the Seed Beetle Callosobruchus maculatus.

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    Ahmed Sayadi

    Full Text Available Despite their unparalleled biodiversity, the genomic resources available for beetles (Coleoptera remain relatively scarce. We present an integrative and high quality annotated transcriptome of the beetle Callosobruchus maculatus, an important and cosmopolitan agricultural pest as well as an emerging model species in ecology and evolutionary biology. Using Illumina sequencing technology, we sequenced 492 million read pairs generated from 51 samples of different developmental stages (larvae, pupae and adults of C. maculatus. Reads were de novo assembled using the Trinity software, into a single combined assembly as well as into three separate assemblies based on data from the different developmental stages. The combined assembly generated 218,192 transcripts and 145,883 putative genes. Putative genes were annotated with the Blast2GO software and the Trinotate pipeline. In total, 33,216 putative genes were successfully annotated using Blastx against the Nr (non-redundant database and 13,382 were assigned to 34,100 Gene Ontology (GO terms. We classified 5,475 putative genes into Clusters of Orthologous Groups (COG and 116 metabolic pathways maps were predicted based on the annotation. Our analyses suggested that the transcriptional specificity increases with ontogeny. For example, out of 33,216 annotated putative genes, 51 were only expressed in larvae, 63 only in pupae and 171 only in adults. Our study illustrates the importance of including samples from several developmental stages when the aim is to provide an integrative and high quality annotated transcriptome. Our results will represent an invaluable resource for those working with the ecology, evolution and pest control of C. maculatus, as well for comparative studies of the transcriptomics and genomics of beetles more generally.

  9. De Novo Transcriptome Assembly and Characterization for the Widespread and Stress-Tolerant Conifer Platycladus orientalis.

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    Xian-Ge Hu

    Full Text Available Platycladus orientalis, of the family Cupressaceae, is a widespread conifer throughout China and is extensively used for ecological reforestation, horticulture, and in medicine. Transcriptome assemblies are required for this ecologically important conifer for understanding genes underpinning adaptation and complex traits for breeding programs. To enrich the species' genomic resources, a de novo transcriptome sequencing was performed using Illumina paired-end sequencing. In total, 104,073,506 high quality sequence reads (approximately 10.3 Gbp were obtained, which were assembled into 228,948 transcripts and 148,867 unigenes that were longer than 200 nt. Quality assessment using CEGMA showed that the transcriptomes obtained were mostly complete for highly conserved core eukaryotic genes. Based on similarity searches with known proteins, 62,938 (42.28% of all unigenes, 42,158 (28.32%, and 23,179 (15.57% had homologs in the Nr, GO, and KOG databases, 25,625 (17.21% unigenes were mapped to 322 pathways by BLASTX comparison against the KEGG database and 1,941 unigenes involved in environmental signaling and stress response were identified. We also identified 43 putative terpene synthase (TPS functional genes loci and compared them with TPSs from other species. Additionally, 5,296 simple sequence repeats (SSRs were identified in 4,715 unigenes, which were assigned to 142 motif types. This is the first report of a complete transcriptome analysis of P. orientalis. These resources provide a foundation for further studies of adaptation mechanisms and molecular-based breeding programs.

  10. Cytogenetic findings in 303 Mexican patients with de novo acute myeloblastic leukemia.

    Science.gov (United States)

    Arana-Trejo, R M; Gómez-Morales, E; Rubio-Borja, M E; Kassack-Ipiña, J J; Cervantes-Peredo, A; Guerrero-Rivera, S; González-Llaven, J; Gutiérrez-Romero, M; Pizzuto-Chávez, J; Kofman-Alfaro, S

    1997-01-01

    In this report we show the chromosomal changes seen in a group of 303 Mexican patients with de novo Acute Myeloblastic Leukemia (AML). Two hundred forty-two patients were diagnosed and treated at two hospitals affiliated with the Instituto Mexicano del Seguro Social (IMSS). These are the Centro Medico Nacional Siglo XXI and Centro Medico La Raza Hospitals; the remaining 61 patients were diagnosed and treated at the Hospital General de Mexico (HGM). Clonal abnormalities were detected in 75.6% of the patients; this result agrees with what has been reported in other large series of AML studies. The incidence of changes per hospital was similar in patients from the IMSS hospitals (72-75%), while an increase was seen in patients from the HGM (85.2%). The chromosomal changes seen in this study in order of frequency were: t(15;17)[18.8%], t(9;22)[9.2%], miscellaneous chromosomal changes (mainly rearrangements of chromosomes 1,2,3,12y17)[8.2%], abnormalities of 16q22 [7.3%], t(8;21)[6.3%], -7/del(7q)[5.6%], t(6;9)[5.3%], and abnormalities of 11q23 [4.6%]. We reported an increase in the incidence of certain types of chromosomal changes seen in cases of AML, in comparison with reports from other countries. These differences could be due to methodological variations, although ethnic, socioeconomic and nutritional differences must not be disregarded. We support this finding when comparing distribution of changes in the population of patients seen in the IMSS hospitals with those from the HGM; the main difference lies in the socioeconomic level.

  11. Identification of putative cis-regulatory elements in Cryptosporidium parvum by de novo pattern finding

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    Kissinger Jessica C

    2007-01-01

    Full Text Available Abstract Background Cryptosporidium parvum is a unicellular eukaryote in the phylum Apicomplexa. It is an obligate intracellular parasite that causes diarrhea and is a significant AIDS-related pathogen. Cryptosporidium parvum is not amenable to long-term laboratory cultivation or classical molecular genetic analysis. The parasite exhibits a complex life cycle, a broad host range, and fundamental mechanisms of gene regulation remain unknown. We have used data from the recently sequenced genome of this organism to uncover clues about gene regulation in C. parvum. We have applied two pattern finding algorithms MEME and AlignACE to identify conserved, over-represented motifs in the 5' upstream regions of genes in C. parvum. To support our findings, we have established comparative real-time -PCR expression profiles for the groups of genes examined computationally. Results We find that groups of genes that share a function or belong to a common pathway share upstream motifs. Different motifs are conserved upstream of different groups of genes. Comparative real-time PCR studies show co-expression of genes within each group (in sub-sets during the life cycle of the parasite, suggesting co-regulation of these genes may be driven by the use of conserved upstream motifs. Conclusion This is one of the first attempts to characterize cis-regulatory elements in the absence of any previously characterized elements and with very limited expression data (seven genes only. Using de novo pattern finding algorithms, we have identified specific DNA motifs that are conserved upstream of genes belonging to the same metabolic pathway or gene family. We have demonstrated the co-expression of these genes (often in subsets using comparative real-time-PCR experiments thus establishing evidence for these conserved motifs as putative cis-regulatory elements. Given the lack of prior information concerning expression patterns and organization of promoters in C. parvum we

  12. De Novo and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects.

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    James R Priest

    2016-04-01

    Full Text Available Congenital heart disease (CHD has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD. In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W. We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08. Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6, cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1, and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06. In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk.

  13. De Novo and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects

    Science.gov (United States)

    Priest, James R.; Osoegawa, Kazutoyo; Mohammed, Nebil; Nanda, Vivek; Kundu, Ramendra; Schultz, Kathleen; Girirajan, Santhosh; Scheetz, Todd; Waggott, Daryl; Haddad, Francois; Reddy, Sushma; Bernstein, Daniel; Burns, Trudy; Steimle, Jeffrey D.; Yang, Xinan H.; Moskowitz, Ivan P.; Hurles, Matthew; Lifton, Richard P.; Nickerson, Debbie; Bamshad, Michael; Eichler, Evan E.; Mital, Seema; Sheffield, Val; Quertermous, Thomas; Gelb, Bruce D.; Portman, Michael; Ashley, Euan A.

    2016-01-01

    Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk. PMID:27058611

  14. Synergistic interactions between Drosophila orthologues of genes spanned by de novo human CNVs support multiple-hit models of autism.

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    Stuart J Grice

    2015-03-01

    Full Text Available Autism spectrum disorders (ASDs are highly heritable and characterised by deficits in social interaction and communication, as well as restricted and repetitive behaviours. Although a number of highly penetrant ASD gene variants have been identified, there is growing evidence to support a causal role for combinatorial effects arising from the contributions of multiple loci. By examining synaptic and circadian neurological phenotypes resulting from the dosage variants of unique human:fly orthologues in Drosophila, we observe numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large de novo copy number variants (CNVs. These CNVs were found in the genomes of individuals with autism, including a patient carrying a 22q11.2 deletion. We first demonstrate that dosage alterations of the unique Drosophila orthologues of candidate genes from de novo CNVs that harbour only a single candidate gene display neurological defects similar to those previously reported in Drosophila models of ASD-associated variants. We then considered pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human de novo CNV. For three of four CNVs with complete orthologous relationships, we observed significant synergistic effects following the simultaneous dosage change of gene pairs drawn from a single CNV. The phenotypic variation observed at the Drosophila synapse that results from these interacting genetic variants supports a concordant phenotypic outcome across all interacting gene pairs following the direction of human gene copy number change. We observe both specificity and transitivity between interactors, both within and between CNV candidate gene sets, supporting shared and distinct genetic aetiologies. We then show that different interactions affect divergent synaptic processes, demonstrating distinct molecular aetiologies. Our

  15. Receptor-Type Guanylyl Cyclase at 76C (Gyc76C) Regulates De Novo Lumen Formation during Drosophila Tracheal Development

    Science.gov (United States)

    Patel, Unisha

    2016-01-01

    Lumen formation and maintenance are important for the development and function of essential organs such as the lung, kidney and vasculature. In the Drosophila embryonic trachea, lumena form de novo to connect the different tracheal branches into an interconnected network of tubes. Here, we identify a novel role for the receptor type guanylyl cyclase at 76C (Gyc76C) in de novo lumen formation in the Drosophila trachea. We show that in embryos mutant for gyc76C or its downsteam effector protein kinase G (PKG) 1, tracheal lumena are disconnected. Dorsal trunk (DT) cells of gyc76C mutant embryos migrate to contact each other and complete the initial steps of lumen formation, such as the accumulation of E-cadherin (E-cad) and formation of an actin track at the site of lumen formation. However, the actin track and E-cad contact site of gyc76C mutant embryos did not mature to become a new lumen and DT lumena did not fuse. We also observed failure of the luminal protein Vermiform to be secreted into the site of new lumen formation in gyc76C mutant trachea. These DT lumen formation defects were accompanied by altered localization of the Arf-like 3 GTPase (Arl3), a known regulator of vesicle-vesicle and vesicle-membrane fusion. In addition to the DT lumen defect, lumena of gyc76C mutant terminal cells were shorter compared to wild-type cells. These studies show that Gyc76C and downstream PKG-dependent signaling regulate de novo lumen formation in the tracheal DT and terminal cells, most likely by affecting Arl3-mediated luminal secretion. PMID:27642749

  16. Illumina TruSeq synthetic long-reads empower de novo assembly and resolve complex, highly-repetitive transposable elements.

    Science.gov (United States)

    McCoy, Rajiv C; Taylor, Ryan W; Blauwkamp, Timothy A; Kelley, Joanna L; Kertesz, Michael; Pushkarev, Dmitry; Petrov, Dmitri A; Fiston-Lavier, Anna-Sophie

    2014-01-01

    High-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or complex genomic arrangements. While TEs strongly affect genome function and evolution, most current de novo assembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly-parallel library preparation and local assembly of short read data and which achieve lengths of 1.5-18.5 Kbp with an extremely low error rate ([Formula: see text]0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organism Drosophila melanogaster (reference genome strain y; cn, bw, sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long-reads, and likely other methods that generate long-reads, offer a powerful approach to improve de novo assemblies of whole genomes.

  17. Tumores malignos de novo en el trasplante hepático experiencia de un centro en la Argentina

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    Florencia Antinucci

    2015-02-01

    Full Text Available El objetivo del presente trabajo ha sido evaluar la incidencia y las características clínicas de los tumores aparecidos de novo en los pacientes sometidos a trasplante hepático, como así también su supervivencia. Para ello, analizamos en forma retrospectiva los 168 trasplantes hepáticos realizados en 159 pacientes adultos en el período mayo 2006 hasta mayo 2014, encontrando una incidencia de neoplasia de novo de 7.5% (n = 12. La edad media en el momento del diagnóstico fue de 63 ± 7 años. Las neoplasias más frecuentes fueron las de piel no melanoma y adenocarcinomas. El 50% de las neoplasias se desarrollaron en el segundo y tercer año postrasplante. El tipo de inmunosupresión no influyó en el tipo de tumor; sin embargo, debemos destacar que la mayor parte de los pacientes recibieron tacrolimus, micofenolato y/o corticoides. El tiempo medio de seguimiento tras el diagnóstico del tumor fue 25 ± 29 meses (0-76, y la tasa de mortalidad fue de un 41% (5/12 pacientes IC95%,15-72.La supervivencia global luego del trasplante a 1 y 5 años, calculada por análisis de Kaplan-Meier, fue de 83 y 55%, respectivamente. Los tumores de novo son frecuentes luego del trasplante hepático y presentan un patrón evolutivo diferente al de la población general. Teniendo en cuenta esta evolución más agresiva, es fundamental el seguimiento periódico en estos pacientes para realizar un diagnóstico lo más precoz posible.

  18. Facilitating best practices in collecting anomalous scattering data for de novo structure solution at the ESRF Structural Biology Beamlines

    Science.gov (United States)

    de Sanctis, Daniele; Oscarsson, Marcus; Popov, Alexander; Svensson, Olof; Leonard, Gordon

    2016-01-01

    The constant evolution of synchrotron structural biology beamlines, the viability of screening protein crystals for a wide range of heavy-atom derivatives, the advent of efficient protein labelling and the availability of automatic data-processing and structure-solution pipelines have combined to make de novo structure solution in macromolecular crystallography a less arduous task. Nevertheless, the collection of diffraction data of sufficient quality for experimental phasing is still a difficult and crucial step. Here, some examples of good data-collection practice for projects requiring experimental phasing are presented and recent developments at the ESRF Structural Biology beamlines that have facilitated these are illustrated. PMID:26960128

  19. Vanillin-bioconversion and bioengineering of the most popular plant flavor and its de novo biosynthesis in the vanilla orchid.

    Science.gov (United States)

    Gallage, Nethaji J; Møller, Birger Lindberg

    2015-01-01

    In recent years, biotechnology-derived production of flavors and fragrances has expanded rapidly. The world's most popular flavor, vanillin, is no exception. This review outlines the current state of biotechnology-based vanillin synthesis with the use of ferulic acid, eugenol, and glucose as substrates and bacteria, fungi, and yeasts as microbial production hosts. The de novo biosynthetic pathway of vanillin in the vanilla orchid and the possible applied uses of this new knowledge in the biotechnology-derived and pod-based vanillin industries are also highlighted.

  20. Mental retardation and verbal dyspraxia in a new patient with de novo creatine transporter (SLC6A8) mutation.

    Science.gov (United States)

    Battini, Roberta; Chilosi, Anna; Mei, Davide; Casarano, Manuela; Alessandrì, M Grazia; Leuzzi, Vincenzo; Ferretti, Giovanni; Tosetti, Michela; Bianchi, M Cristina; Cioni, Giovanni

    2007-08-01

    We report on a 9.5-year-old Italian boy affected by creatine transporter deficit (CT1), due to a de novo mutation in SLC6A8 gene. The patient was investigated by means of a comprehensive neuropsychological protocol and presented with an unusual alteration of speech and expressive-language function, associated with mental retardation, that differed from CT1 patients described to date. In particular, he exhibited a developmental apraxia of speech (DAS) with motor planning and execution deficit, while receptive language was consistent with his mental age.

  1. De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells

    OpenAIRE

    Tadokoro, Yuko; Ema, Hideo; Okano, Masaki; Li, En; Nakauchi, Hiromitsu

    2007-01-01

    DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34−/low, c-Kit+, Sca-1+, lineage marker− (CD34− KSL) cells, a fraction of mouse bone marrow cells hi...

  2. HBV-INFECTION DE NOVO AFTER ORTHOTOPIC LIVER TRANSPLANTATION: CLINICAL AND VIROLOGICAL CHARACTERISTICS, ASSESSMENT OF ANTIVIRUS THERAPY EFFECTIVENESS

    Directory of Open Access Journals (Sweden)

    O. I. Malomuzh

    2012-01-01

    Full Text Available We studied 11 cases of HBV-infection de novo in patients after orthotopic liver transplantation performed because of non-viral cirrhosis. Serum НBeAg, was revedled in all patients. In most cases clinical course of HBV-infection was benign. Treatment with entecavir was more effective than lamivudin, and brought to НBsAg elimination in 4 patients. Treatment with lamivudin led to descrease viral load in all patients and HBsAg elimination in one case. 

  3. Huntington's disease-like and ataxia syndromes: identification of a family with a de novo SCA17/TBP mutation

    DEFF Research Database (Denmark)

    Bech, Sara; Petersen, Thor; Nørremølle, Anne;

    2010-01-01

    The autosomal dominant spinocerebellar ataxias, commonly referred to as SCAs, are clinically and genetically heterogeneous neurodegenerative disorders. Twenty-eight genetic subtypes have been identified, of which 7 are caused by expansion of a CAG trinucleotide repeat that encodes a polyglutamine...... with a Huntington's disease-like phenotype without the HD-gene mutation and 178 patients with genetically unclassified cerebellar ataxia for the mutation in TBP. A 33-year old woman presenting with an HD like phenotype with a de novo 54 CAG/CAA repeat expansion was identified. Her normal allele included 38 repeats......-like phenotypes and ataxia syndromes, also in isolated cases....

  4. Hematopoietic specification from human pluripotent stem cells: current advances and challenges toward de novo generation of hematopoietic stem cells.

    Science.gov (United States)

    Slukvin, Igor I

    2013-12-12

    Significant advances in cellular reprogramming technologies and hematopoietic differentiation from human pluripotent stem cells (hPSCs) have already enabled the routine production of multiple lineages of blood cells in vitro and opened novel opportunities to study hematopoietic development, model genetic blood diseases, and manufacture immunologically matched cells for transfusion and cancer immunotherapy. However, the generation of hematopoietic cells with robust and sustained multilineage engraftment has not been achieved. Here, we highlight the recent advances in understanding the molecular and cellular pathways leading to blood development from hPSCs and discuss potential approaches that can be taken to facilitate the development of technologies for de novo production of hematopoietic stem cells.

  5. Surviving cells after treatment with gemcitabine or 5-fluorouracil for the study of de novo resistance of pancreatic cancer.

    Science.gov (United States)

    Liu, Qing-Hua; Zhang, Jing; Zhao, Chen-Yan; Yu, Dang-Hui; Bu, Hai-Ji; Chen, Ying; Ni, Can-Yong; Zhu, Ming-Hua

    2012-01-01

    One of the hallmarks of pancreatic cancer is its inherent insensitivity to chemotherapy. This study was undertaken to develop a cell model for the study of de novo resistance of pancreatic cancer. The surviving pancreatic cancer cells after a 3-day exposure to gemcitabine or 5-fluorouracil followed by another 7-day recovery were potentially drug-resistant. They had similar morphology and comparable growth and tumorigenic potentials to their untreated parental cells. Repeated subculture affected the cell-cycle profile and growth characteristics of the surviving cells. Our data suggest that surviving pancreatic cancer cells after drug treatment are a useful model for exploring intrinsic resistance.

  6. Vanillin - Bioconversion and Bioengineering of the most popular plant flavour and its de novo biosynthesis in the vanilla orchid.

    Science.gov (United States)

    Gallage, Nethaji J; Moeller, Birger Lindberg

    2014-09-30

    During recent years, biotechnology derived production of flavours and fragrances have expanded rapidly. The world's most popular flavour vanillin is no exception. This review outlines the current state of biotechnology-based vanillin synthesis with the use of ferulic acid, eugenol and glucose as substrates and bacteria, fungi and yeasts as microbial production hosts. The elucidated de novo biosynthetic pathway of vanillin in the vanilla orchid and the possible applied uses of this new knowledge in the biotechnology derived and pod-based vanillin industries are also highlighted.

  7. KeyPathwayMiner - De-novo network enrichment by combining multiple OMICS data and biological networks

    DEFF Research Database (Denmark)

    Baumbach, Jan; Alcaraz, Nicolas; Pauling, Josch K.

    We tackle the problem of de-novo pathway extraction. Given a biological network and a set of case-control studies, KeyPathwayMiner efficiently extracts and visualizes all maximal connected sub-networks that contain mainly genes that are dysregulated, e.g., differentially expressed, in most cases...... studied. The exact quantities for ``mainly'' and ``most'' are modeled with two easy-to-interpret parameters that allow the user to control the number of outliers (not dysregulated genes/cases) in the solutions. We developed two slightly varying models that fall into the class of NP-Hard optimization...

  8. SSP: an interval integer linear programming for de novo transcriptome assembly and isoform discovery of RNA-seq reads.

    Science.gov (United States)

    Safikhani, Zhaleh; Sadeghi, Mehdi; Pezeshk, Hamid; Eslahchi, Changiz

    2013-01-01

    Recent advances in the sequencing technologies have provided a handful of RNA-seq datasets for transcriptome analysis. However, reconstruction of full-length isoforms and estimation of the expression level of transcripts with a low cost are challenging tasks. We propose a novel de novo method named SSP that incorporates interval integer linear programming to resolve alternatively spliced isoforms and reconstruct the whole transcriptome from short reads. Experimental results show that SSP is fast and precise in determining different alternatively spliced isoforms along with the estimation of reconstructed transcript abundances. The SSP software package is available at http://www.bioinf.cs.ipm.ir/software/ssp.

  9. Três casos de hipertensão e Fístula Arteriovenosa Renal: com uma fístula de novo Tres casos de hipertención y Fístula Arteriovenosa Renal: con una fístula de novo Three cases of hypertension and Renal Arteriovenous fistula with a de novo fistula

    Directory of Open Access Journals (Sweden)

    Natalia Correa Vieira Melo

    2009-05-01

    Full Text Available Fístula Arteriovenosa Renal (FAVR é uma causa rara e potencialmente reversível de hipertensão e insuficiência renal e/ou cardíaca. O tratamento da FAVR visa preservar o máximo de parênquima renal e, concomitantemente, erradicar os sintomas e efeitos hemodinâmicos decorrentes da FAVR. No presente estudo, serão relatados três casos de FAVR, incluindo um caso de FAVR idiopática de novo, que se apresentaram com hipertensão e insuficiência renal e/ou cardíaca, e descrever a terapêutica adotada e os resultados obtidos.La Fístula Arteriovenosa Renal (FAVR es una causa rara y potencialmente reversible de hipertensión e insuficiencia renal y/o cardíaca. El tratamiento de la FAVR busca preservar el máximo de parénquima renal y, concomitantemente, erradicar los síntomas y efectos hemodinámicos resultantes de la FAVR. En el presente estudio, se relatarán tres casos de FAVR, incluyendo un caso de FAVR idiopática de novo, que se presentaron con hipertensión e insuficiencia renal y/o cardíaca, y describir la terapéutica adoptada y los resultados obtenidos.The Renal Arteriovenous Fistula (RAVF is a rare and potentially reversible cause of hypertension and kidney and/or heart failure. The treatment of RAVF aims at preserving the most of the renal parenchyma and, concomitantly, eradicating the symptoms and hemodynamic effects caused by the RAVF. The present study reports three cases of RAVF, including one case of a de novo idiopathic RAVF, which presented with hypertension and kidney and/or heart failure and describes the therapeutic measures used to treat these patients as well as the outcomes.

  10. Attach great importance to the occurance and prevention of the de novo malignancy after liver transplantation with 5 cases in a single center%重视肝移植术后新发肿瘤的发生与防治一附单中心5例报告

    Institute of Scientific and Technical Information of China (English)

    章笑忠; 王文涛; 杨俭; 雷建勇; 严律南

    2011-01-01

    tation in our centre and analyze the probable cause of the malignancy by comparing with previous research outcome. Methods We retrospectively analyzed the five patients with de novo malignancy after liver transplantation in our centre and the probable risk factors of the de novo malignancy. All patients with a diagnosis of de novo malignancy after transplantation were determined by pathological examination and were analyzed for patient demographics (age,gender) .indication for the liver transplantation,personal history ( smoking or alcohol abuse) ,family history of malignancy,liver function condition before transplantation,immunosuppresion treatment,type of de novo malignancy, the interval from OLT to the diagnosis of malignancy and the treatment for the malignancy. Results Five patients in all had developed malignancy of colon cancer,lung cancer,esophagus cancer,lymphoma and nasopharyngeal cancer. The median age of the patients at the time of diagnosis of the malignancy was 49 y. The interval from OLT to the diagnosis of malignancy ranges from 17-66 months with the median interval of 44 months. Conclusions De novo maligancy after OLT was related to gender,smoking and the relatively low immune function caused by immunosuppression agents. Promoting the cessation of the alcohol and smoking is essential and the high-risk recipients immunosuppression dosage should be properly reduced especially in the patients who have already been diagnosed as malignancy.%目的 新发肿瘤是肝移植术后常见并发症,是影响肝移植受体晚期死亡的重要原因之一,本文结合5例患者的临床资料和相关关文献对肝移植术后新发肿瘤及其相关危险因素进行阐述.方法 回顾性分析5名病例术前相关资料(年龄、性别、原发肝病、嗜烟酒史及癌症家族史)以及术后情况(移植术后免疫抑制治疗方案、急性排斥反应发生与否、新发肿瘤类型、癌症发生时间、癌症治疗及其预后),分析新发

  11. TUMEFAÇÃO CRANIANA DE NOVO – UM DESAFIO DIAGÓSTICO

    Directory of Open Access Journals (Sweden)

    Ana Maria Ferreira

    2016-07-01

    Full Text Available Introdução: A coleção líquida subaponevrótica é uma tume- fação craniana, benigna, que surge semanas após o nascimen- to, decorrente provavelmente de um parto traumático. O diag- nóstico é desafiante uma vez que há poucos casos descritos na literatura. O tratamento recomendado é conservador, já que na maioria dos casos há resolução espontânea. Caso Clínico: Descreve-se um caso de uma lactente de 2 meses com antecedentes de parto distócico por ventosa e de um cefalohematoma occipitoparietal direito com reabsorção es- pontânea no período neonatal. Observou-se, de novo e com 6 dias de evolução, uma tumefação occipitoparietal direita com 5-6 cm de maior diâmetro, de volume oscilante, consistência mole, móvel nos planos superficiais, transluzente, indolor e sem si- nais inflamatórios, permanecendo a criança clinicamente bem. O estudo analítico, radiografia do crânio e ecografia transfon- tanelar não tinham alterações de relevo. A ecografia de partes moles mostrou uma coleção líquida, sugerindo a possibilidade de um cefalohematoma. Foi feita punção aspirativa com saída de líquido sero-hemático, sem qualquer crescimento bacteriano. Ponderando-se a possibilidade de uma malformação congénita foi realizado RMN CE que evidenciou uma coleção liquida subga- leal. Após revisão da literatura, o diagnóstico de coleção liquida subaponevrótica revelou-se o mais provável e o tratamento tem sido conservador. Comentários: Apesar do impacto da sua apresentação clí- nica, trata-se de uma entidade benigna, pouco descrita na lite- ratura, sendo por isso fundamental que os prestadores de cui- dados de saúde estejam aptos para um diagnóstico imediato e um acompanhamento adequado, que passa por medidas de vigilância até à completa resolução ao fim de algumas semanas.

  12. Synthesis and Characterization of Metal-Organic Frameworks (MOFs) That Are Difficult to Access De Novo

    Science.gov (United States)

    Karagiaridi, Olga

    Metal-organic frameworks (MOFs) are a class of intriguing hybrid materials, comprised of metal-based nodes joined by organic linkers into a crystalline, porous, three-dimensional lattice. Their signature properties (well-defined surfaces, tailorability and ultra-high porosity) render them promising candidates for many applications, including, but not limited to, gas storage, gas separation, catalysis and sensing. One of the greatest challenges associated with MOF synthesis lies in the fact that obtaining a desired MOF structure that is tailored to perform a specific application is often not trivial. Traditional synthetic pathways termed "de novo synthesis" (typically one-pot reactions between the MOF structural building blocks under solvothermal conditions) often give rise to side products that do not possess the desired structure. To circumvent this problem, we have studied in depth two powerful MOF synthetic techniques -- solvent-assisted linker exchange (SALE) and transmetalation. These are heterogeneous reactions of parent MOF crystals with concentrated solutions of organic linkers and inorganic metal salts, respectively, that lead to the replacement of the linkers or metal nodes within the parent MOFs by the desired components, while the overall framework topology is preserved. The projects described in this dissertation have aimed to apply these techniques to transform simple (unfunctionalized) and easy to synthesize representative materials from various MOF systems to structurally and functionally interesting daughter products. Examples include synthesis of MOFs that are energetically "unfavorable", extension of MOF cages by longer linker incorporation, functionalization of MOF pores and endowment of MOFs with permanent and persistent porosity. Through these projects, we have been able to formulate a set of rules that can be applied to predict the successful outcome of SALE. Since the allure of MOFs lies in their applications, expanding the range of

  13. The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion.

    Directory of Open Access Journals (Sweden)

    Katharina Wimmer

    2011-11-01

    Full Text Available Long interspersed (L1 and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the -100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as -0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA-based mutation analysis protocols, resulting in improved detection compared to gDNA-based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16-23(18 within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1:c.1642-1_1642 in intron 14(10c, NM_000267.3(NF1:c.2835_2836 in exon 21(16, and NM_000267.3(NF1:c.4319_4320 in exon 33(25. Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites

  14. De novo Transcriptome Analysis of Sinapis alba in Revealing the Glucosinolate and Phytochelatin Pathways

    Science.gov (United States)

    Zhang, Xiaohui; Liu, Tongjin; Duan, Mengmeng; Song, Jiangping; Li, Xixiang

    2016-01-01

    Sinapis alba is an important condiment crop and can also be used as a phytoremediation plant. Though it has important economic and agronomic values, sequence data, and the genetic tools are still rare in this plant. In the present study, a de novo transcriptome based on the transcriptions of leaves, stems, and roots was assembled for S. alba for the first time. The transcriptome contains 47,972 unigenes with a mean length of 1185 nt and an N50 of 1672 nt. Among these unigenes, 46,535 (97%) unigenes were annotated by at least one of the following databases: NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO), and Clusters of Orthologous Groups of proteins (COGs). The tissue expression pattern profiles revealed that 3489, 1361, and 8482 unigenes were predominantly expressed in the leaves, stems, and roots of S. alba, respectively. Genes predominantly expressed in the leaf were enriched in photosynthesis- and carbon fixation-related pathways. Genes predominantly expressed in the stem were enriched in not only pathways related to sugar, ether lipid, and amino acid metabolisms but also plant hormone signal transduction and circadian rhythm pathways, while the root-dominant genes were enriched in pathways related to lignin and cellulose syntheses, involved in plant-pathogen interactions, and potentially responsible for heavy metal chelating, and detoxification. Based on this transcriptome, 14,727 simple sequence repeats (SSRs) were identified, and 12,830 pairs of primers were developed for 2522 SSR-containing unigenes. Additionally, the glucosinolate (GSL) and phytochelatin metabolic pathways, which give the characteristic flavor and the heavy metal tolerance of this plant, were intensively analyzed. The genes of aliphatic GSLs pathway were predominantly expressed in roots. The absence of aliphatic GSLs in leaf tissues was due to the shutdown of BCAT4, MAM1, and CYP79F1 expressions. Glutathione was extensively

  15. De Novo Transcriptome Assembly of the Chinese Swamp Buffalo by RNA Sequencing and SSR Marker Discovery.

    Directory of Open Access Journals (Sweden)

    Tingxian Deng

    Full Text Available The Chinese swamp buffalo (Bubalis bubalis is vital to the lives of small farmers and has tremendous economic importance. However, a lack of genomic information has hampered research on augmenting marker assisted breeding programs in this species. Thus, a high-throughput transcriptomic sequencing of B. bubalis was conducted to generate transcriptomic sequence dataset for gene discovery and molecular marker development. Illumina paired-end sequencing generated a total of 54,109,173 raw reads. After trimming, de novo assembly was performed, which yielded 86,017 unigenes, with an average length of 972.41 bp, an N50 of 1,505 bp, and an average GC content of 49.92%. A total of 62,337 unigenes were successfully annotated. Among the annotated unigenes, 27,025 (43.35% and 23,232 (37.27% unigenes showed significant similarity to known proteins in NCBI non-redundant protein and Swiss-Prot databases (E-value < 1.0E-5, respectively. Of these annotated unigenes, 14,439 and 15,813 unigenes were assigned to the Gene Ontology (GO categories and EuKaryotic Ortholog Group (KOG cluster, respectively. In addition, a total of 14,167 unigenes were assigned to 331 Kyoto Encyclopedia of Genes and Genomes (KEGG pathways. Furthermore, 17,401 simple sequence repeats (SSRs were identified as potential molecular markers. One hundred and fifteen primer pairs were randomly selected for amplification to detect polymorphisms. The results revealed that 110 primer pairs (95.65% yielded PCR amplicons and 69 primer pairs (60.00% presented polymorphisms in 35 individual buffaloes. A phylogenetic analysis showed that the five swamp buffalo populations were clustered together, whereas two river buffalo breeds clustered separately. In the present study, the Illumina RNA-seq technology was utilized to perform transcriptome analysis and SSR marker discovery in the swamp buffalo without using a reference genome. Our findings will enrich the current SSR markers resources and help spearhead

  16. De novo transcriptome analysis of Sinapis alba in revealing the glucosinolate and phytochelatin pathways

    Directory of Open Access Journals (Sweden)

    Xiaohui eZhang

    2016-03-01

    Full Text Available Sinapis alba is an important condiment crop and can also be used as a phytoremediation plant. Though it has important economic and agronomic values, sequence data and the genetic tools are still rare in this plant. In the present study, a de novo transcriptome based on the transcriptions of leaves, stems and roots was assembled for S. alba for the first time. The transcriptome contains 47,972 unigenes with a mean length of 1,185 nt and an N50 of 1,672 nt. Among these unigenes, 46,535 (97% unigenes were annotated by at least one of the following databases: NCBI non-redundant (Nr, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG pathway, Gene Ontology (GO, and Clusters of Orthologous Groups of proteins (COGs. The tissue expression pattern profiles revealed that 3,489, 1,361 and 8,482 unigenes were predominantly expressed in the leaves, stems and roots of S. alba, respectively. Genes predominantly expressed in the leaf were enriched in photosynthesis- and carbon fixation-related pathways. Genes predominantly expressed in the stem were enriched in not only pathways related to sugar, ether lipid and amino acid metabolisms but also plant hormone signal transduction and circadian rhythm pathways, while the root-dominant genes were enriched in pathways related to lignin and cellulose syntheses, involved in plant-pathogen interactions, and potentially responsible for heavy metal chelating and detoxification. Based on this transcriptome, 14,727 simple sequence repeats (SSRs were identified, and 12,830 pairs of primers were developed for 2,522 SSR-containing unigenes. Additionally, the glucosinolate (GSL and phytochelatin metabolic pathways, which give the characteristic flavor and the heavy metal tolerance of this plant, were intensively analyzed. The genes of aliphatic GSLs pathway were predominantly expressed in roots. The absence of aliphatic GSLs in leaf tissues was due to the shutdown of BCAT4, MAM1 and CYP79F1 expressions. Glutathione was

  17. Study Protocol: A Pilot Study to Determine the Safety and Efficacy of Induction-Therapy, De Novo MPA and Delayed mTOR-Inhibition in Liver Transplant Recipients with Impaired Renal Function. PATRON-Study

    Directory of Open Access Journals (Sweden)

    Schnitzbauer Andreas A

    2010-09-01

    Full Text Available Abstract Background Patients undergoing liver transplantation with preexisting renal dysfunction are prone to further renal impairment with the early postoperative use of Calcineurin-inhibitors. However, there is only little scientific evidence for the safety and efficacy of de novo CNI free "bottom-up" regimens in patients with impaired renal function undergoing liver transplantation. This is a single-center study pilot-study (PATRON07 investigating safety and efficacy of CNI-free, "bottom-up" immunosuppressive (IS strategy in patients undergoing liver transplantation (LT with renal impairment prior to LT. Methods/Design Patients older than 18 years with renal impairment at the time of liver transplantation eGFR 1.5 mg/dL will be included. Patients in will receive a CNI-free combination therapy (basiliximab, MMF, steroids and delayed Sirolimus. Primary endpoint is the incidence of steroid resistant acute rejection within the first 30 days after LT. The study is designed as prospective two-step trial requiring a maximum of 29 patients. In the first step, 9 patients will be included. If 8 or more patients show no signs of biopsy proven steroid resistant rejection, additional 20 patients will be included. If in the second step a total of 27 or more patients reach the primary endpoint the regimen is regarded to be safe and efficient. Discussion If a CNI-free-"bottom-up" IS strategy is safe and effective, this may be an innovative concept in contrast to classic top-down strategies that could improve the patient short and long-time renal function as well as overall complications and survival after LT. The results of PATRON07 may be the basis for a large multicenter RCT investigating the new "bottom-up" immunosuppressive strategy in patients with poor renal function prior to LT. http://www.clinicaltrials.gov-identifier: NCT00604357

  18. Identification of potential inhibitors for AIRS from de novo purine biosynthesis pathway through molecular modeling studies - a computational approach.

    Science.gov (United States)

    Rao, R Guru Raj; Biswal, Jayashree; Dhamodharan, Prabhu; Kanagarajan, Surekha; Jeyaraman, Jeyakanthan

    2016-10-01

    In cancer, de novo pathway plays an important role in cell proliferation by supplying huge demand of purine nucleotides. Aminoimidazole ribonucleotide synthetase (AIRS) catalyzes the fifth step of de novo purine biosynthesis facilitating in the conversion of formylglycinamidine ribonucleotide to aminoimidazole ribonucleotide. Hence, inhibiting AIRS is crucial due to its involvement in the regulation of uncontrollable cancer cell proliferation. In this study, the three-dimensional structure of AIRS from P. horikoshii OT3 was constructed based on the crystal structure from E. coli and the modeled protein is verified for stability using molecular dynamics for a time frame of 100 ns. Virtual screening and induced fit docking were performed to identify the best antagonists based on their binding mode and affinity. Through mutational studies, the residues necessary for catalytic activity of AIRS were identified and among which the following residues Lys35, Asp103, Glu137, and Thr138 are important in determination of AIRS function. The mutational studies help to understand the structural and energetic characteristics of the specified residues. In addition to Molecular Dynamics, ADME properties, binding free-energy, and density functional theory calculations of the compounds were carried out to find the best lead molecule. Based on these analyses, the compound from the NCI database, NCI_121957 was adjudged as the best molecule and could be suggested as the suitable inhibitor of AIRS. In future studies, experimental validation of these ligands as AIRS inhibitors will be carried out.

  19. Sequencing and de novo analysis of Crassostrea angulata (Fujian oyster from 8 different developing phases using 454 GSFlx.

    Directory of Open Access Journals (Sweden)

    Ji Qin

    Full Text Available Research on the mechanism for early development of shellfish, such as body plan, shell formation, settlement and metamorphosis is currently an active research field. However, studies were still limited and not deep enough because of the lack of genomic resources such as genome or transcriptome sequences. In the present research, de novo transcriptome sequencing was performed for Crassostrea angulata, the most economically important cultured oyster species in China, at eight early developmental stages using the 454 sequencing technology. A total of 555,215 reads were produced with an average length of 309 nucleotides that were then assembled into 10,462 contigs. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Six unique sequences related to settlement, metamorphosis and growth were subsequently analyzed by real-time PCR. Given the lack of whole genome information for oysters, transcriptome and de novo analysis of C. angulata from the eight different developing phases will provide important and useful information on early development mechanism and help genetic breeding of shellfish.

  20. De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth.

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    Michael A Fisher

    Full Text Available A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >10(6 de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains--ΔserB, ΔgltA, ΔilvA, and Δfes--were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions can be sustained by sequences designed de novo.

  1. A unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures.

    Science.gov (United States)

    McDaniel, Jonathan R; Radford, D Christopher; Chilkoti, Ashutosh

    2013-08-12

    Elastin-like polypeptides (ELPs) are stimulus-responsive peptide polymers that exhibit inverse temperature phase transition behavior, causing an ELP to aggregate above its inverse transition temperature (T(t)). Although this property has been exploited in a variety of biotechnological applications, de novo design of ELPs that display a specific T(t) is not trivial because the T(t) of an ELP is a complex function of several variables, including its sequence, chain length, polypeptide concentration, and the type and concentration of cosolutes in solution. This paper provides a quantitative model that predicts the T(t) of a family of ELPs (Val-Pro-Gly-Xaa-Gly, where Xaa = Ala and/or Val) from their composition, chain length, and concentration in phosphate buffered saline. This model will enable de novo prediction of the amino acid sequence and chain length of ELPs that will display a predetermined T(t) in physiological buffer within a specified concentration regime, thereby greatly facilitating the design of new ELPs for applications in medicine and biotechnology.

  2. Mandibulofacial dysostosis in a patient with a de novo 2;17 translocation that disrupts the HOXD gene cluster.

    Science.gov (United States)

    Stevenson, David A; Bleyl, Steven B; Maxwell, Teresa; Brothman, Arthur R; South, Sarah T

    2007-05-15

    Treacher Collins syndrome (TCS) is the prototypical mandibulofacial dysostosis syndrome, but other mandibulofacial dysostosis syndromes have been described. We report an infant with mandibulofacial dysostosis and an apparently balanced de novo 2;17 translocation. She presented with severe lower eyelid colobomas requiring skin grafting, malar and mandibular hypoplasia, bilateral microtia with external auditory canal atreasia, dysplastic ossicles, hearing loss, bilateral choanal stenosis, cleft palate without cleft lip, several oral frenula of the upper lip/gum, and micrognathia requiring tracheostomy. Her limbs were normal. Chromosome analysis at the 600-band level showed a 46,XX,t(2;17)(q24.3;q23) karyotype. Sequencing of the entire TCOF1 coding region did not show evidence of a sequence variation. High-resolution genomic microarray analysis did not identify a cryptic imbalance. FISH mapping refined the breakpoints to 2q31.1 and 17q24.3-25.1 and showed the 2q31.1 breakpoint likely affects the HOXD gene cluster. Several atypical findings and lack of an identifiable TCOF1 mutation suggest that this child has a provisionally unique mandibulofacial dysostosis syndrome. The apparently balanced de novo translocation provides candidate loci for atypical and TCOF1 mutation negative cases of TCS. Based on the agreement of our findings with one previous case of mandibulofacial dysostosis with a 2q31.1 transocation, we hypothesize that misexpression of genes in the HOXD gene cluster produced the described phenotype in this patient.

  3. De novo transcriptomes of olfactory epithelium reveal the genes and pathways for spawning migration in japanese grenadier anchovy (Coilia nasus.

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    Guoli Zhu

    Full Text Available BACKGROUND: Coilia nasus (Japanese grenadier anchovy undergoes spawning migration from the ocean to fresh water inland. Previous studies have suggested that anadromous fish use olfactory cues to perform successful migration to spawn. However, limited genomic information is available for C. nasus. To understand the molecular mechanisms of spawning migration, it is essential to identify the genes and pathways involved in the migratory behavior of C. nasus. RESULTS: Using de novo transcriptome sequencing and assembly, we constructed two transcriptomes of the olfactory epithelium from wild anadromous and non-anadromous C. nasus. Over 178 million high-quality clean reads were generated using Illumina sequencing technology and assembled into 176,510 unigenes (mean length: 843 bp. About 51% (89,456 of the unigenes were functionally annotated using protein databases. Gene ontology analysis of the transcriptomes indicated gene enrichment not only in signal detection and transduction, but also in regulation and enzymatic activity. The potential genes and pathways involved in the migratory behavior were identified. In addition, simple sequence repeats and single nucleotide polymorphisms were analyzed to identify potential molecular markers. CONCLUSION: We, for the first time, obtained high-quality de novo transcriptomes of C. nasus using a high-throughput sequencing approach. Our study lays the foundation for further investigation of C. nasus spawning migration and genome evolution.

  4. A de novo microdeletion of SEMA5A in a boy with autism spectrum disorder and intellectual disability.

    Science.gov (United States)

    Mosca-Boidron, Anne-Laure; Gueneau, Lucie; Huguet, Guillaume; Goldenberg, Alice; Henry, Céline; Gigot, Nadège; Pallesi-Pocachard, Emilie; Falace, Antonio; Duplomb, Laurence; Thevenon, Julien; Duffourd, Yannis; St-Onge, Judith; Chambon, Pascal; Rivière, Jean-Baptiste; Thauvin-Robinet, Christel; Callier, Patrick; Marle, Nathalie; Payet, Muriel; Ragon, Clemence; Goubran Botros, Hany; Buratti, Julien; Calderari, Sophie; Dumas, Guillaume; Delorme, Richard; Lagarde, Nathalie; Pinoit, Jean-Michel; Rosier, Antoine; Masurel-Paulet, Alice; Cardoso, Carlos; Mugneret, Francine; Saugier-Veber, Pascale; Campion, Dominique; Faivre, Laurence; Bourgeron, Thomas

    2016-06-01

    Semaphorins are a large family of secreted and membrane-associated proteins necessary for wiring of the brain. Semaphorin 5A (SEMA5A) acts as a bifunctional guidance cue, exerting both attractive and inhibitory effects on developing axons. Previous studies have suggested that SEMA5A could be a susceptibility gene for autism spectrum disorders (ASDs). We first identified a de novo translocation t(5;22)(p15.3;q11.21) in a patient with ASD and intellectual disability (ID). At the translocation breakpoint on chromosome 5, we observed a 861-kb deletion encompassing the end of the SEMA5A gene. We delineated the breakpoint by NGS and observed that no gene was disrupted on chromosome 22. We then used Sanger sequencing to search for deleterious variants affecting SEMA5A in 142 patients with ASD. We also identified two independent heterozygous variants located in a conserved functional domain of the protein. Both variants were maternally inherited and predicted as deleterious. Our genetic scre