16 research outputs found
Multidimensional Crude Feedstock Profiling
No full textThe efficacy and safety of biopharmaceutical products, combined with their ability to address previously untreated conditions, has made the biopharmaceutical field the fastest growing part of the industry. Due to their unique properties, monoclonal antibodies (mAbs) and therapeutic enzymes represent the most used macromolecules for biopharmaceutical development. However, currently the purification of mAbs and enzymes is a target of process optimisation, since it constitutes the bottleneck of the biopharmaceutical manufacturing. Recently, a model-based hybrid process development was proposed for rationally synthesising and designing a protein purification process. In this approach, a combination of high throughput experimentation for physicochemical protein characterisation and thermodynamic parameter regression can be used for an in silico optimisation of a proposed downstream process design. The main goal of this project is first to determine the physicochemical parameters of crude protein mixtures by applying three-dimensional liquid chromatography in two modes of operation, isocratic and gradient elusion. Secondly, to determine the isotherms for thermodynamic parameter regression and finally to systematically compare the determined thermodynamic parameters from the two modes of operation. The experimental scheme consisted of gradient chromatofocusing (gCF) on strong ion exchange column, hydrophobic interaction chromatography (HIC) in gradient and isocratic mode and size exclusion chromatography (SEC) or Matrix assessed laser desorption/ionisation mass spectrometry (MALDI-TOF MS). Moreover, Fourier transform and exponentially modified Gaussian peak fitting was used for the deconvolution of the resulted chromatograms. Finally, an analytical solution for the linear part of the HIC isotherm was proposed for parameter regression and correlation of isocratic and gradient parameters. The results revealed that the experimental methodology as well as the data analysis method proposed, are suitable for the physicochemical characterisation of the crude protein component mixtures as well for the determination and correlation of isocratic and gradient parameters. The determined physicochemical and thermodynamic parameters can be used to predict the protein behaviour and interactions with the chromatographic columns, while in combination with the determined isotherm parameters can be used for an in silico optimisation of a DSP design.Bioprocess Engineering GroupBiotechnologyApplied Science
GATA4 mutations are a cause of neonatal and childhood-onset diabetes
Full text linkThis is the author accepted manuscript. The final version is available from the American Diabetes Association via the DOI in this record.The GATA family zinc finger transcription factors GATA4 and GATA6 are known to play important roles in the development of the pancreas. In mice, both Gata4 and Gata6 are required for pancreatic development. In humans, GATA6 haploinsufficiency can cause pancreatic agenesis and heart defects. Congenital heart defects also are common in patients with GATA4 mutations and deletions, but the role of GATA4 in the developing human pancreas is unproven. We report five patients with deletions (n = 4) or mutations of the GATA4 gene who have diabetes and a variable exocrine phenotype. In four cases, diabetes presented in the neonatal period (age at diagnosis 1-7 days). A de novo GATA4 missense mutation (p.N273K) was identified in a patient with complete absence of the pancreas confirmed at postmortem. This mutation affects a highly conserved residue located in the second zinc finger domain of the GATA4 protein. In vitro studies showed reduced DNA binding and transactivational activity of the mutant protein. We show that GATA4 mutations/deletions are a cause of neonatal or childhood-onset diabetes with or without exocrine insufficiency. These results confirm a role for GATA4 in normal development of the human pancreas.The research leading to these results received funding from the European Community’s 7th Framework Programme (FP7/2007-2013) under grant agreement number FP7-PEOPLE-ITN-2008 (Marie Curie Initial Training Network, Biology of Liver and Pancreatic Development and Disease) and grant agreement number 223211 (Collaborative European Effort to Develop Diabetes Diagnostics), Ministerio de Economía y Competitividad (SAF2011-27086), Diabetes UK (ref. 11/0004193), and the Wellcome Trust. S.E. and A.T.H. are employed as core members of staff within the National Institute for Health Research–funded Exeter Clinical Research Facility. S.E., J.F., and A.T.H. are Wellcome Trust Senior Investigators and A.T.H. is a National Institute for Health Research Senior Investigator. M.B. and W.M. are supported by National Science Center, Poland (NCN) grant 2011/01/M/NZ5/02815 and by Innovative Economy Operational Programme–Activity 1.2 (the TEAM Programme coordinated by the Foundation for Polish Science)
Recessively inherited LRBA mutations cause autoimmunity presenting as neonatal diabetes
Full text linkThis is the author accepted manuscript. The final version is available from the American Diabetes Association via the DOI in this record.The following erratum was published on 5 January 2018 at DOI 10.2337/db18-er03b
Erratum. Recessively Inherited LRBA Mutations Cause Autoimmunity Presenting as Neonatal Diabetes. Diabetes 2017;66:2316–2322
Matthew B. Johnson; Elisa De Franco; Hana Lango Allen; Aisha Al Senani; Nancy Elbarbary; Zeynep Siklar; Merih Berberoglu; Zineb Imane; Alireza Haghighi; Zahra Razavi; Irfan Ullah; Saif Alyaarubi; Daphne Gardner; Sian Ellard; Andrew T. Hattersley; Sarah E. Flanagan
In the article listed above, Ayla Güven, of the Pediatric Endocrinology Clinic, Göztepe Educational and Research Hospital, Istanbul, Turkey, was erroneously omitted from the author list. Dr. Güven recruited patients, provided clinical information, and contributed to discussion.
The authors apologize for this unfortunate omission. The online version of the article (https://doi.org/10.2337/db17-0040) has been updated to correct this omission.Young-onset autoimmune diabetes associated with additional autoimmunity usually reflects a polygenic predisposition, but rare cases result from monogenic autoimmunity. Diagnosing monogenic autoimmunity is crucial for patients' prognosis and clinical management. We sought to identify novel genetic causes of autoimmunity presenting with neonatal diabetes (NDM) (diagnosis <6 months). We performed exome sequencing in a patient with NDM and autoimmune lymphoproliferative syndrome and his unrelated, unaffected parents and identified compound heterozygous null mutations in LRBA Biallelic LRBA mutations cause common variable immunodeficiency-8; however, NDM has not been confirmed in this disorder. We sequenced LRBA in 169 additional patients with diabetes diagnosed <1 year without mutations in the 24 known NDM genes. We identified recessive null mutations in 8 additional probands, of which, 3 had NDM (<6 months). Diabetes was the presenting feature in 6 of 9 probands. Six of 17 (35%) patients born to consanguineous parents and with additional early-onset autoimmunity had recessive LRBA mutations. LRBA testing should be considered in patients with diabetes diagnosed <12 months, particularly if they have additional autoimmunity or are born to consanguineous parents. A genetic diagnosis is important as it can enable personalized therapy with abatacept, a CTLA-4 mimetic, and inform genetic counseling.This work was supported by a Wellcome Trust Senior Investigator Award to S.E. and A.T.H. (grant 098395/Z/12/Z). A.T.H. is a National Institute for Health Research Senior Investigator. E.D.F. is a Naomi Berrie Fellow in Diabetes Research. S.E.F. has a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 105636/Z/14/Z). Additional support came from the University of Exeter and the National Institute for Health Research Exeter Clinical Research Facility
Expanding the Clinical Spectrum Associated With GLIS3 Mutations
Full text linkThis is the author accepted manuscript. The final version is available from the Endocrine Society via the DOI in this record.CONTEXT: GLIS3 (GLI-similar 3) is a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2-type zinc finger domains. The protein is expressed early in embryogenesis and plays a critical role as both a repressor and activator of transcription. Human GLIS3 mutations are extremely rare. OBJECTIVE: The purpose of this article was determine the phenotypic presentation of 12 patients with a variety of GLIS3 mutations. METHODS: GLIS3 gene mutations were sought by PCR amplification and sequence analysis of exons 1 to 11. Clinical information was provided by the referring clinicians and subsequently using a questionnaire circulated to gain further information. RESULTS: We report the first case of a patient with a compound heterozygous mutation in GLIS3 who did not present with congenital hypothyroidism. All patients presented with neonatal diabetes with a range of insulin sensitivities. Thyroid disease varied among patients. Hepatic and renal disease was common with liver dysfunction ranging from hepatitis to cirrhosis; cystic dysplasia was the most common renal manifestation. We describe new presenting features in patients with GLIS3 mutations, including craniosynostosis, hiatus hernia, atrial septal defect, splenic cyst, and choanal atresia and confirm further cases with sensorineural deafness and exocrine pancreatic insufficiency. CONCLUSION: We report new findings within the GLIS3 phenotype, further extending the spectrum of abnormalities associated with GLIS3 mutations and providing novel insights into the role of GLIS3 in human physiological development. All but 2 of the patients within our cohort are still alive, and we describe the first patient to live to adulthood with a GLIS3 mutation, suggesting that even patients with a severe GLIS3 phenotype may have a longer life expectancy than originally described.This work was supported by the Wellcome Trust. A.T.H. and S.E. are Wellcome Trust Senior Investigators and A.T.H. is an National Institute for Health Research Senior Investigator. The views expressed are those of the author and do not reflect the official policy of the Department of the Army, the Department of Defense or the U.S. Government. - See more at: http://press.endocrine.org/doi/10.1210/jc.2015-1827#sthash.nuJ86hHb.dpu
Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability
No full textPubMedID: 25015100Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severityduetocompensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibitedsevere kinetic defects (RAI ? 0.01). There was no correlation between CSG and RAI (r2 = 0.05, P = 0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison ofCSGwith RSI detected a highly significant correlation (r2 = 0.74,P = 0.002).We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity. ©The Author 2014.8Oxford University of Oxford: 2Institute UCLH Biomedical Research Centre Royal Devon and Exeter NHS Foundation Trust Wellcome Trust: 095101/Z/10/Z1Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, Oxford OX3 7LE, UK, 2Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter EX2 5DW, UK, 3Macleod Diabetes and Endocrine Centre and, 4Molecular Genetics Laboratory, Royal Devon and Exeter NHS Foundation Trust, Exeter EX2 5DW, UK, 5Department of Paediatric Endocrinology, Diyarbakir Children State Hospital, Diyarbakir 21100, Turkey, 6Division of Pediatric Endocrinology, Dr. Sadi Konuk Education and Research Hospital, Bakirkoy, Istanbul 34147, Turkey, 7Pediatric Endocrine Division, Queen Rania Al Abdullah Hospital for Children, King Hussein Medical Center, Royal Medical Services, Amman 11814, Jordan and 8Oxford NIHR Biomedical Research Centre, Churchill Hospital, Oxford OX3 7LE, UKThis work was supported by the Wellcome Trust (grant number 095101/Z/10/Z to A.L.G.). A.L.G. is a Wellcome Trust Senior Fellow in Basic and Biomedical Science. A.T.H. and S.E. are Wellcome Trust Senior Investigators. A.T.H. is a National Institute for Health Research (NIHR) Senior Investigator. A.T.H. is funded by the NIHR Exeter Clinical Research Facility. This article presents independent research supported by the NIHR Exeter Clinical Research Facility. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust
Nanoscale Mixed Ion-Electron Conducting NASICON-type Thin-Films: Lithium Titanium Phosphate via Atomic Layer Deposition
No full textThe attached data encompasses the raw data files and processed XPS data for the article “Nanoscale Mixed Ion-Electron Conducting NASICON-type Thin-Films: Lithium Titanium Phosphate via Atomic Layer DepositionThis work was supported by Murata Integrated Passive Solutions, the U.S. Department of Energy, and the National Science Foundation. Murata supported the evaluation and testing of ALD solid electrolyte materials for application in 3D solid state capacitor structures. Under the Office of Science, Office of Basic Energy Sciences, Grant DE-SC0021070, DOE supported the synthesis of Li-containing titanium phosphates with composition varied through a dopant-supercycle process. A.T.H. and J.C. acknowledge support from the Center for Enhanced Nanofluidic Transport (CENT), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences, under Award # DE-SC0019112. D.R.F was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE 1840340. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation
Adiposity-Related Heterogeneity in Patterns of Type 2 Diabetes Susceptibility Observed in Genome-Wide Association Data
Full text linkOBJECTIVE-This study examined how differences in the BMI distribution of type 2 diabetic case subjects affected genome-wide patterns of type 2 diabetes, association mid considered the implications for the etiological heterogeneity of type 2 diabetes.RESEARCH DESIGN AND METHODS-We reanalyzed data from the Wellcome Trust Case Control Consortium genome-wide association scan (1,924 case subjects, 2,938 control Subjects: 393,453 single-nucleotide polymorphisms [SNPs]) after stratifying case subjects (into "obese" and "nonobese") according to median BMI (30.2 kg/m(2)). Replication. of signals in which alternative case-ascertainment strategies generated marked effect size heterogeneity in type 2 diabetes association signal was sought in additional samples.RESULTS-In the "obese-type 2 diabetes" scan, FTO variants had the strongest type 2 diabetes effect, (rs8050136: relative risk [RR] 1.49 [95% CI 1.34-1.66], P = 1.3 X 10(-13)), with only weak evidence for TCF7L2 (rs7901695 RR 1.21 [1.09-1.35], P = 0.001). reversed in the "nonobese" scan, with FTO This situation was association undetectable (RR 1.07 [0.97-1.19], P = 0.19) and TCF7L2 predominant (RR 1.53 [1.37-1.71], P = 1.3 X 10(-14)). These patterns, confirmed by replication, generated strong combined evidence for between-stratum effect size heterogeneity (FTO: P-DIFF = 1.4 X 10(-7); TCF7L2: P-DIFF = 4.0 X 10(-6)). Other signals displaying evidence of effect size heterogeneity in the gencome-wide analyses (on chromosomes 3, 12, 15, and 18) did not, replicate. Analysis of the current list of type 2 diabetes susceptibility variants revealed nominal evidence for effect size heterogeneity for the SLC30A8 locus alone (RRobese 1.08 [1.01-1.15]; RRnonobese 1.18 [1.10-1.27]: P-DIFF = 0.04).CONCLUSIONs-This study demonstrates the impact of differences in case ascertainment on the power to detect and replicate genetic associations in genome-wide association studies. These data reinforce the notion that there is substantial etiological heterogeneity within type 2 diabetes. Diabetes 58:505-510, 2009</p
Dominant ER Stress-Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts
Full text linkThis is the author accepted manuscript. The final version is available from American Diabetes Association via the DOI in this record.Neonatal diabetes is frequently part of a complex syndrome with extrapancreatic features: 18 genes causing syndromic neonatal diabetes have been identified to date. There are still patients with neonatal diabetes who have novel genetic syndromes. We performed exome sequencing in a patient and his unrelated, unaffected parents to identify the genetic etiology of a syndrome characterized by neonatal diabetes, sensorineural deafness, and congenital cataracts. Further testing was performed in 311 patients with diabetes diagnosed before 1 year of age in whom all known genetic causes had been excluded. We identified 5 patients, including the initial case, with three heterozygous missense mutations in WFS1 (4/5 confirmed de novo). They had diabetes diagnosed before 12 months (2 before 6 months) (5/5), sensorineural deafness diagnosed soon after birth (5/5), congenital cataracts (4/5), and hypotonia (4/5). In vitro studies showed that these WFS1 mutations are functionally different from the known recessive Wolfram syndrome-causing mutations, as they tend to aggregate and induce robust endoplasmic reticulum stress. Our results establish specific dominant WFS1 mutations as a cause of a novel syndrome including neonatal/infancy-onset diabetes, congenital cataracts, and sensorineural deafness. This syndrome has a discrete pathophysiology and differs genetically and clinically from recessive Wolfram syndrome.A.T.H. and S.E. are the recipients of a Wellcome Trust Senior Investigator award (grant WT098395/Z/12/Z). A.T.H. is employed as a core member of staff within the National Institute for Health Research–funded Exeter Clinical Research Facility and is a National Institute for Health Research Senior Investigator. E.D.F. is a Naomi Berrie Fellow in Diabetes Research. S.E.F. has a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 105636/Z/14/Z). This work was partly supported by grants from National Institutes of Health (DK020579 and UL1 TR000448) to F.U
A Type 1 Diabetes Genetic Risk Score can identify patients with GAD65 autoantibody positive type 2 diabetes that rapidly progress to insulin therapy
Full text linkThis is the author accepted manuscript. The final version is available from American Diabetes Association via the DOI in this record.Objective
Progression to insulin therapy in clinically diagnosed type 2 diabetes is highly variable. GAD65 autoantibodies (GADA) are associated with faster progression, but their predictive value is limited. We aimed to determine if a Type 1 Diabetes Genetic Risk Score (T1DGRS) could predict rapid progression to insulin treatment over and above GADA testing.
Research Design and Methods
We examined the relationship between T1DGRS, GADA (negative or positive) and rapid insulin requirement (within 5 years) using Kaplan-Meier survival analysis and Cox regression in 8,608 participants with clinical type 2 diabetes (onset >35 years, treated without insulin for ≥6 months). T1DGRS was analyzed both continuously (as standardized scores) and categorized based on previously reported centiles of a type 1 diabetes population (50th (high)).
Results
In GADA positive participants (3.3%), those with higher T1DGRS progressed to insulin more quickly: Probability of insulin requirement at five years [95% CI]: 47.9%[35.0%,62.78%] (high T1DGRS) vs 27.6%[20.5%,36.5%] (medium T1DGRS) vs 17.6%[11.2%,27.2%] (low T1DGRS), p=0.001. In contrast T1DGRS did not predict rapid insulin requirement in GADA negative participants (p=0.4). In Cox regression analysis with adjustment for age of diagnosis, BMI and cohort, T1DGRS was independently associated with time to insulin only in the presence of GADA: hazard ratio per SD increase 1.48 (1.15,1.90), p=0.002.
Conclusions
A Type 1 Diabetes Genetic Risk Score alters the clinical implications of a positive GADA test in patients with clinical type 2 diabetes, and is independent of and additive to clinical features.The Wellcome Trust United Kingdom Type 2 Diabetes Case Control Collection (GoDARTS) was funded by The Wellcome Trust (084727/Z/08/Z, 085475/Z/08/Z, 085475/B/08/Z) and as part of the EU IMI-SUMMIT program. GADA assessment in GoDARTS and DCS was funded by EU Innovative Medicines Initiative 115317 (DIRECT), resources of which are composed of financial contributions from the European Union's Seventh Framework Programme (FP7/2007-2013), and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies in kind contribution. The DCS cohort was partially funded by the Netherlands Organization for Health Research and Development (Priority Medicines Elderly Programme 113102006). The Diabetes Alliance for Research in England (DARE) study was funded by the Wellcome Trust and supported by the Exeter NIHR Clinical Research Facility. The MASTERMIND study was funded by the UK Medical Research Council (MR/N00633X/) and supported by the NIHR Exeter Clinical Research Facility. The PRIBA study was funded by the National Institute for Health Research (U.K.) (DRF-2010-03-72) and supported by the NIHR Exeter Clinical Research Facility.
B.M.S and A.T.H. are supported by the NIHR Exeter Clinical Research Facility. T.J.M. is a National Institute for Health Research Senior Clinical Senior Lecturer. E.R.P. is a Wellcome Trust New Investigator (102820/Z/13/Z). A.T.H. is a Wellcome Trust Senior Investigator and NIHR Senior Investigator. R.A.O is supported by a Diabetes UK Harry Keen Fellowship (16/0005529). A.G.J. is supported by an NIHR Clinician Scientist award (CS-2015-15-018)
