By increasing the PCR amplification regime to 34 cycles, we have demonstrated that it is possible routinely to analyse ,100 pg DNA. The success rate was not improved (without impairing quality) by increasing cycle number further. Compared to amplification of 1 ng DNA at 28 cycles, it was shown that increased imbalance of heterozygotes occurred, along with an increase in the size (peak area) of stutters. The analysis of mixtures by peak area measurement becomes increasingly difficult as the sample size is reduced. Laboratory-based contamination cannot be completely avoided, even when analysis is carried out under stringent conditions of cleanliness. A set of guidelines that utilises duplication of results to interpret profiles originating from picogram levels of DNA is introduced. We demonstrate that the duplication guideline is robust by applying a statistical theory that models three key parameters -namely the incidence of allele drop-out, laboratory contamination and stutter. The advantage of the model is that the critical levels for each parameter can be calculated. This information may be used (for example) to determine levels of contamination that can be tolerated within the strategy employed. In addition we demonstrate that interpreting one banded loci, where allele dropout could have occurred, using LR 5 1/2f was conservative provided a that the band was low in peak area. Furthermore, we demonstrate that an apparent mis-match between crime-stain and a suspect DNA profile does not necessarily result in an exclusion. The method used is complex, yet can be converted into an expert system. We envisage this to be the next step.
Highlights d Vitamin-D-binding protein (DBP) is highly expressed in pancreatic a cells d Glucagon secretion and insulin tolerance are altered in mice lacking DBP d DBP-null a cells possess an abnormal actin cytoskeleton and are dysfunctional d DBP levels are decreased in a cells of donors with late-onset type 1 diabetes
In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a $C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathwaylspecific regulatory gene redD. The segment contained most of an ORF (red2) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in Red2 hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in red2 provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential uppromoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of red2 in the hnrb mutant, allows the accumulation of a threshold level of the RedD protein.
Aims/hypothesis Enterovirus (EV) infection of pancreatic islet cells is one possible factor contributing to type 1 diabetes development. We have reported the presence of EV genome by PCR and of EV proteins by immunohistochemistry in pancreatic sections. Here we explore multiple human virus species in the Diabetes Virus Detection (DiViD) study cases using innovative methods, including virus passage in cell cultures. Methods Six recent-onset type 1 diabetes patients (age 24–35) were included in the DiViD study. Minimal pancreatic tail resection was performed under sterile conditions. Eleven live cases (age 43–83) of pancreatic carcinoma without diabetes served as control cases. In the present study, we used EV detection methods that combine virus growth in cell culture, gene amplification and detection of virus-coded proteins by immunofluorescence. Pancreas homogenates in cell culture medium were incubated with EV-susceptible cell lines for 3 days. Two to three blind passages were performed. DNA and RNA were extracted from both pancreas tissue and cell cultures. Real-time PCR was used for detecting 20 different viral agents other than EVs (six herpesviruses, human polyomavirus [BK virus and JC virus], parvovirus B19, hepatitis B virus, hepatitis C virus, hepatitis A virus, mumps, rubella, influenza A/B, parainfluenza 1–4, respiratory syncytial virus, astrovirus, norovirus, rotavirus). EV genomes were detected by endpoint PCR using five primer pairs targeting the partially conserved 5′ untranslated region genome region of the A, B, C and D species. Amplicons were sequenced. The expression of EV capsid proteins was evaluated in cultured cells using a panel of EV antibodies. Results Samples from six of six individuals with type 1 diabetes (cases) and two of 11 individuals without diabetes (control cases) contained EV genomes (p<0.05). In contrast, genomes of 20 human viruses other than EVs could be detected only once in an individual with diabetes (Epstein–Barr virus) and once in an individual without diabetes (parvovirus B19). EV detection was confirmed by immunofluorescence of cultured cells incubated with pancreatic extracts: viral antigens were expressed in the cytoplasm of approximately 1% of cells. Notably, infection could be transmitted from EV-positive cell cultures to uninfected cell cultures using supernatants filtered through 100 nm membranes, indicating that infectious agents of less than 100 nm were present in pancreases. Due to the slow progression of infection in EV-carrying cell cultures, cytopathic effects were not observed by standard microscopy but were recognised by measuring cell viability. Sequences of 5′ untranslated region amplicons were compatible with EVs of the B, A and C species. Compared with control cell cultures exposed to EV-negative pancreatic extracts, EV-carrying cell cultures produced significantly higher levels of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP1). Conclusions/interpretation Sensitive assays confirm that the pancreases of all DiViD cases contain EVs but no other viruses. Analogous EV strains have been found in pancreases of two of 11 individuals without diabetes. The detected EV strains can be passaged in series from one cell culture to another in the form of poorly replicating live viruses encoding antigenic proteins recognised by multiple EV-specific antibodies. Thus, the early phase of type 1 diabetes is associated with a low-grade infection by EVs, but not by other viral agents. Graphical abstract
C-peptide declines in type 1 diabetes although many long-duration patients retain low, but detectable levels. Histological analyses confirm that beta cells can remain following type 1 diabetes onset. We explored the trends observed in C-peptide decline in UK Genetic Resource Investigating Diabetes (UK GRID) cohort (N=4,079), with beta cell loss in pancreas donors from the network for Pancreatic Organ donors with Diabetes (nPOD) biobank and the Exeter Archival Diabetes Biobank (EADB) (combined N=235), stratified by recently reported age at diagnosis endotypes (< 7, 7-12, ≥ 13 years) across increasing diabetes durations. The proportion of individuals with detectable C-peptide declined beyond the first year after diagnosis, but this was most marked in the youngest age group (< 1 year duration: age < 7 years: 18/20 (90%), 7-12 years: 107/110 (97%), ≥ 13 years: 58/61 (95%) versus. 1-5 years post diagnosis: < 7 years: 172/522 (33%), 7-12 years: 604/995 (61%), ≥ 13 years: 225/289 (78%)). A similar profile was observed in beta cell loss, with those diagnosed at younger ages experiencing more rapid loss of islets containing insulin-positive (insulin+) beta cells < 1 year post diagnosis: age < 7 years: 23/26 (88%), 7-12 years: 32/33 (97%), ≥ 13 years: 22/25 (88%) versus. 1-5 years post diagnosis: < 7 years: 1/12 (8.3%) ,7-12 years: 7/13 (54%), ≥ 13 years: 7/8 (88%)). These data should be considered in the planning and interpretation of intervention trials designed to promote beta cell retention and function.
Introductory paragraph Gene expression is tightly regulated with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function 1 . This silencing is largely controlled by non-coding elements and their disruption might cause human disease 2 . We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo variants affecting a 42bp conserved region encompassed by a regulatory element in intron 2 of Hexokinase 1 ( HK1 ). HK1 is widely expressed across all tissues except for liver and pancreatic beta-cells and is thus termed a “disallowed gene” in these specific tissues. We demonstrated that the variants result in a loss of repression of HK1 in pancreatic beta-cells, thereby causing insulin secretion and congenital hyperinsulinism. Using epigenomic data accessed from public repositories, we demonstrated that these variants reside within a regulatory region that we determine to be critical for cell-specific silencing. Importantly, this has revealed a disease mechanism for non-coding variants that cause inappropriate expression of a disallowed gene.
Aims Misclassification of diabetes is common due to an overlap in the clinical features of type 1 and type 2 diabetes. Combined diagnostic models incorporating clinical and biomarker information have recently been developed that can aid classification, but they have not been validated using pancreatic pathology. We evaluated a clinical diagnostic model against histologically defined type 1 diabetes. Methods We classified cases from the Network for Pancreatic Organ donors with Diabetes (nPOD) biobank as type 1 (n = 111) or non-type 1 (n = 42) diabetes using histopathology. Type 1 diabetes was defined by lobular loss of insulincontaining islets along with multiple insulin-deficient islets. We assessed the discriminative performance of previously described type 1 diabetes diagnostic models, based on clinical features (age at diagnosis, BMI) and biomarker data [autoantibodies, type 1 diabetes genetic risk score (T1D-GRS)], and singular features for identifying type 1 diabetes by the area under the curve of the receiver operator characteristic (AUC-ROC). Results Diagnostic models validated well against histologically defined type 1 diabetes. The model combining clinical features, islet autoantibodies and T1D-GRS was strongly discriminative of type 1 diabetes, and performed better than clinical features alone (AUC-ROC 0.97 vs. 0.95; P = 0.03). Histological classification of type 1 diabetes was concordant with serum C-peptide [median < 17 pmol/l (limit of detection) vs. 1037 pmol/l in non-type 1 diabetes; P < 0.0001]. Conclusions Our study provides robust histological evidence that a clinical diagnostic model, combining clinical features and biomarkers, could improve diabetes classification. Our study also provides reassurance that a C-peptidebased definition of type 1 diabetes is an appropriate surrogate outcome that can be used in large clinical studies where histological definition is impossible.
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