A three-gene panel that distinguishes benign from malignant thyroid nodules.

Reliable preoperative diagnosis of malignant thyroid tumors remains challenging because of the inconclusive cytological examination of fine-needle aspiration biopsies. Although numerous studies have successfully demonstrated the use of high-throughput molecular diagnostics in cancer prediction, the application of microarrays in routine clinical use remains limited. Our aim was, therefore, to identify a small subset of genes to develop a practical and inexpensive diagnostic tool for clinical use. We developed a two-step feature selection method composed of a linear models for microarray data (LIMMA) linear model and an iterative Bayesian model averaging model to identify a suitable gene set signature. Using one public dataset for training, we discovered a three-gene signature dipeptidyl-peptidase 4 (DPP4), secretogranin V (SCG5) and carbonic anhydrase XII (CA12). We then evaluated the robustness of our gene set using three other independent public datasets. The gene signature accuracy was 85.7, 78.8 and 85.7%, respectively. For experimental validation, we collected 70 thyroid samples from surgery and our three-gene signature method achieved an accuracy of 94.3% by quantitative polymerase chain reaction (QPCR) experiment. Furthermore, immunohistochemistry in 29 samples showed proteins expressed by these three genes are also differentially expressed in thyroid samples. Our protocol discovered a robust three-gene signature that can distinguish benign from malignant thyroid tumors, which will have daily clinical application.

The neuroendocrine protein 7B2 suppresses the aggregation of neurodegenerative disease-related proteins.

Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded ß-sheet-rich proteins, including amyloid-ß (Aß)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aß(1-42), Aß(1-40), and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aß(1-42) and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aß(1-42)-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aß plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.

Network analysis of endogenous gene expression profiles after polyethyleneimine-mediated DNA delivery.

BACKGROUND: DNA delivery systems, which transport exogenous DNA to cells, have applications that include gene therapy, tissue engineering and medical devices. Although the cationic nonviral DNA carrier polyethyleneimine (PEI) has been widely studied, the molecular factors and pathways underlying PEI-mediated DNA transfer remain largely unknown, preventing the design of more efficient delivery systems. METHODS: HEK 293 T cells were treated with polyplexes formed with PEI and pEGFPLuc encoding for green fluorescent protein (GFP). Transfected cells expressing GFP were flow-separated from treated, untransfected cells. Gene expression profiles were obtained using Affymetrix HG-U133 2.0 microarrays and differentially expressed genes were identified using R/Bioconductor. Gene network analysis using EGAN (exploratory gene association network) bioinformatics tools was then used to find interaction among genes and enriched gene ontology (GO) terms related to transfection. Genes identified by this method were perturbed using pharmacologic activators or inhibitors to assess their effect on DNA transfer. RESULTS: Microarray analysis comparing transfected cells to untransfected cells revealed 215 genes to be differentially expressed, with the majority enriched to GO processes including metabolism, response to stimulus, cell cycle, biological regulation and cellular component organization or biogenesis pathways. Gene network analysis revealed a coordinated induction of RAP1A, SCG5, PGAP1, ATF3 and NEB genes implicated in cell stress, cell cycle and cytoskeletal processes. Altering pathways with pharmacologic agents confirmed the potential role of RAP1A, SCG5 and ATF3 in transfection. CONCLUSIONS: Microarray and gene network analyses of the sorted, transfected cell population can identify potential mediators of transfection, providing a basis for the design of improved delivery systems.

The neuroendocrine protein 7B2 is intrinsically disordered.

The small neuroendocrine protein 7B2 has been shown to be required for the productive maturation of proprotein convertase 2 (proPC2) to an active enzyme form; this action is accomplished via its ability to block aggregation of proPC2 into nonactivatable forms. Recent data show that 7B2 can also act as a postfolding chaperone to block the aggregation of a number of other proteins, for example, α-synuclein. To gain insight into the mechanism of action of 7B2 in blocking protein aggregation, we performed structural studies of this protein using gel filtration chromatography, intrinsic tryptophan fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Gel filtration studies indicated that 7B2 exists as an extended monomer, eluting at a molecular mass higher than that expected for a globular protein of similar size. However, chemical cross-linking showed that 7B2 exhibits concentration-dependent oligomerization. CD experiments showed that both full-length 27 kDa 7B2 and the C-terminally truncated 21 kDa form lack appreciable secondary structure, although the longer protein exhibited more structural content than the latter, as demonstrated by intrinsic and ANS fluorescence studies. NMR spectra confirmed the lack of structure in native 7B2, but a disorder-to-order transition was observed upon incubation with one of its client proteins, α-synuclein. We conclude that 7B2 is a natively disordered protein whose function as an antiaggregant chaperone is likely facilitated by its lack of appreciable secondary structure and tendency to form oligomers.

Dynamic modulation of prohormone convertase 2 (PC2)-mediated precursor processing by 7B2 protein: preferential effect on glucagon synthesis.

The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.

Frequent epigenetic inactivation of the chaperone SGNE1/7B2 in human gliomas.

In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in low- and high-grade astrocytomas compared to normal brain tissue. Pyrosequencing was performed to confirm the methylation status of this CpG island in 89 astrocytic gliomas of different malignancy grades and six glioma cell lines. Hypermethylation of SGNE1/7B2 was significantly more frequent in diffuse low-grade astrocytomas as well as secondary glioblastomas and anaplastic astrocytomas as compared to primary glioblastomas. mRNA expression analysis by real-time RT-PCR indicates that SGNE1/7B2 expression is downregulated in astrocytic gliomas compared to white matter samples. Treatment of glioma cells with the demethylating agent 5-aza-2'-deoxycytidine restores the transcription of SGNE1/7B2. Overexpression of SGNE1/7B2 in T98G, A172 and U373MG glioblastoma cells significantly suppressed focus formation and led to a significant increase in apoptotic cells as determined by flow cytometric analysis in T98G cells. In summary, we have identified SGNE1/7B2 as a novel target silenced by DNA methylation in astrocytic gliomas. The high incidence of this alteration and the significant effects of SGNE1/7B2 on the growth and apoptosis of glioblastoma cells provide a first proof for a functional implication of SGNE1/7B2 inactivation in the molecular pathology of gliomas.

Peptides Derived From Insulin Granule Proteins Are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice.

The antigenic peptides processed by ß-cells and presented through surface HLA class I molecules are poorly characterized. Each HLA variant (e.g., the most common being HLA-A2 and HLA-A3) carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring ß-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of ß-cells, we analyzed the HLA-A3-restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1-50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neoepitopes, generated either via peptide cis-splicing or mRNA splicing (e.g., secretogranin-5 [SCG5]-009). As reported for HLA-A2-restricted peptides, several epitopes originated from ß-cell granule proteins (e.g., SCG3, SCG5, and urocortin-3). Similarly, H-2Kd-restricted CD8+ T cells recognizing the murine orthologs of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes.

TNFR and LTbetaR agonists induce follicle-associated epithelium and M cell specific genes in rat and human intestinal epithelial cells.

M cells assist mucosal immune surveillance by transcytosis of particles to underlying lymphoid tissue, but the mechanisms of M cell differentiation are poorly understood. To develop a better defined cell culture model of M cell differentiation, we treated human (Caco-2BBe) and rat (IEC-6) intestinal epithelial cell lines with lymphotoxin beta receptor (LTbetaR) and TNF receptor (TNFR) agonists. Treated cells were studied for regulation of genes associated with M cell and follicle-associated epithelium (FAE). We found that LTbetaR and TNFR agonists induce transcription of FAE-specific genes (Ccl20 and Lamb3) in Caco2-BBe cells and IEC-6 cells as well as rodent M cell specific genes such as Sgne-1/Scg5, Cldn4, and Gp2. The cytokines have distinct but complementary effects; TNFR agonists mainly induced FAE-specific genes, while the LTbetaR agonist induced M cell specific genes. The combination of cytokines showed additive induction of the FAE-associated Ccl20, Lamb3 and a surprising induction of CD137/Tnfrsf9. On the other hand TNF agonists appeared to suppress expression of some LTbetaR-induced genes. Functionally, cytokine treatment led to the reorganization of microvilli and Claudin-4 redistribution. These studies suggest complex interactions between these cytokines in the context of either inflammation or tissue differentiation.

7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity.

The prohormone convertase PC2, which is thought to mediate the proteolytic conversion of many peptide hormones, has recently been shown to interact with the neuroendocrine-specific polypeptide 7B2 in Xenopus intermediate lobe (Braks, J. A. M., and G. J. M. Martens. Cell. 78:263. 1994). In the present work we have stably transfected neuroendocrine cell lines with rat 7B2 constructs and found that overexpression of 27 kD 7B2 greatly facilitates the kinetics of maturation of proPC2, both in AtT-20/PC2 cells and in Rin5f cells. The half-life of conversion of proPC2 was reduced from 2.7 to 1.7 h in AtT-20/PC2 cells stably transfected with 27 kD 7B2 cDNA. The previously proposed "chaperone" domain was not sufficient for this facilitation event; however, a construct corresponding to the 21-kD 7B2 protein (which represents the naturally occurring maturation product) functioned well. A 7B2 construct in which maturation of 27 kD 7B2 to its 21-kD form was blocked was unable to facilitate maturation of proPC2. To correlate effects on PC2 maturation with the actual generation of PC2 enzymatic activity, a similar transfection of 21 kD 7B2 was performed using CHO cells previously amplified for the expression of proPC2. Enzymatic activity cleaving the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC was highly correlated with the expression of immunoreactive 21 kD 7B2 in the conditioned medium; medium obtained from the parent cell line was completely inactive. Enzymatic activity was identified as PC2 on the basis of inhibition by the carboxy-terminal peptide of 7B2, which has previously been shown to represent a potent and specific PC2 inhibitor. Taken together, our in vivo results indicate that the interesting secretory protein 7B2 is a bifunctional molecule with an amino-terminal domain involved in proPC2 transport as well as activation.

Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding.

Among the members of the prohormone convertase (PC) family, PC2 has a unique maturation pattern: it is retained in the ER for a comparatively long time and its propeptide is cleaved in the TGN/ secretory granules rather than in the ER. It is also unique by its association with the neuroendocrine protein 7B2. This interaction results in the facilitation of proPC2 maturation and in the production of activatable proPC2 from CHO cells. In the present study, we have investigated the mechanism of this interaction. ProPC2 binds 7B2 in the ER, but exits this compartment much more slowly than 7B2. We found that proPC2 was also slow to acquire the capacity to bind 7B2, whereas 7B2 could bind proPC2 rapidly after synthesis. This indicated that proPC2 folding was the limiting step in the formation of the complex. Indeed, sensitivity of native proPC2 to N-glycanase F digestion and inhibition of proPC2 folding supported the notion that 7B2 is not involved in the early steps of proPC2 folding, and that proPC2 must fold before binding 7B2. Under experimental conditions that prevent propeptide cleavage, 7B2 expression increased proPC2 transport to the Golgi. This increase exhibited the same kinetics as the facilitation of the removal of the propeptide. Finally, proPC2 activation could be reconstituted in Golgi- enriched subcellular fractions. In vitro, 7B2 was required for proPC2 activation at an acidic pH. Taken together, our results demonstrate that rather than promoting proPC2 folding, 7B2 acts as a helper protein involved in proPC2 transport and is required in the proPC2 activation process. We propose, therefore, that 7B2 stabilizes proPC2 in a conformation already competent for these two events.

SGNE1/7B2 is epigenetically altered and transcriptionally downregulated in human medulloblastomas.

In a genome-wide screen using differential methylation hybridization (DMH), we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in medulloblastomas compared to fetal cerebellum. Bisulfite sequencing and combined bisulfite restriction assay were performed to confirm the methylation status of this CpG island in primary medulloblastomas and medulloblastoma cell lines. Hypermethylation was detected in 16/23 (70%) biopsies and 7/8 (87%) medulloblastoma cell lines, but not in non-neoplastic fetal (n=8) cerebellum. Expression of SGNE1 was investigated by semi-quantitative competitive reverse transcription-polymerase chain reaction and found to be significantly downregulated or absent in all, but one primary medulloblastomas and all cell lines compared to fetal cerebellum. After treatment of medulloblastoma cell lines with 5-aza-2'-deoxycytidine, transcription of SGNE1 was restored. No mutation was found in the coding region of SGNE1 by single-strand conformation polymorphism analysis. Reintroduction of SGNE1 into the medulloblastoma cell line D283Med led to a significant growth suppression and reduced colony formation. In summary, we have identified SGNE1 as a novel epigenetically silenced gene in medulloblastomas. Its frequent inactivation, as well as its inhibitory effect on tumor cell proliferation and focus formation strongly argues for a significant role in medulloblastoma development.

7B2 prevents unfolding and aggregation of prohormone convertase 2.

Prohormone convertase 2 (PC2) requires interaction with the neuroendocrine protein 7B2 for the production of an activatable zymogen; the mechanism for this effect is unknown. 7B2 could act proactively to generate an activation-competent form of pro-PC2 during synthesis, or block spontaneous generation of activation-incompetent forms. We here demonstrate that addition of exogenous recombinant 7B2 to CHO cells expressing pro-PC2 prevented the unfolding and aggregation of secreted PC2 forms in a dose-dependent manner, as assessed by aggregation assays, activity assays, cross-linking experiments, and sucrose density gradients. Intracellular pro-PC2 was also found to exist in part as higher-order oligomers that were reduced in the presence of coexpressed 7B2. 7B2 addition did not result in the acquisition of enzymatic competence unless added before or very rapidly after pro-PC2 secretion, indicating that an activation-competent structure cannot be maintained in the absence of 7B2. Velocity sedimentation experiments showed that addition of extracellular 7B2 solubilized three different PC2 species from a precipitable aggregate: two activatable pro-PC2 species, the intact zymogen and a zymogen with a partially cleaved propeptide, and an inactive 66-kDa form. Our results suggest that 7B2 possesses chaperone activity that blocks partially unfolded pro-PC2 forms from losing catalytic competence and then aggregating. The loss of the catalytically competent conformer appears to represent the earliest indicator of pro-PC2 unfolding and is followed on a slower time scale by the appearance of aggregates. Because 7B2 expression is not confined to areas expressing pro-PC2, 7B2 may represent a general intracellular and extracellular secretory chaperone.

Overexpression of Scg5 increases enzymatic activity of PCSK2 and is inversely correlated with body weight in congenic mice.

BACKGROUND: The identification of novel genes is critical to understanding the molecular basis of body weight. Towards this goal, we have identified secretogranin V (Scg5; also referred to as Sgne1), as a candidate gene for growth traits. RESULTS: Through a combination of DNA microarray analysis and quantitative PCR we identified a strong expression quantitative trait locus (eQTL) regulating Scg5 expression in two mouse chromosome 2 congenic strains and three additional F2 intercrosses. More importantly, the eQTL was coincident with a body weight QTL in congenic mice and Scg5 expression was negatively correlated with body weight in two of the F2 intercrosses. Analysis of haplotype blocks and genomic sequencing of Scg5 in high (C3H/HeJ, DBA/2J, BALB/cByJ, CAST/EiJ) and low (C57BL/6J) expressing strains revealed mutations unique to C57BL/6J and possibly responsible for the difference in mRNA abundance. To evaluate the functional consequence of Scg5 overexpression we measured the pituitary levels of 7B2 protein and PCSK2 activity and found both to be increased. In spite of this increase, the level of pituitary alpha-MSH, a PCSK2 processing product, was unaltered. CONCLUSION: Together, these data support a role for Scg5 in the modulation of body weight.

Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours.

INTRODUCTION: Chromogranin (Cg) and secretogranin (Sg) are members of the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In recent publications we have presented generation of region-specific antibodies against CgA and CgB and also development of several region-specific radioimmunoassays for measurements of specific parts of the Cgs. In this study we describe generation of antibodies against SgII, SgIII, SgV and the proconvertases PC1/3 and PC2 and development of radioimmunoassays for measurements of these proteins. MATERIALS AND METHODS: Peptides homologous to defined parts of the secretogranin and proconvertase molecules were selected and synthesised. Antibodies were raised, radioimmunoassays were developed and circulating levels of the proteins in plasma samples from 22 patients with neuroendocrine tumours were measured in the assays. RESULTS: Increased plasma concentrations were recorded in 11, 4 and 3 of the patients with the SgII 154-165 (N-terminal secretoneurin), the SgII 172-186 (C-terminal Secretoneurin) and the SgII 225-242 assays respectively. The SgIII, SgV, PC1/3 and PC2 assays failed to detect increased concentrations in any of the patients. CONCLUSION: Increased concentrations of SgII, especially the N-terminal part of secretoneurin could be measured in plasma from patients with endocrine pancreatic tumours and in this case this assay was quite comparable to measurements of CgA and CgB. Even though secretoneurin was not as frequently increased as CgA and CgB in patients with carcinoid tumours or pheochromocytoma it may be a useful marker for endocrine pancreatic tumours.

Prohormone convertases 1/3, 2, furin and protein 7B2 (Secretogranin V) in endocrine cells of the human pancreas.

Prohormone convertases (PCs) are proteinases that cleave inactive prohormones to biologically active peptides. Seven PCs have been identified; two of them, PC1/3 and PC2, have only been localized in neuroendocrine (NE) tissues; a third, furin, in both endocrine and exocrine tissues. We have studied the immunoreactivity of PC1/3, PC2 and furin in the four major NE cell types of the human pancreas by using double immunofluorescence techniques. The study also included the expression of NE secretory protein 7B2 (secretogranin V), a member of the granin family, which influences the function of PC2. The results showed that the three PCs and 7B2 were expressed only in endocrine pancreas, furin also in exocrine cells. Insulin (B) cells harboured PC1/3 and PC2, but not furin. Glucagon (A) cells were immunoreactive to all three PCs; all glucagon cells expressed PC2, but one subpopulation showed PC1/3 immunoreactivity and another furin. Only a few somatostatin (D) cells contained PC2, but no other proconvertase. Pancreatic polypeptide (PP) cells were non-reactive to all three PCs. 7B2 occurred only in insulin and glucagon cells. A varying co-localization pattern was observed between PCs and between PCs and 7B2, with the exception of PC1/3 and furin which were not co-localized. In conclusion, our study shows that PCs are localized in insulin and glucagon cells and do seem to be important in these cell types for processing of hormone and other protein precursors, especially chromogranins, but for the two other major cell types probably other enzymes are of importance.

7B2 chaperone knockout in APP model mice results in reduced plaque burden.

Impairment of neuronal proteostasis is a hallmark of Alzheimer's and other neurodegenerative diseases. However, the underlying molecular mechanisms leading to pathogenic protein aggregation, and the role of secretory chaperone proteins in this process, are poorly understood. We have previously shown that the neural-and endocrine-specific secretory chaperone 7B2 potently blocks in vitro fibrillation of Aß42. To determine whether 7B2 can function as a chaperone in vivo, we measured plaque formation and performed behavioral assays in 7B2-deficient mice in an hAPPswe/PS1dE9 Alzheimer's model mouse background. Surprisingly, immunocytochemical analysis of cortical levels of thioflavin S- and Aß-reactive plaques showed that APP mice with a partial or complete lack of 7B2 expression exhibited a significantly lower number and burden of thioflavin S-reactive, as well as Aß-immunoreactive, plaques. However, 7B2 knockout did not affect total brain levels of either soluble or insoluble Aß. While hAPP model mice performed poorly in the Morris water maze, their brain 7B2 levels did not impact performance. Since 7B2 loss reduced amyloid plaque burden, we conclude that brain 7B2 can impact Aß disposition in a manner that facilitates plaque formation. These results are reminiscent of prior findings in hAPP model mice lacking the ubiquitous secretory chaperone clusterin.

Highly deleterious variations in COX1, CYTB, SCG5, FK2, PRL and PGF genes are the potential adaptation of the immigrated African ostrich population.

Because of variable inconvenient living conditions in some places around the world, it is difficult to collect reliable physiological data for ostriches. Therefore, this study aims to provide a comprehensive in silico insight for the nature of polymorphism of important genetic loci that are related to physiological and reproductive traits. Sixty-nine mature ostriches ranging over half of Iraq were screened. Six exonic genetic loci, including cytochrome c oxidase I (COX1), cytochrome b (CYTB), secretogranin V (SCG5), feather keratin 2-like (FK2), prolactin (PRL) and placenta growth factor (PGF) were genotyped by PCR-single stranded conformation polymorphism (SSCP). Thirty-six novel SNPs, including seventeen nonsynonymous (ns) SNPs, were observed. Several computational software programs were utilized to assess the extent of the nsSNPs on their corresponding proteins structure, function and stability. The results showed several deleterious functional and stability changes in almost all the proteins studied. The total severity of each missense mutation was evaluated and compared with other nsSNPs accumulatively. It is evident from the extensive cumulative in silico computation that both p.E34D and p.E60K in PGF have the highest deleterious effect. The cumulative predictions from the present study are an impressive guide for the genotypes of African ostriches, which bypassed the expensive protocols for wet laboratory screening, to identify the effects of variants. To the best of our knowledge, this is the first investigation of its kind on the analyses and prediction outcome of missense mutations in African ostrich populations. The highly deleterious nsSNPs in the placenta growth factor are possible adaptive mutations which might be associated with adaptation in extreme and new environments. The flow and protocol of the computational predictions can be extended for various wild animals to identify the molecular nature of adaptations.

Conventional and Neo-antigenic Peptides Presented by ß Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors.

Although CD8+ T-cell-mediated autoimmune ß cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by ß cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known ß cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by ß cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.

Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice.

The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of two genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase [1]; this generally requires 1-2 months of work for each construct. The single 'knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection (using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knockout vector. This approach leads to inactivation of the gene of interest [2]. Recently, an in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing [3]. An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition. The DNA is then purified, and subsequently electroporated into bacteria. The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events. In the present study, we have modified this system to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs. Our approach led from genomic clone to completed construct ready for transfection in a matter of days. The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs [4-8], simplifying this technically demanding method.

Differences of pancreatic expression of 7B2 between C57BL/6J and C3H/HeJ mice and genetic polymorphisms at its locus (Sgne1).

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.