Utility of large consanguineous family-based model for investigating the genetics of type 2 diabetes mellitus

Objectives

This study examined the utility of a family-based model for replicating the results of genome-wide association studies (GWAS) of type 2 diabetes (T2D).

Methods and results

In a total of 232 members of a large consanguineous Omani Arab pedigree (age: 16–80 years), there were 27 diabetics and 50 prediabetics (17 with impaired fasting glucose and 33 with impaired glucose tolerance). All 232 individuals underwent anthropometric and biochemical investigations and genotyped for 14 known common gene variants of modest effect on T2D risk. Power analysis at a LOD score of 3, gave 80% power to locate a single specific locus that accounts for 52% of the total phenotypic variation. Measured genotype analysis (MGA) was used to determine heritability of various quantitative traits (QTs) which ranged 25–56%. Using MGA, some common gene variants were found to have little (< 5%) but significant impact on the heritability of T2D related QTs [KCNJ11 (rs5219), p = 0.004]; [IGF2BP2 (rs4402960), p = 0.02]; [SLC30A8 (rs13266634), p = 0.05]; [CAPN10 (rs2975760), p = 0.031]; [FTO (rs8050136), p = 0.023]; [FTO (rs9939609), p = 0.018] and [SLC30A8 (rs13266634), p = 0.05]. Sib-TDT analysis showed that some gene variants were significantly associated with T2D risk but didn't reach the level of significance after Bonferroni correction [KCNJ11 (rs5219), p = 0.047] and [CAPN10 (rs41266971), p = 0.035].

Conclusion

We have demonstrated that, in principle, a family-based model with minor limitations could be used to replicate some of the results of large GWAS case–control studies. This model could successfully be applied for the future discovery, by deep sequencing, of rare gene variants.     

Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene

The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5–7 days in the disease development as compared to wild-type plants.     

An Analysis of Regulatory T-Cell and Th-17 Cell Dynamics during Cytomegalovirus Replication in Solid Organ Transplant Recipients

Background

CMV-specific T-cells are crucial to control CMV-replication post-transplant. Regulatory T-cells (T-regs) are associated with a tolerant immune state and may contribute to CMV-replication. However, T-cell subsets such as T-regs and IL-17 producing T-cells (Th-17) are not well studied in this context. We explored T-regs and Th-17 frequencies during CMV-replication after transplantation.

Methods

We prospectively evaluated 30 transplant patients with CMV-viremia. We quantified CMV-specific CD4⁺ and CD8⁺ T-cells, T-regs (CD4⁺CD25⁺FoxP3⁺) and Th-17 frequencies using flow-cytometry and followed patients requiring anti-viral treatment. Two subsets were compared: anti-viral treatment requirement (n = 20) vs. spontaneous clearance of viremia (n = 10).

Results

Higher initial CMV-specific CD4⁺ T-cells and lower T-regs were observed in patients with spontaneous clearance (p = 0.043; p = 0.021 respectively). Using a ratio of CMV-specific CD4⁺ T-cells to T-regs allowed prediction of viral clearance with 80% sensitivity and 90% specificity (p = 0.001). One month after stop of treatment, the same correlation was observed in patients protected from CMV-relapse. The ratio of CMV-specific CD4⁺ T-cells to T-regs allowed prediction of relapse with 85% sensitivity and 86% specificity (p = 0.004). Th-17 responses were not correlated with virologic outcomes.

Conclusions

This study provides novel insights into T-regs and Th-17 subpopulations during CMV-replication after transplantation. These preliminary data suggest that measurement of CMV-specific CD4⁺ T-cells together with T-regs has value in predicting spontaneous clearance of viremia and relapse.     

Small interfering RNAs that deplete the cellular translation factor eIF4H impede mRNA degradation by the virion host shutoff protein of herpes simplex virus

The herpes simplex virus (HSV) virion host shutoff (Vhs) protein is an endoribonuclease that accelerates decay of many host and viral mRNAs. Purified Vhs does not distinguish mRNAs from nonmessenger RNAs and cuts target RNAs at many sites, yet within infected cells it is targeted to mRNAs and cleaves those mRNAs at preferred sites including, for some, regions of translation initiation. This targeting may result in part from Vhs binding to the translation initiation factor eIF4H; in particular, several mutations in Vhs that abrogate its binding to eIF4H also abolish its mRNA-degradative activity, even though the mutant proteins retain endonuclease activity. To further investigate the role of eIF4H in Vhs activity, HeLa cells were depleted of eIF4H or other proteins by transfection with small interfering RNAs (siRNAs) 48 h prior to infection or mock infection in the presence of actinomycin D. Cellular mRNA levels were then assayed 5 h after infection. In cells transfected with an siRNA for the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase, wild-type HSV infection reduced beta-actin mRNA levels to between 20 and 30% of those in mock-infected cells, indicative of a normal Vhs activity. In contrast, in cells transfected with any of three eIF4H siRNAs, beta-actin mRNA levels were indistinguishable in infected and mock-infected cells, suggesting that eIF4H depletion impeded Vhs-mediated degradation. Depletion of the related factor eIF4B did not affect Vhs activity. The data suggest that eIF4H binding is required for Vhs-induced degradation of many mRNAs, perhaps by targeting Vhs to mRNAs and to preferred sites within mRNAs.     

Eicosanoids mediate insect hemocyte migration

Hemocyte migration toward infection and wound sites is an essential component of insect defense reactions, although the biochemical signal mechanisms responsible for mediating migration in insect cells are not well understood. Here we report on the outcomes of experiments designed to test the hypotheses that (1) insect hemocytes are able to detect and migrate toward a source of N-formyl-Met-Leu-Phe (fMLP), the major chemotactic peptide from Escherichia coli and (2) that pharmaceutical modulation of eicosanoid biosynthesis inhibits hemocyte migration. We used primary hemocyte cultures prepared from fifth-instar tobacco hornworms, Manduca sexta in Boyden chambers to assess hemocyte migration toward buffer (negative control) and toward buffer amended with fMLP (positive control). Approximately 42% of negative control hemocytes migrated toward buffer and about 64% of positive control hemocytes migrated toward fMLP. Hemocyte migration was inhibited (by >40%) by treating hornworms with pharmaceutical modulators of cycloxygenase (COX), lipoxygenase and phospholipase A2 (PLA2) before preparing primary hemocyte cultures. The influence of the COX inhibitor, indomethacin, and the glucocorticoid, dexamethasone, which leads to inhibition of PLA2, was expressed in a dose-dependent way. The influence of dexamethasone was reversed by injecting arachidonic acid (precursor to eicosanoid biosynthesis) into hornworms before preparing primary hemocyte cultures. The saturated fatty acid, palmitic acid, did not reverse the inhibitor effect. These findings support both our hypotheses, first that insect hemocytes can detect and respond to fMLP, and second, that insect hemocyte migration is mediated by eicosanoids.     

Adamantinoma: A clinicopathological review and update

Background

Matrix metalloproteinases (MMPs) play an important role in the modeling and remodeling of the extracellular matrix in both physiologic and pathologic states and thus plays an important role in tumor progression. Human collagenase-3 (MMP-13) is a member of matrix metalloproteinase family of enzymes that was originally identified in breast carcinomas and subsequently detected during fetal ossification and in arthritic processes.

Aim

The present study was designed to investigate the expression MMP-13 and to correlate its expression with clinicopathological parameters in chondrosarcoma of the jaws.

Methods

Archival tumor tissues from 11 patients with chondrosarcoma of the jaws were analyzed by immunohistochemistry for the expression of MMP-13. Clinical information was obtained through the computerized retrospective database from the tumor registry between 1998 to 2006.

Results

Eight of 11 cases (72.8 %) of chondrosarcomas showed a positive reaction for MMP-13, whereas two cases of normal cartilage were negative for this collagenase. As regard the clinicopathological parameters, there was no correlation between MMP-13 expression and sex, age and tumor site. While, there were significant associations between MMP-13 expression and both of mitotic counts and necrosis. On the other hand, there was a significant difference between low and high grade tumors (P < 0.05) regarding MMP-13 expression. Also, there was no significant correlation between MMP-13 expression in primary lesions and their local recurrence.

Conclusion

MMP-13 is expressed in the majority of chondrosarcoma of the jaws. It is also noteworthy that the expression of MMP-13 may be related to tumor biological aggressiveness and used to aid in predicting patient's poor prognosis.     

The mutation process of microsatellites during the polymerase chain reaction.

We build a mathematical model for the mutation process of microsatellites during polymerase chain reaction (PCR) using the theory of branching processes. Based on the model, we develop a method to estimate the mutation rate of microsatellites per PCR cycle and the probability of expansion by maximizing a quasi-likelihood of the observed data. We show by simulations that the proposed estimation method can accurately recover the relationship between the mutation rate and number of repeat units. The theoretical basis for the proposed method is also given. We apply the method to experimental data on poly-A and poly-CA repeats.     

Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG₁ – κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/μL and 2.1 ng/μL in spiked buffer samples and 28.7 ng/μL and 110 ng/μL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.     

Visualizing the Transport of Porcine Reproductive and Respiratory Syndrome Virus in Live Cells by Quantum Dots-Based Single Virus Tracking

Quantum dots(QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time. The porcine reproductive and respiratory syndrome virus(PRRSV) is a small virus with the virion size of 40–60 nm which causes great economic losses to the swine industry worldwide. A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies. In this study, we labeled the PRRSV with QDs using the streptavidin–biotin labeling system and monitored the viral infection process in live cells. Our results indicated that the labeling method had negligible effect on viral infectivity. We also observed that prior to the entry, PRRSV vibrated on the plasma membrane, and entered the cells via endosome mediated cell entry pathway. Viruses moved in a slow–fast–slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell. Our results also showed that once inside the cell, PRRSV moved along the microtubule,microfilament and vimentin cytoskeletal elements. During the transport process, virus particles also made contacts with non-muscle myosin heavy chain Ⅱ-A(NMHC Ⅱ-A), visualized as small spheres in cytoplasm. This study can facilitate the application of QDs in virus infection imaging, especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism.