Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

Background

Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation.

Results

To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells.

Conclusions

We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.

     

Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells

Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.     

Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.     

Stem cell therapy: A novel approach for myocardial infarction

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Until recently, it was thought that myocardium was not able to repair itself, but studies have now shown that resident cardiac stem cells have regenerative capacity, and stem cell therapy may be a novel approach for cardiac muscle repair and regeneration. Stem cell-derived paracrine factors have been shown to regulate ventricular remodeling, inflammation, apoptosis, cardiomyocytes regeneration, and neovascularization in regions of infarcted cardiac tissue. In this review, we summarize the evidence from cellular, animal, and clinical studies supporting the potential clinical significance of stem cell therapy as a novel therapeutic approach for the treatment of MI.     

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum “XerumFree? XF205” (XF); (3) CnT-20 medium supplemented with “XerumFree? XF205” (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ?Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ?Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ?Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.     

Ameliorative Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats

Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine – induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered.     

UV-B effect on constituents of Azolla caroliniana

Changes in growth and ultrastructure of Azolla caroliniana in response to elevated UV-B radiation were investigated. Exposure of plants to UV-B radiation for 1, 8, 16, 24 and 48 h exhibited a significant decrease in biomass and relative growth rate. This decrease resulted in an increase in doubling time over the control. Also, Chl a and b contents were significantly decreased especially after 16 h. The reduction was accompanied by a decrease in 5-aminolaevulinic acid content (precursor of chlorophyll). On the other hand, contents of carotenoid and UV-absorbing phenolic compounds (flavonoids and anthocyanins) were increased.     

Viral gastroenteritis associated with genogroup II norovirus among U.S. military personnel in Turkey, 2009

The present study demonstrates that multiple NoV genotypes belonging to genogroup II contributed to an acute gastroenteritis outbreak at a US military facility in Turkey that was associated with significant negative operational impact. Norovirus (NoV) is an important pathogen associated with acute gastroenteritis among military populations. We describe the genotypes of NoV outbreak occurred at a United States military facility in Turkey. Stool samples were collected from 37 out of 97 patients presenting to the clinic on base with acute gastroenteritis and evaluated for bacterial and viral pathogens. NoV genogroup II (GII) was identified by RT-PCR in 43% (16/37) stool samples. Phylogenetic analysis of a 260 base pair fragment of the NoV capsid gene from ten stool samples indicated the circulation of multiple and rare genotypes of GII NoV during the outbreak. We detected four GII.8 isolates, three GII.15, two GII.9 and a sole GII.10 NoV. Viral sequences could be grouped into four clusters, three of which have not been previously reported in Turkey. The fact that current NoV outbreak was caused by rare genotypes highlights the importance of norovirus strain typing. While NoV genogroup II is recognized as causative agent of outbreak, circulation of current genotypes has been rarely observed in large number of outbreaks.