Reverse genetics generation of chimeric infectious Junin/Lassa virus is dependent on interaction of homologous glycoprotein stable signal peptide and G2 cytoplasmic domains

The Arenaviridae are a diverse and globally distributed collection of viruses that are maintained primarily by rodent reservoirs. Junin virus (JUNV) and Lassa virus (LASV) can both cause significant outbreaks of severe and often fatal human disease throughout their respective areas of endemicity. In an effort to improve upon the existing live attenuated JUNV Candid1 vaccine, we generated a genetically homogenous stock of this virus from cDNA copies of the virus S and L segments by using a reverse genetics system. Further, these cDNAs were used in combination with LASV cDNAs to successfully generate two recombinant Candid1 JUNV/LASV chimeric viruses (via envelope glycoprotein [GPC] exchange). It was found that while the GPC extravirion domains were readily exchangeable, homologous stable signal peptide (SSP) and G2 transmembrane and cytoplasmic tail domains were essential for correct GPC maturation and production of infectious chimeric viruses. The switching of the JUNV and LASV G1/G2 ectodomains within the Candid1 vaccine background did not alter the attenuated phenotype of the vaccine strain in a lethal mouse model. These recombinant chimeric viruses shed light on the fundamental requirements of arenavirus GPC maturation and may serve as a strategy for the development of bivalent JUNV and LASV vaccine candidates.     

Site-1 protease is essential to growth plate maintenance and is a critical regulator of chondrocyte hypertrophic differentiation in postnatal mice

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.     

Effects of aging on growth factors gene and protein expression in the dorsal and ventral lobes of rat prostate

We hypothesize that various growth factors and their receptors gene and protein are modulated in dorsal and ventral lobes of aging prostate. To test this hypothesis, TGFbeta1, TGFbeta2 TGFbeta3, TGFbetaR-I, TGFbetaR-II, TGFalpha, EGF, EGFR, KGF and KGFR gene and protein expression were analyzed in dorsal and ventral lobes of aging rat prostates (1, 3, 6, 9, 12, 18, 24, and 28/30 months). KGF gene expression was very weak or absent in 1, 3, and 6 month old rat dorsal and ventral lobes of prostate whereas it re-expressed in 9, 12, 18, 24 and 30 month old rat prostate. All growth factors and their receptors expect KGF and EGFR were mainly localized in epithelium of ventral and dorsal lobes of aging rat prostates. EGF, TGFalpha, TGFbeta1, and TGFbetaR-I protein expression was lacking in stroma of dorsal and ventral lobes of 1, 3, 6, 9, 12/18 months old rat prostates. However, EGF, TGFbeta1 and TGFbetaR-I proteins re-expressed in stroma of 24 and 28 months old rat prostates. KGF protein expression was lacking in epithelium of dorsal and ventral lobes of all aging rat prostates. This is the first report to demonstrate differential gene and protein expression of growth factors in dorsal and ventral lobes is associated with aging rat prostate, suggesting their role in pathogenesis of prostatic diseases with aging.     

A repertoire of biomarkers helps in detection and assessment of therapeutic response in epithelial ovarian cancer

In epithelial ovarian cancer (EOC), the cancer antigen 125 (CA-125) has been conventionally used to help in diagnosis and assessment of response to treatment. Currently, YKL-40 (Tyrosine–Lysine–Leucine-40) and circulating cell-free DNA are being evaluated for possession of similar ability. In this study, we aimed to assess the ability of a repertoire of potential biomarkers in detecting and assessing therapeutic response, in advanced EOC. Blood levels of CA-125, YKL-40, total cell-free DNA (CFDNA), cell-free nuclear DNA (CFnDNA), and cell-free mitochondrial DNA (CFmDNA) levels were measured in 100 untreated patients of advanced EOC from November 2009 to June 2011, and again on treatment completion from the 20 patients who appeared for follow-up analysis. Significantly, higher proportion of untreated patients had serum CA-125 >3 times upper limit of normal (ULN) (90.0 %; P < 0.0001) and plasma YKL-40 >ULN (77.0 %; P < 0.0001), both of which significantly decreased, Posttherapy. posttherapy, CFDNA (P < 0.0001), and CFnDNA (P < 0.0001) levels significantly decreased as compared to pretreatment levels. Positive and significant correlations existed between pretherapy CFDNA and CFnDNA [Spearman rho (ρ) = 1.000; P < 0.0001], and also with CFmDNA (ρ = 0.301; P = 0.002), separately between CFnDNA and CFmDNA (ρ = 0.303; P = 0.002), as well as between plasma YKL-40 and patient age (ρ = 0.353; (P < 0.0001). On treatment completion, CFDNA and CFnDNA levels showed positive and significant correlation (ρ = 1.000; P < 0.0001). Therefore serum CA-125 and plasma YKL-40 aid detection and assessment of therapeutic response, in advanced EOC. CFDNA and CFnDNA help in estimating extent of therapeutic response in advanced EOC.     

Tobacco Smoking,Alcohol Drinking,Diabetes, Low Body Mass Index and the Risk of Self-Reported Symptoms of Active Tuberculosis: Individual Participant Data (IPD) Meta-Analyses of 72,684 Individuals in 14 High Tuberculosis Burden Countries

Background

The effects of multiple exposures on active tuberculosis (TB) are largely undetermined. We sought to establish a dose-response relationship for smoking, drinking, and body mass index (BMI) and to investigate the independent and joint effects of these and diabetes on the risk of self-reported symptoms of active TB disease.

Methods and Findings

We analyzed 14 national studies in 14 high TB-burden countries using self-reports of blood in cough/phlegm and cough lasting > = 3 weeks in the last year as the measures of symptoms of active TB. The random effect estimates of the relative risks (RR) between active TB and smoking, drinking, diabetes, and BMI<18.5 kg/m² were reported for each gender. Floating absolute risks were used to examine dyads of exposure. Adjusted for age and education, the risks of active TB were significantly associated with diabetes and BMI<18.5 kg/m² in both sexes, with ever drinking in men and with ever smoking in women. Stronger dose-response relationships were seen in women than in men for smoking amount, smoking duration and drinking amount but BMI<18.5 kg/m² showed a stronger dose-response relationship in men. In men, the risks from joint exposures were statistically significant for diabetics with BMI<18.5 kg/m² (RR = 6.4), diabetics who smoked (RR = 3.8), and diabetics who drank alcohol (RR = 3.2). The risks from joint risk factors were generally larger in women than in men, with statistically significant risks for diabetics with BMI<18.5 kg/m² (RR = 10.0), diabetics who smoked (RR = 5.4) and women with BMI<18.5 kg/m² who smoked (RR = 5.0). These risk factors account for 61% of male and 34% of female estimated TB incidents in these 14 countries.

Conclusions

Tobacco, alcohol, diabetes, and low BMI are significant individual risk factors but in combination are associated with triple or quadruple the risk of development of recent active TB. These risk factors might help to explain the wide variation in TB across countries.     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

Kinetics,Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.     

HS‐SPME‐GC‐FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2‐acetyl‐1‐pyrroline

A rapid micro‐scale solid‐phase micro‐extraction (SPME) procedure coupled with gas‐chromatography with flame ionized detector (GC‐FID) was used to extract parts per billion levels of a principle basmati aroma compound “2‐acetyl‐1‐pyrroline” (2‐AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre‐incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2‐AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 μg/kg. Along with 2‐AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)?2‐hexenal, pentadecanal, 4‐hydroxy‐2‐butanone, n‐hexanal, 2–6‐nonadienal, 3‐methoxy‐2(5H) furanone and 2‐acetyl‐1‐pyridine and octanal. High recovery of 2‐AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2‐AP production by B. cereus. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1356–1363, 2014