Isolation and characterization of enteroaggregative,enterotoxigenic, diffusely adherent <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella</Emphasis> Worthington from human diarrhoeic faecal samples in Kashmir and Secunderabad,India

Seven hundred and thirty-five diarrhoeic faecal samples from children were investigated for presence of enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), diffusely adherent E. coli (DAEC) and Salmonella spp. by polymerase chain reaction (PCR) and bacterial culture. Out of 675 samples from Kashmir, 55 isolates were obtained, which carried at least one virulence gene studied. Out of the 55 isolates, 36 (65.45%) were EAEC, 18 (32.72%) were ETEC while only one isolate (1.81%) was DAEC. All the EAEC isolates were found to be typical as they possessed aggR gene. Six (16.66%) EAEC isolates carried the astA gene. Out of the 18 ETEC isolates, 13 carried the elt gene alone, four possessed both the elt and est genes and the remaining one harboured the est gene alone. Five ETEC isolates also possessed astA gene. Nineteen EAEC isolates belonged to 10 different serogroups. Serogroup O153 was most frequent. The ETEC isolates also belonged to 10 different serogroups of which O159 was most predominant. Out of 224 E. coli isolates from 60 samples of Secunderabad, 27 isolates carried at least one virulence gene. Out of 27 isolates 22 (81.48%) were typical EAEC, three (11.11%) were ETEC and two (7.4%) were DAEC. Fifteen EAEC isolates belonged to seven different serogroups with O86 as most frequent. Four EAEC isolates also possessed the astA gene. All the three ETEC isolates harboured elt gene only and belonged to three different serogroups. Two isolates of Salmonella Worthington were obtained from only two samples in Kashmir.     

The Effects of Rosuvastatin on the Serum Cortisol,Serum Lipid,and Serum Mevalonic Acid Levels in the Healthy Indian Male Population

In this open-label, balanced, randomized, placebo-controlled, parallel study, healthy male volunteers were randomly divided into two groups. Each group received either a single oral dose of rosuvastatin 20 mg or placebo. Estimations were done at predose on day 1 of dosing (baseline) and 24 h postdose after days 7 and 14. Serum cortisol and serum lipid levels were estimated using enzyme-linked immunosorbent assay kits and serum mevalonic acid (MVA) levels were measured using validated liquid chromatography–tandem mass spectrometry method. Rosuvastatin produced a statistically significant (P < 0.05) decrease in total cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and triglycerides. However, the increase in high-density lipoprotein cholesterol and decrease in cortisol and MVA were not statistically significant when compared to the placebo-treated group. The study showed that rosuvastatin at a dose of 20 mg/day for a period of 14 days was very potent as cholesterol-lowering agent, without any significant change in serum cortisol level in the healthy Indian male population.     

Chromate reduction by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> MCMB-821,isolated from the pristine habitat of alkaline crater lake

The Cr(VI)-reducing bacterial strain MCMB-821 was isolated from the alkaline crater lake of Lonar and was identified as Burkholderia cepacia. MCMB-821 was resistant to 1,000-ppm Cr(VI) and reduced 98% of the 75 ppm Cr(VI) within 36 h at pH 9.0 in the presence of 2% salt and lactose as the electron donor. The chromate-reducing efficiency of MCMB-821 was comparable under both aerobic as well as anaerobic conditions. Electron paramagnetic resonance spectroscopy data suggested that MCMB-821 reduced Cr(VI) to Cr(III) via the formation of transient Cr(V) intermediate. The chromate-reducing ability of MCMB-821 was suppressed in the presence of membrane inhibitors and enhanced in the presence of 2,4-dinitrophenol, suggesting the involvement of electron transport chain in the Cr(VI) bioreduction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Thyroid Hormone Treatments Differentially Affect the Temperature Kinetics Properties of FoF1 ATPase and Succinate Oxidase as well as the Lipid/Phospholipid Profiles of Rat Kidney Mitochondria: A Correlative Study

Effect of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodo-l-thyronine (T₃) or replacement therapy (T_R) with T₃ + l-thyroxine (T₄) on the temperature kinetics properties of FoF₁ adenosine triphosphatase (ATPase, ATP synthase, H⁺-translocating ATP synthase EC 3.6.3.14) and succinate oxidase (SO) and on the lipid/phospholipid makeup of rat kidney mitochondria were examined. Tx lowered ATPase activity, which T₃ treatment restored. SO activity was unchanged in Tx but decreased further by T₃ treatment. T_R restored both activities. The energies of ATPase activation in the high and low temperature ranges (E _H and E _L) increased in the Tx and T₃ animals with decrease in phase transition temperature (Tt). T_R restored E _H and E _L but not Tt to euthyroid levels. E _H and E _L of SO decreased in Tx animals. T₃ and T_R restored E _H whereas E _L was restored only in the T_R group; Tt increased in both groups. Total phospholipid and cholesterol contents decreased significantly in Tx and T₃-treated animals. In Tx animals, sphingomyelin (SPM) and phosphatidylcholine (PC) components decreased, while phosphatidylserine (PS) and diphosphatidylglycerol components increased. T₃ and T_R treatments caused decreases in SPM, phosphatidylinositol and PS. PC and phosphatidylethanolamine (PE) increased in the T₃ group. T_R resulted in increased lysophospolipids and PE. Changes in kinetic parameters of the two enzymes were differently correlated with specific phospholipid components. Both T₃ and T_R regimens were unable to restore normal membrane structure-function relationships.     

USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168*

During DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs H2A and ultraviolet radiation (UVR)-induced γH2AX monoubiquitination, and decreases the levels of pBmi1, Bmi1, RNF168 and BRCA1. The effect of USP7 disruption was recapitulated by siRNA-mediated USP7 depletion. The USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of monoubiquitinated H2A (uH2A) and polyubiquitinated H2AX/A, and subsequently affects UVRIF and IRIF of BRCA1 as well as the IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective mutant, prevents UVR-induced RNF168 degradation. The USP7 mutant is unable to cleave Ub-conjugates of RNF168 in vivo. Importantly, ectopic expression of RNF168, or both RNF8 and RNF168 together in USP7-disrupted cells, significantly rescue the formation of UVRIF and IRIF of polyubiquitinated H2A and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168.     

Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34

A putative epoxide hydrolase-encoding gene was identified from the genome sequence of Cupriavidus metallidurans CH34. The gene was cloned and overexpressed in Escherichia coli with His(6)-tag at its N-terminus. The epoxide hydrolase (CMEH) was purified to near homogeneity and was found to be a homodimer, with subunit molecular weight of 36 kDa. The CMEH had broad substrate specificity as it could hydrolyze 13 epoxides, out of 15 substrates tested. CMEH had high specific activity with 1,2-epoxyoctane, 1,2-epoxyhexane, styrene oxide (SO) and was also found to be active with meso-epoxides. The enzyme had optimum pH and temperature of 7.5 and 37°C respectively, with racemic SO. Biotransformation of 80 mM SO with recombinant whole E. coli cells expressing CMEH led to 56% ee(P) of (R)-diol with 77.23% conversion in 30 min. The enzyme could hydrolyze (R)-SO, ～2-fold faster than (S)-SO, though it accepted both (R)- and (S)-SO with similar affinity as K(m)(R) and K(m)(S) of CMEH were 2.05±0.42 and 2.11±0.16 mM, respectively. However, the k(cat)(R) and k(cat)(S) for the two enantiomers of SO were 4.80 and 3.34 s(-1), respectively. The wide substrate spectrum exhibited by CMEH combined with the fast conversion rate makes it a robust biocatalyst for industrial use. Regioselectivity studies with enantiopure (R)- and (S)-SO revealed that with slightly altered regioselectivity, CMEH has a high potential to synthesize an enantiopure (R)-PED, through an enantioconvergent hydrolytic process.     

A geographically-restricted but prevalent Mycobacterium tuberculosis strain identified in the West Midlands Region of the UK between 1995 and 2008

Background

We describe the identification of, and risk factors for, the single most prevalent Mycobacterium tuberculosis strain in the West Midlands region of the UK.

Methodology/Principal Findings

Prospective 15-locus MIRU-VNTR genotyping of all M. tuberculosis isolates in the West Midlands between 2004 and 2008 was undertaken. Two retrospective epidemiological investigations were also undertaken using univariable and multivariable logistic regression analysis. The first study of all TB patients in the West Midlands between 2004 and 2008 identified a single prevalent strain in each of the study years (total 155/3,056 (5%) isolates). This prevalent MIRU-VNTR profile (32333 2432515314 434443183) remained clustered after typing with an additional 9-loci MIRU-VNTR and spoligotyping. The majority of these patients (122/155, 79%) resided in three major cities located within a 40 km radius. From the apparent geographical restriction, we have named this the “Mercian” strain. A multivariate analysis of all TB patients in the West Midlands identified that infection with a Mercian strain was significantly associated with being UK-born (OR = 9.03, 95%CI = 4.56–17.87, p<0.01), Black Caribbean (OR = 5.68, 95%CI = 2.96–10.91, p<0.01) resident in Wolverhampton (OR = 9.29, 95%CI = 5.69–15.19, p<0.01) and negatively associated with age >65 years old (OR = 0.25, 95%CI = 0.09–0.67, p<0.01). A second more detailed investigation analyzed a cohort of 82 patients resident in Wolverhampton between 2003 and 2006. A significant association with being born in the UK remained after a multivariate analysis (OR = 9.68, 95%CI = 2.00–46.78, p<0.01) and excess alcohol intake and cannabis use (OR = 6.26, 95%CI = 1.45–27.02, p = .01) were observed as social risk factors for infection.

Conclusions/Significance

The continued consistent presence of the Mercian strain suggests ongoing community transmission. Whilst significant associations have been found, there may be other common risk factors yet to be identified. Future investigations should focus on targeting the relevant risk groups and elucidating the biological factors that mediate continued transmission of this strain.     

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein.