Purification and characterization of a thermoalkalophilic xylanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Summary An extracellular xylanase was purified to homogeneity from the culture filtrate of a thermophilic Bacillus sp. The molecular weight of the purified xylanase was 44 kDa, as analysed by SDS/PAGE. The enzyme reaction followed Michaelis–Menten kinetics with K_m^app and V_max values of 0.025 mg/ml and 450 U/mg protein, respectively, as obtained from a Lineweaver–Burk plot. The xylanase contained no other enzyme activity except for the hydrolysis of xylan substrate. The optimal temperature of the enzyme assay was 50 °C. The optimum pH for the xylanase activity was at three peaks 6.5, 8.5 and 10.5, respectively and the enzyme was stable over a broad range of pH from pH 6 to 10.5. Metal ions tested with demetalized enzyme had no effect, with the exception of Hg²⁺ and Pb²⁺ (both strong inhibitors). Inhibition of the enzyme activity by N-bromosuccinimide (amino acid modifier) indicated the role of tryptophan residues in the catalytic function of the enzyme. Due to these outstanding properties, the xylanase of Bacillussp. finds potential applications in biopulping, biobleaching and de-inking of recycled paper and other industrial processes.     

Functional characterization of N-terminal nucleotide binding domain (NBD-1) of a major ABC drug transporter Cdr1p of Candida albicans: uncommon but conserved Trp326 of Walker B is important for ATP binding

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.     

Sorafenib Inhibition of Hepatic Stellate Cell Proliferation in Tumor Microenvironment of Hepatocellular Carcinoma: A Study of the Sorafenib Mechanisms

We investigated the mechanism and effects of sorafenib on hepatic stellate cell (HSC) viability and in the liver tumor microenvironment. The expression of α-smooth muscle actin (α-SMA) was measured immunocytochemically in the LX2 cells treated with differing concentrations of sorafenib. Changes in the platelet-derived growth factor (PDGF)-BB and tumor growth factor (TGF)-β1 concentrations were detected in the LX2 supernatant using an enzyme-linked immunosorbent assay (ELISA). Expressions of the extracellular signal-regulated kinase 1 (ERK₁), ERK₂, and Akt signaling pathways were measured using a western blot assay. The LX2 cells were cocultured with HepG2 cells for 24 h to observe their effects on HepG2 cell invasive ability. (1) After treatment with various concentrations of sorafenib for 12, 24, 36, or 48 h, MTT assay showed that the viability of the treated LX2 cells was lower than in the controls. (2) As sorafenib concentration and time of exposure increased, α-SMA expression became weaker in the treated cells. (3) The PDGF-BB and TGF-β1 concentrations decreased with higher concentration, and longer exposures under the same sorafenib concentration. (4) The ERK₁, ERK₂, and Akt expressions were identical between the treated and the control groups, but their phosphorylated expression decreased with increased concentrations of sorafenib. (5) The invasive ability of the HepG2 cells induced by the LX2 gradually decreased as sorafenib concentrations increased. Sorafenib suppressed α-SMA expression, inhibited PDGF-dependent signaling pathways in HSCs, downregulated the PDGF-BB and TGF-β1 expression in the HSCs supernatant, and restrained viability of the HSCs, resulting in suppressed proliferation and invasion in the HepG2 cells.     

Genetic factors affecting allelic recombination at the histidine-3 locus ofNeurospora crassa

Summary In addition to the generec-4, other genetic factors affect the frequency of allelic recombination in thehis-3 locus. One dominant factor, designated asrec-6 ⁺, in association withrec-4 ⁺ causes greater reduction in prototrophic frequency than obtained withrec-4 ⁺ alone. The action ofrec 6 ⁺ in crosses recessive homozygous forrec-4 is not established at the present. The effect ofrec-6 ⁺ is recognised only with onehis-3 allele but not with another. Interaction ofrec-4 ⁺ orrec-4 with other genetic factors can give approximately ten fold variation in the prototrophic frequencies obtained with a pair of alleles. It is suggested that the control of the rate of mutations during meiosis might be one of the roles of the recombination genes.     

Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: novel compounds against aspergillosis

Ten 4-aryl-1,4-dihydropyridine and three 4-aryl-1,2,3,4-tetrahydropyrimidin-2-one derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus and Candida albicans. Although none of the three compounds belonging to pyrimidin-2-one series showed any activity against two pathogens, two of the compounds of the dihydropyridine series, that is, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate and dimethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate, exhibited significant activity against A. fumigatus in disc diffusion, microbroth dilution and percent spore germination inhibition assays. The most active diethyl dihydropyridine derivative exhibited a MIC value of 2.92 microg/disc in disc diffusion and 15.62 microg/ml in microbroth dilution assays. The MIC(90) value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml. The diethyl dicarboxylate derivative of dihydropyridine also exhibited appreciable activity against C. albicans. The in vitro toxicity of the most active diethyl dihydropyridine derivative was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes even at a concentration of 625 microg/ml. The standard drug amphotericin B exhibited 100% lysis of erythrocytes at a concentration almost 16 times less than the safer concentration of the most active dihydropyridine derivative.     

Mechanoluminescence of SrAl2O4:Eu,Dy nanophosphors induced by low impact velocity

The present paper reports the impulsive excitation of mechanoluminescence (ML) in Sr_0.97Al₂O₄:Eu_0.01,Dy_0.02 nanophosphors prepared using a combustion technique. The phosphors are characterized using X‐ray powder diffraction (XRD), high‐resolution transmission electron microscopy (HRTEM) and photoluminescence (PL). The XRD results show that the samples exhibit a monoclinic α‐phase in the crystal structure. The space group of SrAl₂O₄:Eu,Dy nanophosphors is monoclinic P2₁. The PL and ML spectra of SrAl₂O₄:Eu,Dy nanophosphors are excited using light with a wavelength of 365 nm and emission is found at 516 nm. The prepared nanophosphors exhibits an intense ML that can be seen in daylight with the naked eye. When a sample powder is deformed impulsively by the impact of a moving piston, the ML intensity initially increases linearly with time, attains a peak value, I_m, at time t_m, and then decreases with time. The peak ML intensity, I_m, and total ML intensity, I_T, increase linearly with applied pressure and impact velocity. The ML intensity decreases with successive impacts of load onto the phosphors, and the diminished ML intensity can be approximately recovered by UV irradiation. The activation energy using thermoluminescence is found to be 0.57 eV for SrAl₂O₄:Eu,Dy nanophosphors. Copyright © 2016 John Wiley & Sons, Ltd.     

Extended spectrum beta-lactamase and metallo beta-lactamase production among <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolated from different clinical samples in a tertiary care hospital in Kathmandu,Nepal

Background

Extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production in Klebsiella pneumoniae and Escherichia coli are the commonest modes of drug resistance among these commonly isolated bacteria from clinical specimens. So the main purpose of our study was to determine the burden of ESBL and MBL production in E. coli and K. pneumoniae isolated from clinical samples. Further, the antimicrobial susceptibility patterns of E. coli and K. pneumoniae were also determined.

Methods

A cross-sectional study was conducted at Om Hospital and Research Centre, Kathmandu, Nepal by using the E. coli and K. pneumoniae isolated from different clinical samples (urine, pus, body fluids, sputum, blood) from May 2015 to December 2015. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Extended spectrum beta-lactamase production was detected by combined disc method using ceftazidime and ceftazidime/clavulanic acid discs and cefotaxime and cefotaxime/clavulanic acid discs. Similarly, metallo beta-lactamase production was detected by combined disc assay using imipenem and imipenem/ethylenediaminetetracetate discs. Bacteria showing resistance to at least three different classes of antibiotics were considered multidrug resistant (MDR).

Results

Of total 1568 different clinical samples processed, 268 (17.1%) samples were culture positive. Among which, E. coli and K. pneumoniae were isolated from 138 (51.5%) and 39 (14.6%) samples respectively. Of the total isolates 61 (34.5%) were ESBL producers and 7 (4%) isolates were found to be MBL producers. High rates of ESBL production (35.9%) was noted among the clinical isolates from outpatients, however no MBL producing strains were isolated from outpatients. Among 138 E. coli and 39 K. pneumoniae, 73 (52.9%) E. coli and 23 (59%) K. pneumoniae were multidrug resistant. The lowest rates of resistance was seen toward imipenem followed by piperacillin/tazobactam, amikacin and cefoperazone/sulbactam.

Conclusions

High rate of ESBL production was found in the E. coli and K. pneumoniae isolated from outpatients suggesting the dissemination of ESBL producing isolates in community. This is very serious issue and can’t be neglected. Regular monitoring of rates of ESBL and MBL production along with multidrug resistance among clinical isolates is very necessary.

     

Helix, sheet, and polyproline II frequencies and strong nearest neighbor effects in a restricted coil library

A central issue in protein folding is the degree to which each residue's backbone conformational preferences stabilize the native state. We have studied the conformational preferences of each amino acid when the amino acid is not constrained to be in a regular secondary structure. In this large but highly restricted coil library, the backbone preferentially adopts dihedral angles consistent with the polyproline II conformation rather than alpha or beta conformations. The preference for the polyproline II conformation is independent of the degree of solvation. In conjunction with a new masking procedure, the frequencies in our coil library accurately recapitulate both helix and sheet frequencies for the amino acids in structured regions, as well as polyproline II propensities. Therefore, structural propensities for alpha-helices and beta-sheets and for polyproline II conformations in unfolded peptides can be rationalized solely by local effects. In addition, these propensities are often strongly affected by both the chemical nature and the conformation of neighboring residues, contrary to the Flory isolated residue hypothesis.     

Ribonuclease L and metal-ion–independent endoribonuclease cleavage sites in host and viral RNAs

Ribonuclease L (RNase L) is a metal-ion–independent endoribonuclease associated with antiviral and antibacterial defense, cancer and lifespan. Despite the biological significance of RNase L, the RNAs cleaved by this enzyme are poorly defined. In this study, we used deep sequencing methods to reveal the frequency and location of RNase L cleavage sites within host and viral RNAs. To make cDNA libraries, we exploited the 2′, 3′-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion–independent endoribonucleases. We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells. Using these methods, we identified (i) discrete regions of hepatitis C virus and poliovirus RNA genomes that were profoundly susceptible to RNase L and other single-strand specific endoribonucleases, (ii) RNase L-dependent and RNase L-independent cleavage sites within ribosomal RNAs (rRNAs) and (iii) 2′, 3′-cyclic phosphates at the ends of 5S rRNA and U6 snRNA. Monitoring the frequency and location of metal-ion–independent endoribonuclease cleavage sites within host and viral RNAs reveals, in part, how these enzymes contribute to health and disease.     

Deaths from Symptomatically Identifiable Furious Rabies in India: A Nationally Representative Mortality Survey

Background

It is estimated that India has more deaths from rabies than any other country. However, existing estimates are indirect and rely on non-representative studies.

Methods and Principal Findings

We examined rabies deaths in the ongoing Million Death Study (MDS), a representative survey of over 122,000 deaths in India that uses enhanced types of verbal autopsy. We estimated the age-specific mortality rates of symptomatically identifiable furious rabies and its geographic and demographic distributions. A total of 140 deaths in our sample were caused by rabies, suggesting that in 2005 there were 12,700 (99% CI 10,000 to 15,500) symptomatically identifiable furious rabies deaths in India. Most rabies deaths were in males (62%), in rural areas (91%), and in children below the age of 15 years (50%). The overall rabies mortality rate was 1.1 deaths per 100,000 population (99%CI 0.9 to 1.4). One third of the national rabies deaths were found in Uttar Pradesh (4,300) and nearly three quarters (8,900) were in 7 central and south-eastern states: Chhattisgarh, Uttar Pradesh, Odisha, Andhra Pradesh, Bihar, Assam, and Madhya Pradesh.

Conclusions and Significance

Rabies remains an avoidable cause of death in India. As verbal autopsy is not likely to identify atypical or paralytic forms of rabies, our figure of 12,700 deaths due to classic and clinically identifiable furious rabies underestimates the total number of deaths due to this virus. The concentrated geographic distribution of rabies in India suggests that a significant reduction in the number of deaths or potentially even elimination of rabies deaths is possible.