Conservation of cysteine residues in fungal histidine acid phytases

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.     

Environmentally friendly protocol for the determination of sitagliptin phosphate in pharmaceutical preparations and biological fluids using l-tyrosine as a fluorescence probe

A responsive spectrofluorometric method was developed for the determination of sitagliptin phosphate using l -tyrosine as a fluorescence probe. The fluorescence intensity of l -tyrosine was quenched with sitagliptin phosphate. The fluorescence intensity was recorded at 307 nm using a 272 nm excitation wavelength. The calibration plot between fluorescence intensity and the concentration of drug was linear in the range of 0.1 to 2.0 mM with a good correlation value of 0.997. The limit of detection and quantification were established to be 3.7 × 10⁻⁴ and 1.23 × 10⁻³ mM, respectively. Commonly used excipients did not interfere with sitagliptin phosphate measurement. The proposed method was used to measure the sitagliptin phosphate in its standard type, dosage form, and biological samples. The percent recovery ranged from 97.41–103.36%. The static quenching was shown to be responsible for quenching as indicated by the Stern–Volmer plot. The method was validated using ICH guidelines and profitably applied for the content uniformity test, resulting in a high percent recovery and small relative standard deviation. The proposed approach is effortless, susceptible, selective, economic, and provides a high precision and accuracy, and can be used to determine sitagliptin phosphate in the pharmaceutical industry.     

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.     

New antiprotozoal agents: Synthesis and biological evaluation of different 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives

In an endeavor to develop efficacious antiprotozoal agents 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives (5–14) were synthesized, characterized and biologically evaluated for antiprotozoal activity. The compounds were screened in vitro against the HM1: IMSS strain of Entamoeba histolytica and NF54 chloroquine-sensitive strain of Plasmodium falciparum. Among the synthesized compounds six exhibited promising antiamoebic activity with IC₅₀ values (0.14–1.26 μM) lower than the standard drug metronidazole (IC₅₀ 1.80 μM). All nine compounds exhibited antimalarial activity (IC₅₀ range: 1.42–19.62 μM), while maintaining a favorable safety profile to host red blood cells. All the compounds were less effective as an antimalarial and more toxic (IC₅₀ range: 14.67–81.24 μM) than quinine (IC₅₀: 275.6 ± 16.46 μM) against the human kidney epithelial cells. None of the compounds exhibited any inhibitory effect on the viability of Anopheles arabiensis mosquito larvae.     

Synthesis,structure–activity relationship and molecular docking of 3-oxoaurones and 3-thioaurones as acetylcholinesterase and butyrylcholinesterase inhibitors

The present study describes efficient and facile syntheses of varyingly substituted 3-thioaurones from the corresponding 3-oxoaurones using Lawesson’s reagent and phosphorous pentasulfide. In comparison, the latter methodology was proved more convenient, giving higher yields and required short and simple methodology. The structures of synthetic compounds were unambiguously elucidated by IR, MS and NMR spectroscopy. All synthetic compounds were screened for their inhibitory potential against in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Molecular docking studies were also performed in order to examine their binding interactions with AChE and BChE human proteins. Both studies revealed that some of these compounds were found to be good inhibitors against AChE and BChE.     

New data on the genus Chrysotoxum Meigen (Diptera: Syrphidae) from North-East Turkey,Armenia, Azerbaijan and Iran including descriptions of three new species

New data and records of the genus Chrysotoxum Meigen, 1803 are reported, arising from taxonomic and faunistic examination of adult specimens collected from 1920 to 2011 from four northeastern provinces of Turkey (Erzurum, Bayburt, Kars, and Artvin), and from the neighboring countries of Armenia, Azerbaijan and Iran. Three new species are described: Chrysotoxum antennalis Vuji?, Nedeljkovi? &; Hayat sp. n., C. clausseni Vuji?, Nedeljkovi? &; Hayat sp. n. and C. persicum Vuji?, Nedeljkovi? &; Hayat sp. n. The first two are known only from northeastern Turkey, and the third also occurs in Armenia, Azerbaijan and Iran. These new species have in common an antenna with the basoflagellomere being shorter than the scape and pedicel together.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:4C264678-8E47-4DE0-AC7D-91DABC597BCD     

Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis

F₄₂₀H₂:NADP⁺ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP⁺ to NADPH by transferring a hydride from the reduced F₄₂₀ cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon wtFno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP⁺ and the F₄₂₀ precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of wtFno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased k_cat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs₄₂₀ versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s^?1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH₂ to NADP⁺. Therefore, Fno is a dynamic enzyme that regulates NADPH production.     

Effect of 28-homobrassinolide on the drought stress-induced changes in photosynthesis and antioxidant system of <Emphasis Type="Italic">Brassica juncea</Emphasis> L.

To investigate the effect of exogenously applied 28-homobrassinolide (HBL) on drought-stressed plants, photosynthesis and antioxidant systems were examined in Indian mustard (Brassica juncea L.). Seedlings of Indian mustard were subjected to drought stress for 7 days at the 8–14 (DS1)/15–21 (DS2) days’ stage of growth and then returned to normal conditions of growth. These seedlings were sprayed with HBL (0.01 μM) at the 30-day stage and were sampled at 60 days to assess the changes in growth, photosynthesis and antioxidant enzymes. Plants exposed to stress at either of the stages of growth exhibited a significant decrease in growth and photosynthesis. The exposure of plants to stress at an earlier stage (DS1) was more inhibitory than that at a later stage (DS2). However, the follow-up treatment with HBL significantly improved the values of these parameters and also overcame the inhibitory effect of water stress. The activity of antioxidant enzymes [catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7) and superoxide dismutase (E.C. 1.15.1.1)] and proline content in leaves exhibited an increase in response to both the treatment factors, where their interaction had an additive effect. It was, therefore, concluded that the elevated antioxidant system, at least in part, was responsible for amelioration of the drought stress.     

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Transforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.

Methods

Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.

Results

TGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.

Conclusions

RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.     

Cholera outbreak in Forcibly Displaced Myanmar National (FDMN) from a small population segment in Cox’s Bazar,Bangladesh, 2019

BackgroundBangladesh experienced a sudden, large influx of forcibly displaced persons from Myanmar in August 2017. A cholera outbreak occurred in the displaced population during September-December 2019. This study aims to describe the epidemiologic characteristics of cholera patients who were hospitalized in diarrhea treatment centers (DTCs) and sought care from settlements of Forcibly Displaced Myanmar Nationals (FDMN) as well as host country nationals during the cholera outbreak.MethodsDiarrhea Treatment Center (DTC) based surveillance was carried out among the FDMN and host population in Teknaf and Leda DTCs hospitalized for cholera during September-December 2019.ResultsDuring the study period, 147 individuals with cholera were hospitalized. The majority, 72% of patients reported to Leda DTC. Nearly 65% sought care from FDMN settlements. About 47% of the cholera individuals were children less than 5 years old and 42% were aged 15 years and more. Half of the cholera patients were females. FDMN often reported from Camp # 26 (45%), followed by Camp # 24 (36%), and Camp # 27 (12%). Eighty-two percent of the cholera patients reported watery diarrhea. Some or severe dehydration was observed in 65% of cholera individuals. Eighty-one percent of people with cholera received pre-packaged ORS at home. About 88% of FDMN cholera patients reported consumption of public tap water. Pit latrine without water seal was often used by FDMN cholera individuals (78%).ConclusionVigilance for cholera patients by routine surveillance, preparedness, and response readiness for surges and oral cholera vaccination campaigns can alleviate the threats of cholera.