Stigmatellin Y – An anti-biofilm compound from Bacillus subtilis BR4 possibly interferes in PQS–PqsR mediated quorum sensing system in Pseudomonas aeruginosa

Hitherto this is the first report pertaining to production of biofilm inhibitory compound(s) (BIC) from Bacillus subtilis BR4 against Pseudomonas aeruginosa (ATCC 27853) coupled with production optimization. In order to achieve this, combinations of media components were formulated by employing statistical tools such as Plackett–Burman analysis and central composite rotatable design (CCRD). It was evident that at 35 ml L^?1 glycerol and 3.8 g L^?1 casamino acid, anti-biofilm activity and production of extracellular protein significantly increased by 1.5-fold and 1.2-fold, respectively. These results corroborate that the combination of glycerol and casamino acid plays a key role in the production of BIC. Further, metabolic profiling of BIC was carried out using liquid chromatography/tandem mass spectrometry (LC–MS/MS) based on m/z value. The presence of Stigmatellin Y was predicted with monoisotopic neutral mass of 484.2825 Da. In support of optimization study, higher production of BIC was confirmed in the optimized-media-grown BR4 (OPT-BR4) than in the ideal-media-grown BR4 (ID-BR4) by LC–MS/MS analysis. PqsR in P. aeruginosa is a potential target for anti-virulent therapy. Molecular docking study has revealed that Stigmatellin Y interacts with PqsR in the similar orientation like a cognate signal (PQS) and synthetic inhibitor. In addition, Stigmatellin Y was found to exhibit interaction with four more amino acid residues of PqsR to establish strong affinity. Stigmatellin Y thus might play a role of competitor for PQS to distract PQS–PqsR mediated communication in P. aeruginosa. The present investigation thus paves new avenues to develop anti-Pseudomonas virulent therapy.     

Calculation of protein ionization equilibria with conformational sampling: pK(a) of a model leucine zipper, GCN4 and barnase.

The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pK(a) calculations, yet the idea has been tested occasionally. This is the first report of systematic comparison of pK(a) estimates computed from long multiple MD simulations and NMR ensembles. As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used. A variety of conformational averaging and titration curve-averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pK(a) calculations. Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution. Remarkable improvement of the accuracy of pK(a) predictions was achieved by the use of multiple MD simulations. By using multiple trajectories the absolute error in pK(a) predictions for the model leucine zipper was reduced to as low as approximately 0.25 pK(a) units. The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pK(a) calculations, are discussed.     

Mutagenicity of the human bladder carcinogen 4-aminobiphenyl to the bladder of Muta ™Mouse transgenic mice

The human carcinogen 4-aminobiphenyl (4AB) was evaluated in the Muta ™Mouse transgenic mouse mutation assay. A single oral dose of 75 mg/kg induced 6.9-, 1.8- and 2.2-fold increases in the mutation frequency (MF) in the bladder, liver and bone marrow, respectively. Ten daily oral doses of 10 mg/kg 4AB increased the MF in the bladder, liver and bone marrow by 13.7-, 4.8- 2.4-fold the control value, respectively. The repeat dosing protocol was therefore more sensitive than the single dose protocol. Assessment of DNA samples prepared from pooled liver homogenates clearly indicated the increase in MF observed in the individual liver samples obtained from both groups of 4AB-treated mice.     

Schistosoma mansoni: histochemical analysis of the postacetabular gland secretion of cercariae

Contents of the funduses and ducts of the postacetabular glands of Schistosoma mansoni cercariae, the secreted deposits, and the surface film were compared by their histochemical reactions. Techniques for carbohydrate-containing substances, neutral and acid mucosubstance, proteins and amino acids, and enzymes were used. The secretion reacted differently before (within the glands) and after (in secreted deposits) emission.Before emission, the postacetabular gland contents reacted as a neutral mucosubstance containing periodate-engendered and periodate-reactive aldehydes rich in vic-glycols or their substituted amines, probably hexoses other than glucose, such as fucose or galactose. No reactions of significance were observed for acid groups or for glycogen or lipids. In this state, the secretion is termed mucigen.After emission, the secretion stained not only as mucigen, but also as acid mucosubstance, apparently sialomucin. After emission, it is termed mucin.It is probable that acid radicals were present in mucigen but were masked stearically by the presence of adjacent neutral radicals or basic proteins. The surface film reacted as both a neutral and an acid mucosubstance. Evidence suggested that the film itself was neutral and that the reaction for acid mucosubstance was from an overlay of mucin secreted from the postacetabular glands.Proteins and amino acids, especially arginine, and some tyrosine and tryptophan were indicated in mucigen and in mucin by the histochemical tests. There was no histochemical evidence of enzymes. Secretion of the postacetabular glands is concluded from histochemical reactions, as from earlier chromatographic data (Stirewalt and Evans 1960), to be a carbohydrate-protein-lipid complex.     

DNA lesions,chromosomal aberrations and G2 delay in CHO cells cultured in medium containing bromo- or chloro-deoxyuridine

Experiments have been performed to investigate whether BrdUrd- and CldUrd-substituted DNA contains lesions causing a delay in cell-cycle progression and induction of chromosomal aberrations. The presence of lesions has been determined directly by alkaline sucrose gradient and nucleoid sedimentation analysis and indirectly by screening for induced chromosomal aberrations. The influence of inhibitors of DNA repair (caffeine and 3-aminobenzamide) or DNA synthesis (hydroxyurea) on the frequencies of such aberrations has been estimated. It is found that BrdUrd and CldUrd are cytotoxic when present in DNA. No randomly located DNA breaks could be detected under neutral conditions, but BrdUrd-substituted DNA was found to contain numerous alkali-labile sites. CldUrd at high concentrations causes G₂ delay, similar to the action of known DNA-damaging agents. The extent of delay depends on the pattern of incorporation of the analogue, i.e., incorporation for two cell cycles causes the longest delay, growth for 12 h in CldUrd followed by 12 h in dThd-containing medium causes a lesser delay and the delay is not significant when the cells are incubated in the analogue for only 12 h prior to fixation. Numerous chromatid type aberrations are present in cells incubated at the highest CldUrd concentration, and their induction follows the pattern of induction of G₂ delay, indicating the sharing of a common lesion. Caffeine, 3-aminobenzamide and hydroxyurea increase the number of chromosomal aberrations when added 2 h before fixation. The significance of these results is discussed.     

First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia

Environmental bacteria belonging to various families were isolated from polluted water collected from ten different sites in Tunisia. Sites were chosen near industrial and urban areas known for their high degree of pollution. The aim of this study was to investigate cross-resistance between heavy metals (HM), i.e., silver, mercury and copper (Ag, Hg, and Cu), and antibiotics. In an initial screening, 80 isolates were selected on ampicillin, and 39 isolates, retained for further analysis, could grow on a Tris-buffered mineral medium with gluconate as carbon source. Isolates were identified based on their 16S rRNA gene sequence. Results showed the prevalence of antibiotic resistance genes, especially all isolates harbored the bla_TEM gene. Some of them (15.38%) harbored bla_SHV. Moreover, several were even ESBLs and MBLs-producers, which can threaten the human health. On the other hand, 92.30%, 56.41%, and 51.28% of the isolates harbored the heavy metals resistance genes silE, cusA, and merA, respectively. These genes confer resistance to silver, copper, and mercury. A cross-resistance between antibiotics and heavy metals was detected in 97.43% of our isolates.     

Energy adaptive response during parthanatos is enhanced by PD98059 and involves mitochondrial function but not autophagy induction

Parthanatos is a programmed necrotic demise characteristic of ATP (adenosine triphosphate) consumption due to NAD⁺ (nicotinamide adenine dinucleotide) depletion by poly(ADP-ribose) polymerase 1 (PARP1)-dependent poly(ADP-ribosyl)ation on target proteins. However, how the bioenergetics is adaptively regulated during parthanatos, especially under the condition of macroautophagy deficiency, remains poorly characterized. Here, we demonstrated that the parthanatic inducer N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) triggered ATP depletion followed by recovery in mouse embryonic fibroblasts (MEFs). Notably, Atg5^−/− MEFs showed great susceptibility to MNNG with disabled ATP-producing capacity. Moreover, the differential energy-adaptive responses in wild-type (WT) and Atg5^−/− MEFs were unequivocally worsened by inhibition of AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and mitochondrial activity. Importantly, Atg5^−/− MEFs disclosed diminished SIRT1 and mitochondrial activity essential to the energy restoration during parthanatos. Strikingly, however, parthanatos cannot be exasperated by bafilomycin A1 and MNNG neither provokes microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and p62 elimination, suggesting that parthanatos does not induce autophagic flux. Intriguingly, we reported unexpectedly that PD98059, even at low concentration insufficient to inhibit MEK, can promote mitochondrial activity and facilitate energy-restoring process during parthanatos, without modulating DNA damage responses as evidenced by PARP1 activity, p53 expression, and γH2AX (H2A histone family, member X (H2AX), phosphorylated on Serine 139) induction. Therefore, we propose that Atg5 deficiency confers an infirmity to overcome the energy crisis during parthanatos and further underscore the deficits in mitochondrial quality control, but not incapability of autophagy induction, that explain the vulnerability in Atg5-deficient cells. Collectively, our results provide a comprehensive energy perspective for an improved treatment to alleviate parthanatos-related tissue necrosis and disease progression and also provide a future direction for drug development on the basis of PD98059 as an efficacious compound against parthanatos.     

Use of a novel anti-proliferative compound coated on a biopolymer to mitigate platelet-derived growth factor-induced proliferation in human aortic smooth muscle cells: comparison with sirolimus

Drug eluting stents (DES) have become a common mode of treatment for stenosis in coronary arteries. However, currently, the use of sirolimus/paclitaxel-coated DES has come under scrutiny, because of their pro-thrombotic effects leading to potential adverse outcomes in the long run. We have previously documented that d-threo-1-phenyl-2-decanoylamino-3-morholino propanol (D-PDMP); an inhibitor of glucosylceramide synthase and lactosylceramide (LacCer) synthase markedly inhibited platelet-derived growth factor (PDGF)-induced cell proliferation. We have fabricated DES wherein, D-PDMP or sirolimus was coated on to a double layer of poly (lactic-co-glycolic acid) on a bare metal stent. The in vitro release of D-PDMP from biopolymer and its consequent effect on PDGF induced proliferation and apoptosis was assessed in human aortic smooth muscle cells (ASMC). D-PDMP was released from biopolymers in a dose-dependent fashion and was accompanied with a decrease in PDGF-induced cell proliferation, but not apoptosis. In contrast, sirolimus markedly increased apoptosis in these cells in addition to inhibiting proliferation. Our mechanistic studies revealed that D-PDMP, but not sirolimus decreased the cellular level of glucosyl and lactosylceramide that accompanied inhibition of PDGF-induced cell proliferation. Our short-term (14 days) in vivo studies in rabbits also attested to the safety and biocompatibility of the D-PDMP coated stents. Our data reveal the superiority of D-PDMP coated biopolymers over sirolimus coated biopolymers in mitigating ASMC proliferation. Such D-PDMP coated stents may be useful for localized delivery of drug to mitigate neo-vascular hyperplasia and other proliferative disorders. Yong-Dan Tang, Ambarish Pandey, Subbu S. Venkatraman, and Subroto Chatterjee contributed equally to this work.     

Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy

Adipose tissue secretes a variety of bioactive factors, which can regulate cardiomyocyte hypertrophy via reactive oxygen species (ROS). In the present study we investigated whether apelin affects ROS-dependent cardiac hypertrophy. In cardiomyocytes apelin inhibited the hypertrophic response to 5-HT and oxidative stress induced by 5-HT- or H₂O₂ in a dose-dependent manner. These effects were concomitant to the increase in mRNA expression and activity of catalase. Chronic treatment of mice with apelin attenuated pressure-overload-induced left ventricular hypertrophy. The prevention of hypertrophy by apelin was associated with increased myocardial catalase activity and decreased plasma lipid hydroperoxide, as an index of oxidative stress. These results show that apelin behaves as a catalase activator and prevents cardiac ROS-dependent hypertrophy.