Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

Bacterial peptidoglycan (PG or murein) is a single, large, covalently cross‐linked macromolecule and forms a mesh‐like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre‐existing cross‐links for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross‐bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross‐link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.     

Rubidium chloride tolerant callus cultures of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accumulate more potassium and cross tolerate to other salts

Callus cultures from salt tolerant (CSR-10) and susceptible (Swarnadhan) varieties of Oryza sativa L. were established in Murashige and Skoog’s (MS) medium containing lethal concentrations (50 mM) of rubidium chloride (RbCl) as a selective agent. While 95–100% cells were viable in callus cultures grown without RbCl, viability was 75% in 50 mM RbCl selected cultures. Growth of RbCl selected calli in presence of salt was comparable to that of callus grown without it. Cells tolerant to RbCl showed more vacuoles and accumulated more K⁺ in comparison with their corresponding controls. Suspension cultures were established and uptake of ⁸⁶Rb⁺ was measured at 10 and 20 min intervals, which revealed a linear relationship between the absorption of K⁺ and time. Callus cultures (560-day-old) tolerant to 50 mM RbCl regenerated shoots with 35–40% frequencies in both the varieties, but the same age-old callus grown in the medium devoid of RbCl did not show any organogenesis. Callus cultures that are tolerant to 50 mM RbCl when exposed to 25 mM LiCl, 50 mM NaCl, 50 mM KCl and 25 mM CsCl also exhibited cross tolerance in both the varieties. This is the first time that a callus line of rice resistant to RbCl was raised and shown to accumulate a major cation K⁺ and also an increased influx of it.     

Serum levels of interleukin-13 and interferon-gamma from adult patients with asthma in Mysore

Serum protein analysis for noninvasive quantification of airway inflammation in asthma is a promising research tool in the field of lung diseases. Cytokines are believed to have major role in inflammatory process of the airways of the lung. There is an imbalance between T-helper (Th)-2 cells, which secrete interleukin (IL)-4 and interleukin (IL)-13, and Th1 cells, which secrete interferon (IFN)-gamma in asthma. To test the hypothesis that serum IL-13 and IL-4 levels may be elevated whereas IFN-gamma would be decreased in this cohort of patients, a property that could make them possible candidate biomarkers in determining asthma occurrence and severity, we measured concentrations of IL-4, IL-13 and IFN-gamma in serum samples of 88 subjects (44 normal, 12 with mild asthma, 16 with moderate asthma, and 16 with severe asthma). Serum Levels of IL-4, IL-13, and IFN-gamma were determined by an enzyme-linked immune-sorbent assay (ELISA). Median serum level of IFN-gamma in asthmatic patients was 8.0pg/ml, while it was 11.4pg/ml in healthy controls. However, the difference was not significant. Among the different age groups in whom IFN-gamma was assessed, the highest median value in both cases and controls was observed in the age group of 31-40years. The median serum level of IL-13 was 40.0pg/ml in asthmatic patients and 58.25pg/ml in healthy controls. The difference was not significant. On subgroup analysis, no significant difference of IFN-gamma and IL-13 between asthma of different severities was observed. The study also revealed nonsignificant difference of serum cytokines with the duration of asthma, number of allergens, and severity of sensitization. Normal serum levels of IFN-gamma and IL-13 in asthmatic patients suggest their neutral role in the inflammatory process; however, more studies are required to establish the effect of these cytokines in adulthood asthma in different ethnic populations.     

Characterization and Antioxidant Property of Probiotic and Synbiotic Yogurts

The effect of a prebiotic (fructooligosaccharides) or a synbiotic components (prebiotic and probiotic) on the viability, proteolysis and antioxidant properties of probiotic and synbiotic yogurt during 28?days of storage at 4?°C has been investigated. Yogurt starters in conjunction with either probiotic bacteria Lactobacillus plantarum CFR 2194, Lactobacillus fermentum CFR 2192 and/or fructooligosaccharides (FOS) were used for yogurt preparation. Titratable acidity and pH of all yogurt samples followed a similar pattern of increase or decrease during storage. Proteolysis in synbiotic yogurts was found to be significantly (P?<?0.05) higher in comparison with that of control. The addition of prebiotics had no effect (P?=?0.17888) on the viability of yogurt starters during cold storage. No observable changes in the viability of probiotic cultures in probiotic groups. However, supplementation of FOS affected the growth significantly (P?<?0.05) in promoting the growth of L. plantarum and L. fermentum. Antioxidant activities, the index of nutritional value of yogurt, were monitored. Results showed that the DPPH-radical-scavenging activity (85?%) in synbiotic yogurt containing L. plantarum and FOS was significantly higher (P?<?0.05) in comparison with that of control yogurt (72?%). Total phenolics and the ferric reducing power were highest in synbiotic yogurts in comparison with that of other test samples during the entire period of storage. Addition of selected probiotics with FOS thus resulted in an improved functionality of yogurt.     

Pyridine derivatives as anticancer lead compounds with Fatty Acid Synthase as the target: An in silico-guided in vitro study

For the past few decades, structure-based drug discovery (SBDD) has become an inevitable technique in the drug development process for screening hit compounds against therapeutic targets. Here, we have successfully used the SBDD approach viz. virtual high-throughput screening to identify potential inhibitors against the Ketoacyl synthase (KS) domain of Fatty acid synthase (FASN). Overexpression of FASN, and subsequent enhancement of de novo lipogenesis is a key survival strategy of cancer cells. Hence, targeting lipid metabolism using FASN inhibitors has been considered as a promising method to induce metabolic stress, thereby posing a survival disadvantage to cancer cells. In the present study, we have successfully identified eight FASN inhibitors from Asinex Elite database by implementing in silico tools. Five of the hit compounds share a common ring structure, which enables characteristic binding interactions with FASN-KS. Among them, in vitro validation showed that SFA 22637550 possesses significant FASN inhibitory activity and antiproliferative effect in human cancer cells of various origins. The maximum sensitivity was exhibited towards HepG2 hepatocellular carcinoma cells (IC50 = 28 µM). The mode of cell death was found to be apoptosis with a significant increase in SubG0 population without affecting any other phases of the cell cycle. The current study puts forward an excellent core structure for the development of potent FASN inhibitors for successfully targeting cancer cell metabolism, thereby causing selective cell death.     

Studies of Cream Seeded Carioca Beans (Phaseolus vulgaris L.) from a Rwandan Efficacy Trial: In Vitro and In Vivo Screening Tools Reflect Human Studies and Predict Beneficial Results from Iron Biofortified Beans

Iron (Fe) deficiency is a highly prevalent micronutrient insufficiency predominantly caused by a lack of bioavailable Fe from the diet. The consumption of beans as a major food crop in some populations suffering from Fe deficiency is relatively high. Therefore, our objective was to determine whether a biofortified variety of cream seeded carioca bean (Phaseolus vulgaris L.) could provide more bioavailable-Fe than a standard variety using in-vivo (broiler chicken, Gallus gallus) and in-vitro (Caco-2 cell) models. Studies were conducted under conditions designed to mimic the actual human feeding protocol. Two carioca-beans, a standard (G4825; 58μg Fe/g) and a biofortified (SMC; 106μg Fe/g), were utilized. Diets were formulated to meet the nutrient requirements of Gallus gallus except for Fe (33.7 and 48.7μg Fe/g, standard and biofortified diets, respectively). In-vitro observations indicated that more bioavailable-Fe was present in the biofortified beans and diet (P<0.05). In-vivo, improvements in Fe-status were observed in the biofortified bean treatment, as indicated by the increased total-body-Hemoglobin-Fe, and hepatic Fe-concentration (P<0.05). Also, DMT-1 mRNA-expression was increased in the standard bean treatment (P<0.05), indicating an upregulation of absorption to compensate for less bioavailable-Fe. These results demonstrate that the biofortified beans provided more bioavailable Fe; however, the in vitro results revealed that ferritin formation values were relatively low. Such observations are indicative of the presence of high levels of polyphenols and phytate that inhibit Fe absorption. Indeed, we identified higher levels of phytate and quercetin 3–glucoside in the Fe biofortified bean variety. Our results indicate that the biofortified bean line was able to moderately improve Fe-status, and that concurrent increase in the concentration of phytate and polyphenols in beans may limit the benefit of increased Fe-concentration. Therefore, specific targeting of such compounds during the breeding process may yield improved dietary Fe-bioavailability. Our findings are in agreement with the human efficacy trial that demonstrated that the biofortified carioca beans improved the Fe-status of Rwandan women. We suggest the utilization of these in vitro and in vivo screening tools to guide studies aimed to develop and evaluate biofortified staple food crops. This approach has the potential to more effectively utilize research funds and provides a means to monitor the nutritional quality of the Fe-biofortified crops once released to farmers.     

Deltamethrin resistant alleles predominate in Rhipicephalus sanguineus sensu lato in South India

Experimental and Applied Acarology - Brown dog tick, Rhipicephalus sanguineus sensu lato, an important ectoparasite transmitting several pathogens, is the most common tick species infesting dogs....     

A synergistic role for two predicted inner membrane proteins of Escherichia coli in cell envelope integrity

The bacterial cytoplasmic membrane is a principal site of protein translocation, lipid and peptidoglycan biogenesis, signal transduction, transporters and energy generating components of the respiratory chain. Although 25–30% of bacterial proteomes consist of membrane proteins, a comprehensive understanding of their influence on fundamental cellular processes is incomplete. Here, we show that YciB and DcrB, two small cytoplasmic membrane proteins of previously unknown functions, play an essential synergistic role in maintaining cell envelope integrity of Escherichia coli. Lack of both YciB and DcrB results in pleiotropic cell defects including increased levels of lipopolysaccharide, membrane vesiculation, dynamic shrinking and extension of the cytoplasmic membrane accompanied by lysis and cell death. The stalling of an abundant outer membrane lipoprotein, Lpp, at the periplasmic face of the inner membrane leads to lethal inner membrane–peptidoglycan linkages. Additionally, the periplasmic chaperone Skp contributes to yciB dcrB mutant cell death by possibly mistargeting stalled porins into the inner membrane. Consistent with the idea of a compromised envelope in the yciB dcrB mutant, multiple envelope stress response systems are induced, with Cpx signal transduction being required for growth. Taken together, our results suggest a fundamental role for YciB and DcrB in cell envelope biogenesis.