Sequential interactions of silver–silica nanocomposite (Ag–SiO2NC) with cell wall,metabolism and genetic stability of Pseudomonas aeruginosa,a multiple antibiotic‐resistant bacterium

The study was carried out to understand the effect of silver–silica nanocomposite (Ag–SiO₂NC) on the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, a multiple drug‐resistant bacterium. Bacterial sensitivity towards antibiotics and Ag–SiO₂NC was studied using standard disc diffusion and death rate assay, respectively. The effect of Ag–SiO₂NC on cell wall integrity was monitored using SDS assay and fatty acid profile analysis, while the effect on metabolism and genetic stability was assayed microscopically, using CTC viability staining and comet assay, respectively. Pseudomonas aeruginosa was found to be resistant to β‐lactamase, glycopeptidase, sulfonamide, quinolones, nitrofurantoin and macrolides classes of antibiotics. Complete mortality of the bacterium was achieved with 80 μg ml^?1 concentration of Ag–SiO₂NC. The cell wall integrity reduced with increasing time and reached a plateau of 70% in 110 min. Changes were also noticed in the proportion of fatty acids after the treatment. Inside the cytoplasm, a complete inhibition of electron transport system was achieved with 100 μg ml^?1 Ag–SiO₂NC, followed by DNA breakage. The study thus demonstrates that Ag–SiO₂NC invades the cytoplasm of the multiple drug‐resistant P. aeruginosa by impinging upon the cell wall integrity and kills the cells by interfering with electron transport chain and the genetic stability.

Significance and Impact of Study

Although the synthesis, structural characteristics and biofunction of silver nanoparticles are well understood, their application in antimicrobial therapy is still at its infancy as only a small number of microorganisms are tested to be sensitive to nanoparticles. A thorough knowledge of the mode of interaction of nanoparticles with bacteria at subcellular level is mandatory for any clinical application. The present study deals with the interactions of Ag–SiO2NC with the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, which would contribute substantially in strengthening the therapeutic applications of silver nanoparticles.     

Insulin‐like growth factors and their potential role in cardiac epigenetics

Cardiovascular disease (CVD) constitutes a major public health threat worldwide, accounting for 17.3 million deaths annually. Heart disease and stroke account for the majority of healthcare costs in the developed world. While much has been accomplished in understanding the pathophysiology, molecular biology and genetics underlying the diagnosis and treatment of CVD, we know less about the role of epigenetics and their molecular determinants. The impact of environmental changes and epigenetics in CVD is now emerging as critically important in understanding the origin of disease and the development of new therapeutic approaches to prevention and treatment. This review focuses on the emerging role of epigenetics mediated by insulin like‐growth factors‐I and ‐II in major CVDs such as heart failure, cardiac hypertrophy and diabetes.     

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.     

Spaceflight‐induced enhancement of 2‐keto‐L‐gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis

Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l^?1 h^?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m³ fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.

Significance and Impact of the Study

KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening.     

Quantification of plant surface metabolites by matrix‐assisted laser desorption–ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves

The localization of metabolites on plant surfaces has been problematic because of the limitations of current methodologies. Attempts to localize glucosinolates, the sulfur‐rich defense compounds of the order Brassicales, on leaf surfaces have given many contradictory results depending on the method employed. Here we developed a matrix‐assisted laser desorption–ionization (MALDI) mass spectrometry protocol to detect surface glucosinolates on Arabidopsis thaliana leaves by applying the MALDI matrix through sublimation. Quantification was accomplished by spotting glucosinolate standards directly on the leaf surface. The A. thaliana leaf surface was found to contain approximately 15 nmol of total glucosinolate per leaf with about 50 pmol mm^?2 on abaxial (bottom) surfaces and 15–30 times less on adaxial (top) surfaces. Of the major compounds detected, 4‐methylsulfinylbutylglucosinolate, indol‐3‐ylmethylglucosinolate, and 8‐methylsulfinyloctylglucosinolate were also major components of the leaf interior, but the second most abundant glucosinolate on the surface, 4‐methylthiobutylglucosinolate, was only a trace component of the interior. Distribution on the surface was relatively uniform in contrast to the interior, where glucosinolates were distributed more abundantly in the midrib and periphery than the rest of the leaf. These results were confirmed by two other mass spectrometry‐based techniques, laser ablation electrospray ionization and liquid extraction surface analysis. The concentrations of glucosinolates on A. thaliana leaf surfaces were found to be sufficient to attract the specialist feeding lepidopterans Plutella xylostella and Pieris rapae for oviposition. The methods employed here should be easily applied to other plant species and metabolites.     

Novel Y‐chromosome polymorphisms in Chinese domestic yak

Y‐chromosome‐specific haplotypes (Y‐haplotypes) constructed using single nucleotide polymorphisms (Y‐SNPs) in the MSY (male‐specific region of the Y‐chromosome) are valuable in population genetic studies. But sequence variants in the yak MSY region have been poorly characterized so far. In this study, we screened a total of 16 Y‐chromosome‐specific gene segments from the ZFY, SRY, UTY, USP9Y, AMELY and OFD1Y genes to identify Y‐SNPs in domestic yaks. Six novel Y‐SNPs distributed in the USP9Y (g.223C>T), UTY19 (g.158A>C and g.169C>T), AMELY2 (g.261C>T), OFD1Y9 (g.165A>G) and SRY4 (g.104G>A) loci, which can define three Y‐haplotypes (YH1, YH2 and YH3) in yaks, were discovered. YH1 was the dominant and presumably most ancient haplotype based on the comparison of UTY19 locus with other bovid species. Interestingly, we found informative UTY19 markers (g.158A>C and g.169C>T) that can effectively distinguish the three yak Y‐haplotypes. The nucleotide diversity was 1.7 × 10^?4 ± 0.3 × 10^?4, indicating rich Y‐chromosome diversity in yaks. We identified two highly divergent lineages (YH1 and YH2 vs. YH3) that share similar frequencies (YH1 +  YH2: 0.82–0.89, YH3: 0.11–0.18) among all three populations. In agreement with previous mtDNA studies, we supported the hypothesis that the two highly divergent lineages (YH1 and YH2 vs. YH3) derived from a single gene pool, which can be explained by the reunion of at least two paternal populations with the divergent lineages already accumulated before domestication. We estimated a divergence time of 408 110 years between the two divergent lineages, which is consistent with the data from mitochondrial DNA in yaks.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Loss of GluN2A‐containing NMDA receptors impairs extra‐dimensional set‐shifting

Glutamate neurotransmission via the N‐methyl‐d ‐aspartate receptor (NMDAR) is thought to mediate the synaptic plasticity underlying learning and memory formation. There is increasing evidence that deficits in NMDAR function are involved in the pathophysiology of cognitive dysfunction seen in neuropsychiatric disorders and addiction. NMDAR subunits confer different physiological properties to the receptor, interact with distinct intracellular postsynaptic scaffolding and signaling molecules, and are differentially expressed during development. Despite these known differences, the relative contribution of individual subunit composition to synaptic plasticity and learning is not fully elucidated. We have previously shown that constitutive deletion of GluN2A subunit in the mouse impairs discrimination and re‐learning phase of reversal when exemplars are complex picture stimuli, but spares acquisition and extinction of non‐discriminative visually cued instrumental response. To investigate the role of GluN2A containing NMDARs in executive control, we tested GluN2A knockout (GluN2A^KO), heterozygous (GluN2A^HET) and wild‐type (WT) littermates on an attentional set‐shifting task using species‐specific stimulus dimensions. To further explore the nature of deficits in this model, mice were tested on a visual discrimination reversal paradigm using simplified rotational stimuli. GluN2A^KO were not impaired on discrimination or reversal problems when tactile or olfactory stimuli were used, or when visual stimuli were sufficiently easy to discriminate. GluN2A^KO showed a specific and significant impairment in ventromedial prefrontal cortex‐mediated set‐shifting. Together these results support a role for GluN2A containing NMDAR in modulating executive control that can be masked by overlapping deficits in attentional processes during high task demands.