Small molecule sensitization to TRAIL is mediated via nuclear localization,phosphorylation and inhibition of chaperone activity of Hsp27

The small chaperone protein Hsp27 confers resistance to apoptosis, and therefore is an attractive anticancer drug target. We report here a novel mechanism underlying the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitizing activity of the small molecule {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, an inactive analog of the phosphoinositide 3-kinase inhibitor inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, in HeLa cells that are refractory to TRAIL-induced apoptosis. On the basis of the fact that {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 is derived from {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, itself derived from quercetin, and earlier findings indicating that quercetin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 affected Hsp27 expression, we investigated whether {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511 sensitized cancer cells to TRAIL via a conserved inhibitory effect on Hsp27. We provide evidence that upon treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, Hsp27 is progressively sequestered in the nucleus, thus reducing its protective effect in the cytosol during the apoptotic process. {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511-induced nuclear translocation of Hsp27 is linked to its sustained phosphorylation via activation of p38 kinase and MAPKAP kinase 2 and the inhibition of PP2A. Furthermore, Hsp27 phosphorylation leads to the subsequent dissociation of its large oligomers and a decrease in its chaperone activity, thereby further compromising the death inhibitory activity of Hsp27. Furthermore, genetic manipulation of Hsp27 expression significantly affected the TRAIL sensitizing activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511, which corroborated the Hsp27 targeting activity of {"type":"entrez-nucleotide","attrs":{"text":"LY303511","term_id":"1257646067","term_text":"LY303511"}}LY303511. Taken together, these data indicate a novel mechanism of small molecule sensitization to TRAIL through targeting of Hsp27 functions, rather than its overall expression, leading to decreased cellular protection, which could have therapeutic implications for overcoming chemotherapy resistance in tumor cells.     

Rational Design of Broad Spectrum Antibacterial Activity Based on a Clinically Relevant Enoyl-Acyl Carrier Protein (ACP) Reductase Inhibitor

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. {"type":"entrez-nucleotide","attrs":{"text":"CG400549","term_id":"34399433","term_text":"CG400549"}}CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including {"type":"entrez-nucleotide","attrs":{"text":"CG400549","term_id":"34399433","term_text":"CG400549"}}CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of {"type":"entrez-nucleotide","attrs":{"text":"CG400549","term_id":"34399433","term_text":"CG400549"}}CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.     

LncRNA‐AK137033 inhibits the osteogenic potential of adipose‐derived stem cells in diabetic osteoporosis by regulating Wnt signaling pathway via DNA methylation

ObjectivesBone tissue engineering based on adipose‐derived stem cells (ASCs) is expected to become a new treatment for diabetic osteoporosis (DOP) patients with bone defects. However, compared with control ASCs (CON‐ASCs), osteogenic potential of DOP‐ASCs is decreased, which increased the difficulty of bone reconstruction in DOP patients. Moreover, the cause of the poor osteogenesis of ASCs in a hyperglycemic microenvironment has not been elucidated. Therefore, this study explored the molecular mechanism of the decline in the osteogenic potential of DOP‐ASCs from the perspective of epigenetics to provide a possible therapeutic target for bone repair in DOP patients with bone defects.Materials and methodsAn animal model of DOP was established in mice. CON‐ASCs and DOP‐ASCs were isolated from CON and DOP mice, respectively. {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 small interfering RNA (SiRNA) and an {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 overexpression plasmid were used to regulate the expression of {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 in CON‐ASCs and DOP‐ASCs in vitro. Lentiviruses that carried shRNA‐{"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 or {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 cDNA were used to knockdown or overexpress {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033, respectively, in CON‐ASCs and DOP‐ASCs in vivo. Hematoxylin and eosin (H&E), Masson''s, alizarin red, and alkaline phosphatase (ALP) staining, micro‐computed tomography (Micro‐CT), flow cytometry, qPCR, western blotting, immunofluorescence, and bisulfite‐specific PCR (BSP) were used to analyze the functional changes of ASCs.ResultsThe DOP mouse model was established successfully. Compared with CON‐ASCs, {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 expression, the DNA methylation level of the sFrp2 promoter region, Wnt signaling pathway markers, and the osteogenic differentiation potential were decreased in DOP‐ASCs. In vitro experiments showed that {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 silencing inhibited the Wnt signaling pathway and osteogenic ability of CON‐ASCs by reducing the DNA methylation level in the sFrp2 promoter region. Additionally, overexpression of {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 in DOP‐ASCs rescued these changes caused by DOP. Moreover, the same results were obtained in vivo.ConclusionsLncRNA‐{"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 inhibits the osteogenic potential of DOP‐ASCs by regulating the Wnt signaling pathway via modulating the DNA methylation level in the sFrp2 promoter region. This study provides an important reference to find new targets for the treatment of bone defects in DOP patients.     

Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6

Background

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.

Methods

We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 and the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.

Results

OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, a blocker of DAG synthesis, inhibited acute HPV whereas {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125"}}U73343, the inactive form of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, had no effect on HPV.

Conclusion

These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.     

Upregulation of the novel lncRNA U731166 is associated with migration,invasion and vemurafenib resistance in melanoma

Our previous work using a melanoma progression model composed of melanocytic cells (melanocytes, primary and metastatic melanoma samples) demonstrated various deregulated genes, including a few known lncRNAs. Further analysis was conducted to discover novel lncRNAs associated with melanoma, and candidates were prioritized for their potential association with invasiveness or other metastasis‐related processes. In this sense, we found the intergenic lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 (ENSG00000230454) and decided to explore its effects in melanoma. For that, we silenced the lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 expression using shRNAs in a melanoma cell line. Next, we experimentally investigated its functions and found that migration and invasion had significantly decreased in knockdown cells, indicating an essential association of lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 for cancer processes. Additionally, using naïve and vemurafenib‐resistant cell lines and data from a patient before and after resistance, we found that vemurafenib‐resistant samples had a higher expression of lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166. Also, we retrieved data from the literature that indicates lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 may act as a mediator of RNA processing and cell invasion, probably inducing a more aggressive phenotype. Therefore, our results suggest a relevant role of lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 in metastasis development. We also pointed herein the lncRNA {"type":"entrez-nucleotide","attrs":{"text":"U73166","term_id":"1613889","term_text":"U73166"}}U73166 as a new possible biomarker or target to help overcome clinical vemurafenib resistance.     

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.     

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

Pathogenic Klebsiella pneumoniae, resistant to beta-lactam and quinolone drugs, is widely recognized as important bacteria causing array of diseases. The resistance property is obtained by acquisition of plasmid encoded bla_TEM, bla_SHV, bla_CTX-M, QNRA, QNRB and QNRS genes. The aim of this study was to document the prevalence and association of these resistant genes in K. pneumoniae infecting patients in India. Approximately 97 and 76.7 % of the 73 K. pneumoniae isolates showed resistance towards beta-lactam and quinolone drugs respectively. Bla genes were detected in 74 % of K. pneumoniae isolates; with prevalence in the following order: bla_TEM > bla_SHV > bla_CTXM. QNR genes were detected in 67 % samples. Chi-square analysis revealed significant association between presence of bla and qnr genes in our study (P value = 0.000125). Sequence analysis of some bla_TEM, bla_SHV, bla_CTX-M and QNRB PCR products revealed presence of bla_TEM1 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"JN193522","term_id":"343796760","term_text":"JN193522"}}JN193522), bla_TEM116 ({"type":"entrez-nucleotide","attrs":{"text":"JN193523","term_id":"343796762","term_text":"JN193523"}}JN193523 and {"type":"entrez-nucleotide","attrs":{"text":"JN193524","term_id":"343796764","term_text":"JN193524"}}JN193524), bla_SHV11, bla_CTXM72 variants ({"type":"entrez-nucleotide","attrs":{"text":"JF523199","term_id":"329024835","term_text":"JF523199"}}JF523199) and QNRB1 ({"type":"entrez-nucleotide","attrs":{"text":"JN193526","term_id":"343796802","term_text":"JN193526"}}JN193526 and {"type":"entrez-nucleotide","attrs":{"text":"JN193527","term_id":"343796804","term_text":"JN193527"}}JN193527) in our samples.     

CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger,protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels

Inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca²⁺ homeostasis. However, the Ca²⁺ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca²⁺ levels are modulated by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 on neuronal Ca²⁺ homeostasis using cameleon-based mitochondrial Ca²⁺ and cytosolic Ca²⁺ ([Ca²⁺]_i) live imaging. We observed that NCLX-driven mitochondrial Ca²⁺ exchange occurs in cortical neurons under basal conditions as {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 induced a decrease in [Ca²]_i concomitant with a Ca²⁺ accumulation inside the mitochondria. In turn, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 also inhibited mitochondrial Ca²⁺ efflux after the stimulation of acetylcholine receptors. In contrast, {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 strongly prevented depolarization-induced [Ca²⁺]_i increase by blocking voltage-gated Ca²⁺ channels (VGCCs), whereas it did not induce depletion of ER Ca²⁺ stores. Moreover, mitochondrial Ca²⁺ overload was reduced as a consequence of diminished Ca²⁺ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca²⁺ overload by {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 modulates cytosolic and mitochondrial Ca²⁺ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.     

Virtual Screening of compounds to 1-deoxy-Dxylulose 5-phosphate reductoisomerase (DXR) from Plasmodium falciparum

The 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) protein (Gen Bank ID {"type":"entrez-protein","attrs":{"text":"AAN37254.1","term_id":"23497715","term_text":"AAN37254.1"}}AAN37254.1) from Plasmodium falciparum is a potential drug target. Therefore, it is of interest to screen DXR against a virtual library of compounds (at the ZINC database) for potential binders as possible inhibitors. This exercise helped to choose 10 top ranking molecules with ZINC00200163 [N-(2,2di methoxy ethyl)-6-methyl-2, 3, 4, 9-tetrahydro-1H-carbazol-1-amine] a having good fit (-6.43 KJ/mol binding energy) with the target protein. Thus, ZINC00200163 is identified as a potential molecule for further comprehensive characterization and in-depth analysis.     

First report,morphological and molecular characterization of Xiphinema elongatum and X. pachtaicum (Nematoda,Longidoridae) from Ethiopia

A total of six soil samples were collected around rhizosphere of citrus plants during 2010 from Melkassa Agricultural Research Center experimental station, Ethiopia. From these samples two most important ecto-plant parasitic nematodes of the genus Xiphinema were found and analysed. The genus Xiphinema is a large group of the phylum nematoda which constitutes more than 260 species. They are polyphagous root- ectoparasites of many crop plants and some species of this genus cause damage by direct feeding on root tips and transmit nepoviruses. The delimitation and discrimination of two species in the genus is presented, described herein as Xiphinema elongatum and Xiphinema pachtaicum. Morphological and morphometric data were done using light microscopy and results of both species were fit within the previously described nematode species of Xiphinema elongatum and Xiphinema pachtaicum. 18S rDNA were analysed using Bayesian inference (BI) method to reconstruct phylogenetic relationships of the studied Xiphinema sp. ({"type":"entrez-nucleotide","attrs":{"text":"KP407872","term_id":"806981416","term_text":"KP407872"}}KP407872 Xiphinema elongatum and {"type":"entrez-nucleotide","attrs":{"text":"KP407873","term_id":"806981417","term_text":"KP407873"}}KP407873 Xiphinema pachtaicum) with other Xiphinema species. The 18S rDNA sequence of Xiphinema pachtaicum was alike to previously described species from the GenBank but Xiphinema elongatum exhibited very small levels of nucleotides differences (0.4%) which might be possible intra-specific divergence. Though this region of rDNA has less resolution on complex species, its combination with morphological and morphometric analyses, suggests these species as Xiphinema elongatum and Xiphinema pachtaicum with the GenBank accession number of {"type":"entrez-nucleotide","attrs":{"text":"KP407872","term_id":"806981416","term_text":"KP407872"}}KP407872 and {"type":"entrez-nucleotide","attrs":{"text":"KP407873","term_id":"806981417","term_text":"KP407873"}}KP407873, respectively. Short notes, morphological measurements, illustrations, and molecular data are given to these species. These species are reported for the first time from Ethiopia and it provides new geographical information of these organisms.     

Hydrolyzing Proficiency of Keratinases in Feather Degradation

The keratinase degrade highly rigid, cross linked structural polypeptides with different efficiency depending on the type of source. Two newly isolated strains of Bacillus subtilis (RSE163 and RSE165; NCBI Accession no {"type":"entrez-nucleotide","attrs":{"text":"JQ887983","term_id":"453198585","term_text":"JQ887983"}}JQ887983 and {"type":"entrez-nucleotide","attrs":{"text":"JQ887982","term_id":"453198574","term_text":"JQ887982"}}JQ887982) were found to be efficient keratinase producers with unusual catalytic activity result in different morphological changes in degradation pattern of feather, confirmed by their scanned electron micrographs. Maximum keratinolytic activity of both the strains B. subtilis RSE163 and RSE165 were found to be 366 ± 15.79 and 194 ± 7.26 U after 72 h of incubation. While the disulphide reductase activity of RSE163 and RSE165 estimated 0.24 ± 0.05 and 0.15 ± 0.03 U/ml of enzyme after 24 h of incubation. A total of 16 free amino acids of variable concentration were also analyzed in the cell free supernatant of hydrolyzed feather from two strains. Present study demonstrates the action of two different keratinases in feather degradation.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0477-5) contains supplementary material, which is available to authorized users.     

Streptomyces antibioticalis, a Novel Species from a Sanitary Landfill Soil

A novel isolate belonging to the genus Streptomyces, strain SL-4^T, was isolated from soil sample collected from a sanitary landfill, New Delhi, India. The taxonomic status of this isolate was studied by polyphasic approach including morphological, physiological and chemo-taxonomic characterization. Spore chains of SL-4^T were open loops, hooks or extended spirals of wide diameter (retinaculiperti). The cell wall peptidoglycan of the isolate SL-4^T contained L,L-diaminopimelic acid, suggesting that the strain has a cell wall of chemotype-I. The polar lipid profile of the isolate was of Type II, with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The 16SrRNA gene sequence similarity between SL-4^T and its phylogenetic relatives Streptomyces atrovirens NRRLB 16357^T ({"type":"entrez-nucleotide","attrs":{"text":"DQ026672","term_id":"68137786","term_text":"DQ026672"}}DQ026672), S. albogriseolus NRRLB 1305^T ({"type":"entrez-nucleotide","attrs":{"text":"AJ494865","term_id":"21742835","term_text":"AJ494865"}}AJ494865), S viridodiastaticus NBRC 13106^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184317","term_id":"90960133","term_text":"AB184317"}}AB184317), S. caelestis NRRL 2418^T ({"type":"entrez-nucleotide","attrs":{"text":"X80824","term_id":"587528","term_text":"X80824"}}X80824), S. flavoviridis NBRC 12772^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184842","term_id":"90960663","term_text":"AB184842"}}AB184842), S. pilosus NBRC 12807^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184161","term_id":"90959977","term_text":"AB184161"}}AB184161) and S. longispororuber NBRC 13488^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184440","term_id":"90960256","term_text":"AB184440"}}AB184440) was 99.65, 99.65, 99.64, 99.23, 99.15, 99.14 and 99.13 % respectively. Subsequent DNA–DNA hybridization experiments with the test strain and its clade members showed 55.27, 44.27, 36.86, and 15.65 % relatedness between SL-4^T and its relatives S. atrovirens,S. albogriseolus, S. viridodiastaticus and S. longispororuber respectively. The genotypic and phenotypic data was analyzed to verify possibility of the isolate SL-4^T representing novel member of the genus Streptomyces, for which the name S. antibioticalis is being proposed. The type strain is SL-4^T (=CCM 7434^T=MTCC 8588^T).     

Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes

Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes.     

Prevalence,antimicrobial resistance profile,and characterization of multi-drug resistant bacteria from various infected wounds in North Egypt

Multi-drug resistant (MDR) bacteria associated with wounds are extremely escalating. This study aims to survey different wounds in Alexandria hospitals, North Egypt, to explore the prevalence and characteristics of MDR bacteria for future utilization in antibacterial wound dressing designs. Among various bacterial isolates, we determined 22 MDR bacteria could resist different classes of antibiotics. The collected samples exhibited the prevalence of mono-bacterial infections (60%), while 40% included poly-bacterial species due to previous antibiotic administration. Moreover, Gram-negative bacteria showed dominance with a ratio of 63.6%, while Gram-positive bacteria reported 36.4%. Subsequently, the five most virulent bacteria were identified following the molecular approach by 16S rRNA and physiological properties using the VITEK 2 automated system. They were deposited in GenBank as Staphylococcus haemolyticus MST1 ({"type":"entrez-nucleotide","attrs":{"text":"KY550377","term_id":"1304487819","term_text":"KY550377"}}KY550377), Pseudomonas aeruginosa MST2 ({"type":"entrez-nucleotide","attrs":{"text":"KY550378","term_id":"1304487820","term_text":"KY550378"}}KY550378), Klebsiella pneumoniae MST3 ({"type":"entrez-nucleotide","attrs":{"text":"KY550379","term_id":"1304487821","term_text":"KY550379"}}KY550379), Escherichia coli MST4 ({"type":"entrez-nucleotide","attrs":{"text":"KY550380","term_id":"1304487822","term_text":"KY550380"}}KY550380), and Escherichia coli MST5 ({"type":"entrez-nucleotide","attrs":{"text":"KY550381","term_id":"1304487823","term_text":"KY550381"}}KY550381). In terms of isolation source, S. haemolyticus MST1 was isolated from a traumatic wound, while P. aeruginosa MST2 and E. coli MST4 were procured from hernia surgical wounds, and K. pneumoniae MST3 and E. coli MST5 were obtained from diabetic foot ulcers. Antibiotic sensitivity tests exposed that K. pneumoniae MST3, E. coli MST4, and E. coli MST5 are extended-spectrum β-lactamases (ESBLs) bacteria. Moreover, S. haemolyticus MST1 belongs to the methicillin-resistant coagulase-negative staphylococcus (MRCoNS), whereas P. aeruginosa MST2 exhibited resistance to common empirical bactericidal antibiotics. Overall, the study provides new insights into the prevalent MDR bacteria in Egypt for further use as specific models in formulating antibacterial wound dressings.     

Comparative genomics of Riemerella anatipestifer reveals genetic diversity

Background

Riemerella anatipestifer is one of the most important pathogens of ducks. However, the molecular mechanisms of R. anatipestifer infection are poorly understood. In particular, the lack of genomic information from a variety of R. anatipestifer strains has proved severely limiting.

Results

In this study, we present the complete genomes of two R. anatipestifer strains, RA-CH-1 (2,309,519 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP003787","term_id":"403311769","term_text":"CP003787"}}CP003787) and RA-CH-2 (2,166,321 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP004020","term_id":"441482619","term_text":"CP004020"}}CP004020). Both strains are from isolates taken from two different sick ducks in the SiChuang province of China. A comparative genomics approach was used to identify similarities and key differences between RA-CH-1 and RA-CH-2 and the previously sequenced strain RA-GD, a clinical isolate from GuangDong, China, and ATCC11845.

Conclusion

The genomes of RA-CH-2 and RA-GD were extremely similar, while RA-CH-1 was significantly different than ATCC11845. RA-CH-1 is 140,000 bp larger than the three other strains and has 16 unique gene families. Evolutionary analysis shows that RA-CH-1 and RA-CH-2 are closed and in a branch with ATCC11845, while RA-GD is located in another branch. Additionally, the detection of several iron/heme-transport related proteins and motility mechanisms will be useful in elucidating factors important in pathogenicity. This information will allow a better understanding of the phenotype of different R. anatipestifer strains and molecular mechanisms of infection.