New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Molecular dissection of Mitogillin, a fungal ribotoxin

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are small (~17 kDa) basic ribosome-inactivating proteins (RIPs) which catalytically inactivate the large ribosomal subunits of all organisms tested so far; they act as specific ribonucleases by hydrolyzing one single phosphodiester bond in the universally conserved α-sarcin/ricin loop (SRL) of 23 - 28S rRNAs and are among the most potent inhibitors of protein synthesis known. The site of cleavage occurs between G4325 and A4326 (rat ribosome numbering), which is conserved among the large subunit rRNAs of all living species. Amino acid sequence comparison of fungal ribotoxins and guanyl/purine ribonucleases has identified domains or residues likely to be involved in ribonucleolytic activity or cleavage specificity. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing hydroxylamine-induced point mutations into a recombinant Metmature mitogillin gene (mitogillin with a Met codon at the N-terminus and no leader sequence) constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an E.coli expression vector, the proteins over-expressed and purified to homogeneity, and their activities examined by in vitro ribonucleolytic assays. These studies identified the His49Tyr, Glu95Lys, Arg120Lys, and His136Tyr mutations as having profound impact on the ribonucleolytic activities of mitogillin suggesting that these residues are key components of the active site contributing to the catalytic activities of mitogillin. Fifteen deletion mutants (each 4 to 8 amino acid deletions) in motifs of mitogillin lacking amino acid sequence homology with guanyl/purine ribonucleases were constructed by site-directed mutagenesis. Analyses of the purified mutant proteins identified the lysine rich region loop 4 (L4) region and β sheet 1-loop 1-β sheet 2 (B1-L1-B2) domain in fungal ribotoxins as contributing to ribosome-targeting and modulating the catalytic activity of the toxin; these regions show strong sequence similarity to ribosomal proteins and elongation factors. Nine mutant mitogillins that were capable of digesting polyinosinic acid [poly(I)] but unable to cleave the SRL in rabbit reticulocyte ribosomal RNA were isolated after screening E. coli encoding partially degenerate oligonucleotide sequences in the lysine rich L4 region (Lys106-Phe107-Asp108-Ser 109-Lys110-Lys111-Pro112-Lys 113) of the mitogillin gene. Further investigation by site-directed mutagenesis indicated that Lys111 plays an important role in substrate recognition; a Lys111Gln change markedly reduced the ability of mitogillin to specifically cleave the rabbit ribosomal RNA or a 35-mer oligoribonucleotide mimicking the SRL. In addition, a variant mitogillin with an Asn7Ala substitution in the B1-L1-B2 domain exhibited elevated ribonucleolytic activity and reduced substrate specificity, suggesting the involvement of this domain in substrate selection. This mutational study of mitogillin taken together with the recently published X-ray structure of restrictocin (a close relative of mitogillin) supports the hypothesis that the specific cleavage properties of ribotoxins are the result of natural genetic engineering of T1- like ribonucleases in which the ribosomal targeting elements of ribosome-associated proteins were inserted into nonessential regions of the nuclease protein.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Construction of a multiplex promoter reporter platform to monitor Staphylococcus aureus virulence gene expression and the identification of usnic acid as a potent suppressor of psm gene expression

As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm

Autophagy-Dependent Reactivation of Epstein-Barr Virus Lytic Cycle and Combinatorial Effects of Autophagy-Dependent and Independent Lytic Inducers in Nasopharyngeal Carcinoma

Autophagy, a conserved cellular mechanism, is manipulated by a number of viruses for different purposes. We previously demonstrated that an iron-chelator-like small compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle by activating the ERK1/2-autophagy axis in epithelial cancers. Here, we aim to identify the specific stage of autophagy required for EBV lytic reactivation, determine the autophagy dependency of EBV lytic inducers including histone deacetylase inhibitor (HDACi) and C7/iron chelators, for EBV lytic reactivation and measure the combinatorial effects of these types of lytic inducers in nasopharyngeal carcinoma (NPC). Inhibition of autophagy initiation by 3-MA and autolysosome formation by chloroquine demonstrated that only autophagy initiation is required for EBV lytic reactivation. Gene knockdown of various autophagic proteins such as beclin-1, ATG5, ATG12, ATG7, LC3B, ATG10, ATG3 and Rab9, revealed the importance of ATG5 in EBV lytic reactivation. 3-MA could only abrogate lytic cycle induction by C7/iron chelators but not by HDACi, providing evidence for autophagy-dependent and independent mechanisms in EBV lytic reactivation. Finally, the combination of C7 and SAHA at their corresponding reactivation kinetics enhanced EBV lytic reactivation. These findings render new insights in the mechanisms of EBV lytic cycle reactivation and stimulate a rational design of combination drug therapy against EBV-associated cancers

In vitro and in vivo activity of a novel antifungal small molecule against Candida infections

Candida is the most common fungal pathogen of humans worldwide and has become a major clinical problem because of the growing number of immunocompromised patients, who are susceptible to infection. Moreover, the number of available antifungals is limited, and antifungal-resistant Candida strains are emerging. New and effective antifungals are therefore urgently needed. Here, we discovered a small molecule with activity against Candida spp. both in vitro and in vivo. We screened a library of 50,240 small molecules for inhibitors of yeast-to-hypha transition, a major virulence attribute of Candida albicans. This screening identified 20 active compounds. Further examination of the in vitro antifungal and anti-biofilm properties of these compounds, using a range of Candida spp., led to the discovery of SM21, a highly potent antifungal molecule (minimum inhibitory concentration (MIC) 0.2 - 1.6 mu g/ml). In vitro, SM21 was toxic to fungi but not to various human cell lines or bacterial species and was active against Candida isolates that are resistant to existing antifungal agents. Moreover, SM21 was relatively more effective against biofilms of Candida spp. than the current antifungal agents. In vivo, SM21 prevented the death of mice in a systemic candidiasis model and was also more effective than the common antifungal nystatin at reducing the extent of tongue lesions in a mouse model of oral candidiasis. Propidium iodide uptake assay showed that SM21 affected the integrity of the cell membrane. Taken together, our results indicate that SM21 has the potential to be developed as a novel antifungal agent for clinical use

Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance

Silver (Ag) has been used as an antimicrobial agent since a long time, but its molecular mechanism of action was not elucidated due to technical challenges. Here, the authors develop a mass spectrometric approach to identify the Ag-proteome in Staphylococcus aureus, and capture a molecular snapshot of the dynamic bactericidal mode of action of Ag through targeting multiple biological pathways

Autophagy-Dependent Reactivation of Epstein-Barr Virus Lytic Cycle and Combinatorial Effects of Autophagy-Dependent and Independent Lytic Inducers in Nasopharyngeal Carcinoma

Autophagy, a conserved cellular mechanism, is manipulated by a number of viruses for different purposes. We previously demonstrated that an iron-chelator-like small compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle by activating the ERK1/2-autophagy axis in epithelial cancers. Here, we aim to identify the specific stage of autophagy required for EBV lytic reactivation, determine the autophagy dependency of EBV lytic inducers including histone deacetylase inhibitor (HDACi) and C7/iron chelators, for EBV lytic reactivation and measure the combinatorial effects of these types of lytic inducers in nasopharyngeal carcinoma (NPC). Inhibition of autophagy initiation by 3-MA and autolysosome formation by chloroquine demonstrated that only autophagy initiation is required for EBV lytic reactivation. Gene knockdown of various autophagic proteins such as beclin-1, ATG5, ATG12, ATG7, LC3B, ATG10, ATG3 and Rab9, revealed the importance of ATG5 in EBV lytic reactivation. 3-MA could only abrogate lytic cycle induction by C7/iron chelators but not by HDACi, providing evidence for autophagy-dependent and independent mechanisms in EBV lytic reactivation. Finally, the combination of C7 and SAHA at their corresponding reactivation kinetics enhanced EBV lytic reactivation. These findings render new insights in the mechanisms of EBV lytic cycle reactivation and stimulate a rational design of combination drug therapy against EBV-associated cancers