Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events.

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

Actinoplanes oblitus sp. nov., producing the glycopeptide antibiotic A477.

In 1973, Eli Lilly and Company described the filamentous actinomycete producing the glycopeptide antibiotic A477 as an Actinoplanes species on the basis of its morphological and physiological features and deposited it as NRRL 3884T. In this paper, we report that the phylogenetic analysis based on the 16S rRNA gene sequence and the whole genome phylogenomic study indicate that NRRL 3884T forms a distinct monophyletic line within the genus Actinoplanes, being most closely related to Actinoplanes octamycinicus NBRC 14524T [99.6 % 16S rRNA gene similarity, 89.4 % average nucleotide identity (ANI), 46.0 % digital DNA-DNA hybridization (dDDH)] and Actinoplanes ianthinogenes NBRC 13996T (98.8 % 16S rRNA gene similarity, 89.0 % ANI, 47.0 % dDDH). NRRL 3884T forms an extensively branched, non-fragmented vegetative mycelium; either sterile aerial hyphae or regular subglobose sporangia are formed depending on cultivation conditions. The cell wall contains meso-2,6-diaminopimelic acid and 2,6-diamino-3-hydroxypimelic acid and the diagnostic sugars are glucose, mannose and ribose with a minor amount of rhamnose. The predominant menaquinone (MK) is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H8). Mycolic acids are absent. The diagnostic phospholipids are diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids are anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0, with moderate amounts of anteiso-C15 : 0 and iso-C17 : 0. The genomic G+C content is 71.5 mol%. Significant differences in the genomic, morphological, chemotaxonomic and biochemical data between NRRL 3884T and the two most closely related Actinoplanes type strains clearly demonstrate that NRRL 3884T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes oblitus sp. nov. is proposed. The type strain is NRRL 3884T (=DSM 116196T).

Laboratory scale cultivation of Salinispora tropica in shake flasks and mechanically stirred bioreactors.

OBJECTIVE: Marine actinomycetes from the genus Salinispora have an unexploited biotechnological potential. To accurately estimate their application potential however, data on their cultivation, including biomass growth kinetics, are needed but only incomplete information is currently available. RESULTS: This work provides some insight into the effect of temperature, salinity, nitrogen source, glucose concentration and oxygen supply on growth rate, biomass productivity and yield of Salinispora tropica CBN-440T. The experiments were carried out in unbaffled shake flasks and agitated laboratory-scale bioreactors. The results show that the optimum growth temperature lies within the range 28-30 °C, salinity is close to sea water and the initial glucose concentration is around 10 g/L. Among tested nitrogen sources, yeast extract and soy peptone proved to be the most suitable. The change from unbaffled to baffled flasks increased the volumetric oxygen transfer coefficient (kLa) as did the use of agitated bioreactors. The highest specific growth rate (0.0986 h-1) and biomass productivity (1.11 g/L/day) were obtained at kLa = 28.3 h-1. A further increase in kLa was achieved by increasing stirrer speed, but this led to a deterioration in kinetic parameters. CONCLUSIONS: Improvement of S. tropica biomass growth kinetics of was achieved mainly by identifying the most suitable nitrogen sources and optimizing kLa in baffled flasks and agitated bioreactors.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.

Classification of Actinoplanes sp. ATCC 33076, an actinomycete that produces the glycolipodepsipeptide antibiotic ramoplanin, as Actinoplanes ramoplaninifer sp. nov.

Strain ATCC 33076, which produces the antibiotic ramoplanin, was isolated from a soil sample collected in India, and it was classified as a member of the genus Actinoplanes on the basis of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain forms a distinct clade within the genus Actinoplanes, and it is most closely related to Actinoplanes deccanensis IFO 13994T (98.71 % similarity) and Actinoplanes atraurantiacus Y16T (98.33 %). The strain forms an extensively branched substrate mycelium; the sporangia are formed very scantily and are globose with irregular surface. Spores are oval and motile. The cell wall contains meso-diaminopimelic acid and the diagnostic sugars are xylose and arabinose. The predominant menaquinone is MK-9(H6), with minor amounts of MK-9(H4) and MK-9(H2). Mycolic acids are absent. The diagnostic phospholipids are phosphatidylethanolamine, hydroxyphosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids are anteiso-C17 : 0 and iso-C16 : 0, followed by iso-C15 : 0 and moderate amounts of anteiso-C15 : 0, iso-C17 : 0 and C18 : 1ω9c. The genomic DNA G+C content is 71.4 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 33076 and closely related type strains, clearly demonstrated that strain ATCC 33076 represents a novel species of the genus Actinoplanes, for which the name Actinoplanes ramoplaninifer sp. nov. is proposed. The type strain is ATCC 33076T (=DSM 105064T=NRRL B-65484T).

Classification of Nonomuraea sp. ATCC 39727, an actinomycete that produces the glycopeptide antibiotic A40926, as Nonomuraea gerenzanensis sp. nov.

Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic dalbavancin), was isolated from a soil sample collected in India, and it was originally classified as a member of the genus Actinomadura on the base of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicates that the strain forms a distinct clade within the genus Nonomuraea, and it is most closely related to Nonomuraea angiospora DSM 43173T (98.72 % similarity) and Nonomuraea jabiensis A4036T (98.69 %). The strain forms an extensively branched substrate mycelium and aerial hyphae that form spiral chains of spores with ridged surfaces. The cell wall contains meso-diaminopimelic acid and the whole-cell sugars are glucose, ribose, galactose, mannose and madurose (madurose as the diagnostic sugar). The N-acyl type of muramic acid is acetyl. The predominant menaquinone is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H0). The polar-lipid profile includes diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylmethylethanolamine, phosphatidylinositol and a series of uncharacterized phospholipids, glycolipids and phosphoglycolipids. The major cellular fatty acids are iso-C16 : 0 and 10-methyl C17 : 0. The genomic DNA G+C content is 71.2 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 39727 and closely related type strains, clearly demonstrated that strain ATCC 39727 represents a novel species of the genus Nonomuraea, for which the name Nonomuraea gerenzanensis sp. nov. is proposed. The type strain is ATCC 39727T ( = DSM 100948T).

Relationship between glycopeptide production and resistance in the actinomycete Nonomuraea sp. ATCC 39727.

Glycopeptides and ß-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the ß-lactam-sensitive D,D-peptidase/D,D-carboxypeptidase encoded by vanYn, the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanYn gene dosage and expressed vanHatAatXat from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanYn, complementing a vanYn mutant, or duplicating vanYn had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYn activity. The heterologous expression of vanHatAatXat increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanYn-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.

High expression levels of the B cell chemoattractant CXCL13 in rheumatoid synovium are a marker of severe disease.

OBJECTIVE: The B cell chemoattractant chemokine ligand 13 (CXCL13) is emerging as a new biochemical marker in RA. This study was undertaken to dissect the relationship between CXCL13 expression levels in the synovium and clinico-pathological variables relevant to RA pathogenesis and outcome. METHODS: Synovial tissues from 71 RA patients were evaluated by immunohistochemistry. Thirty paired samples were used for comparative gene expression analysis by quantitative real-time PCR. CXCL13 levels were analysed in relation to cellular, molecular and clinical features of inflammation, lymphocyte activation and joint damage. RESULTS: In patients with early disease (<12 months duration), CXCL13 expression correlated significantly with synovial markers of local disease activity and systemic inflammation. Such correlation was less evident in established RA. Notably, the association with lymphocyte infiltration and with expression of B/T cell-related activation and proliferation genes, such as activation-induced cytidine deaminase, IFN-γ and IL-2, remained highly significant independent of disease duration and local disease activity. Patients featuring the highest levels of CXCL13 were more frequently ACPA positive and IgG ACPA titres were increased in the high CXCL13 expression group. Furthermore, the frequency of erosive disease on radiographs was significantly higher in the upper tertile of CXCL13 expression (P = 0.01 with adjustment for disease duration and ACPA). Accordingly, synovial CXCL13 and the local receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) ratio significantly co-varied (ρ = 0.52, P < 0.01), independent of the level of local inflammation. CONCLUSION: Synovial CXCL13 appears to be a marker of a more severe pattern of RA disease, characterized by increased lymphocyte activation and bone remodelling beyond the level of conventional markers of inflammation.

The Impact of Heterologous Regulatory Genes from Lipodepsipeptide Biosynthetic Gene Clusters on the Production of Teicoplanin and A40926.

StrR-like pathway-specific transcriptional regulators (PSRs) function as activators in the biosynthesis of various antibiotics, including glycopeptides (GPAs), aminoglycosides, aminocoumarins, and ramoplanin-like lipodepsipeptides (LDPs). In particular, the roles of StrR-like PSRs have been previously investigated in the biosynthesis of streptomycin, novobiocin, GPAs like balhimycin, teicoplanin, and A40926, as well as LDP enduracidin. In the current study, we focused on StrR-like PSRs from the ramoplanin biosynthetic gene cluster (BGC) in Actinoplanes ramoplaninifer ATCC 33076 (Ramo5) and the chersinamycin BGC in Micromonospora chersina DSM 44151 (Chers28). Through the analysis of the amino acid sequences of Ramo5 and Chers28, we discovered that these proteins are phylogenetically distant from other experimentally investigated StrR PSRs, although all StrR-like PSRs found in BGCs for different antibiotics share a conserved secondary structure. To investigate whether Ramo5 and Chers28, given their phylogenetic positions, might influence the biosynthesis of other antibiotic pathways governed by StrR-like PSRs, the corresponding genes (ramo5 and chers28) were heterologously expressed in Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727, which produce the clinically-relevant GPAs teicoplanin and A40926, respectively. Recombinant strains of NRRL B-16726 and ATCC 39727 expressing chers28 exhibited improved antibiotic production, although the expression of ramo5 did not yield the same effect. These results demonstrate that some StrR-like PSRs can "cross-talk" between distant biosynthetic pathways and might be utilized as tools for the activation of silent BGCs regulated by StrR-like PSRs.

Streptomyces spp. as efficient expression system for a D,D-peptidase/D,D-carboxypeptidase involved in glycopeptide antibiotic resistance.

BACKGROUND: VanYn, encoded by the dbv7 gene (also known as vanYn) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727. This filamentous actinomycete is an uncommon microorganism, difficult-to-handle but biotechnologically valuable since it produces the glycopeptide antibiotic A40926, which is the precursor of the second-generation dalbavancin in phase III of clinical development. In order to investigate VanYn role in glycopeptide resistance in the producer actinomycete an appropriate host-vector expression system is required. RESULTS: The cloning strategy of vanYn gene (G-C ratio 73.3%) in the expression vector pIJ86 yielded a recombinant protein with a tag encoding for a histidine hexamer added at the C-terminus (C-His6-vanYn) or at the N-terminus (N-His6-vanYn). These plasmids were used to transform three Streptomyces spp., which are genetically-treatable high G-C content Gram-positive bacteria taxonomically related to the homologous producer Nonomuraea sp.. Highest yield of protein expression and purification (12 mg of protein per liter of culture at 3 L bioreactor-scale) was achieved in Streptomyces venezuelae ATCC 10595, that is a fast growing streptomyces susceptible to glycopeptides. VanYn is a transmembrane protein which was easily detached and recovered from the cell wall fraction. Purified C-His6-VanYn showed d,d-carboxypeptidase and d,d-dipeptidase activities on synthetic analogs of bacterial peptidoglycan (PG) precursors. C-His6-VanYn over-expression conferred glycopeptide resistance to S. venezuelae. On the contrary, the addition of His6-tag at the N-terminus of the protein abolished its biological activity either in vitro or in vivo assays. CONCLUSIONS: Heterologous expression of vanYn from Nonomuraea sp. ATCC 39727 in S. venezuelae was successfully achieved and conferred the host an increased level of glycopeptide resistance. Cellular localization of recombinant VanYn together with its enzymatic activity as a d,d-peptidase/d,d-carboxypeptidase agree with its role in removing the last d-Ala from the pentapeptide PG precursors and reprogramming cell wall biosynthesis, as previously reported in glycopeptide resistant pathogens.

Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins.

BACKGROUND: A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity. RESULTS: In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full)--immunodominant antigens of Mycobacterium tuberculosis--were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone. CONCLUSIONS: We reached the goal of purifying large quantities of soluble antigens effective in generating immunological response against M. tuberculosis by a robust, controlled, scalable and economically feasible production process.

Cross-Talking of Pathway-Specific Regulators in Glycopeptide Antibiotics (Teicoplanin and A40926) Production.

Teicoplanin and A40926 (natural precursor of dalbavancin) are clinically relevant glycopeptide antibiotics (GPAs) produced by Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727. Their biosynthetic enzymes are coded within large biosynthetic gene clusters (BGCs), named tei for teicoplanin and dbv for A40926, whose expression is strictly regulated by pathway-specific transcriptional regulators (PSRs), coded by cluster-situated regulatory genes (CSRGs). Herein, we investigated the "cross-talk" between the CSRGs from tei and dbv, through the analysis of GPA production levels in A. teichomyceticus and N. gerenzanensis strains, with knockouts of CSRGs cross-complemented by the expression of heterologous CSRGs. We demonstrated that Tei15* and Dbv4 StrR-like PSRs, although orthologous, were not completely interchangeable: tei15* and dbv4 were only partially able or unable to cross-complement N. gerenzanensis knocked out in dbv4 and A. teichomyceticus knocked out in tei15*, implying that the DNA-binding properties of these PSRs are more different in vivo than it was believed before. At the same time, the unrelated LuxR-like PSRs Tei16* and Dbv3 were able to cross-complement corresponding N. gerenzanensis knocked out in dbv3 and A. teichomyceticus knocked out in tei16*. Moreover, the heterologous expression of dbv3 in A. teichomyceticus led to a significant increase in teicoplanin production. Although the molecular background of these events merits further investigations, our results contribute to a deeper understanding of GPA biosynthesis regulation and offer novel biotechnological tools to improve their production.

A novel promising laccase from the psychrotolerant and halotolerant Antarctic marine Halomonas sp. M68 strain.

Microbial communities inhabiting the Antarctic Ocean show psychrophilic and halophilic adaptations conferring interesting properties to the enzymes they produce, which could be exploited in biotechnology and bioremediation processes. Use of cold- and salt-tolerant enzymes allows to limit costs, reduce contaminations, and minimize pretreatment steps. Here, we report on the screening of 186 morphologically diverse microorganisms isolated from marine biofilms and water samples collected in Terra Nova Bay (Ross Sea, Antarctica) for the identification of new laccase activities. After primary screening, 13.4 and 10.8% of the isolates were identified for the ability to oxidize 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the dye azure B, respectively. Amongst them, the marine Halomonas sp. strain M68 showed the highest activity. Production of its laccase-like activity increased six-fold when copper was added to culture medium. Enzymatic activity-guided separation coupled with mass spectrometry identified this intracellular laccase-like protein (named Ant laccase) as belonging to the copper resistance system multicopper oxidase family. Ant laccase oxidized ABTS and 2,6-dimethoxy phenol, working better at acidic pHs The enzyme showed a good thermostability, with optimal temperature in the 40-50°C range and maintaining more than 40% of its maximal activity even at 10°C. Furthermore, Ant laccase was salt- and organic solvent-tolerant, paving the way for its use in harsh conditions. To our knowledge, this is the first report concerning the characterization of a thermo- and halo-tolerant laccase isolated from a marine Antarctic bacterium.

Occurrence of vanHAX and Related Genes beyond the Actinobacteria Phylum.

Clinically relevant glycopeptide antibiotics remain among the most successful classes of natural antibacterials. This success, however, is endangered by the spread of glycopeptide resistance genes, also known as van genes. Thus, it is important to trace and comprehend possible routes of van gene dissemination. In the current work, we present a comprehensive bioinformatic analysis aimed at mapping the occurrence of van genes beyond the Actinobacteria phylum-the most likely natural reservoir of van genes. We show that two additional classes of Gram-positive bacteria, Erysipelotrichia and Ktedonobacteria, as well as one class of Gram-negative bacteria, Anaerolineae, carry van genes. Additionally, we demonstrate that various new genera belonging to the classes Clostridia and Bacilli also carry van genes. The majority of discovered van loci are co-localized with MGE-related genes of various types. Finally, we propose a phylogeny-based scenario for the spread of van genes, unraveling a network of consequential horizontal gene transfer events linking the phylum Actinobacteria with the five other bacterial classes carrying van genes.

Risk of prostate cancer is not associated with levels of C-reactive protein and other commonly used markers of inflammation.

Most population-based studies studied the association between inflammation and prostate cancer (PCa) by assessing C-reactive protein (CRP). As these findings have shown inconsistent results, we aimed to also study different markers that have been commonly taken as indications of inflammation. A cohort based on four groups of men (n = 34,891), according to age at cohort entry (45, 55, 65 and 75 years), with measurements of glucose, triglycerides, total cholesterol, haptoglobin, albumin, hemoglobin and leukocytes were selected from the Apolipoprotein Mortality Risk database. A total of 17,937 men had measurements of non-high-sensitive CRP. Multivariate Cox proportional hazard models were used to analyze associations between inflammatory markers and PCa. A total of 49 of 12,063 men developed PCa in the age 45 group, whereas 207 of 9,940, 472 of 8,266 and 276 of 3,618 were diagnosed in the age 55, 65 and 75 groups, respectively. Mean follow-up time was 7.5 years (SD: 3.9). No markers showed an association with PCa risk, nor was there a trend by quartiles or an indication for different PCa risks by strata of hypercholesterolemia, hyperglycemia and hypertriglyceridemia status. The studied markers were not found to be associated with PCa risk. These null findings might be due to methodological issues; however, it is unlikely that strong and long-lasting associations between inflammation and PCa risk were missed as this was a large database with long follow-up. This indicates need for international consensus on appropriate inflammatory markers in the context of cancer that may be practically applied in large studies.

Low levels of apolipoprotein A-I and HDL are associated with risk of prostate cancer in the Swedish AMORIS study.

BACKGROUND: A detailed analysis of lipid profiles, using apolipoproteins, has not yet been conducted for prostate cancer (PCa). Since several etiological pathways have been proposed for PCa and lipids, we aimed to study this in a large Swedish cohort with 1,469 primary prostate cancers. METHODS: A cohort (n = 69,735) of all men aged 35 years or older, whose levels of triglycerides (TG) (mmol/L), total cholesterol (mmol/L), glucose (mmol/L), LDL (mmol/L), HDL (mmol/L), apoB (g/L), and apoA-I (g/L) were measured at baseline, was selected from the Apolipoprotein MOrtality RISk (AMORIS) database. About 2,008 men developed PCa. Multivariate Cox proportional hazard models were used to analyze associations between lipid components and PCa. RESULTS: ApoA-I and HDL were inversely associated with PCa risk (e.g., HR for HDL: 0.93 (95% CI: 0.81-1.07), 0.88 (0.76-1.01), 0.81 (0.70-0.94), for second, third, and fourth quartiles compared with the first quartile; with p for trend: 0.004; HR for apoA-I: 1.00 (0.88-1.13), 0.93 (0.82-1.05), 0.88 (0.77-0.99),), for second, third, and fourth quartiles compared with the first quartile; with p for trend: 0.022). ApoB, LDL, and non-HDL were not associated with PCa risk. CONCLUSIONS: Our results show that low HDL and ApoA-I as well as increased lipid ratios are related to increased PCa risk. Experimental studies are required to tease out the underlying biological mechanisms linking these lipid components to PCa.

Genomic Insights into the Distribution and Phylogeny of Glycopeptide Resistance Determinants within the Actinobacteria Phylum.

The spread of antimicrobial resistance (AMR) creates a challenge for global health security, rendering many previously successful classes of antibiotics useless. Unfortunately, this also includes glycopeptide antibiotics (GPAs), such as vancomycin and teicoplanin, which are currently being considered last-resort drugs. Emerging resistance towards GPAs risks limiting the clinical use of this class of antibiotics-our ultimate line of defense against multidrug-resistant (MDR) Gram-positive pathogens. But where does this resistance come from? It is widely recognized that the GPA resistance determinants-van genes-might have originated from GPA producers, such as soil-dwelling Gram-positive actinobacteria, that use them for self-protection. In the current work, we present a comprehensive bioinformatics study on the distribution and phylogeny of GPA resistance determinants within the Actinobacteria phylum. Interestingly, van-like genes (vlgs) were found distributed in different arrangements not only among GPA-producing actinobacteria but also in the non-producers: more than 10% of the screened actinobacterial genomes contained one or multiple vlgs, while less than 1% encoded for a biosynthetic gene cluster (BGC). By phylogenetic reconstructions, our results highlight the co-evolution of the different vlgs, indicating that the most diffused are the ones coding for putative VanY carboxypeptidases, which can be found alone in the genomes or associated with a vanS/R regulatory pair.

Genomic-Led Discovery of a Novel Glycopeptide Antibiotic by Nonomuraea coxensis DSM 45129.

Glycopeptide antibiotics (GPAs) are last defense line drugs against multidrug-resistant Gram-positive pathogens. Natural GPAs teicoplanin and vancomycin, as well as semisynthetic oritavancin, telavancin, and dalbavancin, are currently approved for clinical use. Although these antibiotics remain efficient, emergence of novel GPA-resistant pathogens is a question of time. Therefore, it is important to investigate the natural variety of GPAs coming from so-called "rare" actinobacteria. Herein we describe a novel GPA producer-Nonomuraea coxensis DSM 45129. Its de novo sequenced and completely assembled genome harbors a biosynthetic gene cluster (BGC) similar to the dbv BGC of A40926, the natural precursor to dalbavancin. The strain produces a novel GPA, which we propose is an A40926 analogue lacking the carboxyl group on the N-acylglucosamine moiety. This structural difference correlates with the absence of dbv29-coding for an enzyme responsible for the oxidation of the N-acylglucosamine moiety. Introduction of dbv29 into N. coxensis led to A40926 production in this strain. Finally, we successfully applied dbv3 and dbv4 heterologous transcriptional regulators to trigger and improve A50926 production in N. coxensis, making them prospective tools for screening other Nonomuraea spp. for GPA production. Our work highlights genus Nonomuraea as a still untapped source of novel GPAs.

Novel mechanism of glycopeptide resistance in the A40926 producer Nonomuraea sp. ATCC 39727.

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d-alanyl-d-alanine of lipid II with d-alanyl-d-lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide A40926, which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward A40926 during vegetative growth was 4 microg/ml, but this increased to ca. 20 microg/ml during A40926 production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for A40926 production contains a gene, vanY (ORF7), potentially encoding an enzyme capable of removing the terminal d-Ala residue of pentapeptide peptidoglycan precursors. Analysis of UDP-linked peptidoglycan precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala and only traces of the pentapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala-d-Ala. This suggested a novel mechanism of glycopeptide resistance in Nonomuraea sp. ATCC 39727 that was based on the d,d-carboxypeptidase activity of vanY. Consistent with this, a vanY-null mutant of Nonomuraea sp. ATCC 39727 demonstrated a reduced level of glycopeptide resistance, without affecting A40926 productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae, resulted in higher levels of glycopeptide resistance.