Bioprospecting from marine sediments of New Brunswick, Canada : exploring the relationship between total bacterial diversity and actinobacteria diversity

Actinomycetes are an important resource for the discovery of natural products with therapeutic properties. Bioprospecting for actinomycetes typically proceeds without a priori knowledge of the bacterial diversity present in sampled habitats. In this study, we endeavored to determine if overall bacterial diversity in marine sediments, as determined by 16S rDNA amplicon pyrosequencing, could be correlated with culturable actinomycete diversity, and thus serve as a powerful tool in guiding future bioprospecting efforts. Overall bacterial diversity was investigated in eight marine sediments from four sites in New Brunswick, Canada, resulting in over 44,000 high quality sequences (x = 5610 per sample). Analysis revealed all sites exhibited significant diversity (H' = 5.4 to 6.7). Furthermore, statistical analysis of species level bacterial communities (D = 0.03) indicated community composition varied according to site and was strongly influenced by sediment physiochemical composition. In contrast, cultured actinomycetes (n = 466, 98.3% Streptomyces) were ubiquitously distributed among all sites and distribution was not influenced by sediment composition, suggesting that the biogeography of culturable actinomycetes does not correlate with overall bacterial diversity in the samples examined. These actinomycetes provide a resource for future secondary metabolite discovery, as exemplified by the antimicrobial activity observed from preliminary investigation

Discovery of a New Natural Product and a Deactivation of a Quorum Sensing System by Culturing a “Producer” Bacterium With a Heat-Killed “Inducer” Culture

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of “inducer” microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS

Comparison of two total RNA extraction protocols using the marine gorgonian coral Pseudopterogorgia elisabethae and its symbiont Symbiodinium sp.

Marine invertebrates such as soft corals are important sources of secondary metabolites with promising biomedical applications and commercial value. RNA isolation in conjunction with reverse-transcriptase polymerase chain reaction (RT-PCR) are valuable tools utilized to study the molecular elements involved in secondary metabolite production and functional genomics. Two total RNA extraction protocols were compared using fresh tissue and flash frozen preparations from the coral Pseudopterogorgia elisabethae and from its symbiont Symbiodinium sp. isolated using RNeasy minicolumns (Qiagen\uae) and Trizol reagent (Invitrogen\uae). In general, higher yields were obtained by using Trizol reagent when compared to RNeasy. No significant differences were observed in RNA yield when live or flash frozen tissue was used. However, flash frozen holobiont tissue isolated by Trizol resulted in the highest RNA yield of all preparations analyzed. To conclude, both protocols are suitable for RNA isolation. Trizol is recommended if higher yields are the primary concern, but RNeasy is recommended if time is an issue

A microbial community analysis of the octocoral Eunicea fusca

Abstract While there is a significant and growing body of knowledge describing the microbial communities of marine invertebrates such as sponges, there are very few such studies focused on octocorals. The octocoral Eunicea fusca is common on reefs in various regions of the Caribbean and has been the subject of natural product investigations. As part of an effort to describe the microbial community associated with octocorals, a culture-independent analysis of the bacterial community of E. fusca was conducted. Specifically, a 16S rDNA clone library analysis was performed to provide baseline data. A total of 40 bacteria members from 11 groups were found. In general, Proteobacteria were the dominant group with a total of 24 species and \u3b1-Proteobacteria represented the highest percentage of bacteria associated with E. fusca (27.5%). Other prominent groups observed were Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, \u3b4-Proteobacteria, Lentisphaerae and Nitrospirae. This is the first analysis of bacterial populations associated with the gorgonian E. fusca

Suppression Subtractive Hybridization PCR Isolation of cDNAs from a Caribbean Soft Coral

Transcriptomic studies of marine organisms are still in their infancy. A partial, subtracted expressed sequence tag (EST) library of the Caribbean octocoral Erythropodium caribaeorum and the sea fan Gorgonia ventalina has been analyzed in order to find novel genes or differences in gene expression related to potential secondary metabolite production or symbioses. This approach entails enrichment for potential non- housekeeping genes using the suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) method. More than 500 expressed sequence tags (ESTs) were generated after cloning SSH products, which yielded at least 53 orthologous groups of proteins (COGs) and Pfam clusters, including transcription factors (Drosophila Big Brother), catalases, reverse transcriptases, ferritins and various hypothetical protein sequences. A total of 591 EST sequences were deposited into GenBank [dbEST: FL512138 - FL512331, and GH611838]. The results represent proof of concept for enrichment of unique transcripts over housekeeping genes, such as actin or ribosomal genes, which comprised approximately 17% of the total dataset. Due to the gene and sequence diversity of some ESTs, such sequences can find utility as molecular markers in current and future studies of this species and other soft coral biogeography, chemical ecology, phylogenetics, and evolution

Comparative Microbiome and Metabolome Analyses of the Marine Tunicate Ciona intestinalis from Native and Invaded Habitats

Massive fouling by the invasive ascidian Ciona intestinalis in Prince Edward Island (PEI, Canada) has been causing devastating losses to the local blue mussel farms. In order to gain first insights into so far unexplored factors that may contribute to the invasiveness of C. intestinalis in PEI, we undertook comparative microbiome and metabolome studies on specific tissues from C. intestinalis populations collected in invaded (PEI) and native regions (Helgoland and Kiel, Germany). Microbial community analyses and untargeted metabolomics revealed clear location- and tissue-specific patterns showing that biogeography and the sampled tissue shape the microbiome and metabolome of C. intestinalis. Moreover, we observed higher microbial and chemical diversity in C. intestinalis from PEI than in the native populations. Bacterial OTUs specific to C. intestinalis from PEI included Cyanobacteria (e.g., Leptolyngbya sp.) and Rhodobacteraceae (e.g., Roseobacter sp.), while populations from native sampling sites showed higher abundances of e.g., Firmicutes (Helgoland) and Epsilonproteobacteria (Kiel). Altogether 121 abundant metabolites were putatively annotated in the global ascidian metabolome, of which 18 were only detected in the invasive PEI population (e.g., polyketides and terpenoids), while six (e.g., sphingolipids) or none were exclusive to the native specimens from Helgoland and Kiel, respectively. Some identified bacteria and metabolites reportedly possess bioactive properties (e.g., antifouling and antibiotic) that may contribute to the overall fitness of C. intestinalis. Hence, this first study provides a basis for future studies on factors underlying the global invasiveness of Ciona species

Synthesis of (+)-(R)-Tiruchanduramine

The absolute stereochemistry of the marine alkaloid (+)-(R)-tiruchanduramine was established via a convergent total synthesis in six steps and 15.5% overall yield from Fmoc-D-Dab(Boc)-OH

Gaps and barriers in health-care provision for co-morbid diabetes and chronic kidney disease: a cross-sectional study

Patients with diabetes and chronic kidney disease (CKD) are a complex subset of the growing number of patients with diabetes, due to multi-morbidity. Gaps between recommended and received care for diabetes and chronic kidney disease (CKD) are evident despite promulgation of guidelines. Here, we document gaps in tertiary health-care, and the commonest patient-reported barriers to health-care, before exploring the association between these gaps and barriers.This cross-sectional study recruited patients with diabetes and CKD (eGFR < 60 mL/min/1.73 m2) across 4 large hospitals. For each patient, questionnaires were completed examining clinical data, recommended care, and patient-reported barriers limiting health-care. Descriptive statistics, subgroup analyses by CKD stage and hospital, and analyses examining the relationship between health-care gaps and barriers were performed.308 patients, of mean age 66.9 (SD 11.0) years, and mostly male (69.5%) and having type 2 diabetes (88.0%), participated. 49.1% had stage 3, 24.7% stage 4 and 26.3% stage 5 CKD. Gaps between recommended versus received care were evident: 31.9% of patients had an HbA1c ≥ 8%, and 39.3% had a measured blood pressure ≥ 140/90 mmHg. The commonest barriers were poor continuity of care (49.3%), inadequate understanding/education about CKD (43.5%), and feeling unwell (42.6%). However, barriers associated with a failure to receive items of recommended care were inadequate support from family and friends, conflicting advice from and poor communication amongst specialists, the effect of co-morbidities on self-management and feeling unmotivated (all p < 0.05).Barriers to health-care varied across CKD stages and hospitals. Barriers associated with a deviation from recommended care were different for different items of care, suggesting that specific interventions targeting each item of care are required.C. Lo, H. Teede, G. Fulcher, M. Gallagher, P. G. Kerr, S. Ranasinha, G. Russell, R. Walker and S. Zounga

Dark Matter Direct Detection with Non-Maxwellian Velocity Structure

The velocity distribution function of dark matter particles is expected to show significant departures from a Maxwell-Boltzmann distribution. This can have profound effects on the predicted dark matter - nucleon scattering rates in direct detection experiments, especially for dark matter models in which the scattering is sensitive to the high velocity tail of the distribution, such as inelastic dark matter (iDM) or light (few GeV) dark matter (LDM), and for experiments that require high energy recoil events, such as many directionally sensitive experiments. Here we determine the velocity distribution functions from two of the highest resolution numerical simulations of Galactic dark matter structure (Via Lactea II and GHALO), and study the effects for these scenarios. For directional detection, we find that the observed departures from Maxwell-Boltzmann increase the contrast of the signal and change the typical direction of incoming DM particles. For iDM, the expected signals at direct detection experiments are changed dramatically: the annual modulation can be enhanced by more than a factor two, and the relative rates of DAMA compared to CDMS can change by an order of magnitude, while those compared to CRESST can change by a factor of two. The spectrum of the signal can also change dramatically, with many features arising due to substructure. For LDM the spectral effects are smaller, but changes do arise that improve the compatibility with existing experiments. We find that the phase of the modulation can depend upon energy, which would help discriminate against background should it be found.Comment: 34 pages, 16 figures, submitted to JCAP. Tables of g(v_min), the integral of f(v)/v from v_min to infinity, derived from our simulations, are available for download at http://astro.berkeley.edu/~mqk/dmdd