Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk

Metagenomic Analysis of RNA Viruses in a Fresh Water Lake

Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic) and possibly be involved in the spread of emerging and pandemic diseases

New Abundant Microbial Groups in Aquatic Hypersaline Environments

We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach

Ocean currents shape the microbiome of Arctic marine sediments

Prokaryote communities were investigated on the seasonally stratified Alaska Beaufort Shelf (ABS). Water and sediment directly underlying water with origin in the Arctic, Pacific or Atlantic oceans were analyzed by pyrosequencing and length heterogeneity-PCR in conjunction with physicochemical and geographic distance data to determine what features structure ABS microbiomes. Distinct bacterial communities were evident in all water masses. Alphaproteobacteria explained similarity in Arctic surface water and Pacific derived water. Deltaproteobacteria were abundant in Atlantic origin water and drove similarity among samples. Most archaeal sequences in water were related to unclassified marine Euryarchaeota. Sediment communities influenced by Pacific and Atlantic water were distinct from each other and pelagic communities. Firmicutes and Chloroflexi were abundant in sediment, although their distribution varied in Atlantic and Pacific influenced sites. Thermoprotei dominated archaea in Pacific influenced sediments and Methanomicrobia dominated in methane-containing Atlantic influenced sediments. Length heterogeneity-PCR data from this study were analyzed with data from methane-containing sediments in other regions. Pacific influenced ABS sediments clustered with Pacific sites from New Zealand and Chilean coastal margins. Atlantic influenced ABS sediments formed another distinct cluster. Density and salinity were significant structuring features on pelagic communities. Porosity co-varied with benthic community structure across sites and methane did not. This study indicates that the origin of water overlying sediments shapes benthic communities locally and globally and that hydrography exerts greater influence on microbial community structure than the availability of methane

Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean

As part of an ongoing survey of microbial community gene expression in the ocean, we sequenced and compared ~38 Mbp of community transcriptomes and ~157 Mbp of community genomes from four bacterioplankton samples, along a defined depth profile at Station ALOHA in North Pacific subtropical gyre (NPSG). Taxonomic analysis suggested that the samples were dominated by three taxa: Prochlorales, Consistiales and Cenarchaeales, which comprised 36–69% and 29–63% of the annotated sequences in the four DNA and four cDNA libraries, respectively. The relative abundance of these taxonomic groups was sometimes very different in the DNA and cDNA libraries, suggesting differential relative transcriptional activities per cell. For example, the 125 m sample genomic library was dominated by Pelagibacter (~36% of sequence reads), which contributed fewer sequences to the community transcriptome (~11%). Functional characterization of highly expressed genes suggested taxon-specific contributions to specific biogeochemical processes. Examples included Roseobacter relatives involved in aerobic anoxygenic phototrophy at 75 m, and an unexpected contribution of low abundance Crenarchaea to ammonia oxidation at 125 m. Read recruitment using reference microbial genomes indicated depth-specific partitioning of coexisting microbial populations, highlighted by a transcriptionally active high-light-like Prochlorococcus population in the bottom of the photic zone. Additionally, nutrient-uptake genes dominated Pelagibacter transcripts, with apparent enrichment for certain transporter types (for example, the C4-dicarboxylate transport system) over others (for example, phosphate transporters). In total, the data support the utility of coupled DNA and cDNA analyses for describing taxonomic and functional attributes of microbial communities in their natural habitats.Gordon and Betty Moore FoundationUnited States. Dept. of EnergyNational Science Foundation (U.S.) (Science and Technology Center Award EF0424599

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to 300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m 2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified