Diversity, distribution, and expression of opsin genes in freshwater lakes.

Microbial rhodopsins are widely distributed in aquatic environments and may significantly contribute to phototrophy and energy budgets in global oceans. However, the study of freshwater rhodopsins has been largely limited. Here, we explored the diversity, ecological distribution, and expression of opsin genes that encode the apoproteins of type I rhodopsins in humic and clearwater lakes with contrasting physicochemical and optical characteristics. Using metagenomes and metagenome-assembled genomes, we recovered opsin genes from a wide range of taxa, mostly predicted to encode green light-absorbing proton pumps. Viral opsin and novel bacterial opsin clades were recovered. Opsin genes occurred more frequently in taxa from clearwater than from humic water, and opsins in some taxa have nontypical ion-pumping motifs that might be associated with physicochemical conditions of these two freshwater types. Analyses of the surface layer of 33 freshwater systems revealed an inverse correlation between opsin gene abundance and lake dissolved organic carbon (DOC). In humic water with high terrestrial DOC and light-absorbing humic substances, opsin gene abundance was low and dramatically declined within the first few meters, whereas the abundance remained relatively high along the bulk water column in clearwater lakes with low DOC, suggesting opsin gene distribution is influenced by lake optical properties and DOC. Gene expression analysis confirmed the significance of rhodopsin-based phototrophy in clearwater lakes and revealed different diel expressional patterns among major phyla. Overall, our analyses revealed freshwater opsin diversity, distribution and expression patterns, and suggested the significance of rhodopsin-based phototrophy in freshwater energy budgets, especially in clearwater lakes.

Expanded Phylogenetic Diversity and Metabolic Flexibility of Mercury-Methylating Microorganisms.

Methylmercury is a potent bioaccumulating neurotoxin that is produced by specific microorganisms that methylate inorganic mercury. Methylmercury production in diverse anaerobic bacteria and archaea was recently linked to the hgcAB genes. However, the full phylogenetic and metabolic diversity of mercury-methylating microorganisms has not been fully unraveled due to the limited number of cultured experimentally verified methylators and the limitations of primer-based molecular methods. Here, we describe the phylogenetic diversity and metabolic flexibility of putative mercury-methylating microorganisms by hgcAB identification in publicly available isolate genomes and metagenome-assembled genomes (MAGs) as well as novel freshwater MAGs. We demonstrate that putative mercury methylators are much more phylogenetically diverse than previously known and that hgcAB distribution among genomes is most likely due to several independent horizontal gene transfer events. The microorganisms we identified possess diverse metabolic capabilities spanning carbon fixation, sulfate reduction, nitrogen fixation, and metal resistance pathways. We identified 111 putative mercury methylators in a set of previously published permafrost metatranscriptomes and demonstrated that different methylating taxa may contribute to hgcA expression at different depths. Overall, we provide a framework for illuminating the microbial basis of mercury methylation using genome-resolved metagenomics and metatranscriptomics to identify putative methylators based upon hgcAB presence and describe their putative functions in the environment.IMPORTANCE Accurately assessing the production of bioaccumulative neurotoxic methylmercury by characterizing the phylogenetic diversity, metabolic functions, and activity of methylators in the environment is crucial for understanding constraints on the mercury cycle. Much of our understanding of methylmercury production is based on cultured anaerobic microorganisms within the Deltaproteobacteria, Firmicutes, and Euryarchaeota. Advances in next-generation sequencing technologies have enabled large-scale cultivation-independent surveys of diverse and poorly characterized microorganisms from numerous ecosystems. We used genome-resolved metagenomics and metatranscriptomics to highlight the vast phylogenetic and metabolic diversity of putative mercury methylators and their depth-discrete activities in thawing permafrost. This work underscores the importance of using genome-resolved metagenomics to survey specific putative methylating populations of a given mercury-impacted ecosystem.

The utility of long-term blood pressure variability for cardiovascular risk prediction in primary care.

OBJECTIVES: Blood pressure (BP) is a long-established risk factor for cardiovascular disease (CVD). SBP is used in all widely used cardiovascular risk scores for clinical decision-making. Recently, within-person BP variability has been shown to be a major predictor of CVD. We investigated whether cardiovascular risk scores could be improved by incorporating BP variability with standard risk factors. METHODS: We used cohort data on patients aged 40-74 on 1 January 2005, from English general practices contributing to the Clinical Practice Research Datalink, a research database derived from electronic health records. Data were linked to hospital episodes and mortality data. SBP variability independent of the mean was calculated across up to six clinic visits. We divided data geographically into derivation and validation data sets. In the derivation data set, we developed a reference model, incorporating risk factors used in previous scores and an index model, incorporating the same factors and BP variability. We calculated model validation statistics in the validation data set including calibration ratio and c-statistic. RESULTS: In the derivation data set, BP variability was associated with CVD, independently of other risk factors (P = 0.005). However, in the validation data set, both models had similar c-statistic (0.7415 and 0.7419, respectively), R (31.8 and 32.0, respectively) and calibration ratio (0.938 and 0.940, respectively). CONCLUSION: The association of BP variability with CVD is statistically significant in a large data set but does not substantially improve the performance of a cardiovascular risk score.

Freshwater carbon and nutrient cycles revealed through reconstructed population genomes.

Although microbes mediate much of the biogeochemical cycling in freshwater, the categories of carbon and nutrients currently used in models of freshwater biogeochemical cycling are too broad to be relevant on a microbial scale. One way to improve these models is to incorporate microbial data. Here, we analyze both genes and genomes from three metagenomic time series and propose specific roles for microbial taxa in freshwater biogeochemical cycles. Our metagenomic time series span multiple years and originate from a eutrophic lake (Lake Mendota) and a humic lake (Trout Bog Lake) with contrasting water chemistry. Our analysis highlights the role of polyamines in the nitrogen cycle, the diversity of diazotrophs between lake types, the balance of assimilatory vs. dissimilatory sulfate reduction in freshwater, the various associations between types of phototrophy and carbon fixation, and the density and diversity of glycoside hydrolases in freshwater microbes. We also investigated aspects of central metabolism such as hydrogen metabolism, oxidative phosphorylation, methylotrophy, and sugar degradation. Finally, by analyzing the dynamics over time in nitrogen fixation genes and Cyanobacteria genomes, we show that the potential for nitrogen fixation is linked to specific populations in Lake Mendota. This work represents an important step towards incorporating microbial data into ecosystem models and provides a better understanding of how microbes may participate in freshwater biogeochemical cycling.

Building a local community of practice in scientific programming for life scientists.

In this paper, we describe why and how to build a local community of practice in scientific programming for life scientists who use computers and programming in their research. A community of practice is a small group of scientists who meet regularly to help each other and promote good practices in scientific programming. While most life scientists are well trained in the laboratory to conduct experiments, good practices with (big) data sets and their analysis are often missing. We propose a model on how to build such a community of practice at a local academic institution, present two real-life examples, and introduce challenges and implemented solutions. We believe that the current data deluge that life scientists face can benefit from the implementation of these small communities. Good practices spread among experimental scientists will foster open, transparent, and sound scientific results beneficial to society.

Current practice of usual clinic blood pressure measurement in people with and without diabetes: a survey and prospective 'mystery shopper' study in UK primary care.

OBJECTIVES: Hypertension trials and epidemiological studies use multiple clinic blood pressure (BP) measurements at each visit. Repeat measurement is also recommended in international guidance; however, little is known about how BP is measured routinely. This is important for individual patient management and because routinely recorded readings form part of research databases. We aimed to determine the current practice of BP measurement during routine general practice appointments. DESIGN: (1) An online cross-sectional survey and (2) a prospective 'mystery shopper' study where patients agreed to report how BP was measured during their next appointment. SETTING: Primary care. PARTICIPANTS: Patient charity/involvement group members completing an online survey between July 2015 and January 2016. 334 participants completed the prospective study (51.5% male, mean age=59.3 years) of which 279 (83.5%) had diabetes. PRIMARY OUTCOME: Proportion of patients having BP measured according to guidelines. RESULTS: 217 participants with (183) and without diabetes (34) had their BP measured at their last appointment. BP was measured in line with UK guidance in 63.7% and 60.0% of participants with and without diabetes, respectively. Initial pressures were significantly higher in those who had their BP measured more than once compared with only once (p=0.016/0.089 systolic and p<0.001/p=0.022 diastolic, in patients with/without diabetes, respectively). CONCLUSIONS: Current practice of routine BP measurement in UK primary care is often concordant with guidelines for repeat measurement. Further studies are required to confirm findings in broader populations, to confirm when a third repeat reading is obtained routinely and to assess adherence to other aspects of BP measurement guidance.

Contrasting patterns of genome-level diversity across distinct co-occurring bacterial populations.

To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single-cell genomes and a 5-year metagenomic time series. The single-cell genomes grouped into 15 monophyletic clusters (termed "tribes") that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI Actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.

Ecophysiology of Freshwater Verrucomicrobia Inferred from Metagenome-Assembled Genomes.

Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, and yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions and greatly expand knowledge of the genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly)saccharide degraders in freshwater, uncovered interesting genomic features for this lifestyle, and suggested their adaptation to nutrient availabilities in their environments. Verrucomicrobia populations differ significantly between the two lakes in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially derived allochthonous carbon sources of the two ecosystems, respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humus-rich environment. Notably, most epilimnion genomes have large numbers of so-called "Planctomycete-specific" cytochrome c-encoding genes, which exhibited distribution patterns nearly opposite to those seen with glycoside hydrolase genes, probably associated with the different levels of environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes represent a major step toward understanding the role, ecophysiology, and distribution of Verrucomicrobia in freshwater. IMPORTANCE Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called "Planctomycete-specific" cytochrome c-encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.

Blood pressure variability and cardiovascular disease: systematic review and meta-analysis.

OBJECTIVE:  To systematically review studies quantifying the associations of long term (clinic), mid-term (home), and short term (ambulatory) variability in blood pressure, independent of mean blood pressure, with cardiovascular disease events and mortality. DATA SOURCES:  Medline, Embase, Cinahl, and Web of Science, searched to 15 February 2016 for full text articles in English. ELIGIBILITY CRITERIA FOR STUDY SELECTION:  Prospective cohort studies or clinical trials in adults, except those in patients receiving haemodialysis, where the condition may directly impact blood pressure variability. Standardised hazard ratios were extracted and, if there was little risk of confounding, combined using random effects meta-analysis in main analyses. Outcomes included all cause and cardiovascular disease mortality and cardiovascular disease events. Measures of variability included standard deviation, coefficient of variation, variation independent of mean, and average real variability, but not night dipping or day-night variation. RESULTS:  41 papers representing 19 observational cohort studies and 17 clinical trial cohorts, comprising 46 separate analyses were identified. Long term variability in blood pressure was studied in 24 papers, mid-term in four, and short-term in 15 (two studied both long term and short term variability). Results from 23 analyses were excluded from main analyses owing to high risks of confounding. Increased long term variability in systolic blood pressure was associated with risk of all cause mortality (hazard ratio 1.15, 95% confidence interval 1.09 to 1.22), cardiovascular disease mortality (1.18, 1.09 to 1.28), cardiovascular disease events (1.18, 1.07 to 1.30), coronary heart disease (1.10, 1.04 to 1.16), and stroke (1.15, 1.04 to 1.27). Increased mid-term and short term variability in daytime systolic blood pressure were also associated with all cause mortality (1.15, 1.06 to 1.26 and 1.10, 1.04 to 1.16, respectively). CONCLUSIONS:  Long term variability in blood pressure is associated with cardiovascular and mortality outcomes, over and above the effect of mean blood pressure. Associations are similar in magnitude to those of cholesterol measures with cardiovascular disease. Limited data for mid-term and short term variability showed similar associations. Future work should focus on the clinical implications of assessment of variability in blood pressure and avoid the common confounding pitfalls observed to date. SYSTEMATIC REVIEW REGISTRATION:  PROSPERO CRD42014015695.

Chronic renal disease is not chronic kidney disease: implications for use of the QRISK and Joint British Societies risk scores.

BACKGROUND: Chronic kidney disease (CKD) is a major risk factor for cardiovascular disease (CVD) and European guidelines advocate assessment of CVD risk. QRISK and JBS3 risk calculators do not use the consensus definition of CKD stages 3-5 but instead use a definition referring to renal pathologies and CKD stages 4 and 5. Consequently, there is potential for doctors to misclassify their patients when using these risk calculators. OBJECTIVES: To quantify the number of people who may be affected by such misclassifications. METHODS: Database analysis using the Clinical Practice Research Datalink (CPRD).We identified 2512053 adults aged 25-84 without prior history of CVD on 1st January 2014. We identified those with 'chronic renal disease' and/or CKD by searching medical event history data. RESULTS: The study population was 48.7% male with mean age of 50.2 years. A total of 80718 had diagnostic READ codes for CKD stages 3, 4 or 5. Of these, 6585 individuals (8.2%) were classified as having 'chronic renal disease' according to the updated QRISK 2014, up from 3365 according to QRISK 2013. Whilst the updated QRISK definition of 'chronic renal disease' in total identified 62% more people than previously and had improved sensitivity for CKD stages 3 to 5, sensitivity remained poor (8.16%; 95% CI: 7.97-8.35%). CONCLUSION: Misuse of risk scores by general practitioners could result in clinically important differences in risk estimates. Users of risk scores should recognize the potential for error and developers should aim to label risk factors more clearly.

Applying quality improvement methods in a hyperbaric oxygen program: reducing unnecessary glucose testing.

OBJECTIVE: To describe the implementation of a quality improvement (QI) project that aimed at improving and standardizing glucose checks on patients with diabetes undergoing hyperbaric oxygen (HBO2) therapy. METHODS: This is a prospective cohort study. Following the Model for Improvement, nurses and physicians ran several Plan-Do-Study-Act (PDSA) cycles over a four-month period, with multiple iteration and testing changes. They developed and implemented a nurse-led protocol that was tested prospectively. RESULTS: Compared to the pre-protocol baseline (N = 332), glucose checks per session guided by the protocol decreased by 37.7% (2.84 vs. 1.77 per session, P⟨0.001). Compliance with the new protocol was higher than compliance with the existing protocol (97.3% to 84.2%, P⟨0.001). There were no cases of a symptomatic hypoglycemic event after the implementation of the protocol. CONCLUSIONS: A quality improvement project implemented by a multidisciplinary team in a hyperbaric practice was feasible and has improved the management of diabetic patients undergoing HBO2 therapy. Considering how the hyperbaric community values the culture of safety and considering the feasibility of this project, more QI training and projects in hyperbaric programs should be performed.

The incidence of hypoglycemia during HBO2 therapy: A retrospective review.

BACKGROUND: Hypoglycemia is concerning in patients with diabetes undergoing hyperbaric oxygen (HBO2) therapy. We aimed to estimate the incidence, risk factors and a pretreatment glucose threshold of HBO2-associated hypoglycemia. METHODS: We retrospectively evaluated a patient cohort undergoing HBO2 therapy. We calculated the area under the curve (AUC) and odds ratio (OR) with 95% confidence interval (CI) adjusting for patients' age, gender, diabetes type, insulin use, body mass index, hemoglobin A1c and HBO2 treatment time. RESULTS: During 77 months, 3,136 HBO2 sessions were performed on patients with diabetes. In-chamber glucose was higher than pre-HBO2 glucose in 1,708/3,136 sessions (54%). The incidence of hypoglycemia (defined as ≤ 70 mg/dL) during or immediately after HBO2 treatment was 1.5% (0.8-2.1%). Hypoglycemia that was symptomatic or severe was rare. A glucose value pre-HBO2 of 150 mg/dL best predicted the risk of subsequent hypoglycemia (AUC 0.80; 95% CI, 0.75-0.86). Type 1 diabetes was independently associated with increased risk of hypoglycemia (OR 3.69; 95% CI, 1.67, 8.19) whereas insulin use was not. CONCLUSIONS: In patients with diabetes undergoing HBO2, severe hypoglycemia is rare and occurs more frequently in Type 1 diabetes. Pre-HBO2 glucose values may be used to predict subsequent hypoglycemia and reduce the need for routine glucose monitoring during and after HBO2.

Participant- and study-related characteristics predicting treatment completion and study retention in an adolescent smoking cessation trial.

PURPOSE: To determine which factors predict smoking cessation treatment completion and retention among adolescents. METHODS: In a multisite, randomized, controlled trial, the efficacy of motivational interviewing was compared with structured brief advice for smoking cessation and reduction in adolescents (n = 355) aged 14-18 years (55% female, 45% black, 12% Hispanic). Treatment spanned 12 weeks, with follow-up assessments at 24 weeks. Treatment completion was defined as completion of all five counseling sessions. Study retention was defined as completing the 24-week assessment. Participant and study variables served as predictors of treatment completion and retention. RESULTS: In all, 79% of participants completed all five counseling sessions and the same percent completed the 24-week assessment. Black race, precontemplation stage to cut back, and shorter length of time between the baseline assessment and the first counseling session were significantly associated with treatment completion. For every 7.5-day delay in starting treatment after the baseline visit, there was a 50% decrease in the odds of completing all five treatment sessions. Retention at 24 weeks was predicted by black race, younger age, greater maternal education, expectations of graduating college, and structured brief advice intervention. CONCLUSIONS: High rates of treatment completion and study retention can be achieved in a multisession, behavioral intervention for adolescent smoking cessation. Findings suggest that treatment should begin soon after the intake session to maximize treatment completion. Enhanced efforts to retain older adolescents and youth with lower academic goals and lower family income will be important in future studies.

Providers' experiences caring for adolescents who smoke cigarettes.

OBJECTIVE: To describe providers' experiences screening for and counseling adolescent patients who smoke cigarettes. DESIGN: Eight qualitative focus groups were conducted with 51 health care providers in primary care settings. Focus groups were video- and audiotaped; tapes were transcribed for coding by an interdisciplinary team using the constant comparative method. MAIN OUTCOME MEASURES: Providers reported experiences screening for and managing adolescent patients who reported smoking cigarettes. RESULTS: Providers expressed confidence in their ability to screen adolescent patients for tobacco use, particularly as part of regularly scheduled preventive and medical visits. Providers reported difficulty balancing screening for smoking with their concern for maintaining rapport with their adolescent patients. In addition, providers reported that adolescent smoking patterns differed from those of adults, and consequently, providers were not certain at what level of smoking an adolescent required intervention. Furthermore, providers were unclear regarding what interventions were recommended for and effective with adolescents. CONCLUSION: Providers are interested in adolescent evidence-based screening methods and cessation interventions that are supportive of a nonjudgmental and empathic approach to caring for adolescent smokers, particularly those with irregular and situational smoking patterns.