PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions

Feeding Mixed Grass-Clover Silages with Elevated Sugar Contents to Dairy Cows

Grasses with high sugar content (WSC) have been claimed to increase feed intake and milk production and at the same time give more efficient N utilisation and thus reduce pollution (e.g. Miller et al., 2001). In an ongoing EU-supported project (“Sweetgrass”), we have grown the new varieties of perennial ryegrasses and fed them to dairy cows. Results from the first year’s experiment when pure ryegrass silages made from standard or high-sugar varieties were fed, showed small differences in feed intake, milk production and N partitioning. In order to test the principle, it was therefore decided to increase the difference in sugar content in the following experiment by mixing sucrose into the silage before feeding

Profitability of organic and conventional dairy production with different dietary proportions of high-quality grass silage

Profitability in milk production was calculated using diets with three different proportions of high-quality grass silage. The treatments consisted of the same feeds, but differed in the dietary proportion of forage: low (L), medium (M) and high (H), representing one conventional and two organic diets. The calculations were based on results in feed intake and milk production from a large dairy cow experiment. In the calculations, three different districts with different conditions for farming were used as models, and calculations were performed on two different herd sizes. In addition, current financial supports were both included and excluded in the calculations. The results showed only minor differences in profitability between using in average 60% or 70% of dry matter (DM) of silage in diets used in organic production. In conventional production, it was profitable to increase the average dietary proportion of silage from 50% to 60% of DM when the prices of concentrate and grains were as high as at the present. The results showed clear economic benefits of increasing the proportion of high-quality silage in conventional Scandinavian dairy cow diets up to levels similar to the standards of the organic production system

Whole Crop Silage From Barley Fed in Combination with Red Clover Silage to Dairy Cows

Grass silage is the basic feed in Swedish dairy cow rations. The nitrogen utilisation in this type of diet is, however, low. A combination of forage legume protein and whole crop silage carbohydrates might be a solution to this problem. From other countries in Northern Europe the experience from feeding barley whole crop silage in combination with legumes is that it is possible to maintain a reasonably high milk production and at the same time have a good protein utilisation (Kristensen, 1992)

Use of pepper spray in policing : retrospective study of situational characteristics and implications for violent situations

Pepper spray (OC) is a policing tool aimed to prevent or stop aggressive behavior by quickly and temporarily incapacitate without injuring. To date, few studies have investigated OC’s operational usefulness and limitations. OC reduced violent behavior in 93% of the 936 incidents investigated. However, the operative range was often <2 m and it took between 3 and 5 s of spraying before obtaining effect, partly owing to the difficulties of hitting a small, sometimes erratically moving target. Collateral hits were noted in 24% of the incidents, whereof 90% were other officers. Noteworthy, in 21% of incidents officers put themselves at large personal risk by using OC at close range against people armed with lethal weapons. Hence, OC emerges as a suitable tool for handling low threat situations but lacks key traits to ensure safe and efficient policing of high threat situations, e.g., handling armed assailants

Safety of the Deferral of Coronary Revascularization on the Basis of Instantaneous Wave-Free Ratio and Fractional Flow Reserve Measurements in Stable Coronary Artery Disease and Acute Coronary Syndromes.

OBJECTIVES: The aim of this study was to investigate the clinical outcomes of patients deferred from coronary revascularization on the basis of instantaneous wave-free ratio (iFR) or fractional flow reserve (FFR) measurements in stable angina pectoris (SAP) and acute coronary syndromes (ACS). BACKGROUND: Assessment of coronary stenosis severity with pressure guidewires is recommended to determine the need for myocardial revascularization. METHODS: The safety of deferral of coronary revascularization in the pooled per-protocol population (n = 4,486) of the DEFINE-FLAIR (Functional Lesion Assessment of Intermediate Stenosis to Guide Revascularisation) and iFR-SWEDEHEART (Instantaneous Wave-Free Ratio Versus Fractional Flow Reserve in Patients With Stable Angina Pectoris or Acute Coronary Syndrome) randomized clinical trials was investigated. Patients were stratified according to revascularization decision making on the basis of iFR or FFR and to clinical presentation (SAP or ACS). The primary endpoint was major adverse cardiac events (MACE), defined as the composite of all-cause death, nonfatal myocardial infarction, or unplanned revascularization at 1 year. RESULTS: Coronary revascularization was deferred in 2,130 patients. Deferral was performed in 1,117 patients (50%) in the iFR group and 1,013 patients (45%) in the FFR group (p < 0.01). At 1 year, the MACE rate in the deferred population was similar between the iFR and FFR groups (4.12% vs. 4.05%; fully adjusted hazard ratio: 1.13; 95% confidence interval: 0.72 to 1.79; p = 0.60). A clinical presentation with ACS was associated with a higher MACE rate compared with SAP in deferred patients (5.91% vs. 3.64% in ACS and SAP, respectively; fully adjusted hazard ratio: 0.61 in favor of SAP; 95% confidence interval: 0.38 to 0.99; p = 0.04). CONCLUSIONS: Overall, deferral of revascularization is equally safe with both iFR and FFR, with a low MACE rate of about 4%. Lesions were more frequently deferred when iFR was used to assess physiological significance. In deferred patients presenting with ACS, the event rate was significantly increased compared with SAP at 1 year.info:eu-repo/semantics/publishedVersio

Performance of magnetic resonance imaging-based prostate cancer risk calculators and decision strategies in two large European medical centres

Objectives: To compare the performance of currently available biopsy decision support tools incorporating magnetic resonance imaging (MRI) findings in predicting clinically significant prostate cancer (csPCa). Patients and Methods: We retrospectively included men who underwent prostate MRI and subsequent targeted and/or systematic prostate biopsies in two large European centres. Available decision support tools were identified by a PubMed search. Performance was assessed by calibration, discrimination, decision curve analysis (DCA) and numbers of biopsies avoided vs csPCa cases missed, before and after recalibration, at risk thresholds of 5%–20%. Results: A total of 940 men were included, 507 (54%) had csPCa. The median (interquartile range) age, prostate-specific antigen (PSA) level, and PSA density (PSAD) were 68 (63–72) years, 9 (7–15) ng/mL, and 0.20 (0.13–0.32) ng/mL2, respectively. In all, 18 multivariable risk calculators (MRI-RCs) and dichotomous biopsy decision strategies based on MRI findings and PSAD thresholds were assessed. The Van Leeuwen model and the Rotterdam Prostate Cancer Risk Calculator (RPCRC) had the best discriminative ability (area under the receiver operating characteristic curve 0.86) of the MRI-RCs that could be assessed in the whole cohort. DCA showed the highest clinical utility for the Van Leeuwen model, followed by the RPCRC. At the 10% threshold the Van Leeuwen model would avoid 22% of biopsies, missing 1.8% of csPCa, whilst the RPCRC would avoid 20% of biopsies, missing 2.6% of csPCas. These multivariable models outperformed all dichotomous decision strategies based only on MRI-findings and PSAD. Conclusions: Even in this high-risk cohort, biopsy decision support tools would avoid many prostate biopsies, whilst missing very few csPCa cases. The Van Leeuwen model had the highest clinical utility, followed by the RPCRC. These multivariable MRI-RCs outperformed and should be favoured over decision strategies based only on MRI and PSAD.</p

Oxygen-deficient water zones in the Baltic Sea promote uncharacterized Hg methylating microorganisms in underlying sediments

Human-induced expansion of oxygen-deficient zones can have dramatic impacts on marine systems and its resident biota. One example is the formation of the potent neurotoxic methylmercury (MeHg) that is mediated by microbial methylation of inorganic divalent Hg (Hg-II) under oxygen-deficient conditions. A negative consequence of the expansion of oxygen-deficient zones could be an increase in MeHg production due to shifts in microbial communities in favor of microorganisms methylating Hg. There is, however, limited knowledge about Hg-methylating microbes, i.e., those carrying hgc genes critical for mediating the process, from marine sediments. Here, we aim to study the presence of hgc genes and transcripts in metagenomes and metatranscriptomes from four surface sediments with contrasting concentrations of oxygen and sulfide in the Baltic Sea. We show that potential Hg methylators differed among sediments depending on redox conditions. Sediments with an oxygenated surface featured hgc-like genes and transcripts predominantly associated with uncultured Desulfobacterota (OalgD group) and Desulfobacterales (including Desulfobacula sp.) while sediments with a hypoxic-anoxic surface included hgc-carrying Verrucomicrobia, unclassified Desulfobacterales, Desulfatiglandales, and uncharacterized microbes. Our data suggest that the expansion of oxygen-deficient zones in marine systems may lead to a compositional change of Hg-methylating microbial groups in the sediments, where Hg methylators whose metabolism and biology have not yet been characterized will be promoted and expand

The under-ice microbiome of seasonally frozen lakes

Compared to the well-studied open water of the “growing” season, under-ice conditions in lakes are characterized by low and rather constant temperature, slow water movements, limited light availability, and reduced exchange with the surrounding landscape. These conditions interact with ice-cover duration to shape microbial processes in temperate lakes and ultimately influence the phenology of community and ecosystem processes. We review the current knowledge on microorganisms in seasonally frozen lakes. Specifically, we highlight how under-ice conditions alter lake physics and the ways that this can affect the distribution and metabolism of auto- and heterotrophic microorganisms. We identify functional traits that we hypothesize are important for understanding under-ice dynamics and discuss how these traits influence species interactions. As ice coverage duration has already been seen to reduce as air temperatures have warmed, the dynamics of the under-ice microbiome are important for understanding and predicting the dynamics and functioning of seasonally frozen lakes in the near future