Effects of dietary fat, nitrate, and 3-nitrooxypropanol and their combinations on methane emission, feed intake, and milk production in dairy cows.

The objective of the present study was to investigate the effect of individual and combined use of dietary fat, nitrate, and 3-nitrooxypropanol (3-NOP) on dairy cows' enteric methane (CH4) emission and production performance. Twenty-four primiparous and 24 multiparous Danish Holstein cows (111 ± 44.6 d in milk; mean ± standard deviation) were included in an incomplete 8 × 8 Latin square design with six 21-d periods. Dietary treatments were organized in a 2 × 2 × 2 factorial arrangement aiming for 2 levels of FAT (30 or 63 g of crude fat/kg of dry matter [DM]; LF or HF, respectively), 2 levels of NITRATE (0 or 10 g of nitrate/kg of DM; UREA or NIT, respectively), and 2 levels of 3-NOP (0 or 80 mg/kg DM; BLANK or NOP, respectively). Treatments were included in ad libitum-fed partial mixed rations in bins that automatically measured feed intake and eating behavior. Additional concentrate was offered as bait in GreenFeed units used for measurement of gas emission. For total DM intake (DMI), a FAT × NITRATE interaction showed that DMI, across parities and levels of 3-NOP, was unaffected by separate fat supplementation, but reduced by nitrate with 4.6% and synergistically decreased (significant 2-way interaction) with 13.0% when fat and nitrate were combined. Additionally, 3-NOP decreased DMI by 13.4% and the combination of 3-NOP with fat and nitrate decreased DMI in an additive way (no significant 3-way interaction). The decreasing effects on DMI were more pronounced in multiparous cows than in primiparous cows. For treatments with largest reductions in DMI, eating behavior was altered toward more frequent, but smaller meals, a slower eating rate and increased attempts to visit unassigned feed bins. Energy-corrected milk (ECM) yield increased by 6.3% with fat supplementation, whereas ECM yield did not differ among diets including nitrate (FAT × NITRATE interaction). Cows supplemented with 3-NOP had 9.0% lower ECM yield than cows fed no 3-NOP. Based on three 2-way interactions including FAT, NITRATE, and 3-NOP, the combined use of the additives resulted in antagonistic effects on CH4 reduction. A 6% to 7% reduction in CH4 yield (CH4/kg of DMI) could be ascribed to the effect of fat, a 12% to 13% reduction could be ascribed to the effect of nitrate and an 18% to 23% reduction could be ascribed to the effect of 3-NOP. Hence, no combinations of additives resulted in CH4 yield-reductions that were greater than what was obtained by separate supplementation of the most potent additive within the combination. The CH4 yield reduction potential of additives was similar between parities. Increased apparent total-tract digestibility of organic matter (OM) in cows fed combinations including nitrate or 3-NOP was a result of a NITRATE × 3-NOP interaction. Apparent total-tract digestibility of OM was also increased by fat supplementation. These increases reflected observed decreases in DMI. In conclusion, combined use of fat, nitrate, and 3-NOP in all combinations did not result in CH4 reductions that were greater than separate supplementation of the most potent additive within the combination (3-NOP > nitrate > fat). Additionally, separate supplementation of some additives and combined use of all additives reduced DMI.

The effect of feeding and visiting behavior on methane and hydrogen emissions of dairy cattle measured with the GreenFeed system under different dietary conditions.

The objectives were to investigate the effect of feeding and visiting behavior of dairy cattle on CH4 and H2 production measured with voluntary visits to the GreenFeed system (GF) and to determine whether these effects depended on basal diet (BD) and 3-nitrooxypropanol (3-NOP) supplementation. The experiment involved 64 lactating dairy cattle (146 ± 45 d in milk at the start of trial; mean ± SD) in 2 overlapping crossover trials, each consisting of 2 measurement periods. Cows within block were randomly allocated to 1 of 3 types of BD: a grass silage-based diet consisting of 30% concentrates and 70% grass silage (DM basis), a grass silage- and corn silage-mixed diet consisting of 30% concentrates, 42% grass silage, and 28% corn silage (DM basis), or a corn silage-based diet consisting of 30% concentrates, 14% grass silage, and 56% corn silage (DM basis). Each type of BD was subsequently supplemented with 0 and 60 mg 3-NOP/kg DM in one crossover, or 0 and 80 mg 3-NOP/kg DM in the other crossover. Diets were provided in feed bins which automatically recorded feed intake and feeding behavior, with additional concentrate fed in the GF. All visits to the GF that resulted in a spot measurement of both CH4 and H2 emission were analyzed in relation to feeding behavior (e.g., meal size and time interval to preceding meal) as well as GF visiting behavior (e.g., duration of visit). Feeding and GF visiting behavior was related to CH4 and H2 production measured with the GF, in particular the meal size before a GF measurement and the time interval between a GF measurement and the preceding meal. Relationships between gas production and both feeding and GF visiting behavior were affected both by type of BD and 3-NOP supplementation. With an increase of the time interval between a GF measurement and the preceding meal, CH4 production decreased with 0 mg 3-NOP/kg DM but increased with 60 and 80 mg 3-NOP/kg DM, whereas type of BD did not affect these relationships. In contrast, CH4 production increased with 0 mg 3-NOP/kg DM but decreased with 60 and 80 mg 3-NOP/kg DM upon an increase in the size of the meal preceding a GF measurement. With an increase of the time interval between a GF measurement and the preceding meal, or with a decrease of the size of the meal preceding a GF measurement, H2 production decreased for all treatments, although the effect was generally somewhat stronger for 60 and 80 mg 3-NOP/kg DM than for 0 mg 3-NOP/kg DM. Hence, the timing of GF measurements next to feeding and GF visiting behavior are essential when assessing the effect of dietary treatment on the production of CH4 and H2 in a setting where a spot sampling device such as a GF is used and where the measurements depend on voluntary visits from the cows.

Long-term effects of 3-nitrooxypropanol on methane emission and milk production characteristics in Holstein Friesian dairy cows.

The objective was to determine the long-term effect of 3-nitrooxypropanol (3-NOP) on CH4 emission and milk production characteristics from dairy cows receiving 3-NOP in their diet for a full year, covering all lactation stages of the dairy cows. Sixty-four late-lactation Holstein Friesian cows (34% primiparous) were blocked in pairs, based on expected calving date, parity, and daily milk yield. The experiment started with an adaptation period of 1 week followed by a covariate period of 3 weeks in which all cows received the same basal diet and baseline measurements were performed. Directly after, cows within a block were randomly allocated to 1 of 2 dietary treatments: a diet containing on average 69.8 mg 3-NOP/kg DM (total ration level, corrected for intake of non-supplemented GreenFeed bait) and a diet containing a placebo. Forage composition as well as forage to concentrate ratio altered with lactation stage (i.e., dry period and early, mid, and late lactation). Diets were provided as a total mixed ration and additional bait was fed in GreenFeed units which were used for emission measurements. Supplementation of 3-NOP did not affect total dry matter intake (DMI), body weight or body condition score, but resulted in a 6.5% increase in the yields of energy-corrected milk and fat- and protein-corrected milk (FPCM). Furthermore, milk fat and protein as well as feed efficiency were increased upon 3-NOP supplementation. Overall, a reduction of 21%, 20%, and 27% was achieved for CH4 production (g/d), yield (g/kg DMI), and intensity (g/kg FPCM), respectively, upon 3-NOP supplementation. The CH4 mitigation potential of 3-NOP was affected by the lactation stage dependent diet to which 3-NOP was supplemented. On average, a 16%, 20%, 16%, and 26% reduction in CH4 yield (g/kg DMI) was achieved upon 3-NOP supplementation for the dry period, and early, mid, and late lactation diet, respectively. The CH4 mitigation potential of 3-NOP was affected by the length of 3-NOP supplementation within a lactation stage dependent diet and by variation in diet composition within a lactation stage dependent diet as a result of changes in grass and corn silage silos. In conclusion, 3-NOP reduced CH4 emission from cows receiving 3-NOP for a year, with a positive impact on production characteristics. The CH4 mitigation potential of 3-NOP was influenced by diet type, diet composition, and nutrition value, and the efficacy of 3-NOP appeared to decline over time but not continuously. Associated with changes in diet composition, increased efficacy of 3-NOP was observed at the start of the trial, at the start of a new lactation, and, importantly, at the end of the trial. These results suggests that diet composition has a large effect on the efficacy of 3-NOP, perhaps even larger than the week of supplementation after first introduction of 3-NOP. Further studies are needed to clarify the long-term effect of 3-NOP on CH4 emission and to further investigate the effect that variation in diet composition may have on the mitigation potential of 3-NOP.

Gas exchange, rumen hydrogen sinks, and nutrient digestibility and metabolism in lactating dairy cows fed 3-nitrooxypropanol and cracked rapeseed.

Fat in the form of cracked rapeseed and 3-nitrooxypropanol (3-NOP, market as Bovaer) were fed alone or in combination to 4 Danish Holstein multicannulated dairy cows, with the objective to investigate effects on gas exchange, dry matter intake (DMI), nutrient digestion, and nutrient metabolism. The study design was a 4 × 4 Latin square with a 2 × 2 factorial treatment arrangement with 2 levels of fat supplementation; 33 g of crude fat per kg of dry matter (DM) or 64 g of crude fat per kg of DM for low and high fat diets, respectively, and 2 levels of 3-NOP; 0 mg/kg DM or 80 mg/kg DM. In total, 4 diets were formulated: low fat (LF), high fat (HF), 3-NOP and low fat (3LF), and 3-NOP and high fat (3HF). Cows were fed ad libitum and milked twice daily. The adaptation period lasted 11 d, followed by 5 d with 12 diurnal sampling times of digesta and ruminal fluid. Thereafter, gas exchange was measured for 5 d in respiration chambers. Chromic oxide and titanium dioxide were used as external flow markers to determine intestinal nutrient flow. No interactions between fat supplementation and 3-NOP were observed for methane yield (g/kg DM), total-tract digestibility of nutrients or total volatile fatty acid (VFA) concentration in the rumen. Methane yield (g/kg DMI) was decreased by 24% when cows were fed 3-NOP. In addition, 3-NOP increased carbon dioxide and hydrogen yield (g/kg DM) by 6% and 3,500%, respectively. However, carbon dioxide production was decreased when expressed on a daily basis. Fat supplementation did not affect methane yield but tended to reduce methane in percent of gross energy intake. A decrease (11%) in DMI was observed, when cows were fed 3-NOP. Likely, the lower DMI mediated a lower passage rate causing the tendency to higher rumen and total-tract neutral detergent fiber digestibility, when the cows were fed 3-NOP. Total VFA concentrations in the rumen were negatively affected both by 3-NOP and fat supplementation. Furthermore, 3-NOP caused a shift in the VFA fermentation profile, with decreased acetate proportion and increased butyrate proportion, whereas propionate proportion was unaffected. Increased concentrations of the alcohols methanol, ethanol, propanol, butanol, and 2-butanol were observed in the ruminal fluid when cows were fed 3-NOP. These changes in rumen metabolites indicate partial re-direction of hydrogen into other hydrogen sinks, when methanogenesis is inhibited by 3-NOP. In conclusion, fat supplementation did not reduce methane yield, whereas 3-NOP reduced methane yield, irrespective of fat level. However, the concentration of 3-NOP and diet composition and resulting desired mitigation effect must be considered before implementation. The observed reduction in DMI with 80 mg 3-NOP/kg DM was intriguing and may indicate that a lower dose should be applied in a Northern European context; however, the mechanism behind needs further investigation.

A meta-analysis of effects of 3-nitrooxypropanol on methane production, yield, and intensity in dairy cattle.

Ruminants, particularly dairy and beef cattle, contribute to climate change through mostly enteric methane emissions. Several mitigating options have been proposed, including the feed additive 3-nitrooxypropanol (3-NOP). The objectives of this study were to explain the variability in the mitigating effect of 3-NOP and to investigate the interaction between diet composition and 3-NOP dose, using meta-analytical approaches. Data from 13 articles (14 experiments) met the selection criteria for inclusion in the meta-analysis, and 48 treatment means were used for the analysis. Mean differences were calculated as 3-NOP treatment mean minus control treatment mean and then expressed as a percentage of the control mean. Three types of models were developed: (1) one including 3-NOP dose, overall mean, and individual covariate; (2) a combination of neutral detergent fiber (NDF), 3-NOP dose, and overall mean; and (3) one selected model from all combinations of up to 5 covariates, which were compared using a leave-one-out cross validation method. Models including only 3-NOP dose resulted in a significant reduction of 32.7%, 30.9%, and 32.6% for CH4 production (g/d), yield (g/kg dry matter intake), and intensity (g/kg energy-corrected milk), respectively, at an average 3-NOP dose of 70.5 mg/kg dry matter (DM). The greater the NDF content in the diet, the lower the reduction efficiency for a given 3-NOP dose. For 10 g/kg DM increase in NDF content from its mean (329 g of NDF/kg of DM) the 3-NOP effect on CH4 production was impaired by 0.633%, the 3-NOP effect on CH4 yield by 0.647%, and the 3-NOP effect on CH4 intensity by 0.723%. The analysis based on leave-one-out cross validation showed an increase in NDF and crude fat content reduces efficacy of 3-NOP and an increase in 3-NOP dose increases efficacy. A 1% (10 g/kg) DM decrease in dietary NDF content from its mean may increase the efficacy of 3-NOP in reducing CH4 production by 0.915%. A 1% (10 g/kg DM) decrease in dietary crude fat content from its mean enhances the efficacy of 3-NOP on CH4 production by 3.080% at a given dose and NDF level. For CH4 yield, next to 3-NOP dose, dietary NDF content and dietary crude fat content were included in the selected model, but also dietary starch content with an opposite direction to NDF and crude fat. The effect of 3-NOP dose on CH4 intensity was similar to its effect on CH4 production, whereas the effect of dietary NDF content was slightly lower. Expanding the previously published models with the newly available data published from trials since then improved model performance, hence demonstrating the value of regularly updating meta-analyses if a wider range of data becomes available.

The role of expert focus echocardiography during the COVID-19 pandemic.

BACKGROUND: Focus Echocardiography has routinely been used to offer quick diagnosis in critical care environments, predominantly by clinicians with limited training. During the COVID-19 pandemic, international guidance recommended all echocardiography scans were performed as focus studies to limit operator viral exposure in both inpatient and outpatient settings. The aim of this study was to assess the effectiveness of eFoCUS, a focus scan performed by fully trained echocardiographers following a minimum dataset plus full interrogation of any pathology found. METHODS: All diagnostic echocardiograms, performed by fully trained echocardiographers during an 8-week period during the first UK COVID-19 wave, were included. The number of images acquired was compared in the following categories: admission status, COVID status, image quality, indication, invasive ventilation, pathology found, echocardiographer experience, and whether eFoCUS was deemed adequate to answer the clinical question. RESULTS: In 87.4% of the 698 scans included, the operator considered that the eFOCUS echo protocol, with additional images when needed, was sufficient to answer the clinical question on the request. Echocardiographer experience did not affect the number of images acquired. Less images were acquired in COVID-19 positive patients compared to negative/asymptomatic (38 ± 12 vs. 42 ± 12, p = .001), and more images were required when a valve pathology was identified. CONCLUSION: eFoCUS echocardiography is an effective protocol for use during the COVID-19 pandemic. It provides sufficient diagnostic information to answer the clinical question but differs from standard focus/limited protocols by enabling the identification and interrogation of significant pathology and incidental findings, preventing unnecessary repeat scans and viral exposure of operators.

Global ensemble projections reveal trophic amplification of ocean biomass declines with climate change.

While the physical dimensions of climate change are now routinely assessed through multimodel intercomparisons, projected impacts on the global ocean ecosystem generally rely on individual models with a specific set of assumptions. To address these single-model limitations, we present standardized ensemble projections from six global marine ecosystem models forced with two Earth system models and four emission scenarios with and without fishing. We derive average biomass trends and associated uncertainties across the marine food web. Without fishing, mean global animal biomass decreased by 5% (±4% SD) under low emissions and 17% (±11% SD) under high emissions by 2100, with an average 5% decline for every 1 °C of warming. Projected biomass declines were primarily driven by increasing temperature and decreasing primary production, and were more pronounced at higher trophic levels, a process known as trophic amplification. Fishing did not substantially alter the effects of climate change. Considerable regional variation featured strong biomass increases at high latitudes and decreases at middle to low latitudes, with good model agreement on the direction of change but variable magnitude. Uncertainties due to variations in marine ecosystem and Earth system models were similar. Ensemble projections performed well compared with empirical data, emphasizing the benefits of multimodel inference to project future outcomes. Our results indicate that global ocean animal biomass consistently declines with climate change, and that these impacts are amplified at higher trophic levels. Next steps for model development include dynamic scenarios of fishing, cumulative human impacts, and the effects of management measures on future ocean biomass trends.

Methane mitigation potential of 3-nitrooxypropanol in lactating cows is influenced by basal diet composition.

The objective of this study was to investigate whether the CH4 mitigation potential of 3-nitrooxypropanol (3-NOP) in dairy cattle was affected by basal diet (BD) composition. The experiment involved 64 Holstein-Friesian dairy cows (146 ± 45 d in milk at the start of trial; mean ± SD) in 2 overlapping crossover trials, each consisting of 2 measurement periods. Cows were blocked according to parity, d in milk, and milk yield, and randomly allocated to 1 of 3 diets: a grass silage-based diet (GS) consisting of 30% concentrates and 70% grass silage (DM basis), a grass silage- and corn silage-mixed diet (GSCS) consisting of 30% concentrates, 42% grass silage, and 28% corn silage (DM basis), or a corn silage-based diet (CS) consisting of 30% concentrates, 14% grass silage, and 56% corn silage (DM basis). Two types of concentrates were formulated, viz. a concentrate for the GS diet and a concentrate for the CS diet, to meet the energy and protein requirements for maintenance and milk production. The concentrate for the GSCS diet consisted of a 50:50 mixture of both concentrates. Subsequently, the cows within each type of BD received 2 treatments in a crossover design: either 60 mg of 3-NOP/kg of DM (NOP60) and a placebo with 0 mg of 3-NOP/kg of DM (NOP0) in one crossover or 80 mg of 3-NOP/kg of DM (NOP80) and NOP0 in the other crossover. Diets were provided as total mixed ration in feed bins, which automatically recorded feed intake. Additional concentrate was fed in the GreenFeed system that was used to measure emissions of CH4 and H2. The CS diets resulted in a reduced CH4 yield (g/kg DMI) and CH4 intensity (g/kg milk). Feeding 3-NOP resulted in a decreased DMI. Milk production and composition did not differ between NOP60 and NOP0, whereas milk yield and the yield of major components decreased for NOP80 compared with NOP0. Feed efficiency was not affected by feeding 3-NOP. Interactions between BD and supplementation of 3-NOP were observed for the production (g/d) and yield (g/kg DMI) of both CH4 and H2, indicating that the mitigating effect of 3-NOP depended on the composition of the BD. Emissions of CH4 decreased upon 3-NOP supplementation for all BD, but the decrease in CH4 emissions was smaller for GS (-26.2% for NOP60 and -28.4% for NOP80 in CH4 yield) compared with both GSCS (-35.1% for NOP60 and -37.9% for NOP80 for CH4 yield) and CS (-34.8% for NOP60 and -41.6% for NOP80 for CH4 yield), with no difference between the latter 2 BD. Emissions of H2 increased upon 3-NOP supplementation for all BD, but the H2 yield (g/kg DMI) increased 3.16 and 3.30-fold, respectively, when NOP60 and NOP80 were supplemented to GS, and 4.70 and 4.96 fold, respectively, when NOP60 and NOP80 were supplemented to CS. In conclusion, 3-NOP can effectively decrease CH4 emissions in dairy cows across diets, but the level of CH4 mitigation is greater when supplemented in a corn silage-based diet compared with a grass silage-based diet.

Active travelling to school is not associated with increased total daily physical activity levels, or reduced obesity and cardiovascular/pulmonary health parameters in 10-12-year olds: a cross-sectional cohort study.

BACKGROUND/OBJECTIVES: Childhood obesity has increased enormously. Several lifestyle factors have been implicated, including decreased physical activity, partially involving a decline in active travel to school. We aimed to establish the association between school transport mode and physical activity levels of primary 6 and 7 children (aged 10-12). Secondary outcomes were body mass index standard deviation scores, blood pressure levels and lung function. SUBJECTS/METHODS: A cross-sectional study was conducted with a total number of 432 children from three primary schools in North East Scotland. Actigraph accelerometers were used to provide objective measures of physical activity. Ninety-two children in primary 6 and 90 children in primary 7 (40 in common) had adequate data. Modes of transport to school were assessed by a questionnaire. Two hundred and seventeen children in primary 6 and one hundred and sixty-five in primary 7 returned adequate questionnaires. Children who used active transport modes for >70% of their journeys to school over the week were coded as active travellers and <30% were coded as passive travellers. All children also had height, weight, blood pressure levels and lung function measured. RESULTS: Children who lived further away from school, and in more expensive properties were more likely to travel passively to school. Actively commuting children (70% walking) had significantly higher activity levels than passive commuters during the 30 min that encompassed their journey to and from school. However, there were no significant differences between active and passive school travellers in total daily physical activity, BMI SDS, and both systolic and diastolic blood pressure and lung function. CONCLUSIONS: There was no evidence that more days of active travel to school had a significant influence on total physical activity, obesity and related health parameters. Public health interventions promoting active travel to school may have limited success in quelling the childhood obesity epidemic.

Skeletal health, redox balance and gastrointestinal functionality in dairy cows: connecting bugs and bones.

This research reflection examines the physiological links between redox balance, skeletal health and gastrointestinal functionality in dairy cows. With the increase in demand of animal products caused by the growth in human population, the dairy industry needs to develop and implement innovative strategies which are profitable, sustainable and cow friendly. Redox balance, skeletal heath and gastrointestinal functionality are three key physiological systems that are often seen as independent entities. In this research reflection we intend to stress that the antioxidant system, bone health and the microbiome are intimately intertwined. Antioxidants are crucial for the maintenance of redox homeostasis and optimal immune function. Optimal gastrointestinal functionality is important to maintain animal performance, health and welfare. In particular, the intestinal microbiome is increasingly seen as a driver of health and disease. Vitamin D metabolism is pivotal not only for optimal skeletal health, but in light of all the extra-skeletal effect of vitamin D, it is the foundation for optimal productive life. It makes sense to ask the question 'how are redox balance and the microbiome involved in the modulation of bone health and immune function?' In other words, are bugs and bones connected in dairy cows! The existing data available in the literature suggests that this might be the case. The characterization of the interactions between redox balance, skeletal health and the microbiome, will allow the development of a multisystem biological approach to refine nutritional interventions to improve dairy cattle health, welfare and productive longevity.

Estimating contributions of pelagic and benthic pathways to consumer production in coupled marine food webs.

Pelagic and benthic systems usually interact, but their dynamics and production rates differ. Such differences influence the distribution, reproductive cycles, growth rates, stability and productivity of the consumers they support. Consumer preferences for, and dependence on, pelagic or benthic production are governed by the availability of these sources of production and consumer life history, distribution, habitat, behavioural ecology, ontogenetic stage and morphology. Diet studies may demonstrate the extent to which consumers feed on prey in pelagic or benthic environments. But they do not discriminate benthic production directly supported by phytoplankton from benthic production recycled through detrital pathways. The former will track the dynamics of phytoplankton production more closely than the latter. We develop and apply a new analytical method that uses carbon (C) and sulphur (S) natural abundance stable isotope data to assess the relative contribution of pelagic and benthic pathways to fish consumer production. For 13 species of fish that dominate community biomass in the northern North Sea (estimated >90% of total biomass), relative modal use of pelagic pathways ranged from <25% to >85%. Use of both C and S isotopes as opposed to just C reduced uncertainty in relative modal use estimates. Temporal comparisons of relative modal use of pelagic and benthic pathways revealed similar ranking of species dependency over 4 years, but annual variation in relative modal use within species was typically 10%-40%. For the total fish consumer biomass in the study region, the C and S method linked approximately 70% and 30% of biomass to pelagic and benthic pathways, respectively. As well as providing a new method to define consumers' links to pelagic and benthic pathways, our results demonstrate that a substantial proportion of fish biomass, and by inference production, in the northern North Sea is supported by production that has passed through transformations on the seabed.

An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model.

Percutaneous Device Closure of Paravalvular Leak: Combined Experience From the United Kingdom and Ireland.

BACKGROUND: Paravalvular leak (PVL) occurs in 5% to 17% of patients following surgical valve replacement. Percutaneous device closure represents an alternative to repeat surgery. METHODS: All UK and Ireland centers undertaking percutaneous PVL closure submitted data to the UK PVL Registry. Data were analyzed for association with death and major adverse cardiovascular events (MACE) at follow-up. RESULTS: Three hundred eight PVL closure procedures were attempted in 259 patients in 20 centers (2004-2015). Patient age was 67±13 years; 28% were female. The main indications for closure were heart failure (80%) and hemolysis (16%). Devices were successfully implanted in 91% of patients, via radial (7%), femoral arterial (52%), femoral venous (33%), and apical (7%) approaches. Nineteen percent of patients required repeat procedures. The target valve was mitral (44%), aortic (48%), both (2%), pulmonic (0.4%), or transcatheter aortic valve replacement (5%). Preprocedural leak was severe (61%), moderate (34%), or mild (5.7%) and was multiple in 37%. PVL improved postprocedure (P<0.001) and was none (33.3%), mild (41.4%), moderate (18.6%), or severe (6.7%) at last follow-up. Mean New York Heart Association class improved from 2.7±0.8 preprocedure to 1.6±0.8 (P<0.001) after a median follow-up of 110 (7-452) days. Hospital mortality was 2.9% (elective), 6.8% (in-hospital urgent), and 50% (emergency) (P<0.001). MACE during follow-up included death (16%), valve surgery (6%), late device embolization (0.4%), and new hemolysis requiring transfusion (1.6%). Mitral PVL was associated with higher MACE (hazard ratio [HR], 1.83; P=0.011). Factors independently associated with death were the degree of persisting leak (HR, 2.87; P=0.037), New York Heart Association class (HR, 2.00; P=0.015) at follow-up and baseline creatinine (HR, 8.19; P=0.001). The only factor independently associated with MACE was the degree of persisting leak at follow-up (HR, 3.01; P=0.002). CONCLUSION: Percutaneous closure of PVL is an effective procedure that improves PVL severity and symptoms. Severity of persisting leak at follow-up is independently associated with both MACE and death. Percutaneous closure should be considered as an alternative to repeat surgery.

Factors influencing ruminal bacterial community diversity and composition and microbial fibrolytic enzyme abundance in lactating dairy cows with a focus on the role of active dry yeast.

The objective of the current study was to employ a DNA-based sequencing technology to study the effect of active dry yeast (ADY) supplementation, diet type, and sample location within the rumen on rumen bacterial community diversity and composition, and to use an RNA-based method to study the effect of ADY supplementation on rumen microbial metabolism during high-grain feeding (HG). Our previous report demonstrated that the supplementation of lactating dairy cows with ADY attenuated the effect of subacute ruminal acidosis. Therefore, we used samples from that study, where 16 multiparous, rumen-cannulated lactating Holstein cows were randomly assigned to 1 of 2 dietary treatments: ADY (Saccharomyces cerevisiae strain Y1242, 80 billion cfu/animal per day) or control (carrier only). Cows received a high-forage diet (77:23, forage:concentrate), then were abruptly switched to HG (49:51, forage:concentrate). Rumen bacterial community diversity and structure were highly influenced by diet and sampling location (fluid, solids, epimural). The transition to HG reduced bacterial diversity, but epimural bacteria maintained a greater diversity than fluid and solids. Analysis of molecular variance indicated a significant separation due to diet × sampling location, but not due to treatment. Across all samples, the analysis yielded 6,254 nonsingleton operational taxonomic units (OTU), which were classified into several phyla: mainly Firmicutes, Bacteroidetes, Fibrobacteres, Tenericutes, and Proteobacteria. High forage and solids were dominated by OTU from Fibrobacter, whereas HG and fluid were dominated by OTU from Prevotella. Epimural samples, however, were dominated in part by Campylobacter. Active dry yeast had no effect on bacterial community diversity or structure. The phylum SR1 was more abundant in all ADY samples regardless of diet or sampling location. Furthermore, on HG, OTU2 and OTU3 (both classified into Fibrobacter succinogenes) were more abundant with ADY in fluid and solids than control samples. This increase with ADY was paralleled by a reduction in prominent Prevotella OTU. Metatranscriptomic profiling of rumen microbiome conducted on random samples from the HG phase showed that ADY increased the abundance of the cellulase endo-ß-1,4-glucanase and had a tendency to increase the hemicellulase α-glucuronidase. In conclusion, the shift from high forage to HG and sampling location had a more significant influence on ruminal bacterial community abundance and structure compared with ADY. However, evidence suggested that ADY can increase the abundance of some dominant anaerobic OTU belonging to F. succinogenes and phylum SR1. Further, microbial mRNA-based evidence suggested that ADY can increase the abundance of a specific microbial fibrolytic enzymes.

Prolongation of atrio-ventricular node conduction in a rabbit model of ischaemic cardiomyopathy: Role of fibrosis and connexin remodelling.

Conduction abnormalities are frequently associated with cardiac disease, though the mechanisms underlying the commonly associated increases in PQ interval are not known. This study uses a chronic left ventricular (LV) apex myocardial infarction (MI) model in the rabbit to create significant left ventricular dysfunction (LVD) 8weeks post-MI. In vivo studies established that the PQ interval increases by approximately 7ms (10%) with no significant change in average heart rate. Optical mapping of isolated Langendorff perfused rabbit hearts recapitulated this result: time to earliest activation of the LV was increased by 14ms (16%) in the LVD group. Intra-atrial and LV transmural conduction times were not altered in the LVD group. Isolated AVN preparations from the LVD group demonstrated a significantly longer conduction time (by approximately 20ms) between atrial and His electrograms than sham controls across a range of pacing cycle lengths. This difference was accompanied by increased effective refractory period and Wenckebach cycle length, suggesting significantly altered AVN electrophysiology post-MI. The AVN origin of abnormality was further highlighted by optical mapping of the isolated AVN. Immunohistochemistry of AVN preparations revealed increased fibrosis and gap junction protein (connexin43 and 40) remodelling in the AVN of LVD animals compared to sham. A significant increase in myocyte-non-myocyte connexin co-localization was also observed after LVD. These changes may increase the electrotonic load experienced by AVN muscle cells and contribute to slowed conduction velocity within the AVN.

Effects of dietary supplementation with 3-nitrooxypropanol on enteric methane production, rumen fermentation, and performance in young growing beef cattle offered a 50:50 forage:concentrate diet.

There is an urgent requirement internationally to reduce enteric methane (CH4) emissions from ruminants to meet greenhouse gas emissions reduction targets. Dietary supplementation with feed additives is one possible strategy under investigation as an effective solution. The effects of the CH4 inhibitor 3-nitrooxypropanol (3-NOP) at reducing CH4 emissions in beef have been shown mainly in adult cattle consuming backgrounding and high-energy finishing diets. In this study, the effects of dietary supplementation of young growing (≤6 mo) beef cattle with 3-NOP were examined in a 50:50 forage:concentrate diet. A total of 68 Dairy × Beef (Aberdeen Angus and Hereford dairy cross) male calves (≤6 mo of age at the start of experiment, body weight: 147 ±â€…38 kg) underwent a 3-wk acclimatization period and were then assigned to one of two treatments in a completely randomized block design. Dietary treatments were (1) control, placebo (no 3-NOP), and (2) 3-NOP applied at 150 mg kg-1 DM. Calves were fed a partial mixed ration for 12 wk. Body weight was recorded weekly and feed intake daily using the Calan Broadbent feeding system. Methane and hydrogen emissions were measured using the GreenFeed system. Total weight gained, dry matter intake (DMI), and average daily gain were not affected by 3-NOP (P > 0.05) supplementation. On average, the inclusion of 3-NOP decreased (P < 0.001) CH4 emissions: g d-1; g kg-1 DMI; by 30.6% and 27.2%, respectively, during the study with a greater reduction occurring over time. Incorporating 3-NOP into beef cattle diets is an efficient solution to decrease CH4 emissions during indoor feeding and when offered 50:50 forage:concentrate diet.

Enteric methane (CH4) is a by-product from the fermentation of feed in the digestive tract of cattle. The production of CH4 is responsible for the loss of 2% to 12% of the animal's gross energy intake. A potent greenhouse gas, CH4 from ruminant systems accounts for 30% of international anthropogenic CH4 emissions. As a result, a significant effort has been made internationally to reduce CH4 emissions from ruminants in order to achieve reductions in global greenhouse gas emissions. The supplementation of additives in the feed has been demonstrated to be an effective strategy in reducing CH4 emitted from livestock. The purpose of this research was to investigate the effects of supplementing young growing cattle with the CH4 inhibitor, 3-nitrooxypropanol (3-NOP), consuming a 50:50 forage:concentrate diet. A total of 68 Dairy × Beef (Aberdeen Angus and Hereford dairy cross) male calves (≤6 mo of age at the start of the experiment) were assigned to one of two treatments: control (no 3-NOP) and 3-NOP. Animals received their diets for 12 wk. Animal performance was recorded weekly, with CH4 and hydrogen (H2) emissions recorded daily. Dry matter intake and animal performance were not affected by the inclusion of 3-NOP. Over the duration of this study, the inclusion of 3-NOP decreased daily CH4 emissions by 30.6%, with a 227% increase in daily H2 emissions.

A glucan-particle based tularemia subunit vaccine induces T-cell immunity and affords partial protection in an inhalation rat infection model.

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising ß-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community.

Effect of 3-nitrooxypropanol on enteric methane emissions of feedlot cattle fed with a tempered barley-based diet with canola oil.

A dose-response experiment was designed to examine the effect of 3-nitrooxypropanol (3-NOP) on methane (CH4) emissions, rumen function and performance of feedlot cattle fed a tempered barley-based diet with canola oil. Twenty Angus steers of initial body weight (BW) of 356 ±â€…14.4 kg were allocated in a randomized complete block design. Initial BW was used as the blocking criterion. Cattle were housed in individual indoor pens for 112 d, including the first 21 d of adaptation followed by a 90-d finishing period when five different 3-NOP inclusion rates were compared: 0 mg/kg dry matter (DM; control), 50 mg/kg DM, 75 mg/kg DM, 100 mg/kg DM, and 125 mg/kg DM. Daily CH4 production was measured on day 7 (last day of starter diet), day 14 (last day of the first intermediate diet), and day 21 (last day of the second intermediate diet) of the adaptation period and on days 28, 49, 70, 91, and 112 of the finisher period using open circuit respiration chambers. Rumen digesta samples were collected from each steer on the day prior to chamber measurement postfeeding, and prefeeding on the day after the chamber measurement, for determination of rumen volatile fatty acids (VFA), ammonium-N, protozoa enumeration, pH, and reduction potential. Dry matter intake (DMI) was recorded daily and BW weekly. Data were analyzed in a mixed model including period, 3-NOP dose and their interaction as fixed effects, and block as a random effect. Our results demonstrated both a linear and quadratic (decreasing rate of change) effect on CH4 production (g/d) and CH4 yield (g/kg DMI) as 3-NOP dose increased (P < 0.01). The achieved mitigation for CH4 yield in our study ranged from approximately 65.5% up to 87.6% relative to control steers fed a finishing feedlot diet. Our results revealed that 3-NOP dose did not alter rumen fermentation parameters such as ammonium-N, VFA concentration nor VFA molar proportions. Although this experimental design was not focused on the effect of 3-NOP dose on feedlot performance, no negative effects of any 3-NOP dose were detected on animal production parameters. Ultimately, the knowledge on the CH4 suppression pattern of 3-NOP may facilitate sustainable pathways for the feedlot industry to lower its carbon footprint.

Livestock methane (CH4) is the main source of greenhouse gases (GHGs) in agriculture, contributing to 11.6% of global GHGs emissions from human-related activities. Therefore, mitigating CH4 emissions from ruminant animals is a great opportunity for meeting the current climate targets. In this experiment, increasing inclusion rates of a promising CH4-mitigating compound, 3-nitrooxypropanol (3-NOP, from 50 to 125 mg of 3-NOP/kg of dry matter [DM]), were added to a barley-based feedlot diet containing 25 ppm of monensin and 7% fat (DM-basis) and fed to Angus steers. Under these conditions, increasing inclusion rate of 3-NOP reduced both production and yield of CH4 by up to 90%. Rumen fermentation, feed intake, and average daily gain were not affected by the 3-NOP dose. Our results on the potential CH4 suppression of 3-NOP may assist the feedlot industry towards sustainability by lowering its GHG output.

Molecular recognition of chymotrypsin by the serine protease inhibitor ecotin from Yersinia pestis.

Resistance to antibiotics is a problem not only in terms of healthcare but also biodefense. Engineering of resistance into a human pathogen could create an untreatable biothreat pathogen. One such pathogen is Yersinia pestis, the causative agent of plague. Previously, we have used a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. The serine protease inhibitor ecotin was identified as one such target. We have carried out mutational analyses in the closely related Yersinia pseudotuberculosis, validating that the ecotin gene is a virulence-associated gene in this bacterium. Y. pestis ecotin inhibits chymotrypsin. Here, we present the structure of ecotin in complex with chymotrypsin to 2.74 Å resolution. The structure features a biologically relevant tetramer whereby an ecotin dimer binds to two chymotrypsin molecules, similar to what was observed in related serine protease inhibitor structures. However, the vast majority of the interactions in the present structure are distinctive, indicating that the broad specificity of the inhibitor for these proteases is based largely on its capacity to recognize features unique to each of them. These findings will have implications for the development of small ecotin inhibitors for therapeutic use.

CD4+ T-cell immunity to the Burkholderia pseudomallei ABC transporter LolC in melioidosis.

Burkholderia pseudomallei causes melioidosis, a disease with a wide range of possible outcomes, from seroconversion and dormancy to sepsis and death. This spectrum of host-pathogen interactions poses challenging questions about the heterogeneity in immunity to B. pseudomallei. Models show protection to be dependent on CD4(+) cells and IFN-γ, but little is known about specific target antigens. Having previously implicated the ABC transporter, LolC, in protective immunity, we here use epitope prediction, HLA-binding studies, HLA-transgenic models and studies of T cells from seropositive individuals to characterize HLA-restricted LolC responses. Immunized mice showed long-lasting memory to the protein, whereas predictive algorithms identified epitopes within LolC that subsequently demonstrated strong HLA class II binding. Immunization of HLA-DR transgenics with LolC stimulated T-cell responses to four of these epitopes. Furthermore, the responsiveness of HLA transgenics to LolC revealed a hierarchy supportive of HLA polymorphism-determined differential susceptibility. Seropositive human donors of diverse HLA class II types showed T-cell responses to LolC epitopes, which are conserved among Burkholderia species including Burkholderia cenocepacia, associated with life-threatening cepacia complex in cystic fibrosis patients and Burkholderia mallei, which causes glanders. These findings suggest a role for LolC epitopes in multiepitope vaccine design for melioidosis and related diseases.