Astrocyte-specific Ca2+ activity: Mechanisms of action, experimental tools, and roles in ethanol-induced dysfunction.

Astrocytes are a subtype of non-neuronal glial cells that reside in the central nervous system. Astrocytes have extensive peripheral astrocytic processes that ensheathe synapses to form the tripartite synapse. Through a multitude of pathways, astrocytes can influence synaptic development and structural maturation, respond to neuronal signals, and modulate synaptic transmission. Over the last decade, strong evidence has emerged demonstrating that astrocytes can influence behavioral outcomes in various animal models of cognition. However, the full extent of how astrocytes influence brain function is still being revealed. Astrocyte calcium (Ca2+) signaling has emerged as an important driver of astrocyte-neuronal communication allowing intricate crosstalk through mechanisms that are still not fully understood. Here, we will review the field's current understanding of astrocyte Ca2+ signaling and discuss the sophisticated state-of-the-art tools and approaches used to continue unraveling astrocytes' interesting role in brain function. Using the field of pre-clinical ethanol (EtOH) studies in the context of alcohol use disorder, we focus on how these novel approaches have helped to reveal an important role for astrocyte Ca2+ function in regulating EtOH consumption and how astrocyte Ca2+ dysfunction contributes to the cognitive deficits that emerge after EtOH exposure in a rodent model.

Determinants of exposure to endocrine disruptors following hurricane Harvey.

Hurricane Harvey was a category four storm that induced catastrophic flooding in the Houston metropolitan area. Following the hurricane there was increased concern regarding chemical exposures due to damage caused by flood waters and emergency excess emissions from industrial facilities. This study utilized personal passive samplers in the form of silicone wristbands in Houston, TX to both assess chemical exposure to endocrine disrupting chemicals (EDCs) immediately after the hurricane and determine participant characteristics associated with higher concentrations of exposure. Participants from the Houston-3H cohort (n = 172) wore a wristband for seven days and completed a questionnaire to determine various flood-related and demographic variables. Bivariate and multivariate analysis indicated that living in an area with a high Area Deprivation Index (ADI) (indicative of low socioeconomic status), identifying as Black/African American or Latino, and living in the Houston neighborhoods of Baytown and East Houston were associated with increased exposure to EDCs. These results provide evidence of racial/ethnic and socioeconomic injustices in exposure to EDCs in the Houston Metropolitan Area. Since the multiple regression models conducted did not fully explain exposure (0.047 < R2 < 0.34), more research is needed on the direct sources of EDCs within this area to create effective exposure mitigation strategies.

Adaptive immune response ranking is associated with reproductive phenotypes in grazing dairy cows divergent in genetic merit for fertility traits.

A strong adaptive immune response has been reported to have positive effects on fertility; therefore, we investigated antibody- and cell-mediated adaptive immune responses (AMIR and CMIR, respectively) and their associations with reproductive phenotypes using a population of animals that differed in their estimated genetic merit for fertility traits (fertility breeding value; FertBV). Holstein-Friesian heifers (n = 528) grazed on pasture in 4 herds based on age. These herds included 277 heifers of positive (POS) FertBV and 251 of negative (NEG) FertBV. The adaptive immune response (IR) was evaluated before puberty at 7.5 mo of age and used to rank animals as high, average, or low for AMIR, CMIR, and overall IR (combined CMIR and AMIR). The animals were studied from 12 wk of age through to the end of their second lactation to measure growth, puberty, and timing and success of fertility phenotypes, including those related to ovulation and pregnancy. Initial analysis indicated no difference in fertility outcomes between cows ranked as high or average for AMIR (n = 55, n = 407, respectively), CMIR (n = 87, n = 354, respectively), and IR (n = 29, n = 470, respectively), so these groups were pooled as HiAv-IR. Proportions of heifers of POS FertBV were similar within HiAv and low categories across AMIR (0.52 and 0.58, respectively), CMIR (0.51 and 0.59, respectively), and IR (0.53 and 0.48, respectively). Heifers with HiAv-IR had a greater average daily weight gain from 13 to 52 wk of age (661 g, 95% confidence interval 652, 669 vs. 619 g, 95% confidence interval 591, 647) and tended to be younger at puberty (371 d, 95% confidence interval 366, 377 vs. 385 d, 95% confidence interval 369, 401) than low-IR heifers. Low-CMIR cows of a NEG FertBV had a >40 d longer calving to first ovulation interval during their first lactation compared with HiAv-CMIR NEG FertBV cows. Low-CMIR cows also had decreased pregnancy rates at both 3 wk (25% ± 7% vs. 42% ± 3%; least squares means ± standard error) and 6 wk (33% ± 7% vs. 54% ± 3%; least squares means ± standard error) into the seasonal breeding period during their first lactation, compared with HiAv-CMIR cows. In summary, although the number of POS and NEG FertBV cows was similar in each immune group; interaction effects between FertBV and immune ranking on reproductive phenotypes are evident when cows were ranked by the overall IR. There were also associations between dairy cows' CMIR ranking and ability to return to estrus and become pregnant early in the breeding period, which will have benefits in seasonal breeding systems.

Markers of human endometrial hypoxia can be detected in vivo and ex vivo during physiological menstruation.

STUDY QUESTION: Can markers of human endometrial hypoxia be detected at menstruation in vivo? SUMMARY ANSWER: Our in vivo data support the presence of hypoxia in menstrual endometrium of women during physiological menstruation. WHAT IS KNOWN ALREADY: Current evidence from animal models and human in vitro studies suggests endometrial hypoxia is present at menstruation and drives endometrial repair post menses. However, detection of human endometrial hypoxia in vivo remains elusive. STUDY DESIGN, SIZE, DURATION: We performed a prospective case study of 16 women with normal menstrual bleeding. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductively aged female participants with a regular menstrual cycle underwent objective measurement of their menstrual blood loss using the alkaline haematin method to confirm a loss of <80 ml per cycle. Exclusion criteria were exogenous hormone use, an intrauterine device, endometriosis or fibroids >3 cm. Participants attended for two MRI scans; during days 1-3 of menstruation and the early/mid-secretory phase of their cycle. The MRI protocol included dynamic contrast-enhanced MRI and T2* quantification. At each visit, an endometrial sample was also collected and hypoxia-regulated repair factor mRNA levels (ADM, VEGFA, CXCR4) were quantified by RT-qPCR. MAIN RESULTS AND THE ROLE OF CHANCE: Women had reduced T2* during menstrual scans versus non-menstrual scans (P = 0.005), consistent with menstrual hypoxia. Plasma flow (Fp) was increased at menstruation compared to the non-menstrual phase (P = 0.0005). Laboratory findings revealed increased ADM, VEGF-A and CXCR4 at menstruation on examination of paired endometrial biopsies from the menstrual and non-menstrual phase (P = 0.008; P = 0.03; P = 0.009). There was a significant correlation between T2* and these ex vivo hypoxic markers (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study examined the in vivo detection of endometrial hypoxic markers at specific timepoints in the menstrual cycle in women with a menstrual blood loss <80 ml/cycle and without significant uterine structural abnormalities. Further research is required to determine the presence of endometrial hypoxia in those experiencing abnormal uterine bleeding with and without fibroids/adenomyosis. WIDER IMPLICATIONS OF THE FINDINGS: Heavy menstrual bleeding (HMB) is a common, debilitating condition. Understanding menstrual physiology may improve therapeutics. To our knowledge, this is the first in vivo data supporting the presence of menstrual hypoxia in the endometrium of women with normal menstrual bleeding. If aberrant in those with HMB, these non-invasive tests may aid diagnosis and facilitate personalized treatments for HMB. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by Wellbeing of Women grant RG1820, Wellcome Trust Fellowship 209589/Z/17/Z and undertaken in the MRC Centre for Reproductive Health, funded by grants G1002033 and MR/N022556/1. H.O.D.C. has clinical research support for laboratory consumables and staff from Bayer AG and provides consultancy advice (but with no personal remuneration) for Bayer AG, PregLem SA, Gedeon Richter, Vifor Pharma UK Ltd, AbbVie Inc; Myovant Sciences GmbH. H.O.D.C. receives royalties from UpToDate for articles on abnormal uterine bleeding. TRIAL REGISTRATION NUMBER: N/A.

Next generation secondary electron detector with energy analysis capability for SEM.

We report the working of a novel detector design based on a Bessel Box (BB) electron energy analyser in a scanning electron microscope (SEM). We demonstrate the application of our detector for elemental identification through Auger electron detection in an SEM environment and its potential as a complementary technique to energy dispersive X-ray (EDX) spectroscopy. We also demonstrate energy-filtered secondary electron imaging of a copper-on-silicon sample using an electron pass energy of 12 eV. LAY DESCRIPTION: Advancements in the field of the Scanning Electron Microscopy have been one of the major nanotechnology enablers. A Scanning Electron Microscope (SEM) generates a magnified image of the sample by bombarding it with an electron beam and detecting the electrons that scatter off the surface along with the electrons that are generated in the sample. Conventional detectors such as the Everhart-Thornley detector (ET) or through-the-lens (TTL) detectors, either offer little to no energy analysis (ET) or limited energy filtering capability (e.g the low-pass energy filter in TTL). This information is crucial to interpret the image of the sample under study. What is needed is a smart and compact detector that can detect electrons and furnish energy inside the SEM chamber. Here, we report a novel secondary electron (SE) detector design with energy analysis capability for use in scanning electron microscopes. The detector is based on the design of a Bessel Box (BB) energy analyser. We have designed and experimentally tested it in an SEM environment. The band-pass filter action of the detector enables the BB to be operated at a selected energy and allows a narrow window of energies to be detected for generating energy-filtered images.

An Evaluation of the Hybrid Model for Predicting Surgery Duration.

The degree of accuracy in surgery duration estimation directly impacts on the quality of planned surgical lists. Model selection for the prediction of surgery duration requires technical expertise and significant time and effort. The result is often a collection of viable models, the performance of which varies across different strata of the surgical population. This paper proposes a prediction framework to be used after a comprehensive model selection process has been completed for surgery duration prediction. The framework produces a partition of the surgical cases and a "hybrid model" that allocates different predictors from the collection of viable models to different parts of the surgical population. The intention is a flexible prediction process that can reassign models and adapt as surgical processes change. The framework is tested via a simulation study, and its utility is demonstrated by predicting surgery durations for Ear, Nose and Throat surgeries in a New Zealand hospital. The results indicate that the hybrid model is effective, performing better than standard model selection in two of the three simulation studies, and marginally worse when the selected model was the true underlying process.

Introduction of an Alcohol-Related Electronic Screening and Brief Intervention (eSBI) Program to Reduce Hazardous Alcohol Consumption in Namibia's Antiretroviral Treatment (ART) Program.

Alcohol is the most widely abused substance in Namibia and is associated with poor adherence and retention in care among people on antiretroviral therapy (ART). Electronic screening and brief interventions (eSBI) are effective in reducing alcohol consumption in various contexts. We used a mixed methods approach to develop, implement, and evaluate the introduction of an eSBI in two ART clinics in Namibia. Of the 787 participants, 45% reported some alcohol use in the past 12 months and 25% reported hazardous drinking levels. Hazardous drinkers were more likely to be male, separated/widowed/divorced, have a monthly household income > $1000 NAD, and report less than excellent ART adherence. Based on qualitative feedback from participants and providers, ART patients using the eSBI for the first time found it to be a positive and beneficial experience. However, we identified several programmatic considerations that could improve the experience and yield in future implementation studies.

Transcriptomic analysis of circulating neutrophils in metabolically stressed peripartal grazing dairy cows.

The high metabolic demand during the transition into lactation places cows at greater risk of metabolic and infectious disease than at any other time in their lactation cycle. Additionally, a change occurs in the innate immune response during this period, which contributes to increased risk of disease. In the current study, we compared the transcriptomes of neutrophils from dairy cows divergent in their metabolic health post-calving. Cows (n = 5 per risk group) were selected from a parent experiment (n = 45 cows). Those with high or low concentrations of plasma nonesterified fatty acids, plasma ß-hydroxybutyrate, and liver triacylglycerol in both wk 1 and 2 were deemed to be at "high risk" (HR) or "low risk" (LR) of metabolic dysfunction, respectively. Circulating neutrophils were isolated at 3 time points during the transition period (d 0 and wk 1 and 4 post-calving), and gene expression was analyzed using RNA sequencing. Differential gene expression between the risk groups was determined using edgeR (http://bioconductor.org), and pathway analysis was conducted using Ingenuity Pathway Analysis (Ingenuity Systems, Qiagen, Valencia, CA). Statistical analysis indicated no interaction between risk and week. Therefore, the overall effect of risk was analyzed across all time points. In total, 3,500 genes were differentially expressed between the HR and LR cows (false discovery rate < 0.05). Of these, 2,897 genes were identified by Ingenuity Pathway Analysis and used for pathway analysis. Of the relevant pathways identified, neutrophils isolated from HR cows showed downregulation of genes involved in the recruitment of granulocytes, interferon signaling, and apoptosis, and upregulation of genes involved in cell survival. The results indicate that metabolically stressed cows had reduced neutrophil function during the peripartum period, highlighting a potential relationship between subclinical metabolic disease and innate immune function that suggests that metabolic health negatively affects the innate immune system and may contribute to the state of immunosuppression during the peripartum period. In this way, the metabolic stress among the HR cows may reduce their ability to combat infection during the transition period.

Widespread prevalence of a CREBRF variant amongst Maori and Pacific children is associated with weight and height in early childhood.

OBJECTIVE: Investigating a large and ethnically diverse cohort from the Pacific region, we aimed to replicate and extend the recently reported findings that a CREBRF genetic variant is strongly associated with body mass index in Samoans. METHODS: A birth cohort of more than six thousand children was utilised. In this study, genotyping of two markers (rs12513649 and rs373863828) was undertaken in Maori, Pacific, European and Asian individuals in the cohort. RESULTS: We report that these CREBRF genetic variants are not confined to Samoans but are prevalent in all other Pacific populations sampled, including Maori. We found that the rs373863828 variant was significantly associated with growth at 4 years of age. On average, we observed allele-specific increases in weight (P=0·004, +455 g, s.e. 0.158), height (P=0·007, +0·70 cm, s.e. 0.26) and waist circumference (P=0·004, +0·70 cm, s.e. 0.24) at 4 years of age. The rs373863828 variant was not associated with birth weight (P=0·129). CONCLUSIONS: We replicated the finding that a CREBRF variant is associated with increased body mass. We then built on the original findings by demonstrating the prevalence of the rs12513649 and rs373863828 variants in multiple Pacific population groups and by demonstrating that the rs373863828 variant is associated with growth in early childhood. Pacific population groups experience a disproportionately high burden of obesity, starting in early childhood. This new knowledge offers potential for evidence-based interventions aimed at establishing healthy growth trajectories from the earliest possible age.

Early morphologic changes in trapeziometacarpal joint bones with osteoarthritis.

OBJECTIVE: Characterising the morphological differences between healthy and early osteoarthritic (EOA) trapeziometacarpal (TMC) joints is important for understanding osteoarthritis onset, and early detection is important for treatment and disease management. This study has two aims: first, to characterise morphological differences between healthy and EOA TMC bones. The second aim was to determine the efficacy of using a statistical shape model (SSM) to detect early signs of osteoarthritis (OA). METHODS: CT image data of TMC bones from 22 asymptomatic volunteers and 47 patients with EOA were obtained from an ongoing study and used to generate a SSM. A linear discriminant analysis (LDA) classifier was trained on the principal component (PC) weights to characterise features of each group. Multivariable statistical analysis was performed on the PC to investigate morphologic differences. Leave-one-out classification was performed to evaluate the classifiers performance. RESULTS: We found that TMC bones of EOA subjects exhibited a lower aspect ratio (P = 0.042) compared with healthy subjects. The LDA classifier predicted that protrusions (up to 1.5 mm) at the volar beak of the first metacarpal were characteristic of EOA subjects. This was accompanied with widening of the articular surface, deepening of the articular surface, and protruding bone growths along the concave margin. These characteristics resulted in a leave-one-out classification accuracy of 73.9% (95% CI [61.9%, 83.8%]), sensitivity of 89.4%, specificity of 40.9%, and precision of 75.9%. CONCLUSION: Our findings indicate that morphological degeneration is well underway in the EOA TMC joint, and shows promise for a clinical tool that can detect these features automatically.

A randomized controlled trial protocol assessing the effectiveness, safety and cost-effectiveness of methotrexate vs. ciclosporin in the treatment of severe atopic eczema in children: the TREatment of severe Atopic eczema Trial (TREAT).

BACKGROUND: Oral systemic immunomodulatory medication is regularly used off-licence in children with severe atopic eczema. However, there is no firm evidence regarding the effectiveness, safety, cost-effectiveness and impact on quality of life from an adequately powered randomized controlled trial (RCT) using systemic medication in children. OBJECTIVES: To assess whether there is a difference in the speed of onset, effectiveness, side-effect profile and reduction in flares post-treatment between ciclosporin (CyA) and methotrexate (MTX), and also the cost-effectiveness of the drugs. Treatment impact on quality of life will also be examined in addition to whether FLG genotype influences treatment response. In addition, the trial studies the immune-metabolic effects of CyA and MTX. METHODS: Multicentre, parallel group, assessor-blind, pragmatic RCT of 36 weeks' duration with a 24-week follow-up period. In total, 102 children aged 2-16 years with moderate-to-severe atopic eczema, unresponsive to topical treatment will be randomized (1 : 1) to receive MTX (0·4 mg kg-1 per week) or CyA (4 mg kg-1 per day). RESULTS: The trial has two primary outcomes: change from baseline to 12 weeks in Objective Severity Scoring of Atopic Dermatitis (o-SCORAD) and time to first significant flare following treatment cessation. CONCLUSIONS: This trial addresses important therapeutic questions, highlighted in systematic reviews and treatment guidelines for atopic eczema. The trial design is pragmatic to reflect current clinical practice.

Acceptability of latent tuberculosis testing of migrants in a college environment in England.

OBJECTIVES: The majority of tuberculosis (TB) cases in England occur from reactivation of latent tuberculosis infection (LTBI) in the settled migrant population. The National Institute for Health and Clinical Excellence recommends that new entrants from high-incidence countries are screened to detect LTBI. This article seeks to describe an outreach programme and testing for LTBI in an innovative setting-ESOL (English for Speakers of Other Languages) classes at a community college (CC) with evaluation of acceptability. STUDY DESIGN: Partnership working with mixed methods used for evaluation of acceptability. METHODS: A pre-existing network from the local TB partnership designed an outreach intervention and screening for LTBI among students from an ESOL programme at a CC. Screening for LTBI with interferon gamma release assay was the culmination of a programme of health improvement activities across the college. Any student on the ESOL programme younger than the age of 35 years and resident in the UK for less than 5 years was eligible for testing. LTBI testing was carried out on-site, and the experience was evaluated by questionnaires to staff, students and partners. A facilitated debrief among the partners gave further data. RESULTS: A total of 440 eligible students were tested. One hundred and seventy-two student feedback questionnaires were completed, and 36 partner questionnaires were received with 18 CC staff responding. Students, tutors and healthcare professionals found the setting acceptable with some concerns about insufficient resource for timely follow-up. CONCLUSIONS: Students, tutors, community organisations and health professionals found the exercise worthwhile and the method and setting acceptable. There were resource issues for the clinical team in follow-up of students with positive results for such a large screening event. Unexpected barriers were found by the CC as this kind of activity was not recognised for external quality review purposes. There were concerns about reputational loss and stigma of being involved in a TB project. As current initiatives aim to divert workload from stretched general practice surgeries, this may be an important addition to primary care screening.

Accelerating wheat breeding for end-use quality with multi-trait genomic predictions incorporating near infrared and nuclear magnetic resonance-derived phenotypes.

KEY MESSAGE: Using NIR and NMR predictions of quality traits overcomes a major barrier for the application of genomic selection to accelerate improvement in grain end-use quality traits of wheat. Grain end-use quality traits are among the most important in wheat breeding. These traits are difficult to breed for, as their assays require flour quantities only obtainable late in the breeding cycle, and are expensive. These traits are therefore an ideal target for genomic selection. However, large reference populations are required for accurate genomic predictions, which are challenging to assemble for these traits for the same reasons they are challenging to breed for. Here, we use predictions of end-use quality derived from near infrared (NIR) or nuclear magnetic resonance (NMR), that require very small amounts of flour, as well as end-use quality measured by industry standard assay in a subset of accessions, in a multi-trait approach for genomic prediction. The NIR and NMR predictions were derived for 19 end-use quality traits in 398 accessions, and were then assayed in 2420 diverse wheat accessions. The accessions were grown out in multiple locations and multiple years, and were genotyped for 51208 SNP. Incorporating NIR and NMR phenotypes in the multi-trait approach increased the accuracy of genomic prediction for most quality traits. The accuracy ranged from 0 to 0.47 before the addition of the NIR/NMR data, while after these data were added, it ranged from 0 to 0.69. Genomic predictions were reasonably robust across locations and years for most traits. Using NIR and NMR predictions of quality traits overcomes a major barrier for the application of genomic selection for grain end-use quality traits in wheat breeding.

Technical note: Evaluation of endogenous control gene expression in bovine neutrophils by reverse-transcription quantitative PCR using microfluidics gene expression arrays.

Reverse-transcription quantitative-PCR (RT-qPCR) is commonly used for assessing the cellular response to changes in physiologic and pathologic conditions. The selection of stable endogenous control genes is an important step of any RT-qPCR study, as expression can vary depending on the experimental environment. Our objective was to identify endogenous control genes in circulating neutrophils isolated from cows during the peripartum period. To do this, we used microfluidics gene expression arrays (Fluidigm, San Francisco, CA) for RT-qPCR analysis. Selection of the endogenous control genes was based on previous research investigating gene expression in neutrophils. The selected genes included ACTB, B2M, G6PD, GAPDH, GCH1, GOLGA5, OSBPL2, PGK1, RPL13A, RPL19, RPS9, SDHA, SMUG1, SNRPA, TBP, UXT, and YWHAZ. Four genes (GAPDH, GOLGA5, PGK1, and UXT) did not provide satisfactory quantification results using the selected method and were therefore excluded from the analyses. The suitability of the remaining 13 genes for use as endogenous control genes was assessed using geNorm and Normfinder. The gene pair with the greatest stability using geNorm was RPL13A and RPL19, whereas Normfinder ranked RPL19 and YWHAZ as the most stable pair. The 2 genes deemed most suitable for the experimental design were RPL19 and YWHAZ, which were selected for subsequent gene expression analysis. This study highlights that genes used as endogenous controls for relative quantification should be assessed on an experimental basis, even if the genes have been used in previous research.

Strategies to gain body condition score in pasture-based dairy cows during late lactation and the far-off nonlactating period and their interaction with close-up dry matter intake.

In pasture-based systems, cows are generally thinner at the end of lactation than cows fed total mixed rations and, as a result, over-feeding of metabolizable energy (ME) during the far-off nonlactating period is a standard management policy to achieve optimum calving body condition score (BCS). An alternative would be to manage cows to gain BCS through late lactation, such that cows ended lactation close to optimum calving BCS and maintenance of BCS through to calving. We sought to quantify the effect of moderate or excessive ME intakes during the far-off nonlactating period in cows that had been managed to gain or maintain BCS through late lactation and whether the far-off management strategy interacted with close-up level of feeding. Effects on milk production and circulating indicators of energy balance and metabolic health in early lactation were evaluated. A herd of 150 cows was randomly assigned to 1 of 2 feeding levels in late lactation to achieve a low and high BCS at the time of dry-off (approximately 4.25 and 5.0 on a 10-point scale). Following dry-off, both herds were managed to achieve a BCS of 5.0 one month before calving; this involved controlled feeding (i.e., maintenance) and over-feeding of ME during the far-off dry period. Within each far-off feeding-level treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement (i.e., 25 cows/treatment). Body weight and BCS were measured weekly before and after calving, and milk production was measured weekly until wk 7 postcalving. Blood samples were collected weekly for 4 wk pre-calving and 5 wk postcalving, and on d 0, 1, 2, 3, and 4 relative to calving, and analyzed for indicators of energy balance (e.g., blood fatty acids, ß-hydroxybutyrate), calcium status, and inflammatory state. No interaction was observed between far-off and close-up feeding levels. Over-feeding of ME to low BCS cows during the far-off nonlactating period reduced blood fatty acid and ß-hydroxybutyrate concentrations in early lactation, and increased blood albumin to globulin ratio compared with cows that were dried off close to recommended calving BCS and control-fed during the far-off dry period. Cows consuming 65% of their ME requirements during the close-up period had lower fatty acids and ß-hydroxybutyrate in early lactation, but produced less milk, particularly during the first 21 d of lactation, had more than 3-fold greater concentration of haptoglobin immediately postcalving, and had a lower blood cholesterol concentration and albumin to globulin ratio, when compared with cows offered 90 or 120% of their ME requirements. Collectively, these measurements indicate that a severe restriction (<70% of ME requirements) during the close-up nonlactating period increases the risk of disease in early lactation and reduces milk production. In summary, far-off over-feeding of ME to cows that needed to gain BCS did not influence peripartum metabolic health in grazing dairy cows, but restricting cows below 70% ME requirements during the close-up transition period resulted in a blood profile indicative of greater inflammation.

Effect of circulating exosomes from transition cows on Madin-Darby bovine kidney cell function.

The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of ß-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of ß-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 103 Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.

Far-off and close-up dry matter intake modulate indicators of immunometabolic adaptations to lactation in subcutaneous adipose tissue of pasture-based transition dairy cows.

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (-1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off nutritional management.

Effects of precalving body condition and prepartum feeding level on gene expression in circulating neutrophils.

Extensive metabolic, physiological, and immunological changes are associated with calving and the onset of lactation. As a result, cows transitioning between pregnancy and lactation are at a greater risk of metabolic and infectious diseases. The ability of neutrophils to mount an effective immune response to an infection is critical for its resolution, and increasing evidence indicates that precalving nutrition affects postpartum neutrophil function. The objectives of the current study were to investigate the effect of 2 precalving body condition scores (BCS; 4 vs. 5 on a 10-point scale) and 2 levels of feeding (75 vs. 125% of estimated maintenance requirements) on gene expression in circulating neutrophils. We isolated RNA from the neutrophils of cows (n = 45) at 5 time points over the transition period: precalving (-1 wk), day of calving (d 0), and postcalving at wk 1, 2, and 4. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 dynamic array), were used to quantify the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using repeated measures ANOVA with Tukey-Kramer multiple-testing correction to determine treatment effects among weeks. Precalving BCS altered the inflammatory state of neutrophils, with significant increases in overall gene expression of antimicrobial peptides (BNBD4 and DEFB10) and the anti-inflammatory cytokine IL10, and significantly decreased expression of proinflammatory cytokine IL23A in thinner cows (BCS 4) compared with cows calving at BCS 5. Feeding level had a time-dependent effect on gene expression; for example, increased expression of genes involved in leukotriene synthesis (PLA2G4A and ALOX5AP) occurred only at 1 wk postcalving in cows overfed (125% of requirements) precalving compared with those offered 75% of maintenance requirements. Results indicate that precalving body condition and changes in prepartum energy lead to altered gene expression of circulating neutrophils, highlighting the importance of transition cow nutrition for peripartum health.

Determination of the normal reference interval for anti-Müllerian hormone (AMH) in bitches and use of AMH as a potential predictor of litter size.

Anti-Müllerian hormone (AMH) is a reliable endocrine marker of ovarian reserve in many species with extensive literature in both humans and cattle. However, there are no known hormonal predictors of ovarian reserve and potential reproductive performance in the bitch. A prospective cohort study was performed involving 155 intact bitches of various ages (range 1.2-7.6 years) and breeds that were presented for routine breeding management over a one-year period. All bitches were artificially inseminated with frozen or fresh semen using the transcervical insemination (TCI) technique. AMH concentrations were measured using a commercially available canine AMH ELISA (Ansh Labs® , Texas, USA), which we validated prior to performing the study. The reference interval (RI) for AMH for all bitches in the study, regardless of body weight, was 2.9-21.1 ng/ml. There was a significant effect of bitch size and age on AMH concentrations. The RI for giant breeds was significantly (p < .01) lower (1.75-15.6 ng/ml) than small-sized (5.6-24.2 ng/ml), medium-sized (4.3-23.7 ng/ml) and large-sized (4.3-21.0 ng/ml) bitches. The mean AMH concentration in bitches less than 4 years of age was 12.4 ng/ml, whereas the mean AMH concentration in bitches older than 4 years of age was 10.5 ng/ml (p < .05). For each additional year of age above 1 year of age, AMH concentrations fell by 0.5 ng/ml. There was no effect of AMH concentration on the whelping rate. Smaller breeds had smaller litters (and higher AMH concentrations), but within each breed size category, bitches with higher AMH concentrations had significantly larger litter sizes (p < .01). For each 1 ng/ml increase in AMH, litter size increased by 0.3 pups/litter. In conclusion, we determined a normal reference interval for AMH in bitches based on body weight using a canine-specific assay. In agreement with findings in humans and other species, we found that there is a decline in AMH concentrations with advancing age in bitches. Finally, the significant positive correlation between AMH concentrations and litter size indicates that AMH may be a useful management tool for the selection of bitches in breeding programmes.

A Literature Review on Validated Simulations of the Surgical Services.

The surgical department is a critical unit that oversees multiple surgical-based clinical pathways and works with various other units in a hospital. This department faces numerous challenges relating to variability in demand and management of resources. The aim of this article is to review the application of validated simulation models on hospital-wide surgical services. Each of these models is broadly classified by (i) simulation method and (ii) level of detail given to the management of "patient pathways" and "staff workflows". We remark that very few studies have given attention to the management of staff workflows in their validated simulation models.