Randomized clinical trial of ketoprofen or ceftiofur for treatment of metritis in dairy cows.

Our objectives were to compare the efficacy of ketoprofen or ceftiofur for treatment of metritis in dairy cows considering subsequent health, production, and reproduction. Cows from 2 commercial dairy farms in Ontario, Canada were examined with a Metricheck device 3 times per week from 2 to 14 d in milk (DIM). Cows with metritis (fetid vaginal discharge; n = 193) were blocked by parity and fever (rectal temperature ≥39.5°C or <39.5°C) and within each block per farm, randomly assigned to receive 3 mg/kg BW of ketoprofen (KET) or 2.2 mg/kg of ceftiofur hydrochloride (CEF), once a day for 3 d. Day of enrollment was considered study d 0. Rectal temperature and attitude were evaluated in cows with metritis on study d 0, 3, 4, 7, 10, and 13, and vaginal discharge was evaluated on study d 4, 7, 10, and 13. Body condition was scored at enrollment and 35 DIM, and serum concentration of haptoglobin was measured at d 0, 2, 4, and 7. Cows with rectal temperature ≥39.5°C or a depressed attitude on d 3 were classified as clinical failure and received treatment with ceftiofur for 3 d (KET), or 2 additional days (CEF), to a maximum of 5 d of treatment with ceftiofur. At 35 ± 3 DIM cows were examined for uterine involution by transrectal palpation, purulent vaginal discharge (PVD) by Metricheck, and endometritis by endometrial cytology. Time to onset of cyclicity was assessed by serum progesterone (P4) measurements at 28, 42, and 56 DIM. Contemporary cows from the same farms without metritis (NOMET; n = 1,043) were used for comparison. Data were analyzed with mixed linear or logistic regression or Cox's proportional hazard models, including herd as a random effect. The proportion of clinical resolution of metritis on d 3 (96% vs. 92%), of cows with fever (from d 3 to d 13 after enrollment) or fetid discharge (from d 4 to d 13 after enrollment), and the number of medical treatments (3.1 vs. 3.3) were not different between CEF and KET, respectively. Cows in KET received fewer antibiotic treatments than cows in CEF (0.3 vs. 3.1). Uterine involution, the prevalence of PVD (50% vs. 47%) and subclinical endometritis (6.6% vs. 4.3%), and the proportion of cyclic cows (82% vs. 86%) did not differ between CEF and KET. Cows in KET had greater serum haptoglobin concentration from d 2 to 7 after enrollment. The incidence of mastitis, lameness, or displaced abomasum to 60 DIM and subclinical ketosis to 21 DIM did not differ among CEF, KET, and NOMET. There were no differences in median days to first AI (CEF = 68 d; 95% CI: 65-70; KET = 69 d; 95% CI: 68-72; NOMET = 69 d; 95% CI: 68-70), and median days to pregnancy (CEF = 118 d; 95% CI: 92-145; KET = 113 d; 95% CI: 90-135; NOMET = 105 d; 95% CI: 101-109), pregnancy at first AI at 33 d after insemination (CEF = 42%; KET = 41%; NOMET = 41%), pregnancy loss after first AI (CEF = 8%; KET = 11%; NOMET = 8%), hazard of pregnancy or hazard of culling up to 300 DIM. Milk yield was not different between CEF and KET during the first 10 weeks, but lesser in KET at wk 2 and 4 and CEF at wk 2, 4, and 6 than in NOMET. In this pilot-scale study, given early detection, we did not detect differences in subsequent health, milk yield, or reproductive performance in cows with metritis initially treated for 3 d with CEF or KET. Additional, larger studies are warranted.

Progesterone profiles in postpartum dairy cows with inflammatory disorders.

The objective of this prospective cohort study was to determine if progesterone (P4) profiles differed between dairy cows with or without inflammatory disorders early postpartum. A total of 708 cows from 2 commercial herds were enrolled 3 wk before parturition and examined for clinical health disorders (retained placenta, metritis, displaced abomasum, mastitis, or lameness) until 5 wk postpartum. Serum haptoglobin (Hp) was measured in blood at 2 and 6 (±2) DIM, metritis was assessed at 4, 8, 11, and 15 DIM, and purulent vaginal discharge and endometritis (≥6% polymorphonuclear cells in endometrial cytology sampled by cytobrush) were assessed at 35 ± 3 DIM. As Hp ≥0.8 g/L or endometritis were associated with ovarian dysfunction in previous studies, cows with serum Hp ≥0.8 g/L at either time point and endometritis, regardless of clinical disease, were classified as the cohort with inflammatory disorders (INFLAM; n = 139). Clinically healthy cows without difficult calving or twin birth, with Hp <0.8 g/L at both sampling times, without endometritis, and BCS ≥3.00 (1 to 5 scale) were classified as healthy (n = 133). Cows with only one of the 2 conditions (high Hp or endometritis) were excluded. Cohorts had serum P4 measured twice weekly from 35 to 70 (±3) DIM, and the first detected luteal phase (LP) during the sampling period was defined as the interval from onset of luteal activity (P4 increase to ≥1 ng/mL) until decline of P4 to <1 ng/mL. The odds of prolonged LP (≥21 d), average LP length, peak P4, and time to P4 decline (hazard rate) were analyzed using multivariable mixed logistic, linear, or Cox proportional hazard regression models including INFLAM status, parity, sampling day (when applicable), and herd as a random effect considering the covariates of season, milk yield at first DHIA test, and DIM at onset of cyclicity or LP length (when applicable). Cows with INFLAM had greater odds of prolonged LP (LSM ± SEM; 67% vs. 37% ± 7), greater average LP length (17 vs. 15 ± 2 d), lesser P4 at d 4 (4.6 vs. 5.5 ± 0.3 ng/mL) and d 7 (6.0 vs. 7.7 ± 0.3 ng/mL) of the LP, and lesser peak P4 (6.9 vs. 8.2 ± 0.3 ng/mL) during the LP than healthy cows. Status of INFLAM was associated with time to P4 decline in multiparous but not primiparous cows; the LP of INFLAM multiparous cows was less likely to have luteolysis (P4 decline) by d 14 [adjusted hazard ratio (AHR) and 95% CI: 0.54; 0.31 to 0.94] or by d 21 (AHR: 0.32; 0.12 to 0.84) than in healthy multiparous cows. In conclusion, postpartum cows with markers of systemic inflammation at wk 1 and uterine inflammation at wk 5 had altered luteal function (prolonged LP and lower P4 concentrations) before first breeding, which is a possible pathway linking postpartum health disorders and infertility.

Angiographic and Procedural Characteristics in Frail Older Patients with Non-ST Elevation Acute Coronary Syndrome.

Background: Angiographic and procedural characteristics stratified by frailty status are not known in older patients with non-ST elevation acute coronary syndrome (NSTEACS). We evaluated angiographic and procedural characteristics in older adults with NSTEACS by frailty category, as well as associations of baseline and residual SYNTAX scores with long-term outcomes. Methods: In this study, 271 NSTEACS patients aged ≥75 years underwent coronary angiography. Frailty was assessed using the Fried criteria. Angiographic analysis was performed using QAngio® XA Medis in a core laboratory. Major adverse cardiovascular events (MACE) consisted of all-cause mortality, MI, stroke or transient ischaemic attack, repeat unplanned revascularisation and significant bleeding. Results: Mean (±SD) patient age was 80.5 ± 4.9 years. Compared with robust patients, patients with frailty had more severe culprit lesion calcification (OR 5.40; 95% CI [1.75-16.8]; p=0.03). In addition, patients with frailty had a smaller mean improvement in culprit lesion stenosis after percutaneous coronary intervention (50.6%; 95% CI [45.7-55.6]) than robust patients (58.6%; 95% CI [53.5-63.7]; p=0.042). There was no association between frailty phenotype and completeness of revascularisation (OR 0.83; 95% CI [0.36-1.93]; p=0.67). A high baseline SYNTAX score (≥33) was associated with adjusted (age and sex) 5-year MACE (HR 1.40; 95% CI [1.08-1.81]; p=0.01), as was a high residual SYNTAX score (≥8; adjusted HR 1.22; 95% CI [1.00-1.49]; p=0.047). Conclusion: Frail adults presenting with NSTEACS have more severe culprit lesion calcification. Frail adults were just as likely as robust patients to receive complete revascularisation. Baseline and residual SYNTAX score were associated with MACE at 5 years.

Arginine anchor points govern H3 tail dynamics.

Chromatin is dynamically reorganized spatially and temporally, and the post-translational modification of histones is a key component of this regulation. The basic subunit of chromatin is the nucleosome core particle, consisting of two copies each of the histones H2A, H2B, H3, and H4 around which ∼147 base pairs of DNA wrap. The intrinsically disordered histone termini, or tails, protrude from the core and are heavily post-translationally modified. Previous studies have shown that the histone tails exist in dynamic ensembles of DNA-bound states within the nucleosome. Histone tail interactions with DNA are involved in nucleosome conformation and chromatin organization. Charge-modulating histone post-translational modifications (PTMs) are poised to perturb the dynamic interactions between histone tails and DNA. Arginine side chains form favorable interactions with DNA and are sites of charge-modulating PTMs such as citrullination. Our current focus is on the H3 tail, the longest histone tail. Four arginine residues are relatively evenly spaced along the H3 tail sequence, suggesting multivalent interactions with DNA poised for regulation by PTMs. In this study, we use NMR nuclear spin relaxation experiments to investigate the contribution of arginine residues to H3 tail dynamics within the nucleosome core particle. By neutralizing arginine via mutation to glutamine, we begin to work towards a comprehensive understanding of the contribution of individual residues to H3 tail dynamics. We find that neutralization of arginine residues results in increased regional mobility of the H3 tails, with implications for understanding the direct effects of arginine citrullination. Altogether, these studies support a role for dynamics within the histone language and emphasize the importance of charge-modulating histone PTMs in regulating chromatin dynamics, starting at the level of the basic subunit of chromatin.

Preparation of Recombinant Histones and Widom 601 DNA for Reconstitution of Nucleosome Core Particles.

Expression and purification of individual histone proteins and amplification and purification of DNA are the initial steps toward reconstituting nucleosome core particles. Histone proteins are expressed in E. coli, extracted from inclusion bodies, and purified using ion-exchange chromatography. DNA containing the 147 base pair Widom 601 sequence is amplified in bacteria using a plasmid containing multiple copies of this strong nucleosome positioning sequence. Following alkaline lysis of bacteria, DNA is extracted using phenol and chloroform, released from the vector via restriction enzyme digestion, and purified in subsequent precipitation and ion-exchange chromatography steps. Here, we describe a combination of two protocols: one to express and purify recombinant human histones and the other to amplify and purify Widom 601 DNA.

Nucleosome Core Particle Reconstitution with Recombinant Histones and Widom 601 DNA.

Reconstitution of nucleosomes from recombinant histones and DNA is a widely used tool for studying nucleosome structure, dynamics, and interactions. Preparation of reconstituted nucleosomes allows for the study of nucleosomes with defined compositions. Here, we describe methods for refolding recombinant human histones, reconstituting nucleosome core particles with 147 bp Widom 601 DNA, and purification via sucrose gradient.

Cross-sectional study of antimicrobial use and treatment decision for preweaning Canadian dairy calves.

Antimicrobials should be used prudently in farm animals to prevent the development of resistant bacteria in both humans and animals. The objective of this study was to investigate Canadian dairy producers' practices for antimicrobial use in the treatment of disease in preweaning dairy calves. In-person questionnaires were administered to 144 dairy producers across 5 provinces in Canada between July 2019 and August 2020. Almost all (96%) producers used antimicrobials to treat calves with respiratory disease, but only 27% indicated they had a written treatment protocol for respiratory disease. Most (95%) of these protocols for respiratory disease were developed with input from the herd veterinarian. Seventy-four percent of producers used antimicrobials to treat calf diarrhea, with 37% of producers having a written treatment protocol for calf diarrhea with input from the herd veterinarian. The combinations of signs adopted by the producers for antimicrobial treatment in calf respiratory disease and diarrhea were evaluated based on findings from other studies. More than half (56%) of producers who used antimicrobials for calf respiratory disease decided to use antimicrobials by evaluating multiple clinical signs. Eighty-two percent of producers who used antimicrobials for calf diarrhea made decisions based on systemic signs of disease, presence of bloody stool, no response to previous treatment, or on the recommendation from the herd veterinarian. Producers with a written treatment protocol had 3 to 7 times greater odds of using antimicrobials based on multiple signs or systemic signs of disease compared with those without a protocol. Further research may investigate other calf management practices related to decision-making by producers in using antimicrobials to improve antimicrobial stewardship on dairy farms.

Histone H3 and H4 tails play an important role in nucleosome phase separation.

Chromatin organization and its dynamic regulation are crucial in governing the temporal and spatial accessibility of DNA for proper gene expression. Disordered chains of nucleosomes comprise the basis of eukaryotic chromatin, forming higher-level organization across a range of length scales. Models of chromatin organization involving phase separation driven by chromatin-associating proteins have been proposed. More recently, evidence has emerged that nucleosome arrays can phase separate in the absence of other protein factors, yet questions remain regarding the molecular basis of chromatin phase separation that governs this dynamic nuclear organization. Here, we break chromatin down into its most basic subunit, the nucleosome core particle, and investigate phase separation using turbidity assays in conjunction with differential interference contrast microscopy. We show that, at physiologically-relevant concentrations, this fundamental subunit of chromatin undergoes phase separation. Individually removing the H3 and H4 tails abrogates phase separation under the same conditions. Taking a reductionist approach to investigate H3 and H4 tail peptide interactions in-trans with DNA and nucleosome core particles supports the direct involvement of these tails in chromatin phase separation. These results provide insight into fundamental mechanisms underlying phase separation of chromatin, which starts at the level of the nucleosome core particle, and support that long-range inter-nucleosomal interactions are sufficient to drive phase separation at nuclear concentrations. Additionally, our data have implications for understanding crosstalk between histone tails and provide a lens through which to interpret the effect of histone post-translational modifications and sequence variants. STATEMENT OF SIGNIFICANCE: Emerging models propose that chromatin organization is based in phase separation, however, mechanisms that drive this dynamic nuclear organization are only beginning to be understood. Previous focus has been on phase separation driven by chromatin-associating proteins, but this has recently shifted to recognize a direct role of chromatin in phase separation. Here, we take a fundamental approach in understanding chromatin phase separation and present new findings that the basic subunit of chromatin, the nucleosome core particle, undergoes phase separation under physiological concentrations of nucleosome and monovalent salt. Furthermore, the histone H3 and H4 tails are involved in phase separation in a manner independent of histone-associating proteins. These data suggest that H3 and H4 tail epigenetic factors may modulate chromatin phase separation.

Acetylcysteine has No Mechanistic Effect in Patients at Risk of Contrast-Induced Nephropathy: A Failure of Academic Clinical Science.

Contrast-induced nephropathy (CIN) is a major complication of imaging in patients with chronic kidney disease (CKD). The publication of an academic randomized controlled trial (RCT; n = 83) reporting oral (N)-acetylcysteine (NAC) to reduce CIN led to > 70 clinical trials, 23 systematic reviews, and 2 large RCTs showing no benefit. However, no mechanistic studies were conducted to determine how NAC might work; proposed mechanisms included renal artery vasodilatation and antioxidant boosting. We evaluated the proposed mechanisms of NAC action in participants with healthy and diseased kidneys. Four substudies were performed. Two randomized, double-blind, placebo-controlled, three-period crossover studies (n = 8) assessed the effect of oral and intravenous (i.v.) NAC in healthy kidneys in the presence/absence of iso-osmolar contrast (iodixanol). A third crossover study in patients with CKD stage III (CKD3) (n = 8) assessed the effect of oral and i.v. NAC without contrast. A three-arm randomized, double-blind, placebo-controlled parallel-group study, recruiting patients with CKD3 (n = 66) undergoing coronary angiography, assessed the effect of oral and i.v. NAC in the presence of contrast. We recorded systemic (blood pressure and heart rate) and renal (renal blood flow (RBF) and glomerular filtration rate (GFR)) hemodynamics, and antioxidant status, plus biomarkers of renal injury in patients with CKD3 undergoing angiography. Primary outcome for all studies was RBF over 8 hours after the start of i.v. NAC/placebo. NAC at doses used in previous trials of renal prophylaxis was essentially undetectable in plasma after oral administration. In healthy volunteers, i.v. NAC, but not oral NAC, increased blood pressure (mean area under the curve (AUC) mean arterial pressure (MAP): mean difference 29 h⋅mmHg, P = 0.019 vs. placebo), heart rate (28 h⋅bpm, P < 0.001), and RBF (714 h⋅mL/min, 8.0% increase, P = 0.006). Renal vasodilatation also occurred in the presence of contrast (RBF 917 h⋅mL/min, 12% increase, P = 0.005). In patients with CKD3 without contrast, only a rise in heart rate (34 h⋅bpm, P = 0.010) and RBF (288 h⋅mL/min, 6.0% increase, P = 0.001) occurred with i.v. NAC, with no significant effect on blood pressure (MAP rise 26 h⋅mmHg, P = 0.156). Oral NAC showed no effect. In patients with CKD3 receiving contrast, i.v. NAC increased blood pressure (MAP rise 52 h⋅mmHg, P = 0.008) but had no effect on RBF (151 h⋅mL/min, 3.0% increase, P = 0.470), GFR (29 h⋅mL/min/1.73m², P = 0.122), or markers of renal injury. Neither i.v. nor oral NAC affected plasma antioxidant status. We found oral NAC to be poorly absorbed and have no reno-protective effects. Intravenous, not oral, NAC caused renal artery vasodilatation in healthy volunteers but offered no protection to patients with CKD3 at risk of CIN. These findings emphasize the importance of mechanistic clinical studies before progressing to RCTs for novel interventions. Thousands were recruited to academic clinical trials without the necessary mechanistic studies being performed to confirm the approach had any chance of working.

Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility.

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.

Controlled trial of the effect of negative dietary cation-anion difference prepartum diets on milk production, reproductive performance, and culling of dairy cows.

Our objective was to assess the effects of feeding negative dietary cation-anion difference (DCAD) prepartum diets on milk production, reproductive performance, and culling. Cows from 4 commercial farms in Ontario, Canada were enrolled in a pen-level controlled trial from November 2017 to April 2019. Close-up pens (1 per farm) with cows 3 wk before calving were randomly assigned to a negative DCAD (TRT; -108 mEq/kg of dry matter; target urine pH 6.0-6.5) or a control diet (CON; +105 mEq/kg of dry matter with a placebo supplement). Each pen was fed TRT or CON for 3 mo (1 period), and then switched to the other treatment for the next period (4 periods per farm). Data from 15 experimental units (8 pen treatments in TRT and 7 in CON), with a total of 1,086 observational units (cows), were included. The effect of treatment on milk yield at the first 3 milk recording tests of lactation was assessed with linear regression models accounting for repeated measures. The risk of pregnancy at first artificial insemination and culling by 30, 60, and 305 d in milk (DIM) were analyzed with logistic regression models, and effects on time to first AI, pregnancy, and culling were assessed with Cox proportional hazards models. All models included treatment, parity, and their interactions, accounting for pen-level randomization and clustering of animals within farm with random effects, giving 10 degrees of freedom for treatment effects. Multiparous cows fed TRT produced more milk at the first (42.0 vs. 38.8 ± 1.2 kg/d) and second (44.2 vs. 41.7 ± 1.3 kg/d) milk tests. However, multiparous cows fed TRT tended to have 0.2 percentage units less milk fat content at these tests. Although multiparous cows fed TRT tended to have greater energy-corrected milk at the first test (least squares means ± standard error: TRT = 46.1 ± 0.9 vs. CON = 43.8 ± 1 kg/d), there were no differences observed in energy-corrected milk at the second or third tests. In primiparous cows, there was no effect of treatment on milk production. Multiparous cows fed TRT had greater pregnancy to first insemination (TRT = 42 ± 3 vs. CON = 32 ± 4%) and tended to have shorter time to pregnancy [hazard ratio (HR) = 1.20; 95% CI: 0.96-1.49]. In primiparous cows fed TRT, time to pregnancy was increased (HR = 0.76; 95% CI: 0.59-0.99). Culling by 30 DIM tended to be less in TRT (3.3 ± 1.1%) than CON (5.5 ± 1.8%). No effect of treatment on culling by 305 DIM was detected in primiparous cows, but in multiparous cows, the TRT diets decreased the odds of culling (21.3 ± 1.9 vs. 31.7 ± 2.8%) and daily risk of culling to 305 DIM (HR = 0.64; 95% CI: 0.46 to 0.89). Under commercial herd conditions, prepartum negative DCAD diets improved milk production and reproductive performance, and reduced culling risk in multiparous cows. In primiparous cows, TRT diets had no effect on milk yield or culling, but increased the time to pregnancy. Our results suggest that negative DCAD diets should be targeted to multiparous cows.

Controlled trial of the effect of negative dietary cation-anion difference on postpartum health of dairy cows.

The objective of this study was to assess the effects of feeding negative dietary cation-anion difference (DCAD) dry cow diets on postpartum health. Cows from 4 commercial dairy farms in Ontario, Canada, were enrolled in a pen-level controlled trial from November 2017 to April 2019. Close-up pens (1 per farm), with cows 3 wk before expected calving, were randomly assigned to a negative DCAD [TRT; -108 mEq/kg of dry matter (DM); target urine pH 6.0-6.5] or a control diet (CON; +105 mEq/kg of DM with a placebo supplement). Each pen was fed TRT or CON for 3 mo (1 period) then switched to the other treatment for the next period, with 4 periods per farm. Urine pH was measured weekly until calving, and body condition score (BCS) was measured at enrollment and at 5 wk postpartum. Data from 15 experimental units [8 TRT and 7 CON, with 1,086 (TRT: n = 681; CON: n = 405) observational units (cows)] that received the assigned diet for >1 wk were included. The incidence of milk fever (MF), retained placenta (RP), metritis, hyperketonemia (blood ß-hydroxybutyrate >1.2 mmol/L, measured weekly in wk 1 and 2), clinical mastitis within 30 DIM (MAST), displaced abomasum (DA) within 30 d in milk (DIM), purulent vaginal discharge (PVD, assessed once at wk 5), and number of disease events (≥1 or ≥2) were analyzed with logistic regression models with treatment, parity, BCS, and their interactions, accounting for pen-level randomization and clustering of animals within farm with random effects, giving 10 degrees of freedom to test treatment effects. Multiparous cows fed TRT had greater blood calcium between 1 and 4 DIM than multiparous cows fed CON, and the prevalence of subclinical hypocalcemia (total Ca ≤2.14 mmol/L) was lesser when fed TRT compared with CON (d 1: 73 ± 6% vs. 93 ± 4%; d 2: 65 ± 7% vs. 90 ± 5%), with no differences between treatments detected in primiparous cows. We detected interactions of treatment and BCS at enrollment for MF in multiparous cows and of treatment and parity for ≥2 disease events. Overconditioned (BCS ≥3.75) multiparous cows had reduced incidence of MF when fed TRT (TRT: 2 ± 1%, vs. CON: 13 ± 8%). We detected no treatment effects on RP, metritis, hyperketonemia, or PVD incidence. Cows fed TRT had lesser incidence of DA (1.7 ± 0.7% vs. 3.6 ± 1.6%) and tended to have lesser incidence of MAST compared with CON (1.8% ± 0.6% vs. 4.4 ± 1.4%). No treatment effect was detected on ≥1 disease events (TRT: 38 ± 7%, vs. CON: 42 ± 8%); however, multiparous cows on TRT were less likely to have ≥2 disease events than cows on CON (14 ± 4% vs. 23 ± 6%). Under commercial herd conditions, feeding prepartum diets with negative DCAD improved several measures of postpartum health.

Evolutionary Conservation of Structural and Functional Coupling between the BRM AT-Hook and Bromodomain.

The BAF chromatin remodeling complex is critical for genome regulation. The central ATPase of BAF is either BRM or BRG1, both of which contain a C-terminal bromodomain, known to associate with acetylated lysines. We have recently demonstrated that in addition to acetyl-lysine binding, the BRG1/BRM bromodomain can associate with DNA through a lysine/arginine rich patch that is adjacent to the acetyl-lysine binding pocket. Flanking the bromodomain is an AT-hook separated by a short, proline-rich linker. We previously found that the AT-hook and bromodomain can associate with DNA in a multivalent manner. Here, we investigate the conservation of this composite module and find that the AT-hook, linker, and lysine/arginine rich bromodomain patch are ancient, conserved over ~1 billion years. We utilize extensive mutagenesis, NMR spectroscopy, and fluorescence anisotropy to dissect the contribution of each of these conserved elements in association of this module with DNA. Our results reveal a structural and functional coupling of the AT-hook and bromodomain mediated by the linker. The lysine/arginine rich patch on the bromodomain and the conserved elements of the AT-hook are critical for robust affinity for DNA, while the conserved elements of the linker are dispensable for overall DNA affinity but critical for maintaining the relative conformation of the AT-hook and bromodomain in binding to DNA. This supports that the coupled action of the AT-hook and bromodomain are important for BAF activity.

The Clean pilot study: evaluation of an environmental hygiene intervention bundle in three Tanzanian hospitals.

BACKGROUND: Healthcare associated infections (HAI) are estimated to affect up to 15% of hospital inpatients in low-income countries (LICs). A critical but often neglected aspect of HAI prevention is basic environmental hygiene, particularly surface cleaning and linen management. TEACH CLEAN is an educational intervention aimed at improving environmental hygiene. We evaluated the effectiveness of this intervention in a pilot study in three high-volume maternity and newborn units in Dar es Salaam, Tanzania. METHODS: This study design prospectively evaluated the intervention as a whole, and offered a before-and-after comparison of the impact of the main training. We measured changes in microbiological cleanliness [Aerobic Colony Counts (ACC) and presence of Staphylococcus aureus] using dipslides, and physical cleaning action using gel dots. These were analysed with descriptive statistics and logistic regression models. We used qualitative (focus group discussions, in-depth interviews, and semi-structured observation) and quantitative (observation checklist) tools to measure why and how the intervention worked. We describe these findings across the themes of adaptation, fidelity, dose, reach and context. RESULTS: Microbiological cleanliness improved during the study period (ACC pre-training: 19%; post-training: 41%). The odds of cleanliness increased on average by 1.33 weekly during the pre-training period (CI = 1.11-1.60), and by 1.08 (CI = 1.03-1.13) during the post-training period. Cleaning action improved only in the pre-training period. Detection of S. aureus on hospital surfaces did not change substantially. The intervention was well received and considered feasible in this context. The major pitfalls in the implementation were the limited number of training sessions at the hospital level and the lack of supportive supervision. A systems barrier to implementation was lack of regular cleaning supplies. CONCLUSIONS: The evaluation suggests that improvements in microbiological cleanliness are possible using this intervention and can be sustained. Improved microbiological cleanliness is a key step on the pathway to infection prevention in hospitals. Future research should assess whether this bundle is cost-effective in reducing bacterial and viral transmission and infection using a rigorous study design.

Validation of a point-of-care handheld blood total calcium analyzer in postpartum dairy cows.

Our objective was to validate a point-of-care handheld blood total calcium analyzer (Ca meter, CM; TD-5220 Vet Ca2+, TaiDoc, New Taipei, Taiwan) to estimate circulating Ca concentrations in postpartum dairy cows. Whole blood was collected from 251 multiparous cows between 1 and 4 d in milk from 2 commercial dairy herds in Ontario, Canada. Blood total calcium concentration (tCa) was analyzed in whole blood, fresh plasma, and thawed plasma, and compared with tCa results from thawed serum analyzed in a diagnostic laboratory (using a Cobas Calcium Gen 2 kit, Roche Diagnostics, Indianapolis, IN) as the reference test (RT). Lin's concordance correlation coefficient (ßrho;) and Bland-Altman (B-A) plots were assessed to evaluate the agreement between the RT and CM results in each type of sample. Receiver operating characteristic curve analyses were used to describe the accuracy of each test against the categorized RT results (at a cut-point of ≤2.14 mmol/L). Samples where the meter gave a nonquantitative result ("high" or "low"; thawed plasma: 3/247; fresh plasma: 6/100; and whole blood: 20/98) were not included in the ßrho; and B-A analyses. Lin's correlation coefficients demonstrated poor agreement between tests (thawed plasma: ßrho; = 0.16; fresh plasma: ßrho; = 0.21; and whole blood: ßrho; = 0.23). Fresh plasma (using a cut-point of 2.55 mmol/L as measured on the CM) had the greatest diagnostic sensitivity (72%), specificity (86%), and accuracy (77%) for determining subclinical hypocalcemia, but that would still misclassify 23% of samples. In addition to substantial variability, the B-A plots revealed bias with changing concentrations of calcium. Because of low sensitivity on whole blood (58%) or thawed plasma (56%), measurement with the CM is not recommended on these types of samples. This rapid and low-cost meter was not sufficiently accurate to quantify blood Ca concentration, but when used with fresh plasma it might be useful as a screening tool for subclinical hypocalcemia.

Transfer of hepatocellular microRNA regulates cytochrome P450 2E1 in renal tubular cells.

BACKGROUND: Extracellular microRNAs enter kidney cells and modify gene expression. We used a Dicer-hepatocyte-specific microRNA conditional-knock-out (Dicer-CKO) mouse to investigate microRNA transfer from liver to kidney. METHODS: Dicerflox/flox mice were treated with a Cre recombinase-expressing adenovirus (AAV8) to selectively inhibit hepatocyte microRNA production (Dicer-CKO). Organ microRNA expression was measured in health and following paracetamol toxicity. The functional consequence of hepatic microRNA transfer was determined by measuring the expression and activity of cytochrome P450 2E1 (target of the hepatocellular miR-122), and by measuring the effect of serum extracellular vesicles (ECVs) on proximal tubular cell injury. In humans with liver injury we measured microRNA expression in urinary ECVs. A murine model of myocardial infarction was used as a non-hepatic model of microRNA release. FINDINGS: Dicer-CKO mice demonstrated a decrease in kidney miR-122 in the absence of other microRNA changes. During hepatotoxicity, miR-122 increased in kidney tubular cells; this was abolished in Dicer-CKO mice. Depletion of hepatocyte microRNA increased kidney cytochrome P450 2E1 expression and activity. Serum ECVs from mice with hepatotoxicity increased proximal tubular cell miR-122 and prevented cisplatin toxicity. miR-122 increased in urinary ECVs during human hepatotoxicity. Transfer of microRNA was not restricted to liver injury -miR-499 was released following cardiac injury and correlated with an increase in the kidney. INTERPRETATION: Physiological transfer of functional microRNA to the kidney is increased by liver injury and this signalling represents a new paradigm for understanding the relationship between liver injury and renal function. FUNDING: Kidney Research UK, Medical Research Scotland, Medical Research Council.

The effect of prepartum negative dietary cation-anion difference and serum calcium concentration on blood neutrophil function in the transition period of healthy dairy cows.

Our objectives were to assess the effects of a diet with a negative dietary cation-anion difference (DCAD) before calving on phagocytosis (Pc) and oxidative burst (OB) function of circulating neutrophils, and to determine the associations of serum ionized (iCa) and total calcium (tCa) concentrations with Pc and OB in transition dairy cows. We hypothesized that multiparous cows fed a negative DCAD diet prepartum would have greater iCa and tCa, and thus improved Pc and OB. From 3 wk before expected parturition until calving, 38 healthy multiparous cows from 3 farms were assigned to negative DCAD treatment (TRT; -100 mEq/kg of diet dry matter; n = 21) or a control (CON; 95 mEq/kg of dry matter; n = 17) diet. Each farm was on one treatment or the other at a time, but all farms contributed cows to both groups. Urine pH was measured weekly and in TRT was 6.1 ± 0.8 with 80% of 50 samples <7 and 74% ≤ 6.5. Phagocytosis, OB, iCa, and tCa were measured at d -7, 1, and 4 relative to calving. Median fluorescence intensity for Pc (MFIP) and OB (MFIOB), and the shift of percentage of cells active for Pc (PPc) and OB (POB) were measured in isolated, stimulated neutrophils via flow cytometry. Outcomes were assessed with mixed linear regression models accounting for repeated measures. There were no differences between treatments in the 4 neutrophil function outcomes. Although MFIOB varied over time, there were no interactions of treatment with time for any outcome. Serum ionized and tCa did not differ between TRT and CON. The least squares means ± standard deviation for iCa were: d -7, 1.23 ± 0.12 vs. 1.21 ± 0.12; d 1, 1.07 ± 0.12 vs. 1.02 ± 0.12; d 4, 1.16 ± 0.12 vs. 1.17 ± 0.12 mmol/L for TRT and CON, respectively; and for tCa: d -7 2.39 ± 0.25 vs 2.44 ± 0.31; d 1, 2.01 ± 0.25 vs 1.97 ± 0.31; d 4, 2.33 ± 0.25 vs 2.32 ± 0.31 mmol/L, respectively. The proportion of blood samples with tCa <2.15mmol/L at d -7, 1 and 4 was 5, 76, and 13%, respectively, with no differences between TRT and CON. Correlations of iCa or tCa with each of the 4 polymorphonuclear leukocyte (PMN) function outcomes were weak (r < |0.3|). We did not observe the hypothesized differences in aspects of innate immunity in clinically healthy multiparous cows fed a negative DCAD. We underline that cows that experienced clinical disease were excluded from this study, which is important for interpretation of the results.

Multi trace element profiling in pathogenic and non-pathogenic fungi.

Maintaining appropriate levels of trace elements during infection of a host is essential for microbial pathogenicity. Here we compared the uptake of 10 trace elements from 3 commonly-used laboratory media by 3 pathogens, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, and a model yeast, Saccharomyces cerevisiae. The trace element composition of the yeasts, C. albicans, C. neoformans and S. cerevisiae, grown in rich (YPD) medium, differed primarily in P, S, Fe, Zn and Co. Speciation analysis of the intracellular fraction, which indicates the size of the organic ligands with which trace elements are complexed, showed that the ligands for S were similar in the three fungi but there were significant differences in binding partners for Fe and Zn between C. neoformans and S.cerevisiae. The profile for Cu varied across the 3 yeast species. In a comparison of C. albicans and A. fumigatus hyphae, the former showed higher Fe, Cu, Zn and Mn, while A. fumigatus contained higher P, S Ca and Mo. Washing C. albicans cells with the cell-impermeable chelator, EGTA, depleted 50-90 % of cellular Ca, suggesting that a large proportion of this cation is stored in the cell wall. Treatment with the cell wall stressor, Calcofluor White (CFW), alone had little effect on the elemental profile whilst combined Ca + CFW stress resulted in high cellular Cu and very high Ca. Together our data enhance our understanding of trace element uptake by pathogenic fungi and provide evidence for the cell wall as an important storage organelle for Ca.

Direct Detection of miR-122 in Hepatotoxicity Using Dynamic Chemical Labeling Overcomes Stability and isomiR Challenges.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.

The conserved metalloprotease invadolysin is present in invertebrate haemolymph and vertebrate blood.

We identified invadolysin, a novel essential metalloprotease, for functions in chromosome structure, cell proliferation and migration. Invadolysin also plays an important metabolic role in insulin signalling and is the only protease known to localise to lipid droplets, the main lipid storage organelle in the cell. In silico examination of the protein sequence of invadolysin predicts not only protease and lipase catalytic motifs, but also post-translational modifications and the secretion of invadolysin. Here we show that the protease motif of invadolysin is important for its role in lipid accumulation, but not in glycogen accumulation. The lipase motif does not appear to be functionally important for the accumulation of lipids or glycogen. Post-translational modifications likely contribute to modulating the level, localisation or activity of invadolysin. We identified a secreted form of invadolysin in the soluble fraction of invertebrate hemolymph (where we observe sexually dimorphic forms) and also vertebrate plasma, including in the extracellular vesicle fraction. Biochemical analysis for various post-translational modifications demonstrated that secreted invadolysin is both N- and O-glycosylated, but not apparently GPI-linked. The discovery of invadolysin in the extracellular milieu suggests a role for invadolysin in normal organismal physiology.