"Bowel on the Bench": Proof of Concept of a Three-Stage, In Vitro Fermentation Model of the Equine Large Intestine.

The intestinal microbiota of the horse, an animal of huge economic and social importance worldwide, is essential to the health of the animal. Understanding the intestinal ecosystem and its dynamic interaction with diet and dietary supplements currently requires the use of experimental animals, with consequent welfare and financial constraints. Here, we describe the development and assessment, using multiple analytical platforms, of a three-vessel, continuous-flow, in vitro model of the equine hindgut. After inoculation of the model with fresh horse feces, the bacterial communities established in each vessel had a taxonomic distribution similar to that of the source animal. Short-chain fatty acid (SCFA) and branched-chain fatty acid (BCFA) production within the model at steady state was consistent with the expected bacterial function, although higher concentrations of some SCFA/BCFA relative to those in the ex vivo gut content were apparent. We demonstrate the intermodel repeatability and the ability of the model to capture some aspects of individual variation in bacterial community profiles. The findings of this proof-of-concept study, including recognition of the limitions of the model, support its future development as a tool for investigating the impact of disease, nutrition, dietary supplementation, and medication on the equine intestinal microbiota.IMPORTANCE The equine gut model that we have developed and describe has the potential to facilitate the exploration of how the equine gut microbiota is affected by diet, disease, and medication. It is a convenient, cost-effective, and welfare-friendly alternative to in vivo research models.

Effects of 24-week prebiotic intervention on self-reported upper respiratory symptoms, gastrointestinal symptoms, and markers of immunity in elite rugby union players.

OBJECTIVES: Elite rugby union players face numerous physiological and psychological stressors which can increase upper respiratory and gastrointestinal illness risk, and in turn can compromise training and competitive performance. This study aimed to investigate the effect of daily prebiotic supplementation on upper respiratory symptoms, gastrointestinal symptoms, and markers of immune function in elite rugby union players. METHODS: Thirty-three elite rugby union players were randomly assigned to consume a prebiotic (2.8 g/day galactooligosaccharide) or placebo (2.8 g/day maltodextrin), daily for 168 days under double-blind conditions. Participants completed daily and weekly questionnaires for self-reported upper respiratory and gastrointestinal symptoms respectively. Blood and saliva samples were collected at 0, 84, and 168 days for assessment of plasma TNF-α and CRP, and saliva IgA respectively. RESULTS: The prebiotic group experienced a 2-day reduction in upper respiratory symptom duration (P = 0.045). Gastrointestinal symptom severity and incidence were lower in the prebiotic group compared to the placebo group (P < 0.001, P = 0.041) respectively. Salivary immunoglobulin A secretion rate was 42% greater in the prebiotic group compared to the placebo group at day 168 (P = 0.004), no differences in CRP and TNF-α were found (P > 0.05). CONCLUSION: A 168-day dietary prebiotic intervention reduced the duration of upper respiratory symptoms and reduced the incidence and severity of gastrointestinal symptoms in elite rugby union players. These findings suggest that seasonal prebiotic interventions may be beneficial for reducing illness in elite rugby union players, improving their availability to train and compete.Key pointsElite athletes are susceptible to upper respiratory symptoms and gastrointestinal symptoms which may impact upon training availability and competition performance.For the first time, this study shows that a dietary prebiotic intervention can reduce the duration of upper respiratory symptoms by 2 days in elite rugby union players.Dietary prebiotic supplementation can improve the incidence and severity of gastrointestinal symptoms experienced by elite rugby union players.Prebiotic supplementation was able to increase salivary IgA secretion after 168 days.These findings can inform practice suggesting that seasonal prebiotic use has the potential to modulate immune function and reduce illness in elite rugby union, which may improve a player's availability to train and compete.The mechanisms by which prebiotics reduce URS and GIS require further research exploration.

Validation of the UICC 8th edition staging of oral cavity squamous cell carcinoma in a UK cohort.

The eighth edition of the Union for International Cancer Control (UICC) staging manual was recently introduced. The staging of oral cavity squamous cell carcinoma saw changes in relation to depth of invasion and extra-nodal extension. We aimed to evaluate this system and its prognostic ability in a UK cohort. A retrospective review was undertaken of patients diagnosed with squamous cell carcinoma (SCC) of the oral cavity between January 2009 and December 2013. Data were collected on demographics, histology, and recurrence-free (RFS) and five-year overall survival (OS). Patients were staged using both the seventh and eighth editions of the UICC staging manual. Stage-specific survival analysis was performed using the Kaplan-Meier method. A total of 191 records were reviewed and 87 were included in the analysis. The mean (range) age was 60 (37-88) years, and 53% were male. The tongue was the most common site (51%). Using the seventh edition patients were staged as stage I=30, II=14, III=7, IVa=35, and IVb=1. Applying the eighth edition, 26 patients (30%) were upstaged (I=24, II=15, III=14, IVa=17, IVb=17). Ten were upstaged based on pT and 16 on pN status. Both staging manuals showed statistically significant discrimination between stages for both OS and RFS. Patients upstaged from stage IVa in the seventh edition had significantly worse OS in the new system (p=0.043). Both staging systems discriminated accurately between stages. Patients upstaged in stage IVa showed significantly worse OS suggesting improved prognostication with the eighth edition and the changes introduced.

COVID-19 social-distancing measures altered the epidemiology of facial injury: a United Kingdom-Australia comparative study.

The purpose of this study was to undertake a retrospective cross-sectional analysis to compare the frequency and characteristics of facial injury presentations at a UK and an Australian tertiary referral hospital during the implementation of COVID-19 social-distancing measures. The primary predictor variables were a heterogeneous set of factors grouped into logical categories: demographics, injury mechanisms and site, and management. The primary outcome variable was the presentation of a hard or soft tissue facial injury. A descriptive statistical analysis was undertaken on the assembled data. The study found a clinical and statistically significant reduction in the frequency (absolute number) of facial injuries at each study site. In addition, a striking similarity common in both countries was an increase in the number of facial injuries due to falls and a reduction in facial injuries due to interpersonal violence. Conservative (non-operative) management of facial injury increased at both sites. The implementation of COVID-19 social-distancing public health measures, which aimed to limit community transmission of the coronavirus, had a secondary serendipitous effect of reducing the frequency of facial injury presentations and altering their epidemiological characteristics at both a UK and Australian tertiary referral hospital.

Gene expression profiling: classification of mice with left ventricle systolic dysfunction using microarray analysis.

OBJECTIVE: We tested the hypothesis that a set of differentially expressed genes could be used to classify mice according to cardiovascular phenotype after prolonged catecholamine stress. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: One hundred seventy-three male mice were studied: wild-type (WT) C57, WT FVB, WT B6129SF2/J, and beta2 adrenergic receptor knockout. INTERVENTIONS: Mice of each genotype were randomly assigned to 14-day infusions of isoproterenol (120 microg/g/day) or no treatment. Approximately half of the animals underwent left ventricle pressure volume loop analysis. The remaining animals were killed for extraction of messenger RNA from whole heart preparations for microarray analysis. MEASUREMENTS AND MAIN RESULTS: We observed that WT FVB and beta2 adrenergic receptor knockout mice developed systolic dysfunction in response to continuous catecholamine infusion, whereas WT C57 mice developed diastolic dysfunction. Using these mice as the derivation cohort, we identified a set of 83 genes whose differential expression correlated with left ventricle systolic dysfunction. The gene set was then used to accurately classify mice from a separate group (WT B6129SF2/J) into the cohort that developed left ventricle systolic dysfunction after catecholamine stress. CONCLUSIONS: The differential expression pattern of 83 genes can be used to accurately classify mice according to physiological phenotype after catecholamine stress.

Fungicide selective for basidiomycetes.

Concentrations of 2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiin lower than 8 parts per million prevented mycelial growth of a number of Basidiomycetes. By contrast, mycelial growth of various other fungi-Phycomycetes, Ascomycetes, and Deuteromycetes-was 50 percent inhibited only by concentrations of 32 ppm or higher. Two exceptions to this pattern of selective fungitoxicity were found:an isolate of Rhizoctonia solani was not as sensitive as other Basidiomycetes, and the deuteromycete Verticillium alboatrum was inhibited by lower concentrations than affected other fungi in this group. Spore germination of two Basidiomycetes, Uromyces phaseoli and Ustilago nuda, was inhibited 95 percent or more at 10 ppm.

A guide to treatment planning in complex older adults.

There are a growing number of older patients who are retaining their natural teeth. Though the majority of these patients remain independent, many are affected by frailty, multi-morbidity or dementia. The complexities associated with dementia have led to guidelines being produced by the FGDP, Dementia friendly dentistry, although other features of ageing can similarly increase the risk of dental disease and the consequent complexity, safety and suitability of providing treatment. Prevention of dental disease is crucial for older patients as the features of ageing may make the risk of treatment greater than that of younger patients. Conscious sedation or general anaesthesia, typically provided by a specialist dental service, may be required to facilitate treatment, though these approaches may have significant short and long-term impacts on older patients. Clinical guidelines and legislation are available to assist in decision-making for patients who may lack mental capacity, yet for patients who are able to consent for treatment, a comprehensive discussion as part of an informed consent process remains crucial to determine the most appropriate approach to care provision.

The provision of surgical tracheostomies by maxillofacial surgeons in the UK: time for a dedicated tracheostomy team?

Surgical tracheostomy is a commonly provided service by surgical teams for patients in intensive care where percutaneous dilatational tracheostomy is contraindicated. A number of factors may interfere with its provision on shared emergency operating lists, potentially prolonging the stay in intensive care. We undertook a two-part project to examine the factors that might delay provision of surgical tracheostomy in the intensive care unit. The first part was a prospective audit of practice within the University Hospital Coventry. This was followed by a telephone survey of oral and maxillofacial surgery units throughout the UK. In the intensive care unit at University Hospital Coventry, of 39 referrals, 21 (53.8%) were delayed beyond 24 hours. There was a mean (standard deviation) time to delay of 2.2 days (0.9 days) and the most common cause of delay was surgeon decision, accounting for 13 (61.9%) delays. From a telephone survey of 140 units nationwide, 40 (28.4%) were regularly involved in the provision of surgical tracheostomies for intensive care and 17 (42.5%) experienced delays beyond 24 hours, owing to a combination of theatre availability (76.5%) and surgeon availability (47.1%). There is case for having a dedicated tracheostomy team and provisional theatre slot to optimise patient outcomes and reduce delays. We aim to implement such a move within our unit and audit the outcomes prospectively following this change.

A novel protective effect of erythropoietin in the infarcted heart.

Erythropoietin (EPO) has been shown to protect neurons from ischemic stroke, but can also increase thrombotic events and mortality rates in patients with ischemic heart disease. We reasoned that benefits of EPO might be offset by increases in hematocrit and evaluated the direct effects of EPO in the ischemic heart. We show that preconditioning with EPO protects H9c2 myoblasts in vitro and cardiomyocytes in vivo against ischemic injury. EPO treatment leads to significantly improved cardiac function following myocardial infarction. This protection is associated with mitigation of myocyte apoptosis, translating into more viable myocardium and less ventricular dysfunction. EPO-mediated myocyte survival appears to involve Akt activation. Importantly, cardioprotective effects of EPO were seen without an increase in hematocrit (eliminating oxygen delivery as an etiologic factor in myocyte survival and function), demonstrating that EPO can directly protect the ischemic and infarcted heart.

Preferential regulation of protein synthesis initiation complex formation by purine nucleotides.

A comparison of the affinities of eukaryotic initiation factor 2 and eukaryotic elongation factor 1 for GTP and GDP, and of the responses of initiation and elongation complex formation to various GTP mol fractions indicated that the initiation reaction was more sensitive to changes in the GTP: GDP ratio. In vitro regulation of the GTP: GDP ratio by the adenylate energy charge, a sensitive control parameter, also demonstrated a preference for regulation of formation of initiation complexes when compared to elongation complexes. These studies suggest that, based on the availability of energy, initiation is the rate-limiting step in the overall protein synthetic process.

Nucleotide regulation of a eukaryotic protein synthesis initiation complex;.

Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M. GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [(3)H]GDP or [(3)h]gtp from the necleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis.

Regulation of ternary (Met-tRNAf - GTP - eukaryotic initiation factor 2) protein synthesis initiation complex formation by the adenylate energy charge.

Formation of the ternary [Met-tRNAf - GTP - eukaryotic initiation factor 2] protein synthesis initiation complex in rabbit reticulocyte ribosomal eluates is dependent on the GTP: GDP ratio and on the adenylate energy charge. The elements controlling ternary initiation complex formation have been studied in a reconstituted system contianing eukaryotic initiation factor 2 and nucleoside diphosphate kinase purified from the ribosomal eluate. The concentration of GTP required for half maximal formation of the ternary initiation complex is 2.5 - 10(6) M; GDP is a potent competitive inhibitor with Ki equals 3.4 - 10(7) M. Sensitive control of ternary initiation complex formation by the adenylate energy charge occurs through nucleoside diphosphate kinase regulation of the GTP : GDP ratio. Over a wide range of GTP : GDP ratios, 50% of maximal ternary initiation complex formation is observed at an adenylate energy charge of 0.85-0.90 resembling that seen in the unfractionated system. Small changes in adenylate energy charge near this value result in significant changes in the extent of ternary initiation complex formation. Since GTP is continually hydrolyzed to GDP during protein synthesis and since GDP is a competitive inhibitor of GTP binding to several of the protein factors necessary for mRNA translation, the synthetic process provides sensitive control by product inhibition. Ribosome-associated nucleoside diphosphate kinase control of GTP regeneration in response to the adenylate energy charge provides one mechanism for linking protein synthesis to the nutrient state and energy charge of the cell.

Comparison of the interaction of cyclic nucleotide-dependent protein kinases with mononucleosomes and free histones.

Arginine-rich histones H2A, H2B, H3 and H4 contain two regions of interaction with cyclic nucleotide-dependent protein kinases: a substrate phosphorylation site and a region which noncompetitively inhibits cyclic nucleotide binding to the protein kinases. We have compared the interaction of cyclic nucleotide-dependent protein kinases with these two sites in histones which are organized in nucleosome structures with the interaction of the enzymes with free histones. Whereas histones in solution are readily phosphorylated by cyclic GMP-dependent protein kinase and the catalytic subunit of cyclic AMP-dependent protein kinase, mononucleosomes are not phosphorylated by these enzymes. Histones extracted from mononucleosomes can be phosphorylated, indicating that the lack of phosphorylation of nucleosomes is not due to covalent modification of the histones but to their organization within the nucleosome structure. Whereas histones in solution are effective noncompetitive inhibitors of cyclic GMP binding to cyclic GMP-dependent protein kinase and of cyclic AMP binding to the regulatory subunits of cyclic AMP-dependent protein kinase, mononucleosomes do not affect cyclic nucleotide binding. These studies indicate that histones which are organized in nucleosome structures are neither substrates nor modifiers of cyclic nucleotide-dependent protein kinases.

The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.

Myocardial gene transfer and overexpression of beta2-adrenergic receptors potentiates the functional recovery of unloaded failing hearts.

BACKGROUND: Mechanical assistance of the failing left ventricle (LV) can lead to functional recovery after a period of unloading, including restoration of beta-adrenergic receptor (betaAR) inotropic reserve. We tested whether prolonged LV unloading of failing rabbit hearts by use of a heterotopic transplantation technique could lead to recovery and whether adenoviral gene transfer of a beta2AR transgene (Adv-beta2AR) could alter this process. METHODS AND RESULTS: Heart failure was induced by coronary artery ligation in adult New Zealand White rabbits. After 4 weeks, failing hearts were heterotopically transplanted into recipient rabbits, allowing normal coronary perfusion but complete LV unloading. We also placed an LV latex balloon for remote access and in vivo physiological analysis. We found that there was reversal of signaling and functional abnormalities after 30 days of unloading. In another set of failing hearts, we randomly delivered, at the time of transplantation, either 2x10(11) viral particles of Adv-beta2AR or saline via the coronary arteries. Sham-operated animals with nonfailing hearts served as controls. After 5 days of unloading, in vivo LV contractility (LV dP/dt(max)) and relaxation (LV dP/dt(min)) were significantly decreased in saline-treated failing hearts compared with control nonfailing hearts (P<0.05). In failing hearts treated with Adv-beta2AR, however, LV dP/dt(max) and LV dP/dt(min) were improved in response to higher preloads (P<0.05) and betaAR stimulation (P<0.01). CONCLUSIONS: Heterotopic transplantation in the rabbit does allow recovery of the failing heart, and beta2AR overexpression acutely enhances this functional improvement. Accordingly, genetic manipulation of betaAR signaling may represent a novel molecular adjunct to mechanical assistance to facilitate functional myocardial recovery.

A nuclear protein is required for thyroid hormone receptor binding to an inhibitory half-site in the epidermal growth factor receptor promoter.

The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.

A non-randomized controlled stepped wedge trial to evaluate the effectiveness of a multi-level mammography intervention in improving appointment adherence in underserved women.

BACKGROUND: Considerable racial and socio-economic disparities exist in breast cancer. In spite of the existence of numerous evidence-based interventions (EBIs) aimed at reducing breast cancer screening barriers among the underserved, there is a lack of uptake or sub-optimal uptake of EBIs in community and clinical settings. This study evaluates a theoretically based, systematically designed implementation strategy to support adoption and implementation of a patient navigation-based intervention, called Peace of Mind Program (PMP), aimed at improving breast cancer screening among underserved women. METHODS/DESIGN: The PMP will be offered to federally qualified health centers and charity clinics in the Greater Houston area using a non-randomized stepped wedge design. Due to practical constraints of implementing and adopting in the real-world, randomization of start times and blinding will not be used. Any potential confounding or bias will be controlled in the analysis. Outcomes such as appointment adherence, patient referral to diagnostics, time to diagnostic referral, patient referral to treatment, time to treatment referral, and budget impact of the intervention will be assessed. Assessment of constructs from the consolidated framework for implementation research (CFIR) will be assessed during implementation and at the end of the study (sustainment) from each participating clinic. Data will be analyzed using descriptive statistics (chi-square tests) and generalized estimating equations (GEE). DISCUSSION: While parallel group randomized controlled trials (RCT) are considered the gold standard for evaluating EBI efficacy, withholding an effective EBI in practice can be both unethical and/or impractical. The stepped wedge design addresses this issue by enabling all clinics to eventually receive the EBI during the study and allowing each clinic to serve as its own control, while maintaining strong internal validity. We expect that the PMP will prove to be a feasible and successful strategy for reducing appointment no-shows in underserved women. CLINICAL TRIALS REGISTRATION NUMBER: NCT02296177.

Comparative analysis of intestinal tract models.

The human gut is a complex ecosystem occupied by a diverse microbial community. Modulation of this microbiota impacts health and disease. The definitive way to investigate the impact of dietary intervention on the gut microbiota is a human trial. However, human trials are expensive and can be difficult to control; thus, initial screening is desirable. Utilization of a range of in vitro and in vivo models means that useful information can be gathered prior to the necessity for human intervention. This review discusses the benefits and limitations of these approaches.

Heterotopic transplantation as a model to study functional recovery of unloaded failing hearts.

OBJECTIVES: Recent studies have demonstrated cardiac improvement in patients supported with a ventricular assist device, suggesting that reverse remodeling and myocardial recovery are possible. We developed an animal model of cardiac unloading by adapting a heterotopic transplantation technique and used it to examine the pattern of functional recovery in the left ventricle of the failing heart. METHODS: Heart failure was induced in adult New Zealand rabbits by coronary artery ligation with subsequent myocardial infarction. Animals undergoing sham operation served as a control group. After 4 weeks or 3 months, failing hearts were transplanted into the necks of recipient rabbits. A left ventricular latex balloon connected to subcutaneous tubing allowed repeated physiologic analysis on days 1 and after transplantation and then every 5 days until day 30. RESULTS: Contractility (left ventricular dP/dt(max)) and relaxation (left ventricular dP/dt(min)) were significantly lower in transplanted postinfarction hearts as compared to control hearts immediately after transplantation. Both left ventricular dP/dt(max) and left ventricular dP/dt(min) responses to increased preload and to beta-adrenergic stimulation progressively improved to a significantly higher level after 30 days of left ventricular unloading for the hearts that were transplanted 4 weeks after myocardial infarction. However, this functional improvement was not detected in failing hearts transplanted 3 months after infarction. CONCLUSIONS: This model of cardiac unloading appears at least partially to mimic conditions of ventricular assist devices. If performed early in the development of heart failure, it permits improvement of contractile dysfunction and restoration of cardiac responsiveness to mechanical and beta-adrenergic stimulation. Therefore this model may constitute a novel alternative in the study of reverse remodeling in unloaded failing hearts.