Livestock microbial landscape patterns: Retail poultry microbiomes significantly vary by region and season.

Microbes play key roles in animal welfare and food safety but there is little understanding of whether microbiomes associated with livestock vary in space and time. Here we analysed the bacteria associated with the carcasses of the same breed of 28 poultry broiler flocks at different stages of processing across two climatically similar UK regions over two seasons with 16S metabarcode DNA sequencing. Numbers of taxa types did not differ by region, but did by season (P = 1.2 × 10-19), and numbers increased with factory processing, especially in summer. There was also a significant (P < 1 × 10-4) difference in the presences and abundances of taxa types by season, region and factory processing stage, and the signal for seasonal and regional differences remained highly significant on final retail products. This study therefore revealed that both season and region influence the types and abundances of taxa on retail poultry products. That poultry microbiomes differ in space and time should be considered when testing the efficacy of microbial management interventions designed to increase animal welfare and food safety: these may have differential effects on livestock depending on location and timing.

Reversal of warfarin anticoagulation using prothrombin complex concentrate at 25 IU kg-1 : results of the RAPID study.

BACKGROUND: Real-world studies of the emergency reversal of warfarin using 4-factor prothrombin complex concentrate (PCC) report unwarranted delays. The delay to receiving PCC was ≥ 8 h in 46·7% of patients with warfarin-associated bleeding (PWAB) treated with a variable PCC dosing protocol in our retrospective audit. OBJECTIVE: To report the impact of a simplified PCC dosing protocol on the interval to reversal of anticoagulation. METHODS: We developed a PCC dosing protocol standardising the initial PCC dose and simplifying dosing calculations. Study end points were the proportion of PWAB achieving international normalised ratio (INR) ≤1·5 and treated within 8 h of presentation, respectively. RESULTS: Of 17, 15 (88·2%) PWABs achieved a post-treatment INR ≤ 1·5; 14 of 17 (82·4%) PWABs were reversed within 8 h. Median intervals between triage and PCC request and PCC request and start of infusion (administration interval) were 126 min (range 39-520) and 30 min (range 5-100), respectively. Compared with the retrospective cohort, RAPID is associated with an improved administration interval (mean 37·7 vs 76 min, P = 0·031) and the proportion of PWABs treated within 30 min (58·8 vs 6·7%, P = 0·009). CONCLUSION: The RAPID protocol reduces unwarranted delays without compromising efficacy.

Association of ACTN3 R577X but not ACE I/D gene variants with elite rugby union player status and playing position.

We aimed to quantify the ACE I/D and ACTN3 R577X (rs1815739) genetic variants in elite rugby athletes (rugby union and league) and compare genotype frequencies to controls and between playing positions. The rugby athlete cohort consisted of 507 Caucasian men, including 431 rugby union athletes that for some analyses were divided into backs and forwards and into specific positional groups: front five, back row, half backs, centers, and back three. Controls were 710 Caucasian men and women. Real-time PCR of genomic DNA was used to determine genotypes using TaqMan probes and groups were compared using χ(2) and odds ratio (OR) statistics. Correction of P values for multiple comparisons was according to Benjamini-Hochberg. There was no difference in ACE I/D genotype between groups. ACTN3 XX genotype tended to be underrepresented in rugby union backs (15.7%) compared with forwards (24.8%, P = 0.06). Interestingly, the 69 back three players (wings and full backs) in rugby union included only six XX genotype individuals (8.7%), with the R allele more common in the back three (68.8%) than controls (58.0%; χ(2) = 6.672, P = 0.04; OR = 1.60) and forwards (47.5%; χ(2) = 11.768, P = 0.01; OR = 2.00). Association of ACTN3 R577X with playing position in elite rugby union athletes suggests inherited fatigue resistance is more prevalent in forwards, while inherited sprint ability is more prevalent in backs, especially wings and full backs. These results also demonstrate the advantage of focusing genetic studies on a large cohort within a single sport, especially when intrasport positional differences exist, instead of combining several sports with varied demands and athlete characteristics.

Limitations to the identification of HIV-associated neurocognitive disorders in clinical practice.

OBJECTIVES: The objective of this study was to establish the level of awareness of HAND among healthcare providers, the screening tools that are currently used in its detection and factors that limit cognitive assessments. METHODS: We distributed a 12-item questionnaire to doctors and nurses who work in the Department of Genitourinary Medicine and Infectious Disease (GUIDE) service and also to doctors who work in the emergency department (ED) at St James Hospital. RESULTS: 35 surveys were collected, 54% (n = 19) from the GUIDE service and 46% (n = 16) from the ED. 82% (n = 29) of participants were doctors from interns to consultants. There was reasonable appreciation among participants with regards the prevalence of neurocognitive impairment (estimated at 29.1% among patients on HAART, and 39.3% among patients not on HAART). Screening tools were rarely used by GUIDE and ED clinicians (25% vs. 15% of the time). The Mini Mental State Examination (MMSE) was previously used by 37% (n = 13) of the group. Very few people had used the HIV Dementia Scale (HIVDS) 6% (n = 2). 34% of respondents felt that 'Orientation in Person, Place and Time was a sufficient screening tool for cognitive assessment'. Lack of time, exposed environment and lack of availability of screening tool were cited as limitations to cognitive screening in the ED environment. CONCLUSIONS: This study examines awareness of HAND among healthcare providers and also reasons for inadequate assessment. There is a need for consensus on screening guidelines. A quick, easy to use and readily available screening tool may have a role in the acute setting in identifying high-risk patients.

AOA Critical Issues Symposium: Leadership and Education During and After COVID-19: Back to the Future or a New Normal.

ABSTRACT: The devastating impact of COVID-19 has reshaped how we lead and train our future surgeons in the field of orthopaedics. Overnight, leaders in our field had to dramatically shift their mindset to continue to lead a hospital, department, journal, or residency or fellowship program in the face of an unprecedented level of adversity in the history of the United States. This symposium discusses the role of physician leadership during and after a pandemic, as well as the adoption of technology for training surgeons in the field of orthopaedics.

Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation.

BACKGROUND: Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood. METHODS: We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice. RESULTS: Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice. CONCLUSION: This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development.

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

High-Resolution Multi-Isotope Imaging Mass Spectrometry (MIMS) Imaging Applications in Stem Cell Biology.

Multi-isotope imaging mass spectrometry (MIMS) allows the measurement of turnover of molecules within intracellular compartments with a spatial resolution down to 30 nm. We use molecules enriched in stable isotopes administered to animals by diet or injection, or to cells through the culture medium. The stable isotopes used are, in general, 15 N, 13 C, 18 O, and 2 H. For stem cell studies, we essentially use 15 N-thymidine, 13 C-thymidine, and 81 Br from BrdU. This protocol describes the practical use of MIMS with specific reference to applications in stem cell research. This includes choice and administration of stable isotope label(s), sample preparation, best practice for high-resolution imaging, secondary ion mass spectrometry using the Cameca NanoSIMS 50L, and methods for robust statistical analysis of label incorporation in regions of interest (ROI). © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Stable isotope labeling of DNA in cultured cells Basic Protocol 2: Stable isotope labeling of DNA in animals Basic Protocol 3: Preparation of Si chips, the general sample support for NanoSIMS analysis Basic Protocol 4: Stable isotope analysis of DNA replication in single nuclei in a population of cells with NanoSIMS Basic Protocol 5: Data reduction and processing.

Applying the British Association of Oral and Maxillofacial Surgeons quality outcomes metrics to a UK oncology and reconstructive surgery service - benchmarking the data.

The British Association of Oral and Maxillofacial Surgery is soon to implement the Quality Outcomes in Oral in Maxillofacial Surgery (QOMS) to provide a platform for quality management across the specialty in the UK. The initial oncology and reconstruction audits for QOMS involves data collection on specific procedures and metrics. The aim of this report is to determine their appropriateness using extant audit datasets in our institution that overlap substantially with the QOMS audits. Pre-existing datasets comprising information on patients treated for oral cavity SCC with curative intent were analysed. Data on surgical margins, lymphadenectomy lymph node yield, delay between surgery and adjuvant radiotherapy, duration of hospital stay, and complications including flap failures were analysed. All statistical analyses were performed with SPSS 25. Run charts describing longitudinal data were generated using SPC for Excel version 6. Twenty three patients (3.1%) of 701 resections had a positive surgical margin reported. Seventeen (4.3%) of patients had less than 18 LNs in the ND specimen analysed. Mean time to start date of adjuvant therapy was 62 days. Only 9% of patients commenced adjuvant therapy within 6 weeks. The median duration of stay was 18 days. In 1153 free flaps a failure rate of 4.3% was identified. A total of 1349 complications (CD I-V) were recorded in the 1111 patients undergoing major surgery with free flap reconstruction. The QOMS selected metrics for oncology and reconstruction are clinically relevant, readily measurable, and likely to be actionable by the surgical team.

The role of human demographic history in determining the distribution and frequency of transferase-deficient galactosaemia mutations.

Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.

Activation of the MAP kinase pathway by FGF-1 correlates with cell proliferation induction while activation of the Src pathway correlates with migration.

FGF regulates both cell migration and proliferation by receptor-dependent induction of immediate-early gene expression and tyrosine phosphorylation of intracellular polypeptides. Because little is known about the disparate nature of intracellular signaling pathways, which are able to discriminate between cell migration and proliferation, we used a washout strategy to examine the relationship between immediate-early gene expression and tyrosine phosphorylation with respect to the potential of cells either to migrate or to initiate DNA synthesis in response to FGF-1. We demonstrate that transient exposure to FGF-1 results in a significant decrease in Fos transcript expression and a decrease in tyrosine phosphorylation of the FGFR-1, p42(mapk), and p44(mapk). Consistent with these biochemical effects, we demonstrate that attenuation in the level of DNA synthesis such that a 1.5-h withdrawal is sufficient to return the population to a state similar to quiescence. In contrast, the level of Myc mRNA, the activity of Src, the tyrosine phosphorylation of cortactin, and the FGF-1-induced redistribution of cortactin and F-actin were unaffected by transient FGF-1 stimulation. These biochemical responses are consistent with an implied uncompromised migratory potential of the cells in response to growth factor withdrawal. These results suggest a correlation between Fos expression and the mitogen-activated protein kinase pathway with initiation of DNA synthesis and a correlation between high levels of Myc mRNA and Src kinase activity with the regulation of cell migration.

FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.

Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.

Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors.

A new class of protein tyrosine kinase inhibitors was identified that is based on an oxindole core (indolinones). Two compounds from this class inhibited the kinase activity of fibroblast growth factor receptor 1 (FGFR1) and showed differential specificity toward other receptor tyrosine kinases. Crystal structures of the tyrosine kinase domain of FGFR1 in complex with the two compounds were determined. The oxindole occupies the site in which the adenine of adenosine triphosphate binds, whereas the moieties that extend from the oxindole contact residues in the hinge region between the two kinase lobes. The more specific inhibitor of FGFR1 induces a conformational change in the nucleotide-binding loop. This structural information will facilitate the design of new inhibitors for use in the treatment of cancer and other diseases in which cell signaling by tyrosine kinases plays a crucial role in disease pathogenesis.

Low molecular weight heparin significantly reduces embolisation after carotid endarterectomy--a randomised controlled trial.

OBJECTIVES: The administration of unfractionated heparin (UFH) prior to carotid clamping during carotid endarterectomy (CEA) transiently increases the platelet aggregation response to arachidonic acid (AA) despite the use of aspirin. We hypothesized that this phenomenon might be reduced by using low molecular weight heparin (LMWH) resulting in fewer emboli in the early post-operative period. METHODS: 183 aspirinated patients undergoing CEA were randomised to 5000 IU UFH (n=91) or 2500 IU LMWH (dalteparin, n=92) prior to carotid clamping. End-points were: transcranial Doppler (TCD) measurement of embolisation, effect on bleeding and platelet aggregation to AA and adenosine 5'-diphosphate (ADP). RESULTS: Patients randomised to UFH had twice the odds of experiencing a higher number of emboli in the first 3h after CEA, than those randomised to LMWH (p=0.04). This was not associated with increased bleeding (mean time from flow restoration to operation end: 23 min (UFH) vs. 24 min (LMWH), p=0.18). Platelet aggregation to AA increased significantly following heparinisation, but was unaffected by heparin type (p=0.90). The platelets of patients randomised to LMWH exhibited significantly lower aggregation to ADP compared to UFH (p<0.0001). CONCLUSIONS: Intravenous LMWH is associated with a significant reduction in post-operative embolisation without increased bleeding. The higher rate of embolisation seen with UFH may be mediated by increased platelet aggregation to ADP, rather than to AA.

Meticillin-resistant Staphylococcus aureus blood-stream infection among patients attending the emergency department of an urban tertiary-referral hospital.

Meticillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen but reports of community-acquired (CA) MRSA are increasing. This study determined the incidence of MRSA-blood-stream infection (BSI) among patients attending the Emergency Department (ED) of an urban tertiary-referral hospital between January 2004 and September 2005, the proportion of cases that were CA or health-care associated (HCA), the epidemiological types of isolates and the presence of pvl genes in CA-MRSA. Eighteen patients presented with MRSA-BSI; 16 cases were categorised as HCA and two as CA. Most patients were male, elderly and lived locally. Two patients (aged <30 years) had no recent previous HC exposure. Only one patient received appropriate empiric antimicrobial therapy. Isolates from patients with HCA-MRSA were similar to the predominant MRSA strain in Irish hospitals. The two CA-MRSA isolates exhibited different epidemiological types; one was pvl-positive. A significant cohort of patients present to the ED with MRSA-BSI. Careful consideration of appropriate empiric antimicrobial therapy for suspected staphylococcal infection is required.

CN- secondary ions form by recombination as demonstrated using multi-isotope mass spectrometry of 13C- and 15N-labeled polyglycine.

We have studied the mechanism of formation CN- secondary ions under Cs+ primary ion bombardment. We have synthesized 13C and 15N labeled polyglycine samples with the distance between the two labels and the local atomic environment of the 13C label systematically varied. We have measured four masses in parallel: 12C, 13C, and two of 12C14N, 13C14N, 12C15N, and 13C15N. We have calculated the 13C/12C isotope ratio, and the different combinations of the CN isotope ratios (27CN/26CN, 28CN/27CN, and 28CN/26CN). We have measured a high 13C15N- secondary ion current from the 13C and 15N labeled polyglycines, even when the 13C and 15N labels are separated. By comparing the magnitude of the varied combinations of isotope ratios among the samples with different labeling positions, we conclude the following: CN- formation is in large fraction due to recombination of C and N; the CO double bond decreases the extent of CN- formation compared to the case where carbon is singly bonded to two hydrogen atoms; and double-labeling with 13C and 15N allows us to detect with high sensitivity the molecular ion 13C15N-.

Weekly dosing with the platelet-derived growth factor receptor tyrosine kinase inhibitor SU9518 significantly inhibits arterial stenosis.

The platelet-derived growth factor (PDGF) ligands and their receptors have been implicated as critical regulators of the formation of arterial lesions after tissue injury. SU9518 (3[5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrol-3-yl]propionic acid) is a novel synthetic indolinone that potently and selectively inhibits the cellular PDGF receptor kinase and PDGF receptor-induced cell proliferation. Inhibition of PDGF receptor phosphorylation in cell-based assays occurs within 5 minutes after drug exposure and persists for >6 hours after drug removal. The pharmacokinetics indicate plasma levels that exceeded the effective concentration required to inhibit the PDGF receptor in cells for up to 8 hours or 7 days after a single oral or subcutaneous administration, respectively. In the rat balloon arterial injury-induced stenosis model, once-daily oral or once-weekly subcutaneous administration of SU9518 reduced intimal thickening of the carotid artery (ratio of neointimal to medial area, 1.94+/-0.38 versus 1.03+/-0.29 [P<0.01] 2.21+/-0.32 versus 1.34+/-0.45 [P<0.01], respectively). These studies provide the rationale to evaluate PDGF receptor tyrosine kinase inhibitors, including inhibitors related to the indolinone, SU9518, for the treatment of arterial restenosis.

Inhibition of angiogenesis by blocking activation of the vascular endothelial growth factor receptor 2 leads to decreased growth of neurogenic sarcomas.

Neurogenic sarcomas are incurable, common malignant human peripheral nerve tumors subject to local recurrence and systemic metastasis. In this study, the vascularity, vascular endothelial growth factor (VEGF) expression, and effects of inhibiting VEGF receptor on growth of neurogenic sarcomas were examined. Vascularization and VEGF expression were 6.4- and 15-fold higher in tumors than in normal nerves. The small molecule inhibitor (SU5416) of VEGF receptor 2 had no effect on neurogenic sarcoma cell lines in vitro, but the growth of a human tumor explant xenograft model was reduced by 54.8% compared to vehicle. Reduction in tumor growth was due to decreased tumor angiogenesis, leading to reduction of tumor cell proliferation and increased apoptosis. Inhibiting VEGF function may therefore be a useful adjuvant therapy for neurogenic sarcomas.