Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth

Inhibition of PHOSPHO1 activity results in impaired skeletal mineralization during limb development of the chick

PHOSPHO1 is a bone specific phosphatase implicated in the initiation of inorganic phosphate generation for matrix mineralization. The control of mineralization is attributed to the actions of tissue-non specific alkaline phosphatase (TNAP). However, matrix vesicles (MVs) containing apatite crystals are present in patients with hypophosphatasia as well as TNAP null (Akp2(-/-)) mice. It is therefore likely that other phosphatases work with TNAP to regulate matrix mineralization. Although PHOSPHO1 and TNAP expression is associated with MVs, it is not known if PHOSPHO1 and TNAP are co-expressed during the early stages of limb development. Furthermore the functional in-vivo role of PHOSPHO1 in matrix mineralization has yet to be established. Here, we studied the temporal expression and functional role of PHOSPHO1 within chick limb bud mesenchymal micromass cultures and also in wild-type and talpid(3) chick mutants. These mutants are characterized by defective hedgehog signalling and the absence of endochondral mineralization. The ability of in-vitro micromass cultures to differentiate and mineralize their matrix was temporally associated with increased expression of PHOSPHO1 and TNAP. Comparable changes in expression were noted in developing embryonic legs (developmental stages 23–36HH). Micromass cultures treated with lansoprazole, a small-molecule inhibitor of PHOSPHO1 activity, or FGF2, an inhibitor of chondrocyte differentiation, resulted in reduced alizarin red staining (P<0.05). FGF2 treatment also caused a reduction in PHOSPHO1 (P<0.001) and TNAP (P<0.001) expression. Expression analysis by whole mount RNA in-situ hybridization, correlated with qPCR micromass data and demonstrated the existence of a tightly regulated pattern of Phospho1 and Tnap expression which precedes mineralization. Treatment of developing embryos for 5-days with lansoprazole completely inhibited mineralization of all leg and wing long bones as assessed by alcian blue/alizarin red staining. Furthermore, long bones of the talpid(3) chick mutant did not express Phospho1 or Tnap whereas flat bones mineralized normally and expressed both phosphatases. In conclusion, this study has disclosed that PHOSPHO1 expression mirrors that of TNAP during embryonic bone development and that PHOSPHO1 contributes to bone mineralization in developing chick long bones

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Post-traumatic growth 2.5 years after the 2011 Joplin, Missouri tornado

In 2011 a major tornado struck Joplin, Missouri, killing 158 and injuring 1,150 individuals. Approximately 2.5 years after this disaster, an online survey of Joplin adult residents (N = 438) was conducted to examine the relationship between disaster experience, post-traumatic stress (PTS) symptoms, communication with family, friends, and neighbors, and post-traumatic growth (PTG). Results indicate that more tornado exposure and tornado PTS symptoms were related to more PTG. In addition, engaging in more communication about the tornado with family, friends, and neighbors was related to more perceived PTG. Implications for social work practice in long-term post-disaster communities include building upon areas of growth and promoting positive interpersonal connections among survivors

Risk factors leading to COVID_19 cases in a Sydney restaurant

OBJECTIVE: To explore the factors associated with the transmission of SARS-CoV-2 to patrons of a restaurant. METHODS: A retrospective cohort design was undertaken, with spatial examination and genomic sequencing of cases. The cohort included all patrons who attended the restaurant on Saturday 25 July 2020. A case was identified as a person who tested positive to a validated specific Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleic acid test. Associations were tested using chi-squared analysis of case versus non-case behaviours. RESULTS: Twenty cases were epidemiologically linked to exposure at the restaurant on 25 July 2020. All cases dined indoors. All cases able to be genomic sequenced were found to have the same unique mutational profile. Factors tested for an association to the outcome included attentiveness by staff, drink consumption, bathroom use and payment by credit card. No significant results were found. CONCLUSION: Indoor dining was identified as a key factor in SARS-CoV-2 transmission, and outdoor dining as a way to limit transmission. Implications for public health: This investigation provides empirical evidence to support public health policies regarding indoor dining

2011 Joplin, Missouri tornado experience, mental health reactions, and service utilization: Cross-sectional assessments at approximately 6 months and 2.5 years post-event.

Introduction. On May 22, 2011 the deadliest tornado in the United States since 1947 struck Joplin, Missouri killing 161 people, injuring approximately 1,150 individuals, and causing approximately $2.8 billion in economic losses. Methods. This study examined the mental health effects of this event through a random digit dialing sample (N = 380) of Joplin adults at approximately 6 months post-disaster (Survey 1) and a purposive convenience sample (N = 438) of Joplin adults at approximately 2.5 years post-disaster (Survey 2). For both surveys we assessed tornado experience, posttraumatic stress, depression, mental health service utilization, and sociodemographics. For Survey 2 we also assessed social support and parent report of child strengths and difficulties. Results. Probable PTSD relevance was 12.63% at Survey 1 and 26.74% at Survey 2, while current depression prevalence was 20.82% at Survey 1 and 13.33% at Survey 2. Less education and more tornado experience was generally related to greater likelihood of experiencing probable PTSD and current depression for both surveys. Men and younger participants were more likely to report current depression at Survey 1. Low levels of social support (assessed only at Survey 2) were related to more probable PTSD and current depression. For both surveys, we observed low rates of mental health service utilization, and these rates were also low for participants reporting probable PTSD and current depression. At Survey 2 we assessed parent report of child (ages 4 to 17) strengths and difficulties and found that child difficulties were more frequent for younger children (ages 4 to 10) than older children (ages 11 to 17), and that parents reporting probable PTSD reported a greater frequency of children with borderline or abnormal difficulties. Discussion. Overall our results indicate that long-term (multi-year) community disaster mental health monitoring, assessment, referral, outreach, and services are needed following a major disaster like the 2011 Joplin tornado