Increased CXCL10 (IP-10) is associated with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models.

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Empowering trainees through understanding learning theory and changes in medical education.

Background: Empowering trainees to think critically about decision making should result in the National Health Service (NHS) being more efficient and cost effective, thereby reducing the wastage of precious NHS resources on unnecessary investigations, treatment, and consequently putting patients at risk. There is a major shift from acquiring knowledge to critical analysis and synthesis of information for decision making. Trainees must understand how healthcare systems function and consequences of their decisions on budgets and patient care. Equally, faculty need to appreciate that their role is changing from information provider to facilitator of learning through feedback and supervision, role modelling, and innovator of learning approaches. Methods: A survey of 100 postgraduate trainees from the Severn Deanery was conducted on SurveyMonkey in March 2020 and January 2021.  The survey consisted of eight questions focusing on trainee responses to participation in clinical decision-making in the inpatient setting. An additional question on communication with the patient was included in the second iteration. Results: With a response rate of 80, only 35% of trainees had their findings regularly verified by the consultant. One third of trainees reported that decisions were made by the consultant without asking their opinion on investigations or management. It was unusual for trainees to have any interaction with patients on consultant ward rounds and to understand the rationale for the requested investigations. Conclusion: The poor consultant trainee interaction represents a serious lost opportunity for experiential learning with real time feedback. Training programmes should support trainees being given opportunities to nurture analytical, problem-solving skills, dealing with uncertainty among other attributes of patient management. Trainees need to become competent through the art of critical thinking and develop a professional identity. Through this they develop confidence and competence leading to better patient outcomes, and prevention of the depletion of healthcare budgets.

Identification and characterization of in vitro expanded hematopoietic stem cells.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development.

We report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9-4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

Hematopoietic stem cells retain functional potential and molecular identity in hibernation cultures.

Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.

Zinc-dependent multimerization of mutant calreticulin is required for MPL binding and MPN pathogenesis.

Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALRdel52) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALRdel52 oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALRdel52 multimers, which was a co-requisite for MPL binding. CALRdel52 variants incapable of binding zinc were unable to homomultimerize, form CALRdel52-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALRdel52-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALRdel52 relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALRdel52-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALRdel52 oncogenic activity by enabling CALRdel52 multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALRdel52 in the treatment of MPNs.

Caesarean section improves neonatal outcomes only from 24 + 0 weeks for periviable breech but not for cephalic infants.

Background: Although caesarean delivery at periviable gestations may minimize birth trauma, it may not necessarily improve perinatal outcomes. The aim of this study was to assess the impact of mode of birth on outcomes for breech versus cephalic presentation at 22 + 0-25 + 6 weeks.Methods: Retrospective cohort study of single, nonanomalous infants at 22 + 0-25 + 6 weeks gestation born at a tertiary center in Australia. Neonatal outcomes were analyzed comparing both breech and cephalic presentation and mode of delivery.Results: Six hundred and eighty eight women fulfilled the inclusion criteria with 39.7% (273/688) breech and 60.3% (415/688) cephalic infants. Survival was 31.5% (86/273) and 38.1% (158/415) in the breech and cephalic cohorts respectively. Vaginal breech infants had reduced odds of survival compared to the vaginal cephalic group (aOR 0.37, 95% CI 0.17-0.75, p < .01) with no difference in survival if delivery occurred by caesarean section. Vaginal breech birth had higher odds of very low Apgar scores, stillbirth, and neonatal death. At 22 + 0-22 + 6 weeks, outcomes were universally fatal. At 24 + 0-24 + 6 and 25 + 0-25 + 6 weeks, vaginal breech birth had lower odds of survival (aOR 0.33, 95% CI 0.13-0.84, p < .05 and aOR 0.10, 95% CI 0.03-0.34, p < .001 respectively) compared to caesarean breech births.Conclusions: Caesarean section improves perinatal outcomes for periviable breech infants > 24 + 0 weeks.

Emollient prescribing formularies in England and Wales: a cross-sectional study.

OBJECTIVE: To identify and compare emollient formularies across all clinical commissioning groups (CCGs) and local health boards (LHBs) in England and Wales. DESIGN: Formularies were retrieved via CCG/LHB websites or Google search (October 2016-February 2017). Data on structure and content were extracted, and descriptive analyses were undertaken. SETTING: 209 English CCGs and 7 Welsh LHBs. MAIN OUTCOME MEASURES: Number and structure of formularies; number, type and name of emollients and bath additive recommendedandnot recommended; and any rationale given. RESULTS: 102formularies were identified, which named 109 emollients and 24 bath additives. Most were structured in an 'order of preference' (63%) and/or formulation (51%) format. Creams and ointments were the most commonly recommended types of emollients, and three ointments were the most commonly recommended specific emollients (71%-79% of formularies). However, there was poor consensus over which emollient should be used first line and 4 out of 10 of the most recommended lotions and creams contained antimicrobials or urea. Patient preference (60%) and/or cost (58%) were the most common reasons given for the recommendations. Of the 82% of formularies that recommend the use of bath additives, 75% did not give any reasons for their recommendation. CONCLUSIONS: Emollient formularies in England and Wales vary widely in their structure, recommendations and rationale. The reasons for such inconsistencies are unclear, risk confusion and make for inequitable regional variation. There is poor justification for multiple different, conflicting formularies.

Nursing management of childhood chickenpox infection.

Chickenpox is an extremely contagious infectious disease caused by varicella zoster virus (VZV). It is a common childhood illness characterised by an itchy vesicular rash and fever, which usually resolves spontaneously without medical intervention. Serious, and rarely fatal, complications can occur, including pneumonia, central nervous system infection, overwhelming secondary bacterial infections, especially with Group A streptococcus, and necrotising fasciitis. Therefore it is crucial that emergency department (ED) nurses can recognise the signs and symptoms that indicate deterioration. This article reviews best practice management of children with chickenpox, gives up-to-date guidance on the safe use of antipyretics, the avoidance of ibuprofen and discusses immunisation against VZV. It also includes implications for nursing practice and a case study that illustrates some of the challenges that ED nurses may encounter.