Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

Managing opioid use disorder in primary care: PEER simplified guideline.

OBJECTIVE: To use the best available evidence and principles of shared, informed decision making to develop a clinical practice guideline for a simplified approach to managing opioid use disorder (OUD) in primary care. METHODS: Eleven health care and allied health professionals representing various practice settings, professions, and locations created a list of key questions relevant to the management of OUD in primary care. These questions related to the treatment setting, diagnosis, treatment, and management of comorbidities in OUD. The questions were researched by a team with expertise in evidence evaluation using a series of systematic reviews of randomized controlled trials. The Guideline Committee used the systematic reviews to create recommendations. RECOMMENDATIONS: Recommendations outline the role of primary care in treating patients with OUD, as well as pharmacologic and psychotherapy treatments and various prescribing practices (eg, urine drug testing and contracts). Specific recommendations could not be made for management of comorbidities in patients with OUD owing to limited evidence. CONCLUSION: The recommendations will help simplify the complex management of patients with OUD in primary care. They will aid clinicians and patients in making informed decisions regarding their care.

Prise en charge du trouble de consommation d'opioïdes en première ligne: Lignes directrices simplifiées de PEER.

OBJECTIF: Utiliser les meilleures données probantes et les meilleurs principes de prise de décision partagée et éclairée à notre disposition pour élaborer des lignes directrices de pratique clinique visant une approche simplifiée de prise en charge du trouble de consommation d'opioïdes (TCO) en première ligne. MÉTHODES: Onze professionnels de la santé et professionnels paramédicaux représentant divers milieux de pratique, professions et lieux ont créé une liste de questions pertinentes à la prise en charge du TCO en première ligne. Ces questions étaient liées au contexte thérapeutique, au diagnostic, au traitement et à la prise en charge des comorbidités dans le TCO. Les questions ont été étudiées par une équipe expérimentée dans l'évaluation des données probantes à l'aide d'une série de revues systématiques d'études randomisées et contrôlées. Les recommandations émises par le comité des lignes directrices reposent sur les revues systématiques. RECOMMANDATIONS: Les recommandations font ressortir le rôle des soins primaires dans le traitement des patients aux prises avec un TCO, de même que les traitements pharmacologiques et psychothérapies et les diverses pratiques de prescription (p. ex. test urinaire de dépistage de drogues et contrats). Aucune recommandation précise n'a pu être faite sur la prise en charge des comorbidités chez les patients aux prises avec un TCO, en raison des données probantes limitées. CONCLUSION: Les recommandations contribueront à simplifier la prise en charge des cas complexes de TCO en première ligne. Elles aideront tant les cliniciens que les patients à prendre des décisions éclairées au sujet de leurs soins.

Identification of a Novel GRHL3/HOPX/Wnt/ß-Catenin Proto-oncogenic Axis in Squamous Cell Carcinoma of the Esophagus.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor long-term prognosis. The molecular mechanisms underlying the initiation and progression of this tumor are largely unknown. The transcription factor GRHL3 functions as a potent tumor suppressor in SCC of skin, head, and neck. This study aims to determine whether GRHL3 also plays a role in the homeostasis of the esophageal epithelium and in the development of ESCC. METHODS: The effects of Grhl3 deletion on squamous epithelial homeostasis in embryos and adult mice were examined using immunohistochemistry, transmission electron microscopy, and real-time polymerase chain reaction. The conditionally deleted mice were subsequently used to determine susceptibility to ESCC. Whole-transcriptome sequencing (RNA-seq) was performed on ESCC in wild-type and Grhl3 deleted animals. To decipher the signaling pathways, real-time polymerase chain reaction, immunohistochemistry, analysis of chromatin immunoprecipitation sequencing, chromatin immunoprecipitation-polymerase chain reaction, and RNA seq datasets were used. Primary human samples were used to validate the findings in the mouse model. RESULTS: Loss of Grhl3 perturbs the proliferation-differentiation balance in the esophageal epithelium, thereby increasing the susceptibility to esophageal carcinogenesis in adult mice. Grhl3 imparts its tumor suppressor function by regulating the expression of HOPX. We have identified the Wnt/ß-catenin pathway as the downstream effectors of GRHL3 and HOPX through our integrated approach using patient-derived ESCC samples and mouse models. CONCLUSIONS: GRHL3 conveys its tumor suppressor function in ESCC through regulating its target gene HOPX, which limits Wnt/ß-catenin signaling. Targeted therapies to inhibit this pathway could be a potential treatment strategy for ESCC patients with reduced GRHL3 expression.

DNA methylation-mediated down-regulation of DNA methyltransferase-1 (DNMT1) is coincident with, but not essential for, global hypomethylation in human placenta.

The genome of extraembryonic tissue, such as the placenta, is hypomethylated relative to that in somatic tissues. However, the origin and role of this hypomethylation remains unclear. The DNA methyltransferases DNMT1, -3A, and -3B are the primary mediators of the establishment and maintenance of DNA methylation in mammals. In this study, we investigated promoter methylation-mediated epigenetic down-regulation of DNMT genes as a potential regulator of global methylation levels in placental tissue. Although DNMT3A and -3B promoters lack methylation in all somatic and extraembryonic tissues tested, we found specific hypermethylation of the maintenance DNA methyltransferase (DNMT1) gene and found hypomethylation of the DNMT3L gene in full term and first trimester placental tissues. Bisulfite DNA sequencing revealed monoallelic methylation of DNMT1, with no evidence of imprinting (parent of origin effect). In vitro reporter experiments confirmed that DNMT1 promoter methylation attenuates transcriptional activity in trophoblast cells. However, global hypomethylation in the absence of DNMT1 down-regulation is apparent in non-primate placentas and in vitro derived human cytotrophoblast stem cells, suggesting that DNMT1 down-regulation is not an absolute requirement for genomic hypomethylation in all instances. These data represent the first demonstration of methylation-mediated regulation of the DNMT1 gene in any system and demonstrate that the unique epigenome of the human placenta includes down-regulation of DNMT1 with concomitant hypomethylation of the DNMT3L gene. This strongly implicates epigenetic regulation of the DNMT gene family in the establishment of the unique epigenetic profile of extraembryonic tissue in humans.

Corticosteroid injections during the COVID-19 pandemic: experience from a UK centre.

AIMS: To describe the incidence of adverse clinical outcomes related to COVID-19 infection following corticosteroid injections (CSI) during the COVID-19 pandemic. To describe the incidence of positive SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) testing, positive SARS-COV2 IgG antibody testing or positive imaging findings following CSI at our institution during the COVID-19 pandemic. METHODS: A retrospective observational study was undertaken of consecutive patients who had CSI in our local hospitals between 1 February and 30June 2020. Electronic patient medical records (EPR) and radiology information system (RIS) database were reviewed. SARS-CoV-2 RT-PCR testing, SARS-COV2 IgG antibody testing, radiological investigations, patient management, and clinical outcomes were recorded. Lung findings were categorized according to the British Society of Thoracic Imaging (BSTI) guidelines. Reference was made to the incidence of lab-confirmed COVID-19 cases in our region. RESULTS: Overall, 1,656 lab-confirmed COVID-19 cases were identified in our upper tier local authority (UTLA), a rate of 306.6 per 100,000, as of 30June 2020. A total of 504 CSI injections were performed on 443 patients between 1 February and 30June 2020. A total of 11 RT-PCR tests were performed on nine patients (2% of those who had CSI), all of which were negative for SARS-CoV-2 RNA, and five patients (1.1%) received an SARS-CoV-2 IgG antibody test, of which 2 (0.5%) were positive consistent with prior COVID-19 infection, however both patients were asymptomatic. Seven patients (1.6%) had radiological investigations for respiratory symptoms. One patient with indeterminate ground glass change was identified. CONCLUSION: The incidence of positive COVID-19 infection following corticosteroid injections was very low in our cohort and no adverse clinical outcomes related to COVID-19 infection following CSI were identified. Our findings are consistent with CSI likely being low risk during the COVID-19 pandemic. The results of this small observational study are supportive of the current multi-society guidelines regarding the judicious use of CSI.Cite this article: Bone Joint Open 2020;1-9:605-611.

A case-sibling assessment of the association between skin pigmentation and other vitamin D-related factors and type 1 diabetes mellitus.

Fair skin pigmentation has been associated with a higher risk of type 1 diabetes mellitus (T1DM). The aim is to compare children with T1DM directly to a sibling in relation to their skin pigmentation in sun-exposed and unexposed sites, past sun exposure and methylation of the VDR gene promoter. The sample consisted of children with T1DM attending a diabetes outpatient clinic and siblings (total n=42). Cutaneous melanin density was estimated using a spectrophotometer. Parental report on past sun exposure was obtained. DNA methylation analysis of the VDR gene promoter was conducted. Matched data analysis was performed comparing each case directly to their sibling. Cases were significantly more likely to have lighter skin pigmentation at the upper arm (AOR 0.69 [95% CI: 0.52, 0.90]; P=0.01). Low infant sun exposure was imprecisely associated with a two-fold increase in T1DM risk (AOR 2.43 [95% CI: 0.91, 6.51]; P=0.08 for under 1 h of winter sun exposure per leisure day). The VDR gene promoter was completely unmethylated in both cases and siblings. The previously demonstrated association between light skin pigmentation and T1DM risk was evident even in this comparison across sibling pairs. Further work on past UVR exposure and related factors such as skin pigmentation is required.

The changing landscape of research funding: challenges for mid-career researchers.

Funding research is a challenge faced by most scientists around the world. Genome Biology has invited four scientists based in three different countries to share their own experience and opinions regarding funding, the difficulties young scientists must overcome, and how the process of securing funding can be improved. In this part, Nick Wong shares his experience in securing funding for his research in Australia.

Oral versus intravenous antibiotics for bone and joint infections: the OVIVA non-inferiority RCT.

BACKGROUND: Management of bone and joint infection commonly includes 4-6 weeks of intravenous (IV) antibiotics, but there is little evidence to suggest that oral (PO) therapy results in worse outcomes. OBJECTIVE: To determine whether or not PO antibiotics are non-inferior to IV antibiotics in treating bone and joint infection. DESIGN: Parallel-group, randomised (1 : 1), open-label, non-inferiority trial. The non-inferiority margin was 7.5%. SETTING: Twenty-six NHS hospitals. PARTICIPANTS: Adults with a clinical diagnosis of bone, joint or orthopaedic metalware-associated infection who would ordinarily receive at least 6 weeks of antibiotics, and who had received ≤ 7 days of IV therapy from definitive surgery (or start of planned curative treatment in patients managed non-operatively). INTERVENTIONS: Participants were centrally computer-randomised to PO or IV antibiotics to complete the first 6 weeks of therapy. Follow-on PO therapy was permitted in either arm. MAIN OUTCOME MEASURE: The primary outcome was the proportion of participants experiencing treatment failure within 1 year. An associated cost-effectiveness evaluation assessed health resource use and quality-of-life data. RESULTS: Out of 1054 participants (527 in each arm), end-point data were available for 1015 (96.30%) participants. Treatment failure was identified in 141 out of 1015 (13.89%) participants: 74 out of 506 (14.62%) and 67 out of 509 (13.16%) of those participants randomised to IV and PO therapy, respectively. In the intention-to-treat analysis, using multiple imputation to include all participants, the imputed risk difference between PO and IV therapy for definitive treatment failure was -1.38% (90% confidence interval -4.94% to 2.19%), thus meeting the non-inferiority criterion. A complete-case analysis, a per-protocol analysis and sensitivity analyses for missing data each confirmed this result. With the exception of IV catheter complications [49/523 (9.37%) in the IV arm vs. 5/523 (0.96%) in the PO arm)], there was no significant difference between the two arms in the incidence of serious adverse events. PO therapy was highly cost-effective, yielding a saving of £2740 per patient without any significant difference in quality-adjusted life-years between the two arms of the trial. LIMITATIONS: The OVIVA (Oral Versus IntraVenous Antibiotics) trial was an open-label trial, but bias was limited by assessing all potential end points by a blinded adjudication committee. The population was heterogenous, which facilitated generalisability but limited the statistical power of subgroup analyses. Participants were only followed up for 1 year so differences in late recurrence cannot be excluded. CONCLUSIONS: PO antibiotic therapy is non-inferior to IV therapy when used during the first 6 weeks in the treatment for bone and joint infection, as assessed by definitive treatment failure within 1 year of randomisation. These findings challenge the current standard of care and provide an opportunity to realise significant benefits for patients, antimicrobial stewardship and the health economy. FUTURE WORK: Further work is required to define the optimal total duration of therapy for bone and joint infection in the context of specific surgical interventions. Currently, wide variation in clinical practice suggests significant redundancy that likely contributes to the excess and unnecessary use of antibiotics. TRIAL REGISTRATION: Current Controlled Trials ISRCTN91566927. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 23, No. 38. See the NIHR Journals Library website for further project information.

Treatment of bone and joint infection usually requires a long course of antibiotics. Doctors usually give these by injection through a vein (intravenously) for the first 4­6 weeks, rather than by mouth (orally). Although intravenous (IV) administration is more expensive and less convenient for patients, most doctors believe that it is more effective. However, there is little evidence to support this. The OVIVA (Oral Versus IntraVenous Antibiotics) trial set out to challenge this assumption. A total of 1054 patients from 26 UK hospitals were randomly allocated to receive the first 6 weeks of antibiotic therapy either intravenously or orally. Irrespective of the route of administration, the choice of antibiotic was left to an infection specialist so as to ensure that the most appropriate antibiotics were given. Patients were followed up for 1 year. Thirty-nine participants were lost to follow-up. Among the remaining 1015 participants, treatment failure occurred in 14.6% of those treated intravenously and 13.2% of those treated with PO antibiotics. This difference could easily have occurred by chance. Even if it was not by chance, the difference does not suggest that PO therapy is associated with worse outcomes than IV therapy and is too small to conclude that PO therapy is better than IV therapy. Participants in the IV group stayed in hospital longer and 10% of them had complications related to the IV line used for administering the antibiotics. In addition, their treatment was, overall, more expensive. We conclude that PO antibiotic therapy has no disadvantages for the early management of bone and joint infection. It is also cheaper and associated with fewer complications.

BAC-based PCR fragment microarray: high-resolution detection of chromosomal deletion and duplication breakpoints.

The introduction of molecular techniques in conjunction with classical cytogenetic methods has in recent years greatly improved the diagnostic potential for chromosomal abnormalities. In particular, microarray-comparative genomic hybridization (CGH) based on the use of BAC clones promises a sensitive strategy for the detection of DNA copy-number changes on a genomewide scale, offering a resolution as high as >30,000 "bands" (as defined by the number of BACs within the currently highest-density BAC array) [Ishkanian et al., 2004]. We have tested the possibility of further increasing this resolution using PCR fragments generated from individual BAC clones. Using this approach, we have efficiently defined the proximal and distal breakpoints in two cytogenetic cases, one duplication and one deletion, to within 5-20 kb. The results support the potential use of BAC-based PCR fragments to further improve the resolution of the microarray-CGH strategy by an order of magnitude.

Learning experience of Chinese nursing students in an online clinical English course: qualitative study.

The low English proficiency of Chinese nurse/nursing students affects their performance when they work in English-speaking countries. However, limited resources are available to help them improve their workplace English, i.e. English used in a clinical setting. To this end, it is essential to look for an appropriate and effective means to assist them in improving their clinical English. The objective of this study is to evaluate the learning experience of Chinese nursing students after they have completed an online clinical English course. Focus group interview was used to explore their learning experience. 100 students in nursing programs at Tung Wah College were recruited. The inclusion criteria were: (1) currently enrolled in a nursing program; and (2) having clinical experience. Eligible participants self-registered for the online English course, and were required to complete the course within 3 months. After that, semi-structured interviews were conducted on students whom completed the whole and less than half of the course. One of the researchers joined each of the interviews as a facilitator and an observer. Thematic analysis was used to analyze the data. Finally, 7 themes emerged from the interviews: technical issues, adequacy of support, time requirement, motivation, clarity of course instruction, course design, and relevancy of the course. Participants had varied opinions on the 2 themes: motivation and relevancy of the course. Overall, results of this study suggest that the online English course helped students improve their English. Factors which support their learning are interactive course design, no time constraint, and relevancy to their work/study. Factors which detracted from their learning are poor accessibility, poor technical and learning support and no peer support throughout the course.

Atypical Spitzoid neoplasms: a review of potential markers of biological behavior including sentinel node biopsy.

Atypical cutaneous melanocytic lesions, including those with Spitzoid features, can be difficult to categorize as benign or malignant. This can lead to suboptimal management, with potential adverse patient outcomes. Recent studies have enhanced knowledge of the molecular and genetic biology of these lesions and, combined with clinicopathological findings, is further defining their biological spectrum, classification, and behavior. Sentinel node biopsy provides important prognostic information in patients with cutaneous melanoma, but its role in the management of melanocytic lesions of uncertain malignant potential (MELTUMP) is controversial. This paper examines the role of molecular testing and sentinel node biopsy in MELTUMPs, particularly atypical Spitzoid tumors.

Specific-site methylation of tumour suppressor ANKRD11 in breast cancer.

ANKRD11 is a putative tumour suppressor gene in breast cancer, which has been shown in our laboratory to be a co-activator of p53. Our data suggest that down-regulation of ANKRD11 is associated with breast tumourigenesis. Breast cancer cell lines treated with DNA demethylating agents resulted in up-regulation of ANKRD11 expression suggesting that promoter DNA methylation may be responsible for its down-regulation. The transcriptional activity of a CpG-rich region 2kb upstream of the transcription initiation site of ANKRD11 was investigated using dual-luciferase reporter assays. The constructs carrying -661 to -571 bp promoter sequence showed significant transcriptional activity. Using the SEQUENOM Epityper Platform, the region between -770 and +399 bp was analysed in 25 breast tumours, four normal breast tissues and five normal blood samples. The region between -770 and -323 bp was shown to be frequently methylated in breast tumours. The methylation patterns of all analysed CpGs in this region were identical in the normal and tumour samples, except for a 19 bp region containing three CpG sites. These sites had significantly higher levels of methylation in tumours (40%) compared to normal samples (6%). Our findings support the role of ANKRD11 as a tumour suppressor gene and suggest that aberrant DNA methylation of three CpGs in a 19 bp region within the ANKRD11 promoter may be responsible for its down-regulation in breast cancer.