Chemical Stability of Lorazepam Oral Solution Repackaged in Plastic Oral Syringes at Room and Refrigerated Temperature.

Purpose: Generic lorazepam oral solution is supplied in a 30 mL multi-dose bottle requiring protection from light and refrigeration, with a beyond use date of 90 days once the bottle is opened. The repackaging of 1 mL doses of lorazepam oral solution into oral syringes allows for facilitated dispensing, yet no available data supports repackaging and storing lorazepam oral solution in syringes. The validation and application of a stability-indicating high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the quantification of lorazepam allowed for the determination of the stability of lorazepam oral solution when stored in oral syringes. Methods: A stability-indicating HPLC-UV method was developed for the quantification of lorazepam in oral solution. The method was validated using guidance from USP < 1225 >. For the stability investigation, 2 mg/mL lorazepam oral solution was aliquoted into clear plastic oral syringes in 1 mLmilliliter doses from 2 multi-dose stock bottles and randomly allocated for storage in room temperature or refrigerated environment. Baseline lorazepam concentrations were measured on the day the study was initiated and designated as 100% initial concentration samples. Subsequent samples were analyzed in triplicate at time points of 24, 48, and 96 hours and 7, 10, 14, 21, 30, and 60 days. Results: The calibration curves on three non-consecutive days met the linearity criteria of R 2 > 0.99. Inter- day and intra-day precision and accuracy (percent relative standard deviation and percent error) were ≤2% over three days. During the stability investigation, percent initial concentration of lorazepam from room and refrigerated syringes remained above 90% for the duration of the study. Conclusion: The stability-indicating HPLC-UV method was successfully applied to the investigation of lorazepam oral solution stability when stored in syringes at room and refrigerated temperatures. The emergent need for use of lorazepam concentrate for inpatients and the restrictions of how the medication is supplied necessitated a need for the evaluation of repackaging into unit dose syringes for immediate availability from automated dispensing cabinets. Lorazepam oral solution stored in clear plastic syringes maintained greater than 90% initial concentration at both room and refrigerated temperatures for 60 days.

Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate-resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage plasticity and phenotypic heterogeneity. Castrate-resistant prostate adenocarcinoma can transition to neuroendocrine (NE) and occasionally to amphicrine, co-expressed luminal and NE, phenotypes. We developed castrate-resistant prostate cancer (CRPC) patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and NE phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor (St/Pr) cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent St/Pr cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from St/Pr cells and self-renewing differentiated luminal cells. Neuroendocrine prostate cancer (NEPC) was composed almost exclusively of self-renewing St/Pr cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine St/Pr subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell (SC) characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate SCs.

Factors Influencing Shared Decision-Making Between Healthcare Providers and Lesbian, Gay, Bisexual, Transgender, and Queer People of Color About Intimate Partner Violence.

Within the lesbian, gay, bisexual, transgender, and queer (LGBTQ) community, people of color (POC) disproportionately experience intimate partner violence (IPV). While shared decision-making (SDM)-a model of patient-provider communication-about IPV could benefit LGBTQ POC, its unique challenges merit consideration. This study identifies key factors affecting SDM between LGBTQ POC and healthcare providers surrounding IPV. LGBTQ POC participants (n = 217) in Chicago and San Francisco completed surveys about demographic information, healthcare utilization, and IPV history. Individual interviews and focus groups were then conducted with a Chicago-based subset of participants (n = 46) who identified as LGBTQ IPV survivors of color. Descriptive analyses were conducted of survey responses while focus group and interview transcripts were analyzed and thematically coded. Although 71% of survey participants experienced IPV, only 35% were asked about IPV in healthcare interactions within the previous year. Focus group and interview participants endorsed encounter-, patient-, and provider-centered factors affecting SDM around IPV. When IPV was discussed, patient-provider trust was essential while concordance of identities could either encourage or discourage IPV disclosure. Patients were hesitant to disclose IPV if they had never discussed their LGBTQ identity with their provider or thought providers would ignore their preferences for addressing IPV. Deterrents to SDM included providers denying the prevalence of IPV among LGBTQ individuals or lacking resources to support LGBTQ IPV survivors of color. This study highlights the identity-driven barriers that LGBTQ POC face in discussing IPV with providers. Utilizing SDM to discuss IPV with LGBTQ POC can better address the diverse health needs of this community. However, its success requires that providers acknowledge the diversity of experiences among this population, promote LGBTQ-inclusive practices, and identify resources welcome to LGBTQ POC.

Single-cell lineage capture across genomic modalities with CellTag-multi reveals fate-specific gene regulatory changes.

Complex gene regulatory mechanisms underlie differentiation and reprogramming. Contemporary single-cell lineage-tracing (scLT) methods use expressed, heritable DNA barcodes to combine cell lineage readout with single-cell transcriptomics. However, reliance on transcriptional profiling limits adaptation to other single-cell assays. With CellTag-multi, we present an approach that enables direct capture of heritable random barcodes expressed as polyadenylated transcripts, in both single-cell RNA sequencing and single-cell Assay for Transposase Accessible Chromatin using sequencing assays, allowing for independent clonal tracking of transcriptional and epigenomic cell states. We validate CellTag-multi to characterize progenitor cell lineage priming during mouse hematopoiesis. Additionally, in direct reprogramming of fibroblasts to endoderm progenitors, we identify core regulatory programs underlying on-target and off-target fates. Furthermore, we reveal the transcription factor Zfp281 as a regulator of reprogramming outcome, biasing cells toward an off-target mesenchymal fate. Our results establish CellTag-multi as a lineage-tracing method compatible with multiple single-cell modalities and demonstrate its utility in revealing fate-specifying gene regulatory changes across diverse paradigms of differentiation and reprogramming.

Defining cardiac functional recovery in end-stage heart failure at single-cell resolution.

Recovery of cardiac function is the holy grail of heart failure therapy yet is infrequently observed and remains poorly understood. In this study, we performed single-nucleus RNA sequencing from patients with heart failure who recovered left ventricular systolic function after left ventricular assist device implantation, patients who did not recover and non-diseased donors. We identified cell-specific transcriptional signatures of recovery, most prominently in macrophages and fibroblasts. Within these cell types, inflammatory signatures were negative predictors of recovery, and downregulation of RUNX1 was associated with recovery. In silico perturbation of RUNX1 in macrophages and fibroblasts recapitulated the transcriptional state of recovery. Cardiac recovery mediated by BET inhibition in mice led to decreased macrophage and fibroblast Runx1 expression and diminished chromatin accessibility within a Runx1 intronic peak and acquisition of human recovery signatures. These findings suggest that cardiac recovery is a unique biological state and identify RUNX1 as a possible therapeutic target to facilitate cardiac recovery.

Pelvic floor imaging in asymptomatic subjects.

AIM: The aim of this work was to determine the range of normal imaging features during total pelvic floor ultrasound (TPFUS) (transperineal, transvaginal, endovaginal and endoanal) and defaecation MRI (dMRI). METHOD: Twenty asymptomatic female volunteers (mean age 36.5 years) were prospectively investigated with dMRI and TPFUS. Subjects were screened with symptom questionnaires (ICIQ-B, St Mark's faecal incontinence score, obstructed defaecation syndrome score, ICIQ-V, BSAQ). dMRI and TPFUS were performed and interpreted by blinded clinicians according to previously published methods. RESULTS: The subjects comprised six parous and 14 nulliparous women, of whom three were postmenopausal. There were three with a rectocoele on both modalities and one with a rectocoele on dMRI only. There was one with intussusception on TPFUS. Two had an enterocoele on both modalities and one on TPFUS only. There were six with a cystocoele on both modalities, one on dMRI only and one on TPFUS only. On dMRI, there were 12 with functional features. Four also displayed functional features on TPFUS. Two displayed functional features on TPFUS only. CONCLUSION: This study demonstrates the presence of abnormal findings on dMRI and TPFUS without symptoms. There was a high rate of functional features on dMRI. This series is not large enough to redefine normal parameters but is helpful for appreciating the wide range of findings seen in health.

Single-cell lineage tracing reveals hierarchy and mechanism of adipocyte precursor maturation.

White adipose tissue is crucial in various physiological processes. In response to high caloric intake, adipose tissue may expand by generating new adipocytes. Adipocyte precursor cells (progenitors and preadipocytes) are essential for generating mature adipocytes, and single-cell RNA sequencing provides new means to identify these populations. Here, we characterized adipocyte precursor populations in the skin, an adipose depot with rapid and robust generation of mature adipocytes. We identified a new population of immature preadipocytes, revealed a biased differentiation potential of progenitor cells, and identified Sox9 as a critical factor in driving progenitors toward adipose commitment, the first known mechanism of progenitor differentiation. These findings shed light on the specific dynamics and molecular mechanisms underlying rapid adipogenesis in the skin.

Dissecting cell identity via network inference and in silico gene perturbation.

Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks1. Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms-mouse and human haematopoiesis, and zebrafish embryogenesis-and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto, an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a. Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation.

Knowledge of human papillomavirus vaccination: A multi-institution, cross-sectional study of allopathic and osteopathic medical students.

Human papillomavirus (HPV) vaccination is a well-established and successful tool for preventing HPV-related cancers. However, vaccine uptake remains low, influenced by patient hesitancy around safety concerns and little opportunity to discuss the vaccine with trusted healthcare providers. We conducted a national, cross-sectional study of allopathic and osteopathic medical students regarding knowledge of HPV vaccination guidelines March-April 2021. Analysis sought to identify gaps in knowledge as well as demographic and academic correlates of knowledge. A total of 718 students participated (response rate = 50.8%). While 92.8% of participants identified the connection between HPV and cervical cancer, lower percentages associated HPV with vaginal/vulvar (67.7%), anal (63.3%), and penile (53.9%) cancers. Low percentages of participants correctly identified age of HPV vaccine eligibility (33.3%) and how many doses are needed for full protection (48.1%). This study identifies specific knowledge gaps in medical students' training on HPV-related cancers and HPV vaccination guidelines. Through addressing these gaps, we may improve HPV vaccine uptake and decrease the incidence of HPV-related cancers.

The association between levator plate integrity and pelvic floor defaecatory dysfunction.

AIMS: Levator ani deficiency has been implicated in anterior pelvic floor pathology but its association with pelvic floor defaecatory dysfunction is less clear. The aim was to examine the relationship of levator ani deficiency with anatomical abnormalities (rectocoele, intussusception, enterocoele, perineal descent) and patient symptoms (bowel, vagina) in patients with pelvic floor defaecatory dysfunction. METHODS: The prospective observational case series of 223 women presenting to a tertiary colorectal pelvic floor unit with defaecatory dysfunction. Each underwent assessment with symptom severity and quality of life (QoL) scores, integrated total pelvic floor ultrasound (PFUS) (transvaginal, transperineal) and defaecation proctography (DP). Rectocoele, intussusception, enterocoele and perineal descent were assessed on both. Levator ani deficiency was scored using endovaginal ultrasound (score 0-18; mild [0-6], moderate [>6-12], severe [>12-18]). RESULTS: The proportion of patients with rectocoele, enterocoele, and intussusception increased with increasing levator ani damage (mild, moderate, severe). There was a weakly positive correlation between size of rectocoele and levator ani deficiency. On PFUS, there was a weakly positive correlation between severity of intussusception and enterocoele with levator ani deficiency. On DP, there was a weakly positive correlation between perineal descent and levator ani deficiency. There was no association between bowel symptom and QoL scores and levator ani deficiency. Vaginal symptoms were associated with levator ani deficiency. CONCLUSIONS: Anatomical abnormalities which are implicated in pelvic floor defaecatory dysfunction (rectocoele, intussusception, enterocoele, perineal descent) were associated with worsening levator ani deficiency. There was no association between bowel symptoms and levator ani deficiency. Vaginal symptoms were associated with levator ani deficiency.

Gene regulatory network reconfiguration in direct lineage reprogramming.

In direct lineage conversion, transcription factor (TF) overexpression reconfigures gene regulatory networks (GRNs) to reprogram cell identity. We previously developed CellOracle, a computational method to infer GRNs from single-cell transcriptome and epigenome data. Using inferred GRNs, CellOracle simulates gene expression changes in response to TF perturbation, enabling in silico interrogation of network reconfiguration. Here, we combine CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct reprogramming paradigm. By linking early network state to reprogramming outcome, we reveal distinct network configurations underlying successful and failed fate conversion. Via in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting their identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations, providing new mechanistic insights into lineage reprogramming.

The impact of AI on research.

Large and complex datasets have made artificial intelligence (AI) an invaluable tool for discovery across biological research. We asked experts how AI has impacted their work. Their experiences and perspectives offer thoughtful insights into potential offered by AI for their fields.

Anniversary reflections: Inspiring discoveries and the future of the field.

Cell Stem Cell was launched in 2007, and this year marks its 15th anniversary. To recognize this occasion, we asked six advisory board members to reflect on inspiring discoveries reported in Cell Stem Cell and how these breakthroughs connect to their vision for the future of the field.

Identification of a retinoic acid-dependent haemogenic endothelial progenitor from human pluripotent stem cells.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. During embryonic development, HSCs derive from haemogenic endothelium (HE) in a NOTCH- and retinoic acid (RA)-dependent manner. Although a WNT-dependent (WNTd) patterning of nascent hPSC mesoderm specifies clonally multipotent intra-embryonic-like HOXA+ definitive HE, this HE is functionally unresponsive to RA. Here we show that WNTd mesoderm, before HE specification, is actually composed of two distinct KDR+ CD34neg populations. CXCR4negCYP26A1+ mesoderm gives rise to HOXA+ multilineage definitive HE in an RA-independent manner, whereas CXCR4+ ALDH1A2+ mesoderm gives rise to HOXA+ multilineage definitive HE in a stage-specific, RA-dependent manner. Furthermore, both RA-independent (RAi) and RA-dependent (RAd) HE harbour transcriptional similarity to distinct populations found in the early human embryo, including HSC-competent HE. This revised model of human haematopoietic development provides essential resolution to the regulation and origins of the multiple waves of haematopoiesis. These insights provide the basis for the generation of specific haematopoietic populations, including the de novo specification of HSCs.

Supervision and assessment in midwifery practice during a global pandemic: A cohort survey.

AIM: To evaluate new practice assessment processes in midwifery placements linked to a United Kingdom university during COVID-19. BACKGROUND: The new regulatory body standards for supervising and assessing practice of student nurses and midwives replaced the former mentorship model. Locally, these were implemented in conjunction with the Practice Assessment Toolkit - a resource developed from the national project exploring grading in midwifery practice. Emergency regulatory standards in response to the global pandemic impacted on student placements and temporarily created greater flexibility in assessing practice. DESIGN: A cohort survey using mixed methods. METHODS: Online questionnaires comprising qualitative and quantitative components targeted each of the four stakeholder groups: second and third-year student midwives, practice supervisors, practice assessors, midwifery academics. Aspects of the assessment process were explored including whether changes in the assessment process had influenced reliability, views of the Practice Assessment Toolkit and grading versus a binary pass/fail approach. RESULTS: Views were mixed about whether the new practice assessment process improved reliability, but the context of the global pandemic was acknowledged. Some clinicians embraced the changes more readily than others, and organisational approaches varied. There was a reliance on students' knowledge and understanding of requirements. Inconsistencies could have a detrimental effect on student learning and the reliability of assessment. Practice assessors relied on a range of practice supervisors' comments to make their decisions. Some participants considered that the separation of these roles enhanced reliability of assessment while others found it challenging. Detachment of students from the assessment process appeared to promote objectivity and honesty, potentially reducing grade inflation. The Practice Assessment Toolkit was useful and assisted reliability, however issues around individual expectations, application and relationships persisted. Most participants were in favour of retaining grading of practice in at least the final year of the midwifery programme. Qualitative themes comprised: Impetus for change; Reliance and reliability; Benefits of detachment; Mind the gap; To grade or not to grade. CONCLUSIONS: This first evaluation of the new practice assessment process suggested it has potential to increase reliability, however this is dependent on individual and institutional understanding and adherence. The context of the global pandemic also influenced implementation and findings. The benefits of using consistent terminology were demonstrated through application of the Practice Assessment Toolkit. Further evidence is presented of the advantages and challenges of grading practice or using the binary approach. Recommendations are made to promote concepts identified in the findings and for future research. TWEETABLE ABSTRACT: The first evaluation of the new NMC practice supervision and assessment process demonstrated its potential to increase reliability but depended on understanding and application. Concurrent use of the Practice Assessment Toolkit enhanced consistency in midwifery student assessment.

Capybara: A computational tool to measure cell identity and fate transitions.

Measuring cell identity in development, disease, and reprogramming is challenging as cell types and states are in continual transition. Here, we present Capybara, a computational tool to classify discrete cell identity and intermediate "hybrid" cell states, supporting a metric to quantify cell fate transition dynamics. We validate hybrid cells using experimental lineage tracing data to demonstrate the multi-lineage potential of these intermediate cell states. We apply Capybara to diagnose shortcomings in several cell engineering protocols, identifying hybrid states in cardiac reprogramming and off-target identities in motor neuron programming, which we alleviate by adding exogenous signaling factors. Further, we establish a putative in vivo correlate for induced endoderm progenitors. Together, these results showcase the utility of Capybara to dissect cell identity and fate transitions, prioritizing interventions to enhance the efficiency and fidelity of stem cell engineering.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.

Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.