Bidirectional multiciliated cell extrusion is controlled by Notch-driven basal extrusion and Piezo1-driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late-stage development, there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional, whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, response to the Notch signal is age dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo1, such that premature activation of Piezo1 leads to early extrusion while blocking Piezo1 leads to MCC maintenance. Distinct mechanisms for MCC loss underlie the importance of their removal during epithelial remodeling.

Novel centriolar defects underlie a primary ciliary dyskinesia phenotype in an adenylate kinase 7 deficient ciliated epithelium.

The skin of Xenopus embryos contains numerous multiciliated cells (MCCs), which collectively generate a directed fluid flow across the epithelial surface essential for distributing the overlaying mucous. MCCs develop into highly specialized cells to generate this flow, containing approximately 150 evenly spaced centrioles that give rise to motile cilia. MCC-driven fluid flow can be impaired when ciliary dysfunction occurs, resulting in primary ciliary dyskinesia (PCD) in humans. Mutations in a large number of genes (~50) have been found to be causative to PCD. Recently, studies have linked low levels of Adenylate Kinase 7 (AK7) gene expression to patients with PCD; however, the mechanism for this link remains unclear. Additionally, AK7 mutations have been linked to multiple PCD patients. Adenylate kinases modulate ATP production and consumption, with AK7 explicitly associated with motile cilia. Here we reproduce an AK7 PCD-like phenotype in Xenopus and describe the cellular consequences that occur with manipulation of AK7 levels. We show that AK7 localizes throughout the cilia in a DPY30 domain-dependent manner, suggesting a ciliary function. Additionally, we find that AK7 overexpression increases centriole number, suggesting a role in regulating centriole biogenesis. We find that in AK7-depleted embryos, cilia number, length, and beat frequency are all reduced, which in turn, significantly decreases the tissue-wide mucociliary flow. Additionally, we find a decrease in centriole number and an increase in sub-apical centrioles, implying that AK7 influences both centriole biogenesis and docking, which we propose underlie its defect in ciliogenesis. We propose that AK7 plays a role in PCD by impacting centriole biogenesis and apical docking, ultimately leading to ciliogenesis defects that impair mucociliary clearance.

Risk Factors and Cardiovascular Events in Orthotopic Liver Transplantation.

BACKGROUND AND AIMS: Patients undergoing liver transplantation often have significant cardiovascular risk factors and may experience cardiac-related morbidity and mortality. The aim of this study was to assess the frequency of cardiovascular risk factors and outcomes in this population, and to identify factors predictive of post-transplant cardiac morbidity and mortality. METHODS: We studied 261 patients who underwent liver transplantation at a single Veterans' Affairs Medical center between 1997 and 2015 to evaluate new cardiovascular events post-transplantation. RESULTS: The cohort consisted of 261 patients (253 men and 8 women) with a mean age of 58.3 (± 6.5 years), mean model for end-stage liver disease score of 18.0 (±7.2), and mean Framingham risk score of 8.1 (± 4.9). After a median follow-up of 82 months a total of 75 (28.7%) patients died, with 13 deaths (17.3%) attributed to a primary cardiovascular event and 9 (12%) deaths due to a coronary-specific event. Coronary events and/ or the need for revascularization post-transplant occurred in 24 (9.2%) patients. The strongest pre-transplant predictors of mortality were age (p=0.01), Framingham risk score (p=0.01), preexisting coronary artery disease (p=0.01), and preexisting dyslipidemia (p=0.01). The strongest post-transplant predictors of mortality were new-onset hypertension (p=0.01) and new-onset diabetes mellitus (p=0.03) post-transplant. CONCLUSIONS: In this cohort of veterans, coronary artery disease was significantly associated with mortality in the post liver transplantation population; however, the majority of deaths after transplant were attributable to other causes.

A Collaborative-Care Telephone-Based Intervention for Depression, Anxiety, and at-Risk Drinking in Primary Care: The PARTNERs Randomized Clinical Trial.

BACKGROUND: Collaborative care (CC) could improve outcomes in primary care patients with common mental conditions. We assessed the effectiveness of a transdiagnostic model of telephone-based CC (tCC) delivered by lay providers to primary care patients with depression, anxiety, or at-risk drinking. METHODS: PARTNERS was a pragmatic trial in 502 primary care adults presenting with depressive symptoms, anxiety symptoms, or at-risk drinking randomized to (1) usual care by primary care providers (PCPs) enhanced with the results of computer-assisted telephone-based assessments (at baseline and 4, 8, and 12 months later) (enhanced usual care [eUC]) or (2) tCC consisting of eUC plus frequent telephone coaching and psychoeducation provided by mental health technicians who also communicated to the PCP recommendations from a psychiatrist for evidence-based pharmacotherapy, psychotherapy, or, when indicated, referrals to mental health services. The primary analysis compared the change on the 9-item Patient Health Questionnaire (PHQ-9) in participants presenting with depression (n = 366) randomized to tCC versus eUC. Secondary analyses compared changes on the Generalized Anxiety Disorder-7 scale (GAD-7) in those presenting with anxiety (n = 298); or change in the number of weekly drinks in those presenting with at-risk drinking (n = 176). RESULTS: There were no treatment or time×treatment effects between tCC and eUC on PHQ-9 scores for patients with depression during the 12-month follow-up. However, there was a treatment effect (tCC > eUC) on GAD-7 scores in those with anxiety and a time×treatment interaction effect on the number of weekly drinks (tCC > eUC) in those with at-risk drinking. CONCLUSION: Implementing transdiagnostic tCC for common mental disorders using lay providers appears feasible in Canadian primary care. While tCC was not better than eUC for depression, there were some benefits for those with anxiety or at-risk drinking. Future studies will need to confirm whether tCC differentially benefits patients with depression, anxiety, or at-risk drinking.

Bidirectional multiciliated cell extrusion is controlled by Notch driven basal extrusion and Piezo 1 driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late stage development there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, receptivity to the Notch signal is age-dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo 1 such that premature activation of Piezo 1 leads to early extrusion while blocking Piezo 1 leads to MCC maintenance. Distinct mechansms for MCC loss underlie the importance of their removal during epithelial remodeling. Summay Statement: Cell extrusion typically occurs unidirectionally. We have identified a single population of multiciliated cells that extrudes bidirectionally: Notch-driven basal extrusion and Piezo 1-mediated apical extrusion.

History of Suicide Attempt and Clozapine Treatment in Veterans With Schizophrenia or Schizoaffective Disorder.

Objective: To evaluate whether a history of suicide attempt increases the odds of receiving clozapine treatment in veterans with schizophrenia or schizoaffective disorder.Methods: Electronic health record data were obtained for veterans with schizophrenia or schizoaffective disorder treated at any US Veterans Affairs Medical Center between January 1, 2000, and January 31, 2021 (N = 134,692). Logistic regression (adjusted and unadjusted) was applied to estimate odds ratios (ORs) for clozapine treatment in suicide attempters relative to nonattempters.Results: 3,407 patients had a documented history of suicide attempt, while 6,867 patients had received clozapine treatment. Also, 9.4% (n = 321) of suicide attempters versus 5.0% (n = 6546) of nonattempters had received clozapine treatment. The odds of being treated with clozapine was approximately 2-fold in patients with a history of suicide attempt in unadjusted (OR = 1.98, 95% CI, 1.76-2.22) and adjusted (OR = 1.91, 95% CI, 1.67-2.15) analyses.Conclusions: Despite the higher odds of clozapine treatment in suicide attempters with schizophrenia or schizoaffective disorder, clozapine was underutilized in the current sample of veterans. Concerted efforts should be made to expand the use of clozapine in patients with schizophrenia or schizoaffective disorder, especially those with a history of suicide attempt.

Espin overexpression causes stereocilia defects and provides an anti-capping effect on actin polymerization.

Stereocilia are actin-based projections of hair cells that are arranged in a step like array, in rows of increasing height, and that constitute the mechanosensory organelle used for the senses of hearing and balance. In order to function properly, stereocilia must attain precise sizes in different hair cell types and must coordinately form distinct rows with varying lengths. Espins are actin-bundling proteins that have a well-characterized role in stereocilia formation; loss of function mutations in Espin result in shorter stereocilia and deafness in the jerker mouse. Here we describe the generation of an Espin overexpressing transgenic mouse line that results in longer first row stereocilia and discoordination of second-row stereocilia length. Furthermore, Espin overexpression results in the misregulation of other stereocilia factors including GNAI3, GPSM2, EPS8, WHRN, and MYO15A, revealing that GNAI3 and GPSM2 are dispensable for stereocilia overgrowth. Finally, using an in vitro actin polymerization assay we show that espin provides an anti-capping function that requires both the G-actin binding WH2 domain as well as either the C-terminal F-actin binding domain or the internal xAB actin-binding domain. Our results provide a novel function for Espins at the barbed ends of actin filaments distinct from its previous known function of actin bundling that may account for their effects on stereocilia growth.

Impact of Lithium on Suicidality in the Veteran Population.

Background: Lithium has known antisuicidal properties making it an important agent to study in veterans with psychiatric conditions, a population at high risk for suicide. Methods: A single-site, retrospective chart review was conducted at a US Department of Veterans Affairs (VA) teaching hospital. Patients taking lithium for at least 6 months were identified using the VA Lithium Lab Monitoring Dashboard. The primary and secondary objectives were to evaluate the change in number of suicide attempts and suicidal ideation from 3 months prior to lithium initiation to 3 months after a 6-month duration of lithium. Results: The review included 98 patients; 47 (47.9%) received concomitant psychotherapy, 50 (51.0%) were taking an antipsychotic, and 29 (29.6%) an additional mood stabilizer. During the 6-month intervention period, 75 (76.5%) patients had a lithium level drawn and 28 were in the therapeutic range. Of the 98 patients, hospitalization for suicide attempt decreased from 4.1% before lithium use to 0% after lithium use for 6 months (P = .045). Hospitalization for suicidal ideation also decreased from 13.3% before lithium use to 1.0% after lithium use for 6 months (P = .0004). Conclusions: We observed a statistically significant reduction in hospitalization for suicide attempts and suicidal ideation in veterans prescribed lithium following nonfatal suicide behavior and suicidal ideation.

Computed Tomography Findings of Complete Branchial Cleft Fistula.

Branchial cleft anomalies are embryonic remnants of the branchial arches and are described as the second most common congenital neck mass. Depending on their extent, these anomalies are classified as a cyst, sinus, or fistula with branchial cysts being the most common. Branchial cysts deriving from the second branchial arch are by far the most common, accounting for approximately 95% of all cases. Complete second branch arch fistulas with both an internal and external opening are a rare variant of this anomaly, and even less have been well-documented on computed tomography (CT) imaging in the literature. We present here a case of a 20-year-old female with CT findings consistent with a complete second branchial arch fistula extending from the tonsillar fossa to the external lateral neck.

Basolateral protein Scribble binds phosphatase PP1 to establish a signaling network maintaining apicobasal polarity.

Scribble, a member of the LAP protein family, contributes to the apicobasal polarity (ABP) of epithelial cells. The LAP-unique region of these proteins, which is essential and sufficient for ABP, includes a conserved Leucine-Rich Repeat (LRR) domain. The major binding partners of this region that could regulate ABP remain unknown. Here, using proteomics, native gel electrophoresis, and site-directed mutagenesis, we show that the concave surface of LRR domain in Scribble participates in three types of mutually exclusive interactions-(i) homodimerization, serving as an auto-inhibitory mechanism; (ii) interactions with a diverse set of polarity proteins, such as Llgl1, Llgl2, EPB41L2, and EPB41L5, which produce distinct multiprotein complexes; and (iii) a direct interaction with the protein phosphatase, PP1. Analogy with the complex between PP1 and LRR domain of SDS22, a well-studied PP1 regulator, suggests that the Scibble-PP1 complex stores a latent form of PP1 in the basolateral cell cortex. Such organization may generate a dynamic signaling network wherein PP1 could be dispatched from the complex with Scribble to particular protein ligands, achieving fast dephosphorylation kinetics.

Tubulin acetylation promotes penetrative capacity of cells undergoing radial intercalation.

Post-translational modification of tubulin provides differential functions to microtubule networks. Here, we address the role of tubulin acetylation on the penetrative capacity of cells undergoing radial intercalation, which is the process by which cells move apically, insert between outer cells, and join an epithelium. There are opposing forces that regulate intercalation, namely, the restrictive forces of the epithelial barrier versus the penetrative forces of the intercalating cell. Positively and negatively modulating tubulin acetylation in intercalating cells alters the developmental timing such that cells with more acetylation penetrate faster. We find that intercalating cells preferentially penetrate higher-order vertices rather than the more prevalent tricellular vertices. Differential timing in the ability of cells to penetrate different vertices reveals that lower-order vertices represent more restrictive sites of insertion. We shift the accessibility of intercalating cells toward more restrictive junctions by increasing tubulin acetylation, and we provide a geometric-based mathematical model that describes our results.

Ciliogenesis and autophagy are coordinately regulated by EphA2 in the cornea to maintain proper epithelial architecture.

PURPOSE: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. METHODS: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. RESULTS: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. CONCLUSION: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.

Building a ciliated epithelium: Transcriptional regulation and radial intercalation of multiciliated cells.

The epidermis of the Xenopus embryo has emerged as a powerful tool for studying the development of a ciliated epithelium. Interspersed throughout the epithelium are multiciliated cells (MCCs) with 100+ motile cilia that beat in a coordinated manner to generate fluid flow over the surface of the cell. MCCs are essential for various developmental processes and, furthermore, ciliary dysfunction is associated with numerous pathologies. Therefore, understanding the cellular mechanisms involved in establishing a ciliated epithelium are of particular interest. MCCs originate in the inner epithelial layer of Xenopus skin, where Notch signaling plays a critical role in determining which progenitors will adopt a ciliated cell fate. Then, activation of various transcriptional regulators, such as GemC1 and MCIDAS, initiate the MCC transcriptional program, resulting in centriole amplification and the formation of motile cilia. Following specification and differentiation, MCCs undergo the process of radial intercalation, where cells apically migrate from the inner layer to the outer epithelial layer. This process involves the cooperation of various cytoskeletal networks, activation of various signaling molecules, and changes in cell-ECM and cell-cell adhesion. Coordination of these cellular processes is required for complete incorporation into the outer epithelial layer and generation of a functional ciliated epithelium. Here, we highlight recent advances made in understanding the transcriptional cascades required for MCC specification and differentiation and the coordination of cellular processes that facilitate radial intercalation. Proper regulation of these signaling pathways and processes are the foundation for developing a ciliated epithelium.

Mechanical stretch scales centriole number to apical area via Piezo1 in multiciliated cells.

How cells count and regulate organelle number is a fundamental question in cell biology. For example, most cells restrict centrioles to two in number and assemble one cilium; however, multiciliated cells (MCCs) synthesize hundreds of centrioles to assemble multiple cilia. Aberration in centriole/cilia number impairs MCC function and can lead to pathological outcomes. Yet how MCCs control centriole number remains unknown. Using Xenopus, we demonstrate that centriole number scales with apical area over a remarkable 40-fold change in size. We find that tensile forces that shape the apical area also trigger centriole amplification based on both cell stretching experiments and disruption of embryonic elongation. Unexpectedly, Piezo1, a mechanosensitive ion channel, localizes near each centriole suggesting a potential role in centriole amplification. Indeed, depletion of Piezo1 affects centriole amplification and disrupts its correlation with the apical area in a tension-dependent manner. Thus, mechanical forces calibrate cilia/centriole number to the MCC apical area via Piezo1. Our results provide new perspectives to study organelle number control essential for optimal cell function.

Veteran adherence to oral versus injectable AUD medication treatment.

INTRODUCTION: AUD medication treatment has been shown to improve outcomes compared with placebo when confined to per-protocol analysis. The same outcomes, however, have not always been maintained in intent-to-treat analysis, thus suggesting adherence may have a significant impact on efficacy outcomes. There is conflicting evidence present in the literature comparing adherence to oral versus injectable AUD pharmacotherapy and a paucity of information in the veteran population on risk factors for low adherence. METHODS: The primary end point of this retrospective chart review was to determine whether adherence rates differ between oral and injectable AUD treatments in veterans during the first year of treatment (at 3, 6, 9, and 12 months) using the portion of days covered model. Secondary end points were to determine differing characteristics between patients with high versus low adherence and compare alcohol-related readmission rates and discontinuation rates between groups. RESULTS: Adherence to injectable extended-release (XR) naltrexone was significantly higher than oral naltrexone at all time points and was significantly higher than disulfiram at 3, 6, and 9 months, but it was not significantly different from acamprosate at any time point. At months 9 and 12, acamprosate had significantly higher adherence compared with oral naltrexone. Patients with higher adherence were seen more frequently in the mental health clinic and had previously tried more AUD medications. The discontinuation rates and alcohol-related admission rates were not significantly different between groups at 1 year. DISCUSSION: XR naltrexone may improve adherence rates compared with oral naltrexone or disulfiram, but not acamprosate based on these outcomes. Patients may have increased adherence if they are seen more often in clinic and have trialed more AUD medications.

Case Report: Concurrent Clonidine Abuse and Opioid Use Disorder.

Clonidine is a centrally acting alpha-2 selective adrenergic receptor agonist used to treat hypertension and to control or prevent withdrawal in patients with opioid and alcohol use disorders. Case reports describe abuse of clonidine alone or in combination with benzodiazepines, methadone, codeine, or heroin. Clonidine reportedly boosts and extends the opioid-related euphoria and reduces the amount of psychoactive drug needed. In this case report, we describe clonidine abuse and withdrawal management in an elderly patient with concurrent opioid use disorder. The usage of clonidine in the treatment of opioid detoxification remains controversial. Clonidine abuse is underestimated and requires more attention among health-care providers who concurrently prescribe clonidine and opioids. With the opioid epidemic becoming increasingly prevalent, physicians and other health-care providers must be vigilant in their opioid prescribing as well as concurrent prescribing of other psychoactive pharmacologic agents.

The primary care assessment and research of a telephone intervention for neuropsychiatric conditions with education and resources study: Design, rationale, and sample of the PARTNERs randomized controlled trial.

While most patients with depression, anxiety, or at-risk drinking receive care exclusively in primary care settings, primary care providers experience challenges in diagnosing and treating these common problems. Over the past two decades, the collaborative care model has addressed these challenges. However, this model has been adopted very slowly due to the high costs of care managers; inability to sustain their role in small practices; and the perceived lack of relevance of interventions focused on a specific psychiatric diagnosis. Thus, we designed an innovative randomized clinical trial (RCT), the Primary Care Assessment and Research of a Telephone Intervention for Neuropsychiatric Conditions with Education and Resources study (PARTNERs). This RCT compared the outcomes of enhanced usual care and a novel model of collaborative care in primary care patients with depressive disorders, generalized anxiety, social phobia, panic disorder, at-risk drinking, or alcohol use disorders. These conditions were selected because they are present in almost a third of patients seen in primary care settings. Innovations included assigning the care manager role to trained lay providers supported by computer-based tools; providing all care management centrally by phone - i.e., the intervention was delivered without any face-to-face contact between the patient and the care team; and basing patient eligibility and treatment selection on a transdiagnostic approach using the same eligibility criteria and the same treatment algorithms regardless of the participants' specific psychiatric diagnosis. This paper describes the design of this RCT and discusses the rationale for its main design features.