Endogenous pannexin1 channels form functional intercellular cell-cell channels with characteristic voltage-dependent properties.

The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exogenous human pannexin1 (hPanx1) cell­cell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cell­cell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single-channel conductance of ∼175 pS, with a substate of ∼35 pS; the latter showed a peculiar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cell­cell channels were also identified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfering RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cell­cell coupling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibitor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the channel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cell­cell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cell­cell channels in different cell types might require special attention.

A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII.

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

When technology cares for people with dementia: A critical review using neuropsychological rehabilitation as a conceptual framework.

Clinicians and researchers have become increasingly interested in the potential of technology in assisting persons with dementia (PwD). However, several issues have emerged in relation to how studies have conceptualized who the main technology user is (PwD/carer), how technology is used (as compensatory, environment modification, monitoring or retraining tool), why it is used (i.e., what impairments and/or disabilities are supported) and what variables have been considered as relevant to support engagement with technology. In this review we adopted a Neuropsychological Rehabilitation perspective to analyse 253 studies reporting on technological solutions for PwD. We analysed purposes/uses, supported impairments and disabilities and how engagement was considered. Findings showed that the most frequent purposes of technology use were compensation and monitoring, supporting orientation, sequencing complex actions and memory impairments in a wide range of activities. The few studies that addressed the issue of engagement with technology considered how the ease of use, social appropriateness, level of personalization, dynamic adaptation and carers' mediation allowed technology to adapt to PWD's and carers' preferences and performance. Conceptual and methodological tools emerged as outcomes of the analytical process, representing an important contribution to understanding the role of technologies to increase PwD's wellbeing and orient future research.

Stretch-Induced Activation of Pannexin 1 Channels Can Be Prevented by PKA-Dependent Phosphorylation.

Pannexin 1 channels located in the cell membrane are permeable to ions, metabolites, and signaling molecules. While the activity of these channels is known to be modulated by phosphorylation on T198, T308, and S206, the possible involvement of other putative phosphorylation sites remains unknown. Here, we describe that the activity of Panx1 channels induced by mechanical stretch is reduced by adenosine via a PKA-dependent pathway. The mechanical stretch-induced activity-measured by changes in DAPI uptake-of Panx1 channels expressed in HeLa cell transfectants was inhibited by adenosine or cAMP analogs that permeate the cell membrane. Moreover, inhibition of PKA but not PKC, p38 MAPK, Akt, or PKG prevented the effects of cAMP analogs, suggesting the involvement of Panx1 phosphorylation by PKA. Accordingly, alanine substitution of T302 or S328, two putative PKA phosphorylation sites, prevented the inhibitory effect of cAMP analogs. Moreover, phosphomimetic mutation of either T302 or S328 to aspartate prevented the mechanical stretch-induced activation of Panx1 channels. A molecular dynamics simulation revealed that T302 and S328 are located in the water-lipid interphase near the lateral tunnel of the intracellular region, suggesting that their phosphorylation could promote conformational changes in lateral tunnels. Thus, Panx1 phosphorylation via PKA could be modulated by G protein-coupled receptors associated with the Gs subunit.

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.

Accreditation's Role in Bolstering Resilience in the Face of the Zika Virus Outbreak.

The Florida Department of Health (Department) received accreditation status as an integrated public health system from the Public Health Accreditation Board (PHAB) in 2 phases: the State Health Office received accreditation in June 2014 and the 67 county health departments received accreditation in March 2016. Six weeks after PHAB awarded accreditation to the Department as an integrated public health system in March 2016, the World Health Organization declared the Zika outbreak in the Americas a Public Health Emergency of International Concern. Even in that short time, integrated public health accreditation, along with the other components of the Department's performance management system, allowed the Department to address this public health emergency, especially in Miami-Dade County, where the impact of Zika was significant. This case report describes the local response in Miami-Dade County and supporting statewide efforts. Public health departments should consider how public health accreditation could strengthen their ability to fulfill their public health mission. This article provides rationale for state and local health departments to seek accreditation.

Statement on gender-affirmative approach to care from the pediatric endocrine society special interest group on transgender health.

PURPOSE OF REVIEW: The purpose of this Position Statement is to emphasize the importance of an affirmative approach to the health care of transgender individuals, as well as to improve the understanding of the rights of transgender youth. RECENT FINDINGS: Transgender youth have optimal outcomes when affirmed in their gender identity, through support by their families and their environment, as well as appropriate mental health and medical care. SUMMARY: The Pediatric Endocrine Society Special Interest Group on Transgender Health joins other academic societies involved in the care of children and adolescents in supporting policies that promote a safe and accepting environment for gender nonconforming/transgender youth, as well as adequate mental health and medical care. This document provides a summary of relevant definitions, information and current literature on which the medical management and affirmative approach to care of transgender youth are based.

Effect of pioglitazone on plasma ceramides in adults with metabolic syndrome.

BACKGROUND: Metabolic syndrome (MetS) appears closely linked with ceramide accumulation, inducing insulin resistance and toxicity to multiple cell types. Animal studies demonstrate that thiazolidinediones (TZDs) reduce ceramide concentrations in plasma and skeletal muscle and support lowering of ceramide levels as a potential mediator of TZDs' mechanism of action in reducing insulin resistance; however, studies in humans have yet to be reported. This study investigated the effects of pioglitazone therapy on plasma ceramides to understand the mechanism by which TZDs improve insulin resistance in MetS. METHODS: Thirty-seven subjects with MetS were studied in a single-centre, randomized, double-blind, placebo-controlled trial comparing pioglitazone to placebo. Data were collected at baseline and after 6 months of therapy. The primary endpoint was the change from baseline in plasma ceramide concentrations. RESULTS: Treatment with pioglitazone for 6 months, compared with placebo, significantly reduced multiple plasma ceramide concentrations: C18:0 (p = 0.001), C20:0 (p = 0.0004), C24 : 1 (p = 0.009), dihydroceramide C18 :0 (p = 0.005), dihydroceramide C24:1 (p = 0.004), lactosylceramide C16:0 (p = 0.02) and the hexosylceramides C16:0 (p = 0.0003), C18 : 0 (p = 0.00001), C22:0 (p = 0.00002) and C24:1 (p = 0.0006). Additionally, significant reductions were found when ceramides were grouped by species: ceramides (p = 0.03), dihydroceramides (p = 0.02), hexosylceramides (p = 0.00001) and lactosylceramides (p = 0.02). The total of all measured ceramides was also significantly reduced (p = 0.001). Following treatment with pioglitazone, the decrease in some ceramide species correlated negatively with the change in insulin sensitivity (dihydroceramide C16:0, r = -0.54; p = 0.02) and positively with total (lactosylceramide C24:0, r = 0.53; p = 0.02) and high molecular weight (lactosylceramide C24:0, r = 0.48; p = 0.05) adiponectin measurements; however, significant associations with changes in liver fat and glycemic control reduction were not found. CONCLUSIONS: Pioglitazone in individuals with MetS induces a potent decrease in plasma ceramides, and some of the changes correlate with changes in insulin resistance and adiponectin levels.

Legislation, medicine, and politics: care for gender diverse youth.

PURPOSE OF REVIEW: A recent increase in legislation in the United States prohibiting gender-affirming care (GAC) for transgender youth follows a wave of its politicization despite support from all pertinent mainstream medical associations. This review describes the standards of GAC for transgender youth, the origins of legislation prohibiting this care, a review of current legislation in the United States and a discussion on the impact on patients, providers, and the medical field. RECENT FINDINGS: A critical evaluation of historical parallels and current organizations supporting this legislation reveals it stems not from concerns within the medical field but from political and religious interests. This intrusion sets a dangerous precedent, undermining evidence-based medicine, providers' ability to practice according to standards of care, and patients' and guardians' autonomy and medical decision-making. This wave of antitrans rhetoric and legislation has resulted in threats to health providers and hospitals, 'moral distress" in providers, and migration of providers and patients from hostile states. SUMMARY: Similar to antiabortion legislation, these legislative efforts will likely result in negative health outcomes and worsening disparities. The medical community must confront these forces directly through an understanding of the political and structural forces at play and adopting strategies to leverage collective power.

Prenatal presentation of a hyperfunctioning thyroid nodule.

OBJECTIVES: Fetal and neonatal hyperthyroidism are most commonly seen in patients whose mothers have Graves' disease. Rarely, it can be caused by non-autoimmune conditions. As these conditions are rare, the workup and treatment is not uniform and can lead to persistent symptoms and long-term negative health effects. CASE PRESENTATION: This report describes a patient with congenital hyperthyroidism from a toxic adenoma presenting with fetal tachycardia. The patient was initially managed medically after birth, but was eventually treated with thyroidectomy. CONCLUSIONS: This case report highlights an additional, important, differential diagnosis for fetal hyperthyroidism when maternal Graves' disease has been ruled out.

Insulin enhances glucose-stimulated insulin secretion in healthy humans.

Islet beta-cells express both insulin receptors and insulin-signaling proteins. Recent evidence from rodents in vivo and from islets isolated from rodents or humans suggests that the insulin signaling pathway is physiologically important for glucose sensing. We evaluated whether insulin regulates beta-cell function in healthy humans in vivo. Glucose-induced insulin secretion was assessed in healthy humans following 4-h saline (low insulin/sham clamp) or isoglycemic-hyperinsulinemic (high insulin) clamps using B28-Asp insulin that could be immunologically distinguished from endogenous insulin. Insulin and C-peptide clearance were evaluated to understand the impact of hyperinsulinemia on estimates of beta-cell function. Preexposure to exogenous insulin increased the endogenous insulin secretory response to glucose by approximately 40%. C-peptide response also increased, although not to the level predicted by insulin. Insulin clearance was not saturated at hyperinsulinemia, but metabolic clearance of C-peptide, assessed by infusion of stable isotope-labeled C-peptide, increased modestly during hyperinsulinemic clamp. These studies demonstrate that insulin potentiates glucose-stimulated insulin secretion in vivo in healthy humans. In addition, hyperinsulinemia increases C-peptide clearance, which may lead to modest underestimation of beta-cell secretory response when using these methods during prolonged dynamic testing.

Gender-Affirming Medical Treatments.

Individuals with gender dysphoria (as defined by Diagnostic and Statistical Manual of Mental Disorders or DSM-V) experience a marked incongruence between the sex assigned at birth and the experienced gender resulting in significant distress or impairment in social, occupational, or other important areas of functioning. For transgender and gender diverse minors, the Endocrine Society recommends a multidisciplinary approach to gender-affirming medical treatment that involves a physician and a mental health provider, also consistent with the World Professional Association for Transgender Health Standard of Care 8th Edition recommendations. This article will outline the role of medical providers in implementing safe and effective gender-affirming medical treatments in youth.

Initial Assessment of VECTRA Three-Dimensional Imaging to Accurately Simulate Breast Volume Changes in Transfeminine Patients: A Mannequin Study.

Background: Methods that aim to accurately measure and predict breast development can be utilized in gender-affirming treatment planning, patient education, and research. Objectives: The authors sought to evaluate whether three-dimensional (3D) stereophotogrammetry accurately measures transfeminine breast volume changes on a masculine frame when simulating anticipated changes in soft tissue after gender-affirming surgical therapy. Then, we describe the innovative application of this imaging modality in a transgender patient to illustrate the potential role of 3D imaging in gender-affirming surgical care. Methods: A 3D VECTRA scanner (Canfield, Fairfield, NJ) was used to measure anthropometric breast measurements. Postoperative changes in breast volume were simulated on a cardiopulmonary resuscitation mannequin using 450 cc MENTOR breast implants (Mentor Worldwide LLC, Irvine, CA). To demonstrate the ability of the VECTRA to accurately simulate transfeminizing augmentation in practice, we describe its use in a 30-year-old transgender female with a 2-year history of gender-affirming hormone therapy, presenting for gender-affirming surgical care. Results: In the mannequin, mean breast volumes were 382 cc on the right (range 375-388 cc), and 360 cc on the left (range 351-366 cc). The average calculated difference in volume between the 2 sides was 22 cc (range 17-31 cc). There were no instances where the left side was calculated to be larger than the right or where the calculated size was smaller than the actual implant size. Conclusions: The VECTRA 3D camera is a reliable and reproducible tool for preoperative assessment, surgical planning, and simulating breast volume changes after gender-affirming surgery.

Modulation of Postprandial Plasma Concentrations of Digestive Hormones and Gut Microbiota by Foods Containing Oat ß-Glucans in Healthy Volunteers.

Cereal ß-glucans are beneficial health ingredients that reduce cholesterolemia and postprandial glycaemia. However, their impact on digestive hormones and gut microbiota is not yet fully established. Two randomized, double-blind, controlled studies were conducted. In the first study, 14 subjects ingested a breakfast with or without ß-glucan from oats (5.2 g). Compared to the control, ß-glucan increased orocecal transit time (p = 0.028) and decreased mean appetite score (p = 0.014) and postprandial plasma ghrelin (p = 0.030), C-peptide (p = 0.001), insulin (p = 0.06), and glucose (p = 0.0006). ß-glucan increased plasma GIP (p = 0.035) and PP (p = 0.018) without affecting leptin, GLP-1, PYY, glucagon, amylin, or 7α-hydroxy-4-cholesten-3-one, a biomarker of bile acid synthesis. In the second study, 32 subjects were distributed into 2 groups to ingest daily foods with (3 g/day) or without ß-glucan for 3 weeks; stools were collected before/after treatment. No changes in fecal microbiota composition/diversity (deep sequencing) were detected with ß-glucans. These results indicate that acute intake of 5 g ß-glucan slows transit time and decreases hunger sensation and postprandial glycaemia without affecting bile-acid synthesis, these changes being associated with decreased plasma insulin, C-peptide, and ghrelin, and increased plasma GIP and PP. However, regular daily intake of 3 g ß-glucan is not sufficient to have an effect on fecal microbiota composition.

A self-guided curriculum on endocrinology standard of care for gender diverse youth, including ethical considerations.

Objective: While the field of pediatric endocrinology, and the American Board of Pediatrics, continues expanding training to include gender-affirming care, many pediatric endocrinology fellowship programs do not have formal curriculum for this patient population. Members of the Pediatric Endocrine Society (PES) that have a special interest in transgender health designed a curriculum based on Endocrine Society practice guidelines to expand the knowledge of gender affirming care for medical trainees' and faculty. Methods: PES members designed a 5-part self-guided educational module series with embedded knowledge questions. Uniquely, medical ethical reflections were included within each module. Participants completed baseline demographic and baseline and follow-up knowledge surveys. Results: Most participants were pediatric endocrinology fellows and 44 % percent (n = 21) completed all study components, including the follow up knowledge survey. Knowledge question data analysis demonstrated knowledge gained in medical management of pubertal youth and surgical interventions. Conclusion: This is the first medical education curriculum in gender-affirming care created by pediatric endocrinologists grounded in the Endocrine Society practice guidelines. This study demonstrates medical knowledge gained in caring for gender diverse youth and is the first to incorporate ethical considerations for this patient population. While initially designed for pediatric endocrinology trainees and faculty, this curriculum may be of great utility for any provider interested in caring for gender diverse youth.

How Cisgender Clinicians Can Help Prevent Harm During Encounters With Transgender Patients.

Transgender people commonly experience discrimination from clinicians, which directly contributes to worse mental and physical health outcomes among this population. This article describes mechanisms by which stigma perpetuates health inequity among transgender patients and highlights its unique effects on transgender patients of color. The article concludes with recommendations to cisgender clinicians on how to help prevent stigmatizing interactions with transgender patients.

Determinants of Bone Mineral Density in Transgender Youth.

Purpose: We aimed to study determinants of bone health in transgender youth in anticipation of or shortly after initiating puberty suppression and/or gender-affirming hormone therapy. Methods: This was a retrospective review of records of transgender adolescents in our institution between June 2014 and June 2019. Dual energy X-ray absorptiometry was used to assess bone mineral density (BMD). Baseline characteristics were collected and included in a multilinear regression model to assess determinants of lumbar spine (LS) BMD Z-scores adjusted for age and height and accounting for race. Welch's t-test was used to compare characteristics across genders. Results: One hundred nineteen patient records were analyzed. Forty-six patients (38.7%) were assigned male at birth (AMAB) and 73 patients (61.3%) were assigned female at birth (AFAB). Mean (±standard deviation [SD]) age (years) was 14.7±2.6 for AMAB and 15.0±2.2 for AFAB. The adjusted LS BMD Z-score was lower in the AMAB population with a mean (+SD) of -0.605±1.42 compared with 0.043±1.09 in AFAB (p=0.010). In a multivariate model, AMAB gender, vitamin D deficiency, and lower body mass index (BMI) z-scores were determinants of lower LS BMD Z-scores (R 2=0.206). Age, race, ethnicity, insurance status, and Tanner stage were not determinants of BMD. However, post hoc analysis did show that pubertal status modified the results. Conclusion: AMAB transgender adolescents have lower BMD compared with AFAB patients, before or shortly after starting puberty suppression and/or gender-affirming hormone therapy. Lower BMI and vitamin D deficiency were determinants of lower BMD. Further studies are needed to explore etiology for bone health discrepancy in this population.

The Evolution of Adolescent Gender-Affirming Care: An Historical Perspective.

While individuals have demonstrated gender diversity throughout history, the use of medication and/or surgery to bring a person's physical sex characteristics into alignment with their gender identity is relatively recent, with origins in the first half of the 20th century. Adolescent gender-affirming care, however, did not emerge until the late 20th century and has been built upon pioneering work from the Netherlands, first published in 1998. Since that time, evolving protocols for gender-diverse adolescents have been incorporated into clinical practice guidelines and standards of care published by the Endocrine Society and World Professional Association for Transgender Health, respectively, and have been endorsed by major medical and mental health professional societies around the world. In addition, in recent decades, evidence has continued to emerge supporting the concept that gender identity is not simply a psychosocial construct but likely reflects a complex interplay of biological, environmental, and cultural factors. Notably, however, while there has been increased acceptance of gender diversity in some parts of the world, transgender adolescents and those who provide them with gender-affirming medical care, particularly in the USA, have been caught in the crosshairs of a culture war, with the risk of preventing access to care that published studies have indicated may be lifesaving. Despite such challenges and barriers to care, currently available evidence supports the benefits of an interdisciplinary model of gender-affirming medical care for transgender/gender-diverse adolescents. Further long-term safety and efficacy studies are needed to optimize such care.