Positive Nipple Margins in Nipple-Sparing Mastectomy: Management of Nipples Containing Cancer or Atypia.

BACKGROUND: Nipple-sparing mastectomy (NSM) is an oncologically safe approach for breast cancer treatment and prevention; however, there are little long-term data to guide management for patients whose nipple margins contain tumor or atypia. METHODS: NSM patients with tumor or atypia in their nipple margin were identified from a prospectively maintained, single-institution database of consecutive NSMs. Patient and tumor characteristics, treatment, recurrence, and survival data were assessed. RESULTS: A total of 3158 NSMs were performed from June 2007 to August 2019. Nipple margins contained tumor in 117 (3.7%) NSMs and atypia only in 164 (5.2%) NSMs. Among 117 nipple margins that contained tumor, 34 (29%) margins contained invasive cancer, 80 (68%) contained ductal carcinoma in situ only, and 3 (3%) contained lymphatic vessel invasion only. Management included nipple-only excision in 67 (57%) breasts, nipple-areola complex excision in 35 (30%) breasts, and no excision in 15 (13%) breasts. Only 23 (24%) excised nipples contained residual tumor. At 67 months median follow-up, there were 2 (1.8%) recurrences in areolar or peri-areolar skin, both in patients with nipple-only excision. Among 164 nipple margins containing only atypia, 154 (94%) nipples were retained. At 60 months median follow-up, no patient with atypia alone had a nipple or areola recurrence. CONCLUSIONS: Nipple excision is effective management for nipple margins containing tumor. No intervention is required for nipple margins containing only atypia. Our results support broad eligibility for NSM with careful nipple margin assessment.

Multi-omic Analysis of Uterine Leiomyomas in Self-Described Black and White Women: Molecular Insights into Health Disparities.

BACKGROUND: Black women are at an increased risk to develop uterine leiomyomas (ULMs) and to experience worse disease prognosis compared to White women. Epidemiological and molecular factors have been identified as underlying these disparities, but there remains a paucity of deep, multi-omic analysis investigating molecular differences in ULMs from Black and White patients. OBJECTIVE: To identify molecular alterations within ULM tissues correlating with patient race by multi-omic analyses of ULMs collected from cohorts of Black and White women. STUDY DESIGN: We performed multi-omic analysis of ULMs from Black (42) and White (47) women undergoing hysterectomy for symptomatic uterine leiomyomata. Our analysis also included application of orthogonal methods to evaluate fibroid biomechanical properties, such as second harmonic generation microscopy, uniaxial compression testing, as well as sheer wave ultrasonography analyses. RESULTS: We found a greater proportion of mediator complex subunit 12 (MED12) mutant ULMs from Black women (>35% increase, Mann Whitney U p = 7E-4). MED12 mutant tumors exhibited elevated abundance of extracellular matrix proteins, including several collagen isoforms, involved in regulation of the core matrisome. Histological analysis of tissue fibrosis using trichrome staining and secondary harmonic generation microscopy confirmed that MED12 mutant tumors are more fibrotic than MED12 wildtype tumors. Using Sheer Wave ultrasonography in a prospectively collected cohort, Black patients had fibroids that were firmer when compared to White patients, even when similar in size. These analyses also uncovered expression quantitative trait loci (eQTL) associated with altered allele frequencies in African and European populations that correlated with differential abundance of several proteins in ULM that are independent of MED12 mutational status, including multiple eQTL mapping to tetratricopeptide repeat protein 38. CONCLUSIONS: Our study shows that Black women have a higher prevalence of ULMs harboring mutations in MED12 and that this mutational status correlates with increased tissue fibrosis in comparison with wildtype ULMs. Our study provides insights into molecular alterations underlying racial disparities in ULMs and improves our understanding of the molecular etiology underlying ULM development within these populations.

Selenium catalysis enables negative feedback organic oscillators.

The construction of materials regulated by chemical reaction networks requires regulatory motifs that can be stacked together into systems with desired properties. Multiple autocatalytic reactions producing thiols are known. However, negative feedback loop motifs are unavailable for thiol chemistry. Here, we develop a negative feedback loop based on the selenocarbonates. In this system, thiols induce the release of aromatic selenols that catalyze the oxidation of thiols by organic peroxides. This negative feedback loop has two important features. First, catalytic oxidation of thiols follows Michaelis-Menten-like kinetics, thus increasing nonlinearity for the negative feedback. Second, the strength of the negative feedback can be tuned by varying substituents in selenocarbonates. When combined with the autocatalytic production of thiols in a flow reactor, this negative feedback loop induces sustained oscillations. The availability of this negative feedback motif enables the future construction of oscillatory, homeostatic, adaptive, and other regulatory circuits in life-inspired systems and materials.

Practical strategies for care of individuals impacted by suicide in the emergency department: A narrative review.

Introduction Suicide is among the leading causes of death and nurses care for survivors of suicide at many points in their grief journey. Every individual who dies by suicide leaves behind multiple affected survivors and how they are cared for immediately following the suicide influences how they cope with the death. The purpose of this article is to make recommendations for the care of survivors of suicide loss in the emergency department. METHODS: A narrative review of the current literature was conducted using the databases CINAHL and ProQuest. Articles focused on survivors of suicide loss and their care in the immediate period after the death were utilized. RESULTS: Four hundred and twenty-nine articles were found. Applying established inclusion and exclusion criteria and quality assessment using the SANSA guideline, 29 were included in the review. DISCUSSION: Three themes were identified: 1. Risks for suicide in survivors of suicide loss; 2. Interventions in the immediate period after suicide loss; and 3. Active suicide postvention as suicide prevention. Emergency department nurses need to have the ability to readily assess and recognize the survivors of suicide loss who are at higher risk for complicated grieving, and providing rapid and immediate services and resources will help promote coping and positive mental health outcomes in survivors.

Disruption of epithelium integrity by inflammation-associated fibroblasts through prostaglandin signaling.

Inflammation-associated fibroblasts (IAFs) are associated with progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial cells is unknown. Here, we developed an in vitro model whereby human colon fibroblasts are induced by specific cytokines and recapitulate key features of IAFs in vivo. When cocultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid expansion and barrier disruption due to swelling and rupture of individual epithelial cells. Colonoids cocultured with IAFs also show increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated by a paracrine pathway involving prostaglandin E2 and its receptor EP4, leading to protein kinase A -dependent activation of the cystic fibrosis transmembrane conductance regulator. EP4-specific chemical inhibitors effectively prevented IAF-induced colonoid swelling and restored normal proliferation and genome stability. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a therapeutic avenue to mitigate inflammation-associated epithelial injury.

Nelfinavir inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

BACKGROUND: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. METHODS: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in greater detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. RESULTS: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 min. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein, ORF26, was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. CONCLUSIONS: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. This is likely because of the effect of nelfinavir on the ISR and thus protein synthesis and accumulation of the essential triplex capsid protein, ORF26. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Longitudinal changes of swallowing safety and efficiency in infants with spinal muscular atrophy who received disease modifying therapies.

BACKGROUND: Dysphagia is a common feature of the natural history of patients with spinal muscular atrophy (SMA). Literature regarding swallowing safety and efficiency is scarce in patients with SMA, particularly in the era of newborn screening programs and disease-modifying therapies. OBJECTIVE: To describe the longitudinal changes of swallowing safety and efficiency in children with SMA who received one or more disease modifying therapies METHODS: Case series of patients with SMA followed at the University of Florida from 1 May 2019 to 31 December 2022 who had two or more videofluoroscopy swallowing studies (VFSS), with the first being within 30 days of their first treatment. Data extracted from the electronic health record included: neuromotor outcomes, VFSS penetration aspiration scores (PAS), presence of abrnormal oral or pharyngeal residue, clinical history, and timing of disease-modifying therapies administration. RESULTS: Seven subjects were included (five male); three were diagnosed via newborn screen. Median age at diagnosis was 10 days (range: 4-250). Median age at initial VFSS was 29 days (range: 9-246), and age at the last VFSS was 26.1 months (range: 18.2-36.2). All subjects received onasemnogene-abeparvovec (OA); four received additional therapies. PAS at diagnosis was abnormal in four subjects. Six subjects required feeding modifications after VFSS results. Of these, three had silent aspiration (PAS 8) and three of them improved after treatment. CONCLUSIONS: Swallowing safety and efficiency can be impaired in patients with SMA despite early treatment. Larger, prospective studies are needed to define optimal timiing of longitudinal instrumental evaluations.

Penetrating colored pencil injury with Clostridium bifermentans pre-septal cellulitis: case report, literature review, and treatment algorithm.

The incidence of penetrating orbital injuries from writing instruments continues to rise in the pediatric population. Such injuries can cause significant visual morbidity and have a lifelong psychosocial impact. While the description of graphite pencil-related orbital trauma management is well demonstrated with over 40 reported cases, a lack of consistent management protocol for colored pencil-related injuries. Here, we report an inadvertent penetrating orbital colored pencil injury with progressive mechanical ptosis and pre-septal cellulitis necessitating urgent orbitotomy, debridement, and washout to reduce inflammatory and infectious burden. The wooden body serves as a nidus for polymicrobial infection, and the unique composition of colored pencil cores may lead to inflammatory processes that require vigilant multidisciplinary surgical and medical management reflected in our literature review.

Therapeutic Play Gym: Feasibility of a Caregiver-Mediated Exercise System for NICU Graduates with Neuromuscular Weakness-A Case Series.

PURPOSE: To describe the feasibility and effect of caregiver-mediated exercise training using a novel Therapeutic Play Gym in 3 neonatal intensive care unit (NICU) graduates with rare neuromuscular diseases. SUMMARY OF KEY POINTS: Caregivers of 3 medically complex, technology-dependent NICU graduates could not access community-based rehabilitation services after discharging from lengthy initial hospitalizations. These children, diagnosed with spinal muscular atrophy type 0, untreated X-linked myotubular myopathy, and untreated nemaline myopathy 3 (NEM3), completed monthly consultations with a pediatric clinical specialist and 3 assessment appointments. The caregivers agreed to administer a progressive Therapeutic Play Gym home exercise program at a minimum frequency of 3×/wk for 6 months. CONCLUSION: A monthly consultative approach was both feasible and effective to safely progress caregiver-mediated home exercise training using a novel Therapeutic Play Gym. Positive training effects emerged in fitness, function, and caregiver-reported quality of life domains. RECOMMENDATIONS FOR CLINICAL PRACTICE: A strong therapist-caregiver alliance can empower families to perform guided training when community resources are limited. More research is needed to see whether this training model is feasible for children with other conditions; for use in hospital, outpatient, or educational settings; and as an adjuvant exercise treatment for children receiving disease-modifying interventions.

Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated. METHODS: Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed. FINDINGS: Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants. INTERPRETATION: Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study. FUNDING: Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

A Decade of Nipple-Sparing Mastectomy: Lessons Learned in 3035 Immediate Implant-Based Breast Reconstructions.

BACKGROUND: Nipple-sparing mastectomy is commonly performed for breast cancer treatment or prevention. The authors present one of the largest breast reconstruction series in the literature. METHODS: A single-institution retrospective review was conducted from 2007 to 2019. RESULTS: The authors' query identified 3035 implant-based breast reconstructions after nipple-sparing mastectomy, including 2043 direct-to-implant and 992 tissue expander-to-implant reconstructions. The overall major complication rate was 9.15%, and the nipple necrosis rate was 1.20%. Therapeutic mastectomy was associated with higher overall complications and explantations compared with prophylactic mastectomy ( P < 0.01). In comparisons of unilateral and bilateral procedures, bilateral mastectomy had an increased risk for complications (OR, 1.46; 95% CI, 0.997 to 2.145; P = 0.05). Tissue-expander reconstructions had higher rates of nipple necrosis (1.9% versus 0.88%; P = 0.015), infection (4.2% versus 2.8%; P = 0.04), and explantation (5.1% versus 3.5%; P = 0.04) compared with direct-to-implant reconstruction. When assessing plane of reconstruction, the authors found similar rates of complications between subpectoral dual-plane and prepectoral reconstruction. There was no difference in complications between reconstruction with acellular dermal matrix or mesh compared with total or partial muscle coverage without acellular dermal matrix/mesh (OR, 0.749; 95% CI, 0.404 to 1.391; P = 0.361). Multivariable regression analysis revealed preoperative radiotherapy (OR, 2.465; 95% CI, 1.579 to 3.848; P < 0.001), smoking (OR, 2.53; 95% CI, 1.581 to 4.054; P < 0.001), and a periareolar incision (OR, 3.657; 95% CI, 2.276 to 5.875; P < 0.001) to be the strongest predictors of complications and nipple necrosis ( P < 0.05). CONCLUSIONS: Nipple-sparing mastectomy and immediate breast reconstruction has a low rate of complications. In this series, radiation therapy, smoking, and incision choice predicted overall complications and nipple necrosis, whereas direct-to-implant reconstruction and acellular dermal matrix or mesh did not increase risk. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Dental hygiene clinical faculty attitudes and likelihood of working during the COVID-19 pandemic.

OBJECTIVE: To identify circumstances that influenced dental hygiene clinical faculty's likelihood of working during the COVID-19 pandemic, and to explore attitudes (responsibilities, personal issues, ethics, and policy statements) about working. METHODS: This cross-sectional study used a purposive sample of dental hygienists (n = 1710) employed in academic clinical settings that held full-time, part-time, or adjunct appointments. The Health Care Workers' Attitudes to Working During a Pandemic survey was utilized. Likelihood scores were calculated based on respondents more or less likely to work under differing circumstances. Frequencies of agreement for attitudes were developed. Correlations were explored between likelihood scores and demographic data. Answers to the open-ended questions were categorized and described. RESULTS: The response rate was 13.5% (225/1710). Only one-third of respondents had a likelihood score of 100%. Factors with the greatest impact on likelihood to work were personal protective equipment. Almost all respondents' attitudes of working indicated that their employer is responsible for providing PPE. Over 90% felt their main responsibility was to themselves/family. Correlation analyses resulted in no significant associations between likelihood scores and age, etc. Faculty are considering leaving their jobs because of pay cuts and/or not feeling safe. CONCLUSION: Likelihood of working for dental hygiene faculty represents several personal and ethical decisions. Most respondents want employers to be responsible for their on-the-job safety. In addition, personal and work-related issues were causes of unease. The pandemic has had far-reaching implications on day-to-day work of dental hygiene clinical faculty as well as on the long-term trajectories and views on employment in higher education.

Using a web platform for equitable distribution of COVID-19 monoclonal antibodies: a case study in resource allocation.

While medical countermeasures in COVID-19 have largely focused on vaccinations, monoclonal antibodies (mAbs) were early outpatient treatment options for COVID-positive patients. In Minnesota, a centralized access platform was developed to offer access to mAbs that linked over 31,000 patients to care during its operation. The website allowed patients, their representative, or providers to screen the patient for mAbs against Emergency Use Authorization (EUA) criteria and connect them with a treatment site if provisionally eligible. A validated clinical risk scoring system was used to prioritize patients during times of scarcity. Both an ethics and a clinical subject matter expert group advised the Minnesota Department of Health on equitable approaches to distribution across a range of situations as the pandemic evolved. This case study outlines the implementation of this online platform and clinical outcomes of its users. We assess the impact of referral for mAbs on hospitalizations and death during a period of scarcity, finding in particular that vaccination conferred a substantially larger protection against hospitalization than a referral for mAbs, but among unvaccinated users that did not get a referral, chances of hospitalization increased by 4.1 percentage points.

Nelfinavir Inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

Background: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) infections aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. Methods: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. Results: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 minutes. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein ORF26 was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. Conclusions: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Safety and efficacy of gene replacement therapy for X-linked myotubular myopathy (ASPIRO): a multinational, open-label, dose-escalation trial.

BACKGROUND: X-linked myotubular myopathy is a rare, life-threatening, congenital muscle disease observed mostly in males, which is caused by mutations in MTM1. No therapies are approved for this disease. We aimed to assess the safety and efficacy of resamirigene bilparvovec, which is an adeno-associated viral vector serotype 8 delivering human MTM1. METHODS: ASPIRO is an open-label, dose-escalation trial at seven academic medical centres in Canada, France, Germany, and the USA. We included boys younger than 5 years with X-linked myotubular myopathy who required mechanical ventilator support. The trial was initially in two parts. Part 1 was planned as a safety and dose-escalation phase in which participants were randomly allocated (2:1) to either the first dose level (1·3 × 1014 vector genomes [vg]/kg bodyweight) of resamirigene bilparvovec or delayed treatment, then, for later participants, to either a higher dose (3·5 × 1014 vg/kg bodyweight) of resamirigene bilparvovec or delayed treatment. Part 2 was intended to confirm the dose selected in part 1. Resamirigene bilparvovec was administered as a single intravenous infusion. An untreated control group comprised boys who participated in a run-in study (INCEPTUS; NCT02704273) or those in the delayed treatment cohort who did not receive any dose. The primary efficacy outcome was the change from baseline to week 24 in hours of daily ventilator support. After three unexpected deaths, dosing at the higher dose was stopped and the two-part feature of the study design was eliminated. Because of changes to the study design during its implementation, analyses were done on an as-treated basis and are deemed exploratory. All treated and control participants were included in the safety analysis. The trial is registered with ClinicalTrials.gov, NCT03199469. Outcomes are reported as of Feb 28, 2022. ASPIRO is currently paused while deaths in dosed participants are investigated. FINDINGS: Between Aug 3, 2017 and June 1, 2021, 30 participants were screened for eligibility, of whom 26 were enrolled; six were allocated to the lower dose, 13 to the higher dose, and seven to delayed treatment. Of the seven children whose treatment was delayed, four later received the higher dose (n=17 total in the higher dose cohort), one received the lower dose (n=7 total in the lower dose cohort), and two received no dose and joined the control group (n=14 total, including 12 children from INCEPTUS). Median age at dosing or enrolment was 12·1 months (IQR 10·0-30·9; range 9·5-49·7) in the lower dose cohort, 31·1 months (16·0-64·7; 6·8-72·7) in the higher dose cohort, and 18·7 months (10·1-31·5; 5·9-39·3) in the control cohort. Median follow-up was 46·1 months (IQR 41·0-49·5; range 2·1-54·7) for lower dose participants, 27·6 months (24·6-29·1; 3·4-41·0) for higher dose participants, and 28·3 months (9·7-46·9; 5·7-32·7) for control participants. At week 24, lower dose participants had an estimated 77·7 percentage point (95% CI 40·22 to 115·24) greater reduction in least squares mean hours per day of ventilator support from baseline versus controls (p=0·0002), and higher dose participants had a 22·8 percentage point (6·15 to 39·37) greater reduction from baseline versus controls (p=0·0077). One participant in the lower dose cohort and three in the higher dose cohort died; at the time of death, all children had cholestatic liver failure following gene therapy (immediate causes of death were sepsis; hepatopathy, severe immune dysfunction, and pseudomonal sepsis; gastrointestinal haemorrhage; and septic shock). Three individuals in the control group died (haemorrhage presumed related to hepatic peliosis; aspiration pneumonia; and cardiopulmonary failure). INTERPRETATION: Most children with X-linked myotubular myopathy who received MTM1 gene replacement therapy had important improvements in ventilator dependence and motor function, with more than half of dosed participants achieving ventilator independence and some attaining the ability to walk independently. Investigations into the risk for underlying hepatobiliary disease in X-linked myotubular myopathy, and the need for monitoring of liver function before gene replacement therapy, are ongoing. FUNDING: Astellas Gene Therapies.

An unbiased proteomic analysis of PAD4 in human monocytes: novel substrates, binding partners and subcellular localizations.

Peptidylarginine deiminase IV (PAD4) post-translationally converts arginine residues in proteins to citrullines and is implicated in playing a central role in the pathogenesis of several diseases. Although PAD4 was historically thought to be a nuclear enzyme, recent evidence has revealed a more complex localization of PAD4 with evidence of additional cytosolic and cell surface localization and activity. However, the mechanisms by which PAD4, which lacks conventional secretory signal sequences, traffics to extranuclear localizations are unknown. In this study, we show that PAD4 was enriched in the organelle fraction of monocytes with evidence of citrullination of organelle proteins. We also demonstrated that PAD4 can bind to several cytosolic, nuclear and organelle proteins that may serve as binding partners for PAD4 to traffic intracellularly. Additionally, cell surface expression of PAD4 increased with monocyte differentiation into monocyte-derived dendritic cells and co-localized with several endocytic/autophagic and conventional secretory pathway markers, implicating the use of these pathways by PAD4 to traffic within the cell. Our results suggest that PAD4 is expressed in multiple subcellular localizations and may play previously unappreciated roles in physiological and pathological conditions. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Disruption of Epithelium Integrity by Inflammation-Associated Fibroblasts through Prostaglandin Signaling: IAFs disrupt colon epithelium via PGE2-EP4.

Inflammation-associated fibroblasts (IAFs) are associated with the progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial function and architecture is unknown. In this study, we developed an in vitro model whereby human colon fibroblasts are induced to become IAFs by specific cytokines and recapitulate key features of IAFs in vivo. When co-cultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid swelling and barrier disruption due to swelling and rupture of individual epithelial cells. Epithelial cells co-cultured with IAFs also exhibit increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated through a paracrine pathway involving prostaglandin E2 (PGE2) and the PGE2 receptor EP4, leading to PKA-dependent activation of the CFTR chloride channel. Importantly, EP4-specific chemical inhibitors effectively prevented colonoid swelling and restored normal proliferation and genome stability of IAF-exposed epithelial cells. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a potential treatment to mitigate inflammation-associated epithelial injury.

Rare intercellular material transfer as a confound to interpreting inner retinal neuronal transplantation following internal limiting membrane disruption.

Intercellular cytoplasmic material transfer (MT) occurs between transplanted and developing photoreceptors and ambiguates cell origin identification in developmental, transdifferentiation, and transplantation experiments. Whether MT is a photoreceptor-specific phenomenon is unclear. Retinal ganglion cell (RGC) replacement, through transdifferentiation or transplantation, holds potential for restoring vision in optic neuropathies. During careful assessment for MT following human stem cell-derived RGC transplantation into mice, we identified RGC xenografts occasionally giving rise to labeling of donor-derived cytoplasmic, nuclear, and mitochondrial proteins within recipient Müller glia. Critically, nuclear organization is distinct between human and murine retinal neurons, which enables unequivocal discrimination of donor from host cells. MT was greatly facilitated by internal limiting membrane disruption, which also augments retinal engraftment following transplantation. Our findings demonstrate that retinal MT is not unique to photoreceptors and challenge the isolated use of species-specific immunofluorescent markers for xenotransplant identification. Assessment for MT is critical when analyzing neuronal replacement interventions.

Diaphragm pacing and independent breathing in individuals with severe Pompe disease.

Introduction: Pompe disease is an inherited disease characterized by a deficit in acid-α-glucosidase (GAA), an enzyme which degrades lysosomal glycogen. The phrenic-diaphragm motor system is affected preferentially, and respiratory failure often occurs despite GAA enzyme replacement therapy. We hypothesized that the continued use of diaphragm pacing (DP) might improve ventilator-dependent subjects' respiratory outcomes and increase ventilator-free time tolerance. Methods: Six patients (3 pediatric) underwent clinical DP implantation and started diaphragm conditioning, which involved progressively longer periods of daily, low intensity stimulation. Longitudinal respiratory breathing pattern, diaphragm electromyography, and pulmonary function tests were completed when possible, to assess feasibility of use, as well as diaphragm and ventilatory responses to conditioning. Results: All subjects were eventually able to undergo full-time conditioning via DP and increase their maximal tolerated time off-ventilator, when compared to pre-implant function. Over time, 3 of 6 subjects also demonstrated increased or stable minute ventilation throughout the day, without positive-pressure ventilation assistance. Discussion: Respiratory insufficiency is one of the main causes of death in patients with Pompe disease. Our results indicate that DP in Pompe disease was feasible, led to few adverse events and stabilized breathing for up to 7 years.