Repair of noise-induced damage to stereocilia F-actin cores is facilitated by XIRP2 and its novel mechanosensor domain.

Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as 'gaps' in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.

Adrenergic Mechanisms of Audiogenic Seizure-Induced Death in a Mouse Model of SCN8A Encephalopathy.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death amongst patients whose seizures are not adequately controlled by current therapies. Patients with SCN8A encephalopathy have an elevated risk for SUDEP. While transgenic mouse models have provided insight into the molecular mechanisms of SCN8A encephalopathy etiology, our understanding of seizure-induced death has been hampered by the inability to reliably trigger both seizures and seizure-induced death in these mice. Here, we demonstrate that mice harboring an Scn8a allele with the patient-derived mutation N1768D (D/+) are susceptible to audiogenic seizures and seizure-induced death. In adult D/+ mice, audiogenic seizures are non-fatal and have nearly identical behavioral, electrographical, and cardiorespiratory characteristics as spontaneous seizures. In contrast, at postnatal days 20-21, D/+ mice exhibit the same seizure behavior, but have a significantly higher incidence of seizure-induced death following an audiogenic seizure. Seizure-induced death was prevented by either stimulating breathing via mechanical ventilation or by acute activation of adrenergic receptors. Conversely, in adult D/+ mice inhibition of adrenergic receptors converted normally non-fatal audiogenic seizures into fatal seizures. Taken together, our studies show that in our novel audiogenic seizure-induced death model adrenergic receptor activation is necessary and sufficient for recovery of breathing and prevention of seizure-induced death.

Myosin-VIIa is expressed in multiple isoforms and essential for tensioning the hair cell mechanotransduction complex.

Mutations in myosin-VIIa (MYO7A) cause Usher syndrome type 1, characterized by combined deafness and blindness. MYO7A is proposed to function as a motor that tensions the hair cell mechanotransduction (MET) complex, but conclusive evidence is lacking. Here we report that multiple MYO7A isoforms are expressed in the mouse cochlea. In mice with a specific deletion of the canonical isoform (Myo7a-ΔC mouse), MYO7A is severely diminished in inner hair cells (IHCs), while expression in outer hair cells is affected tonotopically. IHCs of Myo7a-ΔC mice undergo normal development, but exhibit reduced resting open probability and slowed onset of MET currents, consistent with MYO7A's proposed role in tensioning the tip link. Mature IHCs of Myo7a-ΔC mice degenerate over time, giving rise to progressive hearing loss. Taken together, our study reveals an unexpected isoform diversity of MYO7A expression in the cochlea and highlights MYO7A's essential role in tensioning the hair cell MET complex.

Estimation of Actual and Ideal Bodyweight Using Morphometric Measurements of Miniature, Saddle-Type, and Thoroughbred Horses.

Adding breed type, height, and neck circumference to body length and girth circumference improves bodyweight (BW) estimation in different breeds of horses; however, equations have not been developed for all breed types. The objectives were to develop BW estimation equations for Miniature, saddle-type, and Thoroughbred horses using morphometric measurements. Measurements were collected on adult (≥3 years, nonpregnant) saddle-type (n = 209), adult (n = 249) and juvenile (<3 years, n = 61) Miniatures, and adult Thoroughbreds (n = 100). Personnel determined body condition score (BCS), measured withers height and girth circumference at the third thoracic vertebra, body length from the point of the shoulder to the point of the buttock and to a line perpendicular to the point of the buttock, and neck circumference at the midway point between the poll and withers. Each horse was weighed using a livestock scale. Bodyweight estimations equations were developed using linear regression modeling and log transformation. Mean (±standard deviation) BCS was 6.1 (±0.8), 5.4 (±0.6), 6.0 (±1.0), and 5.0 (±0.6) for adult and juvenile Miniatures, saddle-type, and Thoroughbreds, respectively. Bodyweight estimation equations developed through the current research were within 4% of the scale BW and offered improvements over previous BW estimation equations and weight tapes, which were off by 5%-25%. Owner-estimated BW was within 8%-15% of scale BW. Morphometric measurements were successfully used to develop BW equations for Miniature, saddle-type, and Thoroughbred horses. The equations will assist owners and professionals with managing horse BW and will be added to the Healthy Horse application.

Mechanisms of Hair Cell Damage and Repair.

Sensory hair cells of the inner ear are exposed to continuous mechanical stress, causing damage over time. The maintenance of hair cells is further challenged by damage from a variety of other ototoxic factors, including loud noise, aging, genetic defects, and ototoxic drugs. This damage can manifest in many forms, from dysfunction of the hair cell mechanotransduction complex to loss of specialized ribbon synapses, and may even result in hair cell death. Given that mammalian hair cells do not regenerate, the repair of hair cell damage is important for continued auditory function throughout life. Here, we discuss how several key hair cell structures can be damaged, and what is known about how they are repaired.

LMO7 deficiency reveals the significance of the cuticular plate for hearing function.

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.

Necroptosis and Apoptosis Contribute to Cisplatin and Aminoglycoside Ototoxicity.

Ototoxic side effects of cisplatin and aminoglycosides have been extensively studied, but no therapy is available to date. Sensory hair cells, upon exposure to cisplatin or aminoglycosides, undergo apoptotic and necrotic cell death. Blocking these cell death pathways has therapeutic potential in theory, but incomplete protection and lack of therapeutic targets in the case of necrosis, has hampered the development of clinically applicable drugs. Over the past decade, a novel form of necrosis, termed necroptosis, was established as an alternative cell death pathway. Necroptosis is distinguished from passive necrotic cell death, in that it follows a cellular program, involving the receptor-interacting protein kinase (RIPK) 1 and RIPK3. In this study, we used pharmacological and genetic interventions in the mouse to test the relative contributions of necroptosis and caspase-8-mediated apoptosis toward cisplatin and aminoglycoside ototoxicity. We find that ex vivo, only apoptosis contributes to cisplatin and aminoglycoside ototoxicity, while in vivo, necroptosis as well as apoptosis are involved in both sexes. Inhibition of necroptosis and apoptosis using pharmacological compounds is thus a viable strategy to ameliorate aminoglycoside and cisplatin ototoxicity.SIGNIFICANCE STATEMENT The clinical application of cisplatin and aminoglycosides is limited due to ototoxic side effects. Here, using pharmaceutical and genetic intervention, we present evidence that two types of programmed cell death, apoptosis and necroptosis, contribute to aminoglycoside and cisplatin ototoxicity. Key molecular factors mediating necroptosis are well characterized and druggable, presenting new avenues for pharmaceutical intervention.

Protein Synthesis Inhibition and Activation of the c-Jun N-Terminal Kinase Are Potential Contributors to Cisplatin Ototoxicity.

Cisplatin has been regarded as an effective and versatile chemotherapeutic agent for nearly 40 years. Though the associated dose-dependent ototoxicity is known, the cellular mechanisms by which cochleovestibular hair cell death occur are not well understood. We have previously shown that aminoglycoside ototoxicity is mediated in part by cytosolic protein synthesis inhibition. Despite a lack of molecular similarity, aminoglycosides were shown to elicit similar stress pathways to cisplatin. We therefore reasoned that there may be some role of protein synthesis inhibition in cisplatin ototoxicity. Employing a modification of the bioorthogonal noncanonical amino acid tagging (BONCAT) method, we evaluated the effects of cisplatin on cellular protein synthesis. We show that cisplatin inhibits cellular protein synthesis in organ of Corti explant cultures. Similar to what was found after gentamicin exposure, cisplatin activates both the c-Jun N-terminal kinase (JNK) and mammalian target of rapamycin (mTOR) pathways. In contrast to aminoglycosides, cisplatin also inhibits protein synthesis in all cochlear cell types. We further demonstrate that the multikinase inhibitor sorafenib completely prevents JNK activation, while providing only moderate hair cell protection. Simultaneous stimulation of cellular protein synthesis by insulin, however, significantly improved hair cell survival in culture. The presented data provides evidence for a potential role of protein synthesis inhibition in cisplatin-mediated ototoxicity.

Providing researchers with online access to NHLBI biospecimen collections: The results of the first six years of the NHLBI BioLINCC program.

The National Heart, Lung, and Blood Institute (NHLBI), within the United States' National Institutes of Health (NIH), established the Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC) in 2008 to develop the infrastructure needed to link the contents of the NHLBI Biorepository and the NHLBI Data Repository, and to promote the utilization of these scientific resources by the broader research community. Program utilization metrics were developed to measure the impact of BioLINCC on Biorepository access by researchers, including visibility, program efficiency, user characteristics, scientific impact, and research types. Input data elements were defined and are continually populated as requests move through the process of initiation through fulfillment and publication. This paper reviews the elements of the tracking metrics which were developed for BioLINCC and reports the results for the first six on-line years of the program.

A Leishmania secretion system for the expression of major ampullate spidroin mimics.

Spider major ampullate silk fibers have been shown to display a unique combination of relatively high fracture strength and toughness compared to other fibers and show potential for tissue engineering scaffolds. While it is not possible to mass produce native spider silks, the potential ability to produce fibers from recombinant spider silk fibers could allow for an increased innovation rate within tissue engineering and regenerative medicine. In this pilot study, we improved upon a prior fabrication route by both changing the expression host and additives to the fiber pulling precursor solution to improve the performance of fibers. The new expression host for producing spidroin protein mimics, protozoan parasite Leishmania tarentolae, has numerous advantages including a relatively low cost of culture, rapid growth rate and a tractable secretion pathway. Tensile testing of hand pulled fibers produced from these spidroin-like proteins demonstrated that additives could significantly modify the fiber's mechanical and/or antimicrobial properties. Cross-linking the proteins with glutaraldehyde before fiber pulling resulted in a relative increase in tensile strength and decrease in ductility. The addition of ampicillin into the spinning solution resulted in the fibers being able to inhibit bacterial growth.

Use of the National Heart, Lung, and Blood Institute Data Repository.

BACKGROUND: Research on data sharing from clinical trials has focused on elucidating perceptions, barriers, and attitudes among trialists and study participants with respect to sharing data. However, little information exists regarding utilization or associated publication of articles once clinical trial data have been widely shared. METHODS: We analyzed administrative records of investigator requests for data access, linked publications, and bibliometrics to describe the use of the National Heart, Lung, and Blood Institute data repository. RESULTS: From January 2000 through May 2016, a total of 370 investigators requested data from 1 or more clinical trials. Requests for trial data have been increasing, with 195 investigators (53%) initiating requests during the last 4.4 years of the study period. The predominant reason for requesting data was post hoc secondary analysis of new questions (72%), followed by analytic or statistical approaches to clinical trials (9%) and meta-analyses or pooled study research (7%). Of 172 requests with online project descriptions, only 2 requests were initiated for reanalysis of primary-outcome findings. Data from 88 of 100 available clinical trials were requested at least once, and the median time from repository availability to first request was 235 days. A total of 277 articles were published on the basis of data from 47 trials. Citation metrics from 224 articles indicated that half of the publications have cumulative citations that rank in the top 34% normalized for subject category and year of publication. CONCLUSIONS: Demand for trial data for secondary analysis has been increasing. Requesting data for the a priori purpose of reanalysis or verification of original findings was rare.

Efficiently Maintaining a National Resource of Historical and Contemporary Biological Collections: The NHLBI Biorepository Model.

INTRODUCTION: Reducing costs by improving storage efficiency has been a focus of the National Heart, Lung, and Blood Institute (NHLBI) Biologic Specimen Repository (Biorepository) and Biologic Specimen and Data Repositories Information Coordinating Center (BioLINCC) programs for several years. METHODS: Study specimen profiles were compiled using the BioLINCC collection catalog. Cost assessments and calculations on the return on investments to consolidate or reduce a collection, were developed and implemented. RESULTS: Over the course of 8 months, the NHLBI Biorepository evaluated 35 collections that consisted of 1.8 million biospecimens. A total of 23 collections were selected for consolidation, with a total of 1.2 million specimens located in 21,355 storage boxes. The consolidation resulted in a savings of 4055 boxes of various sizes and 10.2 mechanical freezers (∼275 cubic feet) worth of space. CONCLUSION: As storage costs in a biorepository increase over time, the development and use of information technology tools to assess the potential advantage and feasiblity of vial consolidation can reduce maintenance expenses.

Providing Contemporary Access to Historical Biospecimen Collections: Development of the NHLBI Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC).

The National Heart, Lung, and Blood Institute (NHLBI), within the United States' National Institutes of Health (NIH), established a Biorepository in 1976 that initially archived biospecimens from population-based blood product safety surveys. It was later expanded to biospecimens from clinical and epidemiological studies in heart, lung, and blood disorders. The NHLBI also established a Data Repository in 2000 to store and distribute study data from NHLBI-sponsored research. The NHLBI Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC) was established in 2008 to develop the infrastructure needed to link the contents of these two related NHLBI Repositories, facilitate access to repository resources, and streamline request processes. Three key program subcomponents were developed simultaneously: 1) the linkage of biospecimen electronic inventory records with their clinical or characterization data; 2) the development and implementation of a website with both public-facing information and private processing workspaces; and 3) the development of processes to maximize efficiency via a web-based system while maintaining workflow control, document tracking, and secure processes. The BioLINCC website was launched on October 1, 2009 with eight biospecimen collections and data from 72 research studies. By the end of the fourth online year, 38 biospecimen collections were linked and posted, and data from 108 research studies had been made available for request. The number of registered users by the end of the fourth online year approached 2600, and continues to show a trend towards an increasing rate of new users per year. BioLINCC has fulfilled 381 requests comprising 851 data collections, as well as 600 teaching dataset requests and 75 data renewal agreements. 154 biospecimen requests comprising 147,388 biospecimens were fulfilled or actively in process. We conclude that the BioLINCC program has been successful in its goal to increase the visibility and utilization of NHLBI biospecimen and data repository resources.

Production Assistance for Cellular Therapies (PACT): four-year experience from the United States National Heart, Lung, and Blood Institute (NHLBI) contract research program in cell and tissue therapies.

BACKGROUND: In 2002, the US National Heart, Lung, and Blood Institute (NHLBI) conducted a workshop to determine needs of the cell therapy community. A consensus emerged that improved access to cGMP facilities, regulatory assistance, and training would foster the advancement of cellular therapy. STUDY DESIGN AND METHODS: A 2003 NHLBI request for proposals resulted in four contracts being awarded to three cell-manufacturing facilities (Baylor College of Medicine, University of Minnesota, and University of Pittsburgh) and one administrative center (The EMMES Corporation). As a result, Production Assistance for Cellular Therapies (PACT) was formed. RESULTS: As of October 1, 2008, PACT has received 65 preliminary applications of which 45 have been approved for product manufacture. A variety of cell therapies are represented including T-regulatory cells, natural killer cells, adipose-derived stem cells, cardiac progenitor cells for cardiac disease, hematopoietic progenitor cells (HPCs) for central nervous system applications, cytotoxic T lymphocytes, and dendritic cells. A total of 169 products have been administered under 12 applications and 2 reagents were manufactured and delivered. Fourteen peer-reviewed publications and 15 abstracts have resulted from the PACT project to date. A cell therapy textbook is nearly complete. PACT technical projects have addressed assay development, rapid endotoxin testing, shipping of cell products, and CD34+ HPC isolation from low-volume marrow. Educational Web seminars and on-site training through workshops have been conducted. CONCLUSIONS: PACT is an active and successful cell therapy manufacturing resource in the United States, addressing research and training while forging relationships among academia, industry, and participating institutions.

Results of the Cord Blood Transplantation Study (COBLT): clinical outcomes of unrelated donor umbilical cord blood transplantation in pediatric patients with hematologic malignancies.

Outcomes of unrelated donor cord blood transplantation in 191 hematologic malignancy children (median age, 7.7 years; median weight, 25.9 kg) enrolled between 1999 and 2003 were studied (median follow-up, 27.4 months) in a prospective phase 2 multicenter trial. Human leukocyte antigen (HLA) matching at enrollment was 6/6 (n = 17), 5/6 (n = 58), 4/6 (n = 111), or 3/6 (n = 5) by low-resolution HLA-A, -B, and high-resolution (HR) DRB1. Retrospectively, 179 pairs were HLA typed by HR. The median precryopreservation total nucleated cell (TNC) dose was 5.1 x 10(7) TNC/kg (range, 1.5-23.7) with 3.9 x 10(7) TNC/kg (range, 0.8-22.8) infused. The median time to engraftment (absolute neutrophil count > 500/mm(3) and platelets 50 000/muL) was 27 and 174 days. The cumulative incidence of neutrophil engraftment by day 42 was 79.9% (95% confidence interval [CI], 75.1%-85.2%); acute grades III/IV GVHD by day 100 was 19.5% (95% CI, 13.9%-25.5%); and chronic GVHD at 2 years was 20.8% (95% CI, 14.8%-27.7%). HR matching decreased the probability of severe acute GVHD. The cumulative incidence of relapse at 2 years was 19.9% (95% CI, 14.8%-25.7%). The probabilities of 6-month and 2-year survivals were 67.4% and 49.5%. Unrelated donor cord blood transplantation from partially HLA-mismatched units can cure many children with leukemias. The study was registered at www.clinicaltrials.gov as #NCT00000603.

Characterization of banked umbilical cord blood hematopoietic progenitor cells and lymphocyte subsets and correlation with ethnicity, birth weight, sex, and type of delivery: a Cord Blood Transplantation (COBLT) Study report.

BACKGROUND: The Cord Blood Transplantation (COBLT) Study banking program was initiated in 1996. The study goals were to develop standard operating procedures for cord blood (CB) donor recruitment and banking and to build an ethnically diverse unrelated CB bank to support a transplantation protocol. STUDY DESIGN AND METHODS: The hematopoietic progenitor cell (HPC) and lymphocyte subset (LS) content of approximately 8000 CB units were characterized, and these results were correlated with donor ethnicity, birth weight, gestational age, sex, and type of delivery. RESULTS: There was a significant correlation of CD34+ cell count with colony-forming unit (CFU)-granulocyte-macrophage (r=0.68, p<0.001), CFU-granulocyte-erythroid-macrophage-megakaryocyte (r=0.52, p<0.001), burst-forming unit-erythroid (BFU-E; r=0.61, p<0.001), and total CFUs (r=0.67, p<0.001). Nucleated red blood cell count was significantly correlated with total CD34+ (r=0.56, p<0.001), total CFU (r=0.50, p<0.001), BFU-E (r=0.48, p<0.001), and counts of CD34+ subsets (p<0.001). Caucasian ethnicity was significantly correlated with higher CD3+/CD4+, CD19+, and CD16+/CD56+ LSs. Furthermore, CD34+/CD38- and CD34+/CD61+ CB units (HPC-C) were significantly lower in African American and Asian persons compared to Caucasian and Hispanic persons. Male sex was associated with significantly fewer CD3+/CD4+, CD19+, and CD16+/CD56+ but increased CD3+/CD8+ LSs (p<0.001). Finally, cesarean section was associated with significantly higher total CFU and CD16+/CD56+ but lower CD3+/CD4+, CD3+/CD8+, and CD19+ LSs. CONCLUSION: These results provide a standard and range for uniformly processed HPC-C progenitor cells and LSs. CB progenitor cells and/or LSs may in the future predict for rapidity of engraftment, incidence of graft-versus-host disease, speed and quality of immunore- constitution, graft-versus-tumor effects, and/or success of gene transfection after CB transplantation.