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The cupula is a gelatinous membrane overlying the crista ampullaris of the semicircular canal, important for sensing rotation of the head and critical for normal balance. Recently the zona pellucida like domain containing 1 protein (ZPLD1, also known as cupulin) was identified in the cupula of fish. Here, we describe two new spontaneous mutations in the mouse Zpld1 gene, which were discovered by the circling behavior of mutant mice, an indicator of balance dysfunction. The Zpld1 mutant mice exhibited normal hearing function as assessed by auditory brainstem response (ABR) measurements, and their otolithic organs appeared normal. In the inner ear, Zpld1 mRNA expression was detected only in the hair cells and supporting cells of the crista ampullaris. Normal vestibular sensory evoked potential (VsEP) responses and abnormal vestibulo-ocular reflex (VOR) responses demonstrated that the vestibular dysfunction of the Zpld1 mutant mice is caused by loss of sensory input for rotary head movements (detected by cristae ampullaris) and not by loss of input for linear head translations (detected by maculae of the utricle and saccule). Taken together, these results are consistent with ZPLD1 being an important functional component of the cupula, but not tectorial or otoconial membranes.

Marijuana, a psychoactive drug that activates cannabinoid-1 (CB1) receptors in the brain, is the most prevalently abused illicit drug among American adolescents and young adults. However, the long-term consequences of adolescent exposure to cannabinoids on the brain and behavior remain poorly understood. In both humans and nonhumans, adolescence is characterized by the maturation of the endocannabinoid neurotransmitter system in the prefrontal cortex and striatum-brain regions that underlie choice and decision making and are densely packed with CB1 receptors. In the current study, the effects of chronic WIN 55,212-2 (a CB1 agonist) exposure during adolescence on reversal learning and delay discounting were compared with those of adult-onset exposure using mice. Mice were administered 3.0 mg/kg/day WIN 55,212-2 or vehicle for 21 days beginning in adolescence (postnatal days 28-49) or adulthood (postnatal days 90-111). For the reversal-learning task, there was no difference in errors or omissions to criterion following a reversal in adolescent-exposed mice but adult-exposed mice showed a delay in beginning the reversal, suggesting that adolescents, but not adults, are resilient to this drug. Adult mice given WIN 55,212-2 in adolescence displayed greater impulsivity in the form of preference for smaller-sooner reinforcers over larger-delayed ones in the delay-discounting procedure, but this was seen to a lesser extent with adult-onset exposure. These data underscore the importance of variables related to the timing and duration of exposure as well as the specific and persistent behavioral endpoints affected by chronic cannabinoid administration. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

The morphology of the early ontogenetic stages of cycad foliage may help resolve the relationships between extinct to extant cycad lineages. However, prior to this study, fossil evidence of cycad seedlings was not known. We describe a compression fossil of cycad eophylls with co-occurring fully developed leaves of adult specimens from the early Palaeocene ( ca 63.8 Ma) Castle Rock flora from the Denver Basin, CO, USA and assign it to the fossil genus Dioonopsis (Cycadales) based on leaf morphology and anatomy. The new fossil seedling foliage is particularly important because fully differentiated pinnate leaves of adult plants and the eophylls belong to the same species based on shared epidermal micromorphology, therefore, increasing the number of morphological characteristics that can be used to place Dioonopsis phylogenetically. Significantly, the seedling fossil has a basic foliage structure that is very similar to seedlings of extant cycads, which is consistent with a cycadalean affinity of Dioonopsis. Nevertheless, the set of morphological characters in the seedling and adult specimens of Dioonopsis suggests a distant relationship between Dioonopsis and extant Dioon. This indicates that extinct lineages of cycads were present and widespread during the early Cenozoic (Palaeogene) coupled with the subordinate role of extant genera in the Palaeogene fossil record of cycads.

Objectives Research to date indicates that parents and children residing in food insufficient homes incur a host of negative health outcomes. Recently, studies have suggested that these homes are also at risk of violence between family members. Our objective is to examine the link between household food insufficiency and physical violence in the home using a recent, nationally representative sample, and to determine whether family mental illness and/or substance misuse inform this association. Methods A sample of nearly 50,000 children and families from the 2016 National Survey of Children's Health were employed in the study. Information concerning household access to food, experiences of violence between parents/adults, and associated mental health and substance use risk factors were available in the data. Logistic regression, employed in a hierarchical fashion, was utilized to analyze the data. Results Household food insufficiency was associated with an increased risk of children witnessing physical violence in the home, and this was especially pronounced in the case of moderate-to-severe food insufficiency. Findings also indicated that family mental illness and substance misuse partly attenuated this association and that household food insufficiency was more strongly associated with violence in the home in the absence of mental health and substance use risk factors. Conclusions for Practice Polices aimed at diminishing food insufficiency may have important collateral benefits in the form of reductions in family violence, and these benefits appear to extend to families that are otherwise at low risk of family violence.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

BRCA1 C-terminal domains are found in a specialized group of 23 proteins that function in the DNA damage response to protect genomic integrity. C-terminal domain phosphatase 1 (CTDP1) is the only phosphatase with a BRCA1 C-terminal domain in the human proteome, yet direct participation in the DNA damage response has not been reported. Examination of the CTDP1 BRCA1 C-terminal domain-specific protein interaction network revealed 103 high confidence interactions enriched in DNA damage response proteins, including FANCA and FANCI that are central to the Fanconi anemia DNA repair pathway necessary for the resolution of DNA interstrand crosslink damage. CTDP1 expression promotes DNA damage-induced FANCA and FANCD2 foci formation and enhances homologous recombination repair efficiency. CTDP1 was found to regulate multiple aspects of FANCI activity, including chromatin localization, interaction with γ-H2AX, and SQ motif phosphorylations. Knockdown of CTDP1 increases MCF-10A sensitivity to DNA interstrand crosslinks and double-strand breaks, but not ultraviolet radiation. In addition, CTDP1 knockdown impairs in vitro and in vivo growth of breast cancer cell lines. These results elucidate the molecular functions of CTDP1 in Fanconi anemia interstrand crosslink repair and identify this protein as a potential target for breast cancer therapy., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest.

Background: Neural stem cell (NSC)-based therapies hold great promise for treating diseases of the central nervous system (CNS). However, several fundamental problems still need to be overcome to fully exploit the clinical potential of NSC therapeutics. Chief among them is the limited survival of NSC grafts within hostile microenvironments., Methods: Herein, we sought to engineer NSCs in an effort to increase graft survival within ischemic brain lesions via upregulation of global SUMOylation, a post-translational modification critically involved in mediating tolerance to ischemia/reperfusion., Findings: NSCs overexpressing the SUMO E2-conjugase Ubc9 displayed resistance to oxygen-glucose-deprivation/restoration of oxygen/glucose (OGD/ROG) and enhanced neuronal differentiation in vitro, as well as increased survival and neuronal differentiation when transplanted in mice with transient middle cerebral artery occlusion in vivo., Interpretation: Our work highlights a critical role for SUMOylation in NSC biology and identifies a biological pathway that can be targeted to increase the effectiveness of exogenous stem cell medicines in ischemic stroke. FUND: Intramural Research Program of the NINDS/NIH, the Italian Multiple Sclerosis Foundation (FISM), the Bascule Charitable Trust, NIH-IRTA-OxCam and Wellcome Trust Research Training Fellowships., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Tissue macrophages have an embryonic origin and can be replenished in some tissues under steady-state conditions by blood monocytes. However, little is known about the residency and properties of infiltrating monocytes after an inflammatory challenge. The meninges of the central nervous system (CNS) are populated by a dense network of macrophages that act as resident immune sentinels. Here we show that, following lymphocytic choriomeningitis virus infection, resident meningeal macrophages (MMs) acquired viral antigen and interacted directly with infiltrating cytotoxic T lymphocytes, which led to macrophage depletion. Concurrently, the meninges were infiltrated by inflammatory monocytes that engrafted the meningeal niche and remained in situ for months after viral clearance. This engraftment led to interferon-γ-dependent functional changes in the pool of MMs, including loss of bacterial and immunoregulatory sensors. Collectively, these data indicate that peripheral monocytes can engraft the meninges after an inflammatory challenge, imprinting the compartment with long-term defects in immune function.

We aimed to characterize peripheral blood gene expression profile of penumbra defined as MRI perfusion-diffusion mismatch (PD MM) in peripheral blood of patients with acute ischemic stroke. We studied 23 patients. Perfusion-diffusion mismatch volume was observed to be associated and significantly correlated with the expression of 34 genes including those related to inflammation, SUMOylation, and coagulation; while lipopolysaccharide inhibition was identified to be a candidate upstream regulator of these processes ( z -score -2.38, P  = 0.04). Penumbral volume is correlated with a specific gene expression profile in the peripheral blood characterized by overlap of inflammatory and neuroprotective pathways that are regulated by lipopolysaccharide inhibition.

The capacity for tumor cells to metastasize efficiently is directly linked to their ability to colonize secondary sites. Here we identify Six2, a developmental transcription factor, as a critical regulator of a breast cancer stem cell program that enables metastatic colonization. In several triple-negative breast cancer (TNBC) models, Six2 enhanced the expression of genes associated with embryonic stem cell programs. Six2 directly bound the Sox2 Srr2 enhancer, promoting Sox2 expression and downstream expression of Nanog , which are both key pluripotency factors. Regulation of Sox2 by Six2 enhanced cancer stem cell properties and increased metastatic colonization. Six2 and Sox2 expression correlated highly in breast cancers including TNBC, where a Six2 expression signature was predictive of metastatic burden and poor clinical outcome. Our findings demonstrate that a SIX2/SOX2 axis is required for efficient metastatic colonization, underscoring a key role for stemness factors in outgrowth at secondary sites. SIGNIFICANCE: These findings provide novel mechanistic insight into stemness and the metastatic outgrowth of triple-negative breast cancer cells. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/720/F1.large.jpg., (©2019 American Association for Cancer Research.)

Maternal and fetal sources of thyroid hormone are important for the development of many organ systems. Thyroid hormone deficiency causes variable intellectual disability and hearing impairment in mouse and man, but the basis for this variation is not clear. To explore this variation, we studied two thyroid hormone-deficient mouse mutants with mutations in pituitary-specific transcription factors, POU1F1 and PROP1, that render them unable to produce thyroid stimulating hormone. DW/J-Pou1f1 dw/dw mice have profound deafness and both neurosensory and conductive hearing impairment, while DF/B-Prop1 df/df mice have modest elevations in hearing thresholds consistent with developmental delay, eventually achieving normal hearing ability. The thyroid glands of Pou1f1 mutants are more severely affected than those of Prop1 df/df mice, and they produce less thyroglobulin during the neonatal period critical for establishing hearing. We previously crossed DW/J-Pou1f1 dw/+ and Cast/Ei mice and mapped a major locus on Chromosome 2 that protects against hypothyroidism-induced hearing impairment in Pou1f1 dw/dw mice: modifier of dw hearing (Mdwh). Here we refine the location of Mdwh by genotyping 196 animals with 876 informative SNPs, and we conduct novel mapping with a DW/J-Pou1f1 dw/+ and 129/P2 cross that reveals 129/P2 mice also have a protective Mdwh locus. Using DNA sequencing of DW/J and DF/B strains, we determined that the genes important for thyroid gland function within Mdwh vary in amino acid sequence between strains that are susceptible or resistant to hypothyroidism-induced hearing impairment. These results suggest that the variable effects of congenital hypothyroidism on the development of hearing ability are attributable to genetic variation in postnatal thyroid gland folliculogenesis and function.

In this study we characterized the TCR repertoire profiles in patients with chronic progressive inflammatory neurological disorders including HAM/TSP, associated with human T-cell lymphotropic virus type I (HTLV-I) infection, and multiple sclerosis (MS), an inflammatory, demyelinating disease of the CNS of unknown etiology. We hypothesized that a T-cell receptor (TCR) clonal repertoire 'signature' could distinguish HAM/TSP patients from healthy controls, as well as from patients with a more heterogeneous CNS-reactive inflammatory disease such as MS. In this study, we applied an unbiased molecular technique - unique molecular identifier (UMI) library-based strategy to investigate with high accuracy the TCR clonal repertoire by high throughput sequencing (HTS) technology. cDNA-TCR β-chain libraries were sequenced from 2 million peripheral mononuclear cells (PBMCs) in 14 HAM/TSP patients, 34 MS patients and 20 healthy controls (HC). While HAM/TSP patients showed a higher clonal T-cell expansion compared to MS and HC, increase of the TCR clonal expansion was inversely correlated with the diversity of TCR repertoire in all subjects. In addition, longitudinal analysis of TCR repertoires from HAM/TSP patients demonstrated a correlation of the TCR clonal expansion with HTLV-I proviral load. Surprisingly, MS patients showed a higher diversity of TCR repertoires than other groups. Despite higher TCR clonal expansions in HAM/TSP patients, no disease-specific TCRs were shared among patients. Only non-shared or "private" TCR repertoires was observed. While no clones that shared the same CDR3 amino acid sequences were seen in either HC or MS patients, there was a cluster of related CDR3 amino acid sequences observed for 18 out of 34 MS patients when evaluated by phylogenetic tree analysis. This suggests that a TCR-repertoire signature may be identified in a subset of patients with MS.

Rationale: Food insecurity is a significant public health concern, with implications for community and individual health and well-being. Although a growing body of literature points to the role of neighborhoods in household food insecurity, studies using nationally representative samples to explore interactions between neighborhood risks - including violence and danger - are lacking., Objective: The present study examines whether interactions between physical disorder, low social capital, and violence/danger in the neighborhood have significant implications for the risk of household food insufficiency using a large, nationally representative sample of U.S. children and their families., Method: Data are from the 2016 National Survey of Children's Health, a survey of a cross-sectional weighted probability sample of U.S. children from 0 to 17 years of age. Multinomial logistic regression techniques were used to analyze the data., Results: Neighborhood risk factors interacted to predict household food insufficiency, with the confluence of low social capital and violence/danger yielding the strongest effects., Conclusions: Our findings suggest that food hardship should be addressed within the context of neighborhood revitalization. The risk of food insufficiency among children and families in especially high-risk ecological contexts might be ameliorated with the provision of informal and formal sources of nutrition assistance and support., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: In order to transition to adulthood and independence, youth with spina bifida must assume significant self-management responsibilities including monitoring for shunt malfunction, maintaining intact skin in areas that are insensate, and maintaining proper bowel and bladder function. Validated measures of specific spina bifida self-management skills are lacking and this hampers the ability of clinical personnel to support successful transition for youth with spina bifida., Methods: We developed a self-report measure specific to SB self-management skills consistent with the framework of the Transition Readiness Assessment Questionnaire (TRAQ). To test the predictive validity of the tool we surveyed 90 youth and young adults ages 12-25 with spina bifida attending a multidisciplinary clinic participating in the National Spina Bifida Patient Registry (NSBPR)., Results: Adjusted for age, gender, race, insurance status and lesion level, higher scores on the TRAQ-SB (increased self-management) were negatively associated with urinary incontinence in the past month. Only lesion level, and not TRAQ-SB scores, was a significant predictor of stool incontinence and skin breakdown., Conclusions: Higher TRAQ-SB scores are negatively associated with bladder incontinence in youth with spina bifida. While stool continence and skin breakdown were not associated with TRAQ-SB scores, this relation is complex and may be obfuscated by either reporting bias or outcome measurement bias. To further refine the questionnaire and understand this relationship we need to field it prospectively in the SB network with larger samples. The TRAQ-SB questionnaire, however, does have value in the clinical setting to help promote the acquisition of specific self-management skills among youth with spina bifida.

Hundreds of thousands of cis-regulatory DNA sequences are predicted in vertebrate genomes, but unlike genes themselves, few have been characterized at the functional level or even unambiguously paired with a target gene. Here we serendipitously identified and started investigating the first reported long-range regulatory region for the Nr2f1 (Coup-TFI) transcription factor gene. NR2F1 is temporally and spatially regulated during development and required for patterning and regionalization in the nervous system, including sensory hair cell organization in the auditory epithelium of the cochlea. Analyzing the deaf wanderer (dwnd) spontaneous mouse mutation, we traced back the cause of its associated circling behavior to a 53 kb deletion removing five exons and adjacent intronic regions of the poorly characterized Mctp1 gene. Interestingly, loss of Mctp1 function cannot account for the hearing loss, inner ear dysmorphology and sensory hair cell disorganization observed in dwnd mutants. Instead, we found that the Mctp1 dwnd deletion affects the Nr2f1 gene located 1.4 Mb away, downregulating transcription and protein expression in the embryonic cochlea. Remarkably, the Mctp1 dwnd allele failed to complement a targeted inactivation allele of Nr2f1, and transheterozygotes or Mctp1 dwnd homozygotes exhibit the same morphological defects observed in inner ears of Nr2f1 mutants without sharing their early life lethality. Defects include improper separation of the utricle and saccule in the vestibule not described previously, which can explain the circling behavior that first brought the spontaneous mutation to attention. By contrast, mice homozygous for a targeted inactivation of Mctp1 have normal hearing and inner ear structures. We conclude that the 53 kb Mctp1 dwnd deletion encompasses a long-range cis-regulatory region essential for proper Nr2f1 expression in the embryonic inner ear, providing a first opportunity to investigate Nr2f1 function in postnatal inner ears. This work adds to the short list of long-range regulatory regions characterized as essential to drive expression of key developmental control genes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To reduce nonactionable oximeter alarms by 80% without increasing time infants were hypoxemic (oxygen saturation [SpO 2 ] ≤ 80%) or hyperoxemic (SpO 2  > 95% while on supplemental oxygen)., Study Design: In 2015, a multidisciplinary team at Connecticut Children's Medical Center initiated a quality improvement project to reduce nonactionable oximeter alarms in two referral neonatal intensive care units (NICUs). Changes made through improvement cycles included reduction of the low oximeter alarm limit for specific populations, increased low alarm delay, development of postmenstrual age-based alarm profiles, and updated bedside visual reminders. Manual alarm tallies and electronic SpO 2 data were collected throughout the project., Results: Alarm tallies were collected for 158 patient care hours with SpO 2 data available for 138 of those hours. Mean number of total nonactionable alarms per patient per hour decreased from 9 to 2 (78% decrease) and the mean number of nonactionable low alarms per patient per hour decreased from 5 to 1 (80% decrease). No change was noted in the balancing measures of percentage time with SpO 2  ≤ 80% (mean 4.3%) or SpO 2  > 95% (mean 23.7%)., Conclusion: Through small changes in oximeter alarm settings, including revision of alarm limits, alarm delays, and age-specific alarm profiles, our NICUs significantly reduced nonactionable alarms without increasing hypoxemia., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Probing an individual cell's gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Introduction: Acute sciatica (symptom duration less than 4 weeks), a major cause of pain and disability, is a common presentation to medical practices and hospital emergency departments. Selective CT fluoroscopy transforaminal epidural steroid injection is often used with the hope of reducing pain and improving function. Recently, there has been interest in using systemic corticosteroids in acute sciatica. However, there is limited evidence to inform management of selective CT fluoroscopy transforaminal epidural steroid in subacute and chronic sciatica and there is no evidence in acute sciatica, even though the practice is widespread. There is also limited evidence for the use of systemic corticosteroids in acute sciatica. Furthermore, the management of selective CT fluoroscopy transforaminal epidural steroid versus systemic steroids has never been directly studied., Methods and Analysis: SCIATICA is a pilot/feasibility study of patients with acute sciatica designed to evaluate the feasibility of undertaking a blinded four-arm randomised controlled intervention study of (1) selective CT fluoroscopy transforaminal epidural steroid (arm 1), (2) selective CT fluoroscopy transforaminal epidural saline (arm 2), (3) 15 days tapering dose of oral steroids (arm 3) and (4) a sham epidural and oral placebo control (arm 4). This feasibility study is designed to evaluate head-to-head, route versus pharmacology of interventions. The primary outcome measure is the Oswestry Disability Index (ODI) at 3 weeks. Secondary outcome is the ODI at 48 weeks. Other outcomes include numerical rating scale for leg pain, Pain DETECT Questionnaire, quality of life, medication use, rescue procedures or surgery, and adverse events. Results of outcomes from this randomised controlled trial will be used to determine the feasibility, sample size and power calculations for a large multicentre study., Ethics and Dissemination: The study has been approved by South Eastern Sydney Local Health District Human Research Ethics Committee (HREC/15/331/POHW/586)., Trial Registration Number: NCT03240783; Pre-results., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Though vaccine uptake and public support have risen since the release of the first HPV vaccines, the United States has far lower initiation and completion rates for the HPV vaccine series in comparison to other vaccines indicated for youth. Disparities are even greater in the Appalachian regions. Understanding factors contributing to these discrepancies is vital to improving vaccine rates in Appalachia. A comprehensive literature search identified all articles pertaining to HPV vaccination in children and adolescents living in Appalachia. The final 15 articles were included in a systematic review of the topic.

The intestinal epithelium constitutes a crucial defense to the potentially life-threatening effects of gut microbiota. However, due to a complex underlying vasculature, hypoperfusion and resultant tissue ischemia pose a particular risk to function and integrity of the epithelium. The small ubiquitin-like modifier (SUMO) conjugation pathway critically regulates adaptive responses to metabolic stress and is of particular significance in the gut, as inducible knockout of the SUMO-conjugating enzyme Ubc9 results in rapid intestinal epithelial disintegration. Here we analyzed the pattern of individual SUMO isoforms in intestinal epithelium and investigated their roles in intestinal ischemia/reperfusion (I/R) damage. Immunostaining revealed that epithelial SUMO2/3 expression was almost exclusively limited to crypt epithelial nuclei in unchallenged mice. However, intestinal I/R or overexpression of Ubc9 caused a remarkable enhancement of epithelial SUMO2/3 staining along the crypt-villus axis. Unexpectedly, a similar pattern was found in SUMO1 knockout mice. Ubc9 transgenic mice, but also SUMO1 knockout mice were protected from I/R injury as evidenced by better preserved barrier function and blunted inflammatory responses. PCR array analysis of microdissected villus-tip epithelia revealed a specific epithelial contribution to reduced inflammatory responses in Ubc9 transgenic mice, as key chemotactic signaling molecules such as IL17A were significantly downregulated. Together, our data indicate a critical role particularly of the SUMO2/3 isoforms in modulating responses to I/R and provide the first evidence that SUMO1 deletion activates a compensatory process that protects from ischemic damage.

Electron-induced surface reactions of (η 5 -C 5 H 5 )Fe(CO) 2 Mn(CO) 5 were explored in situ under ultra-high vacuum conditions using X-ray photoelectron spectroscopy and mass spectrometry. The initial step involves electron-stimulated decomposition of adsorbed (η 5 -C 5 H 5 )Fe(CO) 2 Mn(CO) 5 molecules, accompanied by the desorption of an average of five CO ligands. A comparison with recent gas phase studies suggests that this precursor decomposition step occurs by a dissociative ionization (DI) process. Further electron irradiation decomposes the residual CO groups and (η 5 -C 5 H 5 , Cp) ligand, in the absence of any ligand desorption. The decomposition of CO ligands leads to Mn oxidation, while electron stimulated Cp decomposition causes all of the associated carbon atoms to be retained in the deposit. The lack of any Fe oxidation is ascribed to either the presence of a protective carbonaceous matrix around the Fe atoms created by the decomposition of the Cp ligand, or to desorption of both CO ligands bound to Fe in the initial decomposition step. The selective oxidation of Mn in the absence of any Fe oxidation suggests that the fate of metal atoms in mixed-metal precursors for focused electron beam induced deposition (FEBID) will be sensitive to the nature and number of ligands in the immediate coordination sphere. In related studies, the composition of deposits created from (η 5 -C 5 H 5 )Fe(CO) 2 Mn(CO) 5 under steady state deposition conditions, representative of those used to create nanostructures in electron microscopes, were measured and found to be qualitatively consistent with predictions from the UHV surface science studies.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5 , a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin ( hspn )] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13 hspn/hspn mice have malformations similar to those of Dlx5 - / - embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13 hspn/hspn embryos, indicating that the Slc25a13 hspn deletion affects otic-specific enhancers of Dlx5 In addition, transheterozygous Slc25a13 +/hspn Dlx5 +/ - mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13 hspn/hspn and Dlx5 - / - embryos, verifying a cis -acting effect of the Slc25a13 hspn deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13 hspn deleted region failed to phenocopy the defects of Slc25a13 hspn/hspn mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13 hspn/hspn mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear., (Copyright © 2018 by the Genetics Society of America.)

To understand the cellular basis of behavior, it is necessary to know the cell types that exist in the nervous system and their contributions to function. Spinal networks are essential for sensory processing and motor behavior and provide a powerful system for identifying the cellular correlates of behavior. Here, we used massively parallel single nucleus RNA sequencing (snRNA-seq) to create an atlas of the adult mouse lumbar spinal cord. We identified and molecularly characterized 43 neuronal populations. Next, we leveraged the snRNA-seq approach to provide unbiased identification of neuronal populations that were active following a sensory and a motor behavior, using a transcriptional signature of neuronal activity. This approach can be used in the future to link single nucleus gene expression data with dynamic biological responses to behavior, injury, and disease., (Published by Elsevier Inc.)

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker K V channel into excised squid giant axons. We found that total K + currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout ( rda )]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle. SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia., (Copyright © 2018 the authors 0270-6474/18/380843-15$15.00/0.)

Lineage commitment and differentiation of skeletal stem cells/bone marrow stromal cells (SSCs/BMSCs, often called bone marrow-derived "mesenchymal stem/stromal" cells) offer an important opportunity to study skeletal and hematopoietic diseases, and for tissue engineering and regenerative medicine. Currently, many studies in this field have relied on cell lineage tracing methods in mouse models, which have provided a significant advancement in our knowledge of skeletal and hematopoietic stem-cell niches in bone marrow (BM). However, there is a lack of agreement in numerous fundamental areas, including origins of various BM stem-cell niches, cell identities, and their physiological roles in the BM. In order to resolve these issues, we propose a new hypothesis of "paralogous" stem-cell niches (PSNs); that is, progressively altered parallel niches within an individual species throughout the life span of the organism. A putative PSN code seems to be plausible based on analysis of transcriptional signatures in two representative genes that encode Nes-GFP and leptin receptors, which are frequently used to monitor SSC lineage development in BM. Furthermore, we suggest a dynamic paralogous BM niche (PBMN) model that elucidates the coupling and uncoupling mechanisms between BM stem-cell niches and their zones of active regeneration during different developmental stages. Elucidation of these PBMNs would enable us to resolve the existing controversies, thus paving the way to achieving precision regenerative medicine and pharmaceutical applications based on these BM cell resources. Stem Cells 2018;36:11-21., (© 2017 AlphaMed Press.)

The development of mouse genetic tools has made a significant contribution to the understanding of skeletal and hematopoietic stem cell niches in bone marrow (BM). However, many experimental designs (e.g., selections of marker genes, target vector constructions, and choices of reporter murine strains) have unavoidable technological limitations and bias, which lead to experimental discrepancies, data reproducibility issues, and frequent data misinterpretation. Consequently, there are a number of conflicting views relating to fundamental biological questions, including origins and locations of skeletal and hematopoietic stem cells in the BM. In this report, we systematically unravel complicated data interpretations via comprehensive analyses of technological benefits, pitfalls, and challenges in frequently used mouse models and discuss their translational relevance to human stem cell biology. Particularly, we emphasize the important roles of using large human genomic data-informatics in facilitating genetic analyses of mouse models and resolving existing controversies in mouse and human BM stem cell biology., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations of the human ATP6V1B1 gene cause distal renal tubular acidosis (dRTA; OMIM #267300) often associated with sensorineural hearing impairment; however, mice with a knockout mutation of Atp6v1b1 were reported to exhibit a compensated acidosis and normal hearing. We discovered a new spontaneous mutation (vortex, symbol vtx) of Atp6v1b1 in an MRL/MpJ (MRL) colony of mice. In contrast to the reported phenotype of the knockout mouse, which was developed on a primarily C57BL/6 (B6) strain background, MRL-Atp6v1b1vtx/vtx mutant mice exhibit profound hearing impairment, which is associated with enlarged endolymphatic compartments of the inner ear. Mutant mice have alkaline urine but do not exhibit overt metabolic acidosis, a renal phenotype similar to that of the Atpbv1b1 knockout mouse. The abnormal inner ear phenotype of MRL- Atp6v1b1vtx/vtx mice was lost when the mutation was transferred onto the C57BL/6J (B6) background, indicating the influence of strain-specific genetic modifiers. To genetically map modifier loci in Atp6v1b1vtx/vtx mice, we analysed ABR thresholds of progeny from a backcross segregating MRL and B6 alleles. We found statistically significant linkage with a locus on Chr 13 that accounts for about 20% of the hearing threshold variation in the backcross mice. The important effect that genetic background has on the inner ear phenotype of Atp6v1b1 mutant mice provides insight into the hearing loss variability associated with dRTA caused by ATP6V1B1 mutations. Because MRL-Atp6v1b1vxt/vtx mice do not recapitulate the metabolic acidosis of dRTA patients, they provide a new genetic model for nonsyndromic deafness with enlarged vestibular aqueduct (EVA; OMIM #600791)., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Clinical alarm systems have received significant attention in recent years following warnings from hospital accrediting and health care technology organizations regarding patient harm caused by unsafe practices. Alarm desensitization or fatigue from frequent, false, or unnecessary alarms, has led to serious events and even patient deaths. Other concerns include settings inappropriate to patient population or condition, inadequate staff training, and improper use or disabling. Research on human factors in alarm response and of functionality of medical devices will help clinicians develop appropriate policies, practices, and device settings for clinical alarms in neonatal intensive care units., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Inherited Retinal dystrophy (IRD) is a broad group of inherited retinal disorders with heterogeneous genotypes and phenotypes. Next generation sequencing (NGS) methods have been broadly applied for analyzing patients with IRD. Here we report a novel approach to enrich the target gene panel by microdroplet PCR., Methods: This assay involved a primer library which targeted 3071 amplicons from 2078 exons comprised of 184 genes involved in retinal function and/or retinal development. We amplified the target regions using the RainDance target enrichment PCR method and sequenced the products using the MiSeq NGS platform., Results: In this study, we analyzed 82 samples from 67 families with IRD. Bioinformatics analysis indicated that this procedure was able to reach 99% coverage of target sequences with an average sequence depth of reads at 119×. The variants detected by this study were filtered, validated, and prioritized by pathogenicity analysis. Genotypes and phenotypes were correlated by determining a consistent relationship in 38 propands (56.7%). Pathogenic variants in genes related to retinal function were found in another 11 probands (16.4%), but the clinical correlations showed inconsistencies and insufficiencies in these patients., Conclusions: The application of NGS in IRD clinical molecular diagnosis provides a powerful approach to exploring the etiology and pathology in patients. It is important for the clinical laboratory to interpret the molecular findings in the context of patient clinical presentations because accurate interpretation of pathogenic variants is critical for delivering solid clinical molecular diagnosis to clinicians and patients and improving the standard care of patients.

Protein SUMOylation is a dynamic post-translational modification shown to be involved in a diverse set of physiologic processes throughout the cell. SUMOylation has also been shown to play a role in the pathobiology of myriad cancers, one of which is glioblastoma multiforme (GBM). As such, the clinical significance and therapeutic utility offered via the selective control of global SUMOylation is readily apparent. There are, however, relatively few known/effective inhibitors of global SUMO-conjugation. Herein we describe the identification of topotecan as a novel inhibitor of global SUMOylation. We also provide evidence that inhibition of SUMOylation by topotecan is associated with reduced levels of CDK6 and HIF-1α, as well as pronounced changes in cell cycle progression and cellular metabolism, thereby highlighting its putative role as an adjuvant therapy in defined GBM patient populations.

Objective: To examine whether breast-feeding duration and socio-economic status (SES) interact to predict junk food consumption among offspring and whether the interaction differs across racial/ethnic groups., Design: Survey research using a longitudinal panel design. Hierarchical linear regression was used to analyse the data., Setting: In-home interviews with the child's parents over a 5-year period across the USA., Subjects: Approximately 10 000 American children from the Early Childhood Longitudinal Study: Birth Cohort (ECLS-B)., Results: The findings revealed that longer breast-feeding durations correspond to lower levels of junk food consumption, but that this relationship emerges consistently only among low-SES blacks., Conclusions: Efforts to promote breast-feeding among low-SES black women may have the added benefit of reducing their children's junk food intake, and may thereby promote their general health and well-being. Future research should seek to explore the mechanisms by which breast-feeding might benefit the dietary habits of low-SES black children.

Microglia are resident inflammatory cells of the CNS and have important roles in development, homeostasis and a variety of neurologic and psychiatric diseases. Difficulties in procuring human microglia have limited their study and hampered the clinical translation of microglia-based treatments shown to be effective in animal disease models. Here we report the differentiation of human induced pluripotent stem cells (iPSC) into microglia-like cells by exposure to defined factors and co-culture with astrocytes. These iPSC-derived microglia have the phenotype, gene expression profile and functional properties of brain-isolated microglia. Murine iPSC-derived microglia generated using a similar protocol have equivalent efficacy to primary brain-isolated microglia in treatment of murine syngeneic intracranial malignant gliomas. The ability to generate human microglia facilitates the further study of this important CNS cell type and raises the possibility of their use in personalized medicine applications.

A single nucleotide variant (SNV) of the cadherin 23 gene (Cdh23 c.753A ), common to many inbred mouse strains, accelerates age-related hearing loss (AHL) and can worsen auditory phenotypes of other mutations. We used homologous recombination in C57BL/6 NJ (B6N) and 129S1/SvImJ (129S1) embryonic stem cells to engineer mouse strains with reciprocal single base pair substitutions (B6-Cdh23 c.753A>G and 129S1-Cdh23 c.753G>A ). We compared ABR thresholds and cochlear pathologies of these SNV mice with those of congenic (B6.129S1-Cdh23 Ahl+ and 129S1.B6-Cdh23 ahl ) and parental (B6N and 129S1) strain mice. Results verified the protective effect of the Cdh23 c.753G allele, which prevented high frequency hearing loss in B6 mice to at least 18 months of age, and the AHL-inducing effect of the Cdh23 c.753A allele, which worsened hearing loss in 129S1 mice. ABR thresholds differed between 129S-Cdh23 c.753A SNV and 129S1.B6-Cdh23 ahl congenic mice, and a linkage backcross involving these strains localized a Chr 10 QTL contributing to the difference. These results illustrate the large effects that strain background and congenic regions have on the hearing loss associated with Cdh23 c.753 alleles. Importantly, the B6-Cdh23 c.753G strain can be used to eliminate the confounding influence of the Cdh23 c.753A variant in hearing studies of B6 mice and mutant mice on the B6 background.

A growing body of studies has demonstrated that dysregulated splicing profiles constitute pivotal mechanisms for carcinogenesis. In this study, we identified discriminative splicing profiles of colorectal cancer (CRC) cells compared to adjacent normal tissues using deep RNA-sequencing (RNA-seq). The RNA-seq results and cohort studies indicated a relatively high ratio of exon 4-excluded neuro-oncological ventral antigen 1 (Nova1 -4 ) and intron 2-retained SRSF6 (SRSF6 +intron 2 ) transcripts in CRC tissues and cell lines. Nova1 variants exhibited differential effects on eliminating SRSF6 expression in CRC cells by inducing SRSF6 +intron 2 transcripts which were considered to be the putative target of alternative splicing-coupled nonsense-mediated decay mechanism. Moreover, the splicing profile of vascular endothelial growth factor (VEGF)165/VEGF165b transcripts was relevant to SRSF6 expression, which manipulates the progression of CRC calls. These results highlight the novel and hierarchical role of an alternative splicing cascade that is involved in the development of CRC.

Nodding syndrome is an epileptic disorder of unknown etiology that occurs in children in East Africa. There is an epidemiological association with Onchocerca volvulus , the parasitic worm that causes onchocerciasis (river blindness), but there is limited evidence that the parasite itself is neuroinvasive. We hypothesized that nodding syndrome may be an autoimmune-mediated disease. Using protein chip methodology, we detected autoantibodies to leiomodin-1 more abundantly in patients with nodding syndrome compared to unaffected controls from the same village. Leiomodin-1 autoantibodies were found in both the sera and cerebrospinal fluid of patients with nodding syndrome. Leiomodin-1 was found to be expressed in mature and developing human neurons in vitro and was localized in mouse brain to the CA3 region of the hippocampus, Purkinje cells in the cerebellum, and cortical neurons, structures that also appear to be affected in patients with nodding syndrome. Antibodies targeting leiomodin-1 were neurotoxic in vitro, and leiomodin-1 antibodies purified from patients with nodding syndrome were cross-reactive with O. volvulus antigens. This study provides initial evidence supporting the hypothesis that nodding syndrome is an autoimmune epileptic disorder caused by molecular mimicry with O. volvulus antigens and suggests that patients may benefit from immunomodulatory therapies., (Copyright © 2017, American Association for the Advancement of Science.)

Chemical vapor deposition (CVD) is an attractive technique for the metallization of organic thin films because it is selective and the thickness of the deposited film can easily be controlled. However, thermal CVD processes often require high temperatures which are generally incompatible with organic films. In this paper, we perform proof-of-concept studies of photochemical CVD to metallize organic thin films. In this method, a precursor undergoes photolytic decomposition to generate thermally labile intermediates prior to adsorption on the sample. Three readily available Ru precursors, CpRu(CO) 2 Me, (η 3 -allyl)Ru(CO) 3 Br, and (COT)Ru(CO) 3 , were employed to investigate the role of precursor quantum yield, ligand chemistry, and the Ru oxidation state on the deposition. To investigate the role of the substrate chemistry on deposition, carboxylic acid-, hydroxyl-, and methyl-terminated self-assembled monolayers were used. The data indicate that moderate quantum yields for ligand loss (φ ≥ 0.4) are required for ruthenium deposition, and the deposition is wavelength dependent. Second, anionic polyhapto ligands such as cyclopentadienyl and allyl are more difficult to remove than carbonyls, halides, and alkyls. Third, in contrast to the atomic layer deposition, acid-base reactions between the precursor and the substrate are more effective for deposition than nucleophilic reactions. Finally, the data suggest that selective deposition can be achieved on organic thin films by judicious choice of precursor and functional groups present on the substrate. These studies thus provide guidelines for the rational design of new precursors specifically for selective photochemical CVD on organic substrates.

Otitis media (OM), inflammation of the middle ear, is a common cause of hearing loss in children and in patients with many different syndromic diseases. Studies of the human population and mouse models have revealed that OM is a multifactorial disease with many environmental and genetic contributing factors. Here, we report on otitis media-related hearing loss in asj (ages with stiffened joints) mutant mice, which bear a point mutation in the Enpp1 gene. Auditory-evoked brainstem response (ABR) measurements revealed that around 90% of the mutant mice (Enpp1asj/asj) tested had moderate to severe hearing impairment in at least one ear. The ABR thresholds were variable and generally elevated with age. We found otitis media with effusion (OME) in all of the hearing-impaired Enpp1asj/asj mice by anatomic and histological examinations. The volume and inflammatory cell content of the effusion varied among the asj mutant mice, but all mutants exhibited a thickened middle ear epithelium with fibrous polyps and more mucin-secreting goblet cells than controls. Other abnormalities observed in the Enpp1 mutant mice include over-ossification at the round window ridge, thickened and over-calcified stapedial artery, fusion of malleus and incus, and white patches on the inside of tympanic membrane, some of which are typical symptoms of tympanosclerosis. An excessive yellow discharge was detected in the outer ear canal of older asj mutant mice, with 100% penetrance by 5 months of age, and contributes to the progressive nature of the hearing loss. This is the first report of hearing loss and ear pathology associated with an Enpp1 mutation in mice. The Enpp1asj mutant mouse provides a new animal model for studying tympanosclerotic otitis and otitis media with effusion, and also provides a specific model for the hearing loss recently reported to be associated with human ENPP1 mutations causing generalized arterial calcification of infancy and hypophosphatemic rickets., Competing Interests: The authors have declared that no competing interests exist.

Background: We examined recent trends and interhospital variation in use of indomethacin, ibuprofen, and surgical ligation for patent ductus arteriosus (PDA) in very-low-birth-weight (VLBW) infants., Methods: Included in this retrospective study of the Pediatric Hospital Information System database were 13,853 VLBW infants from 19 US children's hospitals, admitted at age < 3 d between 1 January 2005 and 31 December 2014. PDA management and in-hospital outcomes were examined for trends and variation., Results: PDA was diagnosed in 5,719 (42%) VLBW infants. Cyclooxygenase inhibitors and/or ligation were used in 74% of infants with PDA overall, however studied hospitals varied greatly in PDA management. Odds of any cyclooxygenase inhibitor or surgical treatment for PDA decreased 11% per year during the study period. This was temporally associated with improved survival but also with increasing bronchopulmonary dysplasia, periventricular leukomalacia, retinopathy of prematurity, and acute renal failure in unadjusted analyses. There was no detectable correlation between hospital-specific changes in PDA management and hospital-specific changes in outcomes of preterm birth during the study period., Conclusion: Use of cyclooxygenase inhibitors and ligation for PDA in VLBW infants decreased over a 10-y period at the studied hospitals. Further evidence is needed to assess the impact of this change in PDA management.

Laboratory mice (Mus musculus) have become the major model species for inner ear research. The major uses of mice include gene discovery, characterization, and confirmation. Every application of mice is founded on assumptions about what mice represent and how the information gained may be generalized. A host of successes support the continued use of mice to understand hearing and balance. Depending on the research question, however, some mouse models and research designs will be more appropriate than others. Here, we recount some of the history and successes of the use of mice in hearing and vestibular studies and offer guidelines to those considering how to apply mouse models., Competing Interests: Compliance with Ethical Standards Funding Agencies KKO: Washington University Medical School Department of Otolaryngology SAJ: Nebraska Tobacco Settlement Biomedical Research Foundation KRJ: NIH NIDCD RO1 DC004301, R01 DC005827

Desmosomes are prominent adhesive junctions present between many epithelial cells as well as cardiomyocytes. The mechanisms controlling desmosome assembly and remodeling in epithelial and cardiac tissue are poorly understood. We recently identified protein palmitoylation as a mechanism regulating desmosome dynamics. In this study, we have focused on the palmitoylation of the desmosomal cadherin desmoglein-2 (Dsg2) and characterized the role that palmitoylation of Dsg2 plays in its localization and stability in cultured cells. We identified two cysteine residues in the juxtamembrane (intracellular anchor) domain of Dsg2 that, when mutated, eliminate its palmitoylation. These cysteine residues are conserved in all four desmoglein family members. Although mutant Dsg2 localizes to endogenous desmosomes, there is a significant delay in its incorporation into junctions, and the mutant is also present in a cytoplasmic pool. Triton X-100 solubility assays demonstrate that mutant Dsg2 is more soluble than wild-type protein. Interestingly, trafficking of the mutant Dsg2 to the cell surface was delayed, and a pool of the non-palmitoylated Dsg2 co-localized with lysosomal markers. Taken together, these data suggest that palmitoylation of Dsg2 regulates protein transport to the plasma membrane. Modulation of the palmitoylation status of desmosomal cadherins can affect desmosome dynamics., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The solid-state structure of a dimeric β-diketiminate magnesium(II) complex is discussed. The compound, di-μ-iodido-bis-[(-{4-amino-1,5-bis-[2,6-bis-(propan-2-yl)phen-yl]pent-3-en-2-yl-idene}aza-nido-κ 2 N , N ')magnesium(II)] toluene sesquisolvate, [Mg 2 (C 29 H 41 N 2 ) 2 I 2 ]·1.5C 7 H 8 , crystallizes as two independent mol-ecules, each with 2/ m crystallographic site symmetry, located at Wyckoff sites 2 c and 2 d . These have symmetry-equivalent magnesium atoms bridged by μ-iodide ligands with very similar Mg-I distances. The two Mg atoms are located slightly below (∼0.5 Å) the least-squares plane defined by N-C-C-N atoms in the ligand scaffold, and are approximately tetra-hedrally coordinated. One and one-half toluene solvent mol-ecules are disordered with respect to mirror-site symmetry at Wyckoff sites 4 i and 2 a , respectively. In the former case, two toluene mol-ecules inter-act in an off-center parallel stacking arrangement; the shortest C to C' (π-π) distance of 3.72 (1) Å was measured for this inter-action.

Pancreatic ductal adenocarcinomas are highly malignant cancers characterized by extensive invasion into surrounding tissues, metastasis to distant organs, and a limited response to therapy. A main feature of pancreatic ductal adenocarcinomas is desmoplasia, which leads to extensive deposition of collagen I. We have demonstrated that collagen I can induce epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. A hallmark of EMT is an increase in the expression of the mesenchymal cadherin N-cadherin. Previously we showed up-regulation of N-cadherin promotes tumor cell invasion and that collagen I-induced EMT is mediated by two collagen receptors, α2β1-integrin and discoidin domain receptor 1 (DDR1). DDR1 is a receptor-tyrosine kinase widely expressed during embryonic development and in many adult tissues and is also highly expressed in many different cancers. In the signaling pathway initiated by collagen, we have shown proline-rich tyrosine kinase 2 (Pyk2) is downstream of DDR1. In this study we found isoform b of DDR1 is responsible for collagen I-induced up-regulation of N-cadherin and tyrosine 513 of DDR1b is necessary. Knocking down Shc1, which binds to tyrosine 513 of DDR1b via its PTB (phosphotyrosine binding) domain, eliminates the up-regulation of N-cadherin. The signaling does not require a functional SH2 domain or the tyrosine residues commonly phosphorylated in Shc1 but is mediated by the interaction between a short segment of the central domain of Shc1 and the proline-rich region of Pyk2. Taken together, these data illustrate DDR1b, but not DDR1a, mediates collagen I-induced N-cadherin up-regulation, and Shc1 is involved in this process by coupling to both DDR1 and Pyk2., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Interdecadal modes of tropical Pacific ocean-atmosphere circulation have a strong influence on global temperature, yet the extent to which these phenomena influence global climate on multicentury timescales is still poorly known. Here we present a 2,000-year, multiproxy reconstruction of western Pacific hydroclimate from two speleothem records for southeastern Indonesia. The composite record shows pronounced shifts in monsoon rainfall that are antiphased with precipitation records for East Asia and the central-eastern equatorial Pacific. These meridional and zonal patterns are best explained by a poleward expansion of the Australasian Intertropical Convergence Zone and weakening of the Pacific Walker circulation (PWC) between ∼1000 and 1500 CE Conversely, an equatorward contraction of the Intertropical Convergence Zone and strengthened PWC occurred between ∼1500 and 1900 CE. Our findings, together with climate model simulations, highlight the likelihood that century-scale variations in tropical Pacific climate modes can significantly modulate radiatively forced shifts in global temperature.

Adaptor protein (AP) complexes function in the intracellular sorting and vesicular transport of membrane proteins. The clathrin-associated AP-1 complex functions at the trans-Golgi network and endosomes, and some forms of this complex are thought to mediate the sorting of proteins in plasma membranes of polarized epithelial cells. A null mutation of the mouse Ap1g1 gene, which encodes the gamma-1 subunit of the AP-1 complex, causes embryonic lethality when homozygous, indicating its critical importance in early development but precluding studies of its possible roles during later stages. Here, we describe our analyses of a new spontaneous mutation of Ap1g1 named "figure eight" (symbol fgt) and show that it is an in-frame deletion of 6 bp, which results in the elimination of two amino acids of the encoded protein. In contrast to Ap1g1 (-/-) null mice, mice homozygous for the recessive fgt mutation are viable with adult survival similar to controls. Although Ap1g1 is ubiquitously expressed, the phenotype of Ap1g1 (fgt) mutant mice is primarily restricted to abnormalities in sensory epithelial cells of the inner ear, pigmented epithelial cells of the retina, follicular epithelial cells of the thyroid gland, and the germinal epithelium of the testis, suggesting that impaired AP-1 sorting and targeting of membrane proteins in these polarized cells may underlie the observed pathologies. Ap1g1 (fgt) mutant mice provide a new animal model to study the in vivo roles of gamma-1 adaptin and the AP-1 complex throughout development and to investigate factors that underlie its associated phenotypic abnormalities.

Objective: Analysis of the T-cell receptor (TCR) repertoire in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) can reveal antigen-specific immune responses potentially implicated in the disease process. We applied a new unbiased deep-sequencing method for TCR repertoire analysis to accurately measure and compare receptor diversity and clonal expansions within the peripheral and CSF-trafficking T-cell populations of patients with MS and control individuals with idiopathic intracranial hypertension (IIH)., Methods: Paired blood and CSF TCR β-chain libraries from five MS patients and five IIH controls were sequenced, yielding a total of 80 million reads., Results: Although TCR repertoire diversity was greater in the blood and CSF compartments of MS patients when compared with IIH controls, it is notable that the frequency of clonal expansions was also significantly higher in both compartments of MS patients. Highly expanded T-cell clones were enriched in the CSF compartment of MS patients compared to peripheral blood, very few of them were detected in both compartments., Interpretation: Collectively, our data provide a proof of principle that private compartmentalized T-cell expansion exists in the intrathecal space of MS patients.

Objective: To determine whether prenatal risk factors (RFs) that predict cord blood iron status in term newborns also predict iron status of premature newborns., Study Design: Cord blood iron indices from 80 preterm newborns were compared with historical and demographic RFs for developing iron deficiency if born at term., Result: The presence of multiple RFs did not incrementally interfere with cord iron status in preterm newborns. Poorer iron status accompanied being small for gestational age in prematurity, but other RFs, including diabetes, had relatively little impact., Conclusion: Growth-restricted preterm newborns are at risk for poor iron endowment, likely due to uteroplacental insufficiency. Other RFs were less impactful on iron status of premature newborns than in term newborns, likely reflecting that disruptive effects of RFs are more impactful in the third trimester. Understanding RFs for poor iron endowment is important for clinical recognition and treatment of premature babies.

Genetic factors combined with oxidative stress are major determinants of age-related hearing loss (ARHL), one of the most prevalent disorders of the elderly. Dwarf grey mice, Ggt1dwg/dwg, are homozygous for a loss of function mutation of the g-glutamyl transferase 1 gene, which encodes an important antioxidant enzyme critical for the resynthesis of glutathione (GSH). Since GSH reduces oxidative damage, we hypothesized that Ggt1dwg/dwg mice would be susceptible to ARHL. Surprisingly, otoacoustic emissions and cochlear microphonic potentials, which reflect cochlear outer hair cell (OHC) function, were largely unaffected in mutant mice, whereas auditory brainstem responses and the compound action potential were grossly abnormal. These functional deficits were associated with an unusual and selective loss of inner hair cells (IHC), but retention of OHC and auditory nerve fibers. Remarkably, hearing deficits and IHC loss were completely prevented by N-acetyl-L-cysteine, which induces de novo synthesis of GSH; however, hearing deficits and IHC loss reappeared when treatment was discontinued. Ggt1dwg/dwg mice represent an important new model for investigating ARHL, therapeutic interventions, and understanding the perceptual and electrophysiological consequences of sensory deprivation caused by the loss of sensory input exclusively from IHC.