Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder.

PURPOSE: Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype but with limited neuroradiological data and insufficient evidence for causality of the variants. METHODS: Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays and a zebrafish model. RESULTS: We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs, and increased blood lactate. In vitro studies showed that variants within the TARS2301-381 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model. CONCLUSION: We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity.

Imaging the distribution of iron oxide nanoparticles in hypothermic perfused tissues.

PURPOSE: Herein, we evaluate the use of MRI as a tool for assessing iron oxide nanoparticle (IONP) distribution within IONP perfused organs and vascularized composite allografts (VCAs) (i.e., hindlimbs) prepared for cryopreservation. METHODS: Magnetic resonance imaging was performed on room-temperature organs and VCAs perfused with IONPs and were assessed at 9.4 T. Quantitative T1 mapping and T2∗ -weighted images were acquired using sweep imaging with Fourier transformation and gradient-echo sequences, respectively. Verification of IONP localization was performed through histological assessment and microcomputer tomography. RESULTS: Quantitative imaging was achieved for organs and VCAs perfused with up to 642 mMFe (36 mgFe /mL), which is above previous demonstrations of upper limit detection in agarose (35.7mMFe [2 mgFe /mL]). The stability of IONPs in the perfusate had an effect on the quality of distribution and imaging within organs or VCA. Finally, MRI provided more accurate IONP localization than Prussian blue histological staining in this system, wherein IONPs remain primarily in the vasculature. CONCLUSION: Using MRI, we were able to assess the distribution of IONPs throughout organs and VCAs varying in complexity. Additional studies are necessary to better understand this system and validate the calibration between T1 measurements and IONP concentration.

Perceived barriers and difficulties of intermittent catheterization: In Korean patients with spinal dysraphism and their parents.

AIMS: Integrating regular intermittent catheterization (IC) into daily life is essential for good medical outcomes in patients with neurogenic bladders. The goal is to identify long-term IC-related barriers, or difficulties in Korean patients with spinal dysraphism and their parents. METHODS: The data were prospectively collected using questionnaires from spinal dysraphism patients from two sources: an online community, and those visiting the outpatient clinic of Seoul National university hospital. The questions included were barriers in general and school life, respectively. Also, an open question was included regarding suggestions for school managers or the government in order to overcome identified IC related difficulties. RESULTS: A total of 20 patients and 40 parents answered the questionnaire. Common barriers found in general life were related to lack of places, time, or helpers to perform IC. Substantial numbers of adolescent patients also complained that IC could not be adequately performed due to problems related to privacy or lack of understanding at school. However, the number and nature of barriers varied according to the developmental stage and school environments. Almost all IC barriers seemed to be significant in adolescence. Some parents requested that the government provide space and broaden insurance coverage of catheters in order to facilitate IC. CONCLUSIONS: Various perceived barriers were identified in those who need IC and differences were demonstrated over time. Both dedicated space and time are issues. In addition, patients may benefit from emotional support and enhanced communication with community agencies and government to resolve the problems related with privacy.

The Oct1 homolog Nubbin is a repressor of NF-κB-dependent immune gene expression that increases the tolerance to gut microbiota.

BACKGROUND: Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood. RESULTS: We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism. CONCLUSIONS: This study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.

Patterns of recovery from severe mental illness: a pilot study of outcomes.

We performed a pilot study examining the patterns of recovery from severe mental illness in a model integrated service delivery system using measures from the Milestones of Recovery Scale (MORS), a valid and reliable measure of recovery outcomes which ranges from 1 to 8 (8 levels). For purposes of presentation, we constructed an aggregate MORS (6 levels) where the levels are described as follows: (1) extreme risk; (2) unengaged, poorly self-coordinating; (3) engaged, poorly self-coordinating; (4) coping and rehabilitating; (5) early recovery, and (6) self reliant. We analyzed MORS data on individuals followed over time from The Village in Long Beach, California (658 observations). Using Markov Chains, we estimated origin-destination transition probabilities, simulating recovery outcomes for 100 months. Our models suggest that after 12 months only 8% of "extreme risk" clients remain such. Over 40% have moved to "engaged, poorly self-coordinating." After 2 years, almost half of the initial "extreme Risk" clients are "coping/rehabilitating", "early recovery" or "Self reliant." Most gains occur within 2 years.

Monte carlo model of stricture formation in photodynamic therapy of normal pig esophagus.

Photodynamic therapy (PDT) is FDA-approved for use in patients with Barrett's esophagus using porfimer sodium (2 mg per kg) and a recommended light dose of 130 J cm(-1) for high grade dysplasia. Despite uniform drug and light doses, the clinical outcome of PDT is variable. A significant number of PDT cases result in esophageal strictures, a side effect related to excessive energy absorption. The purpose of this project was to model esophageal stricture formation with a Monte Carlo simulation. An original multilayer Monte Carlo computer simulation was developed for esophageal PDT. Optical absorption and scattering coefficients were derived for mucosal and muscle layers of normal porcine esophagus. Porfimer sodium was added to each layer by increasing the absorption coefficient by the appropriate amount. A threshold-absorbed light dose was assumed to be required for stricture formation and ablation. The simulation predicted irreversible damage to the mucosa with a 160 J cm(-1) light dose and damage to the muscle layer with an additional 160 J cm(-1) light dose for a tissue porfimer sodium content of 3.5 mg kg(-1). The simulation accurately modeled photodynamic stricture formation in normal pig in vivo esophageal tissue. This preliminary work suggests that the absorbed light threshold for stricture formation may be between 2 and 4 J per gram of tissue.

Psychometric properties of an assessment for mental health recovery programs.

The concept of recovery can be operationalized from either the point of view of the consumer, or from the perspective of the agency providing services. The Milestones of Recovery Scale (MORS) was created to capture aspects of recovery from the agency perspective. Evidence establishing the psychometric properties of the MORS was obtained in three efforts: Inter-rater reliability using staff at The Village, a multi-service organization serving the homeless mentally ill in Long Beach, California; inter-rater reliability was also obtained from Vinfen Corporation, a large provider of housing services to mentally ill persons in Boston, Massachusetts. A test-retest reliability study was conducted using staff rating of clients at The Village, and evidence for validity was obtained using the Level of Care Utilization System (LOCUS) as a validity measure. The intra-class correlation coefficient for the inter-rater reliability study was r = .85 (CI .81, .89) for The Village and r = .86 (CI .80, .90) for Vinfen Corporation; test-retest reliability was r = .85 (CI .81, .87); and validity coefficients for the LOCUS were at or above r = .49 for all subscales except one. There is sufficient evidence for the reliability and validity of the MORS.

Identification of the parasitoid community associated with an outbreaking gall wasp, Zapatella davisae, and their relative abundances in New England and Long Island, New York.

Gall wasps (Hymenoptera: Cynipidae) are phytophagous insects that often go unnoticed; however, when they are introduced to a new area or released from their natural enemies, they have the capacity to outbreak and cause extensive foliar damage. One such outbreaking pest, Zapatella davisae (Cynipidae: Cynipini), causes significant damage and mortality to black oak, Quercus velutina, in the northeastern United States. In this study, we aimed to identify the parasitoid community associated with Z. davisae, compare differences in percent parasitism of Z. davisae in Cape Cod and Long Island, and determine which parasitoid species contribute most to parasitism in each region. From both locations, we reared parasitoids, identified morphological groups, analyzed percent parasitism rates for each group, and used DNA barcoding to provide species-level identifications. On Long Island, there was nearly 100% parasitism in 2015 followed by a near total collapse of the population in 2016. In contrast, parasitism rates were lower and remained consistent on Cape Cod between 2015 and 2016, which may explain the greater canopy damage observed in that region. Species of Sycophila were the dominant parasitoids, with one species Sycophila nr. novascotiae representing ~65% of reared parasitoids from Long Island, and two species of Sycophila (S. nr. novascotiae and S. foliatae) with near equal representations on Cape Cod. In order to manage an insect pest, it is important to understand factors that influence its mortality and survival. An understanding of how these infestations progress overtime can help predict the impact that newer infestations in Nantucket, MA, and coastal Rhode Island will have on black oak populations and will aid in the management of this rapidly spreading gall wasp pest.

Antagonizing scalloped with a novel vestigial construct reveals an important role for scalloped in Drosophila melanogaster leg, eye and optic lobe development.

Scalloped (SD), a TEA/ATTS-domain-containing protein, is required for the proper development of Drosophila melanogaster. Despite being expressed in a variety of tissues, most of the work on SD has been restricted to understanding its role and function in patterning the adult wing. To gain a better understanding of its role in development, we generated sd(47M) flip-in mitotic clones. The mitotic clones had developmental defects in the leg and eye. Further, by removing the VG domains involved in activation, we created a reagent (VGDeltaACT) that disrupts the ability of SD to form a functional transcription factor complex and produced similar phenotypes to the flip-in mitotic clones. The VGDeltaACT construct also disrupted adult CNS development. Expression of the VGDeltaACT construct in the wing alters the cellular localization of VG and produces a mutant phenotype, indicating that the construct is able to antagonize the normal function of the SD/VG complex. Expression of the protein:protein interaction portion of SD is also able to elicit similar phenotypes, suggesting that SD interacts with other cofactors in the leg, eye, and adult CNS. Furthermore, antagonizing SD in larval tissues results in cell death, indicating that SD may also have a role in cell survival.

Attention deficit hyperactivity disorder: parents' and professionals' perceptions.

Attention deficit hyperactivity disorder (ADHD) is a prevalent childhood psychiatric condition. This study was a qualitative investigation with parents and professionals conducted in two north London boroughs, using focus groups as well as semi-structured and narrative interviews. The aim was to explore parents' and professionals' beliefs regarding the causes of ADHD and their perceptions of service provision. The sample was drawn purposively from GP practices and voluntary support groups. Professionals were recruited via professional networks. Analysis was thematic. It was found that the views of parents and professionals differed. Professionals were more likely to see ADHD as a medical condition, while parents were more likely to see ADHD in association with socio-environmental causes. Delayed diagnosis, inadequate access to information and a lack of co-ordinated care are stated as some of the reasons for parental dissatisfaction with services. Professionals emphasised the need for multidisciplinary input into the management of ADHD. The implications of these findings were that parents often battled with professionals to encourage them to see their viewpoint, access to treatment was influenced by the views of parents and professionals, and noncompliance occurred when parents had different views from professionals.

Efficacy of Two Systemic Insecticides With Stem Gall Wasp, Zapatella davisae (Hymenoptera: Cynipidae) on Black Oak.

Black oak, Quercus velutina Lamarck, is the dominant deciduous tree on Cape Cod, Nantucket, and Martha's Vineyard, Massachusetts, and in recent years it has experienced widespread mortality and severe canopy loss due to infestations of a stem gall wasp, Zapatella davisae Buffington and Melika (Hymenoptera: Cynipidae). A single application of systemic insecticides emamectin benzoate and imidacloprid was found to reduce or prevent further accumulation of Z. davisae damage on infested black oak during a 1-yr trial.

Economic Evaluation of the Juvenile Drug Court/Reclaiming Futures (JDC/RF) Model.

Juvenile drug court (JDC) programs are an increasingly popular option for rehabilitating juvenile offenders with substance problems, but research has found inconsistent evidence regarding their effectiveness and economic impact. While assessing client outcomes such as reduced substance use and delinquency is necessary to gauge program effectiveness, a more comprehensive understanding of program success and sustainability can be attained by examining program costs and economic benefits. As part of the National Cross-Site Evaluation of JDC and Reclaiming Futures (RF), an economic analysis of five JDC/RF programs was conducted from a multisystem and multiagency perspective. The study highlights the direct and indirect costs of JDC/RF and the savings generated from reduced health problems, illegal activity, and missed school days. Results include the average (per participant) cost of JDC/RF, the total economic benefits per JDC/RF participant, and the net savings of JDC/RF relative to standard JDC.

A surgeon who CARES can be safer.

The characteristics of a high-reliability organization are reviewed. Examples of how these characteristics relate to patient safety in surgical practice are illustrated by vignettes. The characteristics discussed include commitment to safety demonstrated to others by the conduct of one's practice; attention focused on one's own performance and the performance of others to the task at hand; rehearsal and proper preparation and contingency planning for procedures; effective communication so that information is accurate, adequate, unambiguous, and confirmed; and sense-making, or an understanding and verification of consistency between what is observed and expected and between what is planned and the premises for those plans.

Recognition and management of autonomic dysreflexia in pediatric spinal cord injury.

BACKGROUND: Autonomic dysreflexia (AD) is a well-documented complication of spinal cord injury (SCI) at or above the T6 level. However, research into AD has focused primarily on the adult. Because research that involves children with SCI is scarce, current guidelines may not be appropriate for children. Therefore, many episodes of AD may be unrecognized or inappropriately treated. To address this issue, Shriners Hospitals for Children undertook the development of a protocol specific to children and adolescents. METHOD: A task force was developed to look at current literature on AD and blood pressure in children. Utilizing this literature and consensus among the task force members, the tools necessary to treat children with SCI at risk for AD were developed. RESULTS: The task force developed several products intended to assist in the recognition and management of AD. These include an event flow sheet for recording incidents of AD, a letter for the child's school or primary care physician that provides a brief summary of AD and the child's baseline blood pressure, and a policy/protocol with 2 age-specific algorithms to standardize interventions across the 3 Shriners Hospitals in the United States with SCI programs (California, Illinois, and Pennsylvania). CONCLUSION: The Shriners Hospitals for Children Task Force on Autonomic Dysreflexia in Children with Spinal Cord Injury has developed several tools specific to children. However, many questions remain to be answered concerning blood pressure norms and the clinical presentation of AD in children.

Imbalanced expression of Vcan mRNA splice form proteins alters heart morphology and cellular protein profiles.

The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan ((tm1Zim)), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan ((tm1Zim)) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.

Immune response in the barrier epithelia: lessons from the fruit fly Drosophila melanogaster.

The barrier epithelia of multicellular organisms frequently come into direct contact with microorganisms and thus need to fulfill the important task of preventing the penetration of pathogens that could cause systemic infections. A functional immune defence in the epithelial linings of the digestive, respiratory and reproductive organs as well as the epidermis/skin of animals is therefore of crucial importance for survival. Epithelial defence reactions are likely to be evolutionarily ancient, and the use of invertebrate animal models, such as insects and nematodes, has been crucial in unravelling the mechanisms underlying epithelial immunity. This review addresses basic questions of epithelial immunity in animals and humans. It focuses on recent developments in the understanding of the immune responses in the fruit fly Drosophila melanogaster and how the innate immune system acts locally in the epidermis and cuticle, tracheae, gut and genital organs. Both basal immune activities in epithelia that are constantly exposed to microbes as well as positive and negative regulation in response to pathogenic organisms are covered. Important immuno-physiological aspects of epithelial defence mechanisms are also discussed, such as wound healing, re-epithelialization and intestinal homeostasis.

Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

Wild-type Drosophila melanogaster as a model host to analyze nitrogen source dependent virulence of Candida albicans.

The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR) flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5) activates two transcription factors (Stp1 and Stp2) to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively). Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.