Redox regulation of insulin degradation by insulin-degrading enzyme

Insulin-degrading enzyme (IDE) is a thiol sensitive peptidase that degrades insulin and amyloid β, and has been linked to type 2 diabetes mellitus and Alzheimer's disease. We examined the thiol sensitivity of IDE using S-nitrosoglutathione, reduced glutathione, and oxidized glutathione to distinguish the effects of nitric oxide from that of the redox state. The in vitro activity of IDE was studied using either partially purified cytosolic enzyme from male Sprague-Dawley rats, or purified rat recombinant enzyme. We confirm that nitric oxide inhibits the degrading activity of IDE, and that it affects proteasome activity through this interaction with IDE, but does not affect the proteasome directly. Oxidized glutathione inhibits IDE through glutathionylation, which was reversible by dithiothreitol but not by ascorbic acid. Reduced glutathione had no effect on IDE, but reacted with partially degraded insulin to disrupt its disulfide bonds and accelerate its breakdown to trichloroacetic acid soluble fragments. Our results demonstrate the sensitivity of insulin degradation by IDE to the redox environment and suggest another mechanism by which the cell's oxidation state may contribute to the development of, and the link between, type 2 diabetes and Alzheimer's disease.     

Sociosexual and Communication Deficits after Traumatic Injury to the Developing Murine Brain

Despite the life-long implications of social and communication dysfunction after pediatric traumatic brain injury, there is a poor understanding of these deficits in terms of their developmental trajectory and underlying mechanisms. In a well-characterized murine model of pediatric brain injury, we recently demonstrated that pronounced deficits in social interactions emerge across maturation to adulthood after injury at postnatal day (p) 21, approximating a toddler-aged child. Extending these findings, we here hypothesized that these social deficits are dependent upon brain maturation at the time of injury, and coincide with abnormal sociosexual behaviors and communication. Age-dependent vulnerability of the developing brain to social deficits was addressed by comparing behavioral and neuroanatomical outcomes in mice injured at either a pediatric age (p21) or during adolescence (p35). Sociosexual behaviors including social investigation and mounting were evaluated in a resident-intruder paradigm at adulthood. These outcomes were complemented by assays of urine scent marking and ultrasonic vocalizations as indices of social communication. We provide evidence of sociosexual deficits after brain injury at p21, which manifest as reduced mounting behavior and scent marking towards an unfamiliar female at adulthood. In contrast, with the exception of the loss of social recognition in a three-chamber social approach task, mice that received TBI at adolescence were remarkably resilient to social deficits at adulthood. Increased emission of ultrasonic vocalizations (USVs) as well as preferential emission of high frequency USVs after injury was dependent upon both the stimulus and prior social experience. Contrary to the hypothesis that changes in white matter volume may underlie social dysfunction, injury at both p21 and p35 resulted in a similar degree of atrophy of the corpus callosum by adulthood. However, loss of hippocampal tissue was greater after p21 compared to p35 injury, suggesting that a longer period of lesion progression or differences in the kinetics of secondary pathogenesis after p21 injury may contribute to observed behavioral differences. Together, these findings indicate vulnerability of the developing brain to social dysfunction, and suggest that a younger age-at-insult results in poorer social and sociosexual outcomes.     

Evaluation of Propionibacterium acnes Isolates Using Contour-clamped Homogeneous Electric Field Gel Electrophoresis

Contour-clamped homogeneous electric field electrophoresis was performed to compare strains ofPropionibacterium acnes isolated from patients with chronic postoperative endophthalmitis. Propionibacterium acnes isolates were obtained from the vitreous humor of nine patients with chronic postoperative endophthalmitis following cataract surgery. In two of the patients, P. acnes isolates were also obtained from the aqueous humor as well as from the vitreous humor. Bacterial DNA was digested using Not I and Spe I restriction endonucleases. The DNA fragments were then subjected to contour-clamped homogeneous electric field electrophoresis and the DNA banding patterns were analysed. Eight nonidentical banding patterns were identified among the nine vitreous isolates of P. acnes. In each of the two cases from which aqueous and vitreous isolates were recovered from the same eye, the banding patterns were identical. Contour-clamped homogeneous electric field electrophoresis is a powerful method to distinguish P. acnes isolates based on DNA banding patterns and could be used in the epidemiological study of clinical processes caused by this organism.     

Comparing the feasibility, acceptability, clinical-, and cost-effectiveness of mental health e-screening to paper-based screening on the detection of depression, anxiety, and psychosocial risk in pregnant women: a study protocol of a randomized, parallel-group, superiority trial

Background

Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in children. Children with JIA are at risk of inflammation of the uvea in the eye (uveitis). Overall, 20% to 25% of paediatric uveitis is associated with JIA. Major risk factors for development of uveitis in JIA are oligoarticular pattern of arthritis, an age at onset of arthritis of less than seven years of age, and antinuclear antibody positivity. In the initial stages of mild to moderate inflammation the uveitis is asymptomatic. This has led to current practice of screening all children with JIA for uveitis. Approximately 12% to 38% of patients with JIA develop uveitis in seven years following onset of arthritis. In 30% to 50% of children with JIA-associated uveitis structural complications are present at diagnosis. Furthermore about 50% to 75% of those with severe uveitis will eventually develop visual impairment secondary to ocular complications such as cataract and glaucoma. Defining the severity of inflammation and structural complications in uveitis patients is now possible following Standardised Uveitis Nomenclature (SUN) guidelines, and modified to incorporate the consensus of end point and outcome criteria into the design of randomised trials. Despite current screening and therapeutic options (pre-biologics) 10% to 15% of children with JIA-associated uveitis may develop bilateral visual impairment and certified legally blind. To date, there remains no controlled trial evidence of benefits of biologic therapy.

Methods/design

This study will randomise 154 patients aged 2 to 18 years with active JIA-associated uveitis (despite methotrexate (MTX) treatment for at least 12 weeks). All participants will be treated for 18 months, with follow up of 3 years from randomisation (continuing on MTX throughout). All participants will receive a stable dose of MTX and in addition either adalimumab (20 mg/0.8 ml for patients <30 kg or 40 mg/0.8 ml for patients weighing 30 kg or more, subcutaneous (s/c) injection every 2 weeks based on body weight), or placebo (0.8 ml as appropriate according to body weight) s/c injection every 2 weeks.

Discussion

This is the first randomised controlled trial that will assess the clinical effectiveness, safety and cost effectiveness of adalimumab in combination with methotrexate for the treatment of juvenile idiopathic arthritis associated uveitis.

Trial registration

ISRCTN10065623     

In vitro studies of closed-loop feedback and electrosensory processing in Apteronotus leptorhynchus

Electrosensory systems comprise extensive feedback pathways. It is also well known that these pathways exhibit synaptic plasticity on a wide-range of time scales. Recent in vitro brain slice studies have characterized synaptic plasticity in the two main feedback pathways to the electrosensory lateral line lobe (ELL), a primary electrosensory nucleus in Apteronotus leptorhynchus. Currently-used slice preparations, involving networks in open-loop conditions, allow feedback inputs to be studied in isolation, a critical step in determining their synaptic properties. However, to fully understand electrosensory processing, we must understand how dynamic feedback modulates neuronal responses under closed-loop conditions. To bridge the gap between current in vitro approaches and more complex in vivo work, we present two new in vitro approaches for studying the roles of closed-loop feedback in electrosensory processing. The first involves a hybrid-network approach using dynamic clamp, and the second involves a new slice preparation that preserves one of the feedback pathways to ELL in a closed-loop condition.     

Density of the waterborne parasite Ceratomyxa shasta and its biological effects on salmon

The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile salmonids in the Pacific Northwest of North America and is limiting recovery of Chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon populations in the Klamath River. We conducted a 5-year monitoring program that comprised concurrent sentinel fish exposures and water sampling across 212 river kilometers of the Klamath River. We used percent mortality and degree-days to death to measure disease severity in fish. We analyzed water samples using quantitative PCR and Sanger sequencing, to determine total parasite density and relative abundance of C. shasta genotypes, which differ in their pathogenicity to salmonids. We detected the parasite throughout the study zone, but parasite density and genetic composition fluctuated spatially and temporally. Chinook and coho mortality increased with density of their specific parasite genotype, but mortality-density thresholds and time to death differed. A lethality threshold of 40% mortality was reached with 10 spores liter(-1) for Chinook but only 5 spores liter(-1) for coho. Parasite density did not affect degree-days to death for Chinook but was negatively correlated for coho, and there was wider variation among coho individuals. These differences likely reflect the different life histories and genetic heterogeneity of the salmon populations. Direct quantification of the density of host-specific parasite genotypes in water samples offers a management tool for predicting host population-level impacts.     

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.