NADPH oxidase 1 promotes hepatic steatosis in obese mice and is abrogated by augmented skeletal muscle mass.

Exercise as a lifestyle modification is a frontline therapy for nonalcoholic fatty liver disease (NAFLD), but how components of exercise attenuate steatosis is unclear. To uncouple the effect of increased muscle mass from weight loss in obesity, myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius (Gastrocn.) mass and reduces hepatic steatosis and hepatic sterol regulatory element binding protein 1 (Srebp1) expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression but not NADPH oxidase 4 (Nox4) in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin (Ins)-mediated SREBP1 expression was significantly increased in either NOX1, NADPH oxidase organizer 1 (NOXO1), and NADPH oxidase activator 1 (NOXA1) or NOX5-transfected HepG2 cells versus ?-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed that hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Taken together, these data suggest that hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.NEW & NOTEWORTHY This study documents a novel mechanism by which changes in body composition, notably increased muscle mass, protect against fatty liver disease. This mechanism involves NADPH oxidase 1 (NOX1), an enzyme that increases superoxide and increases insulin signaling, leading to increased fat accumulation in the liver. NOX1 may represent a new early target for preventing fatty liver to stave off later liver diseases such as cirrhosis or liver cancer.

Prenatal exposure to a low dose of BPS causes sex-dependent alterations to vascular endothelial function in adult offspring.

Background: Bisphenol S (BPS) is among the most commonly used substitutes for Bisphenol A (BPA), an endocrine disrupting chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins. Bisphenols interfere with estrogen receptor (ER) signaling, which modulates vascular function through stimulation of nitric oxide (NO) production via endothelial nitric oxide synthase (eNOS). BPS can cross into the placenta and accumulates in the fetal compartment to a greater extent than BPA, potentially interfering with key developmental events. Little is known regarding the developmental impact of exposure to BPA substitutes, particularly with respect to the vasculature. Objective: To determine if prenatal BPS exposure influences vascular health in adulthood. Methods: At the time of mating, female C57BL/6 dams were administered BPS (250 nM) or vehicle control in the drinking water, and exposure continued during lactation. At 12-week of age, mesenteric arteries were excised from male and female offspring and assessed for responses to an endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) vasodilator. Endothelium-dependent dilation was measured in the presence or absence of L-NAME, an eNOS inhibitor. To further explore the role of NO and ER signaling, wire myography was used to assess ACh responses in aortic rings after acute exposure to BPS in the presence or absence of L-NAME or an ER antagonist. Results: Increased ACh dilation and increased sensitivity to Phe were observed in microvessels from BPS-exposed females, while no changes were observed in male offspring. Differences in ACh-induced dilation between control or BPS-exposed females were eliminated with L-NAME. Increased dilatory responses to ACh after acute BPS exposure were observed in aortic rings from female mice only, and differences were eliminated with inhibition of eNOS or inhibition of ER. Conclusion: Prenatal BPS exposure leads to persistent changes in endothelium-dependent vascular function in a sex-specific manner that appears to be modulated by interaction of BPS with ER signaling.

Multimodal Retinal Imaging and Microperimetry Reveal a Novel Phenotype and Potential Trial End Points in CRB1-Associated Retinopathies.

Purpose: Biallelic crumbs cell polarity complex component 1 (CRB1) mutations can present as Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), or cystic maculopathy. This study reports a novel phenotype of asymptomatic fenestrated slit maculopathy (AFSM) and examines macular volume profile and microperimetry as clinical trial end points in CRB1-associated retinopathies. Methods: Twelve patients from nine families with CRB1 mutation were recruited. Ultra-widefield (UWF) color fundus photography and autofluorescence (AF), spectral-domain optical coherence tomography (SD-OCT), microperimetry, and adaptive optics (AO) imaging were performed. Macular volume profiles were compared with age-matched healthy controls. Genotyping was performed using APEX genotyping microarrays, targeted next-generation sequencing, and Sanger sequencing. Results: We identified one patient with LCA, five patients with RP, and four patients with macular dystrophy (MD) with biallelic CRB1 mutations. Two siblings with compound heterozygote genotype (c.[2843G>A]; [498_506del]) had AFSM characterized by localized outer retinal disruption on SD-OCT and parafoveal cone loss on AO imaging despite normal fundus appearance, visual acuity, and foveal sensitivity. UWF AF demonstrated preserved para-arteriolar retinal pigment epithelium (PPRPE) in all patients with RP. Microperimetry documented preserved central retinal function in six patients. The ratio of perifoveal-to-foveal retinal volume was greater than controls in 89% (8/9) of patients with RP or MD, whereas central subfield and total macular volume were outside normal limits in 67% (6/9). Conclusions: AO imaging was helpful in detecting parafoveal cone loss in asymptomatic patients. Macular volume profile and microperimetry parameters may have utility as CRB1 trials end points. Translational Relevance: Macular volume and sensitivity can be used as structural and functional end points for trials on CRB1-associated RP and MD.

Progressive sector retinitis pigmentosa due to c.440G>T mutation in SAG in an Australian family.

BACKGROUND: Heterozygous c.440 G > T mutation in the S-antigen visual arrestin (SAG) gene has been described as a cause of autosomal dominant retinitis pigmentosa (adRP) in a series of patients of Hispanic origin. This study presents the early and late clinical features and disease progression rates in an Australian family with SAG adRP. MATERIALS AND METHODS: An observational case series of four family members with adRP. They were examined clinically, with multi-modal retinal imaging and electroretinography (ERG) to ascertain phenotype. Disease progression rate was measured using optical coherence tomography (OCT) and fundus autofluorescence (FAF). A retinal dystrophy panel was used for the proband and cascade testing with targeted Sanger sequencing was conducted in other available family members. RESULTS: The proband presented at 36 years of age with profoundly reduced full-field ERG responses despite a sector RP phenotype. This progressed to a classic RP pattern over several decades leaving a small residual island of central visual field. The horizontal span of the residual outer nuclear layer and the area of hyperautofluorescent ring contracted at a rate of 8-11% and 9-14% per year, respectively. DNA sequencing confirmed the segregation of SAG c.440 G > T mutation with disease. CONCLUSION: SAG adRP presents with a reduced full-field ERG response consistent with a rod-cone dystrophy in mid-life despite a sector RP phenotype. Centripetal progression of the disease into the macula can be tracked by OCT and FAF imaging.

Maternal uniparental isodisomy of chromosome 6 unmasks a novel variant in TULP1 in a patient with early onset retinal dystrophy.

Purpose: Inherited retinal dystrophies are a clinically and genetically heterogeneous group of disorders. Molecular diagnosis has proven utility for affected individuals. In this study, we report an individual enrolled in the Australian Inherited Retinal Disease Registry and DNA Bank diagnosed with clinical features overlapping between Leber congenital amaurosis and retinitis pigmentosa. Methods: DNA from the proband was sequenced using a gene panel for inherited retinal disorders, and a single nucleotide polymorphism (SNP) array was conducted to detect the presence of deletions and uniparental disomy. Results: We identified a novel homozygous variant (c.524dupC, p.(Pro176ThrfsTer7)) in TULP1 resulting from maternal uniparental isodisomy of chromosome 6. The patient had clinical features consistent with biallelic pathogenic variants in TULP1, including congenital nystagmus, night blindness, non-recordable electroretinogram, mild myopia, and mild peripheral pigmentary changes in the fundus. Conclusions: This is the first report of uniparental disomy 6 and a homozygous variant in TULP1 associated with a rod-cone dystrophy. Molecular diagnosis of inherited retinal dystrophies is essential to inform the mode of transmission and clinical management, and to identify potential candidates for future gene-specific therapies.

The genetic profile of Leber congenital amaurosis in an Australian cohort.

BACKGROUND: Leber congenital amaurosis (LCA) is a severe visual impairment responsible for infantile blindness, representing ~5% of all inherited retinal dystrophies. LCA encompasses a group of heterogeneous disorders, with 24 genes currently implicated in pathogenesis. Such clinical and genetic heterogeneity poses great challenges for treatment, with personalized therapies anticipated to be the best treatment candidates. Unraveling the individual genetic etiology of disease is a prerequisite for personalized therapies, and could identify potential treatment candidates, inform patient management, and discriminate syndromic forms of disease. METHODS: We have genetically analyzed 45 affected and 82 unaffected individuals from 34 unrelated LCA pedigrees using predominantly next-generation sequencing and Array CGH technology. RESULTS: We present the molecular findings for an Australian LCA cohort, sourced from the Australian Inherited Retinal Disease Registry & DNA Bank. CEP290 and GUCY2D mutations, each represent 19% of unrelated LCA cases, followed by NMNAT1 (12%). Genetic subtypes were consistent with other reports, and were resolved in 90% of this cohort. CONCLUSION: The high resolution rate achieved, equivalent to recent findings using whole exome/genome sequencing, reflects the progression from hypothesis (LCA Panel) to non-hypothesis (RD Panel) testing and, coupled with Array CGH analysis, is a highly effective first-tier test for LCA.

The differential effects of low birth weight and Western diet consumption upon early life hepatic fibrosis development in guinea pig.

Postnatal intake of an energy dense diet, the Western diet (WD), is a strong risk factor for liver fibrosis. Recently, adverse in utero conditions resulting in low birth weight (LBW) have also been associated with postnatal fibrosis development. We assessed the independent and possible synergistic effects of placental insufficiency-induced LBW and postnatal WD consumption on liver fibrosis in early adulthood, with a specific focus on changes in inflammation and apoptosis pathways in association with fibrogenesis. Male LBW (uterine artery ablation) and normal birth weight (NBW) guinea pig pups were fed either a control diet (CD) or WD from weaning to 150 days. Significant steatosis, mild lobular inflammation, apoptosis and mild stage 1 fibrosis (perisinusoidal or portal) were evident in WD-fed offspring (NBW/WD and LBW/WD). In LBW/CD versus NBW/CD offspring, increased transforming growth factor-beta 1 and matrix metallopeptidase mRNA and sma- and Mad-related protein 4 (SMAD4) were present in conjunction with minimal stage 1 portal fibrosis. Further, connective tissue growth factor mRNA was increased and miR-146a expression decreased in LBW offspring, irrespective of diet. Independent of birth weight, WD-fed offspring exhibited increased expression of fibrotic genes as well as elevated inflammatory and apoptotic markers. Moreover, the augmented expression of collagen, type III, alpha 1 and tumor necrosis factor-alpha was associated with increased recruitment of RNA polymerase II and enhanced histone acetylation (K9) to their respective promoters. These data support a role for both LBW and postnatal WD as factors contributing to hepatic fibrosis development in offspring through distinct pathways.

The contribution of Toll-like receptors to placental inflammation in diet-induced maternal obesity.

Toll-like receptor (TLR)-regulated protein kinases and inflammatory cytokines were activated in fetal vascular smooth muscle cells (VSMC) treated with palmitate. Tumor necrosis factor (TNFα) and interleukin-6 (IL6) were increased and correlated with expression of TLRs in the labyrinth placentae of high fat (HF)-fed rats with increased plasma lipids and visceral adiposity. Thus, local induction of TLR signaling via saturated fatty acids (SFA) may in part contribute to placental inflammation in diet-induced maternal obesity.

The Escherichia coli clamp loader can actively pry open the ß-sliding clamp.

Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds ß first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.

Pax genes during neural development and their potential role in neuroregeneration.

Pax genes encode a family of transcription factors that have long been recognised as obligate contributors to embryonic development of the CNS, with evidence obtained from various animal models illustrating phylogenetically conserved functions. Within the CNS, Pax genes play substantial roles in cellular and regional specification, proliferation, progenitor cell maintenance, anti-apoptosis and neural differentiation. This comprehensive review details the critical functions of those Pax genes involved in pre- and post-natal CNS development, provides possible molecular mechanisms by which Pax genes contribute to proliferation and differentiation of neuronal cells, and explains observed changes in Pax gene expression in response to neurotrauma in the mature animal. Knowledge of the ability of individual Pax genes to specify precise lineages within the CNS is beneficial for cell replacement strategies, particularly in the production of "designer" cells for the treatment of neurodegenerative disorders. The manipulation of stem or committed cells so that they express definitive Pax genes may indeed assist in the pursuit of the holy grail of regenerative medicine - that of CNS cell replacement therapies leading to functional repair. We explain here, however, that only the sophisticated and precise use of Pax genes will lead to a successful outcome.

A slow ATP-induced conformational change limits the rate of DNA binding but not the rate of beta clamp binding by the escherichia coli gamma complex clamp loader.

In Escherichia coli, the gamma complex clamp loader loads the beta-sliding clamp onto DNA. The beta clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, gamma complex binds beta and DNA to form a ternary complex. Binding to primed template DNA triggers gamma complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of gamma complex binding beta and DNA. A fluorescence intensity-based beta binding assay was developed in which the fluorescence of pyrene covalently attached to beta increases when bound by gamma complex. Using this assay, an association rate constant of 2.3 x 10(7) m(-1) s(-1) for gamma complex binding beta was determined. The rate of beta binding was the same in experiments in which gamma complex was preincubated with ATP before adding beta or added directly to beta and ATP. In contrast, when gamma complex is preincubated with ATP, DNA binding is faster than when gamma complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote beta binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for gamma complex to bind beta before DNA during the clamp loading reaction cycle.

Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.

Clamp loaders are multisubunit complexes that use the energy derived from ATP binding and hydrolysis to assemble ring-shaped sliding clamps onto DNA. Sliding clamps in turn tether DNA polymerases to the templates being copied to increase the processivity of DNA synthesis. Here, the rate of clamp release during the clamp loading reaction was measured directly for the first time using a FRET-based assay in which the E. coli gamma complex clamp loader (gamma3deltadelta'chipsi) was labeled with a fluorescent donor, and the beta-clamp was labeled with a nonfluorescent quencher. When a beta.gamma complex is added to DNA, there is a significant time lag before the clamp is released onto DNA. To establish what events take place during this time lag, the timing of clamp release was compared to the timing of DNA binding and ATP hydrolysis by measuring these reactions directly side-by-side in assays. DNA binding is relatively rapid and triggers the hydrolysis of ATP. Both events occur prior to clamp release. Interestingly, the temporal correlation data and simple modeling studies indicate that the clamp loader releases DNA prior to the clamp and that DNA release may be coupled to clamp closing. Clamp release is relatively slow and likely to be the rate-limiting step in the overall clamp loading reaction cycle.

In situ human telomerase reverse transcriptase expression pattern in normal and neoplastic ovarian tissues.

Telomerase is a ribonuclear protein reverse transcriptase that maintains telomere length in eukaryotic cells. Activation of telomerase has been implicated in human cellular immortalization and carcinogenesis. Telomerase activity in ovarian neoplasm has been studied using polymerase chain reaction (PCR)-based methods and shown to be correlated with malignancy. However, we believe those results must be interpreted with caution because such studies used a heterogeneous mix of cells, including normal cell type known to express telomerase when activated. The present study used in situ hybridization that allows determination of the type of cells expressing telomerase, as well as the intensity of that expression, in ovarian neoplasms. A total of 75 specimens were studied. Epithelial telomerase reverse transcriptase mRNA expression was detected in 28 of 31 epithelial ovarian carcinomas, 1 of 1 malignant granulosa cell tumor, 7 of 9 serous borderline ovarian tumors, 11 of 11 mucinous borderline ovarian tumors, 4 of 5 serous cystadenofibromas, 2 of 4 serous cystadenomas, 8 of 8 mucinous cystadenomas, and 0 of 6 normal ovaries except the corpus luteum. Telomerase expression is heterogeneously found in both benign and malignant epithelial tissues. We conclude that human telomerase reverse transcriptase mRNA expression does not seem to be a reliable marker for clinical use in differentiating between benign and malignant tumors.