A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity

Karyopherin-β2 (Kapβ2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapβ2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapβ2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapβ2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapβ2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapβ2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapβ2 recapitulate all salient features of Kapβ2 activity. By contrast, Kapβ2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapβ2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.     

Hypocretin release in normal and narcoleptic dogs after food and sleep deprivation,eating, and movement

Hypocretins (orexins) are recently discovered hypothalamic neuropeptides that have been implicated in the etiology of narcolepsy. The normal behavioral functions of these peptides are unclear, although a role in feeding has been suggested. We measured hypocretin-1 (Hcrt-1) in the cerebrospinal fluid of dogs during a variety of behaviors. We found that 48 h without food (24 h beyond normal 24-h fasting period) produced no significant change in Hcrt-1 levels nor did feeding after the deprivation. In contrast, 24 h of sleep deprivation produced on average a 70% increase in Hcrt-1 level compared with baseline levels. The amount of increase was correlated with the level of motor activity during the sleep-deprivation procedure. A 2-h period of exercise in the same dogs produced a 57% increase in Hcrt-1 levels relative to quiet waking levels, with the magnitude of the increase being highly correlated with the level of motor activity. The strong correlation between motor activity and Hcrt-1 release may explain some of the previously reported behavioral, physiological, and pathological phenomena ascribed to the Hcrt system.     

Neuroprotective effects of tanshinones in transient focal cerebral ischemia in mice.

Tanshinones are the major lipid soluble pharmacological constituents of Danshen, the dried roots of Salvia miltiorrhiza Bunge (Labiatae), a well known traditional Chinese medicine used for the treatment of cerebrovascular diseases including stroke. Potential neuroprotective effects of tanshinones IIA (TsIIA) and IIB (TsIIB) were examined in adult mice subjected to transient focal cerebral ischemia caused by middle cerebral artery occlusion (MCAo). Our results revealed that TsIIA (16 mg/kg) readily penetrated the blood brain barrier reaching a peak concentration of 0.41 nmol/g brain wet weight 60 minutes after intraperitoneal injection and decreased slowly over several hours. Twenty-four hours after middle cerebral artery occlusion, brain infarct volume was reduced by 30% and 37% following treatment with TsIIA and TsIIB, respectively. The reduction in brain infarct volume was accompanied by a significant decrease in the observed neurological deficit. Tanshinones or other structurally related compounds may have potential for further development as neuroprotective drugs.     

Impact of Obesity and Body Fat Distribution on Cardiovascular Risk Factors in Hong Kong Chinese

Objective: Body fat distribution has been reported to differentially contribute to the development of cardiovascular risk. We report the relative associations between general and central obesity and risk factors in 2893 Chinese subjects recruited from the Hong Kong population. Research Methods and Procedures: Anthropometric parameters [waist circumference (WC) and BMI], surrogate measures of insulin resistance (fasting plasma glucose and insulin, oral glucose tolerance test, 2 hours glucose and insulin), fasting lipids (total, low‐density lipoprotein‐cholesterol, high‐density lipoprotein‐cholesterol, and triglycerides) and systolic and diastolic blood pressure were measured. General obesity was classified as BMI ≥25.0 kg/m² and central obesity as a WC ≥80 or ≥90 cm in women and men, respectively. Results: A total of 39.2% of the population was found to be obese. Obesity per se increased the levels of the risk factors, but central adiposity contributed to a greater extent to adverse high‐density lipoprotein‐cholesterol, triglyceride, and insulin resistance levels. There was a continuous relationship between increasing obesity, both general and central, and cardiovascular risk, with lowest risk associated with the lowest indices of obesity. In the 1759 nonobese subjects divided into quartiles of BMI or WC, the levels of the cardiovascular risk factors still significantly increased with increasing quartiles of adiposity. Discussion: Central adiposity appears to contribute to a greater extent than general adiposity to the development of cardiovascular risk in this population. The relationship between obesity parameters and risk is a continuum, with risk factors significantly increasing even at levels usually considered nonobese. These observations support the proposed redefinition of overweight and obesity in Asian populations using lower cut‐off points.     

Nuclear morphometry as a biomarker for bronchial intraepithelial neoplasia: correlation with genetic damage and cancer development.

BACKGROUND: Bronchial carcinomas are preceded by epithelial morphologic changes. The variation in interpretation of these grades of intraepithelial neoplasia makes it difficult to determine its natural history and utility of histopathology as a surrogate endpoint biomarker. The objective of this study was to quantitate morphologic changes of intraepitherlial neoplasia and validate its utility through correlation with histopathology, allelic loss, and cancer development. METHODS: Quantitative nuclear morphometry was performed on 47 normal bronchial biopsies and 28 invasive cancer to generate a morphometry index (MI) that was applied to 1,096 bronchial biopsies from 230 volunteers who were current smokers (> or =25 pack-years) and 30 patients who had cancer. In a subset of 631 biopsies, MI was correlated with frequency of loss of heterozygosity at nine chromosomal regions (14 polymorphic markers). RESULTS: A significant correlation was found between MI and allelic loss in six of nine chromosomal regions. As part of patient long-term follow-up, 12 sites that progressed to cancer were identified and had significantly increased MIs relative to nonprogressing sites. Significant overlap in the MIs was found between most grades of intraepithelial neoplasia. CONCLUSIONS: In chemoprevention trials, nuclear morphometry can supplement histopathology as a Surrogate Endpont Biomarker (SEB) because it is quantitative, collelates well with genetic damage, and may predict cancer development.     

Molecular basis of secretin docking to its intact receptor using multiple photolabile probes distributed throughout the pharmacophore

The molecular basis of ligand binding and activation of family B G protein-coupled receptors is not yet clear due to the lack of insight into the structure of intact receptors. Although NMR and crystal structures of amino-terminal domains of several family members support consistency in general structural motifs that include a peptide-binding cleft, there are variations in the details of docking of the carboxyl terminus of peptide ligands within this cleft, and there is no information about siting of the amino terminus of these peptides. There are also no empirical data to orient the receptor amino terminus relative to the core helical bundle domain. Here, we prepared a series of five new probes, incorporating photolabile moieties into positions 2, 15, 20, 24, and 25 of full agonist secretin analogues. Each bound specifically to the receptor and covalently labeled single distinct receptor residues. Peptide mapping of labeled wild-type and mutant receptors identified that the position 15, 20, and 25 probes labeled residues within the distal amino terminus of the receptor, whereas the position 24 probe labeled the amino terminus adjacent to TM1. Of note, the position 2 probe labeled a residue within the first extracellular loop of the receptor, a region not previously labeled, providing an important new constraint for docking the amino-terminal region of secretin to its receptor core. These additional experimentally derived constraints help to refine our understanding of the structure of the secretin-intact receptor complex and provide new insights into understanding the molecular mechanism for activation of family B G protein-coupled receptors.     

Jejunal nutrient sensing is required for duodenal-jejunal bypass surgery to rapidly lower glucose concentrations in uncontrolled diabetes

Gastrointestinal bypass surgeries restore metabolic homeostasis in patients with type 2 diabetes and obesity(1), but the underlying mechanisms remain elusive. Duodenal-jejunal bypass surgery (DJB), an experimental surgical technique that excludes the duodenum and proximal jejunum from nutrient transit(1,2), lowers glucose concentrations in nonobese type 2 diabetic rats(2–5). Given that DJB redirects and enhances nutrient flow into the jejunum and that jejunal nutrient sensing affects feeding(6,7), the repositioned jejunum after DJB represents a junction at which nutrients could regulate glucose homeostasis. Here we found that intrajejunal nutrient administration lowered endogenous glucose production in normal rats through a gut-brain-liver network in the presence of basal plasma insulin concentrations. Inhibition of jejunal glucose uptake or formation of long chain fatty acyl-coA negated the metabolic effects of glucose or lipid, respectively, in normal rats, and altered the rapid (2 d) glucose-lowering effect induced by DJB in streptozotocin (STZ)-induced uncontrolled diabetic rats during refeeding. Lastly, in insulin-deficient autoimmune type 1 diabetic rats and STZ-induced diabetic rats, DJB lowered glucose concentrations in 2 d independently of changes in plasma insulin concentrations, food intake and body weight. These data unveil a glucoregulatory role of jejunal nutrient sensing and its relevance in the early improvement of glycemic control after DJB in rat models of uncontrolled diabetes.     

Molecular Characterization of the Fecal Microbiota in Patients with Nonalcoholic Steatohepatitis – A Longitudinal Study

Background

The human gut microbiota has profound influence on host metabolism and immunity. This study characterized the fecal microbiota in patients with nonalcoholic steatohepatitis (NASH). The relationship between microbiota changes and changes in hepatic steatosis was also studied.

Methods

Fecal microbiota of histology-proven NASH patients and healthy controls was analyzed by 16S ribosomal RNA pyrosequencing. NASH patients were from a previously reported randomized trial on probiotic treatment. Proton-magnetic resonance spectroscopy was performed to monitor changes in intrahepatic triglyceride content (IHTG).

Results

A total of 420,344 16S sequences with acceptable quality were obtained from 16 NASH patients and 22 controls. NASH patients had lower fecal abundance of Faecalibacterium and Anaerosporobacter but higher abundance of Parabacteroides and Allisonella. Partial least-square discriminant analysis yielded a model of 10 genera that discriminated NASH patients from controls. At month 6, 6 of 7 patients in the probiotic group and 4 of 9 patients in the usual care group had improvement in IHTG (P = 0.15). Improvement in IHTG was associated with a reduction in the abundance of Firmicutes (R² = 0.4820, P = 0.0028) and increase in Bacteroidetes (R² = 0.4366, P = 0.0053). This was accompanied by corresponding changes at the class, order and genus levels. In contrast, bacterial biodiversity did not differ between NASH patients and controls, and did not change with probiotic treatment.

Conclusions

NASH patients have fecal dysbiosis, and changes in microbiota correlate with improvement in hepatic steatosis. Further studies are required to investigate the mechanism underlying the interaction between gut microbes and the liver.