Intravoxel Incoherent Motion Diffusion Weighted MR Imaging at 3.0 T: Assessment of Steatohepatitis and Fibrosis Compared with Liver Biopsy in Type 2 Diabetic Patients

ObjectiveTo evaluate the capability of intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) to assess steatohepatitis and fibrosis determined by histopathology in type 2 diabetic patients.MethodsFifty-nine type 2 diabetic patients (49 women, 10 men; mean age, 54 ± 9 years) were submitted to liver biopsy for the evaluation of non-alcoholic fatty liver disease (NAFLD) and underwent DWI on a 3.0T MR system using 10 b values. Institutional approval and patient consent were obtained. Pure molecular-based (D), perfusion-related (D*), and vascular fraction (f) were calculated using a double exponential model and least squares curve fitting. D, D*, and f were compared between patients with and without steatohepatitis and between patients with and without fibrosis. The variables were compared by using the Ranksum test and Student t-test.ResultsSteatohepatitis was observed in 22 patients and fibrosis in 16 patients. A lower D median (0.70 s/mm² vs. 0.83 s/mm², p<0.05) and a lower D* median (34.39 s/mm² vs. 45.23 s/mm², p<0.05) were observed among those with steatohepatitis. A lower D median (0.70 s/mm² vs. 0.82 s/mm², p<0.05) and a lower D* median (35.01 s/mm² vs. 44.76 s/mm², p=0.05) were also observed among those with fibrosis.ConclusionIVIM-DWI has the potential to aid in the characterization of steatohepatitis and fibrosis.     

Supramolecular interaction of 18‐crown‐6 ether with mesalazine and spectrofluorimetric determination of mesalazine in pharmaceutical formulations

The supramolecular interaction of protonated mesalazine (MSZ) and 18‐crown‐6 ether (18C6) has been examined by Ultraviolet–visible, FT‐IR and fluorescence spectroscopy. The formation of the inclusion complex has been confirmed based on the changes of the spectral properties. The MSZ–18C6 host–guest complex formed in (1:1) stoichiometry and the inclusion constant (K = 1.411 × 10² L mol^–1) was ascertained by the typical double reciprocal plots. Furthermore, the thermodynamic parameters (ΔG°, ΔH° and ΔS°) of (MSZ‐18C6) were obtained. Based on the remarkable enhancement of the fluorescence intensity of MSZ produced through complex formation, a simple, accurate, rapid and highly sensitive spectrofluorometric method for the determination of MSZ in aqueous solution in the presence of 18C6 was developed. The measurement of relative fluorescence intensity was carried with excitation at 298 nm, emission 410 nm. All variables affecting the reactions were studied and optimized. Beer's law was obeyed in the concentration range of 0.1–0.9 µg/mL. The absorbance was found to increase linearly with increasing concentration of MSZ. The molar absorptivity, Sandell sensitivity, limit of detection (LOD) and limit of quantification (LOQ) were calculated. The validity of the described method was assessed, and the method was successfully applied to the determination of MSZ in its pharmaceutical formulation. In addition, a solid inclusion complex was synthesized by the coprecipitation method. Copyright © 2015 John Wiley & Sons, Ltd.     

Macrophage autophagy protects against liver fibrosis in mice

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5^fl/fl LysM-Cre mice, referred to as atg5^−/−) and their wild-type (Atg5^fl/fl, referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5^−/− mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5^−/− mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5^−/− mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5^−/− mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5^−/− macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5^−/− mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.     

Lactate Up-Regulates the Expression of Lactate Oxidation Complex-Related Genes in Left Ventricular Cardiac Tissue of Rats

Background

Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex.

Methods/Principal Findings

Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O₂ ^●-/H₂O₂) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O₂ ^●-/H₂O₂ levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O₂ ^●-/H₂O₂.

Conclusions

Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O₂ ^●-/H₂O₂ driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle.     

Child Centred Approach to Climate Change and Health Adaptation through Schools in Bangladesh: A Cluster Randomised Intervention Trial

Background

Bangladesh is one of the most vulnerable countries to climate change. People are getting educated at different levels on how to deal with potential impacts. One such educational mode was the preparation of a school manual, for high school students on climate change and health protection endorsed by the National Curriculum and Textbook Board, which is based on a 2008 World Health Organization manual. The objective of this study was to test the effectiveness of the manual in increasing the knowledge level of the school children about climate change and health adaptation.

Methods

This cluster randomized intervention trial involved 60 schools throughout Bangladesh, with 3293 secondary school students participating. School upazilas (sub-districts) were randomised into intervention and control groups, and two schools from each upazila were randomly selected. All year seven students from both groups of schools sat for a pre-test of 30 short questions of binary response. A total of 1515 students from 30 intervention schools received the intervention through classroom training based on the school manual and 1778 students of the 30 control schools did not get the manual but a leaflet on climate change and health issues. Six months later, a post-intervention test of the same questionnaire used in the pre-test was performed at both intervention and control schools. The pre and post test scores were analysed along with the demographic data by using random effects model.

Results

None of the various school level and student level variables were significantly different between the control and intervention group. However, the intervention group had a 17.42% (95% CI: 14.45 to 20.38, P = <0.001) higher score in the post-test after adjusting for pre-test score and other covariates in a multi-level linear regression model.

Conclusions

These results suggest that school-based intervention for climate change and health adaptation is effective for increasing the knowledge level of school children on this topic.     

Alcohol Induced Alterations to the Human Fecal VOC Metabolome

Studies have shown that excessive alcohol consumption impacts the intestinal microbiota composition, causing disruption of homeostasis (dysbiosis). However, this observed change is not indicative of the dysbiotic intestinal microbiota function that could result in the production of injurious and toxic products. Thus, knowledge of the effects of alcohol on the intestinal microbiota function and their metabolites is warranted, in order to better understand the role of the intestinal microbiota in alcohol associated organ failure. Here, we report the results of a differential metabolomic analysis comparing volatile organic compounds (VOC) detected in the stool of alcoholics and non-alcoholic healthy controls. We performed the analysis with fecal samples collected after passage as well as with samples collected directly from the sigmoid lumen. Regardless of the approach to fecal collection, we found a stool VOC metabolomic signature in alcoholics that is different from healthy controls. The most notable metabolite alterations in the alcoholic samples include: (1) an elevation in the oxidative stress biomarker tetradecane; (2) a decrease in five fatty alcohols with anti-oxidant property; (3) a decrease in the short chain fatty acids propionate and isobutyrate, important in maintaining intestinal epithelial cell health and barrier integrity; (4) a decrease in alcohol consumption natural suppressant caryophyllene; (5) a decrease in natural product and hepatic steatosis attenuator camphene; and (6) decreased dimethyl disulfide and dimethyl trisulfide, microbial products of decomposition. Our results showed that intestinal microbiota function is altered in alcoholics which might promote alcohol associated pathologies.     

High Prevalence of Severe Food Insecurity and Malnutrition among HIV-Infected Adults in Senegal,West Africa

Background

Malnutrition and food insecurity are associated with increased mortality and poor clinical outcomes among people living with HIV/AIDS; however, the prevalence of malnutrition and food insecurity among people living with HIV/AIDS in Senegal, West Africa is unknown. The objective of this study was to determine the prevalence and severity of food insecurity and malnutrition among HIV-infected adults in Senegal, and to identify associations between food insecurity, malnutrition, and HIV outcomes.

Methods

We conducted a cross-sectional study at outpatient clinics in Dakar and Ziguinchor, Senegal. Data were collected using participant interviews, anthropometry, the Household Food Insecurity Access Scale, the Individual Dietary Diversity Scale, and chart review.

Results

One hundred and nine HIV-1 and/or HIV-2 participants were enrolled. The prevalence of food insecurity was 84.6% in Dakar and 89.5% in Ziguinchor. The prevalence of severe food insecurity was 59.6% in Dakar and 75.4% in Ziguinchor. The prevalence of malnutrition (BMI <18.5) was 19.2% in Dakar and 26.3% in Ziguinchor. Severe food insecurity was associated with missing clinic appointments (p = 0.01) and not taking antiretroviral therapy due to hunger (p = 0.02). Malnutrition was associated with lower CD4 cell counts (p = 0.01).

Conclusions

Severe food insecurity and malnutrition are highly prevalent among HIV-infected adults in both Dakar and Ziguinchor, and are associated with poor HIV outcomes. Our findings warrant further studies to determine the root causes of malnutrition and food insecurity in Senegal, and the short- and long-term impacts of malnutrition and food insecurity on HIV care. Urgent interventions are needed to address the unacceptably high rates of malnutrition and food insecurity in this population.     

More stable structure of wheat germ lipase at low pH than its native state

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0–8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.     

Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural,functional and pharmacological approaches

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ß-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme–FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer–monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach.