Enhanced Lipid Oxidation and Maintenance of Muscle Insulin Sensitivity Despite Glucose Intolerance in a Diet-Induced Obesity Mouse Model

Background

Diet-induced obesity is a rising health concern which can lead to the development of glucose intolerance and muscle insulin resistance and, ultimately, type II diabetes mellitus. This research investigates the associations between glucose intolerance or muscle insulin resistance and tissue specific changes during the progression of diet-induced obesity.

Methodology

C57BL/6J mice were fed a normal or high-fat diet (HFD; 60% kcal fat) for 3 or 8 weeks. Disease progression was monitored by measurements of body/tissue mass changes, glucose and insulin tolerance tests, and ex vivo glucose uptake in intact muscles. Lipid metabolism was analyzed using metabolic chambers and ex vivo palmitate assays in intact muscles. Skeletal muscle, liver and adipose tissues were analyzed for changes in inflammatory gene expression. Plasma was analyzed for insulin levels and inflammatory proteins. Histological techniques were used on muscle and liver cryosections to assess metabolic and morphological changes.

Principal Findings/Conclusions

A rapid shift in whole body metabolism towards lipids was observed with HFD. Following 3 weeks of HFD, elevated total lipid oxidation and an oxidative fiber type shift had occurred in the skeletal muscle, which we propose was responsible for delaying intramyocellular lipid accumulation and maintaining muscle’s insulin sensitivity. Glucose intolerance was present after three weeks of HFD and was associated with an enlarged adipose tissue depot, adipose tissue inflammation and excess hepatic lipids, but not hepatic inflammation. Furthermore, HFD did not significantly increase systemic or muscle inflammation after 3 or 8 weeks of HFD suggesting that early diet-induced obesity does not cause inflammation throughout the whole body. Overall these findings indicate skeletal muscle did not contribute to the development of HFD-induced impairments in whole-body glucose tolerance following 3 weeks of HFD.     

In Vitro Culture of Previously Uncultured Oral Bacterial Phylotypes

Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.     

Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/Principal Findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/Significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.     

Full modeling versus summarizing gene-tree uncertainty: Method choice and species-tree accuracy

With the proliferation of species-tree methods, empiricists now have to confront the daunting task of method choice. Such decisions might be made based on theoretical considerations alone. However, the messiness of real data means that theoretical ideals may not hold in practice (e.g., with convergence of complicated MCMC algorithms and computational times that limit analyses to small data sets). On the other hand, simplifying assumptions made by some approaches may compromise the accuracy of species-tree estimates. Here we examine the purported tradeoff between accuracy and computational simplicity for species-tree analysis, focusing on the different ways the approaches treat gene-tree uncertainty. By considering a diversity of species trees, as well as different sampling designs and total sampling efforts, we not only compare the accuracy of species-tree estimates across methods, but we also partition the variation in accuracy across factors to identify their relative importance. This analysis shows that although the method of analysis affects accuracy, other factors - namely, the history of species divergence and aspects of the sampling design - have a larger impact. Despite a full modeling of gene tree uncertainty (e.g., using a Bayesian framework), species-tree estimates may not be accurate, particularly for recent diversification histories. Nevertheless, we demonstrate how factors within the control of the empirical investigator (e.g., decisions about sampling) improve the accuracy of species tree estimates, and more so than the method of analysis. Lastly, with much of the attention on species-tree analyses focused on the discord among loci arising from the coalescent, this work also highlights a previously overlooked key determinant of species-tree accuracy for recent divergences - the level of genetic variation at a locus, which has important implications for improving species-tree estimates in practice.     

Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase

The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium. Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V. These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode. The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process. Cytochrome c nitrite reductase clearly interacts differently with these two substrates. However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration. These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.     

Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor

Aberrant control of cyclin-dependent kinases (CDKs) is a central feature of the molecular pathology of cancer. Iterative structure-based design was used to optimize the ATP- competitive inhibition of CDK1 and CDK2 by O(6)-cyclohexylmethylguanines, resulting in O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino)purine. The new inhibitor is 1,000-fold more potent than the parent compound (K(i) values for CDK1 = 9 nM and CDK2 = 6 nM versus 5,000 nM and 12,000 nM, respectively, for O(6)-cyclohexylmethylguanine). The increased potency arises primarily from the formation of two additional hydrogen bonds between the inhibitor and Asp 86 of CDK2, which facilitate optimum hydrophobic packing of the anilino group with the specificity surface of CDK2. Cellular studies with O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino) purine demonstrated inhibition of MCF-7 cell growth and target protein phosphorylation, consistent with CDK1 and CDK2 inhibition. The work represents the first successful iterative synthesis of a potent CDK inhibitor based on the structure of fully activated CDK2-cyclin A. Furthermore, the potency of O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino)purine was both predicted and fully rationalized on the basis of protein-ligand interactions.     

The isolation and characterization of putative mesenchymal stem cells from the spiny mouse

The bone marrow represents the most common source from which to isolate mesenchymal stem cells (MSCs). MSCs are capable of differentiating into tissues of the three primary lineages and have the potential to enhance repair in damaged organs through the principals of regenerative medicine. Given the ease with which MSCs may be isolated from different species the aim of this study was to isolate and characterize putative bone marrow derived MSCs from the spiny mouse, Acomys cahirinus. MSCs were isolated from the spiny mouse in a traditional manner, and based on plastic adherence, morphology, colony forming unit-fibroblast assays and functional assessment (adipogenic, osteogenic and chondrogenic differentiation potential) a population of putative mesenchymal stem cells from the compact bone of the spiny mouse have been isolated and characterized. Such methodological approaches overcome the lack of species-specific antibodies for the spiny mouse and could be employed for other species where the cost of generating species-specific antibodies is not warranted.     

Immune suppression blocks sodium-sensitive hypertension following recovery from ischemic acute renal failure

The present study determined the effect of immune suppression with mycophenolate mofetil (MMF) on sodium-sensitive hypertension following recovery from ischemia reperfusion (I/R)-induced acute renal failure. Male Sprague-Dawley rats fed 0.4% NaCl chow were subjected to 40 min bilateral I/R or control sham surgery. After 35 days of recovery, when plasma creatinine levels had returned to normal, the rats were switched to 4.0% NaCl chow for 28 days and administered vehicle or MMF (20 mg.kg(-1).day(-1) ip). High-salt mean arterial pressure was significantly higher in I/R rats (144 +/- 16 mmHg) compared with vehicle-treated sham rats (122 +/- 2 mmHg). Treatment of I/R rats with MMF during the period of high salt intake prevented the salt-induced increase in arterial pressure (114 +/- 3 mmHg). Conscious creatinine clearance was lower in I/R rats (0.27 +/- 0.07 ml.min(-1).100 g body wt(-1)) compared with vehicle-treated sham rats (0.58 +/- 0.04 ml.min(-1).100 g body wt(-1)); MMF treatment prevented the decrease in creatinine clearance in I/R rats (0.64 +/- 0.07 ml.min(-1).100 g body wt(-1)). I/R injury also significantly increased glomerular tissue damage and increased the presence of ED-1 positive (macrophages) and S100A4 positive cells (fibroblasts) in the renal interstitium. The I/R rats treated with MMF exhibited a significant reduction in infiltrating macrophages and fibroblasts and decreased histological damage. The present data indicate that infiltrating immune cells mediate or participate in the development of sodium-sensitive hypertension and renal damage in rats apparently recovered from renal I/R injury.     

Vision-Related Quality of Life and Appearance Concerns Are Associated with Anxiety and Depression after Eye Enucleation: A Cross-Sectional Study

Aims

To investigate the association of demographic, clinical and psychosocial variables with levels of anxiety and depression in participants wearing an ocular prosthesis after eye enucleation.

Methods

This cross-sectional study included 195 participants with an enucleated eye who were attending an ophthalmic clinic for prosthetic rehabilitation between July and November 2014. Demographic and clinical data, and self-reported feelings of shame, sadness and anger were collected. Participants also completed the National Eye Institute Visual Function Questionnaire, the Facial Appearance subscale of the Negative Physical Self Scale, and the Hospital Anxiety and Depression Scale. Regression models were used to identify the factors associated with anxiety and depression.

Results

The proportion of participants with clinical anxiety was 11.8% and clinical depression 13.8%. More anxiety and depression were associated with poorer vision-related quality of life and greater levels of appearance concerns. Younger age was related to greater levels of anxiety. Less educated participants and those feeling more angry about losing an eye are more prone to experience depression. Clinical variables were unrelated to anxiety or depression.

Conclusions

Anxiety and depression are more prevalent in eye-enucleated patients than the general population, which brings up the issues of psychiatric support in these patients. Psychosocial rather than clinical characteristics were associated with anxiety and depression. Longitudinal studies need to be conducted to further elucidate the direction of causality before interventions to improve mood states are developed.     

De Novo Loss-of-Function Mutations in SETD5, Encoding a Methyltransferase in a 3p25 Microdeletion Syndrome Critical Region,Cause Intellectual Disability

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations—four nonsense (c.1195A>T [p.Lys399^∗], c.1333C>T [p.Arg445^∗], c.1866C>G [p.Tyr622^∗], and c.3001C>T [p.Arg1001^∗]) and three frameshift (c.2177_2178del [p.Thr726Asnfs^∗39], c.3771dup [p.Ser1258Glufs^∗65], and c.3856del [p.Ser1286Leufs^∗84])—were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.