Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing

Introduction

Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing.

Methods

We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99^th percentile and 176 lean adults (BMI ≤ 15^th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults.

Results and Conclusion

We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (p_TDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.     

Metabolite targeting: development of a comprehensive targeted metabolomics platform for the assessment of diabetes and its complications

Biomarker studies for metabolic disorders like diabetes mellitus (DM) are an important approach towards a better understanding of the underlying pathophysiological mechanisms of diseases (Roberts and Gerszten in Cell Metab 18:43–50, 2013; Wilson et al. in Proteome Res 4:591–598, 2005). Furthermore, screening of potential metabolic biomarkers opens the opportunity of early diagnosis as well as therapy and drug monitoring of metabolic disorders (Rhee et al. in J Clin Invest 10:1–10, 2011; Wang et al. in Nat Med 17:448–458, 2011; Wenk in Nat Rev Drug Discov 4:594–610, 2005). The aim of the present study was to develop methods for the quantitative determination of 74 potential metabolite biomarkers for DM and diabetic nephropathy (DN) in serum. Several studies have shown that the concentrations of many polar metabolites like amino or organic acids are changed in subjects suffering from diabetes (Wang et al. in Nat Med 17:448–458, 2011; Yuan et al. in J Chromatogr B 813:53–58, 2007). Analyzing polar analytes presents a challenge in liquid chromatography (LC) coupled with ESI–MS/MS (Gika et al. in J Sep Sci 31:1598–1608, 2008; Spagou et al. in J Sep Sci 33:716–727, 2010). Considering those reasons we decided to develop a specific HILIC–ESI–QqQ–MS/MS-method for quantitative determination of these polar metabolites. A subsequent method validation was carried out for both HILIC and RP chromatography with respect to the guidelines of the Food and Drug Administration (FDA in Food and Drug Administration: Guidance for industry, bioanalytical method validation, 2001). The HILIC and RP LC–MS methods were successfully validated. Furthermore, the HILIC method presented here was applied to serum samples of GIPR^dn transgenic mice, a diabetic strain developing DN, and non transgenic littermate controls. Significant, diabetes-associated changes were observed for the concentrations of 21 out of 62 metabolites. The new methods described here accurately quantify 74 metabolites known to be regulated in diabetes, allowing for direct comparison between studies and laboratories. Thus, these methods may be highly adoptable in clinical research, providing a starting point for early diagnosis and metabolic screening.     

BcSAK1, a stress-activated mitogen-activated protein kinase, is involved in vegetative differentiation and pathogenicity in Botrytis cinerea

The gene bcsak1, encoding a mitogen-activated protein kinase (MAPK) of Botrytis cinerea, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H(2)O(2) and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, Deltabcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in B. cinerea the stress-activated MAPK cascade is involved in essential differentiation programs.     

Minireview: Recent progress in hemocyanin research

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-? structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.     

Immune Evasion,Stress Resistance,and Efficient Nutrient Acquisition Are Crucial for Intracellular Survival of Candida glabrata within Macrophages

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes'' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.     

Loss of Prolyl Carboxypeptidase in Two-Kidney,One-Clip Goldblatt Hypertensive Mice

It is well documented that angiotensin (Ang) II contributes to kidney disease progression. The protease prolyl carboxypeptidase (PRCP) is highly expressed in the kidney and may be renoprotective by degrading Ang II to Ang-(1-7). The aim of the study was to investigate whether renal PRCP protein expression and activity are altered in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice. Left renal artery was constricted by using 0.12 mm silver clips. Blood pressure was measured using telemetry over the eleven weeks of study period and revealed an immediate increase in 2K1C animals during the first week of clip placement which was followed by a gradual decrease to baseline blood pressure. Similarly, urinary albumin excretion was significantly increased one week after 2K1C and returned to baseline levels during the following weeks. At 2 weeks and at the end of the study, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion and renal fibrosis. Renal PRCP expression and activity were significantly reduced in clipped kidneys. Immunofluorescence revealed the loss of renal tubular PRCP but not glomerular PRCP. In contrast, expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected, while angiotensin converting enzyme was elevated in unclipped kidneys and renin was increased in clipped kidneys. Results suggest that PRCP is suppressed in 2K1C and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PRCP.     

Extinction pattern of Alpine cave bears - new data and climatological interpretation

ABSTRACT

Cave bears have disappeared from the Alps from different altitudes at different times. The temporal progression of the HDEL (Height Dependent Extinction Line) – a compilation of the geologically most recent radiocarbon dates per altitude level – is not consistent with the general cooling of the temperatures from about 45 ka BP. The cave bear sites of the Northern Alps with the most recent radiocarbon ages are not situated in the lowlands but in caves in altitudes of 1,500 m to 1,700 m above sea level (a.s.l.).

Cave bears fed almost exclusively on herbs and leaves. It was assumed that with the general cooling in the OIS 3 since about 45 ka BP also the migration of the alpine elements into the lowlands took place. It could be recognized that the populations in the lower situated cave bear site became earlier extinct than the cave bear population in the higher altitudes.

With new radiocarbon dates, done at the Curt-Engelhorn-Center Archaeometry at the Reiss-Engelhorn-Museen in Mannheim (Germany), the HDEL can be determined much more precisely and the causes of gradual extinction are also better understood.     

Identification of a major recombination hotspot in patients with short stature and SHOX deficiency

Human growth is influenced not only by environmental and internal factors but also by a large number of different genes. One of these genes, SHOX, is believed to play a major role in growth, since defects in this homeobox-containing gene on the sex chromosomes lead to syndromal short stature (Leri-Weill dyschondrosteosis, Langer mesomelic dysplasia, and Turner syndrome) as well as to idiopathic short stature. We have analyzed 118 unrelated patients with Leri-Weill dyschondrosteosis and >1,500 patients with idiopathic short stature for deletions encompassing SHOX. Deletions were detected in 34% of the patients with Leri-Weill dyschondrosteosis and in 2% of the patients with idiopathic short stature. For 27 patients with Leri-Weill dyschondrosteosis and for 6 with idiopathic short stature, detailed deletion mapping was performed. Analysis was performed by polymerase chain reaction with the use of pseudoautosomal polymorphic markers and by fluorescence in situ hybridization with the use of cosmid clones. Here, we show that, although the identified deletions vary in size, the vast majority (73%) of patients tested share a distinct proximal deletion breakpoint. We propose that the sequence present within this proximal deletion breakpoint "hotspot" region predisposes to recurrent breaks.     

SIRT1 and c-Myc Promote Liver Tumor Cell Survival and Predict Poor Survival of Human Hepatocellular Carcinomas

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.