RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.     

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants

Background

Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.

Methodology/Principal Findings

a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10⁻⁷, corrected p = 0.0494; odds ratio (OR)_CT 1.67, 95% confidence interval (CI) 1.22–2.27; OR_TT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.

Conclusions/Significance

Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.     

Occurrence of rhizobia in the gut of the higher termite Nasutitermes nigriceps

Wood-eating termites feed on a diet highly deficient in nitrogen. They must complement their diet with the aid of nitrogen-fixing bacteria. Nitrogen fixation in the gut has been demonstrated, but information about nitrogen-fixing bacteria in pure culture is scarce. From the higher termite Nasutitermes nigriceps the symbiotic bacterial strain M3A was isolated, which thrives in the hindgut contents. The Gram-negative strain exhibited similarities to the species of the genus Ensifer (including Sinorhizobium) on the basis of morphological and physiological/biochemical features. The 16S rRNA gene analysis showed the highest sequence similarity of the isolate M3A to Ensifer adhaerens (>99%; ATCC 33499). The DNA-DNA hybridization revealed a similarity of 66% with E. adhaerens (NCIMB12342(T)). In contrast to the type strain the isolate M3A possesses the capacity to nodulate plant roots. This is the first report on the detailed identification of a rhizobia-related strain from the intestinal tract of animals. Strain M3A has been deposited with two culture collections (DSM10169; ATCC BAA-396).     

Variability in diapause duration in the chestnut weevil: mixed ESS,genetic polymorphism or bet‐hedging?

Within-generation variability in diapause duration can be viewed either as a mixed Evolutionary Stable Strategy (ESS), a genetic polymorphism of pure strategies, or as bet-hedging. Diapause variability expressed by a single genotype that maximizes mean geometric fitness at the cost of mean arithmetic fitness is a bet-hedging strategy. Bet-hedging differs from mixed ESS and stable genetic polymorphism of pure strategies because in these latter the expected pay-offs for all phenotypes are equal. In insects, individuals with a prolonged diapause (long cycle) lose at least one reproductive opportunity and suffer lower survival before reproduction than those with a short diapause (short cycle). If long-cycle individuals compensate this cost by better adult performance, the compensation leads to a trade-off which could result in mixed ESS or genetic polymorphism of pure strategies since the overall fitness of the two morphs may be similar. In this paper, we show that in the chestnut weevil Curculio elephas adult performance, measured as sex ratio, longevity, weight, and realized fecundity of females, are similar in individuals emerged after one and two years. Long-cycle morphs emerge slightly before short-cycle ones but this eventual advantage for fertility probably does not compensate higher larval mortality and missed reproductive opportunity in long-cycle phenotypes. Therefore, the cost associated with prolonged diapause cannot be completely compensated for by a better adult performance. From these results, and previous data, we conclude that variability in diapause duration cycle is better explained as bet-hedging than mixed ESS or genetic polymorphism of pure strategies.     

Synthesis, molecular structure, substitution and C-C coupling reactions of ruthenium complexes containing (η-C9H7)Ru(PPh3) as a molecular unit

The 2-methallyl complex [(η⁵-C₉H₇)Ru(η³-2-MeC₃H₄)(PPh₃)] (3), prepared from [(η⁵-C₉H₇)Ru(PPh₃)₂Cl] (2) and 2-MeC₃H₄MgCl, reacts with HX (X = Cl, CF₃CO₂) in the presence of ethene to give the chiral-at-metal compounds [(η⁵-C₉H₇)Ru(C₂H₄)(PPh₃)X] (4, 5) in nearly quantitative yields. Treatment of 2 with AgPF₆ and ethene affords [(η⁵-C₉H₇)Ru(C₂H₄)(PPh₃)₂]PF₆ (6), which reacts with acetone to give the substitution product [(η⁵-C₉H₇)Ru(OCMe₂)(PPh₃)₂]PF₆ (7). The molecular structure of 7 has been determined crystallographically. Whereas treatment of 4 with CH(CO₂Et)N₂ yields the olefin complex [(η⁵-C₉H₇)Ru{η²-(Z)-C₂H₂(CO₂Et)₂}(PPh₃)Cl] (8), the reactions of 4 and 5 with Ph₂CN₂, PhCHN₂ and (Me₃Si)CHN₂ lead to the formation of the carbeneruthenium(II) derivatives [(η⁵-C₉H₇)Ru(CRR′)(PPh₃)Cl] (9-11) and [(η⁵-C₉H₇)Ru(CRR′)(PPh₃)(κ¹-O₂CCF₃)] (12-14), respectively. Treatment of 9 (R = R′ = Ph), 10 (R = H, R′ = Ph) and 11 (R = H, R′ = SiMe₃) with MeLi produces the hydrido(olefin) complexes [(η⁵-C₉H₇)RuH(η²-CH₂CPh₂)(PPh₃)] (15), [(η⁵-C₉H₇)RuH(η²-CH₂CHPh)(PPh₃)] (18a,b) and [(η⁵-C₉H₇)RuH(η²-CH₂CHSiMe₃)(PPh₃)] (19) via C-C coupling and β-hydride shift. The analogous reactions of 11 with PhLi gives the η³-benzyl compound [(η⁵-C₉H₇)Ru{η³-(Me₃Si)CHC₆H₅}(PPh₃)] (20). The η³-allyl complex [(η⁵-C₉H₇)Ru(η³-1-PhC₃H₄)(PPh₃)] (17) was prepared from 10 and CH₂CHMgBr by nucleophilic attack.     

Differential effects of water and saline intake on water deprivation-induced c-Fos staining in the rat

We studied c-Fos staining in adult male rats after 48 h of water deprivation and after 46 h of water deprivation with 2 h of access to water or physiological saline. Controls were allowed ad libitum access to water and physiological saline. For immunocytochemistry, anesthetized rats were perfused with a commercially available antibody for c-Fos. Dehydration significantly increased plasma vasopressin (AVP), osmolality, plasma renin activity (PRA), hematocrit, and sodium concentration and decreased urinary volume. Fos staining was significantly increased in the median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus (SON), and magnocellular and parvocellular paraventricular nucleus (PVN), as well as the area postrema, nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVL). Rehydration with water significantly decreased AVP levels and Fos staining in the SON, PVN, and RVL and significantly increased Fos expression in the perinuclear zone of the SON, NTS, and parabrachial nucleus. Rehydration with water was associated with decreased urinary sodium concentration and hypotonicity, and hematocrit and PRA were comparable to levels seen after dehydration. After rehydration with saline, plasma osmolality, hematocrit, and PRA were not different from control, but plasma AVP and urinary sodium concentration were increased. In the SON, Fos staining was significantly increased, with a great percentage of the Fos cells also stained for oxytocin compared with water deprivation. Changes in Fos staining were also observed in the NTS, RVL, parabrachial nucleus, and PVN. Rehydration with water or saline produces differential effects on plasma AVP, Fos staining, and sodium concentration.     

miR-155 Inhibition Sensitizes CD4+ Th Cells for TREG Mediated Suppression

Background

In humans and mice naturally occurring CD4⁺CD25⁺ regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.

Principal Findings

DNA-Microarray analyses of human as well as murine conventional CD4⁺ Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4⁺ Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4⁺ Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.

Conclusion

Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4⁺ Th cells to nTreg cell-mediated suppression.     

RGD Surface Functionalization of the Hydrophilic Acrylic Intraocular Lens Material to Control Posterior Capsular Opacification

Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including the patient''s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs have shown that the former has more severe PCO. On the other hand, we have previously demonstrated that the adhesion of LECs is favored on hydrophobic compared to hydrophilic materials. By combining these two facts and contemporary knowledge in PCO development via the EMT pathway, we propose a biomimetically inspired strategy to promote LEC adhesion without de-differentiation to reduce the risk of PCO development. By surface grafting of a cell adhesion molecule (RGD peptide) onto the conventional hydrophilic acrylic IOL material, the surface-functionalized IOL can be used to reconstitute a capsule-LEC-IOL sandwich structure, which has been considered to prevent PCO formation in literature. Our results show that the innovative biomaterial improves LEC adhesion, while also exhibiting similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties compared to the starting material. In addition, compared to the hydrophobic IOL material, our bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression, which is the crucial pathway to induce PCO. The in vitro assays suggest that this biomaterial has the potential to reduce the risk factor of PCO development.