Regulation of lipid metabolism by Dicer revealed through SILAC mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems.     

Serodiagnosis of Tuberculosis in Asian Elephants (Elephas maximus) in Southern India: A Latent Class Analysis

Background

Mycobacterium tuberculosis, a causative agent of chronic tuberculosis disease, is widespread among some animal species too. There is paucity of information on the distribution, prevalence and true disease status of tuberculosis in Asian elephants (Elephas maximus). The aim of this study was to estimate the sensitivity and specificity of serological tests to diagnose M. tuberculosis infection in captive elephants in southern India while simultaneously estimating sero-prevalence.

Methodology/Principal Findings

Health assessment of 600 elephants was carried out and their sera screened with a commercially available rapid serum test. Trunk wash culture of select rapid serum test positive animals yielded no animal positive for M. tuberculosis isolation. Under Indian field conditions where the true disease status is unknown, we used a latent class model to estimate the diagnostic characteristics of an existing (rapid serum test) and new (four in-house ELISA) tests. One hundred and seventy nine sera were randomly selected for screening in the five tests. Diagnostic sensitivities of the four ELISAs were 91.3–97.6% (95% Credible Interval (CI): 74.8–99.9) and diagnostic specificity were 89.6–98.5% (95% CI: 79.4–99.9) based on the model we assumed. We estimate that 53.6% (95% CI: 44.6–62.8) of the samples tested were free from infection with M. tuberculosis and 15.9% (97.5% CI: 9.8 - to 24.0) tested positive on all five tests.

Conclusions/Significance

Our results provide evidence for high prevalence of asymptomatic M. tuberculosis infection in Asian elephants in a captive Indian setting. Further validation of these tests would be important in formulating area-specific effective surveillance and control measures.     

Small interfering RNAs that deplete the cellular translation factor eIF4H impede mRNA degradation by the virion host shutoff protein of herpes simplex virus

The herpes simplex virus (HSV) virion host shutoff (Vhs) protein is an endoribonuclease that accelerates decay of many host and viral mRNAs. Purified Vhs does not distinguish mRNAs from nonmessenger RNAs and cuts target RNAs at many sites, yet within infected cells it is targeted to mRNAs and cleaves those mRNAs at preferred sites including, for some, regions of translation initiation. This targeting may result in part from Vhs binding to the translation initiation factor eIF4H; in particular, several mutations in Vhs that abrogate its binding to eIF4H also abolish its mRNA-degradative activity, even though the mutant proteins retain endonuclease activity. To further investigate the role of eIF4H in Vhs activity, HeLa cells were depleted of eIF4H or other proteins by transfection with small interfering RNAs (siRNAs) 48 h prior to infection or mock infection in the presence of actinomycin D. Cellular mRNA levels were then assayed 5 h after infection. In cells transfected with an siRNA for the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase, wild-type HSV infection reduced beta-actin mRNA levels to between 20 and 30% of those in mock-infected cells, indicative of a normal Vhs activity. In contrast, in cells transfected with any of three eIF4H siRNAs, beta-actin mRNA levels were indistinguishable in infected and mock-infected cells, suggesting that eIF4H depletion impeded Vhs-mediated degradation. Depletion of the related factor eIF4B did not affect Vhs activity. The data suggest that eIF4H binding is required for Vhs-induced degradation of many mRNAs, perhaps by targeting Vhs to mRNAs and to preferred sites within mRNAs.     

Novel Rath peptide for intracellular delivery of protein and nucleic acids

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30 min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant β structure with a small α helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.     

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.     

Raptor-rictor axis in TGFbeta-induced protein synthesis

Transforming growth factor-beta (TGFbeta) stimulates pathological renal cell hypertrophy for which increased protein synthesis is critical. The mechanism of TGFbeta-induced protein synthesis is not known, but PI 3 kinase-dependent Akt kinase activity is necessary. We investigated the contribution of downstream effectors of Akt in TGFbeta-stimulated protein synthesis. TGFbeta increased inactivating phosphorylation of Akt substrate tuberin in a PI 3 kinase/Akt dependent manner, resulting in activation of mTOR kinase. mTOR activity increased phosphorylation of S6 kinase and the translation repressor 4EBP-1, which were sensitive to inhibition of both PI 3 kinase and Akt. mTOR inhibitor rapamycin and a dominant negative mutant of mTOR suppressed TGFbeta-induced phosphorylation of S6 kinase and 4EBP-1. PI 3 kinase/Akt and mTOR regulated dissociation of 4EBP-1 from eIF4E to make the latter available for binding to eIF4G. mTOR and 4EBP-1 modulated TGFbeta-induced protein synthesis. mTOR is present in two multi protein complexes, mTORC1 and mTORC2. Raptor and rictor are part of mTORC1 and mTORC2, respectively. shRNA-mediated downregulation of raptor inhibited TGFbeta-stimulated mTOR kinase activity, resulting in inhibition of phosphorylation of S6 kinase and 4EBP-1. Raptor shRNA also prevented protein synthesis in response to TGFbeta. Downregulation of rictor inhibited serine 473 phosphorylation of Akt without any effect on phosphorylation of its substrate, tuberin. Furthermore, rictor shRNA increased phosphorylation of S6 kinase and 4EBP-1 in TGFbeta-independent manner, resulting in increased protein synthesis. Thus mTORC1 function is essential for TGFbeta-induced protein synthesis. Our data also provide novel evidence that rictor negatively regulates TORC1 activity to control basal protein synthesis, thus conferring tight control on cellular hypertrophy.     

Cyst ornamentation in aquatic invertebrates: a defence against egg-predation

Hydrobiologia - Eggs, including encysted embryos (cysts) of aquatic invertebrates may not only be thick-walled, but also provided with various external ornamentations such as spines and...     

Effect of salinity on competition between the rotifers Brachionus rotundiformis Tschugunoff and Hexarthra jenkinae (De Beauchamp) (Rotifera)

We studied the effect of different concentrations (0, 3, 6, 9 and 12 g l^–1) of sodium chloride at one food level of Chlorella (1×10⁶ cells ml^–1) on competition between the rotifers B. rotundiformis and H. jenkinae, both of which were isolated from a saline lake. The population growth experiments were conducted for 3 weeks. Both the rotifer species did not survive beyond one week at a salinity of 0 g l^–1. Regardless of salt concentration and the presence of a competitor, H. jenkinae reached higher densities than B. rotundiformis. When grown alone, both B. rotundiformis and H. jenkinae showed optimal peak population densities at the salinity of 6 and 9 g l^–1. Since biomass wise, B. rotundiformis was larger than H. jenkinae, it showed a lower numerical abundance. Thus, the maximum peak population densities of B. rotundiformis and H. jenkinae recorded in this study were 107±3 and 203±28 ind. ml^–1. The maximal rates of population increase for B. rotundiformis and H, jenkinae when grown alone were 0.264±0.003 and 0.274±0.004, respectively. Our results also indicated that B. rotundiformis and H. jenkinae coexisted better at a salinity of 6 and 9 g l^–1 of sodium chloride while a salinity of 3 g l^–1 favoured Hexarthra over B. rotundiformis. At 12 g l^–1, both the rotifer species grown alone or together showed lower growth rates compared to those at lower salinity levels. Except 0 g l^–1, in all other salinity treatments, H. jenkinae was a superior competitor to B. rotundiformis.     

Cost of reproduction in selected species of zooplankton (rotifers and cladocerans)

Reproduction is an energetically costly biological process. Among the freshwater zooplankton, rotifers and cladocerans reproduce parthenogenetically and the cost of reproduction can be estimated using the life table data from demographic studies. Reduced probability of future survival or future reproduction as a result of current investment in offspring production (trade-off) is the central theme of the cost hypothesis. Correlations using present reproduction vs. future reproduction (called the reproductive costs) or future survival (called the survival costs) can be used to evaluate the cost hypothesis. In this work sets of correlations were made: (1) between present reproduction (m _x) vs. future survival (l _x+1, l _x+2, l _x+3 etc. for the entire lifespan) (survival costs), and (2) present reproduction (m _x) vs. future reproduction (m _x+1, m _x+2, m _x+3 etc. for the entire reproductive span) (reproductive costs). These correlations were plotted against the cohort age-classes in order to quantify survival and reproductive costs in rotifers (Asplanchna girodi, Brachionus macracanthus, B. variabilis and Platyias quadricornis) and cladocerans (Ceriodaphnia cornuta – one strain maintained on Chlorella and another strain adapted to Microcystis), Daphnia carinata, D. laevis, Moina macrocopa, Pleuroxus aduncus, Scapholeberis kingi and Simocephalus vetulus). All the tested rotifer species showed negative tendency in correlation coefficients (when the data of current reproduction vs. future reproduction and future survival were plotted) for both reproductive and survival costs. However, from the total survival and reproductive costs derived, 84% of the former and 42% of the latter were statistically significant. In cladocerans about 80% of the costs (correlations between current reproduction vs. future survival or future reproduction) were negative suggesting that present reproduction had negatively affected both the further survival and reproduction of test populations. In terms of statistically significant survival costs, the cladocerans showed a trend slightly lower (72%) but comparable to rotifers. The reproductive costs were significant in 45% cases. In our study, the simple statistical correlations detected the trade-offs between reproduction and survival. Thus, in more than 60% cases of both survival and reproductive costs in zooplankton were negative, and our data supported the cost hypothesis, in the majority of cases where reproduction by zooplankton of a given age class caused reduced survival and reproduction of the next age class.     

Interactions between the Anomopod Cladocerans Ceriodaphnia Dubia,C. Cornuta,Simocephalus Vetulus and S. Serrulatus,the Aphanoneurid Worm Aeolosoma sp., and the Fish Skiffia Lermae: Predation or Competition,or Both?

We tested the reciprocal effects of water conditioned by exudates of Aeolosoma sp. (Aphanoneura, Oligochaeta) and fish (Skiffia lermae: Goodeidae) and by the presence of Aeolosoma sp. on the population dynamics of four species of anomopod cladocerans (C. cornuta, C. dubia, S. serrulatus and S. vetulus) at 25 °C for 26 days using Chlorella vulgaris(1 × 10⁶ cells ml^–1) as a basic food for all. We found that, regardless of treatment, C. cornuta and C. dubia had a long initial phase of slow growth, followed by a rapid increase after the second week and that, regardless of treatment, C. dubia had a higher rate of population increase per day than C. cornuta. S. vetulus had a longer lag phase than S. serrulatus. Fish-conditioned water resulted in growth inhibition in S. serrulatus, but had no effect on S. vetulus, while Aeolosoma-conditioned water inhibited growth of S. vetulus but not that of S. serrulatus. Our results suggest a scale of responses of cladocerans to exposure to fish- and worm-conditioned water and to the presence of worms, ranging from a mild stimulation to no effects to inhibited growth. Kairomone-effects on body size of C. cornuta were not significant, but beak length was strongly influenced, and more by fish than by worm kairomones. In S. vetulus, differences in size were not significant. However, individuals grown in the presence of worms had higher biomass while those grown in the presence of fish and worm kairomones showed a decreased weight. Effects of worm exudates and of their live biomass on cladocerans were thus opposite in Simocephalus. In Ceriodaphnia dubia, in contrast, they were additive. Aeolosoma was depressed by all four cladocerans, although worms tried to penetrate the valves of the cladocerans to feed on the tissue inside. In doing so, they suffered significant casualties, especially from blows of the powerful post-abdomen of Simocephalus. The nature of the cladoceran-worm interaction is therefore far from simple: in addition to the 'chemical communication' that is present, it has a predatory component (worms trying to feed on cladocerans) but the reverse might also be true (cladocerans filtering out tissues of disrupted Aeolosoma). Since both worms and cladocerans feed on algae, exploitative competition also seems to be involved. In this, the worms appear to be the inferior partner, although none of the experiments lasted long enough to drive any of the competing partners to extinction.