Metabolic and immunological responses of Drosophila melanogaster to dietary restriction and bacterial infection differ substantially between genotypes in a population

To respond to changing environmental conditions, a population may either shift toward better‐adapted genotypes or adapt on an individual level. The present work aimed to quantify the relevance of these two processes by comparing the responses of defined Drosophila melanogaster populations to different stressors. To do this, we infected two homogeneous populations (isofemale lines), which differ significantly in fitness, and a synthetic heterogeneous population with a specific pathogen and/or exposed them to food restriction. Pectobacterium carotovorum was used to infect Drosophila larvae either fed standard or protein‐restricted diet. In particular, the two homogeneous groups, which diverged in their fitness, showed considerable differences in all parameters assessed (survivorship, protein and lipid contents, phenol‐oxidase (PO) activity, and antibacterial rate). Under fully nutritious conditions, larvae of the homogeneous population with low fitness exhibited lower survivorship and protein levels, as well as higher PO activity and antibacterial rate compared with the fitter population. A protein‐restricted diet and bacterial infection provoked a decrease in survivorship, and antibacterial rate in most populations. Bacterial infection elicited an opposite response in protein and lipid content in both isofemale lines tested. Interestingly, the heterogeneous population showed a complex response pattern. The response of the heterogeneous population followed the fit genotype in terms of survival and antibacterial activity but followed the unfit genotype in terms of PO activity. In conclusion, our results show that defined genotypes exhibit highly divergent responses to varying stressors that are difficult to predict. Furthermore, the responses of heterogeneous populations do not follow a fixed pattern showing a very high degree of plasticity and differences between different genotypes.     

Ornithine decarboxylase activity in regenerating rat adrenal cortex

The activity of ornithine decarboxylase during the regenerating process of adrenal cortex was evaluated in Sprague-Dawley male rats weighing 140–180 g. In this study, 4 groups of rats were enucleated and another 4 groups were sham operated. Animals were maintained at a temperature of 23 ± 1°C and 12:12 h light:dark cycle. At 7 and 11 days post surgery, animals were sacrificed at 0600 h and 1800 h, respectively. Adrenal glands were immediately removed and assayed for ornithine decarboxylase (ODC). The enzyme activity was found to be significantly elevated in enucleated groups as compared to the sham control groups at the 7th and the 11th day of surgery. ODC activity was found to be about 2 times higher at 1800 h by the 7th day and 5 times higher at 1800 h by the 11th day of adrenal surgery when compared to the activity at 0600 h. From these results, it appears that ODC activity not only increases as the regeneration process of adrenal cortex approaches completion, but also becomes more pronounced towards the end of the light period.     

Advances in optical detection strategies for reporter signal measurements

Many recent advances in bioreporter technology focus on challenges related to bioengineering, yet in many applications implementation of optical signal measurement is equally susceptible to improvement. For bioluminescent bioreporters, one area of effort lies in the development of semiconductor chip-based detector modules; this holds great promise for ultra-compact and field-deployable instrumentation, but has not yet had a palpable impact on improved detection limits. Regarding lower detection limits, single-molecule detection techniques have seen their first application to bioreporters, and preliminary results serve as an indication of future promise. Another technique applicable to fluorescent bioreporters is fluorescence flow cytometry, which is rapid, suitable for high-throughput screening, and lends itself to increased analytical specificity through simple algorithmic approaches to data treatment.     

Role of cytokines in alveolar macrophage accessory cell function in HIV-infected individuals.

Mononuclear phagocytes, including alveolar macrophages (AM), can be chronically infected with HIV and thus serve as a reservoir for the virus. Acting as AC during the generation of an immune response, HIV-infected mononuclear phagocytes can facilitate viral T cell infection by several mechanisms, including direct contact of T cells with HIV-infected macrophages as well as cytokine-induced up-regulation of latent T cell infection. Our laboratory has shown that AM from HIV-infected individuals have enhanced AC function compared to normal AM. In this study we explored AM production and secretion of IL-1 beta and IL-6, two cytokines critical for optimal AC function, in normal volunteers and HIV-infected patients. Cultured AM supernatants and lysates were generated in the presence and absence of LPS and standard mitogens. In initial mixing experiments HIV AM supernatants enhanced mitogen-induced T cell proliferation using normal AM as AC significantly more than normal AM supernatants, suggesting that HIV AM secreted more T cell stimulatory factors than normal AM. Neither group could enhance T cell proliferation induced by HIV AM suggesting these cells already secreted optimal levels of these factors. AM from HIV+ individuals produced and secreted more IL-1 beta (measured by ELISA) and IL-6 (measured in a B9 bioassay and by immunoprecipitation) than normal AM both spontaneously and in the presence of low LPS concentrations and mitogens. In some cases depleting HIV AM supernatants of IL-1 beta and IL-6 on immunoaffinity columns abrogated their enhancement properties indicating that these cytokines were important in the observed enhancement. However, in other patients different factors must also be involved as depletion of IL-1 beta and IL-6 in their AM supernatants had no effect on enhancement function. These results show that HIV AM secretory products are important in the enhanced AC function demonstrated by these cells. However, although augmented IL-1 beta and IL-6 secretion likely contribute significantly to this enhancement, other AC secretory factors and/or functions must also be involved.     

Low IGF-1 levels are associated with cardiovascular risk factors in haemodialysis patients

Cardiovascular diseases (CVD) constitute a significant risk and may, in part, explain the high morbidity and mortality rates among haemodialysis (HD) patients. Several studies have implicated reduced insulin like growth factor (IGF-1) levels in the development of CVD. However, it is not clear whether IGF-1, and its relationship with other hormones such as leptin, insulin, and growth hormone (GH), as well as anthropometric variables may explain the high incidence of vascular complications in chronic kidney disease (CKD) patients. This study was designed to measure total serum IGF-1, leptin, insulin and GH levels in CKD patients and in age-matched control subjects and to elucidate the relationship between IGF-1 and GH, leptin, and insulin as well as other known aetiological risk factors for CVD including blood pressure, body mass index (BMI), and age. The study consisted of 50 CKD patients [36 M and 14 F; mean age; 41.8 ± 10.3 years) on maintenance haemodialysis and 50 healthy control subjects (36 M and 14 F; mean age 41.6 ± 10.2 years) matched for age and sex. None of the subject among patients and controls reported either smoking or history of diabetes mellitus. The circulating levels of IGF-1 were significantly lower (P < 0.001) in both male and female patients compared to the control subjects. Moreover, IGF-1 was strongly and inversely correlated with both systolic blood pressure (SBP) (r = −0.360; P < 0.01) and diastolic blood pressure (DBP) (r = −0.512; P < 0.001) in the CKD group, and when the two groups were combined SBP (r = −0.396; P < 0.001) and DBP (r = −0.296; P < 0.01). When adjusted for age, the correlation was more significant, however, when adjusted for BMI no significant correlation was observed between IGF-1 and blood pressure. IGF-1 was inversely correlated with age (r = −0.367; P < 0.01) and BMI (r = −0.310; P < 0.05) in the control group, but not the patient group. In controls and patients, respectively, a positive correlation between leptin and BMI (r = 0.358; P < 0.01; r = 0.640, P < 0.001) was observed. The results show that circulating levels of IGF-1 were significantly lower in CKD patients as compared to healthy normal subjects and were inversely correlated with SBP and DBP independent of age, but not BMI indicative of a strong relationship between cardiovascular risk factors and low IGF-1 levels. Although, the data do not clearly indicate low IGF-1 levels as a cause or an effect of these cardiovascular risk factors, they do point to an interesting relationship between low IGF-1 levels and increased cardiovascular risk factors among CKD patients as compared to age-matched healthy control subjects.     

Morphology and phylogeny of the pseudocolonial dinoflagellates Polykrikos lebourae and Polykrikos herdmanae n. sp

Both the photosynthetic and heterotrophic forms of the only known marine benthic (sand-dwelling) species of Polykrikos, namely P. lebourae, were investigated using light and electron microscopy and molecular phylogenetic analyses. The pseudocolonies usually contained eight integrated zooids and two nuclei. Pseudocolonies consisting of four or five zooids and one nucleus were observed for the first time for this species; some of these reduced pseudocolonies contained plastids, while others were heterotrophic and contained taeniocyst-nematocyst complexes. The ultrastructure of the plastids in P. lebourae did not conform to the organization of thylakoids and enveloping membranes present in the peridinin-containing plastids of other photosynthetic dinoflagellates (i.e. stacks of 3 thylakoids and 3 outer membranes). Instead, the plastids in P. lebourae had thylakoids arranged in pairs and appeared to be enveloped by only two membranes. Molecular phylogenetic data using small subunit rDNA demonstrated that the photosynthetic and heterotrophic forms of P. lebourae represent two distinct clades. The more inclusive clade containing both forms of P. lebourae was most closely related to heterotrophic polykrikoids, namely P. kofoidii. These results led us to conclude that the photosynthetic and heterotrophic forms of P. lebourae are in fact two distinct lineages, and the heterotrophic form is described here as Polykrikos herdmanae n. sp.     

The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure

Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.     

Dinoflagellate,Euglenid, or Cercomonad? The Ultrastructure and Molecular Phylogenetic Position of Protaspis grandis n. sp.

Protaspis is an enigmatic genus of marine phagotrophic biflagellates that have been tentatively classified with several different groups of eukaryotes, including dinoflagellates, euglenids, and cercomonads. This uncertainty led us to investigate the phylogenetic position of Protaspis grandis n. sp. with ultrastructural and small subunit (SSU) rDNA sequence data. Our results demonstrated that the cells were dorsoventrally flattened, shaped like elongated ovals with parallel lateral sides, 32.5-55.0 mum long and 20.0-35.0 mum wide. Moreover, two heterodynamic flagella emerged through funnels that were positioned subapically, each within a depression and separated by a distinctive protrusion. A complex multilayered wall surrounded the cell. Like dinoflagellates and euglenids, the nucleus contained permanently condensed chromosomes and a large nucleolus throughout the cell cycle. Pseudopodia containing numerous mitochondria with tubular cristae emerged from a ventral furrow through a longitudinal slit that was positioned posterior to the protrusion and flagellar apparatus. Batteries of extrusomes were present within the cytoplasm and had ejection sites through pores in the cell wall. The SSU rDNA phylogeny demonstrated a very close relationship between the benthic P. grandis n. sp. and the planktonic Cryothecomonas longipes. These ultrastructural and molecular phylogenetic data for Protaspis indicated that the current taxonomy of Protaspis and Crythecomonas is in need of re-evaluation. The composition and identity of Protaspis is reviewed and suggestions for future taxonomic changes are presented. Problems within the genus Cryothecomonas are highlighted as well, and the missing data needed to resolve ambiguities between the two genera are clarified.     

Phytochemical Profiling and Compound Isolation of Cissus rhombifolia Vahl. Leaves Aqueous Methanolic Extract with the Evaluation of Its Anti-Inflammatory Effect Using Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells

The inflammatory disorders represent a serious health issue. Certain Cissus species possess anti-inflammatory effect. Cissus rhombifolia Vahl. leaves’ anti-inflammatory activities and phytoconstituents are poorly characterized. In this study, 38 constituents were tentatively characterized in Cissus rhombifolia Vahl. leaves’ aqueous methanolic extract (CRLE) using high-performance liquid chromatography combined with mass spectrometry (HPLC/MS) and Proton Nuclear Magnetic Resonance (¹H-NMR). Myricetin, β-amyrin, and alliospiroside A, were isolated from CRLE using column chromatography. The anti-inflammatory effect of CRLE and its isolated compounds were studied in lipopolysaccharide (LPS)-induced RAW 264.7 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) was used to assess how CRLE and its isolated compounds affected cell viability. Further, its effects on the production of intracellular NO, and inflammatory cytokines cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) were assessed by the Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. CRLE and its isolated compounds, myricetin, β-amyrin, and alliospiroside A decreased the NO production. Western blotting was performed to assess the protein expression levels of the inflammatory cytokines inducible nitric oxide synthase (iNOS). Alliospiroside A downregulated IL-6, TNF-α, and COX-2 and inhibited the expression of iNOS. CRLE and its compounds represent effective alternative candidate to treat inflammatory diseases.     

PKA-induced F-actin rearrangement requires phosphorylation of Hsp27 by the MAPKAP kinase MK5

Mitogen-activated protein kinase (MAPK) pathways can play a role in F-actin dynamics. In particular, the p38 MAPK/MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) pathway is involved in F-actin alternations. Previously, we showed that MK5 is implicated in F-actin rearrangement induced by the cAMP/cAMP-dependent protein kinase pathway in PC12 cells, while others found Hsp27 to be a good in vitro MK5 substrate. Here we demonstrate that MK5 can specifically interact with Hsp27 in vivo and can induce phosphorylation at serine residues 78 and 82 in cells. siRNA-mediated depletion of Hsp27 protein levels, as well as overexpression of the non-phosphorylatable Hsp27-3A mutant prevented forskolin-induced F-actin reorganization. While ectopic expression of a constitutive active MK5 mutant was sufficient to induce F-actin rearrangement in PC12 cells, co-expression of Hsp27-3A could ablate this process. Our results imply that MK5 is involved in Hsp27-controlled F-actin dynamics in response to activation of the cAMP-dependent protein kinase pathway. These findings render the MK5/Hsp27 connection into a putative therapeutic target for conditions with aberrant Hsp27 phosphorylation such as metastasis, cardiovascular diseases, muscle atrophy, autoimmune skin disease and neuropathology.