The majority of currently circulating human immunodeficiency virus type 1 clade B viruses fail to prime cytotoxic T-lymphocyte responses against an otherwise immunodominant HLA-A2-restricted epitope: implications for vaccine design

Human immunodeficiency virus type 1 (HIV-1) mutates to escape immune selection pressure, but there is little evidence of selection mediated through HLA-A2, the dominant class I allele in persons infected with clade B virus. Moreover, HLA-A2-restricted responses are largely absent in the acute phase of infection as the viral load is being reduced, suggesting that circulating viruses may lack immunodominant epitopes targeted through HLA-A2. Here we demonstrate an A2-restricted epitope within Vpr (Vpr59-67) that is targeted by acute-phase HIV-1-specific CD8+ T cells, but only in a subset of persons expressing HLA-A2. Individuals in the acute stage of infection with viruses containing the most common current sequence within this epitope (consensus sequence) were unable to mount epitope-specific T-cell responses, whereas subjects infected with the less frequent I60L variant all developed these responses. The I60L variant epitope was a stronger binder to HLA-A2 and was recognized by epitope-specific T cells at lower peptide concentrations than the consensus sequence epitope. These data demonstrate that HLA-A2 is capable of contributing to the acute-phase cytotoxic T-lymphocyte response in infected subjects, but that most currently circulating viruses lack a dominant immunogenic epitope presented by this allele, and suggest that immunodominant epitopes restricted by common HLA alleles may be lost as the epidemic matures.     

Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.     

Structure and topology of microbial communities in the major gut compartments of Melolontha melolontha larvae (Coleoptera: Scarabaeidae)

Physicochemical gut conditions and the composition and topology of the intestinal microbiota in the major gut compartments of the root-feeding larva of the European cockchafer (Melolontha melolontha) were studied. Axial and radial profiles of pH, O2, H2, and redox potential were measured with microsensors. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes in midgut samples of individual larvae revealed a simple but variable and probably nonspecific community structure. In contrast, the T-RFLP profiles of the hindgut samples were more diverse but highly similar, especially in the wall fraction, indicating the presence of a gut-specific community involved in digestion. While high acetate concentrations in the midgut and hindgut (34 and 15 mM) corroborated the presence of microbial fermentation in both compartments, methanogenesis was confined to the hindgut. Methanobrevibacter spp. were the only methanogens detected and were restricted to this compartment. Bacterial 16S rRNA gene clone libraries of the hindgut were dominated by clones related to the Clostridiales. Clones related to the Actinobacteria, Bacillales, Lactobacillales, and gamma-Proteobacteria were restricted to the lumen, whereas clones related to the beta- and delta-Proteobacteria were found only on the hindgut wall. Results of PCR-based analyses and fluorescence in situ hybridization of whole cells with group-specific oligonucleotide probes documented that Desulfovibrio-related bacteria comprise 10 to 15% of the bacterial community at the hindgut wall. The restriction of the sulfate-reducer-specific adenosine-5'-phosphosulfate reductase gene apsA to DNA extracts of the hindgut wall in larvae from four other populations in Europe suggested that sulfate reducers generally colonize this habitat.     

Oxidative stress in the pathogenesis of nonalcoholic fatty liver disease,in rats fed with a choline-deficient diet

Background/Aim : The pathogenesis of Nonalcoholic Fatty Liver Disease remains largely unknown, but oxidative stress seems to be involved. The aim of this study was to evaluate the role of oxidative stress in experimental hepatic steatosis induced by a choline-deficient diet. Methods : Fatty liver disease was induced in Wistar rats by a choline-deficient diet. The animals were randomized into three groups: I (G1) and II (G2), n=6 each - fed with a choline-deficient diet for four and twelve weeks respectively; Group III (control-G3; n=6) - fed with a standard diet for twelve weeks. Samples of plasma and liver were submitted to biochemical, histological and oxidative stress analysis. Variables measured included serum levels of aminotransferases (AST, ALT), cholesterol and triglycerides. Oxidative stress was measured by lucigenin-enhanced luminescence and the concentration of hydroperoxides (CE-OOH-cholesteryl ester) in the liver tissue. Results: We observed moderate macro- and microvesicular fatty change in periportal zones G1 and G2 as compared to controls (G3). In G2, fatty change was more severe. The inflammatory infiltrate was scanty and no fibrosis was seen in any group. There was a significant increase of AST and triglycerides in G1 and G2 as compared to control group G3. The lucigenin-amplified luminescence (cpm/mg/min × 10³) was significantly increased in G1 (1393±790) and G2 (7191±500) as compared to controls (513±170), p <0.05. The concentrations of CE-OOH were higher in G1 (5.7±0.9 nmol/mg protein) as compared to control (2.6±0.7 nmol/mg protein), p <0.05. Conclusion: 1) Oxidative stress was found to be increased in experimental liver steatosis; 2) The production of reactive oxygen species was accentuated when liver steatosis was more severe; 3) The alterations produced by oxidative stress could be an important step in the pathogenesis of nonalcoholic fatty liver disease.     

Characterization of the osteoblast-like cell phenotype under microgravity conditions in the NASA-approved Rotating Wall Vessel bioreactor (RWV)

Weightlessness induces bone loss in humans and animal models. We employed the NASA-approved Rotating Wall Vessel bioreactor (RWV) to develop osteoblast-like cell cultures under microgravity and evaluate osteoblast phenotype and cell function. Rat osteoblast-like cell line (ROS.SMER#14) was grown in the RWV at a calculated gravity of 0.008g. For comparison, aliquots of cells were grown in conventional tissue culture dishes or in Non-Rotating Wall Vessels (N-RWV) maintained at unit gravity. In RWV, osteoblasts showed high levels of alkaline phosphatase expression and activity, and elevated expression of osteopontin, osteocalcin, and bone morphogenetic protein 4 (BMP-4). In contrast, the expression of osteonectin, bone sialoprotein II and BMP-2 were unaltered compared to cells in conventional culture conditions. These observations are consistent with a marked osteoblast phenotype. However, we observed that in RWV osteoblasts showed reduced proliferation. Furthermore, DNA nucleosome-size fragmentation was revealed both morphologically, by in situ staining with the Thymine-Adenine binding dye bis-benzimide, and electrophoretically, by DNA laddering. Surprisingly, no p53, nor bcl-2/bax, nor caspase 8 pathways were activated by microgravity, therefore the intracellular cascade leading to programmed cell death remains to be elucidated. Finally, consistent with an osteoclast-stimulating effect by microgravity, osteoblasts cultured in RWV showed upregulation of interleukin-6 (IL-6) mRNA, and IL-6 proved to be active at stimulating osteoclast formation and resorbing activity in vitro. We conclude that under microgravity, reduced osteoblast life span and enhanced IL-6 expression may result in inefficient osteoblast- and increased osteoclast-activity, respectively, thus potentially contributing to bone loss in individuals subjected to weightlessness.     

119Sn Mössbauer spectroscopic studies of the products of the reaction of triorganotin(IV) derivatives with 6-thiopurine

A structural study of the products of the reaction of R₃Sn^IV derivatives (R = Me, Buⁿ, Ph) with 6-thiopurine, 6-TPH₂, and its sodium salt, 6-TPHNa, has been undertaken using Mössbauer spectroscopy and the point-charge model rationalization of the Mössbauer parameter nuclear quadrupole splitting. The synthetic reactions have been carried out at ca. 0 °C, 20 °C and 50 °C. The Mössbauer spectra of the complexes AlK₃Sn(6-TPH) are consistent with the occurrence of two distinct tin(IV) sites in samples prepared at the lower temperature, while one only site appears by increasing the temperature of the reaction. Two tin sites constantly occur in the products of the reactions involving the Ph₃Sn^IV moiety; the stoichiometry is assumed to be (Ph₃Sn)₃(6-TPH)(6-TP) for the uniquely-formed complex. Solid state polymeric structures with trigonal bipyramidal environments of the tin atoms and planar SnC₃ skeletons have been proposed. The apical ligand atoms have been assumed to be N, S and N, N in the samples showing two individual tin(IV) sites, and N, N when a single site was present.     

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.     

Cytoskeleton-associated, carbohydrate-metabolizing enzymes in maize identified by yeast two-hybrid screening

We have used yeast two-hybrid screens and biochemical methods to identify glycolytic enzymes that interact with subcellular structures in hypoxic maize seedlings. As binding domain-bait fusion constructs, we have cloned actin, cytosolic aldolase, the three sucrose synthase (SUS) isoforms SUS1, SUS3, and SH1 as well as the SNF1-related protein kinase into yeast and identified cytosolic isoforms of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase, tubulin, and mitochondrial porin voltage-dependent anion channel protein (VDAC) as well as protein kinases and proteins involved in ubiquitinylation and proteasome-linked degradation as interacting activation domain-prey clones. The results were further confirmed using overlay blots (VDAC) as well as co-polymerization and co-precipitation assays (tubulin and actin). Some results were obtained that support the idea of metabolite and modification effects on the association, namely guanosine triphosphate (GTP)/MgCl₂ was necessary for the binding of enolase to actin. GAPDH is inactivated upon association with tubulin but then serves to stabilize the microtubules. The findings support the idea of the dynamic formation of locally associated complexes of enzymes involved in sucrose breakdown and glycolysis in plant cells depending on their metabolic state.     

A sampling optimization analysis of soil‐bugs diversity (Crustacea,Isopoda, Oniscidea)

Biological diversity analysis is among the most informative approaches to describe communities and regional species compositions. Soil ecosystems include large numbers of invertebrates, among which soil bugs (Crustacea, Isopoda, Oniscidea) play significant ecological roles. The aim of this study was to provide advices to optimize the sampling effort, to efficiently monitor the diversity of this taxon, to analyze its seasonal patterns of species composition, and ultimately to understand better the coexistence of so many species over a relatively small area. Terrestrial isopods were collected at the Natural Reserve “Saline di Trapani e Paceco” (Italy), using pitfall traps monthly monitored over 2 years. We analyzed parameters of α‐ and β‐diversity and calculated a number of indexes and measures to disentangle diversity patterns. We also used various approaches to analyze changes in biodiversity over time, such as distributions of species abundances and accumulation and rarefaction curves. As concerns species richness and total abundance of individuals, spring resulted the best season to monitor Isopoda, to reduce sampling efforts, and to save resources without losing information, while in both years abundances were maximum between summer and autumn. This suggests that evaluations of β‐diversity are maximized if samples are first collected during the spring and then between summer and autumn. Sampling during these coupled seasons allows to collect a number of species close to the γ‐diversity (24 species) of the area. Finally, our results show that seasonal shifts in community composition (i.e., dynamic fluctuations in species abundances during the four seasons) may minimize competitive interactions, contribute to stabilize total abundances, and allow the coexistence of phylogenetically close species within the ecosystem.     

Spatial Embedding and Wiring Cost Constrain the Functional Layout of the Cortical Network of Rodents and Primates

Mammals show a wide range of brain sizes, reflecting adaptation to diverse habitats. Comparing interareal cortical networks across brains of different sizes and mammalian orders provides robust information on evolutionarily preserved features and species-specific processing modalities. However, these networks are spatially embedded, directed, and weighted, making comparisons challenging. Using tract tracing data from macaque and mouse, we show the existence of a general organizational principle based on an exponential distance rule (EDR) and cortical geometry, enabling network comparisons within the same model framework. These comparisons reveal the existence of network invariants between mouse and macaque, exemplified in graph motif profiles and connection similarity indices, but also significant differences, such as fractionally smaller and much weaker long-distance connections in the macaque than in mouse. The latter lends credence to the prediction that long-distance cortico-cortical connections could be very weak in the much-expanded human cortex, implying an increased susceptibility to disconnection syndromes such as Alzheimer disease and schizophrenia. Finally, our data from tracer experiments involving only gray matter connections in the primary visual areas of both species show that an EDR holds at local scales as well (within 1.5 mm), supporting the hypothesis that it is a universally valid property across all scales and, possibly, across the mammalian class.