Comparison by flow cytometry of immune changes induced in human monocyte-derived dendritic cells upon infection with dengue 2 live-attenuated vaccine or 16681 parental strain

Dengue is an important threat for world-wide public health. Different vaccines are under development, which are currently assessed using a battery of in vitro and in vivo assays before moving on to humans. It is also important to assess vaccine characteristics on human primary cells; among them, dendritic cells, the most efficient antigen-presenting cells, are the first targets of dengue virus infection. In this study, we used flow cytometry to compare the consequences of such an infection by dengue serotype 2 live-attenuated vaccine (LAV2) or its parental strain DEN2 16681 (DEN2). Optimal conditions of infection have first been defined by a mathematical approach, and flow cytometry allowed studying modifications induced in both infected and noninfected dendritic cell populations after surface and intracellular labeling. Both DEN2 and LAV2 increased the expression of the phenotypic markers CD80, CD86, CD40, CD1a, HLA ABC and CD83, demonstrating cellular activation. Stimulated dendritic cells produced tumor necrosis factor-alpha in particular, and, to a lower extent, interleukin 6. Of importance, whereas DEN2 induced cytokine production both in the infected and noninfected populations, LAV2-induced cytokine production was restricted to the infected population. This limited activation triggered by LAV2 would be in agreement with its attenuation. In conclusion, these in vitro experiments using primary human dendritic cells may participate, in combination with other assays, to the evaluation of the immunogenicity and safety of dengue vaccine candidates.     

Mining the NCI antiviral compounds for HIV-1 integrase inhibitors

HIV-1 integrase (IN) is an essential enzyme for effective viral replication and is a validated target for the development of antiretroviral drugs. Currently, there are no approved drugs targeting this enzyme. In this study, we have identified 11 structurally diverse small-molecule inhibitors of IN. These compounds have been selected by mining the moderately active antiviral molecules from a collection of 90,000 compounds screened by the National Cancer Institute (NCI) Antiviral Program. These compounds, which were screened at the NCI during the past 20 years, resulted in approximately 4000 compounds labeled as 'moderately active.' In our study, chalcone 11 shows the most potent activity with an IC(50) of 2+/-1 microM against purified IN in the presence of both Mn(2+) and Mg(2+) as cofactors. Docking simulations using the 11 identified inhibitors as a training set have elucidated two unique binding areas within the active site: the first encompasses the conserved D64-D116-E152 motif, while the other involves the flexible loop region formed by amino acid residues 140-149. The tested inhibitors exhibit favorable interactions with important amino acid residues through van der Waals and H-bonding contacts.     

Gene Structure and Molecular Phylogeny of the Linker Chains from the Giant Annelid Hexagonal Bilayer Hemoglobins

Giant extracellular hexagonal bilayer hemoglobin (HBL-Hb), found only in annelids, is an ∼3500-kDa heteropolymeric structure involved in oxygen transport. The HBL-Hbs are comprised of globin and linker chains, the latter being required for the assembly of the quaternary structure. The linker chains, varying in size from 225 to 283 amino acids, have a conserved cysteine-rich domain within their N-terminal moiety that is homologous to the cysteine-rich modules constituting the ligand binding domain of the low-density lipoprotein receptor (LDLR) protein family found in many metazoans. We have investigated the gene structure of linkers from Arenicola marina, Alvinella pompejana, Nereis diversicolor, Lumbricus terrestris, and Riftia pachyptila. We found, contrary to the results obtained earlier with linker genes from N. diversicolor and L. terrestris, that in all of the foregoing cases, the linker LDL-A module is flanked by two phase 1 introns, as in the human LDLR gene, with two more introns in the 3′ side whose positions varied with the species. In addition, we obtained 13 linker cDNAs that have been determined experimentally or found in the EST database LumbriBASE. A molecular phylogenetic analysis of the linker primary sequences demonstrated that they cluster into two distinct families of linker proteins. We propose that the common gene ancestor to annelid linker genes exhibited a four-intron and five-exon structure and gave rise to the two families subsequent to a duplication event.     

Propolis protects human spermatozoa from DNA damage caused by benzo[a]pyrene and exogenous reactive oxygen species

Many environmental, physiological and genetic factors have been implicated in defective sperm function, the most common cause of infertility. In addition, sperm preparation techniques such as centrifugation, used prior to in vitro fertilization, are associated with the generation of reactive oxygen species (ROS) and an increase in the level of DNA damage. Factors that can offer spermatozoa protection are, therefore, of great importance. This study was designed to examine in vitro the effect of a Chilean propolis ethanolic extract on human spermatozoa treated with benzo[a]pyrene and exogenous reactive oxygen species. Our experimental evidence demonstrated that the natural drug under investigation is able to protect genomic DNA by damage induced by benzo[a]pyrene, hydrogen peroxide (H2O2) and hydrogen peroxide in combination with adenosine 5'-diphosphate (ADP) and ferrous sulfate (FeSO4), determining a significant reduction of the intracellular oxidants. An increase in membrane damage, measured by monitoring the formation of thiobarbituric acid-reactive substances (TBARS) and lactic dehydrogenase (LDH) release, was observed only in sperm treated with H2O2, ADP and FeSO4. The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack, reducing TBARS formation and LDH release. In summary, our results evidence that the protective effect exhibited by this natural compound in human spermatozoa is correlated, at least in part, to the antioxidant capacity of its active components, and suggest that propolis may have a role in protection against male infertility.     

Cytotoxic T cell immunity against the non-immunogenic, murine, hepatocellular carcinoma Hepa1-6 is directed towards the novel alternative form of macrophage colony stimulating factor

Mouse Hepa1-6 hepatocellular carcinoma (HCC) cells were transduced with the membrane form of macrophage colony stimulating factor (mM-CSF). When mM-CSF transduced Hepa1-6 cells were injected subcutaneously into mice, these cells did not form tumors. The spleens of these immunized mice contained cytotoxic CD8⁺ T lymphocytes (CTL) that killed the unmodified Hepa1-6 cells. We show that the alternative form of macrophage colony stimulating factor (altM-CSF) induced CTL-mediated immunity against Hepa1-6 cells. AltM-CSF is restricted to the H-2D^b allele. CTLs killed RMA-S cells loaded with exogenous altM-CSF peptide. Vaccination of mice with dendritic cells pulsed with the altM-CSF peptide stimulated anti-Hepa1-6 CTLs. Hyper-immunization of mice with mM-CSF Hepa1-6 cells showed inflammation of the liver and kidneys. Although altM-CSF was expressed within liver and kidney cells, its intensity was lower than Hepa1-6 cells. AltM-CSF was detected within the human HepG2 cell line. These studies suggest that altM-CSF may be a tumor antigen for HCC.     

Cross-reactions between engineered xylose and galactose pathways in recombinant Saccharomyces cerevisiae

Background

Bioethanol can be produced from sugar-rich, starch-rich (first generation; 1G) or lignocellulosic (second generation; 2G) raw materials. Integration of 2G ethanol with 1G could facilitate the introduction of the 2G technology. The capital cost per ton of fuel produced would be diminished and better utilization of the biomass can be achieved. It would, furthermore, decrease the energy demand of 2G ethanol production and also provide both 1G and 2G plants with heat and electricity. In the current study, steam-pretreated wheat straw (SPWS) was mixed with presaccharified wheat meal (PWM) and converted to ethanol in simultaneous saccharification and fermentation (SSF).

Results

Both the ethanol concentration and the ethanol yield increased with increasing amounts of PWM in mixtures with SPWS. The maximum ethanol yield (99% of the theoretical yield, based on the available C6 sugars) was obtained with a mixture of SPWS containing 2.5% water-insoluble solids (WIS) and PWM containing 2.5% WIS, resulting in an ethanol concentration of 56.5 g/L. This yield was higher than those obtained with SSF of either SPWS (68%) or PWM alone (91%).

Conclusions

Mixing wheat straw with wheat meal would be beneficial for both 1G and 2G ethanol production. However, increasing the proportion of WIS as wheat straw and the possibility of consuming the xylose fraction with a pentose-fermenting yeast should be further investigated.     

Effects of protons and HZE particles on glutamate transport in astrocytes, neurons and mixed cultures

Radiation-induced neurotoxicity is a well-characterized phenomenon. However, the underlying mechanism of this toxicity is poorly understood. In the central nervous system (CNS), excitotoxic mechanisms are implicated in many neurodegenerative disease processes. Pivotal to the excitotoxic pathway is dysfunction of glutamate signaling. We reported previously that exposure to low-LET γ radiation results in altered glutamate transport in neurons and astrocytes. In the present study, we sought to investigate the effects of various particle radiations of differing LET on glutamate transport as a measure of the neurochemical vulnerability of the CNS. NTera2-derived neurons and astrocytes isolated as pure and mixed cultures were exposed to doses of 10 cGy, 50 cGy or 2 Gy of 250 MeV protons, 290 MeV/nucleon carbon ions, or 1000 MeV/nucleon iron ions. Transporter function was assessed at 3 h, 2 days and 7days after exposure. Functional assessment of glutamate transport revealed that neurons and astrocytes respond in a reciprocal manner after exposure to particle radiation. Uptake activity in neurons increased after particle irradiation. This effect was evident as late as our last time (7 days) after exposure (P < 0.05). In astrocytes, transporter activity decreased after exposure. The decrease in uptake observed in astrocytes was evident 7 days after exposure to carbon and iron ions. Uptake in mixed cultures after exposure to all three forms of radiation revealed a muted interactive response suggestive of the individual responses of each cellular phenotype acting in opposition.     

Tiron administration minimizes the toxicity of vanadate but not its insulin mimetic properties in diabetic rats.

Although it has been reported that vanadate is effective in diminishing the expression of diabetes in the rat, the severe toxic side effects noted in the vanadate-treated animals suggest that chronic oral administration of vanadate argues against its use in human diabetes. The present study was conducted to evaluate the effects of the chelator Tiron on the mobilization of vanadium after administration of sodium metavanadate in the drinking water (0.20 mg/ml) of streptozotocin-induced diabetic rats for 35 days. Intraperitoneal treatment with Tiron (300 or 600 mg/kg) was initiated after three weeks of vanadate administration and continued for two weeks. The ameliorative effects of vanadium with respect to diabetes were not diminished by the administration of Tiron, but the accumulation of vanadium in kidney and bone was significantly decreased in the Tiron-treated groups and diabetes associated increases in serum GOT, GPT and cholesterol were diminished with Tiron treatment. It is concluded that the coadministration of metavanadate and Tiron may be of potential value for treatment of diabetes mellitus.     

Seroprevalence of Antibodies against Seal Influenza A(H10N7) Virus in Harbor Seals and Gray Seals from the Netherlands

In the spring and summer 2014, an outbreak of seal influenza A(H10N7) virus infection occurred among harbor seals (Phoca vitulina) off the coasts of Sweden and Denmark. This virus subsequently spread to harbor seals off the coasts of Germany and the Netherlands. While thousands of seals were reported dead in Sweden, Denmark and Germany, only a limited number of seals were found dead in the Netherlands. To determine the extent of exposure of seals in the Netherlands to influenza A/H10N7 virus, we measured specific antibody titers in serum samples from live-captured seals and seals admitted for rehabilitation in the Netherlands by use of a hemagglutination inhibition assay and an ELISA. In harbor seals in 2015, antibodies against seal influenza A(H10N7) virus were detected in 41% (32 out of 78) pups, 10% (5 out of 52) weaners, and 58% (7 out of 12) subadults or adults. In gray seals (Halichoerus grypus) in 2015, specific antibodies were not found in the pups (n = 26), but in 26% (5 out of 19) of the older animals. These findings indicate that, despite apparent low mortality, infection with seal influenza A(H10N7) virus was geographically widespread and also occurred in grey seals.