Isolation and Characterization of a Thermophilic Bacterium Which Oxidizes Acetate in Syntrophic Association with a Methanogen and Which Grows Acetogenically on H(2)-CO(2)

We previously described a thermophilic (60 degrees C), syntrophic, two-membered culture which converted acetate to methane via a two-step mechanism in which acetate was oxidized to H(2) and CO(2). While the hydrogenotrophic methanogen Methanobacterium sp. strain THF in the biculture was readily isolated, we were unable to find a substrate that was suitable for isolation of the acetate-oxidizing member of the biculture. In this study, we found that the biculture grew on ethylene glycol, and an acetate-oxidizing, rod-shaped bacterium (AOR) was isolated from the biculture by dilution into medium containing ethylene glycol as the growth substrate. When the axenic culture of the AOR was recombined with a pure culture of Methanobacterium sp. strain THF, the reconstituted biculture grew on acetate and converted it to CH(4). The AOR used ethylene glycol, 1,2-propanediol, formate, pyruvate, glycine-betaine, and H(2)-CO(2) as growth substrates. Acetate was the major fermentation product detected from these substrates, except for 1,2-propanediol, which was converted to 1-propanol and propionate. N,N-Dimethylglycine was also formed from glycine-betaine. Acetate was formed in stoichiometric amounts during growth on H(2)-CO(2), demonstrating that the AOR is an acetogen. This reaction, which was carried out by the pure culture of the AOR in the presence of high partial pressures of H(2), was the reverse of the acetate oxidation reaction carried out by the AOR when hydrogen partial pressures were kept low by coculturing it with Methanobacterium sp. strain THF. The DNA base composition of the AOR was 47 mol% guanine plus cytosine, and no cytochromes were detected.     

Sex hormone levels in the brain of d-aspartate-treated rats

d-Aspartate (d-Asp) is an endogenous amino acid present in the central nervous system and endocrine glands of various animal taxa. d-Asp is implicated in neurotransmission, physiology of learning, and memory processes. In gonads, it plays a crucial role in sex hormone synthesis. We have investigated the effects of chronic (30 days d-Asp drinking solution) and acute (i.p. injection of 2 μmol/g bw d-Asp) treatments on sex steroid synthesis in rat brain. Furthermore, to verify the direct effect of d-Asp on neurosteroidogenic enzyme activities, brain homogenates were incubated with different substrates (cholesterol, progesterone, or testosterone) with or without the addition of d-Asp. Enzyme activities were measured by evaluating the in vitro conversion rate of (i) cholesterol to progesterone, testosterone, and 17β-estradiol, (ii) progesterone to testosterone and 17β-estradiol, (iii) testosterone to 17β-estradiol. We found that d-Asp oral administration produced an increase of approximately 40% in progesterone, 110% in testosterone, and 35% in 17β-estradiol. Similarly, the results of the acute experiment showed that at 30 min after d-Asp treatment, the progesterone, testosterone, and 17β-estradiol levels increased by 29–35%, and at 8 h they further increased by a 100% increment. In vitro experiments demonstrate that the addition of d-Asp to brain homogenate + substrate induces a significant increase in progesterone, testosterone and 17β-estradiol suggesting that the amino acid upregulates the local activity of steroidogenic enzymes.     

Unfolding studies of tissue transglutaminase

Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.     

Reconstruction of the Evolutionary Dynamics of A(H3N2) Influenza Viruses Circulating in Italy from 2004 to 2012

Background

Influenza A viruses are characterised by their rapid evolution, and the appearance of point mutations in the viral hemagglutinin (HA) domain causes seasonal epidemics. The A(H3N2) virus has higher mutation rate than the A(H1N1) virus. The aim of this study was to reconstruct the evolutionary dynamics of the A(H3N2) viruses circulating in Italy between 2004 and 2012 in the light of the forces driving viral evolution.

Methods

Phylodinamic analyses were made using a Bayesian method, and codon-specific positive selection acting on the HA coding sequence was evaluated.

Results

Global and local phylogenetic analyses showed that the Italian strains collected between 2004 and 2012 grouped into five significant Italian clades that included viral sequences circulating in different epidemic seasons. The time of the most recent common ancestor (tMRCA) of the tree root was between May and December 2003. The tMRCA estimates of the major clades suggest that the origin of a new viral strain precedes the effective circulation of the strain in the Italian population by 6–31 months, thus supporting a central role of global migration in seeding the epidemics in Italy. The study of selection pressure showed that four codons were under positive selection, three of which were located in antigenic sites. Analysis of population dynamics showed the alternation of periods of exponential growth followed by a decrease in the effective number of infections corresponding to epidemic and inter-epidemic seasons.

Conclusions

Our analyses suggest that a complex interaction between the immune status of the population, migrations, and a few selective sweeps drive the influenza A(H3N2) virus evolution. Our findings suggest the possibility of the year-round survival of local strains even in temperate zones, a hypothesis that warrants further investigation.     

Inhibition of In Vivo HIV Infection in Humanized Mice by Gene Therapy of Human Hematopoietic Stem Cells with a Lentiviral Vector Encoding a Broadly Neutralizing Anti-HIV Antibody

Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/γ_c^null mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/γ_c^null mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1_JR-CSF, mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/γ_c^null mice inoculated with equivalent high-titer HIV-1_JR-CSF. These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.While broadly neutralizing human immunodeficiency virus (HIV)-specific antibodies have the capacity to prevent or suppress HIV infection, they are rarely produced by infected individuals, thereby markedly compromising the ability of the humoral response to control HIV infection (reviewed in reference 28). The high degree of sequence variability in the gp120 structure limits the number of highly conserved epitopes available for targeting by neutralizing antibodies (40). In addition, HIV utilizes several mechanisms to shield the limited number of conserved neutralizing epitopes from the potentially potent antiviral effects of HIV envelope-specific antibodies (14). First, the envelope protein is heavily glycosylated, and the linkage of the most immunoreactive envelope peptide structures to poorly immunogenic glycans shields them from antibody binding (37). Second, exposure of neutralizing epitopes not protected from antibody binding by glycosylation is greatly reduced by trimerization of the gp120-gp41 structure (5). Third, susceptibility of other neutralizing epitopes to antibodies is greatly reduced by limiting their accessibility to antibody binding to the brief transient phase of conformational changes that occur only during binding of the envelope protein to its cellular receptors, CD4 and CCR5 or CXCR4 (41). These intrinsic structural features of gp120 greatly reduce the capacity of natural HIV infection or vaccination to generate broadly neutralizing antibodies able to prevent or control infection. Despite these constraints, rare human antibodies with broad anti-HIV neutralizing activity, i.e., 2G12, b12, 2F5, and 4E10, have been isolated (2).The capacity of passive immunization with neutralizing antibodies to prevent infection was suggested by challenge studies demonstrating that transferred neutralizing antibodies protected monkeys from infection by simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) (15). These studies were extended to humans, including several studies that examined the effect of passive immunotherapy using 2G12, 2F5, and 4E10 on inhibition of HIV replication in infected individuals (20). Passive immunotherapy with a triple combination of 2G12, 2F5, and 4E10 delayed viral rebound after the cessation of highly active antiretroviral therapy (HAART), and activity of 2G12 was critical for inhibitory activity by this antibody combination (18). The key role of 2G12 in suppressing HIV replication was supported by the development of viral rebound in parallel with the emergence of HIV isolates resistant to neutralization by 2G12 (19).While HIV infection may be controlled by the lifelong treatment of HIV-infected individuals with periodic infusions of neutralizing-antibody cocktails every few weeks, this is not a practical or cost-effective therapeutic approach. Eliciting these antibodies by vaccination has not been successful. Therefore, we investigated whether we could circumvent the mechanisms that limit the endogenous production of broadly neutralizing HIV-specific antibodies using a molecular genetic approach to generate B cells that secrete these protective antibodies. In a proof-of-concept study, we examined the capacity of a single lentiviral vector to express the heavy and light chains of the 2G12 antibody, a well-studied anti-HIV human antibody that has broad neutralizing activity both against T cell line-adapted and primary HIV isolates (31). The 2G12 antibody was generated by applying murine/human xenohybridoma technology to establish human hybridoma cell lines from B cells isolated from HIV-infected individuals (16), and it targets the high-mannose and/or hybrid glycans of residues 295, 332, and 392 and peripheral glycans from residues 386 and 448 on gp120. In the current study we demonstrated that a lentiviral vector encoding the heavy and light chains of the 2G12 antibody reprogrammed B cells in vitro to secrete 2G12 with functional neutralizing activity. Furthermore, we demonstrated that the 2G12 lentiviral vector genetically modified human hematopoietic stem cells (hu-HSC), enabling them to differentiate in vivo into progeny cells that secreted 2G12 antibody that inhibited the development of in vivo HIV infection in humanized mice.     

Effects of progressive and maximal exercise on plasma levels of adhesion molecules in athletes with sickle cell trait with or without alpha-thalassemia.

The aim of the study was to examine the effects of exercise on soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) in sickle cell trait (SCT) athletes with or without alpha-thalassemia. Six athletes with SCT, seven athletes with both SCT and alpha-thalassemia (SCTAT), and seven control athletes (Cont) performed an incremental and maximal test on cycloergometer. Levels of sICAM-1 and sVCAM-1 were assessed at rest, immediately after the end of exercise, and 1, 2, and 24 h after exercise. Although Cont and SCTAT groups exhibited similar basal plasma levels of inflammatory and adhesion molecules, the SCT group had higher sVCAM-1 basal concentrations. Incremental exercise resulted in a significant increase of sVCAM-1 in all subjects, which remained elevated only in the SCT group during the recovery period. In conclusion, as sVCAM-1 increased with exercise and during the recovery period, our findings support the concept that SCT athletes might be at risk for microcirculatory disturbances and adhesive phenomena developing at rest and several hours after exercise. alpha-Thalassemia might be considered protective among exercising SCT subjects.