Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

BACKGROUND: Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel(®)) to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. METHODS: Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC) viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. RESULTS: Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye). In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected) compared to saline (12 of 12 infected) and a control gel (5 of 7 infected). CONCLUSION: These results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV

Cooperative Transition between Open and Closed Conformations in Potassium Channels

Potassium (K+) ion channels switch between open and closed conformations. The nature of this important transition was revealed by comparing the X-ray crystal structures of the MthK channel from Methanobacterium thermoautotrophicum, obtained in its open conformation, and the KcsA channel from Streptomyces lividans, obtained in its closed conformation. We analyzed the dynamic characteristics and energetics of these homotetrameric structures in order to study the role of the intersubunit cooperativity in this transition. For this, elastic models and in silico alanine-scanning mutagenesis were used, respectively. Reassuringly, the calculations manifested motion from the open (closed) towards the closed (open) conformation. The calculations also revealed a network of dynamically and energetically coupled residues. Interestingly, the network suggests coupling between the selectivity filter and the gate, which are located at the two ends of the channel pore. Coupling between these two regions was not observed in calculations that were conducted with the monomer, which emphasizes the importance of the intersubunit interactions within the tetrameric structure for the cooperative gating behavior of the channel

Investigating Homology between Proteins using Energetic Profiles

Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology

Mining Predicted Essential Genes of Brugia malayi for Nematode Drug Targets

We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression

Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations

Can Insects Develop Resistance to Insect Pathogenic Fungi?

This paper presents new, important information on the microevolution of insect resistance to the insect pathogenic fungus Beauveria bassiana which will have far-reaching implications for the development of insect pathogenic fungi as biological control agents. We placed successive generations of a melanic population of the Greater wax moth, Galleria mellonella, under constant selective pressure from the insect pathogenic fungus, Beauveria bassiana. Enhanced fungal resistance was observed and larvae from the 25th generation were studied in detail to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. There are 3 novel, core findings from the study:1.Antifungal resistance in these insects is pathogen species-specific, and probably arises through trans-generational immune priming. The resistance was less obvious in earlier generations, suggesting subtle cumulative changes that are only fully apparent in the 25th generation. 2.The insect’s fecundity is already pushed close to minimum by its melanic phenotype. Therefore, the additional drain on resources required to boost antifungal defence still more, comes not from further compromising life history traits but via a re-allocation of the insect’s immune defences. Specifically during B. bassiana infection, systemic (fat body and hemocoel) responses, particularly the expression of antimicrobial peptides, are damped down in favour of a tailored repertoire of enhanced responses in the integument (cuticle and epidermis) – the foremost and most important barrier to natural fungal infection. 3.A previously-overlooked range of putative stress-management factors are activated during the specific response of selected insects to B. bassiana. This too occurs primarily in the integument, and contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses during the battle between host and pathogen.No other study to date has examined so many genes in this context. Indeed, we show that the epidermis has a great capacity to express defense and stress-management genes as well as the fat body (which is the main tissue producing antimicrobial peptides and has been the traditional focus of attention). We therefore propose a “be specific / fight locally / de-stress” model to explain resource allocation and defence priorities for insects selected for superior resistance to insect-pathogenic fungi. However, we also show that these insects are less fecund and probably at no evolutionary advantage in the wild, implying that the risk is small of biological control agents failing in the field

Azimuthal Charged-Particle Correlations and Possible Local Strong Parity Violation

Parity-odd domains, corresponding to nontrivial topological solutions of the QCD vacuum, might be created during relativistic heavy-ion collisions. These domains are predicted to lead to charge separation of quarks along the system’s orbital momentum axis. We investigate a three-particle azimuthal correlator which is a P even observable, but directly sensitive to the charge separation effect. We report measurements of charged hadrons near center-of-mass rapidity with this observable in Au+Au and Cu+Cu collisions at √sNN=200  GeV using the STAR detector. A signal consistent with several expectations from the theory is detected. We discuss possible contributions from other effects that are not related to parity violation

Multistrange baryon elliptic flow in Au plus Au collisions at root(NN)-N-S=200 GeV

We report on the first measurement of elliptic flow nu(2)(p(T)) of multistrange baryons Xi(-)+Xi(+) and Omega(-)+Omega(+) in heavy-ion collisions. In minimum-bias Au+Au collisions at root s(NN)=200 GeV, a significant amount of elliptic flow, comparable to other nonstrange baryons, is observed for multistrange baryons which are expected to be particularly sensitive to the dynamics of the partonic stage of heavy-ion collisions. The p(T) dependence of nu(2) of the multistrange baryons confirms the number of constituent quark scaling previously observed for lighter hadrons. These results support the idea that a substantial fraction of the observed collective motion is developed at the early partonic stage in ultrarelativistic nuclear collisions at the Relativistic Heavy Ion Collider