Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 alpha-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with K(D) approximately 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.     

Molecular and morphological examination of Cyrtobagous sp. collected from Argentina, Paraguay, Brazil, Australia, and Florida

Two members of the floating fern genus Salvinia (Salviniaceae), S. minima Baker and S. molesta Mitchell, have established in the United States. Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), long established on Florida S. minima, was released in Texas and Louisiana as a biocontrol agent for both species. Subsequently, sequence analysis of the 28S rRNA D2 expansion domain suggested that the Florida and Brazilian populations (used worldwide for biocontrol) of C. salviniae might constitute two cryptic species. In response, the Brazilian weevil was imported from Australia and released instead onto S. molesta. We sampled C. singularis Hustache and C. salviniae from their native ranges in Brazil, Argentina, and Paraguay and sequenced them (D2) along with Australian and Florida samples. The genetic distance between C. singularis and C. salviniae samples is much greater (almost 5×) than the distance between either the Florida and Brazilian samples or the Brazilian and Argentinean/ Paraguayan C. salviniae samples. Since C. singularis and C. salviniae are cryptic species, the Florida and Brazilian populations (or for that matter Brazilian and Argentinean/Paraguayan) could reasonably be described as demes or ecotypes. Occurrence data indicates that, in parts of their ranges, C. salviniae and C. singularis are not only sympatric but also feed on the same plant species at the same site. While host adaptation (species preferences) likely occurs within local demes, both species seem capable of adapting to the available resource (Salvinia species). Finally, a polymerase chain reaction (PCR) primer was developed to distinguish the Florida and Brazilian/Australian types.     

HIV-1 Tat raises an adjuvant-free humoral immune response controlled by its core region and its ability to form cysteine-mediated oligomers

Proteins are poor immunogens that require an adjuvant to raise an immune response. Here we show that the human immunodeficiency virus, type 1 Tat protein possesses an autoadjuvant property, and we have identified the determinants and the molecular events that are associated with this unusual property. Using a series of chemically synthesized Tat101 derivatives, we show that the core region controls the autoadjuvant phenomenon independently of the B-cell recognition and T-cell stimulation that are associated with epitopes respectively located on the N-terminal region and the cysteine-rich region. We also show that cysteine-mediated oligomerization is a key molecular event of the adjuvant-free antibody response. In particular, a Tat dimer formed by the oxidation of two cysteine residues, at position 34 only, raises an adjuvant-free antibody response that is comparable with that observed with the wild-type protein. Unlike the parent protein, the Tat dimer has no transactivating activity and remains homogeneous for several weeks in solution. This construct might be of value for the design of an adjuvant-free Tat-based vaccine. Furthermore, we suggest that the specific autoadjuvanticity determinant of Tat could be used to provide other proteins with adjuvant-free immunogenicity.     

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.     

Responses and structural recovery of periphytic diatom communities after short-term disturbance in some rivers (Hanoi, Vietnam)

Field transfer experiments of periphytic diatom assemblages developed on artificial substrates were set up to assess the responses of those communities to environmental disturbances. The glass slides were positioned for colonization at the relatively unpolluted site (Red, in the Red River) and at the heavily polluted site (TL, in the To Lich River) in the beginning of the experiment. After a period of 2?weeks, the colonized glass slides were concomitantly transferred from the unpolluted Red site to the heavily polluted TL site and to the moderate polluted site (NT₂, in the Nhue River) and, conversely, from the TL site to the Red site, and then to the NT₂ site. The responses and the adapting capacity of periphytic diatom communities to environmental changes were assessed through the cell density, diversity index, species richness, taxonomic composition, and diatom indices after 2 and 4?weeks of transfer periods. For all transfers except for the transfer from the Red to the TL site in which the growth inhibition of diatom cells was found, the diatom density significantly increased until the end of the experiment. Thus, the diatom communities have expressed their pollution tolerance or sensitivities by changing their composition to adapt themselves to environmental changes. Characteristic species of the Red site (Gyrosigma scalproides, Navicula recens) were replaced by Nitzschia palea, Nitzschia umbonata, Aulacoseira granulate typical species of the NT₂ site, in the biofilm transferred from the Red site to the NT₂ site. The relative abundances of typical diatom species of the Red site proliferated in the biofilm transferred from the TL site to the Red site. The replacement of periphytic diatom communities appeared after the transfer from the second week at the different sites. The slow shift of the species towards the typical species at the TL site could result from the organized structure of diatoms within biofilm before the transfer from the Red site to the TL site. The shifts in values of the Index of Specific Polluosensitivity and Diatom Assemblage Index to organic pollution throughout the experiment indicated the clear sensitivity of these indices to water quality changes.     

Comparison of Boolean analysis and standard phylogenetic methods using artificially evolved and natural mt-tRNA sequences from great apes

Boolean analysis (or BOOL-AN; Jakó et al., 2009. BOOL-AN: A method for comparative sequence analysis and phylogenetic reconstruction. Mol. Phylogenet. Evol. 52, 887-97.), a recently developed method for sequence comparison uses the Iterative Canonical Form of Boolean functions. It considers sequence information in a way entirely different from standard phylogenetic methods (i.e. Maximum Parsimony, Maximum-Likelihood, Neighbor-Joining, and Bayesian analysis). The performance and reliability of Boolean analysis were tested and compared with the standard phylogenetic methods, using artificially evolved - simulated - nucleotide sequences and the 22 mitochondrial tRNA genes of the great apes. At the outset, we assumed that the phylogeny of Hominidae is generally well established, and the guide tree of artificial sequence evolution can also be used as a benchmark. These offer a possibility to compare and test the performance of different phylogenetic methods. Trees were reconstructed by each method from 2500 simulated sequences and 22 mitochondrial tRNA sequences. We also introduced a special re-sampling method for Boolean analysis on permuted sequence sites, the P-BOOL-AN procedure. Considering the reliability values (branch support values of consensus trees and Robinson-Foulds distances) we used for simulated sequence trees produced by different phylogenetic methods, BOOL-AN appeared as the most reliable method. Although the mitochondrial tRNA sequences of great apes are relatively short (59-75 bases long) and the ratio of their constant characters is about 75%, BOOL-AN, P-BOOL-AN and the Bayesian approach produced the same tree-topology as the established phylogeny, while the outcomes of Maximum Parsimony, Maximum-Likelihood and Neighbor-Joining methods were equivocal. We conclude that Boolean analysis is a promising alternative to existing methods of sequence comparison for phylogenetic reconstruction and congruence analysis.     

Investigation of Hepatitis B Virus and Human Immunodeficiency Virus Transmission among Severely Mentally Ill Residents at a Long Term Care Facility

Background

A high prevalence of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections have been reported among persons with severe mental illness. In October, 2009, the Cook County Department of Public Health (CCDPH) initiated an investigation following notification of a cluster of HBV infections among mentally ill residents at a long term care facility (LTCF).

Methods

LTCF staff were interviewed and resident medical records were reviewed. Residents were offered testing for HBV, HCV, and HIV. Serum specimens from residents diagnosed with HBV or HIV infection were sent to the Centers for Disease Control and Prevention (CDC) for analysis.

Results

Eleven newly diagnosed HBV infections were identified among mentally ill residents at the LTCF. Of these 11 infections, 4 serum specimens were available for complete HBV genome sequencing; all 4 genomes were found to be closely related. Four newly diagnosed HIV infections were identified within this same population. Upon molecular analysis, 2 of 4 HIV sequences from these new infections were found to be nearly identical and formed a tight phylogenetic cluster.

Conclusions

HBV and HIV transmission was identified among mentally ill residents of this LTCF. Continued efforts are needed to prevent bloodborne pathogen transmission among mentally ill residents in LTCFs.     

Adolescent expectations of early death predict adult risk behaviors

Only a handful of public health studies have investigated expectations of early death among adolescents. Associations have been found between these expectations and risk behaviors in adolescence. However, these beliefs may not only predict worse adolescent outcomes, but worse trajectories in health with ties to negative outcomes that endure into young adulthood. The objectives of this study were to investigate perceived chances of living to age 35 (Perceived Survival Expectations, PSE) as a predictor of suicidal ideation, suicide attempt and substance use in young adulthood. We examined the predictive capacity of PSE on future suicidal ideation/attempt after accounting for sociodemographics, depressive symptoms, and history of suicide among family and friends to more fully assess its unique contribution to suicide risk. We investigated the influence of PSE on legal and illegal substance use and varying levels of substance use. We utilized the National Longitudinal Study of Adolescent Health (Add Health) initiated in 1994-95 among 20,745 adolescents in grades 7-12 with follow-up interviews in 1996 (Wave II), 2001-02 (Wave III) and 2008 (Wave IV; ages 24-32). Compared to those who were almost certain of living to age 35, perceiving a 50-50 or less chance of living to age 35 at Waves I or III predicted suicide attempt and ideation as well as regular substance use (i.e., exceeding daily limits for moderate drinking; smoking ≥ a pack/day; and using illicit substances other than marijuana at least weekly) at Wave IV. Associations between PSE and detrimental adult outcomes were particularly strong for those reporting persistently low PSE at both Waves I and III. Low PSE at Wave I or Wave III was also related to a doubling and tripling, respectively, of death rates in young adulthood. Long-term and wide-ranging ties between PSE and detrimental outcomes suggest these expectations may contribute to identifying at-risk youth.     

Structural, biochemical, and in vivo characterization of the first virally encoded cyclophilin from the Mimivirus

Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses.