No time to relax: Age-dependent infectivity of cercariae in marine coastal ecosystems.

Age dynamics of the ability of cercariae of two digenean species, Himasthla elongata (Himasthlidae) and Renicola parvicaudatus (Renicolidae), to infect the second intermediate host (SIH), mussels (Mytilus edulis), was investigated experimentally. This is the first study of this kind made on cercariae transmitted in the intertidal of the northern seas. The larvae of all tested ages (from 0.5 to 6 hr) were equally successful in infecting mussels. This finding disagrees with the literature data on cercariae of several freshwater digeneans, which are practically incapable of infecting the SIH during the first 1-3 hr of life. The presence of a time delay before the attainment of the maximum infectivity (TDMI) may be associated with the need for physiological maturation of cercariae in the very beginning of their life in the environment, the need for their broad dispersion, and the prevention of superinfection of the downstream host. The absence of TDMI in the cercariae examined in our study could be associated with the instability of environmental factors in the marine intertidal (wave impact, tidal currents). These factors promote a broad dispersion of cercariae in the intertidal biotope and prevent superinfection of potential SIHs. Biological and behavioural features may also play a role. We hypothesize that the presence or absence of TDMI does not depend on the taxonomic affiliation of the cercariae but is determined by the transmission conditions.

Enriched Environment Induces Sex-Specific Changes in the Adult Neurogenesis, Cytokine and miRNA Expression in Rat Hippocampus.

An enriched environment stimulates adult hippocampal plasticity, but the exact cellular and molecular mechanisms are complex, and thus a matter of debate. We studied the behavior and hippocampal neurogenesis in adult male and female Wistar rats that were housed in an enriched environment (EE) for two months. Both EE males and females performed better than control animals in a Barnes maze, meaning that EE enhances spatial memory. However, the expression levels of neurogenesis markers KI67, DCX, Nestin, and Syn1 increased only in EE females, while in EE males only KI67 and BDNF were higher than in the corresponding control. The number of DCX+ neurons on brain slices increased in the dentate gyrus of EE females only, i.e., the level of adult hippocampal neurogenesis was increased in female but not in male rats. The level of anti-inflammatory IL-10 and signaling pathway components was upregulated in EE females. Of 84 miRNAs tested, in the hippocampi of EE female rats we detected upregulation in the expression levels of 12 miRNAs related to neuronal differentiation and morphogenesis, while in EE males four miRNAs were upregulated and involved in the regulation of cell proliferation/differentiation, and one was downregulated and associated with the stimulation of proliferation. Taken altogether, our results point to sex-specific differences in adult hippocampal plasticity, IL-10 expression, and miRNA profiles induced by an enriched environment.

Radiological and Morphometric Features of Canalis Sinuosus in Russian Population: Cone-Beam Computed Tomography Study.

INTRODUCTION: Cone-beam computed tomography (CBCT) is considered to be the most informative radiographic method for pre- and postoperative analysis of the maxillary anatomy and for avoiding further complication. Canalis sinuosus is one of such structures that damage can go along with bleeding and neurological symptomatology. The aim of the study was to investigate radiological and morphometric features of the canalis sinuosus in Russian population using CBCT technique. MATERIALS AND METHODS: 150 CBCT scans of 61 males and 89 females aged from 24 to 80 years were retrospectively studied with different slice thickness and evaluated with regards to prevalence and diameter among age and gender groups in Russia. RESULTS: CS prevalence in this study was 67%, and CS was most frequently presented in the lateral incisor region (33.5%). Women showed statistically higher CS prevalence (p < 0.01) than the male group, and there was no statistically significant difference observed between occurrence and localization of CS and age groups. CONCLUSION: CBCT examination demonstrated good diagnostic efficiency in CS visualization, and the CS may have variations on its location and prevalence with statistically significant differences between the gender group and without significant differences among age groups and can depend on the population.

Bias in medicine: a survey of medical student attitudes towards HIV-positive and marginalized patients in Russia, 2010.

BACKGROUND: Russia has a substantial HIV epidemic which is poised to escalate in the coming years. The increases in prevalence of HIV will result in increased healthcare needs by a medical system with limited experience with HIV. A healthcare provider's attitude towards a patient plays a significant role in determining the patient's health-related behaviours and medical outcomes. Previous studies have identified negative attitudes of medical students towards people living with HIV. Studying the prevalence of such attitudes is of particular interest, as medical students represent the future workforce and also as the schooling years present a unique opportunity to nurture bias-free healthcare providers. The study measures prevalence of prejudicial attitudes towards HIV-positive and HIV-negative patients who belong to marginalized subgroups. METHODS: The cross-sectional survey was conducted among medical students of a Russian medical university. Of 500 students surveyed, 436 provided sufficient data to be included in the analysis. Prejudicial attitudes were defined as reluctance to provide medical care to a specified hypothetical patient. Nine hypothetical HIV-positive and HIV-negative patients were proposed: physicians, injecting drug users, commercial sex workers, men who have sex with men and a patient HIV-positive due to blood transfusion. A log-binomial regression solved using generalized estimating equations was utilized to identify factors associated with reluctance to treat. RESULTS: Prevalence of reluctance to provide medical care to HIV-positive patients in marginalized subgroups was high (ranging from 26.4% up to 71.9%), compared to a maximum of 7.5% if a patient was an HIV-negative physician. Students in their clinical years reported more negative attitudes than preclinical students. In general, female students were less willing to provide care than their male counterparts. CONCLUSIONS: Prejudicial attitudes about HIV-positive patients and those in marginalized subgroups of the population are prevalent among medical students in Russia. Given the increasing prevalence of HIV in the country, reasons for this hesitance to treat must be identified and addressed. Educational programs for healthcare providers are urgently needed to eliminate bias in the delivery of critically needed medical care. These targeted interventions should be coupled with other programs to eliminate structural barriers to care.

The analysis of near full-length genome sequences of HIV type 1 subtype A viruses from Russia supports the monophyly of major intrasubtype clusters.

The HIV-1 epidemic in Russia has been insufficiently studied, with only 11 complete genome sequences from this country currently available, only three of which are of the locally predominant genetic form, the former Soviet Union (FSU) subtype A variant (A(FSU)). Here we analyze 10 newly derived A(FSU) near full-length genome sequences from Russia. Samples were selected based on phylogenetic clustering in protease-reverse transcriptase in two of the major A(FSU) clusters, V77I(PR) (n=6), widely circulating in Russia and other FSU countries, and A(SP1) (n=4), predominant in St. Petersburg. The phylogenetic analysis shows that the V77I(PR) genomes group in a monophyletic cluster together with 10 previously obtained A(FSU) genome sequences from Uzbekistan, Kazakhstan, Russia, and Cyprus, all bearing the V77I substitution in protease. Similarly, the four A(SP1) genomes group in a monophyletic cluster. These results therefore show that the monophyly of V77I(PR) and A(SP1) A(FSU) clusters is supported in near complete genomes.

Molecular imaging of breast tumors using a near-infrared fluorescently labeled clusterin binding peptide.

Several reports have shown that secreted clusterin (sCLU) plays multiple roles in tumor development and metastasis. Here, we report on a 12-mer sCLU binding peptide (designated P3378) that was identified by screening a phage-display peptide library against purified human sCLU. Differential resonance perturbation nuclear magnetic resonance using P3378 and a scrambled control peptide (designated P3378R) confirmed the P3378-sCLU interaction and demonstrated that it was sequence specific. P3378 and P3378R peptides were conjugated to an Alexa680 near infrared fluorophore (NIRF) and assessed for their tumor homing abilities in in vivo time-domain fluorescence optical imaging experiments using living 4T1 tumor bearing BALB/c mice. When injected in separate animals, both peptides accumulated at the tumor site, however the NIRF-labeled P3378 peptide was retained for a significant longer period of time than the P3378R peptide. Similar observations were made after simultaneously injecting the same tumor-bearing animal with a peptide mixture of P3378 DyLight (DL)680 and the P3378R-DL800. Coinjection of P3378-DL680 with excess unlabeled P3378 blocked tumor accumulation of fluorescent signal while excess P3378R control peptide did not confirming the sequence specificity of the tumor accumulation. Finally, ex vivo fluorescence microscopy of these tumors confirmed the presence of P3378-DL680 in the tumor and its colocalization with CLU. These results confirm the tumor targeting specificity of the P3378 CLU-binding peptide and suggest its usefulness for the in vivo monitoring of solid tumors secreting detectable levels of CLU.

Construction and phenotypic characterization of HIV type 1 functional envelope clones of subtypes G and F.

Subtype G has been estimated to represent the fourth most prevalent clade in the HIV-1 pandemic and subtype F is widely circulating in parts of South America (frequently within BF recombinant forms) and in Romania. However, functional envelope clones of these subtypes are lacking, which are needed for studies on antibody-mediated neutralization, coreceptor usage, and efficiency of viral entry inhibitor drugs. Here we report the construction, neutralization properties, and coreceptor usage of HIV-1 functional envelope clones of subtypes G (n = 15) and F (n = 7). These clones were obtained through RT-PCR amplification of HIV-1 gp160 from plasma RNA, and were used for pseudovirus production. All 15 subtype G-enveloped pseudoviruses were resistant to neutralization by gp120-targeted broadly neutralizing monoclonal antibodies (MAbs) b12 and 2G12, while a majority were neutralized by gp41-targeted MAbs 2F5 and 4E10. With regard to the subtype F envelopes, all seven pseudoviruses were resistant to 2F5 and b12, six were resistant to G12, and six were neutralized by 4E10. Coreceptor usage testing revealed that 21 of 22 envelopes were CCR5-tropic, including all 15 subtype G envelopes, seven of which were from patients with CD4(+) T cell counts <200/ml. These results confirm the broadly neutralizing activity of 4E10 on envelope clones across all tested group M clades, including subtypes G and F, reveal the resistance of most subtype F-enveloped pseudoviruses to broadly neutralizing MAbs b12, 2G12, and 2F5, and suggest that, similarly to subtype C, CXCR4 tropism is uncommon in subtype G, even at advanced stages of infection.

Short communication: Molecular epidemiology of HIV type 1 in the Republic of Dagestan, Russian Federation: virtually uniform circulation of subtype A, former Soviet Union variant, with predominance of the V77I(PR) subvariant.

We examine the distribution of viral genetic forms and the presence of antiretroviral drug resistance mutations in HIV-1 infections in the Republic of Dagestan, in the North Caucasus area of Russia, where a recent large increase in HIV-1 infections has been documented. Samples were collected from 41 HIV-1-infected individuals from Dagestan, most of them from the cities of Derbent (n = 21) and Mahachkala (n = 18). Thirty six were injecting drug users and five were infected by heterosexual contact. None was on antiretroviral drug treatment. HIV-1 protease and a segment of reverse transcriptase were amplified by RT-PCR from plasma RNA and sequenced, and phylogenetic trees were constructed via maximum likelihood. Forty (97.6%) of 41 samples were of subtype A, former Soviet Union variant (A(FSU)), of which 27 (67.5%) clustered with the subvariant containing the V77I substitution in protease (V77I(PR)). Within this cluster, 13 viruses formed a local subcluster, 10 of which were from Derbent. Four viruses clustered with the A(SP2) subcluster, recently identified in St. Petersburg, two with a virus from Georgia and one with a virus from Azerbaijan. No mutations associated with antiretroviral drug resistance were detected. The results, therefore, show the relationship of the HIV-1 epidemic in Dagestan with that of other areas of Russia and of neighboring countries, and reveal the spread of the A(FSU) V77I(PR) variant in the North Caucasus area.

Near full-length genome characterization of a newly identified HIV type 1 subtype F variant circulating in St. Petersburg, Russia.

We report the near full-length genome characterization of an HIV-1 subtype F virus (D88_845) collected in St. Petersburg, Russia, from a 25-year-old Russian woman perinatally infected in 1982. In a Bayesian phylogenetic analysis, the genome sequence branched basally to the subsubtype F1 clade. In partial sequences, D88_845 clustered with 13 other subtype F sequences from Russia, corresponding to gag (n = 2), pol (n = 3), and env (n = 8) segments. At least 11 of these sequences are from samples collected in St. Petersburg from heterosexually infected Russian individuals. In each of these segments, the Russian viruses formed a monophyletic cluster that branched as a sister clade of the F1 subsubtype. One sequence from Belgium branched with D88_845 with a posterior probability of 0.99. This is the first report on the identification and near full-length genome characterization of the subtype F variant circulating in St. Petersburg, which is closely related to, but distinct from, the F1 subsubtype.

Molecular epidemiology of HIV-1 in St Petersburg, Russia: predominance of subtype A, former Soviet Union variant, and identification of intrasubtype subclusters.

OBJECTIVES: To examine HIV-1 genetic diversity in St. Petersburg. METHODS: Partial HIV-1 pol sequences from 102 plasma samples collected in 2006 were analyzed with a Bayesian phylogeny inference method. RESULTS: Subtype A, former Soviet Union (FSU) variant (AFSU), was the predominant clade (89.3%); other clades were subtypes B (9.7%) and F1 (1%). AFSU was predominant both among injecting drug users (98.2%) and heterosexually infected individuals (91.4%), whereas subtype B was more prevalent among homosexual men (75%). Within the AFSU variant, most sequences (93.5%) branched within 1 of 4 strongly supported subclusters. The largest comprised 63% AFSU viruses and was uncommon outside St Petersburg. A second subcluster (17.4% AFSU viruses) corresponds to the variant with the V77I substitution in protease, which is widely circulating in different FSU countries. Two minor subclusters comprised 8.7% and 6.5% AFSU viruses, respectively. There was no correlation between risk exposure and AFSU subclusters. Six of 8 subtype B sequences, 4 of them from homosexual men, grouped in a monophyletic subcluster. CONCLUSIONS: The results of this study show a great predominance of AFSU viruses in St Petersburg and point to a few phylogenetically identifiable introductions as the origin of most current HIV-1 AFSU infections in the city.

Structure dissection of human progranulin identifies well-folded granulin/epithelin modules with unique functional activities.

Progranulin is a secreted protein with important functions in several physiological and pathological processes, such as embryonic development, host defense, and wound repair. Autosomal dominant mutations in the progranulin gene cause frontotemporal dementia, while overexpression of progranulin promotes the invasive progression of a range of tumors, including those of the breast and the brain. Structurally, progranulin consists of seven-and-a-half tandem repeats of the granulin/epithelin module (GEM), several of which have been isolated as discrete 6-kDa GEM peptides. We have expressed all seven human GEMs using recombinant DNA in Escherichia coli. High-resolution NMR showed that only the three GEMs, hGrnA, hGrnC, and hGrnF, contain relatively well-defined three-dimensional structures in solution, while others are mainly mixtures of poorly structured disulfide isomers. The three-dimensional structures of hGrnA, hGrnC, and hGrnF contain a stable stack of two beta-hairpins in their N-terminal subdomains, but showed a more flexible C-terminal subdomain. Interestingly, of the well-structured GEMs, hGrnA demonstrated potent growth inhibition of a breast cancer cell line, while hGrnF was stimulatory. Poorly folded peptides were either weakly inhibitory or without activity. The functionally active and structurally well-characterized human hGrnA offers a unique opportunity for detailed structure-function studies of these important GEM proteins as novel members of mammalian growth factors.

New insights into the origin of the HIV type 1 subtype A epidemic in former Soviet Union's countries derived from sequence analyses of preepidemically transmitted viruses.

The HIV-1 subtype A epidemic affecting injecting drug users (IDU) in former Soviet Union (FSU) countries started dramatically in Odessa, southern Ukraine, in 1995, and is caused by a variant of monophyletic origin, often designated IDU-A. We phylogenetically analyzed one near full-length genome and two partial sequences of three HIV-1 subtype A viruses collected in St. Petersburg, Russia, heterosexually transmitted in 1992-1994. The sequences branched basally to the IDU-A clade, together with eight viruses from Odessa collected in 1993, all presumably acquired heterosexually, and two viruses from the Democratic Republic of Congo. Of all other FSU sequences in databases, only those from three recently collected viruses, one from Ukraine and two from northwestern Russia, at least one of them acquired heterosexually, branched basally to the IDU-A cluster. The results indicate that the FSU IDU-A variant derives from a strain that initially propagated heterosexually in Ukraine and originated in central Africa.

Transforming bivalent ligands into retractable enzyme inhibitors through polypeptide-protein interactions.

The concept of bivalent polypeptides with controllable flexible linkers is demonstrated through the design of a new generation of 'antidote'-reversible inhibitors of thrombin. These molecules contain two binding moieties, each of which in isolation has only a moderate affinity of binding, which are linked together by a flexible peptide bridge. We show that activities of the potent bivalent inhibitors of thrombin can be reversed by the specific, but much weaker, binding of the linker moiety to protein 'antidotes'.

Rational design and selection of bivalent peptide ligands of thrombin incorporating P4-P1 tetrapeptide sequences: from good substrates to potent inhibitors.

The tetrapeptide Phe-Asn-Pro-Arg is a structurally optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or some variants to a C-terminal fragment of hirudin, we were able to generate a series of new bivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P3 and P3' sites of the active-site directed sequence, Phe(P4)-Xaa(P3)-Pro(P2)-Arg(P1)-Pro(P1')-Gln(P2')-Yaa(P3'). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3' sites for bivalent and optimized two-site binding. Very significantly, panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially the catalytic active site of thrombin. Modes of action of the newly discovered bivalent inhibitors are rationalized in light of the allosteric properties of thrombin, especially the interplay between the proteolytic action and regulatory binding occurring at thrombin surfaces remote from the catalytic active site.