Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis.

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures.

Seroprevalence of human alphaherpesvirus 1 and 2 among pregnant women infected or uninfected with Zika virus from Rio de Janeiro, Brazil.

Pregnant women are an important group to be monitored for infection due to the risk of transmitting infections to their babies. Both herpes simples virus (HSV) and Zika virus (ZIKV) are neurotropic viruses that can be transmitted congenitally. In this study, the prevalence and risk factors of HSV among Zika-positive and -negative pregnant women from Rio de Janeiro, Brazil, were evaluated and compared. About 167 serum samples included in our study were from pregnant women with ZIKV infection symptoms, who were attended to in different hospitals in Rio de Janeiro between November 2015 to February 2016. Blood samples collected from 167 pregnant women were used for this study. The presence of HSV antibodies and viremia were evaluated by commercial ELISA and quantitative real-time polymerase chain reaction analyses, respectively. The data obtained from medical records were statistically analyzed. The HSV-1 and HSV-2 prevalence among pregnant women was 80.2% and 12.5% for Zika-positive women and 84.5% and 5.6% for Zika-negative women, respectively. None of the pregnant women exhibited HSV viremia. Age, trimester of gestation, and skin color were associated with HSV-1 and HSV-2 prevalence among the groups studied. HSV-2 was more prevalent in Zika-positive pregnant women than in Zika-negative pregnant women, and this simultaneous infection should be better investigated in future studies.

Evaluation of a Dielectric-Only Transmitarray for Generating Multi-Focusing Near-Field Spots Using a Cluster of Feeds in the Ka-Band.

A transmitarray antenna is evaluated to generate a multi-focusing spot area in the Fresnel region of the antenna in the Ka-band. The antenna is designed to focus the radiated field at a certain point using a central feeding configuration. The number of feeds is increased to create as many focusing spots as feeds. The feeds are placed along an arc defined in the principal planes of the transmitarray, radiating independent near-field spots and providing a solution with a wide-angle spot scanning without an antenna displacement and a high isolation between feeds. To validate this concept, a transmitarray based on dielectric-only cells is designed and simulated under full-wave conditions. Then, this design is manufactured using a 3D printing technique, and the prototype is measured in a planar acquisition range. Measurements are performed for different feed positions in order to validate the multi-focusing capability of the antenna. Measurements and simulations show a high agreement and validate the proposed design technique.

Development of an Anthropomorphic Phantom of the Axillary Region for Microwave Imaging Assessment.

We produced an anatomically and dielectrically realistic phantom of the axillary region to enable the experimental assessment of Axillary Lymph Node (ALN) imaging using microwave imaging technology. We segmented a thoracic Computed Tomography (CT) scan and created a computer-aided designed file containing the anatomical configuration of the axillary region. The phantom comprises five 3D-printed parts representing the main tissues of interest of the axillary region for the purpose of microwave imaging: fat, muscle, bone, ALNs, and lung. The phantom allows the experimental assessment of multiple anatomical configurations, by including ALNs of different size, shape, and number in several locations. Except for the bone mimicking organ, which is made of solid conductive polymer, we 3D-printed cavities to represent the fat, muscle, ALN, and lung and filled them with appropriate tissue-mimicking liquids. Existing studies about complex permittivity of ALNs have reported limitations. To address these, we measured the complex permittivity of both human and animal lymph nodes using the standard open-ended coaxial-probe technique, over the 0.5 GHz-8.5 GHz frequency band, thus extending current knowledge on dielectric properties of ALNs. Lastly, we numerically evaluated the effect of the polymer which constitutes the cavities of the phantom and compared it to the realistic axillary region. The results showed a maximum difference of 7 dB at 4 GHz in the electric field magnitude coupled to the tissues and a maximum of 10 dB difference in the ALN response. Our results showed that the phantom is a good representation of the axillary region and a viable tool for pre-clinical assessment of microwave imaging technology.

Phenotype and function of HBV-specific T cells is determined by the targeted epitope in addition to the stage of infection.

OBJECTIVE: Chronic HBV infection affects more than 250 million people worldwide and remains a global healthcare problem in part because we lack curative treatment. Sustained viral control requires HBV-specific T cells, but these become functionally impaired in chronic infection. Clinical evidence indicates that functional cure of HBV infection by the host immune response is feasible. Developing T cell-based therapies able to achieve functional cure will require identification of the requirements for a successful T cell response against HBV and the relative contribution of individual T cell specificities to HBV control. DESIGN: The phenotype and function of HBV-specific T cells were studied directly ex vivo using fluorochrome-labelled multimers. We studied multiple HBV-specific T cell specificities targeting different HBV proteins in individuals with either an acute self-limiting or chronic HBV infection. RESULTS: We detected strong T cell responses targeting multiple HBV viral proteins in acute self-limiting and low-frequency core and polymerase-specific T cells in chronic infection. Expression of the T cell inhibitory receptor PD-1, as well as T cell differentiation, T cell function and T cell regulation differed by stages and outcomes of infection. In addition, these features differed significantly between T cells targeting different HBV specificities. CONCLUSION: HBV-specific T cells with different target specificities are characterised by distinct phenotypical and functional profiles. These results have direct implications for the design of immunological studies in HBV infection, and are potentially relevant for informing immunotherapeutic approaches to induce functional cure.

Replication Protein A-1 Has a Preference for the Telomeric G-rich Sequence in Trypanosoma cruzi.

Replication protein A (RPA), the major eukaryotic single-stranded binding protein, is a heterotrimeric complex formed by RPA-1, RPA-2, and RPA-3. RPA is a fundamental player in replication, repair, recombination, and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes. Recently, we have showed that T. cruziRPA presents canonical functions being involved with DNA replication and DNA damage response. Here, we found by FISH/IF assays that T. cruziRPA localizes at telomeres even outside replication (S) phase. In vitro analysis showed that one telomeric repeat is sufficient to bind RPA-1. Telomeric DNA induces different secondary structural modifications on RPA-1 in comparison with other types of DNA. In addition, RPA-1 presents a higher affinity for telomeric sequence compared to randomic sequence, suggesting that RPA may play specific roles in T. cruzi telomeric region.

Nationwide overview of the distribution of hepatitis B virus genotypes in Brazil: a 1000-sample multicentre study.

The influence of hepatitis B virus (HBV) genotypes in the natural history of the disease and its response to antiviral treatment have been addressed in many studies. In Brazil, studies on HBV genotype circulation have been restricted to specific population groups and states. Here, we have conducted a nationwide multicentre study with an unprecedented sample size representing all Brazilian regions in an effort to better understand the viral variants of HBV circulating among chronic carriers. Seven HBV genotypes were found circulating in Brazil. Overall, HBV/A was the most prevalent, identified in 589 (58.7 %) samples, followed by HBV/D (23.4 %) and HBV/F (11.3 %). Genotypes E, G, C and B were found in a minor proportion. The distribution of the genotypes differed markedly from the north to the south of the country. While HBV/A was the most prevalent in the North (71.6 %) and Northeast (65.0 %) regions, HBV/D was found in 78.9 % of the specimens analysed in the South region. HBV/F was the second most prevalent genotype in the Northeast region (23.5 %). It was detected in low proportions (7 to 10 %) in the North, Central-West and Southeast regions, and in only one sample in the South region. HBV/E was detected in all regions except in the South, while monoinfection with HBV/G was found countrywide, with the exception of Central-West states. Our sampling covered 24 of the 26 Brazilian states and the Federal District and is the first report of genotype distribution in seven states. This nationwide study provides the most complete overview of HBV genotype distribution in Brazil to date and reflects the origin and plurality of the Brazilian population.

Functional and structural studies of a Phospholipase A2-like protein complexed to zinc ions: Insights on its myotoxicity and inhibition mechanism.

BACKGROUND: One of the main challenges in snakebite envenomation treatment is the development of stable, versatile and efficient anti-venom therapies. Local myotoxicity in accidents involving snakes from the Bothrops genus is still a consequence of serum therapy inefficient neutralization that may lead to permanent sequelae in their victims. One of the classes of toxins that participate in muscle necrosis is the PLA2-like proteins. The aim of this work was to investigate the role of zinc ions in the inhibition of PLA2-like proteins and to advance the current knowledge of their action mechanism. METHODS: Myographic and electrophysiological techniques were used to evaluate the inhibitory effect of zinc ions, isothermal titration calorimetry assays were used to measure the affinity between zinc ions and the toxin and X-ray crystallography was used to reveal details of this interaction. RESULTS: We demonstrated that zinc ions can effectively inhibit the toxin by the interaction with two different sites, which are related to two different mechanism of inhibition: preventing membrane disruption and impairing the toxin state transition. Furthermore, structural study presented here included an additional step in the current myotoxic mechanism improving the comprehension of the allosteric transition that PLA2-like proteins undergo to exert their function. CONCLUSIONS: Our findings show that zinc ions are inhibitors of PLA2-like proteins and suggest two different mechanisms of inhibition for these ions. GENERAL SIGNIFICANCE: Zinc is a new candidate that can assist in anti-venom treatments and can promote the design of new and even more accurate structure-based inhibitors for PLA2-like proteins.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

BACKGROUND: Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. METHODS AND RESULTS: Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. CONCLUSIONS: We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. GENERAL SIGNIFICANCE: LCalA is the first reported calmodulin that binds in vivo telomeric DNA.

A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms.

Envenomation via snakebites is an important public health problem in many tropical and subtropical countries that, in addition to mortality, can result in permanent sequelae as a consequence of local tissue damage, which represents a major challenge to antivenom therapy. Venom phospholipases A2 (PLA2s) and PLA2-like proteins play a leading role in the complex pathogenesis of skeletal muscle necrosis, nevertheless their precise mechanism of action is only partially understood. Recently, detailed structural information has been obtained for more than twenty different members of the PLA2-like myotoxin subfamily. In this review, we integrate the available structural, biochemical and functional data on these toxins and present a comprehensive hypothesis for their myotoxic mechanism. This process involves an allosteric transition and the participation of two independent interaction sites for docking and disruption of the target membrane, respectively, leading to a five-step mechanism of action. Furthermore, recent functional and structural studies of these toxins complexed with ligands reveal diverse neutralization mechanisms that can be classified into at least three different groups. Therefore, the data summarized here for the PLA2-like myotoxins could provide a useful molecular basis for the search for novel neutralizing strategies to improve the treatment of envenomation by viperid snakes.

Structural bases for a complete myotoxic mechanism: crystal structures of two non-catalytic phospholipases A2-like from Bothrops brazili venom.

Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA2s) are among the main components from Bothrops genus venoms and are capable of causing drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from B. brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, two independent sites of interaction were identified between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed of five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors to complement the conventional antivenom administration.

Human Kir2.1 Potassium Channel: Protocols for Cryo-EM Data Processing and Molecular Dynamics Simulations.

Kir channels are potassium (K+) channels responsible for the mechanism of inward rectification, which plays a fundamental role in maintaining the resting membrane potential. There are seven Kir subfamilies, and their opening and closing mechanism is regulated by different regulatory factors. Genetically inherited defects in Kir channels are responsible for several rare human diseases, and for most of them, there are currently no effective therapeutic treatments. High-resolution structural information is not available for several members within the Kir subfamilies. Recently, our group achieved a significant breakthrough by utilizing cryo-EM single-particle analysis to elucidate the first structure of the human Kir2.1 channel. We present here the data processing protocol of the cryo-EM data of the human Kir2.1 channel, which is applicable to the structural determination of other ion channels by cryo-EM single-particle analysis. We also introduce a protocol designed to assess the structural heterogeneity within the cryo-EM data, allowing for the identification of other possible protein structure conformations present in the collected data. Moreover, we present a protocol for conducting all-atom molecular dynamics (MD) simulations for K+ channels, which can be incorporated into various membrane models to simulate different environments. We also propose some methods for analyzing the MD simulations, with a particular emphasis on assessing the local mobility of protein residues.

Structural studies with crotoxin B from Crotalus durissus collilineatus venom suggest a heterodimeric assembly formed by two new isoforms.

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms.

Detection of mixed populations of wild-type and YMDD hepatitis B variants by pyrosequencing in acutely and chronically infected patients.

BACKGROUND: Lamivudine (LAM) is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods. RESULTS: Phylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F) for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P = 0.012). As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20) had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies. CONCLUSIONS: YMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD variants before virological or biochemical breakthrough might contribute to making better therapy choices and thus improving treatment outcome.

Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom.

Two myotoxic and noncatalytic Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A(2) (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56-2.05 Šand belonged to space groups P3(1)21 (braziliantoxin-II), P6(5)22 (braziliantoxin-III) and P2(1) (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A(2) braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A(2).

Modelling level I Axillary Lymph Nodes depth for Microwave Imaging.

PURPOSE: Patient-specific information on the depth of Axillary Lymph Nodes (ALNs) is important for the development of new diagnostic imaging technologies, e.g. Microwave Imaging (MWI), aiming to assess the diagnosis of ALNs during breast cancer staging. Studies about ALNs depth have been presented for treatment planning, but they lack information on sample size and usability of the data to infer the depth of ALNs. The aim of this study was to create a mathematical model that can be used to predict a depth interval where level I ALNs are likely to be located. METHODS: We extracted biometric features of 98 patients who underwent breast Magnetic Resonance Imaging (MRI) to train two types of regression models. We then tested different combination of features to predict ALNs depth and found the best predictor. The final prediction models were then implemented in an algorithm used for MWI and tested with anthropomorphic phantoms of the axillary region. RESULTS: Body Mass Index (BMI) was the feature with best performance to predict ALNs depth with coefficient of determination (R2) ranging from 0.49 to 0.55 and Root Mean Squared Error (RMSE) ranging from 0.68 to 0.91 cm. The proposed model showed satisfactory results in microwave images of patients with different BMIs. CONCLUSIONS: The presented results contribute to the development of reconstruction algorithms for new imaging technologies and to the assessment of ALNs in other medical applications.

Cryo-electron microscopy unveils unique structural features of the human Kir2.1 channel.

We present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen's syndrome. The structural analysis (cryo-electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP2-binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP2. Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel.

Increase in Hepatitis A Cases Linked to Imported Strains to Rio de Janeiro, Brazil: A Cross-Sectional Study.

This study aims to evaluate the epidemiological and molecular features associated with HAV transmission in adults in Rio de Janeiro during a period of increased registered cases of HAV (2017-2018). Socio-epidemiological data and serum samples from anti-HAV IgM+ individuals were obtained. HAV RNA was RT-PCR amplified and sequenced for further phylogenetic and phylogeographic analyses. From fifty-two HAV IgM+ individuals, most were men (78.85%; p = 0.024), aged 20-30 years old (84.61%; p < 0.001), resided in the Rio de Janeiro north zone (31/52; 59.62%; p = 0.001), and are men who have sex with men (MSM) (57.69%; p = 0.002). Sexual practices were more frequent (96%) than others risk factors (food-borne (44%), water-borne (42.31%), and parenteral (34.62%)). Individuals who traveled to endemic regions had a 7.19-fold (1.93-36.04; p < 0.01) increased risk of HAV. Phylogenetic analysis revealed four distinct clades of subgenotype IA, three of them comprised sequences from European/Asian MSM outbreaks and one from Brazilian endemic strains. Bayesian Inference showed that the imported strains were introduced to Brazil during large mass sportive events. Sexual orientation and sexual practices may play a role in acquiring HAV infection. Public policies targeting key populations must be implemented to prevent further dissemination of HAV and other STIs.

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A2 class.

Phospholipases A2 (PLA2s) are enzymes responsible for membrane disruption through Ca(2+) -dependent hydrolysis of phospholipids. Lys49-PLA2s are well-characterized homologue PLA2s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA2s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA2 (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA2 conserves all the residues responsible for Ca(2+) coordination and of the catalytic network, features thought to be fundamental for PLA2 enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA2s. Crystallographic studies of the complex BthTX-II/Ca(2+) show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca(2+) are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA2s than to other Asp49-PLA2s, thus allowing Crotalinae subfamily PLA2s to be classified into two main branches: a catalytic and a myotoxic one.

Antibody dynamics and spontaneous viral clearance in patients with acute hepatitis C infection in Rio de Janeiro, Brazil.

BACKGROUND: The anti-HCV antibody response has not been well characterized during the early phase of HCV infection and little is known about its relationship to the clinical course during this period. METHODS: We analyzed serial anti-HCV antibodies longitudinally obtained from a prospective cohort of 65 patients with acute HCV infection by using a microparticle enzyme immunoassay AxSYM HCV 3.0 (Abbott Diagnostics) during the first 12 months from HCV acquisition in Rio de Janeiro, Brazil. Spontaneous viral clearance (SVC) was defined as undetectable HCV RNA in serum, in the absence of treatment, for three consecutive HCV PCR tests within 12-months of follow-up. RESULTS: Baseline antibody values were similar among patient groups with self-limiting HCV evolution (n = 34) and persistent viremia (n = 31) [median (interquartile range) signal/cut-off ratio (s/co) 78.7 (60.7-93.8) vs. 93.9 (67.8-111.9), p = 0.26]. During 12-months follow-up, patients with acute spontaneous resolving HCV infection showed significantly lower serial antibody response in comparison to individuals progressing to chronic infection [median (interquartile range) s/co 62.7 (35.2-85.0) vs. 98.4 (70.4-127.4), p < 0.0001]. In addition, patients with self-limiting HCV evolution exhibited an expeditious, sharp decline of serial antibody values after SVC in comparison to those measured before SVC [median (interquartile range) s/co 56.0 (25.4-79.3) vs. 79.4 (66.3-103.0), p < 0.0001]. CONCLUSION: Our findings indicate a rapid short-term decline of antibody values in patients with acute spontaneous resolving HCV infection.