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Introduction: The maximum oxygen uptake (V˙O 2 max), the maximum rate of oxygen that can be sustained before the onset of blood lactate accumulation, and the metabolic cost of locomotion are the main physiological factors associated with long-distance running performance. The latter is known as the running economy. Generally, runners reach peak performance in long races between 25 and 30 years of age, with a progressive decline occurring thereafter. However, it is not known whether the running economy is affected or how it is affected by aging., Aim: To investigate the effect of age and years of running experience on the running economy of amateur long-distance runners aged 20-80 years., Methods: Sixty-nine recreational long-distance runners, divided into five age groups according to decade of life, participated in this study: Group 1 (n= 9) 27.2 ± 1.3 years, Group 2 (n= 18) 35.9 ± 2.2 years, Group 3 (n= 17) 43.4 ± 2.8 years, Group 4 (n= 17) 53.0 ± 2.3 years, and Group 5 (n= 8) 65.5 ± 2.9 years. For running economy assessment, oxygen cost (OC) and energy cost (EC) were measured. Furthermore, the participants were interviewed on their running experience., Results: For EC, the two independent variables composing the regression model were age (ß = 0.703, t= 5.443, p < 0.001) and running experience (ß = -0.230, t = -1.785, p= 0.07), and 34% of the energy cost variation can be explained by these two factors. EC and OC were compared among the groups. There were no significant differences between Groups 1 and 2 (p= 0.999), Groups 1 and 3 (p= 1.000), and Groups 1 and 4 (p= 0.528). However, Group 5 had a significantly higher energy cost than Group 1 (p < 0.001), Group 2 (p < 0.001), Group 3 (p < 0.001) and Group 4 (p < 0.001)., Conclusion: The number of years of running experience has a positive effect on running economy, but it is insufficient to overcome the negative effect of the aging process. Furthermore, running economy was significantly worse in participants aged ≥60 years compared with that in younger athletes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Rhipicephalus microplus, popularly known as the cattle tick, is the most important tick of livestock as it is responsible for significant economic losses. The use of chemical acaricides is still the most widely used control method despite its known disadvantages. Vaccination would be a safe alternative for the control of R. microplus and holds advantages over the use of chemical acaricides as it is environmental-friendly and leaves no residues in meat or milk. Two vaccines based on the Bm86 protein were commercialized, TickGARD® and Gavac®, with varying reported efficacies in different countries. The use of other vaccines, such as Tick Vac®, Go-Tick®, and Bovimune Ixovac® have been restricted to some countries. Several other proteins have been analyzed as possible antigens for more effective vaccines against R. microplus, including peptidases, serine proteinase inhibitors, glutathione S-transferases, metalloproteases, and ribosomal proteins, with efficacies ranging from 14% to 96%. Nonetheless, more research is needed to develop safe and efficient tick vaccines, such as the evaluation of the efficacy of antigens against other tick species to verify cross-reactivity and inclusion of additional antigens to promote the blocking of the infection and spreading of tick-borne diseases. This review summarizes the discoveries of candidate antigens for R. microplus tick vaccines as well as the methods used to test their efficacy., (Copyright © 2021. Published by Elsevier GmbH.)

Background: Morphological changes characteristic of femoroacetabular impingement (FAI) are common in soccer players. However, the clinical relevance of such anatomical variations is still not well-defined., Hypothesis: We hypothesized that high alpha angle values and/or acetabular retroversion index (ARI) are correlated with rotational range of motion (ROM) of the hip and that there are clinical-radiological diferences between the dominant lower limb (DLL) and nondominant lower limb (NDLL) in professional soccer players., Study Design: Cross-sectional., Level of Evidence: Level 3., Methods: A total of 59 male professional soccer players (average age 25.5 years, range 18-38 years) were evaluated in the preseason. As main outcome measures, we evaluated the alpha angle and the ARI and hip IR and ER ROM with radiographic analysis., Results: The measurements taken on DLL and NDLL were compared and a significant difference was found between the sides in the ER ( P = 0.027), where the DLL measures were 1.54° (95% CI, 0.18-2.89) greater than the NDLL. There were no significant differences between the sides in the measures of IR ( P > 0.99), total ROM ( P = 0.07), alpha angle ( P = 0.250), and ARI ( P = 0.079). The correlations between the rotation measurements and the alpha angle in each limb were evaluated and the coefficient values showed no correlation; so also between the ARI and rotation measures., Conclusion: Morphological changes of the femur or acetabulum are not correlated with hip IR and ER ROM in male professional soccer players. ER on the dominant side was greater than on the nondominant side. There was no significant difference in the other measurements between sides., Clinical Relevance: In clinical practice, it is common to attribute loss of hip rotational movement to the presence of FAI. This study shows that anatomical FAI may not have a very strong influence on available hip rotational movement in professional soccer athletes.

Background: Throwing athletes present alterations in shoulder rotation range of motion (ROM), but not much is known about the relationship between these alterations and performance measurements in volleyball practitioners., Purpose: To compare the passive ranges of motion of internal rotation (IR), external rotation (ER), and total rotation motion (TRM) of the shoulder in dominant and nondominant limbs of young volleyball athletes and to investigate their relationship with ball speed during serves with and without precision (inside and outside court, respectively). The possible association of anthropometrics and competitive practice time with these velocities was also investigated., Study Design: Cross-sectional study., Methods: Fifty-seven male volleyball athletes (mean age 17.11 ± 1.88 y; weight 74.68 ± 9.7 kg; height 1.87 ± 0.09 cm) were evaluated for shoulder IR and ER with a bubble goniometer and serve speed inside and outside court was measured with a radar gun. Simple and multiple regression analyses were applied to investigate associations of ROM, anthropometrics, and competitive practice time with serve speed., Results: Dominant shoulders had diminished IR ROM compared to nondominant shoulders (59.1º ± 16.7º vs 66.4º ± 16.9º; p < 0.001) as well as diminished TRM (173.5º ± 31.8º vs 179.1º ± 29.9º; p < 0.001). Simple regression showed negative association between dominant ER and serve speed outside the court ( p = 0.004). Positive associations existed between age and serve speed in both conditions ( p < 0.001), BMI and speed inside ( p = 0.009) and outside the court ( p = 0.008), and between competitive practice time and speed inside ( p = 0.008) and outside court ( p = 0.003). However, multiple analysis confirmed only age ( p < 0.001) and BMI to be associated with ball velocities (inside court p = 0.034; outside court p = 0.031)., Conclusion: The results of this study demonstrated that young volleyball athletes presented lower IR and TRM of the shoulder in the dominant upper limb. Age and BMI were directly associated with ball velocities when serving. Passive rotation ROM did not have a relationship with this performance measurement., Level of Evidence: 3b., Competing Interests: The authors have no conflict of interest to declare.

We investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores were produced in solid or liquid Adámek-modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m -2 under artificial UV-B radiation. Our results showed that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in regions where heat and UV-B are limiting environmental factors., Competing Interests: Declaration of competing interest The authors have declared no competing interests., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The purpose of this study was to investigate the effectiveness of adding photobiomodulation therapy and neuromuscular electrical stimulation (NMES) to volleyball athletes' training, focusing on muscle strength and jumping skills. Thirty-six athletes were randomly placed into three groups: control, photobiomodulation therapy, and NMES. The athletes trained to improve their muscle strength and jumping skills. The athletes in the photobiomodulation therapy group were submitted to photobiomodulation therapy (850 nm, continuous, energy density 0.8 J/cm 2 , radiant energy per point 6 J, total radiant energy 36 J) before undergoing strength and plyometric training. The NMES group additionally underwent NMES-based quadriceps femoris muscle strength training (base frequency 1 kHz, frequency modulation 70 Hz, intensity maximum tolerable). The variables analyzed were muscle strength, jumping ability, global impression, and jump frequency; they were measured at baseline and during follow-ups at 6 and 8 weeks. The statistical analysis was conducted on an intention-to-treat basis. The between-group differences and their respective 95% CIs were calculated using linear mixed models by using group, time, and group-versus-time interaction terms. Dominant lower limb strength improved the most in the NMES group compared to the control group (mean difference = 1.4, 95% CI = .5 to 2.4). Non-dominant lower limb strength increased in both the photobiomodulation therapy group (mean difference = 1.1, 95% CI = .3 to 2) and the NMES group (mean difference = 1.9, 95% CI = 1.1 to 2.8) compared to the control group, but the NMES group improved more than the photobiomodulation therapy group (mean difference = 0.8, 95% CI = 0.1 to 1.7). The NMES group had the greatest improvement in global perceived effect scale compared to the control group (mean difference = 1.1, 95% CI = 1 to 2.2). Dominant lower limb strength improved in the NMES group compared to the control group. Non-dominant lower limb strength increased in both the photobiomodulation therapy group and the NMES group compared to the control group, but the NMES group improved significantly more than the photobiomodulation therapy group; the NMES group also improved in the global perceived effect scale compared to the control group. This study found that, for volleyball athletes, photobiomodulation therapy and NMES both promoted benefits in terms of muscle-strength gain. In addition, these benefits were maintained for 2 weeks even after training was interrupted. Dominant lower limb strength improved in the NMES group compared to the control group. Non-dominant lower limb strength increased in both the photobiomodulation therapy group and the NMES group compared to the control group, but the NMES group improved significantly more than the photobiomodulation therapy group; the NMES group also improved in global impression of jumps compared to the control group.

Background: Despite the increasing international popularity of CrossFit, there is a paucity of scientific evidence on the risk of CrossFit-related musculoskeletal injuries (CRMIs)., Purpose: To investigate the incidence (cumulative incidence proportion [IP] and incidence density [ID]) of CRMIs and the association of CRMIs with personal and training characteristics., Study Design: Descriptive epidemiology study., Methods: A prospective, 12-week descriptive epidemiology cohort study was conducted in a convenience sample of CrossFit facilities in a single Brazilian city. Printed baseline questionnaires were distributed to 13 CrossFit boxes. All participants who filled out the questionnaire and consented to participate in the study were invited to respond to an online follow-up questionnaire every 2 weeks to collect data on CrossFit training characteristics and CRMIs. A CRMI was defined as any self-reported musculoskeletal injury or pain that prevented an athlete from exercising for at least 1 day. The IP was defined as the number of new cases divided by the entire population at risk, while the ID was defined as new events divided by the total person-time exposure in hours. Logistic mixed models were developed to investigate the association of CRMIs with personal and training characteristics., Results: A total of 515 CrossFit participants filled out the baseline questionnaire and provided informed consent, and 406 (78.8%) completed at least 1 follow-up measure. There were 133 participants who reported at least 1 CRMI during the study, and a total of 247 unique and new CRMIs were reported over a total estimated person-time exposure to CrossFit of 13,041 hours. The IP was 32.8% (95% CI, 28.4%-37.5%). The ID was 18.9 (95% CI, 16.6-21.3) per 1000 hours of CrossFit exposure. The shoulders (19.0%; n = 47) and lumbar spine (15.0%; n = 37) were most affected. Muscle injuries (45.3%; n = 112) and joint pain (24.7%; n = 61) were the most common CRMI types reported. Switching between prescribed and scaled down training loads (odds ratio [OR], 3.5 [95% CI, 1.7-7.3]) and previous injuries (OR, 3.2 [95% CI, 1.4-7.7]) were risk factors for a CRMI, while CrossFit experience was identified as a protective factor (OR, 0.7 [95% CI, 0.5-1.0])., Conclusion: In this 12-week prospective study, the ID was 18.9 CRMIs per 1000 hours of exposure; switching between training loads and previous injuries was associated with 3.5- and 3.2-fold higher odds, respectively, of sustaining CRMIs., Competing Interests: The authors declared that there are no conflicts of interest in the authorship and publication of this contribution. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2020.)

Bovine cysticercosis, caused by Taenia saginata metacestodes, is the cause of significant economic losses to the meat production chain by condemnation and downgrading of infected carcasses. It is also a public health issue causing human taeniasis. This study evaluated the occurrence of bovine cysticercosis at the meat inspection procedures in slaughterhouses of south and north regions of the Tocantins State in Brazil. Specimens identified as cysts of T. saginata were collected and analyzed by molecular (PCR) and histopathological techniques. The cysts were collected from March to December of 2010 in slaughterhouses located in the cities of Alvorada (South) and Araguaína (North). The frequency of cystic lesions during the study was 0.033% (53/164,091) with 69.81% of calcified lesions and 30.9% of live cysts at meat inspection. From 14 samples submitted to molecular analysis, 28.57% (4/14) were positive for T. saginata . The histopathological analysis of the non- T. saginata samples showed lesions suggestive of granuloma and hydatid disease. The results indicated that the identification of the etiological agent is difficult by macroscopic inspection, emphasizing the need to associate specific diagnostic methods at meat inspection in abattoirs. In addition, species-specific PCR would be an effective tool for diagnosis, monitoring, and identifying cysticercosis, assisting the conventional tests.

Background: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy., Objective: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL)., Methods: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection., Findings: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis., Main Conclusions: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite., Competing Interests: The authors have declared that no competing interests exist.

Objective: To evaluate pain, functional capacity, and quality of life of patients with non-specific chronic low back pain, after home-based exercise therapy with different kinds of supervision., Method: Thirty individuals of both gender, between 18 and 65 years old, performed the proposed exercises three times a week, for eight weeks. Group A (N = 17) performed the exercises after a single supervised session. Group B (N = 13) was supervised once a week at the rehabilitation center. Both groups received a booklet with instructions, and questionnaires to evaluate pain, functional capacity and quality of life; during the initial evaluation, after four and eight weeks., Results: There was an improvement in pain and functional capacity between the initial evaluation and week 4, and the initial evaluation and week 8 in both groups (p <0.05). In the quality of life evaluation, the criteria for pain, functional capacity, and physical aspects had significant improvement after 8 weeks (p <0.05). There was no difference when comparing groups A and B (p >0,05)., Conclusion: Home-based exercise therapy, when performed in a period of eight weeks, using the booklet, was effective for improving level of pain, functional capacity, and quality of life in patients with non-specific chronic low back pain. The weekly supervision did not significantly influence the final outcome between the groups.

Botulism is a well-known intoxication that affects humans and animals. The disease is endemic in cattle in Brazil and recently emerged as an important disease in commercial laying hens and broiler chickens in Europe. Dogs and other animal species can also be affected. Although antitoxins are commonly administered to humans diagnosed with botulism, in animals this is rarely the case and the treatment of botulism is still based only on support therapy. In the present work, we report an outbreak of type C botulism in Brazil that simultaneously affected domestic chickens, dogs and a black-pencilled marmoset (Callithrix penicillata). The successful use of Clostridium botulinum types C and D antitoxin for the treatment of an affected dog is also described., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Weakness of the rotator cuff muscles can lead to imbalances in the strength of shoulder external and internal rotators, change the biomechanics of the glenohumeral joint and predispose an athlete to injury. Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has demonstrated promising results in a variety of health conditions. However few studies addressed its potential approach in the realm of athletics., Hypothesis/purpose: The purpose of this study was to investigate if transcranial direct current stimulation (tDCS) technique increases the isometric muscle strength of shoulder external and internal rotators in handball athletes., Study Design: Randomized, double-blind, placebo-controlled, crossover study., Methods: Eight female handball players aged between 17 and 21 years (Mean=19.65; SD=2.55) with 7.1 ± 4.8 years of experience in training, participating in regional and national competitions were recruited. Maximal voluntary isometric contraction (MVIC) of shoulder external and internal rotator muscles was evaluated during and after 30 and 60 minutes post one session of anodal and sham current (2mA; 0.057mA/cm 2 ) with a one-week interval between stimulations., Results: Compared to baseline, MVIC of shoulder external and internal rotators significantly increased after real but not sham tDCS. Between-group differences were observed for external and internal rotator muscles. Maximal voluntary isometric contraction of external rotation increased significantly during tDCS, and 30 and 60 minutes post-tDCS for real tDCS compared to that for sham tDCS. For internal rotation MVIC increased significantly during and 60 minutes post-tDCS., Conclusions: The results indicate that transcranial direct current stimulation temporarily increases maximal isometric contractions of the internal and external rotators of the shoulder in handball players., Level of Evidence: 2.

The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB). Using methods of enhancing protein stability in solution, we tested different expression, cell lysis, and purification protocols with and without ligand supplementation. The protein stability was evaluated by dynamic light scattering and circular dichroism spectroscopy assays. Best-derived protocols (expression at 18 °C, cell lysis with homogenizer, and three purification steps) were used to produce 20 mg of homogeneous 6xHis-NahB per liter of culture. The secondary and quaternary structures of 6xHis-NahB were assessed by circular dichroism and size-exclusion chromatography experiments, respectively. The enzyme was NAD + -dependent and active at pH 7.0 and 9.4 for the oxidation of the substrate. The Michaelis-Menten parameters determined at pH 7.0 and 25 °C for the substrate and cofactor, presented respective K m values of 6 and 350 μM, and a k cat value of 8.3 s -1 . Furthermore, we identified conditions for the crystallization of 6xHis-NahB. X-ray diffraction data were collected from a single 6xHis-NahB crystal which diffracted to 2.21 Å. The crystal belongs to space group I222, with unit-cell parameters a = 63.62, b = 69.50, and c = 117.47 Å. The tertiary structure of 6xHis-NahB was determined using the molecular replacement method. Further structural refinement is currently underway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Study Design Clinical measurement study. Background Ankle sprain is a common condition. Therefore, translated, culturally adapted, and clinimetrically tested instruments for measuring the ankle are needed. Objectives To translate and cross-culturally adapt the Sports Athlete Foot and Ankle Score (SAFAS) and the global rating of change (GROC) into Brazilian Portuguese. This study also aimed to test the measurement properties of the SAFAS, the GROC, and the existing Brazilian-Portuguese versions of the numeric pain-rating scale (NPRS) and the ankle-hindfoot scale of the American Orthopaedic Foot and Ankle Society (AOFAS) in a group with ankle sprain. Methods The SAFAS and GROC instruments were cross-culturally adapted into Brazilian Portuguese and pretested in this population (n = 18). The measurement properties of the SAFAS, GROC, NPRS, and AOFAS (internal consistency, ceiling and floor effects, construct validity, reproducibility, and responsiveness) were tested in a test-retest design in patients with ankle sprain at baseline (n = 63) and at follow-up periods of 48 to 72 hours (n = 53) and 4 weeks (n = 43) after baseline. Results The SAFAS, GROC, NPRS, and AOFAS yielded considerable internal consistency (Cronbach alpha ranging from .76 to .97), moderate reliability (intraclass correlation coefficient model 2,1 ranging from 0.59 to 0.80), and dubious agreement (percentage of the standard error of measurement ranging from 13% to 22.43%). The highest correlations on construct validity were observed among the 4 SAFAS subscales (Pearson r = 0.75 to 0.86, P<.01), but statistically significant values appeared in the intercorrelation of instruments. There were no observed floor or ceiling effects in any of the instruments. All of their scores of change showed moderate correlation with the GROC (r = -0.48 to 0.30, P<.05) and could be classified as responsive. Conclusion The Brazilian-Portuguese versions of the SAFAS, GROC, NPRS, and AOFAS had acceptable measurement properties in high-performing athletes. J Orthop Sports Phys Ther 2016;46(12):1042-1050. Epub 8 Nov 2016. doi:10.2519/jospt.2016.6218.

In the present investigation we used a recombinant LiD1 toxin, named rLiD1his, from Loxosceles intermedia brown spider to elicit specific antibodies in mice carrying different Human Leukocyte Antigens class II (HLAII) {DRB1.0401 (DR4), DQB1.0601 (DQ6) and DQB1.0302 (DQ8)} as well as in BALB/C and C57BL/6 control mice. All mice strains produced high antibody titers against rLiD1his but DR4 mice antibodies (the lower responder mice) were not able to recognize L. intermedia crude venom. The anti-rLiD1his sera, except from DR4 mice, were able to neutralize dermonecrotic, hemorrhagic and edematogenic effects of rLiD1his in naïve rabbits. Overlapping peptides from the amino acid sequence of LiD1 toxin were prepared by SPOT method and differences in LiD1 epitope recognition were observed using different mice anti-rLiD1his sera. The region (160)DKVGHDFSGNDDISDVGK(177) was recognized by transgenic DQ8 and DQ6 mice sera. Other epitopes were recognized by at least two different animals' sera including (10)MGHMVNAIGQIDEFVNLG(27), (37)FDDNANPEYTYHGIP(51), (70)GLRSATTPGNSKYQEKLV(87) and (259)AAYKKKFRVATYDDN(273). Among these epitopes, the epitopes 37-51 and 160-177 have already been shown in previously studies as good candidates to be used alone or combined with other peptides to induce protective immune response against Loxosceles venoms. The results presented here highlight the importance of HLAII in antibody response and recognition of specific B-cell epitopes of rLiD1his spider toxin according to HLAII type and impact in the epitopic vaccine development against this spider., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Snake venom metalloproteinases are important toxins that play fundamental roles during envenomation. They share a structurally similar catalytic domain, but with diverse hemorrhagic capabilities. To understand the structural basis for this difference, we build and compare two dynamical models, one for the hemorrhagic atroxlysin-I from Bothrops atrox and the other for the non-hemorraghic leucurolysin-a from Bothrops leucurus. The analysis of the extended molecular dynamics simulations shows some changes in the local structure, flexibility and surface determinants that can contribute to explain the different hemorrhagic activity of the two enzymes. In agreement with previous results, the long Ω-loop (from residue 149 to 177) has a larger mobility in the hemorrhagic protein. In addition, we find some potentially-relevant differences at the base of the S1' pocket, what may be interesting for the structure-based design of new anti-venom agents. However, the sharpest differences in the computational models of atroxlysin-I and leucurolysin-a are observed in the surface electrostatic potential around the active site region, suggesting thus that the hemorrhagic versus non-hemorrhagic activity is probably determined by protein surface determinants.

The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD(+). NahI is in family 8 (ALDH8) of the NAD(P)(+)-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress as a hexa-histidine-tagged fusion protein. After purification by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted and, for the first time, KM (1.3±0.3μM) and kcat (0.9s(-1)) values were determined at presumed NAD(+) saturation. NahI is highly specific for its biological substrate and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens' sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).

Study Design: Clinical measurement study., Objectives: To cross-culturally adapt the Anterior Knee Pain Scale (AKPS), the Functional Index Questionnaire (FIQ), and the Pain Severity Scale (PSS) for patellofemoral pain syndrome (PFPS) into Brazilian Portuguese. This study also aimed to test the measurement properties of the AKPS, the FIQ, and the PSS, and the existing Brazilian Portuguese versions of the numeric pain rating scale (NPRS) and the Global Perceived Effect scale in a group with PFPS., Background: PFPS is a common condition. Therefore, translated, culturally adapted, and clinimetrically tested instruments for measuring PFPS are needed., Methods: The AKPS, FIQ, and PSS instruments were cross-culturally adapted into Brazilian Portuguese. The measurement properties of the AKPS, FIQ, PSS, NPRS, and Global Perceived Effect scale (internal consistency, ceiling and floor effects, and construct validity) were tested in 83 patients with PFPS. The reproducibility and responsiveness were tested in 52 patients with PFPS in a test-retest design, with follow-up testing at 48 to 72 hours and at 4 weeks after baseline., Results: The AKPS, the FIQ, and the PSS yielded adequate internal consistency (Cronbach alpha ranging from .75 to .87) and excellent reliability (intraclass correlation coefficients [model 2,1] ranging from 0.90 to 0.97). The AKPS and the PSS yielded very good agreement (standard error of measurement, 2.9% and 3.5%, respectively). The highest correlations were observed among the AKPS, the FIQ, and the PSS (Pearson r>0.60, P<.05). No floor or ceiling effects were observed for any of the instruments. Effect sizes used for measuring internal responsiveness ranged from moderate to high for all measures. The NPRS and the AKPS were the measures with the highest external responsiveness., Conclusion: The Brazilian Portuguese versions of the AKPS, FIQ, PSS, NPRS, and Global Perceived Effect scale have acceptable measurement properties.

Study Design: Clinical measurement., Objectives: To translate, adapt, and test the measurement properties of the Brazilian Portuguese version of the Victorian Institute of Sport Assessment-Patella (VISA-P) questionnaire., Background: It is important to objectively measure symptoms and functional limitations related to patellar tendinopathy using outcome measures that have been validated in the language of the target population. Cross-cultural adaptations are also useful to enhance the understanding of the measurement properties of an assessment tool, regardless of the target language., Methods: The VISA-P questionnaire was translated into Brazilian Portuguese, culturally adapted, and titled VISA-P Brazil. It was then administered on 2 occasions with a 24- to 48-hour interval between them, and a third time after a month of physical therapy treatment. The following measurement properties were analyzed: internal consistency, test-retest reliability, agreement, construct validity, floor and ceiling effects, and responsiveness., Results: The VISA-P Brazil had high internal consistency (Cronbach α = .76; if item deleted, Cronbach α = .69-.78), excellent reliability and agreement (intraclass correlation coefficient = 0.91; 95% confidence interval: 0.85, 0.95; standard error of measurement, 5.2 points; minimal detectable change at the 90% confidence level, 12.2 points), and good construct validity (Pearson r = 0.60 compared to Lysholm). No ceiling and floor effects were detected for the VISA-P Brazil, and the responsiveness, based on 32 patients receiving physical therapy intervention for 1 month, demonstrated a large effect size of 0.97 (95% confidence interval: 0.68, 1.25)., Conclusion: The VISA-P Brazil is a reproducible and responsive tool and can be used in clinical practice and research to assess the severity of pain and disability of patients with patellar tendinopathy.

Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. The nah operon in P. putida, which is present on the 83 kb metabolic plasmid NAH7, codes for enzymes involved in the conversion of naphthalene to salicylate. The enzyme NahF (salicylaldehyde dehydrogenase) catalyzes the last reaction in this pathway. The nahF gene was subcloned into the pET28a(TEV) vector and the recombinant protein was overexpressed in Escherichia coli Arctic Express at 285 K. The soluble protein was purified by affinity chromatography followed by gel filtration. Crystals of recombinant NahF (6×His-NahF) were obtained at 291 K and diffracted to 2.42 Å resolution. They belonged to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 169.47, c = 157.94 Å. The asymmetric unit contained a monomer and a crystallographic twofold axis generated the dimeric biological unit., (© 2012 International Union of Crystallography. All rights reserved.)

Bovine trypsin is a model system for the serine protease class of enzymes, which is an important target for contemporary medicinal chemistry. Some structural and thermodynamic reports are available on its interaction with benzamidine-based compounds but no structural information is available so far on its binding modes to the active principles of the trypanocidal drugs Pentacarinate (pentamidine) and Berenil (diminazene). The crystallographic structures of bovine beta-trypsin in complex with the ligands were determined to a resolution of 1.57 A (diminazene) and 1.70 A (diminazene and pentamidine). The second benzamidine moieties in these inhibitors are bound to the enzyme in different hot spots and only few hydrogen bonds mediate these interactions. Thermodynamic parameters for the association of pentamidine with beta-trypsin reveal that this inhibitor has about 1.3-fold lower affinity than diminazene. Moreover its binding mode resembles other benzamidine-based compounds that assess the aryl binding pocket of the enzyme; however, with almost 2.5-fold higher affinity. This is the first structural evidence of the binding of Berenil and Pentacarinate active principles trypanocidal drugs to serine proteases., (Copyright 2010 Elsevier B.V. All rights reserved.)

Leucurolysin-a (leuc-a) is a class P-I snake-venom metalloproteinase isolated from the venom of the South American snake Bothrops leucurus (white-tailed jararaca). The mature protein is composed of 202 amino-acid residues in a single polypeptide chain. It contains a blocked N-terminus and is not glycosylated. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood. Unlike some other venom fibrinolytic metalloproteinases, leuc-a has no haemorrhagic activity. Leuc-a was sequenced and was crystallized using the hanging-drop vapour-diffusion technique. Crystals were obtained using PEG 6000 or PEG 1500. Diffraction data to 1.80 and 1.60 A resolution were collected from two crystals (free enzyme and the endogenous ligand-protein complex, respectively). They both belonged to space group P2(1)2(1)2(1), with very similar unit-cell parameters (a = 44.0, b = 56.2, c = 76.3 A for the free-enzyme crystal).

Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P2(1)), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 A, beta = 96.81 degrees , and diffract X-rays to 1.8 A resolution.

Interleukin-22 (IL-22) is a cytokine that regulates the production of acute phase proteins of the immunological response. On binding to its cognate receptor (IL-22R1), which is associated to the interleukin-10 receptor 2 (IL-10R2), IL-22 promotes activation of signal transducer and activator of transcription (STAT) pathway and several other cellular responses. A soluble receptor termed interleukin-22 binding protein (IL-22BP) is also able to bind to IL-22 as a natural protein antagonist, and probably provides systemic regulation of IL-22 activity. This inflammatory response system is analyzed here in terms of its molecular physiology and structural assembly. Three-dimensional (3D) model of IL-22 and structural basis of its interactions with the cognate receptors are discussed.

Interleukin-22 (IL-10-related T cell-derived inducible factor/IL-TIF/IL-22) is a novel cytokine belonging to the IL-10 family. Recombinant human IL-22 (hIL-22) was found to activate the signal transducers and activators of transcription factors 1 and 3 as well as acute phase reactants in several hepatoma cell lines, suggesting its involvement in the inflammatory response. The crystallographic structure of recombinant hIL-22 has been solved at 2.0 A resolution using the SIRAS method. Contrary to IL-10, the hIL-22 dimer does not present an interpenetration of the secondary-structure elements belonging to the two distinct polypeptide chains but results from interface interactions between monomers. Structural differences between these two cytokines, revealed by the crystallographic studies, clearly indicate that, while a homodimer of IL-10 is required for signaling, hIL-22 most probably interacts with its receptor as a monomer.