Expression and purification of active shikimate dehydrogenase from Plasmodium falciparum.

Plasmodium falciparum is known to cause severe malaria, current treatment consists in artemisinin-based combination therapy, but resistance can lead to treatment failure. Knowledge concerning P. falciparum essential proteins can be used for searching new antimalarials, among these a potential candidate is shikimate dehydrogenase (SDH), an enzyme part of the shikimate pathway which is responsible for producing endogenous aromatic amino acids. SDH from P. falciparum (PfSDH) is unexplored by the scientific community, therefore, this study aims to establish the first protocol for active PfSDH expression. Putative PfSDH nucleotide sequence was used to construct an optimized expression vector pET28a+PfSDH inserted in E. coli BL21(DE3). As a result, optimal expression conditions were acquired by varying IPTG and temperature through time. Western Blot analysis was applied to verify appropriate PfSDH expression, solubilization and purification started with lysis followed by two-steps IMAC purification. Enzyme activity was measured spectrophotometrically by NADPH oxidation, optimal PfSDH expression occur at 0.1 mM IPTG for 48 hours growing at 37 °C and shaking at 200 rpm, recombinant PfSDH obtained after purification was soluble, pure and its physiological catalysis was confirmed. Thus, this study describes the first protocol for heterologous expression of PfSDH in soluble and active form.

Literature Review on Crotalus durissus terrificus Toxins: From a Perspective of Structural Biology and Therapeutic Applications.

BACKGROUND: The venom of Crotalus durissus terrificus, as well as its fractions, has intrigued research groups worldwide who are working to isolate, characterize, and find possible biotechnological applications. A number of studies have elucidated that these fractions and their derivatives possess pharmacological properties, which can enable the development of new drug prototypes with anti-inflammatory, antinociceptive, antitumor, antiviral, and antiparasitic applications. OBJECTIVE: This review presents a systematic study on Crotalus durissus terrificus, the most notable crotalid subspecies in South America, focusing on the composition, toxicological mechanisms, structural aspects, and applications of the main venom toxins (convulxin, gyroxin, crotamine, crotoxin, and their subunits). CONCLUSION: The authors have found that research on this snake and its toxins is still an area of focus, despite that almost a century has passed since the isolation of crotoxin. Several applications of these proteins in the development of novel drugs and bioactive substances have also been demonstrated.

Sensitivity comparison for the Leishmania spp. detection in different canine tissues using PCR-HRM.

BACKGROUND: Leishmaniasis is a vector-borne disease caused by a parasite protozoon from the genus Leishmania. Among the molecular techniques applied for detecting these parasites, real-time PCR with High Resolution Melting (PCR-HRM) proved advantageous since it simultaneously determines both the presence and species of the pathogen in one step, through amplification and later analysis of curves generated by melting temperature. METHODS: Based on this molecular technique, the goal of this study was to estimate the PCR-HRM sensitivity for Leishmania spp. detection in different canine tissues by evaluating biological samples obtained from popliteal, submandibular, and pre-scapular lymph nodes, from bone marrow and ear pinnae of 28 stray dogs captured in the metropolitan area of Asunción (Paraguay). RESULTS: The rk39 immunochromatographic test showed that 25/28 tested dogs (89%) presented antibodies against L. infantum. In 20/25 dogs that tested positive for rk39 (80%), it was possible to detect Leishmania spp. by PCR-HRM and determine that the species corresponded entirely to L. infantum. Regarding the analysis of different tissues, the parasite was detected in all popliteal lymph node samples, followed by high detection in submandibular (at 95%) and pre-scapular lymph nodes (at 90%), bone marrow (at 85%), and ear pinnae (at 85%). CONCLUSIONS: This study demonstrated that the use of real-time PCR-HRM using the molecular marker hsp70 was a highly sensitive method for simultaneously detecting and identifying Leishmania species in different tissues taken from infected dogs. In addition, the usefulness of ear pinnae as easily accessible tissue for molecular diagnosis was emphasized.

Sensitivity comparison for the Leishmania spp. detection in different canine tissues using PCR-HRM

ABSTRACT Background: Leishmaniasis is a vector-borne disease caused by a parasite protozoon from the genus Leishmania. Among the molecular techniques applied for detecting these parasites, real-time PCR with High Resolution Melting (PCR-HRM) proved advantageous since it simultaneously determines both the presence and species of the pathogen in one step, through amplification and later analysis of curves generated by melting temperature. Methods: Based on this molecular technique, the goal of this study was to estimate the PCR-HRM sensitivity for Leishmania spp. detection in different canine tissues by evaluating biological samples obtained from popliteal, submandibular, and pre-scapular lymph nodes, from bone marrow and ear pinnae of 28 stray dogs captured in the metropolitan area of Asunción (Paraguay). Results: The rk39 immunochromatographic test showed that 25/28 tested dogs (89%) presented antibodies against L. infantum. In 20/25 dogs that tested positive for rk39 (80%), it was possible to detect Leishmania spp. by PCR-HRM and determine that the species corresponded entirely to L. infantum. Regarding the analysis of different tissues, the parasite was detected in all popliteal lymph node samples, followed by high detection in submandibular (at 95%) and pre-scapular lymph nodes (at 90%), bone marrow (at 85%), and ear pinnae (at 85%). Conclusions: This study demonstrated that the use of real-time PCR-HRM using the molecular marker hsp70 was a highly sensitive method for simultaneously detecting and identifying Leishmania species in different tissues taken from infected dogs. In addition, the usefulness of ear pinnae as easily accessible tissue for molecular diagnosis was emphasized.

Antileishmanial activity evaluation of a natural amide and its synthetic analogs against Leishmania (V.) braziliensis: an integrated approach in vitro and in silico.

Leishmaniasis is considered a neglected disease, which makes it an unattractive market for the pharmaceutical industry; hence, efforts in the search for biologically active substances are hampered by this lack of financial motivation. Thus, in the present study, we report the leishmanicidal activity and the possible mechanisms of action of compounds with promising activity against the species Leishmania (V.) braziliensis, the causative agent of the skin disease leishmaniasis. The natural compound 1a (piplartine) and the analog 2a were the most potent against promastigote forms with growth inhibition values for 50% of the parasite population (IC50) = 8.58 and 11.25 µM, respectively. For amastigote forms, the ICa50 values were 1.46 and 16.7 µM, respectively. In the molecular docking study, piplartine showed favorable binding energy (-7.13 kcal/mol) and with 50% inhibition of trypanothione reductase (IC50) = 91.1 µM. Preliminary investigations of the mechanism of action indicate that piplartine increased ROS levels, induced loss of cell membrane integrity, and caused accumulation of lipid bodies after 24 h of incubation at its lowest effective concentration (IC50), which was not observed for the synthetic analog 2a. The mode of action for the leishmanicidal activity of piplartine (1a) was assigned to involve affinity for the trypanothione reductase of Leishmania (V.) braziliensis TR.

Calidad farmacéutica de tabletas de metronidazol y cápsulas de amoxicilina disponibles en el mercado hondureño / Pharmaceutical quality of tablets of metronidazole and capsules of amoxicilin available in the honduran market

INTRODUCCIÓN: el metronidazol, antiparasitario y antimicrobiano, y la amoxicilina, aminopenicilina de amplio espectro, son medicamentos de amplio uso. OBJETIVOS: determinar la calidad farmacéutica de formulaciones de administración oral disponibles en el mercado hondureño, en la ciudad de Tegucigalpa. MÉTODOS: se analizaron productos de cinco marcas de comprimidos de metronidazol 500 mg y cápsulas de amoxicilina 500 mg, respectivamente, todas ellas dentro de su período de validez de uso. Se evaluaron las propiedades tecnológicas y químicas de las tabletas y cápsulas, tales como peso pro-medio, dureza, valoración, uniformidad de contenido, ensayo de disolución, perfil de disolución, área bajo la curva y eficiencia de la disolución. RESULTADOS: no todas las especialidades medicinales contenían la información sobre las condiciones de almacenamiento, respecto a la temperatura, luz y humedad, declarados en los rótulos y prospectos. De acuerdo a la evaluación tecnológica y química, todos los productos evaluados cumplieron con las especificaciones de la farmacopea. Sin embargo, existen diferencias estadísticamente significativas entre los productos según las pruebas de disolución in vitro. La liberación del metronidazol se ajustó al modelo de Hixson Crowell, mientras que la de amoxicilina, en dos formulaciones se ajustó al modelo de Higuchi, y en el resto al modelo de Hixson-Crowell. CONCLUSIONES: se sugiere la necesidad de una mayor vigilancia a los medicamentos que se comercializan en el país con el fin de verificar el cumplimiento de los estándares de calidad.

INTRODUCTION: metronidazole, an antiparasitic and antimicrobial, and amoxicillin, a broad-spectrum aminopenicillin, are widely used drugs. Aims: to determine the pharmaceutical quality of oral administration formulations available in the Honduran market, in the city of Tegucigalpa. METHODS: products from five brand of metronidazole tablets (500 mg) and amoxicillin capsules (500 mg) were analyzed, respectively, all of them within their period of validity of use. For this, the technological, chemical and chemical properties of the tablets and capsules were evaluated, such as average weight, hardness, evaluation of active principle, uniformity of content, dissolution test, dis-solution profile, area under curve and dissolution efficiency. RESULTS: not all medicinal specialties contained information on storage conditions, regarding temperature, light and humidity, stated on the labels and leaflets. According to the technological and chemical evaluation, all the evaluated products satisfied the specifications of the pharmacopeia. However, there are statistically significant differences between the products according to in vitro dissolution tests. The release of metronidazole was adjusted to the Hixson-Crowell model, while that of amoxicillin, in two formulations was adjusted to the Higuchi model, and in the rest to the Hix-son-Crowell model. CONCLUSIONS: The need for greater surveillance of the drugs that are marketed in the country is suggested, in order to verify compliance with quality standards.

Isolation, Biochemical Characterization and Antiparasitic Activity of BmatTX-IV, A Basic Lys49-Phospholipase A2 from the Venom of Bothrops mattogrossensis from Paraguay.

BACKGROUND: Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential. METHODS: A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated. RESULTS: Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 µg/mL against Leishmania infantum promastigotes and of 13.8 µg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 µg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 µg/mL. CONCLUSION: Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity.

Identification of a peptide derived from a Bothrops moojeni metalloprotease with in vitro inhibitory action on the Plasmodium falciparum purine nucleoside phosphorylase enzyme (PfPNP).

There is a growing need for research on new antimalarial agents against Plasmodium falciparum infection, especially in regards to planning molecular architecture for specific molecular targets of the parasite. Thus, a metalloprotease from Bothrops moojeni, known as BmooMPα-I, was explored in this study, through in silico assays, aiming at the development of a peptide generated from this molecule with potential inhibitory action on PfPNP, an enzyme necessary for the survival of the parasite. In order to isolate BmooMPα-I, cation exchange and reverse phase chromatographies were performed, followed by in vitro assays of antiparasitic activity against the W2 strain of P. falciparum. The interactions between BmooMPα-I and PfPNP were evaluated via docking, and the resulting peptide, described as Pep1 BM, was selected according to the BmooMPα-I region demonstrating the best interaction score with the target of interest. The values for the specific activities of the PfPNP reaction were measured using the inorganic phosphate substrate and MESG. The fraction corresponding to BmooMPα-I was identified as fraction 4 in the cation exchange chromatography step, due to proteolytic activity on casein and the presence of a major band at ≅ 23 kDa. BmooMPα-I was able to inhibit in vitro growth of W2 P. falciparum, with an IC50 value of 16.14 µg/mL. Virtual screening with Pep1 BM demonstrated two PfPNP target binding regions, with ΔG values at the interaction interface of -10.75 kcal/mol and -11.74 kcal/mol. A significant reduction in the enzymatic activity of PfPNP was observed in the presence of Pep 1 BM when compared to the assay in the absence of this possible inhibitor. BmooMPα-I showed activity in vitro against W2 P. falciparum. By means of in silico techniques, the Pep 1 BM was identified as having potential binding affinity to the catalytic site of PfPNP and of inhibiting its catalytic activity in vitro.

Psychometric evaluation of a screening question for persistent depressive disorder.

BACKGROUND: About one in five patients with depression experiences a chronic course. Despite the great burden associated with this disease, there is no current screening instrument for Persistent Depressive Disorder (PDD). In the present study, we examine a short screening test, the persistent depression screener (PDS), that we developed for DSM-5 PDD. The PDS is comprised of one question that is administered following an initial self-assessment for depression. METHODS: Ninety patients from an inpatient clinic/day clinic specialized in treating depression completed the PDS. They were also assessed using a structured clinical interview covering the DSM-5 criteria for PDD. Retest reliability was examined after two weeks (n = 69, 77%). RESULTS: In this sample, the prevalence of PDD was 64%. Sensitivity of the PDS was 85% with a positive predictive value of 80%. Specificity was 63%. Positive and negative likelihood ratios were 2.3 and .24, respectively. Agreement between the PDS results and the outcome of the clinical interview was moderate (Cohen's Kappa κ = .48 ([95%-CI .28, .68], p < .001, SE = 0.10)). Prevalence-adjusted bias-adjusted Kappa was PABAK = .53. Retest reliability of the PDS was moderate (Cohen's Kappa κ = .52 ([95%-CI .3, .74], p < .001, SE = 0.11)). CONCLUSIONS: The present study shows that the PDS - when applied following a self-rating depression scale - might be a valid and reliable way to detect PDD. However, the results of the PDS must be confirmed by a diagnostic interview.

Antiprotozoal action of synthetic cinnamic acid analogs.

INTRODUCTION: Leishmaniasis, Chagas disease, and malaria cause morbidity globally. The drugs currently used for treatment have limitations. Activity of cinnamic acid analogs against Leishmania spp., Trypanosoma cruzi, and Plasmodium falciparum was evaluated in the interest of identifying new antiprotozoal compounds. METHODS: In vitro effects of analogs against L. braziliensis, L. infantum chagasi, T. cruzi, and P. falciparum, and hemolytic and cytotoxic activities on NCTC 929 were determined. RESULTS: Three analogs showed leishmanicidal and tripanocidal activity. No antiplasmodial, hemolytic, or cytotoxic activity was observed. CONCLUSIONS: Antiprotozoal activity of analogs against L. infantum braziliensis, L. infantum chagasi, and T. cruzi was demonstrated.

Antiprotozoal action of synthetic cinnamic acid analogs

Abstract INTRODUCTION Leishmaniasis, Chagas disease, and malaria cause morbidity globally. The drugs currently used for treatment have limitations. Activity of cinnamic acid analogs against Leishmania spp., Trypanosoma cruzi, and Plasmodium falciparum was evaluated in the interest of identifying new antiprotozoal compounds. METHODS In vitro effects of analogs against L. braziliensis, L. infantum chagasi, T. cruzi, and P. falciparum, and hemolytic and cytotoxic activities on NCTC 929 were determined. RESULTS Three analogs showed leishmanicidal and tripanocidal activity. No antiplasmodial, hemolytic, or cytotoxic activity was observed. CONCLUSIONS Antiprotozoal activity of analogs against L. infantum braziliensis, L. infantum chagasi, and T. cruzi was demonstrated.

Do 12-week yoga program influence respiratory function of elderly women?

Aging produces several respiratory limitations and reduces tolerance to physical efforts, sometimes leading to pulmonary diseases in the elderly. The literature draws attention to the possible benefits of Yoga practice among the elderly, presenting evidence for significant improvements in quality of life. It was hypothesized that yoga practice can improve respiratory function in the elderly. The effects of a yoga program on pulmonary volumes and respiratory muscle strength were verified in 36 elderly women divided into a yoga group [YG] (63.1 ± 13.3 years of age) and a control group (61.0 ± 6.9 years of age). Maximal inspiratory and expiratory pressure (MIP and MEP) were assessed by a manovacuometer and tidal volume (VT), vital capacity (VC) and minute ventilation (VE) were measured by a ventilometer. The program comprised 65 min sessions, 3 times/week during 12 weeks. The heart rate and respiratory rate decreased significantly in the YG (76-39 ± 8-03 vs. 74-61±10.26 bpm and 18.61 ± 3.15 vs. 16.72 ± 3.12 resp/min, respectively). In the YG, VT and VE increased significantly (0.55 ± 0.22 vs. 0.64 ± 0.2 ml and 9.19 ± 2.39 vs. 10.05 ± 2.11 ml, respectively), as well as VC (1.48 ± 0.45 vs. 2.03 ± 0.72 ml). Improvements were also found in MIP and MEP in the YG (62.17 ± 14.77 vs. 73.06 ± 20.16 cmH2O and 80.56 ± 23.94 vs. 86.39 ± 20.16 cmH2O, respectively). It was concluded that a 12-week yoga program significantly improves pulmonary function of aged women.

Direct structure elucidation by powder X-ray diffraction of a metal-organic framework material prepared by solvent-free grinding.

Powder X-ray diffraction (XRD) has been exploited to establish the structural properties of a porous interpenetrated mixed-ligand metal-organic framework material prepared by solid-state grinding, recognizing that product phases from mechanochemical synthesis are typically microcrystalline powders. The importance of subjecting the powder XRD data to rigorous scrutiny in such applications is emphasized.

Solvent-free synthesis of metal complexes.

Avoiding the use of solvents in synthesis can reduce environmental contamination and even be more convenient than using solvent-based synthesis. In this tutorial review we focus on recent research into the use of mechanochemistry (grinding) to synthesise metal complexes in the absence of solvent. We include synthesis of mononuclear complexes, coordination clusters, spacious coordination cages, and 1-, 2- and 3-dimensional coordination polymers (metal organic frameworks) which can even exhibit microporosity. Remarkably, in many cases, mechanochemical synthesis is actually faster and more convenient than the original solvent-based methods. Examples of solvent-free methods other than grinding are also briefly discussed, and the positive outlook for this growing topic is emphasised.

La perspectiva discursiva en psicología social / The discursive perspective in social psychology

El artículo proporciona una presentación de la Perspectiva Discursiva en Psicología Social. Para ello, realiza un repaso de antecedentes, fundamentos, algunos recursos concretos de la perspectiva discursiva y una aproximación a la lectura que esta perspectiva realiza de la cognición social. A través de estos pasos, el artículo pretende: resaltar la importancia de la aportación teórica, no solamente metodológica de esta perspectiva; subrayar la importancia del lenguaje en la construcción de la realidad, y adentrarse en la comprensión de las posibilidades que esta importancia supone para el quehacer de la propia psicología. (AU)

La perspectiva discursiva en psicología social / The discursive perspective in social psychology

El artículo proporciona una presentación de la Perspectiva Discursiva en Psicología Social. Para ello, realiza un repaso de antecedentes, fundamentos, algunos recursos concretos de la perspectiva discursiva y una aproximación a la lectura que esta perspectiva realiza de la cognición social. A través de estos pasos, el artículo pretende: resaltar la importancia de la aportación teórica, no solamente metodológica de esta perspectiva; subrayar la importancia del lenguaje en la construcción de la realidad, y adentrarse en la comprensión de las posibilidades que esta importancia supone para el quehacer de la propia psicología.