Prevalencia de baja avidez de Inmunoglobulina G anti Toxoplasma gondii y comportamiento de riesgo para toxoplasmosis en embarazada

Introducción: la toxoplasmosis es una infección zoonótica producida por Toxoplasma gondii, protozoo intracelular que puede afectar al hijo de la mujer embarazada y causar severas secuelas por lo que el monitoreo serológico debe ser realizado. Objetivo: determinar la prevalencia de baja avidez IgG anti Toxoplasma gondii y el comportamiento de riesgo para la enfermedad de toxoplasmosis en mujeres que estuvieron embarazadas durante el período 2017-2019 que acudieron al Instituto de Investigaciones en Ciencias de la Salud de la Universidad Nacional de Asunción-Paraguay. Metodología: fueron analizadas 371 fichas de pacientes con serología IgG positiva para toxoplasmosis cuyas muestras fueron procesadas en el Departamento de Producción del Instituto de Investigaciones en Ciencias de la Salud entre los años 2017-2019. Posteriormente, en el año 2020, se realizó 149/371 encuestas digitales de en estas mismas mujeres sobre conocimiento y comportamiento de riesgos para Toxoplasmosis. Resultados: se observó una prevalencia de 18 % de baja avidez para toxoplasmosis. A partir de la encuesta se encontró el 98 % conoce la enfermedad, el 73 % adquirió información durante el embarazo y el 50,3 % recibió orientación de prevención, además, el 65 % refirió como formas de transmisión comer carnes mal cocidas y verduras crudas. En cuanto al comportamiento de riesgo 46 % consume de aguatería, 20 % consume carne a punto medio y 78 % vegetales crudos. El 54 % realiza actividad de cultivo, tienen mascotas como gatos 4,3 %, perros 82 %, además el 9 % refirió dormir con sus mascotas. Conclusión: la prevalencia de baja Avidez en la población estudiada fue del 18 %. Se evidenció algunos comportamientos de riesgo para la toxoplasmosis en las mujeres encuestadas, por lo que se demuestra la necesidad de aplicar programas de prevención primaria en nuestro país.

Introduction: toxoplasmosis is a zoonotic infection caused by Toxoplasma gondii, an intracellular protozoan that can affect children of pregnant women and cause severe sequelae; therefore, serological monitoring should be performed. Objective: to determine the prevalence of low avidity IgG anti-Toxoplasma gondii and the risk behavior for toxoplasmosis disease in pregnant women during the 2017-2019 time period, who attended the Health Sciences Research Institute of the Universidad Nacional de Asuncion - Paraguay. Methodology: a total of 371 patient records with positive IgG serology for toxoplasmosis, whose samples were processed in the Production Department of the Instituto de Investigaciones en Ciencias de la Salud between the years 2017-2019 were analyzed. Subsequently, in 2020, 149/371 digital surveys of the same women were conducted on their knowledge and risk behavior for toxoplasmosis. Results: a low avidity prevalence of 18 % for toxoplasmosis was observed. 98 % knew about the disease, 73 % acquired information during pregnancy, and 50.3 % received preventive orientation. 65 % reported that eating undercooked meat and raw vegetables is a form of disease transmission. Regarding risk behavior, 46 % of the participants consumed poultry, 20 % consumed medium-rare-cooked meat, and 78 % consumed raw vegetables. Fifty-four percent of the patients performed farming activities, 44.3 % had cats as pets, 82 % had dogs, and 9 % slept with their pets. Conclusion: some risk behaviors for toxoplasmosis were evident in the women surveyed, demonstrating the need to implement primary prevention programs in our country.

Prevalencia de baja avidez de Inmunoglobulina G anti Toxoplasma gondii y comportamiento de riesgo para toxoplasmosis en embarazada / Prevalence of low avidity of anti-Toxoplasma gondii immunoglobulin G and risk behavior for toxoplasmosis in pregnant women

Introducción: la toxoplasmosis es una infección zoonótica producida por Toxoplasma gondii, protozoo intracelular que puede afectar al hijo de la mujer embarazada y causar severas secuelas por lo que el monitoreo serológico debe ser realizado. Objetivo: determinar la prevalencia de baja avidez IgG anti Toxoplasma gondii y el comportamiento de riesgo para la enfermedad de toxoplasmosis en mujeres que estuvieron embarazadas durante el período 2017-2019 que acudieron al Instituto de Investigaciones en Ciencias de la Salud de la Universidad Nacional de Asunción-Paraguay. Metodología: fueron analizadas 371 fichas de pacientes con serología IgG positiva para toxoplasmosis cuyas muestras fueron procesadas en el Departamento de Producción del Instituto de Investigaciones en Ciencias de la Salud entre los años 2017-2019. Posteriormente, en el año 2020, se realizó 149/371 encuestas digitales de en estas mismas mujeres sobre conocimiento y comportamiento de riesgos para Toxoplasmosis. Resultados: se observó una prevalencia de 18 % de baja avidez para toxoplasmosis. A partir de la encuesta se encontró el 98 % conoce la enfermedad, el 73 % adquirió información durante el embarazo y el 50,3 % recibió orientación de prevención, además, el 65 % refirió como formas de transmisión comer carnes mal cocidas y verduras crudas. En cuanto al comportamiento de riesgo 46 % consume de aguatería, 20 % consume carne a punto medio y 78 % vegetales crudos. El 54 % realiza actividad de cultivo, tienen mascotas como gatos 4,3 %, perros 82 %, además el 9 % refirió dormir con sus mascotas. Conclusión: la prevalencia de baja Avidez en la población estudiada fue del 18 %. Se evidenció algunos comportamientos de riesgo para la toxoplasmosis en las mujeres encuestadas, por lo que se demuestra la necesidad de aplicar programas de prevención primaria en nuestro país.

Introduction: toxoplasmosis is a zoonotic infection caused by Toxoplasma gondii, an intracellular protozoan that can affect children of pregnant women and cause severe sequelae; therefore, serological monitoring should be performed. Objective: to determine the prevalence of low avidity IgG anti-Toxoplasma gondii and the risk behavior for toxoplasmosis disease in pregnant women during the 2017-2019 time period, who attended the Health Sciences Research Institute of the Universidad Nacional de Asuncion - Paraguay. Methodology: a total of 371 patient records with positive IgG serology for toxoplasmosis, whose samples were processed in the Production Department of the Instituto de Investigaciones en Ciencias de la Salud between the years 2017-2019 were analyzed. Subsequently, in 2020, 149/371 digital surveys of the same women were conducted on their knowledge and risk behavior for toxoplasmosis. Results: a low avidity prevalence of 18 % for toxoplasmosis was observed. 98 % knew about the disease, 73 % acquired information during pregnancy, and 50.3 % received preventive orientation. 65 % reported that eating undercooked meat and raw vegetables is a form of disease transmission. Regarding risk behavior, 46 % of the participants consumed poultry, 20 % consumed medium-rare-cooked meat, and 78 % consumed raw vegetables. Fifty-four percent of the patients performed farming activities, 44.3 % had cats as pets, 82 % had dogs, and 9 % slept with their pets. Conclusion: some risk behaviors for toxoplasmosis were evident in the women surveyed, demonstrating the need to implement primary prevention programs in our country.

Comprehensive inventory of páramo birds and their habitat affinities in a conservation hotspot, the Macizo del Cajas Biosphere Reserve, southern Ecuador

The páramo ecosystem is a significant centre of Andean bird diversity with high concentrations of threatened species. The Macizo del Cajas Biosphere Reserve's páramos are a district of the biogeographic páramo province of northern Andes and are therefore considered a conservation hotspot with representative bird diversity. To enhance regional conservation efforts, comprehensive inventories of bird species that occupy this páramo are required. We present an updated bird inventory for the páramos of Macizo del Cajas and included validated records from eBird and GBIF databases along with records from continuous monitoring across this páramo landscape for five years. We also provide notes on habitat affinity and important new, rare, restricted range, and threatened birds. We report 112 bird species within the reserve, including five endemics, and three globally and 12 nationally threatened species. Finally, we discuss the use of habitat affinities as indicators of biodiversity patterns in páramo to improve conservation tools for key habitats.

El ecosistema de páramo es un centro importante de diversidad de aves andinas con altas concentraciones de especies amenazadas. Los páramos de la Reserva de la Biosfera Macizo del Cajas son un distrito biogeográfico de la provincia del páramo de los Andes del norte y por tanto, son un punto crítico de conservación con una diversidad de aves representativa. Inventarios exhaustivos de la avifauna que ocupa este páramo son requeridos para asegurar esfuerzos de conservación regional. El presente estudio brinda un inventario actualizado de aves de los páramos del Macizo del Cajas. Se incluyen registros verificados desde eBird y GBIF, así como registros de cinco años continuos de monitoreo a través del paisaje de páramo. Además, se incluyen notas acerca de la afinidad de hábitat y registros importantes, nuevos, raros y de aves amenazadas. En total, se reportan 112 especies de aves dentro de la reserva, incluyen cinco endémicas, tres globalmente amenazadas y 12 a escala nacional. Finalmente, se discute el uso de la afinidad de hábitat como indicador de los patrones de biodiversidad en el páramo para mejorar herramientas de conservación para hábitats clave.

T cell immunotherapy mediated by dual-affinity aptamer targeted liposome / 药学学报

Redirecting immune cells to the tumor cells and enhancing its anti-tumor immune response is a very promising cancer treatment strategy. AS1411 aptamers have high affinity for malignant tumors with high nucleolin expression, and cytotoxic T lymphocyte associated protein 4 (CTLA-4) aptamers can specifically bind to CTLA-4, which is expressed by T cells. In this study, a dual-affinity aptamer targeted liposome (Dat. Lipo) was constructed based on AS1411 aptamer and CTLA-4 aptamer, and its immunotherapeutic effect on T cells was studied. After the aptamer was modified with cholesterol, Dat. Lipo was prepared by instillation method; its effect of redirecting T cells was determined by confocal micrographs; its T cell immunotherapy effect was evaluated by cell counting kit-8 (CCK8) and T cell penetration was evaluated by tumor spheroids. The results showed that compared with liposomes loaded with one type aptamer, Dat. Lipo could effectively promote the redirection of T cells to tumor cells; Dat. Lipo had good biosafety and immunotherapeutic effect on MCF-7 and HepG2 cells in a concentration-dependent manner; Dat. Lipo could also promote T cells to infiltrate into the tumor spheroids and enhance the immunotherapy effect of T cells in different dimensions. In summary, Dat. Lipo can use the high affinity of aptamers to redirect T cells to tumor cells, enhance the effect of immunotherapy, and has a promising application prospect in tumor therapy. This study was approved by the Examination Committee of Cancer Hospital of Xiangya School of Medicine, Central South University, Hunan Cancer Hospital.

Prediction of concentration immediately dangerous to life or health of benzene and its derivatives based on quantitative structure-activity relationship / 环境与职业医学

Background With the increasing exposure to hazardous chemicals in the workplace and frequency of occupational injuries and occupational safety accidents, the acquisition of occupational exposure limits of hazardous chemicals is imminent. Objective To obtain more unknown immediately dangerous to life or health (IDLH) concentrations of hazardous chemicals in the workplace by exploring the application of quantitative structure-activity relationship (QSAR) prediction method to IDLH concentrations, and to provide a theoretical basis and technical support for the assessment and prevention of occupational injuries. Methods QSAR was used to correlate the IDLH values of 50 benzene and its derivatives with the molecular structures of target compounds. Firstly, affinity propagation algorithm was applied to cluster sample sets. Secondly, Dragon 2.1 software was used to calculate and pre-screen 537 molecular descriptors. Thirdly, the genetic algorithm was used to select six characteristic molecular descriptors as dependent variables and to construct a multiple linear regression model (MLR) and two nonlinear models using support vector machine (SVM) and artificial neural network (ANN) respectively. Finally, model performance was evaluated by internal and external validation and Williams diagram was drawn to determine the scopes of selected models. Results The ANN model results showed that \begin{document}$ {R}_{\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{i}\mathrm{n}}

Universal chimeric Fcγ receptor T cells with appropriate affinity for IgG1 antibody exhibit optimal antitumor efficacy

Developing universal CARs with improved flexible targeting and controllable activities is urgently needed. While several studies have suggested the potential of CD16a in tandem with monoclonal antibodies to construct universal CAR-T cells, the weak affinity between them is one of the limiting factors for efficacy. Herein, we systematically investigated the impact of Fcγ receptor (FcγR) affinity on CAR-T cells properties by constructing universal CARs using Fcγ receptors with different affinities for IgG1 antibodies, namely CD16a, CD32a, and CD64. We demonstrated that the activities of these universal CAR-T cells on tumor cells could be redirected and regulated by IgG1 antibodies. In xenografted mice, 64CAR chimeric Jurkat cells with the highest affinity showed significant antitumor effects in combination with herceptin in the HER2 low expression U251 MG model. However, in the CD20 high expression Raji model, 64CAR caused excessive activation of CAR-T cells, which resulted in cytokine release syndrome (CRS) and the decline of antitumor activity, and 32CAR with a moderate affinity brought the best efficacy. Our work extended the knowledge about FcγR-based universal CAR-T cells and suggested that only the FcγRCAR with an appropriate affinity can offer the optimal antitumor advantages of CAR-T cells.

Construction and optimization of a novel plasma membrane model for membrane affinity assay based on polydiacetylene vesicles / 药学学报

Most drugs need to interact with cell membrane to reach the biological target, so that membrane affinity assay is an important early screening step in drug discovery. However, at present, the traditional oil-water distribution method is still used, a new, simple and accurate method for membrane affinity assay is urgently needed. In this study, according to the colorimetric principle, a new assay model based on polydiacetylene vesicles was optimized through a series of experiments including different concentrations of vesicle solution, temperature, or pH reaction environment. On this basis, tetracaine hydrochloride, 2-methylimidazole and histamine were used as model drugs to measure the membrane affinity constants and verify the between-batch precision of the optimized assay model (relative standard deviation less than 5%). In addition, polydiacetylene vesicles were stable for up to 180 days, demonstrating the potential application of the assay model. This strategy is simple, stable, reliable, with high reproducibility, low cost and easy to promote, which provided a new tool and a new direction for the high-throughput assay of membrane affinity.

The technologies of small molecule ligand-target protein interaction and high throughput ligand screening / 药学学报

The interaction of drug and target protein is a critical part of new drug discovery. It is the premise for drugs to exert therapeutic effects by targeting specific binding sites of target proteins and thereby affecting its pharmacological activity. Currently, a variety of techniques are exploited to detect the interaction between drug ligands and target proteins. For example, cellular thermal shift assay (CETSA) and differential scanning fluorimetry (DSF) based on thermodynamics, mass spectrometry and nuclear magnetic resonance technology, etc. In addition, high-throughput ligand screening technology provides technical convenience for the search of specific ligand, and is a powerful tool to efficiently identify the interaction between drug ligand and target protein. Here, we summarize the detection techniques of interaction between small molecules and target proteins, and discuss the application of high-throughput ligand screening technology in drug research.

Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity / 中国生物制品学杂志

@#Objective To express dengue virus(DENV)NS2B-NS3 protease in E.coli,optimize the expression conditions and determine the enzyme activity,so as to lay a foundation of screening and discovering of lead compounds targeting DENV.Methods Codon-optimized NS2B-NS3 gene was inserted into pET-28a vector to construct recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3,which was transformed E.coli Rosetta(DE3)competent cells and induced by IPTG to express NS2B-NS3 protease. The optimal expression conditions of NS2B-NS3 protease in E.coli were determined by optimizing induction length,induction temperature and IPTG concentration. NS2B-NS3 protease was isolated and purified by HisTrap~(TM) affinity chromatography column and measured for the protease activity by fluorescence resonance energy transfer(FRET)assay.Results The recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3 was constructed correctly as identified by restriction analysis(NheⅠ/XhoⅠ)and sequencing. The optimal expression conditions of NS2BNS3 protease in E.coli were as follows:induction temperature of 20 ℃,induction length of 10 h and IPTG concentration of0. 2 mmol/L. The purified NS2B-NS3 protease showed a purity of more than 90% with a exhibited a of 20 mg/L,which bound to mouse monoclonal antibody against His-tag specifically and had good hydrolytic activity with a specific activity of 16. 111 U/mg,a K_m of 16. 46 μmol/L and a k_(cat) of 0. 028/s.Conclusion DENV NS2B-NS3 protease with high purity and activity was successfully prepared,which laid an experimental foundation of the establishment of high-throughput screening model for inhibitors targeting NS2B-NS3 protease.

Expression and purification of a peptide fragment of 660 - 1468 amino acids in Neisseria gonorrhoeae NGO2105 protein as well as preparation and identification of its polyclonal antibody / 中华皮肤科杂志

Objective:To prokaryotically express a peptide fragment of 660 - 1468 amino acids in Neisseria gonorrhoeae NGO2105 protein, and to prepare and identify its polyclonal antibody. Methods:The pCold TF-NGO2105 660-1468 aa recombinant plasmid was transformed into the bacterium Escherichia coli BL21 (DE3) for protein expression. After the inclusion body protein was denatured and renatured, the target protein was purified. Then, BALB/c mice were immunized with the target protein to prepare a polyclonal antiserum; the antibody potency was evaluated by enzyme-linked immunosorbent assay, the specificity of the antibody against NGO2105 protein in Neisseria gonorrhoeae was analyzed by Western blot analysis, the affinity of the antiserum with Neisseria gonorrhoeae was analyzed by flow cytometry, and adhesion inhibition assay was performed to evaluate the inhibitory effect of anti-NGO2105 660-1468 aa antibody on the adhesion of Neisseria gonorrhoeae to human cervical epithelial ME-180 cells. Comparisons between different groups were performed by using t test. Results:The NGO2105 660-1468 aa protein was expressed as the inclusion body, and the soluble target protein was obtained by denaturation, renaturation, and purification. After immunization of mice with the target protein, the antiserum titer was 5.12 × 10 6, and flow cytometry showed that the antibody bound well to the Neisseria gonorrhoeae NGO2105 660-1468 aa. Adhesion inhibition assay showed that the anti-NGO2105 660-1468 aa antibody significantly inhibited the adhesion of Neisseria gonorrhoeae to ME-180 cells, and the inhibitory effect was concentration-dependent to some extent, with the adhesion rates of Neisseria gonorrhoeae treated with 20- and 40-fold dilutions of the anti-NGO2105 660-1468 aa antibody being 52.9% and 79.2% respectively, significantly lower than the adhesion rate in the untreated group (100%, t = 8.40, 5.29, P < 0.001, = 0.006, respectively) . Conclusion:The NGO2105 660-1468 aa protein was successfully expressed and purified, and a highly potent polyclonal antibody was prepared, which had a good affinity with Neisseria gonorrhoeae and an adhesion inhibition ability.

Affinity therapy, Novidade no tratamento do autismo / Affinity therapy, Nouveauté dans le traitement de l'autisme / Affinity therapy, Novelty in the treatment of autism

Es constante en el autismo una fijación o una ritualización, una obsesión o una pasión, un interés específico o una aptitud, en resumen, una particularidad, una afinidad. Este punto permite construir una dinámica subjetiva autista: una relación al mundo, al cuerpo, a los otros y al conocimiento. La alienación significante se correlaciona con un apoyo alienante al objeto como una compensación. Para Laurent, es un "órgano suplementario" a partir del cual el sujeto articula y desglosa todo su mundo. La defensa autística es el "regreso del goce sobre un borde". El sujeto procura el apoyo de un doble, en las variadas formas clínicas y con la intervención que resultará, un tratamiento de pulsiones y dinámica vital. Considerando esta defensa: ¿qué tratamiento sería posible con el autismo? ¿Qué apoyo institucional se puede ofrecer? ¿Cómo se podría acompañar al autista? La Affinity therapy nombra lo que sería un tratamiento del autista orientado por los objetos, los intereses específicos, los dobles, las particularidades de cada autista. El artículo muestra el interés de la Affinity therapy, explica la importancia y el peso de esta práctica en el tratamiento del autismo en un trabajo interdisciplinar orientada por el discurso analítico

É frequente no autismo uma fixação ou uma ritualização, uma obsessão ou uma paixão, um interesse ou uma atitude, em resumo, uma particularidade, uma afinidade. Este ponto permite construir uma dinâmica subjetiva autista: uma relação com o mundo, com o corpo, com os outros e com o conhecimento. A alienação significante é correlata de um apoio alienante no objeto como uma compensação. Para Laurent, é um "órgão suplementar" a partir do qual o sujeito articula e separa o seu mundo. A defesa autística é o "retorno do gozo sobre uma borda". O sujeito procura o apoio de um duplo, nas variadas formas clínicas e com a intervenção [do analista?] da qual resultará um tratamento das pulsões e uma dinâmica vital. Considerando esta defesa: qual o tratamento possível do autismo? Qual apoio institucional se pode oferecer? Como acompanhar o autista? A Affinity therapy nomeia o que seria o tratamento do autista orientado pelos objetos, pelos interesses específicos, pelos duplos, pelas particularidades de cada autista. O artigo mostra o interesse da Affinity therapy, explica a importância e o peso desta prática no tratamento do autismo num trabalho interdisciplinar orientada pelo discurso analítico.

Une fixation ou une ritualisation, une obsession ou une passion, un intérêt ou une attitude, bref, une particularité, une affinité, est fréquente. Ce point permet la construction d'une dynamique subjective autistique : un rapport au monde, au corps, aux autres et au savoir. L'aliénation significative est corrélée avec le soutien aliénant sur l'objet comme compensation. Pour Laurent, c'est un « organe supplémentaire ¼ à partir duquel le sujet articule et sépare son monde. La défense autistique est le «retour de la jouissance par-dessus bord ¼. Le sujet cherche l'appui d'un double, dans les différentes formes cliniques et avec l'intervention [de l'analyste ?] qui va déboucher sur un traitement des pulsions et une dynamique vitale. Considérant cette défense : quel est le traitement possible de l'autisme ? Quel soutien institutionnel peut être proposé ? Comment accompagner l'autiste ? L'Affinity therapy nomme ce que serait le traitement des autistes orienté par les objets, par les intérêts spécifiques, par les doubles, par les particularités de chaque autiste. L'article montre l'intérêt de la thérapie par affinité, explique l'importance et le poids de cette pratique dans la prise en charge de l'autisme dans un travail interdisciplinaire guidé par le discours analytique.

A fixation or a ritualization, an obsession or a passion, an interest or an attitude, in short, a particularity, an affinity, is frequent. This point allows the construction of an autistic subjective dynamic: a relationship with the world, with the body, with others and with knowledge. Meaningful alienation is correlated with alienating support on the object as compensation. For Laurent, it is a "supplementary organ" from which the subject articulates and separates his world. The autistic defense is the "return of jouissance over the edge". The subject seeks the support of a double, in the various clinical forms and with the intervention [of the analyst?] which will result in a treatment of the drives and a vital dynamic. Considering this defense: what is the possible treatment of autism? What institutional support can be offered? How to accompany the autistic? Affinity therapy names what would be the treatment of autistic people oriented by objects, by specific interests, by doubles, for the particularities of each autistic person. The article shows the interest of Affinity therapy, explains the importance and weight of this practice in the treatment of autism in an interdisciplinary work guided by analytical discourse.

Identification of a novel PHGDH covalent inhibitor by chemical proteomics and phenotypic profiling

The first rate-limiting enzyme of the serine synthesis pathway (SSP), phosphoglycerate dehydrogenase (PHGDH), is hyperactive in multiple tumors, which leads to the activation of SSP and promotes tumorigenesis. However, only a few inhibitors of PHGDH have been discovered to date, especially the covalent inhibitors of PHGDH. Here, we identified withangulatin A (WA), a natural small molecule, as a novel covalent inhibitor of PHGDH. Affinity-based protein profiling identified that WA could directly bind to PHGDH and inactivate the enzyme activity of PHGDH. Biolayer interferometry and LC-MS/MS analysis further demonstrated the selective covalent binding of WA to the cysteine 295 residue (Cys295) of PHGDH. With the covalent modification of Cys295, WA blocked the substrate-binding domain (SBD) of PHGDH and exerted an allosteric effect to induce PHGDH inactivation. Further studies revealed that with the inhibition of PHGDH mediated by WA, the glutathione synthesis was decreased and intracellular levels of reactive oxygen species (ROS) were elevated, leading to the inhibition of tumor proliferation. This study indicates WA as a novel PHGDH covalent inhibitor, which identifies Cys295 as a novel allosteric regulatory site of PHGDH and holds great potential in developing anti-tumor agents for targeting PHGDH.

Affinity transfer of nanobodies to a CDR3 only single-domain antibody fragment NBL42 / 国际生物医学工程杂志

Objective:To evaluate the potential of a previously identified CDR3 only single-domain antibodies (sdAbs) fragment, NBL42, as a general framework for affinity transfer.Methods:The H3 loops of VHH-A4(A4), VHH-H5(H5), cAb-Lys3(L3) and B6H12 which bind with alliinase, PD-1, lysozyme and CD47, respectively, were grafted into the corresponding loop of NBL42. The genes of the reconstituted CDR3 only sdAbs were synthesized, expressed in E. coliand purified with Ni 2+ column affinity chromatography. The antigen binding and stability of the recombinant CDR3 only sdAbs were assayed by ELISA. Results:The recombinant NBL42-A4CDR3, NBL42-H5CDR3, NBL42-L3CDR3 and NBL42-B6H12CDR3 ran as a single peak at 15, 15, 28 and 16 kDa, respectively, in SDS-PAGE as expected molecular weight. Grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 expressed in a soluble form and specifically bind with alliinase and PD-1, respectively, but lost about 50% of their binding activity. In contrast, the grafted sdAbs NBL42-Lys3CDR3 and NBL42-B6H12CDR3 completely lost their antigen binding capacity. NBL42 sdAbs and grafted sdAbs NBL42-A4CDR3 and NBL42-H5CDR3 retain roughly half of their binding activity after 90 ℃ heat treatment, indicating high stability. The C88Y mutation in NBL42 and the Swiss Mode 3D model predicted that the C88Y residue in FR3 may play a key role in NBL42 stability and CDR3 affinity transfer.Conclusions:The structure of NBL42 has potential as a framework for CDR3 transplantation and affinity transfer.

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

Study on deoxygenation of polymerized bovine hemoglobin and its effects on the molecular stability / 中国输血杂志

【Objective】 To identify the key factors that regulate the physical vacuum stirring deoxygenation process of bovine hemoglobin and evaluate the stabilities of polyHb solution with different oxygenation degree. 【Methods】 The effects of several factors including gas liquid specific phase boundary area, pH values and polyHb concentrations on deoxygenation process were investigated through monitoring the oxygen saturation of polyHb solution. Furthermore, the MetHb contents of the studied polyHb solutions were measured at regular intervals during the experiments to evaluate the molecular stability of the polyHb through monitoring the oxidation degrees of the experimental Hb solutions under the mimic pasteurevirus inactivation conditions. 【Results】 The results showed that the more gas liquid specific phase boundary area could accelerate deoxygenation rate, the better deoxygenation results could be yielded. On the other hand, lower pH value could promote the deoxygenation process of the polyHb solution due to the decreasing of oxygen affinity of the polyhemoglobin molecules. Furthermore, results of investigation about the Hb concentration presented that Hb concentration had no remarkable effects on the deoxygenation of polyHb. In addition, the results from the stability experiments presented that higher degree of deoxygenation could decrease the oxidation process and thus increase the heat tolerance of the polyhemoglobin. 【Conclusion】 The deoxygenation of polyhemglonbin could improve the stability of the polyhemoglobin and the deoxygenation process could be regulated by changing the gas liquid specific phase boundary area and oxygen affinity. This study could provide reference and preliminary basis for the industrialization and development of HBOCs in the future.

Anticuerpos neutralizantes, nuevas pruebas de laboratorio contra el SARS-CoV-2 / Neutralizing antibodies, new laboratory tests against SARS-CoV-2

RESUMEN Introducción: En la presente revisión conoceremos los detalles de esta nueva prueba de laboratorio, utilizada para cuantificar los anticuerpos neutralizantes contra el SARS-CoV-2. Esta prueba diagnóstica comienza a tener un mayor protagonismo, a razón del proceso de infección y vacunación en el mundo, para comprender los misterios del correlato de protección inmunológica. Contenido: Los anticuerpos neutralizantes pueden bloquear la capacidad del virus, para unirse al receptor ACE2 en las células humanas y estos anticuerpos permiten eliminar el efecto de microorganismos invasores. Su actividad se genera por las proteínas situadas en la superficie de los virus, a las que se unen para «bloquear¼ la infección. Los anticuerpos neutralizantes se definen in vitro por su capacidad para bloquear la entrada, fusión o salida del coronavirus, es decir son anticuerpos funcionales. En la actualidad existen diferentes pruebas de laboratorio (pruebas de inmunoensayo de alto rendimiento), que pueden detectar anticuerpos inmunoglobulinas G anti proteína S del SARS-CoV-2 y que se correlacionan con las pruebas de laboratorio idóneas para la determinación de estos anticuerpos. Es crucial que estas pruebas de inmunoensayo de alto rendimiento, sean validadas en su fabricación comparándolas con los métodos gold standard para determinar la presencia de anticuerpos neutralizantes. Perspectiva: Se pretende ampliar el conocimiento de esta nueva prueba, que en un futuro permitirán definir los valores de correlato inmunológico generados por las vacunas o por una infección previa.

ABSTRACT Introduction: In this review we will know the details of this new laboratory test, used to quantify neutralizing antibodies against SARS-CoV-2. This diagnostic test begins to have a greater role, due to the process of infection and vaccination in the world, to understand the mysteries of the correlate of immune protection. Content: Neutralizing antibodies can block the ability of the virus to bind to the ACE2 receptor in human cells and these antibodies allow to eliminate the effect of invading microorganisms. Their activity is generated by proteins on the surface of viruses, which they bind to "block" infection. Neutralizing antibodies are defined in vitro by their ability to block the entry, fusion or exit of the coronavirus, that is, they are functional antibodies. Currently there are different laboratory tests (high-throughput immunoassay tests), which can detect immunoglobulin G anti-protein S antibodies to SARS-CoV-2 and which correlate with the gold standard laboratory tests for the determination of these antibodies. It is crucial that these high-throughput immunoassay tests are manufacturing validated against gold standard methods to determine the presence of neutralizing antibodies. Perspective: This review aims to expand the knowledge of this new test, which in the future will allow defining the immunological correlate values generated by vaccines or by a previous infection.

Seroprevalencia de Brucella canis en perros de un refugio para animales de compañía en Bogotá, Colombia / Seroprevalence of Brucella canis in canines from a dog shelter in Bogotá, Colombia

Resumen | Introducción. El riesgo de infección con Brucella canis en humanos y perros aumenta con la exposición constante a perros portadores asintomáticos. En Colombia hay evidencia de infección con B. canis en personas que conviven con perros. Una preocupación adicional en Bogotá es la falta de información actualizada sobre la prevalencia de la infección en perros destinados a programas de adopción. Objetivo. Establecer la seroprevalencia de la infección por B. canis en perros de un refugio para animales de compañía destinados a la adopción en Bogotá. Materiales y métodos. Se hizo un estudio descriptivo de corte transversal en un refugio para animales de Bogotá. Se detectaron anticuerpos contra B. canis en el suero de 51 perros (28 hembras y 23 machos) mediante una prueba inmunocromatográfica de flujo lateral. Asimismo, los individuos positivos se analizaron con PCR para la detección del ADN de Brucella spp. Resultados. La seroprevalencia de B. canis fue del 1,96 % (1/51). El perro seropositivo correspondió a una hembra asintomática de tres años de edad en la cual no se detectó ADN bacteriano en sangre mediante la PCR. Conclusiones. La seroprevalencia representada por un solo perro con IgG anti-B. canis puede considerarse un riesgo potencial para las poblaciones de perros y humanos, ya que podría tratarse de un animal con infección persistente capaz de diseminar la bacteria.

Abstract | Introduction: The risk of Brucella canis infection in humans and dogs has increased due to the permanent exposure to asymptomatic carrier dogs. In Colombia, there is evidence of B. canis infection in humans living with dogs. In the case of Bogotá, an additional concern is the lack of updated information related to the prevalence of the infection in dogs. Objective: To determine the seroprevalence of infection by B. canis in dogs intended for adoption programs in Bogotá. Materials and methods: By means of a descriptive cross-sectional study carried out in a dog shelter in Bogotá, anti-B. canis IgG antibodies were detected in the serum from 51 dogs (28 females and 23 males) using a lateral-flow immunochromatographic test. Additionally, seropositive animals were analyzed with PCR to detect Brucella spp DNA. Results: Brucella canis seroprevalence was 1.96% (1/51). The seropositive dog was an asymptomatic three-year-old she-dog in which no bacteria DNA was detected in the blood through PCR. Conclusions: The seroprevalence determined in this study represented by a single dog with anti-B. canis IgG can be considered a potential risk both for canine and human populations since this single dog could have a persistent infection capable of spreading the bacteria.

Recombinant expression of black sesame polyphenol oxidase and its enzymatic properties / 生物工程学报

To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO), a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E. coli. Subsequently, the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification. The synthesized Bsppo gene contained 1 752 bp which encodes 585 amino acids with a deduced molecular weight of 65.3 kDa. Transformation of the recombinant vector into E. coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO. The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification. The results demonstrated that the optimal temperature and pH for BsPPO was 25°C and 4.0, respectively. BsPPO exhibited a good stability under low temperature and acidic environment. Low-intensity short-term light exposure increased the activity of BsPPO. Cu²⁺ could improve the activity of BsPPO while Zn²⁺ and Ca²⁺ showed the opposite effect. BsPPO could catalyze the oxidation of monophenols, diphenols, and triphenols, and exhibited good catalytic activity on l-tyrosine and vanillic acid. Moreover, BsPPO exhibited high catalytic activity on black sesame metabolites, including 2-methoxy cinnamic acid, indole-3-carboxylic acid and phloretin. These results may serve as a basis for further characterization of BsPPO.

Characterization of the affinity-tags-regulated (S)-carbonyl reductase 2 towards 2-hydroxyacetophenone reduction / 生物工程学报

The influence of different affinity tags on enzyme characteristics varies. The (S)-carbonyl reductase 2 (SCR2) from Candida parapsilosis can reduce 2-hydroxyacetophenone, which is a valuable prochiral ketones. Different affinity tags, i.e. his-tag, strep-tag and MBP-tag, were attached to the N terminus of SCR2. These tagged SCR2 enzymes, i.e. his6-SCR2, strep-SCR2 and MBP-SCR2, were heterologously expressed in Escherichia coli and purified to study their characteristics towards 2-hydroxyacetophenone reduction. Affinity tags did affect the characteristics of the recombinant SCR2 enzymes. Specifically, affinity tags affect the stability of recombinant SCR2 enzymes: 1) At pH 6.0, the remaining enzyme activities of his6-SCR2 and strep-SCR2 were only 95.2% and 90.0% of the untagged SCR2, while that of MBP-SCR2 was 1.2 times of the untagged SCR2 after incubating for 13 h at 30 °C. 2) The half-life of MBP-SCR2 at 50 °C was 26.6%-48.8% longer than those of strep-SCR2, his6-SCR2 and untagged SCR2. 3) The kcat of MBP-SCR2 was about 1.25-1.45 times of that of small affinity-tagged and untagged SCR2 after storing at -80 °C for 60 d. Structural informatics indicated that the α-helices at the C terminus of MBP-SCR2 contributed to the stability of the N terminus of fusion protein of SCR2. Data from circular dichroism showed that the MBP-tag has some influence on the secondary structure of SCR2, while melting temperature analysis demonstrated that the Tm of the recombinant MBP-SCR2 was about 5 °C higher than that of the untagged SCR2. This study obtained an efficient and stable recombinant SCR2, i.e. the MBP-SCR2. Moreover, this study could serve as a reference for other researchers to evaluate and select appropriate affinity tags for their research.

Analysis of serum anti-EB virus antibody in children with infectious mononucleosis / 中华实用儿科临床杂志

Objective:To understand the profile type of serum Epstein-Barr virus (EBV) antibodies in children with infectious mononucleosis (IM), and to analyze the significance of viral capsid antigen (VCA) IgG antibody affinity in the diagnosis of IM.Methods:Retrospective analysis was performed on the results of the serum anti-EBV antibody profile and plasma EBV nucleic acid test of 150 hospitalized children with IM diagnosed in Beijing Children′s Hospital, Capital Medical University, from May 2016 to May 2019.Anti-EBV antibody profiles, including anti-VCA-IgG, anti-VCA-IgM, anti-early antigen (EA) IgA, anti-EBV nuclear antigen (EBNA) IgG, and anti-VCA-IgG affinity, were detected by enzyme-linked immunosorbent assay (ELISA). Plasma EBV nucleic acids were detected by real-time quantitative PCR.Results:There were mainly two types of anti-EBV antibody profiles in 150 children with IM: (1)130 cases who were positive for anti-VCA-IgM/IgG, negative for anti-EBNA-IgG and positive for anti-VCA-IgG antibodies with low affinity, accounting for 86.7% (130/150 cases), of which 50 cases were positive for anti-early antigen IgA; (2)18 cases who were negative for anti-VCA-IgM, positive for anti-VCA-IgG, negative for anti-EBNA-IgG and positive for anti-VCA-IgG antibody with low affinity, accounting for 12.0% (18/150 cases), of which 5 cases were positive for anti-EA IgA.EBV DNA was measured in 132 children, with a posi-tive rate of 37.9% (50/132 cases).Conclusions:There were several types of serum EBV antibody profiles in children with IM, 12.0% of patients with IM in this study were negative for anti-VCA-IgM, and the diagnosis of IM was confirmed by the affinity of anti-VCA IgG.