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Objective To screen out specific aldosterone(ALD)antibodies using phage display technology and recom-binant antibody technology,providing raw materials for the research and development of ALD diagnostic kits.Methods Five healthy and clean New Zealand white rabbits were selected and immunized for the first time against the diluted ALD-keyhole limpet hemocyanin antigen(2 mg·L-1)using a multi-point injection method on the back,with a dose of 1 mg per rabbit.Immunization was administered again every 2 weeks,with a 50％reduction in dose.Starting from the third immunization,the ear vein blood of the rabbits was collected one week after each immunization.A chemiluminescent plate coated with 0.25 mg·L-1 ALD-bovine serum albumin antigen was used to measure serum titers via indirect and competitive methods.After the 5th immunization,the rabbit with high serum titers and good specificity was selected,and its spleen and bone marrow were removed.The spleen tissue was grinded,and RNA was extracted using TRIzol reagent in one step to obtain gene sequences in the variable region of light chain(VL)and the variable region of heavy chain(VH).The single-chain variable fragment(ScFv)was connected through the linker and constructed into the bacteriophage vector Pcomb3xss;then,it was carried to Escherichia coli TG1 through electrotransformation,and the ALD ScFv phage display library was constructed accordingly.Three to five rounds of enrichment screening were performed against the library.Monoclonal clones,identified by enzyme-linked immunosorbent assay(ELISA)competitive method,were selected for phage supernatant preparation,and a highly competitive clone sequence was obtained.The screened clone sequence was inserted into the pCMV3 expression vector,and the HEK293 cell was transfected using the transient transfection method after the plasmid was extracted.One week later,the supernatant was collected,and its purity and expression were identified by affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Results After the 5th immunization,the serum titers of 5 rabbits were indirectly tested,and the results showed that the serum titers of 4# and 5# white rabbits were still greater than 10,000 after being diluted by 32,000 times.The test results based on the competitive method showed that the ratio of low to high values in the plasma sample of 5#white rabbit was 2∶1,superior to that of other white rabbits.The 5# white rabbit was selected for phage library construction.The VL and VH gene fragments were amplified by conventional polymerase chain reaction,and then bridged into ScFv(VL+VH).The agar gel electrophoresis analysis showed that the size of the band was about 750 bp,which was consistent with the size of the originally designed fragment.ScFv was cleaved and electroporated into Escherichia coli TG1 to construct a phage library with a storage capacity of 4.73 × 108 cfu·mL-1.After 3 rounds of washing,300 monoclonal clones were selected from the outbound petri dishes to prepare monoclonal bacteriophages.The ELISA results showed a positive rate of 100％among the 300 clones,and 42 clones were tested positive for calibration competition,with a screening rate of 14％.The 42 positive clones were further subjected to clinical sample competition testing,and 16 monoclonal strains that met the requirements were screened.The 16 strains were retested,and the results of the two tests were consistent.After sequencing,6 antibody sequences were selected for construction and expression.After purification,SDS-PAGE reduced gel electrophoresis results showed that there were bands at positions 50,000 on the heavy chain and 25,000 on the light chain.Six highly affinitive and competitive rabbit ALD monoclonal antibodies were obtained.Conclusion Six highly affinitive and competitive rabbit ALD antibodies are successfully screened using phage display technology,which provides a reference method for the discovery of small molecule antibodies.The screened AD1 85 and AD277 antibodies show a competitive advantage twice that of the positive control in the competition of calibration and clinical samples,providing a possibility for the development of raw materials for ALD detection kits.
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@#Objective To compare the application of two pretreatment methods,deoxyribonuclease(DNase)treatment and ligand affinity,in detecting the residual amount of free and mispackaged host cell DNA(HCDNA)in recombinant adenoassociated virus(rAAV).Methods Free and mispackaged HCDNA were isolated by DNase treatment and ligand affinity respectively,and then the nucleic acid was extracted and detected by qPCR. The accuracy and reproducibility of two pretreatment methods for detecting HCDNA residues were compared.Results Using DNase treatment,the result of nucleic acid quantitative detection in non-DNase-treated group was the total residual amount of HCDNA,that in DNase-treated group was the amount of mispackaged HCDNA,and the difference between them was the residual amount of free HCDNA. The recovery rates of both the untreated and treated groups were more than 75%,and the RSD of reproducibility was less than 30%. Using affinity extraction method,with the affinity ligand combined with rAAV,the result of recovery rate of mispackaged HCDNA was over 75%,and that of free HCDNA was only 36%.Conclusion DNase treatment method can effectively detect free and mispackaged HCDNA,laying a foundation for further research.
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@#Objective To select and optimize the process parameters of SARS-CoV-2 F61 affinity chromatography by the screening and optimization experiment of Design of Experiment(DoE),in order to obtain the optimal process conditions.Methods Eight process parameters that may affect the experimental response results in affinity chromatography were selected and their level ranges were determined.DoE screening test was used to perform 8 factor 2 level screening tests on the selected process parameters.The response values were detected and the mathematical model was fitted by statistical software.Three key process parameters significantly affecting the key quality attributes were obtained by Pareto diagram analysis(P < 0.05).Then DoE response surface method(RSM)was selected to optimize the key process parameters.First,the full factorial experiment design was completed,the response results were detected to fit the mathematical model,and the bending term P value was analyzed to judge the range of significant factors in the optimal range(bending P < 0.05),then according to the sequential complement of the central composite face-centered design(CCF)experiment,through the detection of the response results to fit the response surface model,the range of optimal conditions was obtained,and the stability of the optimal parameters was finally verified by repeated experiments.Results In the screening experiment,it was found that the significant factors affecting F61 in the affinity chromatography stage were elution buffer pH,elution buffer salt concentration,leaching buffer salt concentration and equilibrium buffer salt concentration.The optimal conditions of key process parameters in affinity chromatography were obtained by CCF.When the pH of elution buffer was 3.2,the elution buffer salt concentration was 0.07 mol/L NaCl,and the leaching buffer salt concentration was 0.31 mol/L NaCl,the yield of F61 reached 95.25%,the residual amount of host cell protein(HCP)was 97.33 ppm,and the monomer purity of the sample was98.51% at the affinity chromatography stage.Conclusion Different types of DoE methods were used to screen and optimize the process parameters of F61 affinity chromatography stage,and the optimal process conditions were obtained,which lays a foundation for the esta-blishment of F61 purification process.
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@#Objective To optimize the expression conditions(expression and induction conditions)and purification methods(non-denaturing and denaturing purification)of recombinant Hq001 protein in salivary glands of Haemaphysalis qinghaiensis.Methods The recombinant plasmid pET-30a-Hq001 was transformed into competent cells E.coil BL21(DE3),E.coil Rosetta(DE3)and E.coil ArcticExpress(DE3)pRARE2 respectively for the selection of an optimal expression strain.The final concentration of IPTG(0,0.5,1.0 mmol/L),induction temperature(20,25 ℃)and induction time(0,2,4,6,8 h)were optimized.The recombinant bacteria expressed under the ideal induction condition were homogenized by French press and the target protein was purified by passing through a Ni-NTA affinity chromatography column under either native(denaturationrenaturation-column chromatography)or denatured conditions(denaturation-column chromatography-renaturation).The purified products were analyzed by 12% SDS-PAGE.Results E.coil BL21(DE3)was proved to be the most suitable strain for the expression of recombinant Hq001 protein.The optimum induction condition was induction with 0.5 mmol/L IPTG for 4 h at 25 ℃.The target protein with a relative molecular mass of approximately 18 800 was obtained by non-denaturing purification method,and the size was consistent with the expectation.Conclusion The recombinant protein rHq001 in salivary glands of Haemaphysalis qinghaiensis can be obtained by the optimized expression conditions and purification methods.
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Ubiquitination, a diverse post-translational modification, is carried out by enzymes including E1-activating enzymes, E2-conjugating enzymes, E3 ligases, and deubiquitinating enzymes (DUBs). Ubiquitin itself possesses 7 lysine residues and N-terminal methionine, allowing for the formation of polyubiquitin chains with different lengths and linkages. These chains exhibit various topologies that can be recognized by proteins containing ubiquitin-binding domain, thereby transmitting distinct cellular signals. To unravel the physiological mechanisms associated with ubiquitin, numerous ubiquitin probes have been developed. This review provides an overview of recent advancements in the field of ubiquitin probes, focusing on activity-based and affinity-based probes. Activity-based probes are designed to covalently bind to DUBs, E1s, or E3s, enabling the identification and characterization of these enzymes. Affinity-based probes, on the other hand, selectively bind to ubiquitin-binding domains, facilitating the identification of proteins that interact with ubiquitin. Moreover, this review comprehensively discusses the synthetic methodologies employed for the acquisition of ubiquitin probes. These includes meticulous discussions on the synthesis of individual monomeric modules, the establishment of isopeptide linkages, as well as the incorporation of reactive functional groups. Additionally, the review explores the emerging area of cell-penetrating ubiquitin probes and highlights their latest applications in living cells. These probes incorporate cell-penetrating peptides to enable their internalization into cells, allowing for direct visualization and manipulation of ubiquitin-modified proteins within their native environment. Overall, this review offers insights into the design, synthesis, and applications of ubiquitin probes, highlighting their significance in elucidating ubiquitin-mediated cellular processes.
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@#Objective To express,purify and identify human monomolecular antibody against Clostridium perfringens type A α-toxin,in order to lay a foundation for the prevention and treatment of various diseases caused by this toxin.Methods The fully human single-chain fragment variable(ScFv) gene against Clostridium perfringens type A was linked with the constant region of light chain and heavy chain of human antibody in different combinations to construct multiple monomolecular antibody expression plasmids against Clostridium perfringens α-toxin,which were expressed in competent E.coLi BL21(DE3).Indirect ELISA was used to detect the immunobinding activity of the monomolecular antibodies,and the monomolecular antibody with the highest immunobinding activity was purified by SepharoSe 4 FF and rProtein-A FF affinity chromatography,The purified products were analyzed by 12% SDS-PAGE.Indirect ELISA was used to detect the immune binding activity of each monomolecular antibody.Results Five recombinant plasmids,PTS-ScFv-CL-CH_2-CH_3,PTS-ScFv-CH_2-CH_3,PTS-ScFv-CL-CH_2,PTS-ScFvCH_2,and PTS-ScFv-CL,were constructed.After transfection into E.coli BL21(DE3) and purification,the corresponding monomolecular antibodies,ScFv-CL-CH_2-CH_3,ScFv-CH_2-CH_3,ScFv-CL-CH_2,ScFv-CH_2,and ScFv-CL,were obtained,which had the relative molecular mass of about 60 000,and the concentrations of about 1 mg/mL.Among them,ScFv-CH_2-CH_3showed the highest immune binding activity,and the A_(450) value reached 3.9,much higher than the other four monomolecular antibodies,with the concentration of about 1 mg/mL and the purity about 86%.Conclusion A fully human monomolecular antibody ScFv-CH_2-CH_3 with high affinity,low immunogenicity and internalization activity was obtained,which lays a foundation for the further study of therapeutic antibody against CPA.
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ObjectiveTo compare the effects of Taxillus chinensis from different hosts with different meridian affinity on bone microstructure and bone metabolism in ovariectomized osteoporotic rats， and investigate its mechanism of action. MethodEighty-eight specific-pathogen-free （SPF）-grade female Sprague-Dawley （SD） rats were selected and randomly divided into 11 groups： sham-operated group， model group， low-， medium- and high-dose groups of T. chinensis from Morus alba （2.5， 5， and 10 g·kg-1）， low-， medium- and high-dose groups of T. chinensis from Cinnamomum cassia （2.5， 5， and 10 g·kg-1）， and low-， medium- and high-dose groups of T. chinensis from C. burmannii （2.5， 5， and 10 g·kg-1）. After 12 weeks of drug intervention， the rats were examined for proximal femur bone density and bone microstructure using dual-energy X-ray absorptiometry （DXA） and micro-computed tomography （Micro-CT）. Histopathological changes in rat femur were observed by the hematoxylin-eosin staining （HE）. Contents of serum estradiol （E2）， bone Gla protein （BGP）， bone alkaline phosphatase （BALP）， tartrate-resistant acid phosphatase 5b （TRACP-5b） and pre-collagen type Ⅰ amino-terminal protopeptide （PINP） were measured by the enzyme-linked immunosorbent assay （ELISA）. Real-time quantitative polymerase chain reaction （Real-time PCR） was employed to detect the messenger ribonucleic acid （mRNA） expressions of bone morphogenetic protein-2 （BMP-2）， Smad1， Smad9 and recombinant runt-related transcription factor 2 （Runx2） in rat humerus. Western blot was used to detect the protein expressions of BMP-2， p-Smad1/5/9 and Runx2 in rat humerus. ResultCompared with that in the sham-operated group， the femur microstructure of rats in the model group was significantly disrupted， with significant decreases in bone mineral density （BMD） value， bone volume fraction （BV/TV）， trabecular number （Tb.N）， and trabecular thickness （Tb.Th） （P<0.01）， and significant increases in trabecular separation （Tb.Sp） and structure model index （SMI） （P<0.01）. The serum levels of BGP， BALP， TRACP-5b and PINP were significantly increased （P<0.05， P<0.01）， and E2 levels were significantly decreased （P<0.01）. The mRNA expressions of BMP-2， Smad1， Smad9， and Runx2 were significantly decreased in rat humerus （P<0.01）， and the protein expressions of BMP-2， p-Smad1/5/9， and Runx2 were significantly reduced （P<0.01）. Compared with the model group， the administration groups of T. chinensis from different hosts all elevated the BMD， BV/TV， Tb.N， Tb.Th， Tb.Sp， and SMI levels in the femur， improved bone microstructure， increased serum E2 levels （P<0.05， P<0.01）， lowered the levels of serum BGP， BALP， TRACP-5b， and PINP， upregulated the mRNA expression of BMP-2， Smad1， and Runx2 and upregulated the mRNA expression levels of Smad9 （P<0.05， P<0.01）， and upregulated the protein expressions levels of BMP-2， p-Smad1/5/9， and Runx2 （P<0.01）. The best effect was observed in the group of T. chinensis from C. cassia. ConclusionT. chinensis from different hosts improved osteoporosis in ovariectomized rats， with the group of T. chinensis from C. cassia being the most potent among the administered groups， and its treatment of osteoporosis may regulate the balance of bone conversion by regulating BMP/Smad signaling pathway.
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ObjectiveTo examine the inhibitory effects of berberine compounds， including columbamine， on acetylcholinesterase from the perspectives of drug-target binding affinity and kinetics and explore the blood-brain barrier （BBB） permeability of these compounds in different multi-component backgrounds. MethodThe median inhibitory concentration （IC50） of acetylcholinesterase by berberine compounds including columbamine was measured using the Ellman-modified spectrophotometric method. The binding kinetic parameters （Koff） of these compounds with acetylcholinesterase were determined using the enzyme activity recovery method. A qualitative analysis of the ability of these components to penetrate the BBB and arrive at the brain tissue in diverse multi-component backgrounds （including medicinal herbs and compound formulas） was conducted using ultra performance liquid chromatography-high resolution mass spectrometry （UPLC-HRMS）. ResultBerberine compounds， including columbamine， exhibited strong inhibition of acetylcholinesterase， with IC50 values in the nanomolar range. Moreover， they displayed better drug-target binding kinetics characteristics （with smaller Koff values） than the positive control of donepezil hydrochloride （P<0.01）， indicating a longer inhibition duration of acetylcholinesterase. Berberine components such as columbamine could penetrate the BBB to arrive at brain tissue in the form of a monomer， as well as in the multi-component backgrounds of Coptis and Phellodendri Chinensis Cortex medicinal extracts and the compound formula Huanglian Jiedutang. ConclusionThese berberine compounds such as columbamine exhibit a strong inhibitory effect on acetylcholinesterase and can arrive at brain tissue in multi-component backgrounds. In the level of pharmacological substance， this supports the clinical efficacy of compound Huanglian Jiedutang in improving Alzheimer's disease， providing data support for elucidating the pharmacological basis of compound Huanglian Jiedutang.
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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.
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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.
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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.
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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.
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Activity-based protein profiling(ABPP)and affinity-based protein profiling(AfBPP)are reliable chemical proteomics techniques that exhibit significant advantages in identifying the direct acting targets of small molecule drugs and toxicants,which can help researchers understand the pharmaco-logical and toxicological mechanisms of active small molecules.This paper introduces ABPP and AfBPP technologies and summarizes the applications of this technique in drug off-target effect and target iden-tification of toxicants in recent years,such as drug off-target effect of crenolanib,BIA 10-2474,orlistat and target identification of VX,fenitrothion,acrolein.It is hoped that this review will give readers a better idea of ABPP/AfBPP and offer a line of thinking for researchers in the fields of pharmacology,toxicology and chemical biology.
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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}}
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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.
ABSTRACT
@#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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.