The value of peripheral blood and pleural effusion T-SPOT.TB combined with pleural effusion adenosine deaminase in diagnosis of tuberculous pleurisy / 中国热带医学

@#Abstract: Objective To explore the relationship between peripheral blood and pleural effusion tuberculosis (TB) infection effector T cells, and to further evaluate the value of combined pleural effusion adenosine deaminase (ADA) for rapid diagnosis of tuberculous pleurisy. Methods The test data of 80 cases of tuberculous pleurisy and 70 cases of nontuberculous pleurisy treated in the Sixth People's Hospital of Nantong City from January 2017 to December 2020 were analyzed. The TBinfected effector T cells were also detected simultaneously in the peripheral blood and the pleural effusion by the T-SPOT technique, and the pleural effusion ADA was detected by the rate method. The subject operating characteristic curve (ROC) was applied to take the optimal pleural effusion ADA threshold to compare the sensitivity and specificity of different critical values. Person phase analysis was applied to analyze the correlation between peripheral blood and pleural effusion T-SPOT.TB. Data of peripheral blood, pleural effusion T-SPOT.TB and ADA were integrated. Results When pleural effusion ADA>45 U/L, the sensitivity and specificity for the diagnosis of tuberculous pleurisy were 50.0% and 94.3%, respectively; when ADA > 25.15 U/ L, the sensitivity and specificity were 80.0% and 72.9%. When ADA > 45 U / L, pleural/ blood T-SPOT.TB spot ratio (spot forming cells, SFCs) > 2 times, the specificity for the diagnosis of tuberculous pleurisy was 100% (highest); when 25.15 U/L< pleural effusion ADA ≤ 45 U/L, pleural/blood T-SPOT.TB spot ratio > 2 times, the specificity for the diagnosis of tuberculous pleurisy was 92.3% (second). When pleural effusion ADA ≤ 25.15 U/L, and the pleural effusion/blood T-SPOT.TB spot number ratio > 2 times, with 83.3% specificity (the lowest of the three groups). Conclusions The level of pleural effusion ADA is one of the most used methods for diagnosing tuberculous pleurisy. Further combination of pleural effusion and blood T-SPOT.TB, if the ratio of pleural effusion / blood T-SPOT. TB spots is greater than 2 times, it can further improve the diagnosis rate of tuberculous pleurisy.

Investigation on the mechanism of anti-drug-induced liver injury and related effector substances of traditional Chinese medicine from the perspective of metabolism / 药学学报

Drug-induced liver injury (DILI) is one of the common clinical adverse drug reactions and remains a major cause of drug restriction, development termination and withdrawal from the pharmaceutical market today. In recent years, a variety of chemical components and metabolites of traditional Chinese medicine (TCM), as well as the endogenous effector substances influenced by metabolism of both, have attracted much attention for their significant hepatoprotective activities. However, the mechanism of TCM against DILI is complex, the related effector substances are still unclear, and its metabolism-related studies are still relatively weak. Therefore, this review summarized the mechanisms of DILI and its treatment by TCM from the perspective of metabolism, and for the first time, innovatively classified the Chinese medicine effector substances into two categories: exogenous (active components and metabolites of TCM) and endogenous (intestinal probiotics and endogenous metabolites), in order to reduce the occurrence of DILI, explore and develop effective anti-drug-induced liver injury effector substances of TCM, and further develop clinical drugs with hepatoprotective effects.

Mecanismos inmunológicos en Cnidaria: un sistema inmune basal de gran complejidad e interés bioprospectivo / Immunological mechanisms in Cnidaria: A highly complex basal immune system with bioprospective interest

Introducción: La protección ante agentes biológicos propios y externos de los cnidarios dependen de la inmunidad innata, la cual consta de tres procesos inmunológicos principales: 1) reconocimiento inmunológico, 2) señalización intracelular, y 3) respuesta efectora. Objetivo: Revisar críticamente el conocimiento actual del repertorio molecular involucrado en la respuesta inmune en cnidarios, así como, su papel en el establecimiento de la simbiosis, y las posibles aplicaciones biotecnológicas de las moléculas involucradas en el proceso de inmunidad. Métodos: Se realizó una revisión de artículos científicos encontrados a través de las bases de datos del NCBI, Google Scholar y Scielo, con palabras claves como inmunidad y/o reconocimiento inmunológico en cnidarios, en una ventana de tiempo de la última década, sin descartar literatura clásica más antigua. Resultados: El reconocimiento inmunológico consiste en receptores inmunológicos que reconocen patrones moleculares e inducen respuestas efectoras como la movilización de moléculas al sitio de la infección, la ingestión microbiana y la formación de moléculas que activan cascadas de señalización. La fase de señalización involucra mediadores de la traducción de señales que activan genes de trascripción, y cascadas de señalización intracelular que inician respuestas de defensa adecuadas. Las respuestas efectoras incluyen la capa superficial del mucus, péptidos antimicrobianos, especies reactivas de oxígeno y la respuesta celular mediada por fagocitosis. Por último, se presenta un esquema y una tabla integral de las vías de respuesta inmune en los cnidario. Conclusiones: La inmunidad en Cnidaria está mediada por mecanismos de defensa complejos integrados por receptores de reconocimiento de patógenos, vías de señalización intracelular, células y moléculas efectoras encargadas de la eliminación del patógeno, y reconocimiento-aceptación de simbiontes. El estudio de compuestos activos del sistema inmune en Cnidaria ha sido poco explorado, sin embargo, el trabajo realizado con otros compuestos presentes en las toxinas de este filo, los sitúa como una fuente importante de moléculas antimicrobianas dignas de un análisis de bioprospección.

Introduction: Cnidarians depend on innate immunity for protection against both their own and external biological agents. It consists of three main immunological processes: 1) immune recognition, 2) intracellular signaling, and 3) effector response. Objective: To critically review current knowledge of the molecular repertoire involved in the immune response in cnidarians, its role in symbiosis, and possible biotechnological applications. Methods: We used keywords such as immunity, and immunological recognition in cnidarians, in the NCBI, Scielo and Google Scholar databases, for the last decade. Results: Cnidarian immune recognition consists of molecular pattern receptors and responses such as the mobilization of molecules to the site of infection, microbial ingestion, and the formation of molecules that activate signaling cascades. The signaling phase involves translation mediators that activate transcriptional genes and intracellular signaling cascades that initiate defenses. Effector responses include surface layer mucus, antimicrobial peptides, reactive oxygen species, and the cellular response mediated by phagocytosis. Conclusions: Immunity in Cnidaria is mediated by complex defense mechanisms composed of pathogen recognition receptors, intracellular signaling pathways, effector cells and molecules responsible for pathogen elimination, and recognition of symbionts. There is a potential for toxin compounds useful as antimicrobial molecules.

Research and reflections on the nitric oxide-donating cardio-cerebrovascular drugs / 中国药科大学学报

@#Nitric oxide (NO) as a signal transduction species and/or effector molecule plays an extremely important role in the cardiovascular, nervous, and immune systems. Insufficient production or abnormal signal transduction of NO is closely associated with onset and progression of many diseases. Studies on direct or indirect regulation of NO levels in vivo and related metabolic pathways have become a hot spot in biomedical and pharmaceutical fields. This review briefly introduces the generation of NO in vivo and its physiological and pathological functions in cardio-cerebrovascular system, with focus on the authors'' research on NO-donating cardio-cerebrovascular and related drugs, aiming to provide some reference for future development of these drugs.

Effect of metformin on bleomycin-induced systemic sclerosis mice by regulating the balance of Teff/regulatory T cells / 中华风湿病学杂志

Objective:To explore the effect and mechanism of different concentrations of metformin on bleomycin (BLM)-induced systemic sclerosis (SSc) mice model.Methods:C57BL/6 mice were divided into the normal group, the model group, the high, the medium and the low metformin (MET) treatment groups randomly. All mice were sacrificed after BLM and metformin treatment for 4 weeks. Local skin was exminedby histopathological staining method to measure the thickness of dermis and collagen, and immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) were used to detect the protein and mRNA levels of Interleukin (IL)-17, forkhead box P3 (Foxp3) and α-smooth muscle actin (α-SMA). Flow cytometry was used to detect the percentage of effector T cell (Teff) and regulatory cells (Treg) in splenic mononuclear cells. The data such as dermal collagen thickness, α-SMA, IL-17, Foxp3, Teff and Treg levels were statistically analyzed by one-way analysis of variance. The data such as dermal collagen thickness, α-SMA, IL-17, Foxp3, Teff and Treg levels were analyzed by one-way analysis of variance, and least significant difference (LSD)- t or Kruskal-Wallis test was used for comparison between groups. Results:Compared with the normal group, remarkable fibrotic lesions appeared in the skin of mice in the model group, and the levels of T-helper cells (Th)1, Th2, Th17, and T follicular helper cells (Tfh) cells were increased, accompanied by a significant decrease in the level of Treg cells. After high-dose metformin treatment, the dermal thickness [(131±25) μm], collagen thickness [(119±18) μm], and α-SMA [(3.0±0.5)/HPH] were significantly reduced( F=14.390, P<0.01; F=40.245, P<0.01; F=44.626, P<0.01). Th1[(27.00±6.68)%], Th17[(0.56±0.20)%], Tfh[(6.4±1.6)%] cells ware significantlyreduced ( F=32.390, P<0.01; F=16.083, P<0.01; F=16.546, P<0.01), and Treg[(11.23±1.52)%] cells were significantly increased ( F=10.171, P<0.01). Conclusion:Metformin can effectively reverse the local skin changesin BLM-induced SSc mouse model, and show immune regulation and anti-fibrosis effects by restoring the Teff/Treg balance.

Identification of the host substrate of a Salmonella typhimurium type three secretion system effector SseK3 and preliminary investigation on its biological function / 中华微生物学和免疫学杂志

Objective:To identify new host substrate of SseK3 and study its biological function.Methods:A yeast two hybrid system (Y2H) was used to identify the potential binding proteins of SseK3 from the Hela cDNA library; the arginine N-acetylglucosamine (Arg-GlcNAc) modification of the substrate protein by SseK3 was detected by co-expression in 293T cells and in vitro activity test; the modification sites of the substrate protein by SseK3 were detected by point mutation; the effect of Arg-GlcNAc modification of the substrate protein on its interaction protein binding ability was detected by immunoprecipitation test. Results:Results of Y2H and gene sequencing showed that Snapin was a new substrate of SseK3. Snapin could be Arg-GlcNAc-modified by SseK3 in vivo and in vitro; the modification sites of Snapin were arginine 119 and arginine 120; Arg-GlcNAc-modified Snapin inhibited its binding with SNAP25. Conclusions:Snapin, a new host substrate protein of SseK3, was successfully screened in this study. The Arg-GlcNAc modification of Snapin by SseK3 was studied, and the effect of this modification on Snapin function was preliminarily studied, which provided theoretical basis for further understanding the function of Arg-GlcNAc modification of bacteria and the mechanism of action in the process of pathogen infection.

Resistencia de plantas a patógenos: una revisión sobre los conceptos de resistencia vertical y horizontal / Plant resistance to pathogens: A review describing the vertical and horizontal resistance concepts

Resumen Para aumentar la resistencia de las plantas a los patógenos se requiere de un trabajointerdisciplinario de las ciencias biológicas y agrícolas. En este escenario, la fitopatología se habeneficiado del encuentro de disciplinas biológicas claves, como la ecología, la evolución y labiología molecular. Sin embargo, este encuentro no ha estado exento de controversia entre losacadémicos de estas disciplinas, quienes a menudo han otorgado diferentes significados a unmismo concepto. Este documento tiene como propósito ofrecer una visión abarcativa y modernade la resistencia de las plantas a los patógenos, enmarcada dentro del concepto clásico deresistencia vertical y horizontal propuesto por Van der Plank. Este concepto tiene implicacionesen áreas centrales para la agricultura, como el fitomejoramiento y la genética vegetal, loque justifica su uso como el marco conceptual indicado. Dentro de este contexto se explicanotros conceptos clásicos de la fitopatología en combinación con descripciones modernas de lainteracción entre plantas y patógenos, y se discute cómo estos conceptos están relacionadoscon la resistencia cuantitativa y de campo. Asimismo, esta revisión tiene un propósito adicionalcomo aporte a la ense˜nanza de la fitopatología en la América de habla hispana, dado que enesta región existe, además de la barrera interdisciplinaria, un factor adicional y persistenteque impide llevar la discusión a un terreno común: la baja competencia lingüística en el idiomainglés en todos los niveles educativos. Esta revisión pretende ser una herramienta de apoyopara los docentes de fitopatología de la región, interesados en explicar los conceptos modernosde resistencia a patógenos en plantas.

Abstract Understanding plant resistance requires an interdisciplinary effort between biolo-gical and agricultural sciences. In this setting, phytopathology has experienced an upsurge of interest from researchers and scholars in the disciplines of ecology, evolution and molecular biology. This encounter did neither avoid misunderstandings among scholars nor the use of similar concepts with different meanings. The purpose of this paper is to offer a modern comprehensive view of plant resistance against pathogens using a classical phytopathology concept as framework: Van der Plank s concept of horizontal and vertical resistance. This concept is used in other agricultural science disciplines (plant breeding and genetics), supporting why it is a proper framework for explaining plant resistance. Within this frame, other classical phytopathologycal concepts are explained in combination with modern model descriptions of plant-pathogen interactions and how all these concepts are related with quantitative and field resistance. This review is written in Spanish because it serves an additional purpose. In the Spanish-speaking America, besides interdisciplinarity, phytopathology as an academic discipline faces another challenge: the students' low-English language proficiency. In this regard, this review intends to become a companion guide for plant-pathology teachers in the region interested in providing an insight into the modern concepts of plant resistance.

A giant step forward: chimeric antigen receptor T-cell therapy for lymphoma / 医学前沿

The combination of the immunotherapy (i.e., the use of monoclonal antibodies) and the conventional chemotherapy increases the long-term survival of patients with lymphoma. However, for patients with relapsed or treatment-resistant lymphoma, a novel treatment approach is urgently needed. Chimeric antigen receptor T (CAR-T) cells were introduced as a treatment for these patients. Based on recent clinical data, approximately 50% of patients with relapsed or refractory B-cell lymphoma achieved complete remission after receiving the CD19 CAR-T cell therapy. Moreover, clinical data revealed that some patients remained in remission for more than two years after the CAR-T cell therapy. Other than the CD19-targeted CAR-T, the novel target antigens, such as CD20, CD22, CD30, and CD37, which were greatly expressed on lymphoma cells, were studied under preclinical and clinical evaluations for use in the treatment of lymphoma. Nonetheless, the CAR-T therapy was usually associated with potentially lethal adverse effects, such as the cytokine release syndrome and the neurotoxicity. Therefore, optimizing the structure of CAR, creating new drugs, and combining CAR-T cell therapy with stem cell transplantation are potential solutions to increase the effectiveness of treatment and reduce the toxicity in patients with lymphoma after the CAR-T cell therapy.

Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis / 亚洲男科学杂志(英文版)

The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development, whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction. We previously demonstrated that in mice, the makorin-2 (Mkrn 2) gene is expressed exclusively in the testis and its deletion leads to male infertility. To understand the potential molecular mechanism, in this study, we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn 2. To identify specific gene(s) involved, we performed digital gene expression profiling (DGE) and pathway analysis via gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22 (PERP) expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels. GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility. Further, Gene Expression Omnibus (GEO) dataset analysis showed that p53, upstream of PERP, was upregulated in oligoasthenoteratozoospermia (OAT). These observations suggest that Mkrn 2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn 2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.

Discovery of bioactive compounds and effector substance based on gut microbiota / 中草药

With the increase of the chronic complex diseases and the growing advantage of traditional Chinese medicine (TCM) in treating chronic complex diseases, Chinese materia media (CMM) became more and more importance in the world. However, the ambiguity of bioactive compounds, effective materials and their corresponding mechanisms has limited the acceptance of CMM in clinic and the internalization of CMM in the world. As a result, the discovery of bioactive compounds and effective materials that are directly associated with therapeutic effects of CMM is one of the most important scientific question in researching of CMM. Hence, in this article, we briefly reviewed current methods for discovery of bioactive compounds and effective materials. Then, with references related to the international research frontier and the characteristics of CMM ingredients, according to the characteristics of most Chinese medicine through oral administration, combined with the previous research basis of our research group, a research model that is based on gut microbiota was investigated to discover bioactive compounds and effective materials. The aim of this research model is to provide new thoughts on discovery of new drugs, the mechanism study of CMM, etc., and eventually promote the modernization and internalization of CMM.

Feasibility of Robot-Assisted Gait Training with an End-Effector Type Device for Various Neurologic Disorders

Robots are being used to assist the recovery of walking ability for patients with neurologic disorders. This study aimed to evaluate the feasibility and functional improvement of training with robot-assisted gait training (RAGT) using the Morning Walk®, an end-effector type robot using footplates and saddle seat support. A total of 189 individuals (65.1% men, 34.9% women; mean age, 53.2 years; age range: 5–87 years) with brain lesions, spinal cord injuries, Parkinson's disease, peripheral neuropathies, and pediatric patients were involved in this retrospectively registered clinical trial. Each participant performed 30 minutes of RAGT, five times a week, for a total of 24 sessions. Failure was defined as an inability to complete all 24 sessions, and the reasons for discontinuation were analyzed. Parameters of Medical Research Council scales and Functional Ambulation Categories were analyzed before and after RAGT training. Among the 189 patients, 22 (11.6%) failed to complete the RAGT. The reasons included decreased cooperation, musculoskeletal pain, saddle seat discomfort, excessive body-weight support, joint spasticity or restricted joint motion, urinary incontinence from an indwelling urinary catheter, and fatigue. Comparison between the pre- and post-training motor and ambulatory functions showed significant improvement. The result of the study indicates that the Morning Walk® is feasible and safe and useful for functional improvement in patients with various neurologic disordersTRIAL REGISTRATION: Clinical Research Information Service Identifier: KCT0003627

Caracterização de dois pares efetor/inibidor associados ao sistema de secreção tipo IV de Xanthomonas citri / Characterization of the two effector/inhibitor pair associated with the type IV secretion system of Xanthomonas citri

O sistema de secreção tipo IV (T4SS) da família de bactérias Xanthomonadaceae transfere efetores (X-Tfes) com a capacidade de matar outras bactérias, conferindo uma vantagem em comunidades bacterianas mistas para colonizar diferentes nichos como o solo ou as superfícies das plantas. Os X-Tfes possuem diferentes domínios putativos com atividades hidrolíticas contra componentes do envelope celular bacteriano do tipo: glicohidrolases, transglicosilases, amidases e lipases. Os X-Tfes por sua atividade biológica inata podem ocasionar dano intracelular para a bactéria que os produz. Para se proteger contra estas atividades, também são produzidas lipoproteínas com função inibitoria (X-Tfis) localizadas no periplasma. Os genes que codificam os X-Tfes e os X-Tfis estão organizados em operons, o que permite gerar os pares efetor/inibidor simultaneamente. Entre os potenciais X-Tfes do fitopatógeno Xanthomonas citri estão Xac1918 e Xac0574. Xac1918 é uma proteína com um domínio da superfamília da lisozima e um domínio conhecido como RTX (Repeats in Toxin) de ligação ao cálcio, enquanto Xac0574 tem um domínio da superfamília da lipase 3. Os seus possíveis inibidores, Xac1917 e Xac0573 respectivamente, apresentam um peptídeo sinal no N-terminal contendo o lipobox representativo das lipoproteínas. As proteínas Xac0574 e Xac0573 são monômeros em solução que formam um complexo estável 1:1, favorecido termodinamicamente (ΔG°= -12 Kcal/mol) com uma constante de dissociação de 2,45 nM, garantindo que a bactéria fique protegida contra os efeitos nocivos de Xac0574 quando é produzida intracelularmente. Xac0574 é uma fosfolipase A1, sem atividade lisofosfolipase, com a capacidade de hidrolisar os três fosfolipídios majoritários que compõem a membrana celular bacteriana, fosfatidilglicerol (PG), cardiolipina e fosfatidiletanolamina (PE), mostrando uma aparente preferência pelo último. A atividade enzimática de Xac0574 explica a forte inibição do crescimento celular em E. coli após da sua indução heteróloga, já que gera uma diminuição de quase 10 vezes da população celular comparada com a cultura não induzida com a mesma construção. Poroutro lado, Xac0573 inibe efetivamente a atividade enzimática de Xac0574 ao formar o complexo, além de não ter atividade fosfolipase nem lisofosfolipase. Foram produzidos cristais da Xac1918 e Xac0573 que difrataram com uma resolução de 3,0 e 2,5 Å, respectivamente. Porém, só foi gerado um modelo de Xac0573. Xac0573 está composta por duas folhas ß antiparalelas com uma topologia característica de ß sanduíche Com uma pequena hélice e duas voltas. Um alinhamento de homólogos de Xac0573 identificou nas extremidades da proteína as regiões conservadas, constituindo duas possíveis interfaces de interação que podem ser as responsáveis por bloquear o acesso dos fosfolipídios ao sítio catalítico ou impedir os rearranjos estruturais de Xac0574 que são necessários para a sua atividade enzimática. Adicionalmente, a topologia da Xac0573 é semelhante do domínio C2, conhecido em eucariotos como domínio de ligação ao lipídio e ao cálcio, e está envolvido em processos de sinalização de segundos mensageiros lipídicos, proteínas de trafego de membranas e mecanismos de fusão de membranas. Nossos resultados apontam para uma nova função biológica do domínio C2 como um inibidor enzimático intracelular em bactérias

The type IV secretion system (T4SS) of the bacteria family Xanthomonadaceae transfers effectors (X-Tfes) with that can kill other bacterial cells, conferring an advantage to the bacterial community during colonization of different niches in the soil or on the plant surface. The X-Tfes possess different putative domains with hydrolytic activity against components of the bacterial cellular envelope, including glycohydrolase, transglycolase, amidase and lipase domain. The innate biological activity of X-Tfes can cause intracellular damage. Therefore, the bacteria that produce them also produce lipoproteins with inhibitor function (X-Tfis) located in the periplasm for their protection. The genes that code for X-Tfes and X-Tfis are organized in operons that allow for their simultaneous expression. Among the X-Tfes of the phytopathogen Xanthomonas citri are Xac1918 and Xac0574. Xac1918 is carries a lysozyme superfamily domain, as well as a domain known as RTX (Repeats in Toxic) predict to bind calcium, while, Xac0574 has a domain belonging to the lipase 3 superfamily. Their possible inhibitors, Xac1917 e Xac0573 respectively, carry an N-terminal signal peptide containing a lipobox found in bacterial lipoproteins. The Xac0574 and Xac0573 proteins are both monomers in solution, They can form a stable 1:1 complex, that is thermodynamically favored (ΔG°= -12 Kcal/mol) with a dissociation constant of 2,45 nM. This affinity ensure that the bacterium is protected against the harmful effects of Xac0574 when it is produced intracellularly. We show that Xac0574 is a phospholipase A1, without lisophospholipase activity, and is able to hydrolyze the three most common phospholipids found in the membranes of Gram negative bacteria, namely phosphatidylglycerol (PG), cardiolipin and phosphatidylethanolamine (PE), presenting an apparent preference for PE. The enzymatic activity of Xac0574 explains the strong inhibition of growth of E. coli cells after its heterologous induction: a nearly 10-fold decrease in the cell population is observed when compared to the non-induced culture with the same construct. On the other hand, Xac0573 effectively inhibits the enzymatic activity of Xac0574. Furthermore, Xac0573 does not possess when forming the complex, besides not having phospholipase nor lysophospholipase activity.Crystals of Xac1918 and Xac0573 were produced which diffracted with to resolution of 3.0 and 2.5 Å, respectively. However, we were able to resolve the structure of only Xac0573. Xac0573 is composed of two anti-parallel sheet that form a ß-sandwich with three small helices. An alignment to Xac0573 homologs identified conserved regions at the ends of the protein that constitute two possible interfaces of interaction that may be responsible for blocking the access of the phospholipids to the catalytic site or impede the structural rearrangements of Xac0574 that are necessary for its enzymatic activity. Additionally, the topology of Xac0573 is similar to that to C2 domains, known in eukaryotes to bind lipids and calcium and to be involved in signaling processes mediated by lipid second messengers, membrane trafficking and membrane fusion mechanisms. Our results point to a new biological function of the C2 domain as an intracellular enzyme inhibitor in bacteria

Human-robot global Simulink modeling and analysis for an end-effector upper limb rehabilitation robot / 生物医学工程学杂志

Robot rehabilitation has been a primary therapy method for the urgent rehabilitation demands of paralyzed patients after a stroke. The parameters in rehabilitation training such as the range of the training, which should be adjustable according to each participant's functional ability, are the key factors influencing the effectiveness of rehabilitation therapy. Therapists design rehabilitation projects based on the semiquantitative functional assessment scales and their experience. But these therapies based on therapists' experience cannot be implemented in robot rehabilitation therapy. This paper modeled the global human-robot by Simulink in order to analyze the relationship between the parameters in robot rehabilitation therapy and the patients' movement functional abilities. We compared the shoulder and elbow angles calculated by simulation with the angles recorded by motion capture system while the healthy subjects completed the simulated action. Results showed there was a remarkable correlation between the simulation data and the experiment data, which verified the validity of the human-robot global Simulink model. Besides, the relationship between the circle radius in the drawing tasks in robot rehabilitation training and the active movement degrees of shoulder as well as elbow was also matched by a linear, which also had a remarkable fitting coefficient. The matched linear can be a quantitative reference for the robot rehabilitation training parameters.

Mechanisms of recombinant adenovirus-mediated SD-HA fusion protein proliferation inhibition and induced apoptosis of K562 cells / 中华血液学杂志

Objective@#To investigate whether fusion protein SD-HA could regulate its downstream signaling molecule activity by competing with the phospho-BCR-ABL Y177 site, and its mechanisms to inhibit proliferation and induce apoptosis of K562 cells.@*Methods@#Co-immunoprecipitation interaction technology analysis of fusion protein SD-HA functioned by potently binding to the phospho-BCR-ABL Y177 site, Ras, MAPK and Akt activities were observed in the Ad5F35-SD-HA-treated cells. Western blot analyses of SD-HA fusion protein on cell membrane receptor pathway to death cascade caspase-8, caspase-3 and PRAP were performed.@*Results@#Exploration into the underlying mechanisms revealed that Ad5F35-SD-HA infection functioned by binding to the phospho-BCR-ABL Y177 site, which lead to a complex with Grb2. competitively disrupted the Grb2 SH2-phospho-BCR-ABL Y177 formation. The fusion protein SD-HA could reduce the activation of Ras and phosphorylation of MAPK (p-MAPK) and the expression level of p-ELK, inhibition of Ras-MAPK signaling pathway; SD-HA fusion protein could reduce p-Akt and Akt substrate p-GSK with inhibition of PI3K-Akt signaling pathway, thereby inhibiting the proliferation of K562 cells. Caspases-8-induced apoptosis signal could be activated by DED protein binding to DED domain of precursor caspases-8.@*Conclusions@#The strategy of fusion protein SD-HA inhibiting-Y177 BCR-ABL and Grb2 binding could be used as a novel entry point for the treatment of chronic myeloid leukemia.

The effects of miR-10a on the immune function of splenic CD4+CD25+ Treg cells in septic mice / 中华急诊医学杂志

Objective To investigate the role of miR-10a in CD4+CD25+Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis.Methods Sepsis mouse model was established by cecal ligation and puncture(CLP).Balb/c mice of clean grade were sacrificed 1,3,5,and 7 days after operation.Blood as well as spleen samples were harvested at given intervals.The splenic CD4+CD25+Treg cells and CD4+T cells were isolated by MACS microbeads.Cells were cultured,and phenotypes were analyzed by flow cytometry.The miR-10a expressed in Treg cells were detected by Real-time PCR.After administration of LV-mmu-miR-10a-5p-inhibition,the immunosuppressive function have been detected.Statistical analyses were performed using one-way analysis of variance (SPSS 19.0,Chicago,USA) test followed by Dunnett-t test to compare among three or more groups or by Student's t-test to compare between two groups.Results The percentages of splenic Tregs (CD4+CD25+/CD4+T) was (7.34±1.2)％ in normal group,and the increase in percentage of Tregs in spleen has been observed in septic mice (P＜0.05).The mean fluorescence intensity (MFI) of Foxp3+Treg was increased in septic mice compared with sham group (P＜0.05).The expression of miR-10a was significantly elevated on CLP 1-7 day (P＜0.05).After down-regulation of miR-10a in septic mice,the percentages of Tregs (CD4+CD25+/CD4+T) was significantly increased in septic mice (P＜0.05),the MFI of Foxp3+Treg was increased in septic mice compared with control group (P＜0.05).The CD4+T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group (P＜0.05).After down-regulation of miR-10a in septic mice,the CD4+T cell proliferative activity was significantly suppressed compared with control group (P＜0.05).Conclusions Treg plays a critical role in immunosuppression in septic mice.Inhibition of miR-10a in vivo could enhence immunesuppression of CD4+CD25+Treg.Therefore miR-10a may participate in the regulation of CD4+CD25+Treg immunosuppression in sepsis and become the target for immunotherapy.

In vitro study of joint intervention of E-cad and Bmi-Ⅰ mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma / 中南大学学报(医学版)

Objective:To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors ofnasopharyngeal carcinoma cells.Methods:Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed,and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously.The integration of target genes were detected by PCR,the expressions of E-cad and Bmi-1 were detected by Western blot.The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay.T-he cell cycle and apoptosis were detected by flow cytometry.The cell migration and invasion were detected by Transwell assay.Results:The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells,the protein expression level of E-cad was up-regulated,and the protein expression level of Bmi-1 was down-regulated.The intervention of E-cad and Bmi-1 didn't affect the proliferation,cell cycle and apoptosis of CNE-2 cells,but it significantly inhibited the migration and invasion ability of CNE-2 cells.Furthermore,the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P＜0.01).Conclusion:The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro,which may lay the preliminary experimental basis for gene therapy of human cancer.

Differences in susceptibility to Lewis lung carcinoma and T lymphocyte subsets of aged mice / 中华老年医学杂志

Objective To investigate the differences in susceptibility to Lewis lung carcinoma and T lymphocyte subsets in the immune microenvironment between young and elderly mice.Methods Six C57/B6 mice at two months(young)and six mice at twelve months(aged)were injected with Lewis lung carcinoma cells at the dose of 1 × 106 in the left armpit to establish a murine model of lung carcinoma.The weight and tumor growth were monitored.Blood samples for routine blood tests were collected after 24 days.The proportions of CD4+ and CD8+T cells in the spleen were detected by flow cytometry,and the infiltration of CD4+,CD8+ T cells and related effector T cells in the tumor microenvironment were determined in the same way.Results The body weight of tumor bearing mice in the aged group was significantly higher than that in the young group(P ＜0.001);The tumor weight in the aged group(5.084±0.528)g was significantly higher than that in the young group(2.963 ±0.378)g(t =3,349,P =0.012);Routine blood tests showed that the numbers of leukocytes and subsets(except mononuclear)in the aged group were significantly lower than in the young group(P ＜0.05);Flow cytometry found that the effector and memory/effector CD4+T cell ratios in the spleen were significantly higher in the aged group than in the young group(P ＜0.001)and the expression of effector and memory/effector CD8+T cells in the tumor microenvironment was also significantly higher than in the young group(P ＜0.05);Quantitative expression values of IL-6 and IL-10 in the tumor microenvironment were 25090±3820 and 10670± 1793 in the aged group and 6252±864 and 3061±451 in the young group,respectively.Moreover,the expression levels of IL-6 and IL-10(t =3.925,P =0.01;t =3.552,P =0.02)in the tumor microenvironment in the aged group were significantly lower than those in the young group.Conclusions Young mice are more susceptible to Lewis lung carcinoma,probably as a result of differences in inflammation and immunity.

Estudos funcionais e bioquímicos sobre o reconhecimento e inibição de efetores de um sistema de secreção tipo IV de Xanthomonas citri subsp. citri. / Functional and biochemical son the recognition and Inhibition of effectors of a Type IV secretion System of Xanthomonas citri subsp. citri

Sistemas de Secreção Tipo IV (T4SSs), normalmente compostos por 12 proteínas (VirB1-VirB11 e VirD4) são tipicamente associados às funções de conjugação bacteriana e transferência de fatores de patogenicidade para células hospedeiras. Mas também, muitas espécies da ordem Xanthomonadales possuem um T4SS associado a matar bactérias. O modelo atual de morte de uma célula-alvo mediada pelo T4SS é baseado na secreção de toxinas denominadas XVIPs ("Xanthomonas VirD4 interacting proteins") ou X-Tfe (Xanthomonadaceae-T4SS effector) no qual cada XVIP/X-Tfe apresenta uma proteína de imunidade cognata denominada X-Tfi (Xanthomonadaceae-T4SS immunity protein). Demonstramos que um XVIP, XAC2609, é secretado através do T4SS de modo que depende de contato célula-célula e do seu domínio XVIPCD ("XVIP conserved domains"). A porção N-terminal de XAC2609 codifica um domínio GH19 que cliva a peptideoglicana de E. coli, mas perde a sua atividade na presença do seu inibidor cognato, o X-Tfi XAC2610. Portanto, XAC2609/XAC2610 formam um par de proteínas efetora/imunidade associado ao T4SS de X. citri. Através de diferentes técnicas de microscopias utilizando a cepa Δxac2610, foi observado que XAC2610 protege o envelope celular de X. citri contra efeitos de autólise celular promovidos pela atividade de XAC2609. Ensaios funcionais baseados nas observações de fenótipos de colônias e de formação de biofilme mostraram que XAC2610 confere imunidade para X. citri contra uma atividade 7 intrínseca de XAC2609. A proteína com o papel de reconhecer os substratos através da interação com os sinais de secreção do T4SS é VirD4. No T4SS de X. citri, existe a hipótese de que o domínio XVIPCD seja o sinal de secreção presente nas XVIPs. Logo, os aspectos bioquímicos e biofísicos da interação VirD4-XVIPCD foram investigados através de experimentos de co-purificação por cromatografia de afinidade e exclusão molecular, RMN e SAXS. Demonstramos que o domínio AAD de VirD4 (VirD4AAD) está associado a interagir especificamente com o domínio XVIPCD de XAC2609 (XAC2609XVIPCD), formando um heterodímero em solução. VirD4AAD é um domínio globular e monomérico e XAC2609XVIPCD é desenovelado mas se enovela concomitante à interação com VirD4AAD. Construções de XAC2609 contendo mutações pontuais no domínio XVIPCD foram utilizadas em ensaios in vivo de secreção pela X. citri e ensaios in vitro de interação com VirD4AAD por titulação monitorada por calorimetria isotérmica (ITC). Através desses experimentos, observamos que uma forte interação entre VirD4AAD-XAC2609XVIPCD é essencial para secreção de XAC2609 via o T4SS. Esses resultados permitem concluir que o domínio XVIPCD é o sinal de secreção dos substratos do T4SS de X. citri e que o AAD confere especificidade à VirD4 por interagir com o XVIPCD. Finalmente, através de ensaios de competições bacterianas entre E. coli e X. citri, foram observados diferentes fenótipos associados à função do T4SS: i) nocautes gênicos das subunidades estruturais VirB5, VirB11 abolem a função do T4SS em X. citri.; ii) nocautes de xac2611, apresentaram uma maior vantagem adaptativa do que a cepa selvagem de X. citri em competições e a expressão epissomal de XAC2611 inibe fortemente a função do T4SS e iii) a atividade ATPásica de VirD4 é essencial para a função do sistema e a expressão de mutantes 8 de VirD4 exerce um fenótipo de dominância negativa sobre a função do T4SS em X. citri

The Type IV secretion System (T4SS) is typically associated with the function of bacterial conjugation and as a pathogenicity factor. T4SSs are normally composed of 12 proteins, VirB1-VirB11 and VirD4. Many species of the order Xanthomonadales possess a T4SS associated with killing bacteria. The current model of the T4SS killing is based on the secretion of toxins denominated XVIPs/X-Tfes (Xanthomonas VirD4 interacting proteins) /(Xanthomonadaceae-T4SS effector) in which each XVIP/X-Tfe has a cognate immunity protein denominated X-Tfi (Xanthomonadaceae-T4SS immunity protein). We demonstrate that an XVIP, XAC2609, is secreted through the T4SS so that it depends on cell-cell contact and its XVIPCD domain ("XVIP conserved domains"). The N-terminal portion of XAC2609 encodes a GH19 domain which cleaves the E. coli peptidoglycan but loses its activity in the presence of its cognate inhibitor, X-Tfi XAC2610. Therefore, XAC2609 /XAC2610 form a pair of effector/immunity proteins associated with X. citri T4SS. By using the X. citri Δxac2610 strain, has been shown through different microscopic techniques that XAC2610 protects the cell envelope of X. citri against the effects of cellular autolysis promoted by XAC2609 activity. Functional assays based on observations of colony phenotypes and biofilm formation has shown that XAC2610 confers immunity to X. citri against an intrinsic activity of XAC2609. VirD4 is the protein that recognizes the substrates through the interaction with the T4SS secretion signals. In the T4SS of X. citri, is hypothesized that the XVIPCD domain is the secretion signal present in the XVIPs. Here, the biochemical and biophysical aspects of the VirD4-XVIPCD interaction were investigated through Pull- Down, Molecular Exclusion Chromatography, NMR and SAXS assays. It has been shown the AAD domain of VirD4 (VirD4AAD) is associated with specifically interacting with the XAC2609XVIPCD domain (XAC2609XVIPCD), forming a heterodimer in solution. VirD4AAD is a globular and monomeric domain while XAC2609XVIPCD is elongated, but upon interaction with VirD4AAD goes through structural compaction process. Constructs of XAC2609 containing point mutations in the XVIPCD domain were used to perform secretion experiments in X. citri and Isothermal titration calorimetry against VirD4AAD. Through these assays, it has been characterized that a strong interaction between VirD4AAD-XAC2609XVIPCD is essential for secretion of XAC2609 via T4SS. Consequently, these results allow concluding that the XVIPCD domain is the secretion signal of X. citri T4SS substrate and the AAD confer specificity to VirD4 by interact with the XVIPCD domains. Finally, bacterial competitions between E. coli and X. citri showed different phenotypes associated with T4SS function: i) virB5, virB11 knockouts abolish the function of T4SS in X. citri.; ii) knockouts of xac2611 exhibited a higher adaptive efficiency than the wild-type X. citri strain in competitions, but the expression of XAC2611 abolishes the function of T4SS in the wild strain of X. citri; iii) The ATPase activity of VirD4 is essential and exerts a negative dominance over the T4SS function in X.citri

Identification of cyclic di-GMP protein receptors: high-throughput screening strategies and experimental verification / 生物工程学报

cyclic di-GMP (c-di-GMP) is a universal second messenger in bacterial cells. It regulates various biological processes such as biofilm development, pathogenicity, motility, exopolysaccharide (EPS) production and cell cycle. The second messenger exerts its function by binding to effectors, such as riboswitches and proteins. However, due to the diverse conformations of c-di-GMP, its effectors are hardly to be predicted by homology search. Identification of c-di-GMP effectors is the initial step to investigate its regulatory function in bacterial signal transduction, however, it remains to be a technically difficult task. Here we reviewed the mechanism of biofilm development controlled by c-di-GMP through binding to various types of protein effectors, and summarized the screening strategies, including genetics analysis, protein pull-down combined with LC/MS/MS identification, DRaCALA systematic screening and molecular docking-based prediction. We also summarized experimental methods for verifying protein-c-di-GMP interaction, including isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis etc. In addition, we discussed the advantages and disadvantages of these strategies and methods. The present review aims to facilitate the future investigations that are focused on regulatory role of novel c-di-GMP effectors.