Construction and characterization of QKI knockout GC1-spg cell strain with CRISPR/CAS9 / 基础医学与临床

Objective To investigate whether QKI protein plays any important role in the process of spermatogene-sis.Constructing GC1-spg cell strain which knocked out QKI by the technology of CRISPR/Cas9,and detecting its effect on the proliferation and differentiation of QKI protein in vitro.Methods The plasmid PX330 was used to construct QKI knockout recombinant plasmid, then transfected it to GC1-spg wild-type cells and selected by puromycin.GC1-spg knock-QKI cell strain was identified by Western blot and gene sequencing; The wild-type and knockout cell strain was cultured normally,then detected the growth curve by cell counting kit(CCK8),and using quantitative PCR to get the changes of meiotic-related gene differentiation.Results QKI knockout GC1-spg cell strain was successfully constructed.Compared with the control group,the growth of QKI knockout cell strain was significantly decreased(P<0.05), and the expression of meiosis related molecular marker gene of c-kit, Mtl5 and Pspa2 was significantly decreased(P<0.05).Conclusions QKI proteins can affect reproductive sper-matogenesis by acting on proliferation and differentiation.

Hereditary spastic paraplegia with SPG30 mutation: A report from North East China

Hereditary spastic paraplegia is a heterogeneous group of genetic neurodegenerative disorders of the nervous system. It is classified into four subtypes based on the mode of inheritance; and among them, most autosomal recessive hereditary spastic paraplegia cases are due to type SPG11 and SPG15 gene mutations. Autosomal recessive hereditary spastic paraplegia cases with SPG30 gene mutation have never been reported in China. Herein, we present our experience with a case of hereditary spastic paraplegia with SPG30 gene mutation in our hospital from North East China. In this patient we detected a missense mutation of c.499 C>T (p.Arg167Cys) in gene KIF1A, a causative gene of type SPG30.

Preparation of Lysozyme-loaded PLGA Microspheres by SPG Membrane Emulsification / 中国药师

Objective:To develop a new preparation process of PLGA microparticles for protein drugs by SPG membrane emulsifi-cation combined with W/O/W double emulsion-solvent technique. Methods:Lysozyme was used as the model drug to prepare the mi-croparticles. The influence of formula factors on the properties of the microparticles was studied, and the physicochemical properties, in vivo compatibility and degradation of the microparticles were investigated as well. Results:The drug loading of lysozyme-loaded mi-croparticles was 35%, the entrapment efficiency was 72. 43% and the average size was 63. 89 μm with PDI of 0. 675. DSC and FTIR showed that lysozyme was entrapped in the microparticles. The microspheres had promising biocompatibility and sustained degradation in vivo. Conclusion:The paper describes a new satisfactory preparation process of PLGA microparticles for protein drugs with good in vitro and in vivo properties.

Use of Staphylococcal Protein-A and Streptococcal Protein-G for Detection of Red Blood Cells (RBC) Antibodies and Comparison with Other Techniques.

Background: Detection of red blood cells antibodies is important for the diagnosis of autoimmune hemolytic anemia, hemolytic disease of newborn, pre-transfusion testing and other problems. The aim of this study was to use Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) as reagents in immunological tests for detecting red blood cells (RBC) antibodies and to compare the method with other techniques. Study Design & Methods: Sera from 60 patients, comprising forty-four anti-D positive sera from pregnant women and 16 from healthy controls were, used for the study. The anti-globulin gel test and the standard Coombs’ test were used to determine RBC antibodies in these sera and the result were compared with that of protein A and protein G tests. Results: With various degree of agglutination all 4 techniques detected the presence of RBC antibodies (anti-D) in the sera from 44 pregnant women, and tested negative for the remaining 16 sera (from healthy controls). The sensitivity and the specificity of the 4 techniques was 100%. Conclusions: This preliminary study demonstrates that both SpA and SpG tests can be used for the detection of RBC antibodies and therefore requires more study and testing before they can become useful standard tests in transfusion medicine.

Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species / Comparación de las técnicas de detección de la reactividad de la proteína de unión de la inmunoglobulina frente a la inmunoglobulina producida por diferentes especies aviarias y mamíferas

The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera).These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.

El fundamento de este estudio radica en el uso de varios ensayos inmunológicos para investigar la reactividad de la proteína de unión de la inmunoglobulina (IBP) frente a las inmunoglobulinas de varias especies aviarias y mamíferas. Las proteínas IBP estudiadas fueron la proteína estafilocócica A (SpA), la proteína estreptocócica G (SpG), la proteína peptoestreptocócica L (SpL), y la proteína recombinante LA (SpLA). Las varias técnicas inmunológicas usadas fueron: la inmunodifusión doble (técnica de Ouchterlony) para examinar las reactividades positivas de la proteína alta; el ensayo por inmunoabsorción ligado a enzimas(ELISA), de tipo directo y competitivo, para examinar la capacidad de realizar uniones positivas de proteína moderada y baja, respectivamente, además del ensayo ELISA 'Sándwich', los análisis inmunoblot, yla purificación de IgG, mediante cromatografía de afinidad, los cuales fueron pruebas sensibles y útiles en el tamizaje y las pruebas de confirmación. La técnica de Ouchterlony mostró que - en comparación con otras proteínas - la SpLA tenía el grado más alto de reactividad con los sueros animales y las inmunoglobulinas purificadas, mientras que la SpL fue la menos reactiva. Con el ELISA directo, la SpL reaccionó con los sueros de mapache, la IgG de conejo, así como con la IgY de palomas y gallinas de Bantam, en tanto con el ELISA directo, la SpA reaccionó con sueros de mofeta, coyote, mapache, mula, asno y seres humanos. ELISA "sándwich" reveló una alta reactividad tanto de SpG como de SpLA, con títulos séricos mamíferos que iban desde 1:32 (suero de mapache) hasta 1:1024 (sueros de mula y de asno). Estos resultados sugieren que la proteína de unión IBP puede usarse en la detección de la inmunoglobulina usando varios ensayos inmunológicos, lo cual es importante para el diagnóstico de enfermedades infecciosas en las poblaciones animales y aviarias bajo estudio, así como para la purificación de inmunoglobulinas.

Thinning of the Corpus Callosum and Cerebellar Atrophy is Correlated with Phenotypic Severity in a Family with Spastic Paraplegia Type 11

BACKGROUND: Mutations in the spatacsin gene are associated with spastic paraplegia type 11 (SPG11), which is the most-common cause of autosomal recessive hereditary spastic paraplegia. Although SPG11 has diverse phenotypes, thinning of the corpus callosum is an important feature. CASE REPORT: Clinical, genetic, and radiological evaluations were undertaken in a large family from Gujarat in North India with hereditary spastic paraplegia, whose affected members presented with varying degrees of spasticity, ataxia, and cognitive impairment. The clinical severity and the degree of corpus callosum and cerebellar atrophy varied among the four affected individuals in the family. Genetic testing of the affected members revealed recessive mutations in the spatacsin gene, consistent with a diagnosis of SPG11. CONCLUSIONS: We believe that the extent of corpus callosum thinning and cerebellar atrophy is correlated with disease severity in affected patients. The addition of extrapyramidal features in the most-affected members suggests that SPG11 exhibits considerable phenotypic heterogeneity.

Community Acquired Methicillin Resistant Staphylococcus Aureus: A Rare Presentation.

Introduction Incidence of community acquired methicillin resistant staphylococcus aureus (CA-MRSA) is increasing. Toxic shock syndrome (TSS), Necrotizing fasciitis (NF), Symmetrical peripheral gangrene (SPG) as a manifestation of CA-MRSA are rare in pediatrics. Case Presentation We report a young boy who presented with TSS, NF and SPG by CA-MRSA following trauma. Conclusion CA-MRSA should be taken into consideration as an etiology for these type of clinical presentations. Early and aggressive surgical and medical intervention are the cornerstone for successful management.

Relativity between sPG and Gastritis of H.pylori / 浙江中医药大学学报

[Objective] To explore the relativity between sPG and gastritis of H.pylori. [Method] Select 73 cases of chronic gastritis correctly diagnosed by gastric microscope and pathology, among which, 36 were chronic atrophic gastritis and 36 chronic superficial gastritis. 49 cases of positive H.pylori were administered with Lizhu Weisanlian tablets, measure the sPG before and after treatment. [Result] In the infected cases of H.pylori, PG Ⅰ was similar between atrophic gastritis and superficial gastritis; for PG Ⅱ value, atrophic gastritis group was more than superficial gastritis group, positive H.pylori patients higher than negative ones; PGR(PG Ⅰ/PG Ⅱ) was lower in atrophic gastritis group than superficial one, so was in positive H.pylori than negative one. After anti-H.pylori treatment, 87.75% patients were removed H.pylori, where the PG Ⅰ rose, PG Ⅱ lowered, PGR rose in atrophic gastritis group; in the superficial one where the H.pylori was not removed, no changes happened to PG Ⅰ, PG Ⅱ and PGR. In superficial gastritis group where the H.pylori was not removed, PGR rose. [Conclusion] H.pylori infection is correlated with sPG. Serum PG Ⅰ, PG Ⅱ and PGR have definite value in early diagnosing H.pylori concerned atrophic gastritis. PGR can be an index to evaluate the removal efficacy of H.pylori.

Characteristics of Natural transformation of C_3 components and regulation of rIFN-? and Sizofiran on pro-C_3 / 中国免疫学杂志

1.Shanghai Institute of Physiology,Chinese Academy of Sciences.Shanghai 200031.2.Central Research Laboratories,Kaken pharmaceutical Co.,Ltd.Tokyo 107.3.Department of Gynecology,Cancer Institute Hospital.Tokyo 117.The natural transformation of C_3 components and regulation of rIFN-? and/or SPG on pro-C_3 were observed by immuno-blotting analysis.Under the condition of sera stored at 4℃,the pe-riod of transformation from pro-C_3 to C_(3(?)) was about 18 days.The highest level of fragment C_3and C_(3b) appeared on day 8 during the degradation of pro-C_3.Level of pro-C_3 could be enhancedby administration of rIFN-? and/or SPG,and the most potent means was to take the method ofrIFN-? and SPG in combination.These results were suggested that so called the third compo-nents of complement in vivo was in pro-C_3 form in reality,and most reagent was to affect on thesynthesis of pro-C_3 instead of other C_3 components.In addition,all obtained results about the lev-els of C_3 components should be considered as an“instantaneous result”due to the C_3 componentsvaried with the store time-course of sera.