Subcellular localization of ataxin-3 and its effect on the morphology of cytoplasmic organoids / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the subcellular localization of ataxin-3 and the effect of polyglutamine (polyQ) expansion mutation on the morphology of mitochondrion, golgi apparatus and endoplasmic reticulum.</p><p><b>METHODS</b>Transient transfection was employed to build cell models expressing wild-type or mutant ataxin-3 proteins. Indirect immunofluorescence was applied to identify markers of organelle membrane. The results were observed under a laser scanning confocal microscope.</p><p><b>RESULTS</b>No co-localization was observed for ataxin-3 protein and mitochondrial marker TOM20, but the percentage of cells with mitochondrial fragmentation has increased in cells expressing mutant ataxin-3 (P<0.05). No co-localization was observed for ataxin-3 protein and golgi marker GM130, and mutant ataxin-3 did not cause golgi fragmentation. Wide type and polyQ-expanded ataxin-3 both showed partial co-localization with ER marker calnexin. The latter showed more overlap with calnexin, and the overlapping signals were mostly located in the places where aggregates were situated.</p><p><b>CONCLUSION</b>PolyQ-expanded ataxin-3 protein may indirectly affect the integrity of mitochondria, but may cause no effect on the structure and functions of golgi apparatus. Endoplasmic reticulum may be another place where extended ataxin-3 protein can induce cytotoxicity in addition to the nucleus.</p>

Genesis, development and application prospect of antibody library: a review / 生物工程学报

Antibodies are immunoglobulins specifically introduced by immunity response of high animals, with the responsibility for recognising and cleaning out specific antigens. Antibody is not only a powerful weapon against pathogen invasion in the organism, but also a tool for specific molecular recognition used in basic scientific research. The diversity of antibody molecules resulted in the concept of antibody library; each individual animal is a natural antibody library. In the post-genome era, in order to fit various "omics", especially for proteomics requirement of high throughput technology, some gene engineering antibody libraries and antibody alternative libraries have been constructed based on phage display technology. Yet, more and more in vitro display systems such as ribosome display, mRNA display have been used for antibody library study, and that present more advantages than phage display. This mini review outlines the genesis, development and application prospect of antibody libraries according to the published reviews and research articles, and offers up to date development and application prospect of antibody library technology.

Strategy for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin / 生物工程学报

We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.

Comparative study of pelvic floor muscle exercise and surgical therapy in women with moderate stress urinary incontinence / 中国实用护理杂志

Objective To study comparatively the clinical efficacy of pelvic floor muscle exercise (PFMlE)and surgical therapy in women with moderate stress urinary incontinence(SUI),in order to provide theoretical reference for finding an appropriate SUI treatment method. Methods 114 cases of women with moderate SUI were randomly divided into the treatment group and the control group. The control group Wag given surgical thempy. while the treatment group was given the PFME treatment. After 12 weeks the clinical efficacy and quality of life of the two groups were evaluated, the cost-benefit was analyzed. Results The total effective rate of treatment group was 86.2%.higher than 83.9% of the control group. Compared with before treatment, the I-QOL scores of the two groups increased significantly, the ICIQ scores of the two groups decreased significantly, and the treatment group was significantly different compared with the control group. The cost-benefit analysis showed that the total medical cost and the spending cost of the total clinical effective rate increased l percent and the I-QOL increased 1 score were significantly lower than that of the control group. Conclusions The PFME therapy of women with moderate SUI is the preferred treatment and it is worthy of promotion and application.

Identification of the small supernumerary marker chromosomes in two patients with Turner syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the small supernumerary marker chromosomes (sSMC) and guide the genetic counseling and medical treatment in two patients with Turner syndrome.</p><p><b>METHODS</b>High resolution GTG and C banding, SRY amplification by PCR and fluorescence in situ hybridization (FISH) on metaphase chromosomes were performed to the two patients.</p><p><b>RESULTS</b>The karyotypes of the two patients were 45, X [29]/46,X, +mar[31] and 45,X[71]/46,X, +mar[29] respectively. SRY test indicated SRY-positive for patient 1, whose sSMC was originated from chromosome Y. The karyotype was confirmed as 45,X[29]/46,X,idic(Y)(q10)[31]. ish idic(Y)(q10)(RP11-115H13x2) (SRY+) by FISH. While in patient 2, the sSMC was originated from chromosome X, whose karyotype was determined as 45, X[71]/46,X, r(X)(p11.23q21)[29]. ish r(X) (p11.23q21)(AL591394.11xAC092268.3).</p><p><b>CONCLUSION</b>Using cytogenetic and molecular cytogenetic analyses, we have identified the sSMCs in two patients with Turner syndrome, which was helpful to the clinical diagnosis and treatment.</p>

Rapid prenatal diagnosis of chromosome aneuploidies in 60 uncultured amniotic fluid samples by fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the feasibility of rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) using uncultured amniotic fluid.</p><p><b>METHODS</b>Bacterial artificial chromosome (BAC) DNA probes were prepared and validated by using cultured peripheral blood. Interphase FISH for chromosomes 13, 18, 21, X and Y was performed in 60 amniotic fluid samples for the rapid prenatal diagnosis of chromosome aneuploidies, and the results were compared with the karyotypes from conventional cytogenetic analysis.</p><p><b>RESULTS</b>Of all 60 cases, 58 were concordant with their karyotypes, and 1 case of inv(9) and another case of t(2,12) were identified by karyotyping. Two cases of trisomy 21 and 1 case of trisomy 18 were detected by FISH and confirmed with conventional cytogenetics (sensitivity=100%). There were no false-positive or false-negative results.</p><p><b>CONCLUSION</b>This evaluation demonstrated that FISH employing BAC DNA probes could accurately and rapidly detect aneuploidies involving the above 5 chromosomes. However, as it does not identify structural chromosome aberrations and aneuploidies involving other chromosomes, it is not a substitute for conventional chromosome analysis, and the negative FISH result should be carefully interpreted.</p>

Mutation screening of the dystrophin gene in 14 Chinese Duchenne/Becker muscular dystrophy patients without gross deletions / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To search for the dystrophin gene mutations of Duchenne muscular dystrophy (DMD) patients without gross deletions, in order to offer accurate genetic counseling and prenatal diagnosis for those families.</p><p><b>METHODS</b>All 79 exons of the dystrophin gene as well as its 5'-UTR and 3'-UTR of 14 Chinese DMD/Becker muscular dystrphy (BMD) patients without detectable gross deletions were screened by denaturing high performance liquid chromatography (DHPLC) and heteroduplex fragments were identified by subsequent sequencing.</p><p><b>RESULTS</b>Seven causative point mutations, including two novel ones, were detected in 7 patients. Fourteen known polymorphisms and 7 unknown intronic variations were also detected. Five mothers of the patients were obligate carriers.</p><p><b>CONCLUSION</b>DHPLC is an efficient way of identifying point mutations and the female carriers in DMD families.</p>

Selection of a novel single-chain variable fragment antibody specifically against a linear epitope of white spot syndrome virus / 生物工程学报

White spot syndrome virus (WSSV) is one of the most important pathogens in shrimp farm throughout the world. Many researches on WSSV have been done, but no efficient approach has been gained to protect and cure the disease. In this study, we constructed a single-chain fragment variable (scFv) antibody library displayed on phage using spleen cells from mice immunized with denatured WSSV. After several rounds of panning respectively against purified intact WSSV virions and purified VP28 expressed in Escherichia coli, five novel scFv antibodies specifically against WSSV were selected, one of which, clone P75E8, recognized a linear epitope. The location in virions of the epitopes recognized by the five scFv clones was determined by immunoelectron microscopy. This study provides a new way to obtain more different antibodies specifically binding to WSSV, and especially provides a new strategy to obtain scFvs against linear epitopes.

Screening of LRRK2 interactants by yeast 2-hybrid analysis / 中南大学学报(医学版)

Objective To isolate and identify the potential binding partners of LRRK2,a gene linked to both dominant familial form and sporadic form of Parkinson's disease,thus to further our knowledge of its function.Methods We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait.The bait amplified by polymerase chain reaction(PCR) was then cloned into a yeast expression plasmid pGBKT7.After being sequenced and analyzed,pGBKT7-bait was transformed into the yeast strain AH109.Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain.Then human fetal brain cDNA library was trarnsformed into that yeast strain.which could express pGBKT7-bait fusion protein.The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade)containing X-a-gal.We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7,respectively.At last,these plasmids isolated from these true positive colonies were analyzed by bioinformatics.Results We obtained 9 true positive colonies,these colonies were sequenced, and we performed sequence Blast in GenBank.Three colonies of the 9 positive colonies were not in open reading-frames.Among other 6 colonies,there were known proteins including spermatid perinuclear RNA-binding protein(STRBP)and BCL2-associated athanogene 5 isoform b(BAG5),as well as unknown proteins including tyrosine phosphatase non-receptor type(PTPN23),1(3)mbt-like 3 isoform b(L3 MBTL3),RALY RNA binding protein-like isoform 1(RALYL),and Homo sapiens mRNA for KIAA1783 protein,partial cds(KIAA 1783).Conclusion True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid.Our screened proteins may provide a new research clue for revealing biological functions of LRRK2,pathogenesis of Parkinson's disease,and other neurodegerations.

p53 Anti-tumor Research in Bel-7402 by Using Human-derived Vector / 生物化学与生物物理进展

In order to study the tumor suppression effect of p53 with CMV enhancer and hTERT promoter mediated by human-derived vector pHrn in liver cancer cell Bel-7402, report plasmid pchEGFP, tumor suppressor plasmids pchp53Arg and pchp53Pro were constructed by inserting expression cassette CMVe+hTERTp+EGFP, CMVe+hTERTp+p53Arg and CMVe+hTERTp+p53Pro into pHrn respectively. 24 h after cell transfection by lipofectamine 2000, GFP expression pattern was analyzed through fluorescence microscope and flow cytometry; RT-PCR and Western blot were taken to study the p53 expression pattern. The cell apoptosis by Hoechst 33258 and Annexin V-FITC/PI staining was also studied. Results show that the expression of GFP and p53 protein in Bel-7402were detected, but apparent cell apoptosis could not be found. The recombinant p53 mediated by human-derived vector could express in Bel-7402, but no significant tumor suppression effect was detected, which might result from the down regulation effect of the wild type p53 on hTERT promoter.

Prenatal diagnosis of prelingual deafness by determination of SLC26A4 gene mutation / 中华妇产科杂志

G mutation of SLC26A4, the parents and the second child were carriers of the same mutation, while the fetus had a wild-type form. Conclusion It is feasible to identify deafness related genes by screening for GJB2 and SLC26A4 mutation, thus providing correct prenatal diagnosis and avoiding deaf delivery of baby.