Objective · To design and build a high-throughput sequencing approach based on targeted panel sequencing (TPS) using for the primary immunodeficiency disease (PID) diagnosis. Methods · By reviewing the literature and querying the relevant databases to determine the known diseasecausing genes of PID, capture probes using for the TPS were designed and customized for all exons and flanking sequences of these genes. A child suspected with PID was diagnosed by the customized TPS. Results · The PID sequencing panel contains a total of 100 known pathogenic genes. The sequencing data of the patient has 16414298 reads. The average coverage depth is 157 X, 98.35% of the target region sequencing depth is greater than 20 X, and 99.97% of the target region sequencing depth is greater than 1 X. Finally, a heterozygous nonsense mutation was found in the exon 2 of the CXCR4 gene (c.1000C>T, p.Arg334*) in the child. The results of Sanger sequencing confirmed the variation in the child and showed that his parents were wildtype at the corresponding sites, indicating the mutation is de novo. Conclusion · This study established a high-throughput sequencing diagnostic approach for PID, with which a case of WHIM syndrome was successfully diagnosed.

OBJECTIVE Apolyclonalantibody-basedhomogeneouschemiluminescenceimmunoas-say was developed and optimized using AlphaLISA technology for the quantitative detection of microcys-tin-LR(MC-LR)inwatersamples.METHODS Thismethodwasbasedonacompetitivemodelin which an immune complex was formed from the ingegral binding of artificial MC-LR antigen-coated lumi-nescene beads,free MC-LR standards or sa mples,antibody and biotinylated second antibody.Next sensor bead were added that approached the i mmune co mplex through biotin-streptavidin interaction. With the exciting light,the energy was passed from the sensor luminescer before a special emission light could be observed.To opti mize the reaction conditions,working dilutions of polyclonal antibody and bioti-nylated second antibody were assayed while the effect of buffer syste ms and ti me of each reaction were evaluated.RESULTS Maininfluencingfactorsoftheassaywerediscussedasworkingdilutionsofpoly-clonal antibody and biotinylated goat anti rabbit IgG,assay buffer and reacting ti me.After opti mization of reaction conditions,MC-LR AlphaLISA could be finished in 40 min,with a sensitivity of 0.006 μg·L-1 and a dynamic range of 0.006 -5 μg·L-1 .The coefficient of variation was below 10% and average recovery was 1 07.7%.Moreover,the cross reactivity rates of MC-RR and MC-RY to MC-LR were 13.2%and0.91%,respectively.CONCLUSION Thismethodishighlysensitiveandspecific,time-saving and quite suitable for high throughput determination of MC-LR water samples.

The DNA structures could be altered or even damaged by exogeous or endogenous factors during cell proliferation. Failure of effective and timely repair will lead to cell cycle arrest or apoptosis. By taking the advantage of the quick proliferation of cancer cells, DNA damage induction, cell cycle arrest and apoptosis promotion have become important strategies for ant-cancer chemotherapy. Previous reports showed that an array of natural compounds inhibit cancer cell proliferation by inducing DNA damage, which have therapeutic potentials for anti-cancer drug research and development.
Animals ; Biological Products ; pharmacology ; therapeutic use ; DNA Damage ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Neoplasms ; drug therapy

Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro.MethodsHuman corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells.ResultsRT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells.ConclusionsThe expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.

Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.

BACKGROUNDVascular control and tissue dissection are crucial steps in successful laparoscopic surgery. Recently, a new commercially available vessel sealing technology, the LigaSure vessel sealing system (Valleylab, Boulder, USA), has been introduced. The aim of the present study was to evaluate the benefits of the LigaSure in laparoscopic nephrectomy.

METHODSFrom January 2005 to March 2010, 170 laparoscopic nephrectomies were performed with the LigaSure vessel sealing system, including simple and radical nephrectomy and nephroureterectomy. In a retrospective study, the laparoscopic operating time, estimated intraoperative blood loss, duration of postoperative drainage, total amount of postoperative drainage, as well as postoperative hospital stay, were recorded and studied.

RESULTSAll 170 laparoscopic nephrectomies using LigaSure were accomplished successfully without conversion to open surgery. There was no severe vascular complication or other serious complications. The mean laparoscopic operating time was 124.2 minutes (range, 14 - 230 minutes); mean blood loss was 148.6 ml (range, 20 - 540 ml); mean time for postoperative drainage was 3.1 days (range, 1 - 7 days); mean amount of postoperative drainage was 206.5 ml (range, 27 - 435 ml) and mean postoperative hospital stay was 6.9 days (range, 3 - 18 days).

CONCLUSIONSLaparoscopic nephrectomy using LigaSure appears technically feasible and easy, and produces satisfactory results. The LigaSure provides a safe and fast way to seal vessels and tissue bundles during nephrectomy.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Laparoscopy ; methods ; Male ; Middle Aged ; Nephrectomy ; methods ; Retrospective Studies ; Young Adult

Objective To investigate the prognostic factors affecting the postoperative survival of patients with invasive bladder cancer,and to predict the survival time of the patients.Methods We retro- spectively analyzed the clinical and follow-up data of 178 patients with invasive bladder cancer treated by radical cystectomy and urinary diversion from 1991 to 2004.A multivariate analysis was performed in these patients by the Cox proportional hazard model.A prognostic index(PI)based on the Cox regression was con- structed.According to the individualized PI,the patients were classified into different hazard groups and the expected survival curve of each patient was calculated.Results Cox regression analysis showed that the factors which influenced the postoperative survival included tumor stage(RR=1.982,P=0.000),grade (RR=1.978,P =0.042),lymph node metastasis(RR=2.142,P=0.048),Tis(RR=6.177,P= 0.000),tumor shape(RR=0.416,P=0.003),number of tumors( RR=1.820,P=0.035),pathological type(RR=2.228,P=0.032),patient age(RR=0.672,P=0.025)and neoadjuvant chemotherapy (RR=0.257,P=0.016).Based on the percentile of PI,patients were classified into 3 prognostic groups; the median survival time of 3 groups were 42.5,22.5 and 7.0 months,respectively.There were significant differences between each 2 among the 3 groups(P＜0.01).Conclusions Neoadjuvant chemotherapy, tumor stage,grade,lymph node metastasis,Tis,shape and number of tumors,pathological type,patient age were important prognostic factors.PI value can be used to predict the prognosis of patients with invasive blad- der cancer.

Objective To study the pathologic and clinical features of malignant rhabdoid tumor of the kidney(MRTK),and to improve the diagnosis and treatment of the disease.Methods The clinical and pathologic data of 5 patients(4 men and 1 woman;mean age,50 years;age range,21-67 years)with MRTK(3 tumors on the left and 2 on the right)were retrospectively analyzed in combination with review of the relevant literature.Of the 5 cases,I was incidentally diagnosed with renal tumor during physical examina- tion;and 3 had gross hematuria,low back pain and discomfort,and abdominal masses.Results Radical nephrectomy was performed in all 5 cases.The tumors averaged 6.5cm in diameter.By NWTS staging,4 ca- ses had stageⅡtumors and 1 case had stageⅢtumor.Pathological features were as follows.Rhabdoid cells were characterized by eccentric nuclei,prominent nucleoli,and abundant cytoplasm containing eosinophilic inclusions that were strongly positive for vimentin and epithelial membrane antigen(EMA).Electron micros- copy showed intermediate filaments and round,irregular fibroid or whorl-like corpuscles in the cytoplasm. Follow-up was available in 4 patients(mean,6.8 months;range,3-24 months).Of them,2 died of metasta- sis or complications 12 months after operation;and 2 were alive without recurrence and metastasis for 6 months.Conclusions MRTK is a rare and morphologically distinctive neoplasm with specific findings of pathological features.The tumor has a poorer prognosis,but comparatively it is better in adults than in adoles- cents.

Pulmonary infection after renal transplantation is a well recognized and prevalent postoperative complication, which can occur at either the early stage or late stage after transplantation. The etiology and this phenomenon and its impacts remains unclear. It may be life-threatening in severe patients. Early diagnosis and treatment are important; meanwhile, the dosage of immunosuppressant should be minimized. Prophylactic management should also be emphasized.
Humans ; Kidney Transplantation ; Pneumonia ; diagnosis ; etiology ; therapy ; Postoperative Complications ; diagnosis ; etiology ; therapy