A novel intracellular nanobody against HPV16 E6 oncoprotein.

Cervical cancer occurs as a result of the persistent infection of high-risk human papillomavirus (HPV). HPV16 oncoproteins E6 and E7 exert different and concerted pro-tumor actions in cell transformation and malignance maintenance in various m echanisms. Nanobody expressed as "intracellular antibodies" (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. In this work, phage-display approach was employed to select the high affinity HPV16 E6-specific nanobody, nanobody Nb9 against HPV16 E6 was selected. Nb9 has high affinity (Kaff =6.3 × 108 M-) and can specifically bind endogenous HPV16 E6 protein in HPV16 positive CaSki and SiHa cells. In Nb9 overexpressed SiHa and CaSki cells, nucleus localization of HPV16 E6 was inhibited, p53 inactivation was prevented and increased apoptosis was observed. Moreover, tumor growth was inhibited in mouse xenograft model. Taken together, our results suggested that nanobody Nb9 could be a useful inhibitor for HPV16 E6 function and particularly appropriate for the treatment of HPV-associated disease.

Generation of mWasabi fluorescent protein-binding nanobodies.

mWasabi is a bright monomeric green fluorescent protein. It can be used as a fusion tag to monitor various biological events, e.g. protein localization. Here we report the selection of camelid-derived single-domain antibody fragments (nanobodies) against mWasabi. In this work, phage-display approach was employed to select the high affinity mWasabi-specific Nb (nanobodies). These nanobodies were able to recognize mWasabi or in a fused fashion with PD1. The interesting binding characteristics of these two mWasabi-specific nanobodies could be valuable for design new tools for cellular tracing or targeting based on the mWasabi-fusing protein in many different biological research fields.

Nanobody against the E7 oncoprotein of human papillomavirus 16.

The persistent infection of high-risk human papillomavirus (HPV) is one of the most common causes of cervical cancer. It is well documented that expression of two oncogenes (E6/E7) plays a key role in tumor progression. HPV16E7 -targeting via nanobody (Nb) therefore could be beneficial for HPV16-associated cancer diagnosis and therapy. In this work, phage-display approach was employed to select the high affinity HPV16E7-specific Nb. Firstly; a high-quality immune library was constructed. After three round of biopanning, high-affinity HPV16 E7-specific nanobodies were retrieved. By phage ELISA and sequencing, four different sequences of anti- HPV16E7 nanobodies were selected. Then recombinant nanobody Nb2 was cloned and expressed in E. coli, and the specificity and thermal stability of purified Nb2 was evaluated. To examine the potential of Nb2 as an inhibitor of E7 function, Nb2 was expressed within HPV16 positive cells. Proliferation assay showed that the intracellular expressed Nb2 as an intrabody can decrease the growth of HPV16-positive cells. The results indicate that Nb2 as an intracellular antibody directed towards HPV oncoprotein E7 has great promise in applications for the therapy of HPV16-associated disease.

Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 promotes lung adenocarcinoma by directly interacting with specificity protein 1.

Metastasis-associated lung adenocarcinoma transcript 1 (malat1) is an oncogenic long non-coding RNA (lncRNA) which has been proven to be associated with various types of tumors. Transcription factor specificity protein 1 (SP1) is overexpressed in many types of cancers. Previously, we observed that malat1 expression level is regulated by SP1 in lung cancer. In the present study, we found that transfection of expression construct of malat1 5' end fragment M5 enhances stability and transcriptional activity of SP1. Various SP1 target genes are also upregulated following overexpression of malat1 M5 in lung adenocarcinoma cells. We also showed that malat1 M5 interacts with the C-terminal domain of SP1 by RNA immunoprecipitation (RIP) assay coupled with UV cross-linking. Malat1-SP1 association results in increase of SP1 stability. In turn, SP1 promotes malat1 transcription, thus forming a positive feedback loop. In conclusion, our data show that in lung adenocarcinoma cells, malat1 interacts with SP1 protein and promotes SP1-mediated transcriptional regulation of SP1 target genes.

Novel heterozygous NOTCH3 pathogenic variant found in two Chinese patients with CADASIL.

NOTCH3 mutations have been described to cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Here, we report 2 CADASIL patients from a Chinese family. Whole genome sequencing was performed on the two CADASIL patients. The novel variant c.128G>C in exon 2 of NOTCH3 was identified and confirmed through PCR-Sanger sequencing (Human Genome Variation Society nomenclature: HGVS: NOTCH3 c.128G>C; p.Cys43Ser). The heterozygous NOTCH3 variant cause a cysteine to serine substitution at codon 43. According to the variant interpretation guideline of American College of Medical Genetics and Genomics (ACMG), this variant was classified as "pathogenic". Other variants in HTRA1, COL4A1 and COL4A2 were also found, they were classified as "benign".

[Laparoscopic percutaneous extraperitoneal closure does not affect vas deferens orientation or testicular volume and perfusion].

OBJECTIVE: To investigate the influence of single-port laparoscopic percutaneous extraperitoneal closure (LPEC) on the orientation of the vas deferens and the volume and perfusion of the testis in pediatric patients undergoing inguinal hernia repair. METHODS: A total of 92 consecutively enrolled boys diagnosed with unilateral inguinal hernia underwent single-port LPEC between June 2013 and June 2014. The orientation of the vas deferens and the testicular volume and perfusion of the patients were ultrasonographically assessed preoperatively and at 1 and 6 months after surgery. RESULTS: All the surgical procedures were performed successfully without conversion or serious perioperative complications. Ultrasonography showed no angulation or distortion of the vas deferens on the surgical side during a six-month follow-up period. Similarly, no obvious changes were observed in the testicular volume or perfusion. CONCLUSIONS: Single-port LPEC is safe and effective in the treatment of pediatric inguinal hernia and does not affect the orientation of the vas deferens or testicular volume and perfusion.

Corrigendum to "Combination of low-energy shock-wave therapy and bone marrow mesenchymal stem cell transplantation to improve the erectile function of diabetic rats" by Hai-Tao Shan et al.

[This corrects the article DOI: 10.4103/1008-682X.184271.].

Combination of low-energy shock-wave therapy and bone marrow mesenchymal stem cell transplantation to improve the erectile function of diabetic rats.

Stem cell transplantation and low-energy shock-wave therapy (LESWT) have emerged as potential and effective treatment protocols for diabetic erectile dysfunction. During the tracking of transplanted stem cells in diabetic erectile dysfunction models, the number of visible stem cells was rather low and decreased quickly. LESWT could recruit endogenous stem cells to the cavernous body and improve the microenvironment in diabetic cavernous tissue. Thus, we deduced that LESWT might benefit transplanted stem cell survival and improve the effects of stem cell transplantation. In this research, 42 streptozotocin-induced diabetic rats were randomized into four groups: the diabetic group (n = 6), the LESWT group (n = 6), the bone marrow-derived mesenchymal stem cell (BMSC) transplantation group (n = 15), and the combination of LESWT and BMSC transplantation group (n = 15). One and three days after BMSC transplantation, three rats were randomly chosen to observe the survival numbers of BMSCs in the cavernous body. Four weeks after BMSC transplantation, the following parameters were assessed: the surviving number of transplanted BMSCs in the cavernous tissue, erectile function, real-time polymerase chain reaction, and penile immunohistochemical assessment. Our research found that LESWT favored the survival of transplanted BMSCs in the cavernous body, which might be related to increased stromal cell-derived factor-1 expression and the enhancement of angiogenesis in the diabetic cavernous tissue. The combination of LESWT and BMSC transplantation could improve the erectile function of diabetic erectile function rats more effectively than LESWT or BMSC transplantation performed alone.

MiR-20a and miR-20b negatively regulate autophagy by targeting RB1CC1/FIP200 in breast cancer cells.

AIMS: RB1CC1/FIP200 was essential for autophagosome formation. Therefore, RB1CC1/FIP200 cellular levels are critical for the activation of the autophagy pathways. Following the screen of miRNAs affecting RB1CC1/FIP200 level and rapamycin-induced autophagy, we discovered miR-20a and miR-20b could regulate autophagy by targeting RB1CC1/FIP200. MAIN METHODS: Inhibitory effect of miR-20a and 20b on basal and rapamycin-stimulated autophagy was demonstrated using various autophagic tests including GFP-LC3 puncta analysis, LC3II/LC3I gel shift and TEM observation. KEY FINDINGS: We discovered RB1CC1/FIP200 as cellular targets of miR-20a and miR-20b. Upon miR-20a and miR-20b overexpression, both mRNA and protein levels of RB1CC1/FIP200 decreased. miR-20a and miR-20b target sequences present in the 3' UTR of RB1CC1/FIP200 mRNAs and introduction of mutations abolished the miR-20a and miR-20b responsiveness. In MCF7 and MDA-MB-231 breast cancer cells, miR-20a and miR-20b over-expression attenuated basal and rapamycin-induced autophagy; while suppression of miR-20a or miR-20b by specific antagomir showed normal rapamycin-induced autophagic activity. SIGNIFICANCE: To our knowledge, this is the first study showing the significance of miR-20a and miR-20b regulating autophagy by targeting RB1CC1/FIP200.

[Effect of platelet-derived growth factor-BB on rat corpus cavernosum smooth muscle cell proliferation, migration and phenotypic modulation].

OBJECTIVE: To study the effect of platelet-derived growth factor-BB (PDGFBBB) on rat corpus cavernosum smooth muscle (CCSM) cell proliferation, migration and phenotypic modulation and explore the underlying mechanisms. METHODS: Wistar rat CCSM cells were obtained through a modified tissue culture method and identified by immunofluorescence assay. The effect of PDGFBB on the proliferation of CCSM cells was investigated using a CCK-8 kit and the optimum PDGFBB concentration for cell treatment was determined. CCSM cells were treated with vehicle or PDGF-BB at the optimum concentration, and the cell migration was examined using scratch assay; the mRNA expression of the transcription factor myocardin and the contractile phenotype markers αSMA and SMMHC in CCSM cells were determined by qRT-PCR at 24 h and 48 h. The protein expression of myocardin in CCSM cells incubated with PDGFBB for 0, 24 and 48 h was examined by Western blotting. RESULT: In CCSM cell culture, 96.5%and 96% of the cells were positive for αSMA and smoothelin, respectively. PDGFBB at different concentrations markedly promoted the proliferation of CCSM cells; the optimum PDGFBB concentration for enhancing cell proliferation was 12.5 ng/mL, which induced the migration of CCSM cells and significantly reduced the mRNA expressions of myocardin, αSMA and SMMHC (P<0.01). Exposure to PDGFBB decreased the protein expression of myocardin as the exposure time extended (within 48 h). CONCLUSION: CCSM cells of a high purity can be obtained by the modified tissue culture method. PDGFBB can promote the proliferation and migration of CCSM cells and cause a phenotypic conversion from the contractile to the synthetic type possibly by down-regulating myocardin.

Stem cell therapy for erectile dysfunction of cavernous nerve injury rats: a systematic review and meta-analysis.

INTRODUCTION: Stem cell treatment is a novel therapeutic strategy for erectile dysfunction (ED) patients with bilateral cavernous nerve injury (CNI). The relative animal studies provide important clues to design pre-clinical studies and clinical studies further in the future. PURPOSE: This study aims to evaluate the effects and influential factors of stem cell transplantation on ED rats with CNI. MATERIALS AND METHODS: We searched PubMed and EBSCO databases published before April 30, 2014 for pre-clinical studies to evaluate the efficacy of stem cell transplantation in the treatment of ED rats with CNI. A systematic review and a planned subgroup analysis were performed to identify whether or not some certain influential factors could bring significant effects on stem cell treatment. RESULTS: 12 studies with 319 rats were enrolled in this meta-analysis. Pooled analysis results confirmed the efficacy of stem cell transplantation. Subgroup analysis results showed that treatment effects were not related to CNI models, follow-up time, stem cell species, stem cell sources, markers and delivery approaches in the transplantation. Uncultured stem cells were poorly effective compared with cultured stem cells. Periprostatic implantation (PPI) with acellular scaffolds could promote cavernous nerve regeneration, but was less effective for smooth muscle cell recovery. Stem cells modified by NGF or BDNF combined with udenafil/bFGF seemed to be more effective than those modified by BDNF alone. CONCLUSION: This meta-analysis shows that stem cell therapy can be performed to recover erectile function. Future studies should focus on nerve restoration and vascular cell recovery. The synergistic actions of multiple growth factors following stem cell transplantation should also be considered as beneficial strategies to obtain preferable effects.

Sp1-mediated transcriptional regulation of MALAT1 plays a critical role in tumor.

BACKGROUND: MALAT1 was discovered as a prognostic marker for lung cancer metastasis and has been found upregulated in many types of tumor, but its transcriptional regulation mechanism in tumors remains unclear. METHODS: A deletion analysis of MALAT1 promoter region was performed to find the cis elements that were critical for the transcriptional activation of MALAT1 gene. Reporter gene assays were employed to analyze the effect of Sp1 on the promoter activity of MALAT1 gene. The binding activity of Sp1 with the promoter of MALAT1 gene was examined by EMSA and ChIP assay. Effects of Sp1 on regulation of MALAT1 were analyzed by RNA interference in vitro and in vivo mouse model. RESULTS: By means of luciferase assay, Sp1 was found to activate the promoter of the human MALAT1 gene. The binding of Sp1 to this region was also detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. Sp1 knockdown also decreased the MALAT1 and inhibited A549 lung cancer cells' growth and invasion in vitro. Furthermore, knockdown of Sp1 also mimicked the inhibition of MALAT1 in A549 lung cancer cells' growth and metastasis in vivo. CONCLUSIONS: Taken together, our data suggest that upregulation of MALAT1 was mediated by the transcription factor Sp1 in A549 lung cancer cells, and Sp1 could be therapeutic target for cancer.

[Clinical study of tacrolimus in postoperative treatment of patients with renal transplantation for diabetic end-stage renal disease].

OBJECTIVE: To compare the efficacy and safety of tacrolimus (FK506)- versus cyclosporine (CsA)-based immunosuppression protocol in the treatment of patients with renal transplantation for diabetic end-stage renal disease. METHODS: A total of 64 patients with end-stage renal disease were randomized into FK506 (n=33) and CsA (n=31) group after cadaveric renal transplantation, the former group adopting small-dose FK506 therapy with low trough concentration, and the latter receiving CsA therapy. The dose of insulin was adjusted according to blood glucose level of the patients after the operation. The incidence of acute rejections, blood glucose level, the dose of insulin and changes in blood pressure, lipid metabolism and liver function were compared between the two groups. RESULTS: The 1-year patient/graft survival rates were 96.97%/93.94% in FK506 group and 96.77%/90.32% in CsA group. Four patients (12.12%) developed acute rejection in the FK506 group, while 11 (35.48%) did in the CsA group during the first year after the operation (P<0.05). The dose of the insulin of the FK506 group (34.35 U/d, 14.09 U/d) was not significantly different from that in the CsA group (28.15 U/d, 13.05 U/d) at the first month and 1 year after the operation (P>0.05). One year after the operation, 21 patients (63.63%) required anti-hypertension treatment, 5 (15.15%) needed anti-hyperlipidemia treatment and 3 (9.09%) had abnormal liver function in FK506 group, while in the CsA group, 28 patients (90.32%) needed anti-hypertension therapy, 13 (41.94%) received anti-hyperlipidemia treatment and 11 (35.48%) showed signs of abnormal liver function, with significant differences between the two groups. CONCLUSIONS: Small-dose FK506 therapy with low concentrations is effective in the postoperative management of patients with renal transplantation for diabetic end-stage renal disease, with only mild adverse effect. FK506 is comparable with CsA in terms of the effect on glucose metabolism of the patients.

Effect of anti-tumor necrosis factor-alpha monoclonal antibody in alleviating renal ischemia-reperfusion injury.

OBJECTIVE: To investigate the effect of anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha mAb) in alleviating renal ischemia-reperfusion injury. METHODS: Fifty normal male Sprague-Dawley rats were randomly divided into 3 groups, namely group A that was subjected to ischemia-reperfusion injury with intravenous administration of anti-TNF-alpha mAb (0.1mg/kg.b.w.) 5 min before reperfusion (treatment group), group B with the same injury followed by saline administration in the same manner (control group), and group C with only anesthetization and leparotomy but not ischemia (sham operation group). Routine assays were performed for testing the levels of blood creatine (Cr), blood urea nitrogen (BUN), plasma TNF-alpha and the cell apoptosis. Ultrastructure of the kidney was also observed. RESULTS: Renal ischemia-reperfusion resulted in significant increase of the levels of Cr, BUN and TNF-alpha in the plasma (P<0.01), but these effects were offset by administration of anti-TNF-alpha mAb (P<0.01). In group B, widespread pathological changes and cell apoptosis were observed in the renal tissue following renal ischemia-reperfusion injury, while similar changes were scarcely visible in group A due to the protective effect of intravenous administration of anti-TNF-alpha mAb 5 min before reperfusion. CONCLUSION: Renal ischemia-reperfusion injury can be alleviated by anti-TNF-alpha mAb treatment.