High expression of NXPH4 correlates with poor prognosis, metabolic reprogramming, and immune infiltration in colon adenocarcinoma.

Background: Colon adenocarcinoma (COAD) is a prevalent gastrointestinal malignant disease with high mortality rate, and identification of novel prognostic biomarkers and therapeutic targets is urgently needed. Although neurexophilin 4 (NXPH4) has been investigated in several tumors, its role in COAD remains unclear. The aim of this study was to explore the prognostic value and potential functions of NXPH4 in COAD. Methods: The expression of NXPH4 in COAD were analyzed using The Cancer Genome Atlas (TCGA) and datasets from the Gene Expression Omnibus (GEO) database. The prognostic value of NXPH4 was determined using Kaplan-Meier analysis and Cox regression analysis. To investigate the possible mechanism underlying the role of NXPH4 in COAD, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were employed. The correlation between NXPH4 expression and immune cell infiltration levels was examined thorough single-sample gene set enrichment analysis (ssGSEA). Furthermore, the competing endogenous RNA (ceRNA) regulatory network that may be involved in NXPH4 in COAD was predicted and constructed through a variety of databases. Results: NXPH4 expression was significantly higher in COAD tissue compared with normal colon tissues. Meanwhile, high expression of NXPH4 was associated with poor prognosis in COAD patients. GO-KEGG and GSEA analyses indicated that NXPH4 was associated with glycolysis and hypoxia pathway, and may promote COAD progression and metastasis by modulating metabolic reprogramming. ssGSEA analysis demonstrated that NXPH4 expression also associated with immune infiltration. Furthermore, we identified various microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) as upstream regulators of NXPH4 in COAD. Conclusions: The present study revealed that high expression of NXPH4 is associated with tumor progression, metabolic reprogramming, and immune infiltration. These findings suggest that NXPH4 could serve as a reliable prognostic biomarker and a promising therapeutic target in COAD.

Prognostic biomarker NRG2 correlates with autophagy and epithelial­mesenchymal transition in breast cancer.

Breast cancer (BRCA) is a leading cause of death in women worldwide, accounting for 31% of female cancer. Autophagy plays a crucial role in cancer progression, however, the function of autophagy-related gene neuroregulatory protein 2 (NRG2) in BRCA and its underlying molecular mechanisms remain unclear. In the present study, the expression of the NRG2 gene in BRCA was significantly down-regulated compared with the normal controls. The low expression level of NRG2 was related to poor survival rate of BRCA. The receiver operating characteristic curve of NRG2 showed a good diagnostic value for distinguishing BRCA from normal tissues (AUC=0.932). GO-KEGG analysis and GSEA enrichment analysis showed that NRG2 and its regulated genes were enriched in autophagy-related and immune-related pathways, and NRG2 was positively correlated with a number of immune cells and immune checkpoint genes. In addition, knockdown of NRG2 significantly promoted the proliferation, invasion and migration of BRCA cells. The autophagy marker, LC3-II and epithelial-mesenchymal transition (EMT) marker, vimentin were increased, while P62 and E-cadherin were decreased in response to NRG2 depletion. The findings of the present study demonstrated that NRG2 acts as a tumor suppressor factor that contributes to the immune escape and anti-tumor immunity inhibition by regulating the pathological process of autophagy and EMT, suggesting that NRG2 could be used as a prognostic biomarker and clinical target for BRCA therapy.

KRT19 is a Promising Prognostic Biomarker and Associates with Immune Infiltrates in Serous Ovarian Cystadenocarcinoma.

Background: Ovarian cancer (OV) is the highest prevalent gynecologic tumor with complicated pathogenesis; high-grade serous ovarian cystadenocarcinoma (HGSOC) is the most epidemiological and malignant subtype of OV. Keratin type I cytoskeleton 19 (KRT19) is an intermediate filament protein which plays essential roles in the maintenance of epithelial cells. However, its role in OV remains largely unknown. Methods: Bioinformatic analysis with various databases was conducted in this study. In details, KRT19 expression was assessed using databases including The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Gene Expression Omnibus (GEO) and Human Protein Atlas (HPA). GO-KEGG and GSEA analysis were performed by R packages. The biological function of KRT19 was analyzed based on the single-cell sequencing information from CancerSEA database. The association of KRT19 expression with immunomodulator and chemokine was predicted via the TISIDB database. Results: The expression of KRT19 was significantly upregulated in ovarian samples compared with normal controls. KRT19 expression was negatively associated with prognosis in OV, and further analysis revealed that KRT19 had promising diagnostic significance in distinguishing OV cancer from normal samples. GO-KEGG and GSEA analysis indicated that KRT19 was associated with multiple biological functions and pathways including epidermis development, apical junction, inflammatory response, and epithelial mesenchymal transition. By using different GEO series, we found that KRT19 was differentially expressed in OV-associated tissues. Furthermore, the increased KRT19 expression was positively correlated with the immune infiltration levels of the most immune cells in OV. Conclusion: This study demonstrated that KRT19 is a promising prognosis and diagnosis biomarker that determines cancer progression and is correlated with tumor immune cells infiltration in OV, suggesting being a molecular target for immunotherapies.

Aqueous extract of Sanghuangporus baumii induces autophagy to inhibit cervical carcinoma growth.

Sanghuangporus baumii, an edible fungus rich in heteropolysaccharides, has been found to have some anti-cervical cancer effects. In the current study, the effects of an aqueous extract of S. baumii on cervical cancer were investigated in a U14 cervical carcinoma cell implanted female Kunming mouse model. An aqueous extract of S. baumii (SHWE) was administered to tumor-bearing mice by gavage for 21 days. SHWE treatment significantly inhibited tumor growth by 67.4% at a dose of 400 mg per kg bodyweight. Transcriptomic results showed that the expression of key genes GABARAP, VMP1, VAMP8 and STX17 which are involved in the autophagy pathway was regulated after SHWE treatment, suggesting that SHWE may induce autophagy in tumors. The results were further confirmed by measuring the LC3II/LC3I ratio using western blotting. Moreover, some differentially expressed genes were involved in the insulin signaling pathway, implying that SHWE induced autophagy by disturbing glucose uptake and utilization in tumors. The analysis of the gut microbiota indicated that SHWE treatment stimulated the proliferation of Akkermansia, a well-known probiotic that presented benefits in metabolic regulation and cancer therapy. In conclusion, SHWE administration modified the gut microbiota, disturbed the glucose metabolism and induced autophagy in tumors, and then inhibited the development of cervical carcinoma in vivo.

Development of neutron activation system on EAST.

A neutron activation system (NAS) has been developed on the Experimental Advanced Superconducting Tokamak (EAST) for the investigation of time-integrated neutron yield. It is a robust neutron diagnostic with high precision and a wide dynamic range. Some high purity materials with a proper nuclear reaction energy threshold and cross section are tested as neutron detectors in the NAS, and they are delivered to the tokamak device by a pneumatic transfer system. The length of the pneumatic pipeline is about 50 m, and the transfer time ranges from 10 s to 20 s. The decay gamma rays of the activated samples are measured with a high-purity germanium spectrometer, and its detection efficiencies are 6.9% at 336 keV and 1.7% at 1779 keV, respectively. Indium and silicon samples with a reaction threshold of 0.3 MeV and 4.0 MeV, respectively, were successfully implemented in the diagnosis of DD and DT neutron yield in the EAST. The neutron flux of the NAS was compared with the count of the fission chamber in the EAST neutral beam injection experiment, and the DD neutron yield evaluated by the NAS ranges from 1.9 × 1012 n/shot to 5.9 × 1014 n/shot during recent experimental campaign.

Improvement of the C-glycosylation Step for the Synthesis of Remdesivir.

The bulk supply of the antiviral C-nucleoside analogue remdesivir is largely hampered by a low-yielding C-glycosylation step in which the base is coupled to the pentose unit. Here, we disclose a significantly improved methodology for this critical transformation. By utilizing diisopropylamine as a cost-effective additive, the addition reaction furnishes an optimal yield of 75% of the desired ribofuranoside adduct, representing the highest yield obtained thus far for this key step. The method proved suitable for hectogram scale synthesis without column chromatographic operations.

Abdominal wall elasticity of children during pneumoperitoneum.

OBJECTIVE: Laparoscopic surgery in children is often hampered by limited working space. This is partially compensated by stretching the child's abdominal wall. The volume of space that can be gained and changes to abdominal wall elasticity with respect to age are unknown. In the current research, we studied the extent of abdominal wall elasticity while establishing pneumoperitoneum in children. METHODS: One hundred and sixty three children less than 18 years of age, undergoing elective laparoscopic surgery were recruited for the study. After induction of general anesthesia with a standardized muscle relaxant dose, a length of 5 cm was marked above, below and lateral to the umbilicus. The length of the marking was measured under increasing intraabdominal pressure (IAP of 0, 4, 6, 8, 10, 12 mmHg). The measurements were repeated to assess the effect of prestretching. The patients were divided into two groups: infants (less than one year of age) and older children (more than one year of age). RESULTS: Depending on the age and axes of the measurements, a child's abdomen stretches up to 17% on average, with induction of pneumoperitoneum. The percentage of stretch tapers off as the IAP approaches peak pressure. As children become older, the longitudinal abdominal wall elasticity decreases, but the transverse abdominal wall elasticity increases. Regardless of age, prestretching results in a statistically significant increase in the elasticity over the transverse and lower sagittal abdominal wall. CONCLUSION: A child's abdominal wall has considerable expandability. The characteristics of elasticity change depending on the axis and age. Prestretching can improve intraabdominal working space. This knowledge is helpful in port position design for minimally invasive surgery in children. LEVEL OF EVIDENCE: Level II.

Construction of the highly oxidized bicyclo[3.2.1]octane CD ring system of aconitine via a late stage enyne cycloisomerization.

A strategy for synthesizing the highly oxidized bicyclo[3.2.1]octane CD rings of aconitine is reported. The key features of the synthesis include a ring-closing metathesis to form the C-ring as well as the application of a ruthenium-catalysed enyne cycloisomerization to assemble the bridged CD ring system of aconitine.

Single-Incision Laparoscopic-Assisted Anorectoplasty Versus Three-Port Laparoscopy in Treatment of Persistent Cloaca: A Midterm Follow-up.

Purpose: The aim of this study was to compare the safety and efficacy of single-incision laparoscopic-assisted anorectoplasty (SILAARP) to that of traditional three-port laparoscopic-assisted anorectoplasty (3-PLAARP) for persistent cloaca (PC) patients with mid to high rectal pouches in midterm follow-up. Materials and Methods: A study comparing 13 SILAARPs and 12 3-PLAARPs performed by a single surgeon from November 2005 to May 2016 was presented (laparoscopy was performed for anorectoplasty, and urethra vaginoplasty was performed perineally). Data including demographics and perioperative and midterm outcomes were retrospectively compared. Operative performances between the groups were compared using the Global Operative Assessment of Laparoscopic Skills (GOALS). Results: The two groups were comparable in terms of demographics. No significant difference was observed between SILAARP and 3-PLAARP groups in terms of median operative time, transfusion rates, intraoperative complications, time to resume diet, postoperative hospital stays, and GOALS scores. Postoperative outcomes included 1 patient with incomplete intestinal obstruction (SILAARP group), 1 patient requiring intermittent catherization to remain dry (3-PLAAR group), 2 patients with occasional urine incontinence (one in each group), and 5 girls with mild mucosal prolapse (2 and 3 in the SILAARP and 3-PLAAR groups, respectively); however, these did not differ significantly between groups. Trocar-related scars of the patient who was undergoing SILAARP were almost invisible. After 18 months follow-up, the rates of soiling and constipation were similar in both groups. Conclusion: The midterm outcomes of SILAARP were comparable to those of 3-PLAARP. It appears to be a safe and effective procedure with better cosmetic results for mid to high rectal pouches in PC children.

Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models.

Gene expression study is widely used to obtain information of the cell activities and phenotypes. To quantify gene expression, measurement of the mRNA copy number is commonly done by quantitative RT-PCR (RT-qPCR). However, proper reference gene is needed for different tissues to normalize the expression level of different genes accurately. In this study, reference gene determination was done for three-dimensional (3D) artificial tissue constructs in hydrogel. Porcine synovium-derived mesenchymal stem cells (SMSCs) and rabbit chondrocytes were cultured in both alginate and agarose hydrogels to set up four different 3D culture systems to form the artificial tissue constructs. The gene expression levels of candidate genes were determined by RT-qPCR and then analyzed by geNorm, Bestkeeper, and Normfinder. For porcine SMSCs, PPIA, and TBP were selected for tissue in alginate scaffold whereas HPRT and TBP were selected for the agarose scaffold system. On the other hand, HPRT, PPIA, and RPL18 were the stable reference genes for rabbit chondrocytes in alginate scaffold while TBP, RPL5, and RPL18 were selected for rabbit chondrocytes in agarose scaffold. This study has further indicated that suitable reference genes are different for each tissue and study purpose. The reference genes are expressed in different stability when a scaffold of different material is used.

Redifferentiation of dedifferentiated chondrocytes by adenoviral vector-mediated TGF-ß3 and collagen-1 silencing shRNA in 3D culture.

Autologous chondrocytes remain one of the most preferable candidates among various therapeutic cell species because of their high efficacy, despite remarkable progress in discovery and development of therapeutic cells for cartilage regenerative medicine to date. However, the essential process of cell expansion via repeated monolayer sub-cultures inevitably induces chondrocytic dedifferentiation. In this study, we aimed to achieve and enhance redifferentiation of dedifferentiated chondrocytes with dual genes of transforming growth factor (TGF)-ß3 and short hairpin RNA (shRNA) that restore chondrocytic phenotype and silence fibrous collagen type I (Col I), respectively. It was hypothesized that gene delivery of the two targets would promote chondrogenesis in chondrocytes, and meanwhile inhibit the expression of the undesired Col I. Three types of recombinant adenoviruses were constructed. Two of them were of single-function vectors with the ability to express either TGF-ß3 (Ad-TGFß3) or shRNA (specific for Col I, Ad-shRNA); the third type was of double-function vectors that encode both TGF-ß3 and anti-Col I shRNA (Ad-double). We infected the dedifferentiated chondrocytes with Ad-double, or co-transduced them with Ad-TGFß3 and Ad-shRNA at the same time (designated as Ad-combination). Data from real-time RT-PCR and histological staining suggested a restoration in the expression of cartilage-specific genes including aggrecan, type II collagen, and cartilage oligomeric matrix protein (COMP); while a significant down-regulation of Col I expression was observed in groups treated with Ad-double and Ad-combination compared to other control groups. These results demonstrated that, by genetic modification, dedifferentiated chondrocytes managed to redifferentiate back to chondrocytic phenotype, which may greatly facilitate cartilage regenerative medicine by providing sufficient number of competent therapeutic cells.

In vitro study of chondrocyte redifferentiation with lentiviral vector-mediated transgenic TGF-ß3 and shRNA suppressing type I collagen in three-dimensional culture.

Chondrocytes are the primary candidate therapeutic cells to cure cartilaginous lesions. Ideally, for transplantation, autologous chondrocytes are isolated from the patient, amplified in vitro, seeded in a scaffold and implanted back. However, significant concerns arise with chondrocyte dedifferentiation during monolayer amplification, whereby cells lose their chondrocytic phenotype by rapidly downregulating the expression of cartilage markers such as type II collagen (Col II) and aggrecan. The accompanying upregulation in type I collagen (Col I) is also problematic, as it leads to unexpected fibrosis and causes such engineered cartilage to lack the desired mechanical strength to make up joint lesions. Transforming growth factor-ß3 (TGF-ß3) has been proved effective in maintaining chondrocytic morphology and promoting total collagen production. In this study, we aimed to deliver the TGF-ß3 gene into dedifferentiated chondrocytes with recombinant lentiviral vectors; by transgenic expression of TGF-ß3, chondrocytic redifferentiation is catalysed. Simultaneously, shRNA targeting Col I was also incorporated into the vector to suppress Col I production. The results indicated that chondrocytes underwent dedifferentiation in monolayer culture in the presence or absence of transgenic TGF-ß3. In three-dimensional culture, effective redifferentiation was managed in the dedifferentiated chondrocytes that were transduced with transgenic TGF-ß3. The incorporation and expression of Col I-targeting shRNA were also effective in reducing Col I production in a post-transcriptional manner.

Optimal construction and delivery of dual-functioning lentiviral vectors for type I collagen-suppressed chondrogenesis in synovium-derived mesenchymal stem cells.

PURPOSE: This study aims to deliver both transforming growth factor ß3 (TGF-ß3) and shRNA targeting type I collagen (Col I) by optimal construction and application of various dual-functioning lentiviral vectors to induce Col I-suppressed chondrogenesis in synovium-derived mesenchymal stem cells (SMSCs). METHODS: We constructed four lentiviral vectors (LV-1, LV-2, LV-3 and LV-4) with various arrangements of the two expression cassettes in different positions and orientations. Col I inhibition efficiency and chondrogenic markers were assessed with qPCR, ELISA and staining techniques. Among the four vectors, LV-1 has two distant and reversely oriented cassettes, LV-2 has two distant and same-oriented cassettes, LV-3 has two proximal and reversely oriented cassettes, and LV-4 has two proximal and same-oriented cassettes. Col I and chondrogenic markers, including type II collagen (Col II), aggrecan and glycosaminoglycan (GAG), were examined in SMSCs cultured in 3-D alginate hydrogel. RESULTS: All of the four vectors showed distinct effects in Col I level as well as diverse inductive efficiencies in upregulation of the cartilaginous markers. Based on real-time PCR results, LV-1 was optimal towards Col I-suppressed chondrogenesis. CONCLUSION: LV-1 vector is competent to promote Col I-suppressed chondrogenesis in SMSCs.

Note: Tangential x-ray diagnosis for investigating fast MHD events in EAST tokamak.

A tangential x-ray diagnosis has been installed in the experimental advanced superconducting tokamakvacuum vessel for the study of fast magnetohydrodynamics (MHD) events. This system is based on absolute x-ray ultraviolet detectors with a collimator which is processed by laser machine. The first experimental results have proved its ability to measure the small-scale and transient MHD perturbations.

Microcavitary hydrogel-mediating phase transfer cell culture for cartilage tissue engineering.

Hydrogels have been widely used as cell-laden vehicles for therapeutic transplantation in regenerative medicine. Although the advantages of biocompatibility and injectability for in situ grafting have made hydrogel a superior candidate in tissue engineering, there remain challenges in long-term efficacy of tissue development using hydrogel, especially when more sophisticated applications are demanded. The major bottleneck lies in environmental constraints for neo-tissue generation in the gel bulk such as proliferation of encapsulated cells (colonies) per se and also accommodation of their endogenously produced extracellular matrices. In this study, we endeavor to develop an innovative tissue engineering system to overcome these drawbacks through a novel microcavitary hydrogel (MCG)-based scaffolding technology and a novel phase transfer cell culture (PTCC) strategy to enable phenotypically bona fide neo-tissue formation in an injectable artificial graft. For this purpose, microspherical cavities are created in cell-encapsulating hydrogel bulk via a retarded dissolution of coencapsulated gelatin microspheres. Based on proliferation and affinity selection, the encapsulated cell colonies adjacent to the gel-cavity interface will spontaneously outgrow the hydrogel phase and sprout into cavities, enabling neo-tissue islets to fill up the voids and further expand throughout the whole system for full tissue regeneration. The design of MCG-PTCC strategy was elicited from an observation of a spontaneous dynamic outgrowth of chondrocytes from the edge of a cell-laden hydrogel construct over prolonged cultivation--a phenomenon named edge flourish. This MCG-PTCC strategy potentially introduce a new application to hydrogels in the field of regenerative medicine through elevation of its role as a cell vehicle to a three-dimensional transplantable growth-guiding platform for further development of newly generated tissues that better fulfill the demanding criteria of scaffolds in therapeutic tissue regeneration.

Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system.

A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-beta3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-beta3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-beta3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.

Effects of combinational adenoviral vector-mediated TGF beta 3 transgene and shRNA silencing type I collagen on articular chondrogenesis of synovium-derived mesenchymal stem cells.

In this study, transgenic effects of combination of transforming growth factor (TGF) beta3 and shRNA silencing type I collagen (Col I) on chondrogenesis of synovium-derived mesenchymal stem cells (SMSCs) were evaluated. SMSCs were infected with recombinant adenoviruses encoding TGF beta 3 (Ad-TGF beta 3) and/or anti-Col I shRNA (Ad-shRNA) separately, simultaneously (Ad-combination), or conjugately (Ad-double, mediated by one vector encoding both). The transduced SMSCs were encapsulated in alginate hydrogel and cultured for 30 days in chondrogenic medium. The expression of cartilaginous extracellular matrix components was investigated by quantitative real-time RT-PCR (qRT-PCR) and histological staining. qRT-PCR showed an up-regulation in chondrocytes marker genes such as type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP) in Ad-TGF beta 3, Ad-double, and Ad-combination groups on day 30. Whereas, Ad-TGF beta 3 treatment induced significant elevation in Col I, which could be largely resisted by anti-Col I shRNA functionality. Histological and immunohistochemical staining results were consistent with our qRT-PCR data. These results demonstrate that the application of combinational adenoviral vector-mediated transgenic TGF beta 3 and shRNA targeting Col I possesses the potential in promoting the chondrogenic differentiation of SMSCs as well as inhibiting the formation of fibrocartilage.

Continuous supply of TGFß3 via adenoviral vector promotes type I collagen and viability of fibroblasts in alginate hydrogel.

In recent years, transforming growth factor-ß3 (TGFß3) has interested more and more researchers with its competence in engineered histogenesis. In the present study we employed recombinant adenoviral vectors to deliver the constitutively active TGFß3 gene to human dermal fibroblasts, which could maintain the continuous secretion of TGFß3 from the cells. The expression of type I collagen in the Ad-TGFß3 group increased significantly in comparison with other three groups: Neg (cells without treatment of the adenovirus), Ad-null (cells with treatment of the adenovirus, without the inserted gene) and Ad-shRNA (cells with treatment of the adenovirus encoding shRNA specific for type I collagen). Additionally, we demonstrated that TGFß3 enhanced the expression of Smad4 while inhibiting that of MMP-9, thus promoting the collagen transcription via the Smad signal transduction pathway and restraining collagen degradation by MMP-9, which contributed to the increasing type I collagen expression level. As type I collagen mediates cell-material interactions by providing anchorage, the viability of encapsulated fibroblasts in Ad-TGFß3 group was significantly higher than that in other three groups. Accordingly, this approach forms an effective way to improve the compatibility of non-adhesive hydrogels containing anchorage-dependent cells.

A dual-functioning adenoviral vector encoding both transforming growth factor-beta3 and shRNA silencing type I collagen: construction and controlled release for chondrogenesis.

Hyaline articular cartilage degeneration is a common clinical syndrome globally, whereas cell-based therapy has proved to be a good solution to such problems. Given that transforming growth factor (TGF-beta3) is helpful in maintaining chondrocytic phenotype or stimulating chondrogenic differentiation of stem cells, vectors containing TGF-beta3 expression cassette can be delivered to therapeutic cells. One problem involved in the application of therapeutic cells in chondrogenesis is the undesirable production of type I collagen in such cells as chondrocytes and synovial mesenchymal stem cells during ex vivo culture, which undermines the mechanical strength of engineered cartilage. RNA interference (RNAi) strategy can be used to knock down its expression to allow better biological and mechanical functions in artificial tissues. In this study, for the first time we report the construction of an adenoviral vector that can express both TGF-beta3 to promote chondrogenesis and short hairpin RNA (shRNA) targeting type I collagen to block its production. This dual-functioning vector (Ad-dual) was found to function well in three model cell types: human fibroblast, osteoblast and porcine chondrocyte in terms of the release of TGF-beta3 protein and down-regulation of type I collagen production. Besides, we also tested its efficacy in porcine synovial mesenchymal stem cells, highlighting its potential applications in cell-based therapy for the treatment of articular cartilage degeneration.

Rac1 GTPase is activated by hepatitis B virus replication--involvement of HBX.

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (betaPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.