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Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. However, host-HEV interactions have yet to be fully understood. Zinc-finger antiviral protein (ZAP) is a novel interferon (IFN)-stimulated gene product that inhibits a variety of viruses in synergy with IFN-β. To evaluate the role of ZAP in HEV infection, its expressions in HEV-infected patients and in cell cultures were measured. We report a significant inhibition of ZAP expression in patients with HEV genotype four acute infection. The expression of ZAP in the HEV life cycle was monitored in cultures of HEV-infected cells. Results indicated that the ZAP level decreased significantly after HEV infection. ZAP over-expression inhibited HEV replication, whereas its knockdown by RNA interference significantly increased HEV RNA. These suggest that ZAP serves as an antiviral in HEV infection. Moreover, silencing ZAP decreased IFN regulatory factor 3 (IRF3) phosphorylation in HEV-infected cells treated with poly(I:C), indicating that ZAP synergizes with IFN-β. In conclusion, ZAP is an important anti-HEV host factor and in synergy with IFN-β, inhibits HEV replication.

Context Chinese herbs such as Cortex Mori [Morus alba L. (Moraceae)] may inhibit human immunodeficiency virus (HIV), but active compounds are unknown. Objective Screening of Cortex Mori and other herbs for anti-HIV active compounds. Materials and methods HIV-1 virus (multiplicity of infection: 20), and herbs (dissolved in dimethyl sulfoxide, working concentrations: 10, 1, and 0.1 mg/mL) such as Cortex Mori, etc., were added to 786-O cells (105 cell/well). Zidovudine was used as a positive control. Cell survival and viral inhibition rates were measured. The herb that was the closest inactivity to zidovudine was screened. Mass spectrometry identified the active compounds in herbs (mobile phase: 0.05% formic acid aqueous solution and acetonitrile, gradient elution, detection wavelength: 210 nm). The effect of the compounds on reverse transcriptase (RT) products were evaluated by real-time PCR. Gene enrichment was used to analyse underlying mechanisms. Results With a dose of 1 mg/mL of Cortex Mori, the cell survival rate (57.94%) and viral inhibition rate (74.95%) were closest to the effect of zidovudine (87.87%, 79.81%, respectively). Neochlorogenic acid, one of the active ingredients, was identified by mass spectrometry in Cortex Mori. PCR discovery total RT products of neochlorogenic acid group (mean relative gene expression: 6.01) significantly inhibited (control: 35.42, p

The recurrent outbreak of coronaviruses and variants underscores the need for broadly reactive antivirals and vaccines. Here, a novel broad-spectrum human antibody named 76E1 was isolated from a COVID-19 convalescent patient and showed broad neutralization activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants and also exhibited the binding breath to peptides containing the epitope from γ- and δ- coronaviruses. 76E1 cross-protects mice from SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and treatment models. The epitope including the fusion peptide and S2’ cleavage site recognized by 76E1 was significantly conserved among α-, β-, γ- and δ- coronaviruses. We uncovered a novel mechanism of antibody neutralization that the epitope of 76E1 was proportionally less exposed in the prefusion trimeric structure of spike protein but could be unmasked by binding to the receptor ACE2. Once the epitope exposed, 76E1 inhibited S2’ cleavage, thus blocked the membrane fusion process. Our data demonstrate a key epitope targeted by broadly-neutralizing antibodies and will guide next-generation epitope-based pan-coronavirus vaccine design.

Background Pulmonary arterial hypertension (PAH) is characterised by remodelling in vascular smooth muscles, and switching from contractile (differentiated) to synthetic (dedifferentiated) phenotype. This study aimed to investigate the effect of a mutated caveolin-1 (Cav1F92A) gene from bone marrow mesenchymal stem cells (rBMSCs) on phenotypic switching in the smooth muscle cells during PAH. Methods Human pulmonary smooth muscle cells (HPASMCs) were treated with monocrotaline (MCT,1 μM), and co-cultured with Cav1F92A gene modified rBMSCs (rBMSCs/Cav1F92A). The nitric oxide (NO) production, cell adhesion, cell viability and inflammatory cytokines expression in rBMSCs was measured to evaluate the survival rate of rBMSCs and the changes of inflammatory cytokines. The concentration of NO/cGMP (nitric oxide/Guanosine-3',5'-cyclic monophosphate), the tumour necrosis factor-alpha (TNF-α), transforming growth factor-beta1 (TGF-β1) mRNA, the expression of contractile smooth muscle cells (SMCs) phenotype markers (thrombospondin-1 and Matrix Gla protein, MGP), the synthetic SMCs phenotype markers (H-caldesmon and smooth muscle gene SM22 alpha, SM22α), cell migration and the morphological changes in rBMSCs/Cav1F92A co-cultured HPASMCs were investigated. Results Cav1F92A increased NO concentration, cell adhesion, cell viability, anti-inflammatory cytokines interleukin-4 (IL-4), and interleukin-10 (IL-10), but decreased the inflammatory cytokines interleukin-1α (IL-1α), interferon-γ (INF-γ) and TNF-α expression in rBMSCs. rBMSCs/Cav1F92A activated the NO/cGMP, down-regulated TNF-α, TGF-β1, thrombospondin-1 and MGP expression, up-regulated SM22α and H-caldesmon expression, restored cell morphology, and inhibited cell migration in MCT treated HPASMCs. Conclusions rBMSCs/Cav1F92A inhibits switching from contractile to synthetic phenotype in HPASMCs. It also inhibits migration and promotes morphological restoration of these cells. rBMSCs/Cav1F92A may be used as a therapeutic modality for PAH.

Hepatitis E virus (HEV) is the major pathogen of viral hepatitis. However, the understanding of the HEV life cycle is limited. In the present study, cells were separately infected with non-enveloped HEV (derived from feces or bile) or quasi-enveloped HEV (derived from the cell culture after serial passages, eHEV) and observed by confocal fluorescence microscopy to investigate the life cycle of HEV. HEV finished its binding and entry into host cells at first 6 h post-inoculation (hpi). Cells inoculated with eHEV showed less infectivity than cells inoculated with non-enveloped HEV. Newly synthesized progeny virions were released into the supernatant of cell cultures from 48 hpi. qRT-PCR and Western blotting results showed that the supernatant's progeny viruses were infectious even after 5 serial passages. These results show the significant difference between non-enveloped HEV and eHEV, which will provide novel insights into the HEV replication cycle. The efficient cell culture of HEV will promote the development of anti-HEV drugs and vaccines. This article is protected by copyright. All rights reserved.

Hepatitis E virus (HEV) infection has become a global concern with high mortality rates among pregnant women, especially those in their third trimester of pregnancy. Estrogen plays an important role in mediating the body, regulating physiological and pathological processes. Estrogen is activated by binding to estrogen receptors (ERs) and mediates rapid signaling events by pathways that involve transmembrane ERs. Our previous study had confirmed that high estrogen levels during pregnancy are associated with high HEV titers. However, the association between HEV infection and estrogen signaling pathways remains unclear. In the present study, the regulation of estrogen signaling pathways by HEV infection was evaluated. Results demonstrated that HEV infection significantly inhibits the cAMP-PKA-CREB and PI3K-AKT-mTOR signaling pathways, but is independent of the Ras-Raf-MEK-ERK signaling pathway. In summary, the increasing estrogen levels and highly activated ERα during pregnancy aggravates HEV replication. The exacerbation of HEV replication, in turn, inhibits ERα expression and suppresses both cAMP-PKA-CREB and PI3K-AKT-mTOR signaling pathways.

Hepatitis E virus (HEV) is the main pathogen of hepatitis worldwide. However, its infection biology and pathogenesis remain largely unknown. Suitable small animal models are required to advance the study of HEV infection. Although an efficient model of genotype 1 (gt1) and gt3 HEV infection has been established in human liver chimeric mice, the infectivity of gt4 HEV infection in mice has not been comprehensively characterized. In this study, immunocompromised BALB/c nude, immunocompetent BALB/c, and C57BL/6 mice were inoculated with either gt3 or gt4 HEV (19 HEV strains, including human, swine, macaque-adapted, and cow HEV strains). Infectivity was identified by viral RNA and antigen detection, inflammation, and histopathological analysis. Then, HEV-infected BALB/c mice were treated with antiviral drugs. Acute HEV infection was established in BALB/c mice inoculated with eight gt4 HEV strains. However, gt3 HEV strains failed to achieve active HEV infection. HEV infection was established in BALB/c nude and regular mice inoculated with gt4 HEV but not in C57BL/6 mice. Gt4 HEV infection resulted in rapid viremia and high titers in feces, sera, and replication sites. HEV infection in mice showed no gender preference. Furthermore, chronic gt4 HEV infection was well imitated in BALB/c mice for 32 weeks and caused liver fibrosis. Conclusions: BALB/c mice have a great potential for reproducing the process of gt4 HEV infection. The successful establishment of a gt4 HEV small-animal model provides an opportunity to further understand HEV infection biology and zoonotic transmission and develop anti-HEV vaccine.

Objective Squamous cell carcinoma is often associated with the deletion or mutation of zinc finger protein 750 (ZNF750), its deletion or mutation is associated with squamous epithelial malignant biological characteristics. The present study is to explore the mechanism of ZNF750 to suppress the tumor malignant process by regulation tumor microenvironment. Methods To evaluate the changes of tumor microenvironment in oral squamous cells carcinoma cell line CAL-27 cell, the expression of angiogenin, vascular endothelial growth factor (VEGF), prolyl hydroxylase 2 (PHD2), G protein signal regulated protein 5 (RGS5), integrin A5 (ITGA5), integrin B1 (ITGB1) and CD44 were detected by Western-blot. The changes of platelet derived growth factor (PDGFB) and tumor vascular marker CD105 (Endoglin) mRNA were estimated by qPCR. The effect of over-expressed ZNF750 on cell viability and lateral migration capacity was investigated by CCK-8 and cell scratch assay in three oral squamous cells carcinoma. Results ZNF750 could effectively inhibit the protein or mRNA expression of angiogenin, VEGF, RGS5 and CD105, repressed the cell adhesion molecules ITGA5, ITGB1 and CD44, but up-regulate the protein or mRNA expression of PHD2 and PDGFB. The cell viability and lateral migration ability of three oral squamous cells carcinoma were reduced by over-expression of ZNF750. Conclusion ZNF750 could modulate the tumor vascular microenvironment to inhibit the oral squamous cells carcinoma malignant progression.

To evaluate whether uterine injury caused by hepatitis E virus (HEV) infection is responsible for adverse pregnancy outcomes. HEV-infected female BALB/c mice were coupled with healthy male BALB/c mice at 0, 7, 14, 21, and 91 dpi to explore the uterine injury caused by HEV infection. Mice were euthanized after 10 days of copulation, and uteruses were collected for HEV RNA and antigen detection and histopathological analysis. Inflammatory responses; apoptosis; and estrogen receptor ɑ (ER-ɑ), endomethal antibody (ERAb), cytokeratin-7 (CK7), vimentin (VIM), and vascular endothelial growth factor (VEGF) expression levels were evaluated. After 10 days of copulation, miscarriage and nonpregnancy, as well as enlarged uteruses filled with inflammatory cytokines, were found in HEV-infected mice. HEV RNA and antigens were detected in the sera and uteruses of HEV-infected mice. Significant endometrial thickness (EMT) thinning, severe inflammatory responses, and aggravated apoptosis in the uteruses of HEV-infected mice that experienced miscarriage might contribute to adverse pregnancy outcomes. Furthermore, significantly suppressed ER-ɑ expression and increased ERAb, CK7, VIM, and VEGF expression levels were found in the uteruses of HEV-infected mice that had miscarried. However, uterine damage recovered after complete HEV clearance, and impaired fertility was improved. EMT injury, severe inflammatory responses, and aggravated apoptosis in the uterus caused by HEV infection are responsible for poor pregnancy outcomes.

Zinc-finger protein 750 (ZNF750) is the potential anti-cancer gene in oral squamous cell carcinoma (OSCC). The present study was to investigate the expression changes of ZNF750 in OSCC tissue and to reveal the induction of altered mRNA expression profiles caused by over-expressed ZNF750 in CAL-27 cell. The expression level of ZNF750 in tissue specimens from OSCC patients was detected by immunohistochemistry. Gene expression profiling was performed using Human Signal Transduction PathwayFinder RT2 Profiler™ PCR Array. The expression changes of 84 key genes representing 10 signal transduction pathways in human following over-expressed ZNF750 in CAL-27 cell was examined. The expression of ZNF750 protein was reduced in OSCC tissues. The R2 PCR Array analysis revealed that 39 of the 84 examined genes that changed at least a two-fold between control and ZNF750 groups. These genes related to oxidative stress, WNT, JAK/STAT, TGFβ, NF-kappaB (NFκB), p53, Notch, Hedgehog, PPAR and Hypoxia signaling. ZNF750 could inhibit the candidate genes ATF4, SQSTM1, HMOX1, CCND1, TNF-alpha, TNFSF10 and FOSL1 but activate CDKN1A and EMP1. Our studies suggest that ZNF750 can regulate signaling pathways that related to proliferation, cell cycle, inflammation and oxidative stress in CAL-27 cell.

Background This study aimed to investigate the effect of bone marrow derived mesenchymal stem cells (rBMSCs) transduced with lentiviral vectors expressing endothelial nitric oxide synthase (eNOS) and/or a mutant caveolin-1(F92A-Cav1), on the pulmonary haemodynamics and structure in a rat model of pulmonary arterial hypertension (PAH). Methods Pulmonary arterial hypertension was induced with monocrotaline (MCT) in 60 adult male Wistar rats prior to delivery of lentiviral vector transduced rBMSCs expressing Cav1, eNOS and/or F92A-Cav1. Changes in pulmonary haemodynamics, right ventricular hypertrophy index (RVHI), and serum nitric oxide (NO) were evaluated. Ultrastructure changes in lung tissues were observed by transmission electron microscopy. Expression of Kruppel-like factor 4 (KLF4), p53, P21, eNOS, and alpha-smooth muscle actin were evaluated by real time PCR, western blotting or immunohistochemistry. Results Treatment of PAH rats with gene modified rBMSCs (eNOS +/- Cav1 F92A) decreased right ventricular systolic pressure and improved pulmonary haemodynamics. The protein of alpha-smooth muscle actin expression was decreased whilst KLF4, p53, P21, eNOS expression, and serum NO concentration was elevated. The survival rate of rats in the treatment groups was also improved, after 35 days of observation. Conclusion Intravenous delivery of rBMSCs expressing eNOS/F92A-Cav1 to PAH rats inhibits pulmonary vascular smooth muscle cell proliferation, and improves pulmonary haemodynamics, vascular remodelling and short-term survival. Activation of KLF4-p53 signalling pathway may be involved in these beneficial effects.

Abtract Background Zinc-finger protein 750 (ZNF750) is a potential tumor suppressor in oral squamous cell carcinoma (OSCC). However, the molecular mechanisms underlying its anti-tumor effect remain elusive in OSCC. This study aimed to elucidate the genes and pathways involved in tumor suppression following the ZNF750 over-expression in OSCC cell line CAL-27 cell by using the genomics approach. Methods The RNA sequence libraries were constructed, and the data were analyzed to identify differentially expressed genes (DEGs) between vector groups and ZNF750 groups (over-expressed ZNF750 in CAL-27 cell). QPCR and western-blot was used to validate differential expression of candidate genes with cell cycle regulation. The cell cycle distribution was analyzed by BrdU staining. Results By RNA sequencing profiling, 7,131 genes were differentially expressed in ZNF750 groups. Among the DEGs, 3,285 genes were upregulated, 3,846 genes were downregulated and 4,507 genes were identified in three main categories (cellular_component, biological process and molecular function) based on the gene ontology (GO) classification. The Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis defined the DEGs could be categorized into 280 pathways and identified the top two most significant pathways involved in spliceosome and cell cycle. Functional categorization and enrichment analysis revealed that most of DEGs involved in binding and catalytic activity, and the cell cycle associated genes were significantly enriched in response to ZNF750 over-expression. ZNF750 induced cell cycle arrest in G0/G1 phase of the cell cycle. Conclusion Data from this study revealed that the cell cycle pathway was a key factor involved in the anti-tumor effect of ZNF750 in CAL-27 cells.

Background Pulmonary arterial hypertension (PAH) is associated with sustained vasoconstriction, inflammation and suppressed apoptosis of smooth muscle cells. Our previous studies have found that rat bone marrow-derived mesenchymal stem cells (rBMSCs) transduced with a mutant caveolin-1(F92A-Cav1) could enhance endothelial nitric oxide synthase (eNOS) activity and improve pulmonary vascular remodeling, but the potential mechanism is not yet fully explored. The present study was to investigate the gene expression profile upon rBMSCs/F92A-Cav1delivered to PAH rat to evaluate the role of F92A-Cav1 in its regulation. Methods PAH was induced with monocrotaline (MCT, 60 mg/kg) prior to delivery of lentiviral vector transduced rBMSCs expressing Cav1 or F92A-Cav1. Gene expression profiling was performed using Rat Signal Transduction PathwayFinder array. The expression changes of 84 key genes representing 10 signal transduction pathways in rat following rBMSCs/F92A-Cav1 treatment was examined. Results Screening with the Rat Signal Transduction PathwayFinder R 2 PCR Array system and subsequent western blot, immunohistochemistry or real time PCR analysis revealed that F92A-Cav1 modified rBMSCs can inhibit the inflammation factors (TNF-alpha, Icam1 and C/EBPdelta), pro-proliferation genes (c-Myc, Bcl2a1d, Notch1and Hey2), oxidative stress gene (Hmox1) and activate cell cycle arrested gene Cdkn1a, ameliorating inflammation and inhibiting cell proliferation in PAH rat. Conclusion rBMSCs/F92A-Cav1 inhibits inflammation and cell proliferation by regulating signaling pathways that related to inflammation, proliferation, cell cycle and oxidative stress.

Background: This study was aimed to investigate whether osteoblasts from diabetic patients have a promoting effect on osteogenesis of human umbilical cord mesenchymal stem cells (HUMSCs). Methods: HUMSCs were co-cultured with osteoblasts of diabetic and non-diabetic patients. Morphological appearance and cytochemical characteristics of the non-diabetic osteoblasts and diabetic osteoblasts were observed by hematoxylin-eosin staining, type I collagen protein expression, alkaline phosphatase (ALP) staining and Alizarin Red S staining. Cell viability, type I collagen protein expression, ALP activity and osteocalcin mRNA expression in HUMSCs were investigated. Results: Compared with negative control group, the cell proliferation, type I collagen protein expression, ALP activity and osteocalcin mRNA were increased in HUMSCs co-cultured with diabetic and non-diabetic osteoblasts (P0.05). Conclusions: Similar to osteoblasts from non-diabetic patients, osteoblasts from diabetic patients also have the ability to promote HUMSCs proliferation, and leading to HUMSCs exhibit some characteristic of osteoblasts.

Zinc-finger protein 750 (ZNF750) encodes a putative C2H2 zinc finger protein and is typically mutated or deleted in squamous cell carcinoma. The role of ZNF750 in oral squamous cell carcinoma (OSCC) remains unknown. The aim of the present study was to investigate the effects of ZNF750 overexpression in CAL-27 cells. Cell viability, and the expression of genes associated with proliferation, differentiation and the epithelial-mesenchymal transition were investigated in CAL-27 cells following ZNF750 overexpression, using Cell Counting kit-8, reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. In addition, scratch wound, invasion and migration assays were performed. Cell viability, matrix metalloproteinase 28 expression, cyclin B1 expression and mesenchymal marker neural cadherin expression were decreased following ZNF750 overexpression compared with the control groups. ZNF750 overexpression induced the differentiation-associated genes late cornified envelope 3A and small proline-rich protein 1A and upregulated the expression of late epidermal differentiation factor Kruppel-like factor 4. Overexpression of ZNF750 in CAL-27 cells resulted in inhibition of cell invasion and migration. Taken together, these data suggest that ZNF750 may inhibit the metastasis of OSCC.

This study was aimed to investigate the effect of down-regulating the CXC chemokine receptor-4 (CXCR4) expression on cell proliferation, invasion and migration of human ovarian cancer cell line SW626. The CXCR4 specific short hairpin RNA (shRNA) plasmid vector was constructed and then transfected into the SW626 cells. The expression of CXCR4 mRNA and protein was detected by real-time RT-PCR and western blot respectively. Cell proliferation was detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cell invasion and migration was assayed in Biocoat Matrigel invasion chambers. The expression level of CXCR4 in SW626 cell transfected with CXCR4-siRNA was inhibited, leading to significant decrease in SW626 cell proliferation, invasion and migration. We conclude that CXCR4 is essential for tumor cell proliferation and invasion. The CXCR4 molecule is a potential therapeutic target to control ovarian cancer cell growth or metastasis.

The aim of this study was to investigate the apoptotic effect of a proteasome inhibitor MG-132 on Tca-8113, a cell line of human tongue squamous cell carcinoma. Tca-8113 cells were treated with 10, 20, and 30μM of MG-132, or 5μM thapsigargin. Apoptosis rate was determined with annexin V/propidium iodide double staining. Expression of E3ubiquitin-protein ligase was determined by ELISA, and Grp78 and caspase-12 mRNA, and Grp78 and caspase-12 protein was assessed by RT-PCR and Western blot, respectively. Apoptosis was observed 18h after MG-132 treatment. The apoptotic rate in the 10, 20, and 30μM MG-132 group was 13.5, 19.6 and 34.7%, respectively, which was higher than in the thapsigargin (8.5%, P

Background Nitric oxide (NO) is generated in endothelial cells by endothelial nitric oxide synthase (eNOS). Caveolin-1 (Cav1) inhibits eNOS function and NO production. Modifying Cav1 scaffold domain, in particular Phenylalanine at position 92 (F92) is critical for the inhibitory actions of Cav1 toward eNOS. The aims of this study were to investigate the effect of enhanced NO production in term of in vitro angiogenesis on rat bone marrow derived mesenchymal stem cells (BMSCs) transduced with a novel bicistronic lentiviral vector co-expressing eNOS and mutant Cav1 (F92A). Methods A bicistronic eNOS/F92-Cav1 lentiviral vector was constructed, and used to transduce rat BMSCs. The expression of eNOS and VEGF protein were confirmed by western-blot. NO production was detected by the greiss assay and in vitro angiogenesis was assessed by matrigel assisted capillary tube formation. The cell viability was evaluated using a Cell Counting Kit (CCK)-8. Results The bicistronic eNOS/F92A-Cav1 lentiviral vector increased eNOS and VEGF protein expression, NO production compared to controls. In vitro capillary formation was increased in eNOS-F92A transduced cells and cell viability was not affected by transduction. Conclusion Transduction of rat BMSCs with an eNOS-F92A-Cav1 lentiviral vector can increase NO production by enhancing eNOS protein expression. The increased NO production did not reduce cell viability. This study demonstrates that genetic modification of BMSCs to enhance NO producton could be applied in stem cell based therapeutic approaches to treat diseases such as pulmonary arterial hypertension (PAH) which is characterized by decreased endothelial NO release.

Exposure of Tca-8113 cells to proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG-132) causing apoptosis is associated with endoplasmic reticulum (ER) stress. X-box-binding protein-1 (XBP1) is an important regulator of a subset of genes active during ER stress, which is related to cell survival and is required for tumor growth. The present study is to evaluate the effect of MG-132 on ROS production, XBP1 gene expression, tumor necrosis factor receptor-associated factor 2 (TRAF2), ASK1 and c-jun protein expression in tongue squamous cell carcinoma cell line Tca-8113 cells. ROS production was measured by reactive oxygen species assay. X-box binding protein-1 (XBP1) mRNA was analyzed by real-time-PCR, TRAF2, ASK1 and c-jun protein were investigated by western blot and immunocytochemistry respectively. The result indicated that ROS production, TRAF2, ASK1 and c-jun were elevated in MG-132 treated cells. Giving ROS scavenger N-acetyl-L-cysteine (NAC) largely prevented the effects of MG-132. Furthermore, treating with MG-132 lead to decreased XBP1 mRNA expression but could not completely block the expression of XBP1. Taken together, these findings provide the evidence that MG-132 induced ER stress lead to Tca-8113 cells apoptosis through ROS generation and TRAF2-ASK1-JNK signal pathway activation.

Objectives: To investigate the effect of a seashell protein Haishengsu (HSS), an extract from a shellfish Tegillarca granosa, on cell growth and the expression of apoptosis genes in leukemia K562 cells. Methods: Cultured K562 cells were treated with HSS at various concentrations (10-40mg/L). The cell cycle, cell growth and the expression of apoptosis suppressor gene bcl-2 and apoptosis promoting gene bax were evaluated. Results: HSS, 20mg/L, inhibited cell cycle in the G0/G1 and S phases. HSS, 20mg/L, also inhibited the growth of K562 cells over time. Expression of bcl-2 gene in the HSS 20mg/L (58.8%±4.7%) and HSS 40mg/L group (26.6%±2.1%) were lower than in the control group (91.0±8.7%, P < 0.01). Expression of bax gene in the HSS 20mg/L (77.7±3.6%) and 40mg/L group (90.6±3.7%) were higher than in the control group (10.9±6.6%, P < 0.01). Conclusion: HSS suppresses leukemia K562 cell growth by inhibiting the G0/G1 and S phases of the cell cycle. It also induces apoptosis in these leukemia cells by reducing the expression of apoptosis suppressor gene bcl-2, and increasing the expression of apoptosis promoting gene bax. Further studies are required to investigate the clinical efficacy of HSS in leukemia.

Background Nitric oxide (NO) plays a role in a number of physiological processes including stem cell differentiation and osteogenesis. Endothelial nitric oxide synthase (eNOS), one of three NO-producing enzymes, is located in a close conformation with the caveolin-1 (CAV-1WT) membrane protein which is inhibitory to NO production. Modification of this interaction through mutation of the caveolin scaffold domain can increase NO release. In this study, we genetically modified equine adipose-derived stem cells (eASCs) with eNOS, CAV-1WT, and a CAV-1F92A (CAV-1WT mutant) and assessed NO-mediated osteogenic differentiation and the relationship with the Wnt signaling pathway. Methods NO production was enhanced by lentiviral vector co-delivery of eNOS and CAV-1F92A to eASCs, and osteogenesis and Wnt signaling was assessed by gene expression analysis and activity of a novel Runx2-GFP reporter. Cells were also exposed to a NO donor (NONOate) and the eNOS inhibitor, l-NAME. Results NO production as measured by nitrite was significantly increased in eNOS and CAV-1F92A transduced eASCs +(5.59 ± 0.22 μM) compared to eNOS alone (4.81 ± 0.59 μM) and un-transduced control cells (0.91 ± 0.23 μM) (p WT transgenes resulted in lower NO production. Canonical Wnt signaling pathway-associated Wnt3a and Wnt8a gene expressions were increased in eNOS-CAV-1F92A cells undergoing osteogenesis whilst non-canonical Wnt5a was decreased and similar results were seen with NONOate treatment. Treatment of osteogenic cultures with 2 mM l-NAME resulted in reduced Runx2, ALP, and Wnt3a expressions, whilst Wnt5a expression was increased in eNOS-delivered cells. Co-transduction of eASCs with a Wnt pathway responsive lenti-TCF/LEF-dGFP reporter only showed activity in osteogenic cultures co-transduced with a doxycycline inducible eNOS. Lentiviral vector expression of canonical Wnt3a and non-canonical Wnt5a in eASCs was associated with induced and suppressed osteogenic differentiation, respectively, whilst treatment of eNOS-osteogenic cells with the Wnt inhibitor Dkk-1 significantly reduced expressions of Runx2 and ALP. Conclusions This study identifies NO as a regulator of canonical Wnt/β-catenin signaling to promote osteogenesis in eASCs which may contribute to novel bone regeneration strategies.