Adenoviral RNA Research Articles

TRIM35 Monoubiquitinates H2B in Cardiac Cells, Implications for Heart Failure.

The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.

Adenovirus Transcriptome in Human Cells Infected with ChAdOx1-Vectored Candidate HIV-1 Vaccine Is Dominated by High Levels of Correctly Spliced HIVconsv1&62 Transgene RNA.

We develop candidate HIV-1 vaccines, of which two components, ChAdOx1.tHIVconsv1 (C1) and ChAdOx1.HIVconsv62 (C62), are delivered by the simian adenovirus-derived vaccine vector ChAdOx1. Aberrant adenovirus RNA splicing involving transgene(s) coding for the SARS-CoV-2 spike was suggested as an aetiology of rare adverse events temporarily associated with the initial deployment of adenovirus-vectored vaccines during the COVID-19 pandemic. Here, to eliminate this theoretically plausible splicing phenomenon from the list of possible pathomechanisms for our HIV-1 vaccine candidates, we directly sequenced mRNAs in C1- and C62-infected nonpermissive MRC-5 and A549 and permissive HEK293 human cell lines. Our two main observations in nonpermissive human cells, which are most similar to those which become infected after the intramuscular administration of vaccines into human volunteers, were that (i) the dominant adenovirus vector-derived mRNAs were the expected transcripts coding for the HIVconsvX immunogens and (ii) atypical splicing events within the synthetic open reading frame of the two transgenes are rare. We conclude that inadvertent RNA splicing is not a safety concern for the two tested candidate HIV-1 vaccines.

The type of SARS-CoV-2 vaccine does not affect ovarian function in assisted reproduction cycle

To assess whether vaccination or the type of vaccine against SARS-CoV-2 affects ovarian function in an assisted reproduction treatment. A retrospective and observational study. University-affiliated private invitro fertilization (IVF) center. Five hundred one patients who had received the complete vaccination schedule. Treatment before and after vaccination. Parameters for both reproductive outcomes and IVF results in patients vaccinated RESULT(S): We included 510 patients, distributed as follows: 13.5% (n = 69) received a viral vector vaccine, either the adenovirus serotype 26 vector vaccine (Ad26.CoV2.S; Janssen; n = 31) or the chimpanzee adenovirus vector vaccine (ChAdOx; AstraZeneca; n = 38). The remaining 86.5% (n = 441) received an messenger RNA vaccine from either Pfizer-BioNTech (n = 336) or Moderna (n = 105). Sample size for the unexposed women was n = 1190. No differences were found in any of the evaluated parameters for both reproductive outcomes and IVF results in patients vaccinated with any adenovirus or messenger RNA vaccine. When we compared the results after vaccination with different types of vaccines between the exposed and unexposed groups, and similar results were obtained in the days of stimulation or the doses of administered follicle stimulating hormone. Finally, the numbers of oocytes were as follows: Johnson & Johnson (9.2 ± 2.6), AstraZeneca (7.7 ± 1.2), Moderna (11.3 ± 1.8), Pfizer (12.6 ± 1.0), and the unvaccinated group (10.2 ± 1.5), P=0.057. These early results suggest no measurable detrimental effect on reproductive outcomes, regardless of the type of vaccine received.

ASPP2 Coordinates ERS-Mediated Autophagy and Apoptosis Through mTORC1 Pathway in Hepatocyte Injury Induced by TNF-α.

Background: Though ASPP2 plays an important role in regulating cell apoptosis and autophagy in case of liver injury, there remains a lack of clarity on the molecular mechanism of ASPP2 regulating autophagy and apoptosis. Methods: A hepatocyte injury model was constructed using HL7702 cell line and TNF-α. The cells were treated by ASPP2 overexpression adenovirus or short hairpin RNA lentivirus and endoplasmic reticulum stress (ERS) or the mammalian target of rapamycin (mTOR) inhibitor or agonist, respectively. The autophagy was detected by means of western blot and Green fluorescent protein-labeled- Microtubule-associated protein light chain 3 (GFP-LC3) plasmid transfection, while the apoptosis was detected through western blot, flow cytometry and TUNEL assay. Besides, the proteins related to ERS and mTOR were detected by western blot. Results: The low level of ASPP2 expression was accompanied by high-level autophagy and low-level apoptosis and vice versa in case of hepatocyte injury induce by TNF-α. By upregulating the proteins related to mTORC1 and ERS, ASPP2 induced apoptosis but inhibited autophagy. However, the effect of ASPP2 on autophagy and apoptosis can be reversed by the use of mTORC1 and ERS interfering agent, which indicates that ASPP2 regulated autophagy and apoptosis through mTORC1and ERS pathway. ERS treatment made no difference to the expression of ASPP2 and mTOR-related proteins, which suggests the possibility that the regulation of ERS on apoptosis and autophagy could occur in the downstream of ASPP2 and mTOR. Conclusion: ASPP2 could inhibit autophagy and induce apoptosis through mTORC1-ERS pathway in case of the hepatocyte injury induce by TNF-α. The role of ASPP2-mTORC1-ERS axis was verified in hepatocyte injury, which suggests the possibility that ASPP2 is an important regulatory molecule for the survival and death of hepatocyte.

Vaccines against COVID-19: Priority to mRNA-Based Formulations.

As of September 2021, twenty-one anti-COVID-19 vaccines have been approved in the world. Their utilization will expedite an end to the current pandemic. Besides the usual vaccine formats that include inactivated viruses (eight approved vaccines) and protein-based vaccines (four approved vaccines), three new formats have been validated: recombinant adenovirus (six approved vaccines), DNA (one approved vaccine), and messenger RNA (mRNA, two approved vaccines). The latter was the fastest (authorized in 2020 in the EU, the USA, and Switzerland). Most Western countries have reserved or use the protein vaccines, the adenovirus vaccines, and mRNA vaccines. I describe here the different vaccine formats in the context of COVID-19, detail the three formats that are chiefly reserved or used in Europe, Canada, and the USA, and discuss why the mRNA vaccines appear to be the superior format.

Olig2 knockdown alleviates hypoxic-ischemic brain damage in newborn rats.

Neuronal damage is an important pathological mechanism in neonatal hypoxic-ischemic brain damage (HIBD). We found in our previous studies that oligodendrocyte transcription factor 2 (Olig2) downregulation was able to increase cell survival in the brain. However, the specific mechanism has yet to be clarified. Sprague-Dawley rats aged 3 d were randomly divided into three groups: the normal control group, the Olig2-RNAi group, and the RNAi-negative control group. The normal control group received no treatment, the Olig2-RNAi group received the Olig2 RNAi adenovirus, and the RNAi-negative control group was given the control adenovirus after the completion of the HIBD model. Infarct lesions and their volumes were observed by triphenyltetrazolium chloride (TTC) staining 3 d after the completion of the adenovirus local injection. The condition of the tissue was characterized by hematoxylin-eosin staining 7 d after the model was established, and cell viability was determined by azure methylene blue staining. Subcellular damage was analyzed by transmission electron microscopy. Rotarod analysis was performed to detect moving behavior ability and an MWM assay was conducted to evaluate the memory. TTC staining showed a smaller brain injury area in the Olig2-RNAi group than in the RNAi-negative control group. Hematoxylin-eosin staining indicated the presence of severe cell injury in the hippocampal region after HIBD, which improved after Olig2 knockdown. Azure methylene blue staining and electron microscopy results suggested that the cells improved after Olig2 knockdown. The rats stayed longer on the rotating rod, and their latency in the water maze test was gradually shortened relative to that of the rats in the Olig2-RNAi negative control group. Olig2 knockdown can promote the repair of hypoxic-ischemic brain damage in newborn rats.

Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.

PDCD4 deficiency ameliorates left ventricular remodeling and insulin resistance in a rat model of type 2 diabetic cardiomyopathy.

ObjectiveDiabetic cardiomyopathy (DCM) is characterized by cardiac remodeling, dysfunction, and insulin resistance; however, the underlying mechanism has not been fully elucidated. Programmed cell death 4 (PDCD4) is a novel inflammation and apoptosis gene, but its role in type 2 DCM remains elusive. We aimed to determine if PDCD4 intervention improves DCM by affecting left ventricular remodeling, function, and insulin resistance.Research design and methodsWe designed a PDCD4-/- rat, established a type 2 diabetes animal model, and constructed a PDCD4 overexpressed adenovirus and PDCD4 small interfer RNA (siRNA) vectors to alter PDCD4 expression in H9c2 cardiomyocytes. Thereafter, glucose levels, lipid metabolism, echocardiography, and extent of myocardial fibrosis, inflammation, and apoptosis were compared in vivo and in vitro.ResultsPDCD4 deficiency improved insulin resistance, cardiac remodeling, and dysfunction in type 2 DCM rats and improved myocardial hypertrophy, fibrosis, inflammation, and apoptosis. Proliferation and transformation of cardiac fibroblasts was reduced via PDCD4 downregulation in vitro under high-glucose stimulation. Furthermore, PDCD4 regulated the myocardial phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) pathway in vivo and in vitro. PDCD4 intervention affected cardiac remodeling, dysfunction, and insulin resistance by influencing fibrosis, inflammation, and apoptosis via the PI3K-AKT pathway in vivo.ConclusionsPDCD4 knockdown protected against left ventricular remodeling, dysfunction, and insulin resistance in type 2 DCM rats. The underlying mechanisms may involve reducing cardiomyocyte apoptosis, inflammation, fibrosis, and normalized PI3K-AKT phosphorylation. To the best of our knowledge, our study is the first to report the effects and underlying mechanisms of PDCD4 in type 2 DCM. These results provide a potential new treatment avenue for improving the prognosis of patients with type 2 DCM.

293-LB: The Suppression of Xanthine Oxidoreductase (XOR) Improves Skeletal Muscle Insulin Resistance

Hyperuricemia is closely associated with insulin resistance. Xanthine oxidoreductase (XOR), a key enzyme known to catalyze purines to uric acid, is expressed in ubiquitous tissues. In addition, XOR expression is upregulated in obesity. Therefore, we speculated that XOR may exacerbate insulin resistance. But, the effects of XOR on insulin sensitivity have not been fully elucidated. Febuxostat is a selective XOR inhibitor. In this study we investigated the effect of suppression of XOR using febuxostat on insulin sensitivity in vivo and in vitro. Male Wistar rats were fed with 60% high fat diet containing with either febuxostat or not, for 4 weeks. Euglycemic-hyperinsulinemic clamp studies were performed after an 8-h fast. Differentiated C2C12 cells were incubated with 0 or 10 μM febuxostat. For Xor silencing experiments, we used the Xor RNAi adenovirus. The glucose infusion rate and insulin stimulated-glucose disposal rate were significantly increased by 12% (P<0.05) and 17% (P<0.05), respectively, in febuxostat fed rats. But the clamp hepatic glucose output was no significant change between two groups. Consistent with the clamp data, the insulin-stimulated phosphorylation of Akt and AMPK were significantly increased by 90% (P<0.05) and 53% (P<0.05), respectively, in skeletal muscle of febuxostat fed rats. In differentiated C2C12 cells, febuxostat treatment significantly increased the insulin-stimulated phosphorylation of Akt and AMPK by 65% (P<0.05) and 43% (P<0.05), respectively. Furthermore, knockdown of XOR with XOR RNAi adenovirus significantly increased the insulin-stimulated phosphorylation of Akt by 60% (P<0.05). These results suggest that the suppression of XOR in skeletal muscle improves the skeletal muscle insulin resistance. In conclusion, our findings demonstrated that suppression of XOR had beneficial effects on skeletal muscle insulin sensitivity in an insulin resistant state. The upregulation of XOR in skeletal muscle might cause the insulin resistance. Disclosure C.T. Moriya: None. H. Satoh: None. S. Kakehi: None. H. Watada: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kissei Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Pfizer Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. Other Relationship; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Kowa Pharmaceutical Europe Co. Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Sanwa Chemical Industry Co. Ltd., Takeda Pharmaceutical Company Limited.

Efficient Generation of an Fah/Rag2 Dual-Gene Knockout Porcine Cell Line Using CRISPR/Cas9 and Adenovirus.

The shortage of human hepatocytes continues to be a significant limitation for the widespread application of hepatocyte transplantation and bioartificial liver (BAL) support therapy. Recombinant activation gene 2 (Rag2) and fumarylacetoacetate hydrolase (Fah)-deficient mice could be highly repopulated with human hepatocytes. However, Fah/Rag2-deficient mice can only produce up to 1 × 108 human hepatocytes per mouse. We hypothesized that 2-10 × 1010 human hepatocytes can be produced per Fah/Rag2-deficient pig, which is an adequate supply for hepatocyte transplantation and BAL therapy. In a novel approach, we used stably transfected Cas9 cells and single-guide RNA adenoviruses containing fluorescent reporters to enrich porcine cells with Fah/Rag2 dual gene mutations. This resulted in the construction of Fah/Rag2 double knockout porcine iliac artery endothelial cells, which were subsequently used for generating Fah/Rag2-deficient pigs.

LOXL2, a copper-dependent monoamine oxidase, activates lung fibroblasts through the TGF-β/Smad pathway.

A previous study demonstrated that Lysyl oxidase‑like2 (LOXL2) serves an essential role in matrix remodeling and fibrogenesis, thus indicating its involvement in fibrosis‑associated diseases. Our previous studies revealed a novel association between LOXL2 expression and pulmonary fibrosis in mice. However, the exact role and mechanisms of LOXL2 in interstitial lung disease remain poorly understood. The present study aimed to detect LOXL2 expression in mice with bleomycin (BLM)‑induced pulmonary fibrosis, and explore the effects of silencing LOXL2 on the proliferation, activation and fibrosis process of mouse lung fibroblasts(MLFs). In addition, the present study investigated the association between LOXL2 and the transforming growth factor-β (TGF‑β)/Smad signaling pathway to identify the mechanism underlying the role of LOXL2 in fibrosis progression. An animal model of pulmonary fibrosis was established by administering an intratracheal injection of 5mg/kg BLM to C57BL/6 mice. ELISA and immunohistochemical examination were used to detect the LOXL2 level in the serum, lung homogenate and pulmonary tissues in mice. Pulmonary tissues of mice were extracted to culture primary MLFs, and a LOXL2 small interfering RNA adenovirus vector was established to silence LOXL2 in MLFs. Cell proliferation was detected using the cell counting kit‑8 assay. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to measure the expression of LOXL2, TGF‑β1, Smad2/3, phosphorylated(p)Smad2/3, Smad4, and Smad7 and Snail in cells. Interleukin‑6(IL‑6) and type1 collagenα1 (COL1A1) in the supernatant of cells were analyzed by ELISA. It was demonstrated that LOXL2 expression was significantly increased in serum, lung homogenate and pulmonary tissues of mice with BLM‑induced pulmonary fibrosis compared with control mice. Furthermore, silencing LOXL2 significantly decreased MLF proliferation, and the levels of IL‑6 and COL1A1 in the supernatant of cells. Furthermore, silencing LOXL2 inhibited the expression of pSmad2/3, Smad4 and Snail, while it promoted Smad7 expression. The present data provides a comprehensive analysis of the LOXL2 in pulmonary fibrosis and indicates prominent roles for LOXL2 in fibrogenesis via regulation of the TGF‑β/Smad signaling pathway.

Effect of heme oxygenase-1 on expressions of hypoxia inducing factor 1 alpha and vascular endothelial growth factor 1 alpha after orthotopic liver transplantation ischemia-reperfusion injury in rats

Objective To explore the effect of heme oxygenase-1 (HO-1)on expressions of hypoxia inducing factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF)and regeneration of hepatic vascular plexus after orthotopic liver transplantation ischemia-reperfusion injury in rats. Methods The experimental study was conducted. According to the random number table, 240 SD rats were divided into the 3 groups, 80 rats in each group. Empty virus group: rats were transfected with the empty virus. Induced group: rats were transfected with HO-1 overexpression adenovirus. Inhibited group: rats were transfected with HO-1 RNAi adenovirus. Rats were made pairs (1: 1) and established rat liver transplantation model according to two cuffs method . Rats with less weight and with heavier weight were respectively chosen as donor rats and recipient rats, and then recieved tail intravenous injection of adenovirus at 24 hours before operation. (1) Detection of transfection efficiency of adenovirus before operation: HO-1 expression of liver tissue of rats in each group was detected by Western blot at 12 and 24 hours after injection of adenovirus. (2) Liver function test of recipient rats after liver transplantation: liver functions of recipient rats [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT)] were detected at 1, 3, 7 and 14 days postoperatively. (3) Pathological histology of liver tissue and injury scores of recipient rats in the 3 groups after liver transplantation: paraffin sections of recipient rats in the 3 groups at postoperative 1 and 14 days were stained by HE staining and observed by light microscope, and were evaluated by Suzuki damage score standard. (4) Relative expressions of HIF-1α, VEGF and HO-1 in liver tissue of recipient rats were detected by Western blot. (5) Von Willebrand factor (vWF) in liver tissue of recipient rats at 14 days postoperatively was detected by immunofluorescence staining and small vessels were counted. Measurement data with normal distribution were represented as ±s. Comparison between groups was analyzed by the independent-sample t test, comparison among groups was done using one-way ANOVA, and pairwise comparison was analyzed by the LSD test. Results (1) Detection of transfection efficiency of adenovirus before operation: the relative expression of HO-1 of liver tissue of rats at 12 and 24 hours preoperatively after injection of adenovirus was 1.08±0.16 and 1.08±0.26 in the empty virus group, 1.18±0.21 and 1.39±0.19 in the induced group, 0.87±0.26 and 0.57±0.12 in the inhibited group, respectively, with statistically significant differences in different time points (F=4.232, 36.513, P 0.05). Suzuki score at 14 day postoperatively in the empty virus group, induced group and inhibited group were respectively 4.0±0.8, 2.9±0.8 and 5.1±1.4, with a statistically significant difference (F=9.776, P 0.05). The relative expressions of VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 1 day postoperatively were 1.21±0.25, 0.55±0.12 in the empty virus group and 2.13±0.40, 0.72±0.12 in the induced group and 0.91±0.22, 0.26±0.07 in the inhibited group, respectively, with statistically significant differences (F=35.158, 39.082, P<0.05). The relative expressions of HIF-1α, VEGF (43 KD), VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 7 days postoperatively were 0.49±0.22, 0.46±0.13, 0.98±0.37, 0.98±0.37 in the empty virus group and 0.83±0.26, 0.63±0.19, 1.60±0.33, 1.49±0.46 in the induced group and 0.24±0.09, 0.30±0.12, 0.64±0.18, 0.75±0.26 in the inhibited group, respectively, with statistically significant differences (F=16.853, 10.021, 20.756, 8.156, P<0.05). (5) Immunofluorescence staining results: number of small vessels at 14 days postoperatively in the empty virus group, induced group and inhibited group was respectively 7.9±2.0, 10.6±1.9 and 7.6 ±1.9, with a statistically significant difference (F=5.921, P<0.05). Conclusion HO-1 could promote expressions of HIF-1α and VEGF in liver tissue after liver transplantation ischemia-reperfusion injury and regeneration of intrahepatic vascular plexus, and it also alleviate bile duct ischemia-reperfusion injury after liver transplantation. Key words: Ischemia-reperfusion injury; Liver transplantation; Heme oxygenase-1; Revascularization

A novel multi-target RNAi adenovirus inhibits hepatoma cell proliferation, migration, and induction of angiogenesis.

The pathogenesis of hepatocellular carcinoma (HCC) is a multi-step process involving many genes. Consequently, single gene targeting therapy has limited efficacy, making combination therapy targeting multiple genes a necessity. Based on our previous findings, we constructed a single vector mediating simultaneous expression of multiple short hairpin RNAs (shRNAs) against human vascular endothelial growth factor receptor 2 (VEGFR2), chemokine C-C motif receptor 1 (CCR1), and epithelial cell adhesion molecule (EpCAM), three genes closely related to HCC progression that act through separate pathways. The shRNA vector efficiently downregulated the mRNA and protein of all three molecules in Huh7 hepatoma cells. The vector also inhibited cell proliferation and migration and reduced angiogenesis. Furthermore, this shRNA vector can be recombined into adenovirus, a gene therapy vector, for better in vivo application. It thus offers a potentially effective future gene therapy approach to treating human liver cancer.

Antiviral activity of an aqueous extract derived from Aloe arborescens Mill. against a broad panel of viruses causing infections of the upper respiratory tract

BackgroundA number of antiviral therapies have evolved that may be effectively administered to treat respiratory viral diseases. But these therapies are very often of limited efficacy or have severe side effects. Therefore there is great interest in developing new efficacious and safe antiviral compounds e.g. based on the identification of compounds of herbal origin. HypothesisSince an aqueous extract of Aloe arborescens Mill. shows antiviral activity against viruses causing infections of the upper respiratory tract in vitro we hypothesised that a product containing it such as Biaron C® could have an antiviral activity too. Study designAntiviral activity of Bioaron C®, an herbal medicinal product consisting of an aqueous extract of Aloe arborescens Mill., Vitamin C, and Aronia melanocarpa Elliot. succus, added as an excipient, was tested in vitro against a broad panel of viruses involved in upper respiratory tract infections. MethodsThese studies included human adenovirus and several RNA viruses and were performed either with plaque reduction assays or with tests for the detection of a virus-caused cytopathic effect. ResultsOur studies demonstrated an impressive activity of Bioaron C® against members of the orthomyxoviridae – influenza A and influenza B viruses. Replication of both analysed influenza A virus strains – H1N1 and H3N2 – as well as replication of two analysed influenza B viruses – strains Yamagatal and Beiying – was significantly reduced after addition of Bioaron C® to the infected cell cultures. In contrast antiviral activity of Bioaron C® against other RNA viruses showed a heterogeneous pattern. Bioaron C® inhibited the replication of human rhinovirus and coxsackievirus, both viruses belonging to the family of picornaviridae and both representing non-enveloped RNA viruses. In vitro infections with respiratory syncytial virus and parainfluenza virus, both belonging to the paramyxoviridae, were only poorly blocked by the test substance. No antiviral activity of Bioaron C® was detected against adenovirus – a non-enveloped DNA virus. ConclusionsThese results represent the first proof of a selective antiviral activity of Bioaron C® against influenza viruses and create basis for further analyses of type and molecular mechanisms of the antiviral activity of this herbal medicine.

Gap Junction-associated Na+ Influx Is Involved in the Mediation of Ca2+ Transients in Neonatal Rat Ventricular Myocytes.

Gap junction (GJ) coupling is involved in the regulation of spontaneous Ca(2+) transients in neonatal rat ventricular myocytes (NRVMs); however, the underlying mechanism(s) remains incompletely clear. In this study, we investigated Na influx mediated by Cx43-associated GJ modulating the spontaneous NRVM excitation. Intracellular Na(+) and Ca(2+) concentrations ([Na(+)](i) and [Ca(2+)](i)) were assessed using CoroNa Green and Fluo-4 fluorescent probes, respectively, by confocal microscopy. GJ function was evaluated by measuring fluorescence recovery after photobleaching. The results showed that [Na(+)](i) and spontaneous Ca(2+) oscillating were significantly decreased by the treatment of NRVMs with GJ uncouplers or adenovirus-mediated Cx43 gene knockdown using short hairpin RNA, while both of the intracellular cation levels were increased by adenovirus-mediated Cx43 overexpression. Neither Na(+) channel blocker (tetrodotoxin) nor the GJ uncoupler (Gap27) or Cx43-short hairpin RNA adenovirus could completely suppress the Ca(2+) transients, but their combination usage could. In addition, Ca(2+) transients disappeared in NRVMs incubated in Na(+)-free, but not in Ca(2+) -free medium, in which the remained transients could be further abolished by Gap27. Collectively, our findings suggest that Cx43-associated GJ function is involved in the regulation of Na(+) influx into cardiomyocytes, which contributes, at least in part, to triggering spontaneous excitation and regulation of cardiomyocyte automaticity.

Effect of alteration of caveolin-1 expression on doxorubicin-induced apoptosis in H9c2 cardiac cells.

Doxorubicin is an anthracycline antibiotic widely used in cancer treatment. Although its antitumor efficacy appears to be dose dependent, its clinical use is greatly restricted by the development of cardiotoxicity associated with apoptosis. Although caveolin-1, the major structural protein in caveolae, can positively or negatively regulate apoptosis depending on the stimulus or cell types, the contribution of caveolin-1 to doxorubicin-induced apoptosis remains unknown. This study was performed to identify the regulatory role of caveolin-1 on doxorubicin-induced apoptosis in H9c2 cardiac cells using a genetic approach. Caveolin-1 knockdown with a short hairpin (sh) RNA adenovirus, but not overexpression of wild-type caveolin-1, resulted in a marked inhibition of doxorubicin-induced caspase-3 cleavage. However, caveolin-1 knockdown tended to protect against doxorubicin-induced decrease in cell viability, but it did not significantly reverse cell death induced by doxorubicin. Doxorubicin stimulated the phosphorylation of p38 and extracellular signal regulated kinase (ERK). Doxorubicin-induced caspase-3 cleavage was inhibited by U0126, a MEK inhibitor or SB203580, a p38 inhibitor. Caveolin-1 knockdown markedly inhibited doxorubicin-induced p-38 phosphorylation but not ERK-mediated p-53 phosphorylation in H9c2 cardiac cells. Our results suggest that reduced caveolin-1 expression plays an anti-apoptotic role in doxorubicin-induced apoptosis but that it is insufficient to prevent such an apoptosis in H9c2 cardiac cells.

Sustained local delivery of oncolytic short hairpin RNA adenoviruses for treatment of head and neck cancer.

Oncolytic adenovirus (Ad)-mediated gene therapy is a promising approach for suppression of primary tumors. Therapeutic efficacy of Ad-mediated gene therapy has been limited by immunogenicity, rapid dissemination of viral progenies into systemic circulation and short duration of biological activity. Polymeric sustained local delivery can overcome many of these challenges to produce a viable therapy with improved outcomes. Silk-elastinlike protein polymer (SELP) hydrogels were used for matrix-mediated delivery of oncolytic Ad, containing short hairpin RNA (shRNA) targeted to C-Met (sh-C-Met), to solid tumors in a nude mouse model of human head and neck cancer. The biological activity of Ad released from SELP hydrogels was examined as a function of time to investigate protective effects on viral activity. Antitumor efficacy and viral distribution were investigated for 3 weeks in tumor-bearing mice. The encapsulation of Ad with SELP hydrogels sustained biological activity longer than Ad alone. Ad in SELP matrix showed 1.5-fold greater antitumor efficacy compared to that of naked Ad in human xenograft tumor models. Histological analysis demonstrated that treatment with Ad in a SELP matrix resulted in apoptosis in a wider area of tumor tissue and higher density of Ad infection compared to Ad administered alone. Matrix-mediated delivery of Ad-containing shRNA with SELP hydrogels enhances therapeutic efficacy by tumor-selective infection, spatiotemporal control and preservation of biologic activity.

Tafazzin knockdown interrupts cell cycle progression in cultured neonatal ventricular fibroblasts

Mutation of the mitochondrial protein tafazzin causes dilated cardiomyopathy in Barth syndrome. Previous studies have shown that tafazzin knockdown promotes hypertrophy of neonatal cardiac myocytes. The current investigation was designed to show whether tafazzin knockdown affects cardiac fibroblast proliferation and collagen secretion, which contribute to fibrosis in dilated cardiomyopathy. In primary cultures of neonatal ventricular fibroblasts (NVFs) transduced with a tafazzin short hairpin RNA adenovirus, tafazzin knockdown increased production of reactive oxygen species and activation of mitogen-activated protein kinases and induced protein and DNA synthesis via cell cycle regulators. It also reduced intracellular ATP, activated AMPK, and caused multinucleation, hypertrophy, and enhanced collagen secretion. We concluded that tafazzin knockdown interrupts the NVF cell cycle and this in turn may contribute to fibrosis and dilated cardiomyopathy in Barth syndrome.

Effects of interleukin-6 in wound healing of human biliary epithelial cells

Objective To investigate the mechanism of interlekin-6 (IL-6) in wound healing of human biliary epithelial cells ( BECs ).Methods BECs were cultured in IL-6 at different concentrations:0 ng/L(0 ng/L group),10 ng/L (10 ng/L group),50 ng/L (50 ng/L group),100 ng/L (100 ng/L group),1000 ng/L ( 1000 ng/L group).The effects of IL-6 on the phosphorylation of signal transducer and activator of transcription 3( STAT3 ) and the expression of trefoil family factors 3 (TFF3) were detected.BECs were divided into untreated group,STAT3-RNAi group (BECs transfected with STAT3 RNAi adenovirus) and Control-RNAi group (BECs transfected with vacant RNAi adenovirus).The effects of IL-6 on the expression of TFF3 were detected after RNAi of STAT3.In vitro wound models were constructed for the untreated group,STAT3-RNAi group and Control-RNAi group,and the effects of IL-6 and TFF3 on BECs of the 3 groups were detected.All data were analyzed by using the Student's t test,analysis of variance or Sidak test.Results The expressions of phosphorylated STAT3 in the 50 ng/L group,100 ng/L group and 1000 ng/L group were 0.240 ± 0.052,0.714 ± 0.124,0.327 ± 0.069,respectively,which were significantly higher than 0.033 ± 0.011 of the 0 ng/L group (q =5.246,17.260,7.451,P ＜ 0.05 ).The contents of mRNA and protein of TFF3 increased as the increase of IL-6 concentration (q =12.045,9.889,P ＜ 0.05).After RNAi of STAT3 of the BECs,the expression of TFF3 decreased when the concentration of IL-6 was 1000 ng/L.The expression of TFF3 of the STAT3-RNAi group was 0.037 ± 0.005,which was significantly lower than 0.267 ± 0.038 of the Control-RNAi group and 0.301 ± 0.042 in the untreated group ( q =12.135,13.929,P ＜ 0.05 ).In the in vitro wound model,the speed of BECs migration in the STAT3-RNAi group was (9.1 ± 1.5 ) μm/h,which was slower than (25.1 ± 3.8 ) μm/h of the Control-RNAi group after 12 hours of interference with IL-6 (q =7.737,P ＜ 0.05 ).The speed of BECs migration of STAT3-RNAi group was (39.2 ± 4.7) μm/h after adding 1 g/L of recombinant TFF,which was significantly faster than that of the Control-RNAi group (q =14.507,P ＜0.05).Conclusion IL-6 promotes cell migration and wound healing by activating STAT3 and up-regulating TFF3 expression. Key words: Biliary epithelial cells; Wound healing; Interleukin-6; Signal transducer and activator of transcription 3; Trefoil family factor 3

Expression and clinical significance of HMGB1 in human liver cancer: Knockdown inhibits tumor growth and metastasis in vitro and in vivo

The high-mobility group box 1 (HMGB1) signaling pathway plays a crucial role in tumorigenesis and progression of many malignant cancers. The present study aimed to investigate the expression and clinical significance of HMGB1 in human primary liver cancer, and further explore the molecular mechanisms of HMGB1 in tumor growth and metastasis. Forty cases of human liver cancer and normal liver tissues were collected. The expression of HMGB1 was assessed using RT-PCR and western blot assays in biopsy samples. The HMGB1 pathway invitro was blocked using transfection of the recombinant small hairpin RNA adenovirus vector rAd5-HMGB1 into the human liver cancer cell line SMMC-7721. The expression of HMGB1, phosphorylated AKT (p-AKT), Ki-67 and matrix metallopeptidase-2 (MMP-2) was detected by Real-PCR and western blot assays. Cell proliferative activities and metastatic capability were determined by MTT and Transwell assays. Cell cycle distribution and apoptosis were detected by flow cytometry. A subcutaneous xenograft tumor model was established, validating the effects of rAd5-HMGB1 on tumor growth invivo. As a consequence, HMGB1 was found to be highly expressed in liver cancer compared with normal tissues, and was positively associated with pathological grade and distant metastases of liver cancer. Knockdown of HMGB1 downregulated the expression of p-AKT, Ki-67 and MMP-2, inhibited the proliferative activities and metastatic potential of SMMC-7721 cells, induced cell cycle arrest and apoptosis, and slowed the growth of xenograft tumors. Altogether, the expression of HMGB1 is closely correlated with pathological grade and distant metastases of liver cancer, and knockdown of HMGB1 inhibits liver cancer growth and metastasis, suggesting that HMGB1 may be involved in liver cancer development and progression through AKT-mediated regulation of Ki-67 and MMP-2 expression, and represent a potential therapeutic target for this aggressive malignancy.