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Background: Colorectal cancer (CRC) is the third most common type of cancer worldwide, and distant metastasis is frequently noted at diagnosis or follow-up. Notably, some patients with CRC can present with distant organ metastasis without any nodal involvement, which was defined as direct distant organ metastasis (DDOM). In this study, we evaluated the prognostic significance of DDOM for patients with CRC., Methods: This study included 325 patients who had undergone primary colorectal cancer resection between August 2008 and December 2021. The patients with and without DDOM were compared (Kaplan-Meier analysis) in terms of overall survival (OS) and time to recurrence. Furthermore, the patients' clinicopathological risk factors and protective factors were analyzed (multivariate Cox proportional hazards model)., Results: Of the 325 patients, 65 (20%) had DDOM (Direct + group) and 260 (80%) did not (Direct - group). The Kaplan-Meier analysis revealed that OS was significantly better in the Direct + group than in the Direct - group (p < 0.01). A subgroup analysis by CRC stage was performed; for the patients with non-stage-IV CRC, the rate of OS was significantly higher in the Direct + group than in the Direct - group (p = 0.02). However, DDOM did not affect the OS of the patients with stage IV CRC. The multivariate analysis indicated DDOM, left colon tumor location, and postoperative adjuvant chemotherapy were significant protective factors for disease-related mortality in the patients with non-stage-IV CRC; by contrast, body mass index, curative resection, and postoperative adjuvant chemotherapy were identified to be significant protective factors in the patients with stage IV CRC., Conclusions: DDOM appears to be significantly associated with improved OS in patients with non-stage-IV CRC but not in those with stage IV CRC. Furthermore, the time to cancer recurrence may not vary significantly between patients with DDOM and those without it., Competing Interests: Declaration of competing interest Drs. Ching-Heng Hsiao, Yen-Liang Li, Kee-Thai Kiu, Min-Hsuan Yen, and Tung-Cheng Chang declare no conflicts of interest or financial ties., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The field of RNA therapeutics has been emerging as the third milestone in pharmaceutical drug development. RNA nanoparticles have displayed motile and deformable properties to allow for high tumor accumulation with undetectable healthy organ accumulation. Therefore, RNA nanoparticles have the potential to serve as potent drug delivery vehicles with strong anti-cancer responses. Herein, we report the physicochemical basis for the rational design of a branched RNA four-way junction (4WJ) nanoparticle that results in advantageous high-thermostability and -drug payload for cancer therapy, including metastatic tumors in the lung. The 4WJ nanostructure displayed versatility through functionalization with an anti-cancer chemical drug, SN38, for the treatment of two different cancer models including colorectal cancer xenograft and orthotopic lung metastases of colon cancer. The resulting 4WJ RNA drug complex spontaneously targeted cancers effectively for cancer inhibition with and without ligands. The 4WJ displayed fast renal excretion, rapid body clearance, and little organ accumulation with undetectable toxicity and immunogenicity. The safety parameters were documented by organ histology, blood biochemistry, and pathological analysis. The highly efficient cancer inhibition, undetectable drug toxicity, and favorable Chemical, Manufacturing, and Control (CMC) production of RNA nanoparticles document a candidate with high potential for translation in cancer therapy., Competing Interests: Declaration of competing interest P.G. has patents P2024-136-8471 and P2022-263-8315 pending to The Ohio State University. P.G. is the consultant, licensor, and grantee of Oxford Nanopore Technologies; as well as the cofounder and consultant of ExonanoRNA, LLC. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Diabetic cardiomyopathy is a common complication of diabetes, resulting in cardiac hypertrophy and heart failure associated with excessive reactive oxygen species and mitochondria-mediated apoptosis generation. Mitogen-activated protein kinase-c-Jun N-terminal kinase (MAPK-JNK), regulated by microRNA (miR)-210, affects mitochondrial function and is activated by advanced glycation end-products (AGE) in cardiac cells. Diallyl trisulfide (DATS), an antioxidant in garlic oil, inhibits stress-induced cardiac apoptosis. This study examined whether DATS enhances miR-210 expression to attenuate cardiac apoptosis. We investigated the DATS-mediated attenuation mechanism of AGE-enhanced cardiac apoptosis by modulating miR-210 and its upstream transcriptional regulator, FoxO3a. We found FoxO3a binding sites in the miR-210 promoter region. Our results indicated that DATS treatment inhibited AGE-induced JNK activation, phosphoprotein c-Jun nuclear transactivation, and cardiac apoptosis and reversed the AGE-induced reduction in cardiac miR-210 levels. The luciferase activity after DATS treatment was significantly lower than that of the control and was reversed following AGE treatment. We also showed that FoxO3a, upregulated by DATS treatment, may bind to the miR-210 promoter to enhance its expression and downregulates JNK expression to attenuate AGE-induced cardiac apoptosis. Oral administration of DATS enhanced FoxO3a expression in the heart and reduced diabetes-induced heart apoptosis. Our findings indicate that DATS mediates AGE-induced cardiac cell apoptosis attenuation by promoting FoxO3a nuclear transactivation to enhance miR-210 expression and regulate JNK activation. Our results suggest that DATS can be used as a cardioprotective agent, and miR-210 is a critical regulator in inhibiting diabetic cardiomyopathy., Competing Interests: Declaration of competing interests The authors declare no competing financial interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Enzyme-labeled secondary antibody is often used to amplify the output signal in the process of antibody detection. However, its preparation process is complex and time-consuming. Herein, we fabricated an innovative hydrophilic rhodamine B-loaded / boronic acid-modified graphene oxide (HRBGO) nanocomposite, used as a substitute of enzyme-labeled second antibody. The synthetic HRBGO was loaded with generous rhodamine B and modified with boronic acid. Therefore, the HRBGO could selectively label the carbohydrate chains of Fc fragment of primary antibody through specific boronate affinity recognition, and then perform signal output and amplification by releasing rhodamine B. To verify the practicability of HRBGO, trastuzumab as a humanized monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2) was selected as model antibody. A glycosylation site-blocked / HER2-immobilized magnetic nanoparticles (GHMN) was also prepared for selectively capturing trastuzumab from complex samples via specific immunoaffinity. Because the glycosylation sites of HER2 can also be labeled with the HRBGO by boronate affinity recognition, these sites were blocked by a masking agent to minimize the background signal. For specific and ultrasensitive detection of trastuzumab, the integration of GHMN and HRBGO was proposed and optimized in detail. Trastuzumab detection based on HRBGO consisted of three steps: specific capture, selective labeling, and output signal. The proposed strategy provided ultrahigh sensitivity with limit of detection of 0.35 fg mL -1 and was successfully applied in the detection of trastuzumab in spiked serum sample with recovery and relative standard deviation in the range of 98.7-103.8% and 3.8-6.0%, respectively. To assess universal applicability, the HRBGO was also successfully used for the determination of anti-SARS-COV2 RBD antibody in human serum sample., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mitochondria are essential for hepatitis B virus (HBV) infection. Moreover, the findings of our previous study indicate that host mitochondrial genetic factors are associated with chronic hepatitis B (CHB) for the Han Chinese. However, in terms of genetic heterogeneity, the impact of mitochondria on host susceptibility to HBV infection in ethnic minorities in China remains unclear. Here, a total of 7070 subjects who had visited the hospital between June 1, 2019, and April 31, 2020, were enrolled for seroprevalence of HBV infection investigation. A total of 220 individuals with CHB (CHBs) and 223 individuals with a trace of HBV infection (spontaneously recovered subjects, SRs) were analyzed for mitochondrial DNA (mtDNA) sequence variations and classified into respective haplogroups. Haplogroup frequencies were compared between CHBs and SRs. Among eight nationalities, Yi nationality patients had the highest HBsAg prevalence rate (27.9% [95% CI: 25.3%-30.5%]) and the lowest vaccination rate (4.9% [95% CI: 3.7%-6.2%]). After adjustment for age and gender, haplogroup F was a risk factor for CHB infection (P = 0.049, OR = 2.079, 95% CI = 1.002-4.31), while D4 had a significant negative correlation with the HBeAg-positive rate (P = 0.024, OR = 0.215, 95% CI = 0.057-0.816). Together with our previous study, the findings indicate that different nationalities have different genetic susceptibility to HBV infection., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: The fibroblast growth factor 21 (FGF21)-adiponectin axis participates in energy hemostasis and obesity-related syndrome. The present study aimed to investigate whether concentrated fish oil (FO) intervention could alleviate non-alcoholic fatty liver disease (NAFLD) via the regulation of the FGF21-adiponectin axis., Methods: In a randomized controlled trial, 61 patients with NAFLD, age 55.9 ± 15.6 y, were randomly divided into two groups: FO (3 g/d; n = 30) and corn oil (CO; 3 g/d; n = 31), which served as the control group., Results: After a 3-mo intervention, there were significant net reductions in serum alanine transaminase (-5.4 ± 14.5 U/L vs. -0.25 ± 4.70 U/L; P = 0.001) and triacylglycerol (-0.70 ± 1.10 mmol/L vs. 0.11 ± 1.04 mmol/L; P = 0.018) levels in the FO group compared with the CO group. Furthermore, the mean changes of FGF21 levels (-16.3 ± 20.1 pg/mL vs. 7.2 ± 32.9 pg/mL; P = 0.002) were significantly decreased, but adiponectin levels (1.14 ± 1.53 μg/mL vs. -0.42 ± 2.04 pg/mL; P = 0.011) were significantly increased in the FO group compared with the CO group. In the animal study, the mice fed the high-fat diet demonstrated characteristics of NAFLD. The administration of FO significantly improved high-fat diet-induced hepatic steatosis, insulin resistance, and inflammation compared with the high-fat control group. In addition, FO improved the sensitivity of FGF21, and stimulated the expression levels of adiponectin in the liver., Conclusions: The present study suggested that FO could potentially ameliorate NAFLD through mediating the FGF21-adiponectin axis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tolvaptan has been approved for the treatment of autosomal dominant polycystic kidney disease and heart failure. However, the role of tolvaptan in patients with abdominal aortic aneurysm (AAA) has not been examined. Human aortic smooth muscle cells (HASMCs) were used as the in vitro model. Via Ang II infusion, experimental AAAs were induced in Apo-E knockout mice. In vitro study showed that tolvaptan suppressed matrix metalloproteinase (MMP) expressions (MMP-2 and MMP-9) and apoptosis in Ang II-stimulated HASMCs. In the Apo-E knockout mice with Ang II-induced AAA, the animals exhibited AAA formation with elastic lamina degradation, dilatation of the suprarenal aorta, increased macrophage infiltration and higher expressions of MMPs. Treatment with a high dose of tolvaptan prevented experimental AAA formation while preserving the elastic lamina structure, reducing inflammatory macrophages, and inhibiting gelatinolytic activity, MMP expressions and apoptosis of SMCs in aorta tissue. Specifically, tolvaptan reduced the expression of receptor-interacting protein kinase 3 (RIP3) and decreased apoptosis of SMCs. Our data demonstrated that tolvaptan reduces experimental AAA formation and dissection by inhibiting destruction of the aortic structure integrity and reducing inflammatory macrophage infiltration, MMP-2 and MMP-9 expressions, and apoptosis of vascular SMCs, indicating tolvaptan may have therapeutic potential for AAA and dissection., (Copyright © 2022. Published by Elsevier Inc.)

Background: The association of obstructive sleep apnea (OSA) with bradycardia is not well-characterized, which may confer significant morbidity and mortality if left untreated. We sought to clarify the prevalence of comorbid OSA and bradycardia, and the effect of continuous positive airway pressure (CPAP) therapy on bradycardia outcomes., Methods: We systematically searched four electronic databases (PubMed, Embase, Cochrane Library, Scopus) for randomized or observational studies reporting the co-prevalence of sleep apnea and bradycardia or evaluated the use of CPAP on the incidence of bradycardias. We used random-effects models in all meta-analyses and evaluated heterogeneity using I 2 ., Results: We included 34 articles from 7204 records, comprising 4852 patients. Among patients with OSA, the pooled prevalence of daytime and nocturnal bradycardia were 25% (95% CI: 18.6 to 32.7) and 69.8% (95% CI: 41.7 to 88.2) respectively. Among patients with bradycardia, the pooled prevalence of OSA was 56.8% (95% CI: 21.5 to 86.3). CPAP treatment, compared to those without, did not significantly reduce the risk of daytime (two randomized trials; RR: 0.50; 95% CI: 0.11 to 2.21) or nocturnal bradycardia (one randomized-controlled trial and one cohort study; RR: 0.76; 95% CI: 0.48 to 1.20)., Conclusions: This meta-analysis demonstrates a high comorbid disease burden between OSA and bradycardia. Future research should explore the treatment effect of CPAP on bradycardia incidence, as compared to placebo., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

To assess the heterogeneity of HBV reverse transcriptase (RT) quasispecies during 10 years of antiviral therapy and their association with antiviral efficacy. Nineteen patients with chronic hepatitis B (CHB) infection receiving nucleos(t)ide analogues (NAs) were enrolled. Based on the antiviral efficacy after 1 year of treatment, 5 patients were grouped into an early virologic response (EVR) group, while 8 patients were grouped into a late virologic response (LVR) group. Furthermore, 6 CHB patients that had undergone antiviral treatment for 10 years were grouped into a virologic breakthrough (VBT) group. The HBV RT from each patient were amplified, cloned, and sequenced. The complexity of the RT gene in the EVR group was significantly higher than that in the LVR (P = 0.0393) and VBT groups (P = 0.0141). Phylogenetic tree analysis showed that the average branch length of the EVR and LVR groups were significantly greater than that of VBT group (P < 0.001). The complexity (at the nucleotide level) of the RT quasispecies was negatively correlated with the corresponding HBV DNA load (P = 0.0163) at one year post-antiviral treatment. Moreover, both the LVR and VBT groups accumulated more deleterious mutations than the EVR group. After 1 year of NAs treatment, the increased HBV quasispecies complexity and evolutionary topologies, coupled with less deleterious mutations, are likely associated with a favorable efficacy during long-term antiviral treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Virus-encoded tumor necrosis factor receptors (vTNFRs) facilitate viral escape from the host immune response during viral propagation. Cyprinid Herpesvirus-2 (CyHV-2) is a double-stranded DNA virus of alloherpesviridae family that causes great economic losses in the aquaculture industry. The present study identified and characterized a novel TNFR homolog termed ORF4 in CyHV-2. ORF4 was identified as a secreted protein and a homolog of herpesvirus entry mediator (HVEM). ORF4 localized to the cytoplasm in infected GiCF cells. ORF4 overexpression enhanced viral propagation, while downregulation of ORF4 via siRNA decreased viral propagation. ORF4 overexpression promoted GiCF proliferation, and its downregulation suppressed CyHV-2-induced apoptosis. GST-pulldown and LC-MS/MS assays identified 44 conditional binding proteins that interact with ORF4 protein, while the GST pulldown test did not support the idea that ORF4 interact with histone H3.3. Taken together, our results contribute to our understanding of the vTNFR function in alloherpesviridae pathogenesis and host immune regulation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Alcoholic liver fibrosis (ALF) is commonly associated with long-term alcohol consumption and the activation of hepatic stellate cells (HSCs). Inhibiting the activation and proliferation of HSCs is a critical step to alleviate liver fibrosis. Increasing evidence indicates that ecto-5'-nucleotidase (CD73) plays a vital role in liver disease as a critical component of extracellular adenosine pathway. However, the regulatory role of CD73 in ALF has not been elucidated. In this study, both ethanol plus CCl 4 -induced liver fibrosis mice model and acetaldehyde-activated HSC-T6 cell model were employed and the expression of CD73 was consistently elevated in vivo and in vitro. C57BL/6 J mice were intraperitoneally injected with CD73 inhibitor Adenosine 5'-(α, β-methylene) diphosphate sodium salt (APCP) from 5th week to the 8th week in the development of ALF. The results showed APCP could inhibit the activation of HSCs, reduce fibrogenesis marker expression and thus alleviate ALF. Silencing of CD73 inhibited the activation of HSC-T6 cells and promoted apoptosis of activated HSC-T6 cells. What's more, the proliferation of HSC-T6 cells was inhibited, which was characterized by decreased cell viability and cycle arrest. Mechanistically, Wnt/β-catenin pathway was activated in acetaldehyde-activated HSC-T6 cells and CD73 silencing or overexpression could regulate Wnt/β-catenin signaling pathway. Collectively, our study unveils the role of CD73 in HSCs activation, and Wnt/β-catenin signaling pathway might be involved in this progression., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dromedary camel hepatitis E virus is a novel HEV that belongs to the family Hepeviridae, and is classified as genotype 7 HEV (HEV-7). Since HEV-7 is transmitted from camels to humans and causes acute hepatitis E, this virus is a non-negligible pathogen for zoonosis, and a vaccine against HEV-7 infection is urgently needed. Here, we first intravenously inoculated HEV-7 to rhesus monkeys to explore the susceptibility, and we established an animal model. We then used virus-like particles (VLPs) of HEV-1 (HEV-1 VLPs) and HEV-3 (HEV-3 VLPs), a candidate hepatitis E vaccine, to intramuscularly inoculate rhesus monkeys. The monkeys elicited IgG antibody titers as high as >1:102,400 against heterologous HEV-7 without any adjuvants. The HEV-1 VLPs and HEV-3 VLPs-immunized monkeys were challenged intravenously with HEV-7, and they were protected completely from the infection, demonstrating that these VLPs could be a usable vaccine against HEV-7 infection. We also observed that HEV-7-infected rhesus monkeys did not show any liver damage during these experiments. Further efforts are necessary to establish an animal model for investigation of the pathogenesis of hepatitis E caused by HEV-7 infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The current differentiation process of human pluripotent stem cells (hPSCs) into cardiomyocytes to enhance the purity of hPSC-derived cardiomyocytes requires some purification processes, which are laborious processes. We developed cell sorting plates, which are prepared from coating thermoresponsive poly(N-isopropylacrylamide) and extracellular matrix proteins. After hPSCs were induced into cardiomyocytes on the thermoresponsive surface coated with laminin-521 for 15 days, the temperature of the cell culture plates was decreased to 8-9 °C to detach the cells partially from the thermoresponsive surface. The detached cells exhibited a higher cardiomyocyte marker of cTnT than the remaining cells on the thermoresponsive surface as well as the cardiomyocytes after purification using conventional cell selection. The detached cells expressed several cardiomyocyte markers, such as α-actinin, MLC2a and NKX2.5. This study suggested that the purification of hPSC-derived cardiomyocytes using cell sorting plates with the thermoresponsive surface is a promising method for the purification of hPSC-derived cardiomyocytes without conventional laborious processes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human induced pluripotent stem cells (hiPSCs) were generated on several biomaterials from human amniotic fluid in completely xeno-free and feeder-free conditions via the transfection of pluripotent genes using a nonintegrating RNA Sendai virus vector. The effect of xeno-free culture medium on the efficiency of the establishment of human amniotic fluid stem cells from amniotic fluid was evaluated. Subsequently, the effect of cell culture biomaterials on the reprogramming efficiency was investigated during the reprogramming of human amniotic fluid stem cells into hiPSCs. Cells cultured in laminin-511, laminin-521, and Synthemax II-coated dishes and hydrogels having optimal elasticity that were engrafted with specific oligopeptides derived from vitronectin could be reprogrammed into hiPSCs with high efficiency. The reprogrammed cells expressed pluripotency proteins and had the capability to differentiate into cells derived from all three germ layers in vitro and in vivo. Human iPSCs could be generated successfully and at high efficiency (0.15-0.25%) in completely xeno-free conditions from the selection of optimal cell culture biomaterials., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Commonly, stem cell culture is based on batch-type culture, which is laborious and expensive. We continuously cultured human pluripotent stem cells (hPSCs) on thermoresponsive dish surfaces, where hPSCs were partially detached on the same thermoresponsive dish by decreasing the temperature of the thermoresponsive dish to be below the lower critical solution temperature for only 30 min. Then, the remaining cells were continuously cultured in fresh culture medium, and the detached stem cells were harvested in the exchanged culture medium. hPSCs were continuously cultured for ten cycles on the thermoresponsive dish surface, which was prepared by coating the surface with poly(N-isopropylacrylamide-co-styrene) and oligovitronectin-grafted poly(acrylic acid-co-styrene) or recombinant vitronectin for hPSC binding sites to maintain hPSC pluripotency. After ten cycles of continuous culture on the thermoresponsive dish surface, the detached cells expressed pluripotency proteins and had the ability to differentiate into cells derived from the three germ layers in vitro and in vivo. Furthermore, the detached cells differentiated into specific cell lineages, such as cardiomyocytes, with high efficiency., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) mediate the recognition of Helicobacter pylori (H. pylori) and initiate the innate immune response to infection. Genetic polymorphisms of TLRs play important roles in gastric carcinogenesis. The aim of this study was to determine the association between single nucleotide polymorphisms (SNPs) in TLR genes and H. pylori infection in the prognosis of gastric cancer (GC). A total of 756 GC patients were included in this study. Nine SNPs (TLR2: rs3804100, rs7696323, and rs10116253; TLR4: rs10983755, rs11536878, rs1927914, and rs7873784; TLR5: rs5744174; and TLR9: rs187084) in TLR genes were genotyped by MassARRAY assay. Kaplan-Meier survival curves and Cox regression were employed to conduct the associations between SNPs in TLRs and the survival of GC. Multivariate Cox regression indicated that patients with the TLR2 rs3804100 TT genotype exhibited worse survival than those with the CC + CT genotype (HR = 1.262, 95% CI: 1.006-1.582). No significant interaction between rs3804100 and H. pylori infection was observed for the prognosis of GC. Our results suggested that the TLR2 rs3804100 polymorphism may be a potential prognostic biomarker for GC independent of the H. pylori infection-related pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We tested the hypothesis that extracorporeal-shock-wave (ECSW)-assisted adipose-derived stromal vascular fraction (SVF) therapy was better than either one for restoring the blood flow in critical limb ischemia (CLI). Adult male-SD rats were categorized into group 1 (sham-operated-control), group 2 (CLI), group 3 [CLI + ECSW (280 impulses/0.10 mJ/mm 2 ) applied to left inguinal area at 3 h after CLI], group 4 [CLI + SVF (1.2 × 10 6 ) implanted into CLI area at 3 h after CLI], group 5 (CLI + ECSW-SVF). In vitro studies showed that ECSW significantly enhanced angiogenesis in human umbilical-vein endothelial cells and carotid-artery ring, and SVF significantly suppressed inflammation (TNF-α/NF-Κb/IL-1ß/MMP-9) in smooth-muscle cells treated by LPS (all p < .001). By day 14 after CLI, the ratio of ischemic/normal blood flow (INBF) was highest in group 1, lowest in group 2, significantly higher in group 5 than in groups 3 and 4, but no difference was shown between the latter two groups (all p < .001). The fibrotic area in CLI region exhibited an opposite pattern of INBF ratio (all p < .0001). Protein (CD31/vWF/eNOS) and cellular (CD31/vWF) expressions and number of small vessels in CLI area exhibited an identical pattern, whilst protein expressions of apoptotic (caspase3/PARP/mitochondrial-Bax) fibrotic/DNA-damaged (Samd3/TFG-ß/γ-H2AX) biomarkers exhibited an opposite pattern to INBF among five groups (all p < .0001). The numbers of angiogenetic cells in CLI region (SDF-1α/VEGF/CXCR4) and endothelial-progenitor cells (C-kit/CD31+//Sca-1/CD31+//CD34/KDR+/VE-cadherin/CD34+) in circulation significantly and progressively increased from groups 2 to 5 (all p < .0001). In conclusion, ECSW-SVF therapy effectively enhanced angiogenesis and restoration of blood flow in CLI area., (Copyright © 2018. Published by Elsevier Inc.)

Sperm cells can be damaged during the semen cryopreservation process, decreasing their fertilizing ability. Physical damage and oxidative stress may occur during the freeze-thawing process. Antioxidants such as the native antioxidant melatonin can potentially improve cryopreservation outcomes. In this study, we added melatonin to cryoprotectant to examine its effect on frozen-thawed human sperm. We found that adding 0.1mM melatonin to cryoprotectant significantly increased sperm viability (24.80 ± 0.46% vs. 20.97 ± 1.27%, P < 0.05) and membrane integrity (P < 0.05), and decreased intracellular reactive oxygen species and lipid peroxidation damage. Furthermore, mRNA levels of the transcription factor NF-E2-related factor-2 and its downstream genes were significantly increased. Resistance to oxidative stress was enhanced and expression of the antiapoptotic gene Bcl-2 was increased by inclusion of 0.1mM melatonin in the cryoprotectant. Moreover, 0.1mM melatonin upregulated the expression of heat shock protein 90 (HSP90), which confers resistance to stressors in frozen-thawed sperm. Results obtained upon addition of inhibitors of melatonin receptors (luzindole and 4-P-PDOT) and an HSP90 inhibitor (geldanamycin) in the cryoprotectant demonstrated that melatonin promoted HSP90 translation via the melatonin receptor MT1 and increased adenosine triphosphate levels, thus increasing the viability of thawed sperm., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A novel cluster of five ferret hepatitis E virus (HEV) strains was detected from nine laboratory ferrets (Mustela putorius furo) imported from a ferret farm in the U.S. Our detection of ferret HEV RNA and anti-HEV antibodies, and alanine aminotransferase (ALT) value assessment indicated that all of the 9 ferrets were infected with ferret HEV, and that the infection exhibited three patterns: sub-clinical infection (n=2), acute hepatitis (n=6) and persistent infection (n=1). Next-generation sequence analyses of the entire genome sequences of the five strains revealed that their nucleotide sequence identities ranged from 99.5% to 99.9%, indicating that genetically similar ferret HEVs had been circulating at this the U.S. ferret farm. In contrast, the strains shared 82% and 89% nucleotide sequence identities with other ferret HEV that isolated from the Netherlands (JN998607) and the U.S. (AB890374), suggesting that these strains form a novel cluster of ferret HEV with diverse genomes depending on the region where their host. Particles with a diameter of ~35nm at a density of 1.201g/cm 3 were observed in the fecal specimens by electron microscopy. There was no evidence that the particles were associated with the cell membrane. The ferret HEV RNA was not constantly detected in urine, suggesting that the excretion of ferret HEV into urine is not a common feature of HEV infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Porcine bocaviruses (PBoVs), new members of the Bocavirus genus, have been identified in swine worldwide. However, the antigenicity and epidemiology of PBoVs are still unclear. Here we used a recombinant baculovirus expression system to express the main capsid protein VP2 of Japan strain JY31b in insect Tn5 cells, and successfully produced the virus-like particles of PBoV (PBoV-LPs). The diameter and densities of the PBoV-LPs were estimated to be 30nm and 1.300g/cm(3), respectively, which were similar to the values for the native virion of PBoV. Antigenic analysis demonstrated that the PBoV-LPs were not cross-reactive with porcine circovirus 2, but were cross-reactive with human bocavirus 1, 2, 3 and 4. An ELISA for detection of anti-PBoV IgG antibodies was established using PBoV-LPs as antigen, which proved to be useful for monitoring PBoV infection in both swine and wild boars. The preliminary epidemiology research showed that 90.7% of pigs and 59.5% of wild boars were positive for the anti-PBoV-IgG, suggesting that both species were also widely infected with PBoV. The seven PBoV strains detected in wild boars separated into four subgroups, demonstrating the genetic diversity of PBoV., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mastitis, which commonly occurs during the postpartum period, is caused by the infection of the mammary glands. The most common infectious bacterial pathogen of mastitis is Staphylococcus aureus (S. aureus) in both human and animals. Brazilin, a compound isolated from the traditional herbal medicine Caesalpinia sappan L., has been shown to exhibit multiple biological properties. The present study was performed to determine the effect of brazilin on the inflammatory response in the mouse model of S. aureus mastitis and to confirm the mechanism of action involved. Brazilin treatment was applied in both a mouse model and cells. After brazilin treatment of cells, Western blotting and qPCR were performed to detect the protein levels and mRNA levels, respectively. Brazilin treatment significantly attenuated inflammatory cell infiltration and inhibited the expressions of TNF-α, IL-1β and IL-6 in a dose-dependent manner. Administration of brazilin in mice suppressed S. aureus-induced inflammatory injury and the production of proinflammatory mediators. This suppression was achieved by reducing the increased expression of TLR2 and regulating the NF-κB and MAPK signaling pathways in the mammary gland tissues and cells with S. aureus-induced mastitis. These results suggest that brazilin appears to be an effective drug for the treatment of mastitis and may be applied as a clinical therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Trichodysplasia spinulosa-associated polyomavirus (TSV) was identified in, and shown to be the probable cause of, trichodysplasia spinulosa, a rare skin disease. To date, serological analyses have revealed that TSV infection is common among adults in the general population of Europe and Australia. However, there have been no reports of TSV in Asia., Objective: To study the prevalence of TSV infection in Japan., Study Design: TSV-VP1 expressed in a recombinant baculovirus expression system in an insect cell line, Tn5, self-assembled into virus-like particles. Overall, 1000 serum samples were examined by enzyme-linked immunosorbent assays using virus-like particles of TSV as antigen. Participants ranged in age from 0 to 94 years., Results: Overall, 629 of 1000 serum samples (62.9%) were positive for anti-TSV antibodies. The seropositive rate increased with age and the seroprevalence of TSV significantly increased from 17.1% (25/146) in children aged from 0 to 4 years to 78.7% (472/600) in adults aged over 20 years (odds ratio = 0.056, 95% confidence interval = 0.035-0.900, P = 0.000, Chi-squared test). TSV seropositivity was not different between sera obtained in 1980 and 2012, and was not associated with sex. Competitive assay demonstrated that TSV antibodies did not cross-react with BK virus or Merkel cell polyomavirus., Conclusions: These results provide evidence that TSV circulates widely in the Japanese population, with primary exposure occurring mainly at early childhood, similar to that previously reported in other countries., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chitosan coated porous titanium alloy implant (CTI) is demonstrated a promising approach to improve osseointegration capacity of pure porous titanium alloy implant (TI). Since chitosan has been demonstrated to exhibit antioxidant activity, we propose CTI may ameliorate the ROS overproduction, thus reverse the poor osseointegration under diabetic conditions, and investigate the underlying mechanisms. Primary rat osteoblasts incubated on the TI and the CTI were subjected to normal serum (NS), diabetic serum (DS), DS + NAC (a potent ROS inhibitor) and DS + LY294002 (a PI3K/AKT-specific inhibitor). In vivo study was performed on diabetic sheep implanted with TI or CTI into the bone defects on crista iliaca. Results showed that diabetes-induced ROS overproduction led to osteoblast dysfunction and apoptosis, concomitant with the inhibition of AKT in osteoblasts on the TI substrate. While CTI stimulated AKT phosphorylation through ROS attenuation, thus reversed osteoblast dysfunction evidenced by improved osteoblast adhesion, increased proliferation and ALP activity, and decreased cytotoxicity and apoptotic rate, which exerted same effect to NAC treatment on the TI. These effects were further confirmed by the improved osseointegration within the CTI in vivo evidenced by Micro-CT and histological examinations. In addition, the aforementioned promotive effects afforded by CTI were abolished by blocking PI3K/AKT pathway with addition of LY294002. These results demonstrate that the chitosan coating markedly ameliorates diabetes-induced impaired bio-performance of TI via ROS-mediated reactivation of PI3K/AKT pathway, which elicits a new surface functionalization strategy for better clinical performance of titanium implant in diabetic patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. Here we describe a general and simple approach to confer tumor tropism to any mPEG-NP. We demonstrate this approach with humanized bispecific antibodies (BsAbs) that can bind to both mPEG molecules on mPEG-NPs and to EGFR or HER2 molecules overexpressed on the surface of cancer cells. Simple mixing of BsAbs with mPEG-NPs can mediate preferential binding of diverse mPEG-NPs to cancer cells that overexpress EGFR or HER2 under physiological conditions and significantly increase cancer cell killing by liposomal doxorubicin to EGFR(+) and HER2(+) cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drug-loaded NPs to antigen-positive human tumors in a mouse model. Anti-mPEG BsAbs offer a simple one-step method to confer tumor specificity to mPEG-NPs for enhanced tumor accumulation and improved therapeutic efficacy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background and Aim: Serum adhesion molecules play a pivotal role in the pathogenesis of sepsis syndrome. This study aimed to evaluate the prognostic value of serum adhesion molecules in patients with severe sepsis and mechanical ventilation (MV) at the emergency department., Methods: Eighty-seven consecutive patients with severe sepsis, including 35 with MV, were evaluated. Serum samples were collected for analysis of serum adhesion molecules. The patients' clinical and laboratory data on admission were also recorded., Results: The maximum 24-h APACHE II and 24-h SOFA scores were significantly higher in the severe sepsis patients requiring MV than in patients without MV (p=0.02 and p<0.001). Mortality rate was significantly higher in severe sepsis patients requiring MV than in patients without MV (40% [14/35] vs. 9.6% [5/52], p=0.001). Both VCAM-1 level (p=0.03) and lactate concentration (p=0.04) on admission had significant differences between survivors and non-survivors in patients requiring MV. In the logistic regression model, only VCAM-1 level (p=0.049) was independently predictive of mortality. By correlation analysis, lactate concentration significantly correlated with the mean VCAM-1 level on admission (γ=0.484, p=0.004). The area under the ROC curve for VCAM-1 level was 0.747 (p=0.02, 95% CI: 0.576-0.918). The cut-off value of VCAM-1 level for predicting hospital mortality in severe sepsis patients receiving MV was 1870ng/mL, with 77% sensitivity and 71% specificity; then the likelihood ratio equals 2.7., Conclusions: In this study, VCAM-1 level is a more powerful outcome predictor of hospital mortality in severe sepsis patients requiring MV than lactate concentration and other conventional parameters on admission. This suggests that increased plasma VCAM-1 concentration may be useful in identifying who are at risk of hospital mortality among severely septic patients requiring MV., (Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background and Aims: The outcomes of Chinese patients undergoing dialysis after coronary revascularization are unknown. We examined the outcomes of Taiwanese dialysis patients after coronary artery bypass grafting (CABG), percutaneous transluminal coronary angioplasty (PTCA), or coronary stenting., Methods: Using data from the National Health Research Institute database, we determined the outcomes of 1,287 dialysis patients who underwent initial coronary revascularization between 1997 and 2008., Results: The 7-year overall survival rates were 69 ± 4%, 68 ± 3%, and 57 ± 2% for the CABG, stent, and PTCA patients (p = 0.001), respectively. After demographic and comorbidity adjustment, hazard ratios (HRs) for all-cause death in the CABG (vs. PTCA) and stent (vs. PTCA) patients were 0.695 (p = 0.015) and 0.721 (p = 0.009). Additionally, no significant difference in all-cause death was found between the CABG and stent patients. Moreover, the ≥65-year-old CABG group patients and the <65-year-old coronary stent group patients showed better survival than the PTCA group patients. Compared with the PTCA and CABG groups, the coronary stent group was significantly associated with a higher risk for recurrent acute myocardial infarction (AMI). Based on age stratification, the ≥65-year-old stent group had a higher risk for recurrent AMI than the PTCA group (HR, 1.562; p = 0.026)., Conclusions: Chinese patients undergoing dialysis who underwent CABG or coronary stenting had better survival than those who underwent PTCA. Moreover, being ≥65 years old, CABG shows better survival compared with PTCA; being <65 years old, coronary stenting show better survival compared with PTCA., (Copyright © 2014 IMSS. Published by Elsevier Inc. All rights reserved.)

Background: Human herpesvirus type 8 (HHV-8) is the aetiologic agent of Kaposi's sarcoma (KS). The incidence of KS in renal transplant patients is much higher than in healthy controls. The risk is even higher among recipients seropositive for HHV-8 before transplantation. Patients with end-stage renal disease (ESRD) are immunocompromised and are candidates for renal transplantation, but HHV-8 seroprevalence in ESRD patients has not been well documented., Objectives: This study aimed to evaluate HHV-8 seroprevalence in ESRD patients in a cohort in Taiwan., Study Design: Blood samples collected from 149 ESRD patients and 149 age- and sex-matched healthy controls were analysed for HHV-8 antibody with immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) and for HHV-8 DNA with polymerase chain reaction., Results: Seropositivity and titres for HHV-8 antibodies with IFA as well as seropositivity with ELISA were significantly greater in ESRD patients than in healthy controls (P=0.006, 0.001 and 0.003, respectively). Patients with a history of taking herbal medicine had significantly greater ELISA positivity than those without such a history (P=0.004). ELISA positives, particularly patients, had much higher IFA antibody titres than ELISA negatives (P<0.0001). Seropositivity in ESRD patients was not related to lymphopaenia, monocytosis, dialysis duration or a history of transfusion. Two diabetic ESRD patients were positive for HHV-8 DNA., Conclusions: ESRD patients had significantly greater HHV-8 seropositivity than healthy controls in Taiwan. This association seems to be related to the geographic location of the cohort and invites further studies for the early association of HHV-8 infection in ESRD patients and risk for KS., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Oral delivery with virus-like particles (VLPs) is advantageous because of the inherited entry pathway from their parental viral capsids, which enables VLP to withstand the harsh and enzymatic environment associated with human digestive tract. However, the repeat use of this system is challenged by the self-immunity. In order to overcome this problem, we engineered the recombinant capsid protein of hepatitis E virus by inserting p18 peptide, derived from the V3 loop of HIV-1 gp120, into the antibody-binding site. The chimeric VLP resembled the tertiary and quaternary structures of the wild type VLP and specifically reacted with an HIV-1 antibody against V3 loop. Different from the wild type VLP, the chimeric VLP was vulnerable to trypsin cleavage although it appeared as intact particle, suggesting that the intermolecular forces of attraction between the recombinant capsid proteins are strong enough to maintain the VLP icosahedral arrangement. Importantly, this VLP containing the V3 loop did not react with anti-HEV antibodies, in correspondence to the mutation at its antibody-binding site. Therefore, the insertion of peptides at the surface antigenic site could allow VLPs to escape pre-existing anti-HEV humoral immunity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Rabies virus (RABV) infection continues to be a global threat to human and animal health, yet no curative therapy has been developed. RNA interference (RNAi) therapy, which silences expression of specific target genes, represents a promising approach for treating viral infections in mammalian hosts. We designed six small interfering (si)RNAs (N473, N580, N783, N796, N799 and N1227) that target the conserved region of the RABV challenge virus standard (CVS)-11 strain nucleoprotein (N) gene. Using a plasmid-based transient expression model, we demonstrated that N796, N580 and N799 were capable of significantly inhibiting viral replication in vitro and in vivo. These three siRNAs effectively suppressed RABV expression in infected baby hamster kidney-21 (BHK-21) cells, as evidenced by direct immunofluorescence assay, viral titer measurements, real-time PCR, and Western blotting. In addition, liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival, compared to a non-liposome-mediated delivery method. Collectively, our results showed that the three siRNAs, N796, N580 and N799, targeting the N gene could potently inhibit RABV CVS-11 reproduction. These siRNAs have the potential to be developed into new and effective prophylactic anti-RABV drugs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hepatitis E virus (HEV), a causative agent of human hepatitis E, is transmitted through an oral-fecal route, often by contaminated drinking water. Serum specimens were collected from 112 non-A, -B, and -C acute hepatitis patients from 1989 to 2004 in Japan. Of these, 24 patients were found to be positive for anti-HEV IgM and diagnosed with acute Hepatitis E. Seventeen of these patients had a clear history of traveling abroad before disease onset and were counted as cases of imported HEV infection. HEV RNA was detected in 16 of these imported cases, and the nucleotide sequences similar to those of HEV detected in India, Bangladesh, and China were identified. By phylogenetic analysis, the remaining imported case appeared to have been imported from India, even though the patient's travel history was uncertain. These results indicated that some sporadic cases of hepatitis E in Japan are caused by imported HEV, and that phylogenetic analyses enable us to identify the country or area where a patient has been infected., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The H5N1 avian influenza virus (AIV) causes widespread infections in bird and human respiratory tracts, and vaccines and drug therapy are limited in their effectiveness. Recent studies of AIV structures have been published and provide new targets for designing antiviral drugs such as antisense oligonucleotides (AS ODNs), which effectively inhibit gene replication. In this study, we designed and synthesized three AS ODNs (NP267, NP628, NP749) that were specific for the RNA binding region of nucleoprotein (NP) based on AIV structure. Results showed that all three AS ODNs could inhibit viral replication in MDCK cells. The NP628 showed the best antiviral effect of all through viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, the liposome mediated NP628 could partially protect the mice from a lethal H5N1 influenza virus challenge. Moreover, the NP628 group had a lower viral titer and lung index in the infected mice when compared with the viral control. Our results showed that AS ODN targeting of the AIV NP gene could potently inhibit AIV H5N1 reproduction, thus, formulating a candidate for an emergent therapeutic drug for the pathogenic H5N1 influenza virus infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

H5N1 avian influenza virus (AIV) causes widespread infections in poultry and wild birds, and has the potential to emerge as a pandemic threat to human. Antisense oligonucleotides (AS ODNs) are highly effective at inhibiting gene replication. Antibody-mediated delivery is a novel approach to target specific cells and tissues. In this study, we designed and synthesized three AS ODNs (PA4, PA492 and PA1203) specific for conserved region of AIV PA protein, and all the three AS ODNs could inhibit viral replication. The PA492 ODN showed the best antiviral effect by viral titers and quantitative RT-PCR in MDCK cells. The fusion protein scFv-tP was constructed as a single chain variable fragment (scFv) against AIV hemaglutinin antigen with a truncated protamine (tP). The results showed that scFv-tP fusion improved the antiviral effectiveness of PA492 in MDCK cells as measured by viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, scFv-tP-delivered PA492 was also found to partially protect mice from lethal H5N1 influenza virus challenge. Using scFv-tP delivery, fluorescein isothiocyanate labeled-PA492 was found to be significantly localized in the lungs, compared to liposome-delivered PA492. Moreover, the fusion protein mediated PA492 had a lower lung index and viral titers in the infected mice as compared with the liposome method. These results provided a potential method for using anti-HA fusion protein for the targeted delivery of AS ODNs against AIV H5N1., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The hemagglutinin antigen (HA) of avian influenza virus (AIV) is an immunogen abundant on the surfaces of infected cells, and can be used as a target for specific antibodies to clear viral infection. Protamine has been demonstrated to deliver DNA into cells effectively. Accordingly, a fusion protein of anti-HA single-chain fragment variable (scFv) and truncated protamine (tP) may be used as a vehicle for delivering the anti-AIV siRNA into the AIV-infected cells for gene therapy. To test this hypothesis, we constructed a novel recombinant plasmid, pET28-scFv-tP, by connecting the genes for anti-H5N1 AIV HA-specific scFv with synthesized oligonucleotides encoding the 22 amino acids of human tP and a linker. Furthermore, the recombinant scFV-tP was expressed and purified, with a yield of 7-8mg of scFv-tP and a purity of >92% from 1L of bacterial culture. Characterization of its bioactivity revealed that scFv-tP recognized HA, similar to its scFv control, in a dose-dependent manner and that the scFv-tP, but not its scFv control, bound to DNA and delivered plasmid and oligonucleotide DNA into the AIV-infected MDCK cells effectively. More importantly, transfection with the mixture of the scFv-tP and plasmid for the NP-specific siRNA significantly inhibited the replication of AIV in MDCK cells, as compared with that transfection with the scFv-plasmid mixture, even with the plasmid in liposome. Our data demonstrated that the recombinant scFv-tP retained the functions of both scFv and tP, and might be potentially used for delivering genetic materials for targeting therapy of AIV infection in vivo., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Recently, nanomaterials coating gained much concern in orthopedic implants such as bone, cartilage, joint, etc. The wear particles would generate from coating in living organism due to corrosion. In this study, we demonstrated that the intraarticular injected anatase TiO2 nanoparticles had a potential toxicological effect on major organs and knee joints of rats. The histopathological changes of heart, lung and liver indicated the dissemination of intraarticular TiO2 nanoparticles from joint cavity to system. In the knee joint, the aggregated TiO2 nanoparticles deposited and resulted in the synovium hypotrophy and lymphocytes and plasma cells infiltration, but had no effects on cartilage. In the TiO2-exposed synovium, the oxidative damage was induced because the glutathione peroxidase (GSH-Px), reduced glutathione (GSH), oxidized glutathione (GSSG), and superoxide dismutase (SOD) levels were highly regulated to counteract over-produced free radicals, i.e. hydrogen peroxide (H2O2). Further, the lipid peroxidation was detected in the synovium though the expression of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin (IL-1beta) was not much interfered. This research suggested that the amounts of nanocoating in the surface of implants should be controlled and standardized.

Objective: This study was conducted to investigate the potential role of intravascular oxidative stress in inexplicable episodes of coronary artery spasm (CAS) during coronary angiography (CAG)., Design and Methods: Serial patients with stable angina scheduled for routine CAG were prospectively evaluated. Patients with clinical evidence of variant angina were excluded. Blood sampling was done immediately before CAG., Results: Inexplicable episodes of CAS were identified during CAG in 7 patients with and 8 patients without significant coronary artery disease (CAD). Compared to those without CAS (n=220), the 15 patients with CAS had significantly increased plasma triglyceride level, white blood cell (WBC) count and superoxide production in whole blood, and reduced plasma high-density lipoprotein (HDL) level. In multivariate analysis, CAS episodes were associated with an elevated baseline WBC count (relative risk, RR, of increasing one tertile, 2.49) and peak whole blood superoxide production (RR, 2.36), and a reduced plasma HDL level (RR, 0.37)., Conclusions: Both an increased WBC count and superoxide production in whole blood preceded and predicted the inexplicable development of CAS during CAG irrespective of the presence of CAD, suggesting that intravascular oxidative stress might be the major mediator. Antioxidants given before CAG may be warranted to prevent CAS episodes.

Objectives: This study aimed to investigate whether baseline serum levels of matrix metalloproteinases (MMPs) could predict long-term prognosis of coronary revascularizations., Designs and Methods: Ninety-one consecutive patients receiving coronary revascularizations (58 percutaneous coronary interventions and 33 coronary artery bypass graft surgeries) for stable coronary artery disease (CAD) were studied. Baseline serum levels of high-sensitive C-reactive protein (hsCRP), MMP-2, -3 and -9 drawn before revascularization were correlated to the clinical adverse events within >12 months after revascularizations., Results: Baseline characteristics were similar between the two groups. There were total 22 major adverse cardiovascular events during a mean period of 27 months. Only baseline serum MMP-9 level independently predicted future cardiovascular events after coronary revascularization either by multivariate analysis (relative risk 3.18, p=0.028) or by Kaplan-Meier analysis (p=0.021)., Conclusions: Baseline serum MMP-9 level predicted the prognosis after coronary revascularizations, suggesting its potential role in risk stratification before revascularization strategies for stable CAD.

Silkworm (Bombyx mori), a model system for Lepidoptera, has contributed enormously to the study of insect immunology especially in humoral immunity. But little is known about the molecular mechanism of immune response in the silkworm. Toll receptors are a group of evolutionarily ancient proteins, which play a crucial role in the innate immunity of both insects and vertebrates. In human, Toll-like receptors (TLRs) are the typical pattern recognition receptors for different kinds of pathogen molecules. Toll-related receptors in Drosophila, however, were thought to function as cytokine receptors in immune response and embryogenesis. We have identified 11 putative Toll-related receptors and two Toll analogs in the silkworm genome. Phylogenetic analysis of insect Toll family and human TLRs showed that BmTolls is grouped with Drosophila Tolls and Anopheles Tolls. These putative proteins are typical transmembrane receptors flanked by the extracellular leucine-rich repeat (LRR) domain and the cytoplasmic TIR domain. Structural prediction of the TIR domain alignment found five stranded sheets and five helices, which are alternatingly joined. Microarray data indicated that BmToll and BmToll-2 were expressed with remarkable enrichment in the ovary, suggesting that they might play a role in the embryogenesis. However, the enriched expression of BmToll-2 and -4 in the midgut suggested that the proteins they encode may be involved in immune defense. Testis-specific expression of BmToll-10 and -11 and BmToLK-2 implies that these may be involved in sex-specific biological functions. The RT-PCR results indicated that 10 genes were induced or suppressed with different degrees after their immune system was challenged by different invaders. Expression profiles of BmTolls and BmToLKs reported here provide insight into their role in innate immunity and development.

Matrix metalloproteinase (MMP) is critical to the progression of atherosclerosis and neointima hyperplasia after vascular injury. We investigated the effects of carvedilol, a pharmacological antioxidant with alpha- and beta-adrenergic blocking activity, on MMP-2 and MMP-9 expression. Vascular injury was induced with the balloon catheters on abdominal aortas of high-cholesterol-fed rabbits. On Day 21, there was significant aortic neointima formation with increased oxidative DNA damage by immunostaining with 8-hydroxy-2'-deoxyguanosine and enhanced MMP-2 and MMP-9 expressions by Western blotting, which were significantly reduced by oral administration of carvedilol (20 mg/kg/day) or probucol (100 mg/kg/day). Vascular expression (by Western blot), activity (by gelatin zymography), and mRNA levels of MMP-2 and MMP-9 were also reduced by carvedilol or probucol. Besides, pretreatment with carvedilol or probucol but not propranolol, a beta-blocker, or prazocin, an alpha-blocker, inhibited tumor necrosis factor-alpha-stimulated expressions and activities of MMP-2 and MMP-9 in human aortic smooth muscle cells. On electrophoretic mobility-shift assay, carvedilol inhibited the binding activities of activator protein-1 and specific protein-1, two major transcription factors for MMP promoter regions. Accordingly, carvedilol, a pharmacological antioxidant, inhibited in vivo and in vitro expression of MMP-2 and MMP-9 properly by modulating the redox-related pathways, suggesting its potential clinical implications.

Increased oxidative stress and vascular inflammation have been shown in patients with cardiac syndrome X (CSX; angina, exercise-induced ischemia, and normal coronary angiogram). This study was conducted to assess the impact of basal superoxide generation by circulating mononuclear cells (MNCs), a contributor to intravascular oxidative stress, and serum inflammatory biomarkers, including high-sensitivity C-reactive protein, homocysteine, soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1, and von Willebrand factor, on the long-term prognosis of CSX. During a mean follow-up of 31.5 +/- 14.2 months (maximum 5 years), a total of 12 events were recorded in 92 consecutive CSX patients. There were no deaths or myocardial infarctions, but 8 hospitalizations for acute coronary syndrome, 3 for stroke, and 1 for congestive heart failure due to left ventricular systolic dysfunction. Under univariate analysis, only basal superoxide generation by MNCs was associated with the risk for cardiovascular event. Based on multivariate analysis, basal superoxide generation by MNCs could still independently predict future events (relative risk for the highest compared to the lowest tertile, 3.87; 95% confidence interval, 1.42-10.54, p = 0.008). These findings demonstrate that long-term prognosis is fair in patients with CSX. Basal superoxide production of MNCs independently predicts future cardiovascular events, suggesting its potential role in measuring disease progression and risk stratification in these patients.

Recombinant hepatitis E virus capsid protein (HEV CP) assembles orally immunogenic virus-like particles (VLP) when expressed in an insect cell system. We used plant expression cassettes, pHEV101 and pHEV110, for transformation of potato to express HEV CP, and 10 independent transgenic lines of HEV101 and 6 lines of HEV110 were obtained. ELISA for HEV CP was performed on tuber extracts. Accumulation of HEV CP in tubers varied from about 5 to 30 microg/g fresh tuber depending on the transgenic plant line. We further compared the expression levels with the yield of tubers for each line. Tuber yield varied less than expression levels, and ranged from about 600 to 1000 g per pot. Although Western blot showed that apparently intact HEV CP accumulated, we observed very limited assembly of virus-like particles in potato tubers. Oral immunization of mice with transgenic potatoes failed to elicit detectable anti-CP antibody response in serum, suggesting that VLP assembly is a key factor in orally delivered HEV CP vaccines.

We made use of 81,635 expressed sequence tags (ESTs) derived from 12 different cDNA libraries of the silkworm, Bombyx mori, inbred strain Dazao (P50), to identify high-quality candidate single nucleotide polymorphisms (SNPs). By PHRAP assembling, 12,980 contigs containing 11,537 contigs assembled by more than one read were obtained, and 101 candidate SNPs and 27 single base insertions/deletions were identified from 117 contigs assembled from 1576 high-quality reads base-called with PHRED and screened on the basis of the neighborhood quality standard (NQS). Simultaneously, we also predicted 40 SNPs in coding regions (cSNPs), of which 26 were predicted to lead to amino acid non-synonymous variations and 14 synonymous substitutions. Also, the 1.66:1 ratio of transition/transversion is different from that of other insects. As the first SNP analysis of a Lepidoptera, B. mori, the single nucleotide polymorphic density is estimated to be 1.3 x 10(-3) by sequence diversity. This analysis shows that expressed sequences from multiple libraries may provide an abundant source of comparative reads to mine for cSNPs from the silkworm genome., (Copyright 2004 Elsevier Ltd.)

Hepatitis E virus (HEV) is an important causative agent of enterically-transmitted hepatitis. Successful vaccine development is crucial in controlling global HEV infection. HEV capsid protein, with 111 amino acids truncated at the N-terminus, was efficiently expressed in the baculovirus expression system. Expressed protein spontaneously assembled into virus-like particles (VLPs) and was released into culture medium. When cynomolgus monkeys were orally inoculated with 10mg of purified rHEV VLPs, serum IgM, IgG, and IgA responses were observed. All these antibody responses were obtained without adjuvants. When the monkeys were challenged with native HEV by intravenous injection, they were protected against infection or developing hepatitis. These results suggested that recombinant HEV (rHEV) VLPs can be a candidate for the oral hepatitis E vaccine.

Extracellular regulated kinases (ERKs)-1 and -2 are members of the MAPK family of protein kinases involved in the proliferation, differentiation and apoptosis of bone cells. The purpose of the present study investigated the biocompatibility role, and signaling pathways of components of mineral trioxide aggregate (MTA) by culturing human osteosarcoma cell line (U2OS) in the presence of materials. Biocompatibility effects were assessed using the MTT assay for mitochondrial enzyme activity. The statistical analysis of the survival rate was performed using one-way analysis of variance (ANOVA) with p<0.05 shown statistical difference. The signaling pathway of MTA-treated U2OS cells were assessed by the western blotting methods. Dose-dependent and time-dependent tests were conducted. The results showed that the survival rates of the MTA extract experimental groups were higher than that of the control group (p<0.05). ERKs activity was dose-dependent, decreasing as the concentrations of the MTA extract decreased, and was time-dependent, decreasing as the treatment time increased. Suppression of ERK pathway by PD98059 resulted in dose-dependent and time-dependent decreases. The findings suggest that MTA is a biocompatible material to U2OS cells, and the ERK kinase pathway plays a signal transduction role in the MTA treated U2OS cells.