Novel STAT3 oligonucleotide compounds suppress tumor growth and overcome the acquired resistance to sorafenib in hepatocellular carcinoma.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and progression of tumors, leading to resistance and poor prognosis. Activation of STAT3 signaling is frequently detected in hepatocellular carcinoma (HCC), but potent and less toxic STAT3 inhibitors have not been discovered. Here, based on antisense technology, we designed a series of stabilized modified antisense oligonucleotides targeting STAT3 mRNA (STAT3 ASOs). Treatment with STAT3 ASOs decreased the STAT3 mRNA and protein levels in HCC cells. STAT3 ASOs significantly inhibited the proliferation, survival, migration, and invasion of cancer cells by specifically perturbing STAT3 signaling. Treatment with STAT3 ASOs decreased the tumor burden in an HCC xenograft model. Moreover, aberrant STAT3 signaling activation is one of multiple signaling pathways involved in sorafenib resistance in HCC. STAT3 ASOs effectively sensitized resistant HCC cell lines to sorafenib in vitro and improved the inhibitory potency of sorafenib in a resistant HCC xenograft model. The developed STAT3 ASOs enrich the tools capable of targeting STAT3 and modulating STAT3 activity, serve as a promising strategy for treating HCC and other STAT3-addicted tumors, and alleviate the acquired resistance to sorafenib in HCC patients. A series of novel STAT3 antisense oligonucleotide were designed and showed potent anti-cancer efficacy in hepatocellular carcinoma in vitro and in vivo by targeting STAT3 signaling. Moreover, the selected STAT3 ASOs enhance sorafenib sensitivity in resistant cell model and xenograft model.

Gut microbiome causal impacts on the prognosis of breast cancer: a Mendelian randomization study.

BACKGROUND: Growing evidence has shown that gut microbiome composition is associated with breast cancer (BC), but the causality remains unknown. We aimed to investigate the link between BC prognosis and the gut microbiome at various oestrogen receptor (ER) statuses. METHODS: We performed a genome-wide association study (GWAS) to analyse the gut microbiome of BC patients, the dataset for which was collected by the Breast Cancer Association Consortium (BCAC). The analysis was executed mainly via inverse variance weighting (IVW); the Mendelian randomization (MR) results were verified by heterogeneity tests, sensitivity analysis, and pleiotropy analysis. RESULTS: Our findings identified nine causal relationships between the gut microbiome and total BC cases, with ten and nine causal relationships between the gut microbiome and ER-negative (ER-) and ER-positive (ER+) BC, respectively. The family Ruminococcaceae and genus Parabacteroides were most apparent among the three categories. Moreover, the genus Desulfovibrio was expressed in ER- BC and total BC, whereas the genera Sellimonas, Adlercreutzia and Rikenellaceae appeared in the relationship between ER + BC and total BC. CONCLUSION: Our MR inquiry confirmed that the gut microbiota is causally related to BC. This further explains the link between specific bacteria for prognosis of BC at different ER statuses. Considering that potential weak instrument bias impacts the findings and that the results are limited to European females due to data constraints, further validation is crucial.

Living near greenness is associated with higher bone strength: A large cross-sectional epidemiological study in China.

BACKGROUND: Living near green spaces may benefit various health outcomes. However, no studies have investigated the greenness-bone linkage in the general population. Moreover, to which extent ambient air pollution (AAP), physical activity (PA), and body mass index (BMI) mediate this relationship remains unclear. We aimed to explore the association between greenness and bone strength and the potential mediating roles of AAP, PA, and BMI in Chinese adults. METHODS: This cross-sectional analysis enrolled 66,053 adults from the China Multi-Ethnic Cohort in 2018-2019. The normalized difference vegetation index (NDVI) and enhanced vegetation index (EVI) were employed to define residential greenness. The calcaneus quantitative ultrasound index (QUI) was used to indicate bone strength. Multiple linear regression models and mediation analyses were used to estimate the residential greenness-bone strength association and potential pathways operating through AAP (represented by PM2.5 [particulate matter <2.5 µm in diameter]), PA, and BMI. Stratification analyses were performed to identify susceptible populations. RESULTS: Higher residential exposure to greenness was significantly associated with an increase in QUI, with changes (95% confidence interval) of 3.28 (3.05, 3.50), 3.57 (3.34, 3.80), 2.68 (2.46, 2.90), and 2.93 (2.71, 3.15) for every interquartile range increase in NDVI500m, NDVI1000m, EVI500m, and EVI1000m, respectively. Sex, urbanicity, annual family income, smoking, and drinking significantly modified the association of greenness-bone strength, with more remarkable associations in males, urban residents, subjects from wealthier families, smokers, and drinkers. For the NDVI500m/EVI500m-QUI relationship, the positive mediating roles of PM2.5 and PA were 6.70%/8.50 and 2.43%/2.69%, respectively, whereas those negative for BMI and PA-BMI were 0.88%/1.06% and 0.05%/0.05%, respectively. CONCLUSION: Living in a greener area may predict higher bone strength, particularly among males, urban residents, wealthier people, smokers, and drinkers. AAP, PA, BMI, and other factors may partially mediate the positive association. Our findings underscore the importance of optimizing greenness planning and management policies.

Untargeted GC-MS-Based Metabolomics for Early Detection of Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant gastrointestinal cancers in the world with a 5-year survival rate of approximately 68%. Although researchers accumulated many scientific studies, its pathogenesis remains unclear yet. Detecting and removing these malignant polyps promptly is the most effective method in CRC prevention. Therefore, the analysis and disposal of malignant polyps is conducive to preventing CRC. METHODS: In the study, metabolic profiling as well as diagnostic biomarkers for CRC was investigated using untargeted GC-MS-based metabolomics methods to explore the intervention approaches. In order to better characterize the variations of tissue and serum metabolic profiles, orthogonal partial least-square discriminant analysis was carried out to further identify significant features. The key differences in tR-m/z pairs were screened by the S-plot and VIP value from OPLS-DA. Identified potential biomarkers were leading in the KEGG in finding interactions, which show the relationships among these signal pathways. RESULTS: Finally, 17 tissue and 13 serum candidate ions were selected based on their corresponding retention time, p-value, m/z, and VIP value. Simultaneously, the most influential pathways contributing to CRC were inositol phosphate metabolism, primary bile acid biosynthesis, phosphatidylinositol signaling system, and linoleic acid metabolism. CONCLUSIONS: The preliminary results suggest that the GC-MS-based method coupled with the pattern recognition method and understanding these cancer-specific alterations could make it possible to detect CRC early and aid in the development of additional treatments for the disease, leading to improvements in CRC patients' quality of life.

Prodigiosin isolated from Serratia marcescens in the Periplaneta americana gut and its apoptosis­inducing activity in HeLa cells.

Serratia marcescens are considered to be abundant and optimal resources for obtaining prodigiosin, which can be isolated from soil, water, plants and air but rarely from insects. In the present study, a strain of Serratia marcescens named WA12­1­18 was isolated from the gut of Periplaneta americana, which was capable of producing high levels of pigment reaching 2.77 g/l via solid fermentation and was identified as prodigiosin by ultraviolet, high performance liquid chromatography (LC), Fourier­transform infrared spectroscopy, LC­mass spectroscopy and nuclear magnetic resonance. The apoptotic tumor cells treated with prodigiosin were examined by 4',6­diamidino­2­phenylindole (DAPI) staining assays and transmission electron microscopy. Flow cytometry (FCM) was utilized to measure the apoptotic rate with Annexin V staining and the expression levels of proteins involved in apoptosis, including B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X (Bax) and caspase­3 were determined by western blot analysis and reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The experimental results revealed that prodigiosin could inhibit the proliferation of HeLa cells and the half­maximal inhibitory concentration values of prodigiosin in HeLa were 2.1, 1.2 and 0.5 µg/ml over 24, 48 and 72 h, respectively. Furthermore, DAPI staining assays and transmission electron microscopy clearly demonstrated that prodigiosin could induce HeLa cell apoptosis. FCM results revealed that the cell apoptotic rates were 19.7±1.4, 23.7±2.4 and 26.2±2.3% following the treatment with 0.5, 1.0 and 2.0 µg/ml prodigiosin for 48 h, respectively. Western blot analysis and RT­qPCR revealed that prodigiosin could activate apoptosis­associated molecules including Bcl­2, Bax and caspase­3. Therefore, the results of the present study demonstrated that the prodigiosin could induce apoptosis in HeLa cells, which may be associated with the upregulation of Bax and caspase­3, the concomitant downregulation of Bcl­2 levels and also triggering the extrinsic apoptotic signaling pathway.

Transduction with Lentiviral Vectors Altered the Expression Profile of Host MicroRNAs.

RNA interference (RNAi) is widely used in gene knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNAs), lentiviral short hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by microRNA (miRNA) microarray analysis that this inhibition was induced by the alteration of host miRNA expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, -216b-5p, 217, and -30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the upregulation of JUN contributed to the dysregulation of miR-216a-5p, -216b-5p, and -217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.IMPORTANCE We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs oversaturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knockout studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which in turn upregulated miR-216a-5p, miR-216b-5p, and miR-217. miR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors, and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.

GP205, a new hepatitis C virus NS3/4A protease inhibitor, displays higher metabolic stability in vitro and drug exposure in vivo.

NS3/4A serine protease is a prime target for direct-acting antiviral therapies against hepatitis C virus (HCV) infection. Several NS3/4A inhibitors have been widely used in clinic, while new inhibitors with better characteristics are still urgently needed. GP205 is a new macrocyclic inhibitor of NS3/4A with low nanomolar activities against HCV replicons of genotypes 1b, 2a, 4a, and 5a, with EC50 values ranging from 1.5 to 12.8 nmol/L. In resistance selection study in vitro, we found resistance-associated substitutions on D168: The activity of GP205 was significantly attenuated against 1b replicon with D168V or D168A mutation, similar as simeprevir. No cross resistance of GP205 with NS5B or NS5A inhibitor was observed. Combination of GP205 with sofosbuvir or daclatasvir displayed additive or synergistic efficacy. The pharmacokinetic profile of GP205 was characterized in rats and dogs after oral administration, which revealed good drug exposure both in plasma and in liver and long plasma half-life. The in vitro stability test showed ideal microsomal and hepatic cells stability of GP205. The preclinical profiles of GP205 support further research on this NS3/4A inhibitor to expand the existing HCV infection therapies.

Effect of Biliary Drainage on the Toxicity and Toxicokinetics of Amanita exitialis in Beagles.

Amatoxin poisoning induces delayed-onset acute liver failure, which are responsible for more than 90% of deaths in mushroom poisoning. It has been postulated from animal and human studies that biliary drainage interrupting enterohepatic amatoxin circulation may affect amatoxin poisoning. Dogs were randomly divided into four groups of six animals each. In 20 mg/kg and 60 mg/kg with biliary drainage groups, after accepting bile drainage operation, beagles were fed Amanita exitialis powder (20 or 60 mg/kg) in starch capsules. In control and bile drainage groups, the beagle dogs were fed with empty capsules. They were assessed for toxicity signs, biochemical and pathological changes, and peptide toxins in plasma, urine and bile. The data were directly compared with those from our published studies on Amanita exitialis-exposed beagles without biliary drainage. Amatoxins were rapidly absorbed and eliminated from plasma after Amanita exitialis ingestion. Amatoxins in 0⁻1-day urine accounted for more than 90% of the total urine excretion, and amatoxins in bile accounted for less than 20% of the total urine and bile excretion. The dogs with biliary drainage showed less severe toxicity signs and biochemical and pathological changes and much lower internal exposure than dogs without biliary drainage. Biliary drainage caused a more than 70% reduction in intestinal amatoxin absorption and could reduce amatoxin absorption from the gastrointestinal tract.

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

[In situ experimental research on natural attenuation of oil pollutants in a gas station].

The natural attenuation rules of oil pollutants are investigated by carrying out a field experiment in a gas station in Beijing, using a set of soil gas sampling and monitoring device designed independently. The samples of soil gas were collected in the field and tested to examine the content of O2, CO2 and VOCs in the unsaturated zone. The results show that after 381 days' natural attenuation, the concentration of the contaminants at the polluted point deceased by 99.2%, while the ratio of BTEX to TVOC decreased from 17.0% to 12.1%. Moreover, the content of O2 decreased while that of CO2 grew as the soil depth increases at G3 point. Further theoretical analysis indicated that: (1) The test results of first-step experiment show that there was a certain level of contamination in G3 area. And after 381 days' natural attenuation, the content of the BTEX at G3 point fell below the environmental standard level, which means the control measures could be removed. (2) The origin of the contamination was proved as short-term sources, and no long-term sources of leakage existed. (3) An effective removal of contaminants in the soil was found under the influence of the natural attenuation. This method could be applied in long-term monitoring for similar contaminated sites. (4) The test of O2and CO2 contents was proved to be an effective approach to determine if the aerobic degradation happens.

[Research of the natural attenuation capacity of oil pollutants based on in-situ experiment].

Based on the results of the in-situ experiment carried out in a Beijing gas station, the ways of natural attenuation were evaluated as both aerobic and anaerobic biodegradation via oxygen and carbon equilibrium calculation. And the research got the rates of the natural attenuation and half-life of the oil pollutants byusing the first-order decay model. Based on the analysis, the capability of monitored natural attenuation and the environmental quality were evaluated. The results show that the actual oxygen consumption of contaminated site is less than that needed theoretically in aerobic degradation of all pollutants, while the amount of CO2 detected is far less than that in the aerobic degradation by theoretical formation. And the half-life of petroleum contaminants in contaminated site is about 50 days. The analysis indicated that: (1) The ways of natural attenuation were proved as both aerobic and anaerobic biodegradation; (2) The contamination of the BTEX tested by the fist-step experiment fell below the environmental standard level after about 250 days' natural attenuation, which means the environmental risk in this gas station had been eliminated and the control measures could be removed.

[Quantitation of HTLV-I proviral load using real-time quantitative PCR with Taqman MGB probe].

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.

Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress.

To gain new insight into the biological function of the human augmenter of liver regeneration (hALR) in HCC, we studied its involvement in radiation-induced damage and recovery of HCC cells. We found that hALR expression was up-regulated in both HCC tissues and multiple hepatoma cell lines and correlated significantly with increased radiation clonogenic survival after radiation treatment. Exogenous expression of hALR increased radiation resistance in SMMC-7721 cells, and the increased survival was accompanied by a decrease in apoptosis and a prolonged G(2)-M arrest after irradiation. Overexpression of ALR significantly increased the mitochondrial membrane potential, inhibited cytochrome c release, and opposed the loss of intracellular ATP levels after radiation. Moreover, knockdown of ALR by siRNA resulted in inhibition of viability in the absence of exogenously added oxidative stress and radiation sensitization in HepG2 cells. Importantly, hALR expression was very low in normal hepatocyte L02 cells, and hALR silencing had a minimal effect on L02 viability and radiation sensitivity. These results suggest that human ALR is important for hepatoma cell viability and involved in the protection of hepatoma cells against irradiation-induced damage by its association with mitochondria. Targeting hALR may be a promising novel approach to enhance the radiosensitivity of hepatoma cancers.

[Efficient expression of exogenous human gp96 gene in tumor cells].

BACKGROUND & OBJECTIVE: Immunization of mice with preparations of heat shock protein(HSP) gp96 isolated from cancer cells has been shown to elicit specific protective cytotoxic T lymphocyte response against cells from which gp96 originate. This phenomenon exploits a new practicable pathway for cancer immunotherapy. But gp96 is generally expressed at low level in cells. Gp96 preparations from limited cells or tissue are difficult to meet the needs of study. So the current study aims to acquire minoclonal cell lines expressing gp96 at high level in order to prepare enough gp96 with high quality. METHODS: The recombinant plasmid pcDNA-hgp96 of human gp96 cDNA was constructed by ligating the fragment of gp96 cDNA into the pcDNA3 plasmid, a eucaryotic expressing vector. Then the recombinant plasmid was transfected into Hela cells and the stable transfectants were selected with G418. The expression level of gp96 of the positive monoclonal cells was detected by Western blot analysis. RESULTS: The recombinant expression plasmid of human gp96 cDNA was successfully constructed. The monoclonal cell lines with stable transfection were obtained. A monoclonal cell line expressing gp96 on high level was selected out. CONCLUSIONS: The monoclonal cell line expressing gp96 at high level has been successfully established, which lays the groundwork for the study of its antitumor immunity.