Association between novel genetic variants of Notch signaling pathway genes and survival of hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Although the Notch pathway plays an important role in formation and progression of hepatocellular carcinoma (HCC), few studies have reported the associations between functional genetic variants and the survival of hepatitis B virus (HBV)-related HCC. METHODS: In the present study, we performed multivariable Cox proportional hazard regression analysis to evaluate associations between 36,101 SNPs in 264 Notch pathway-related genes and overall survival (OS) of 866 patients with HBV-related HCC. RESULTS: It was found that three independent SNPs (NEURL1B rs4868192, CNTN1 rs444927 and FCER2 rs1990975) were significantly associated with the HBV-related HCC OS. The number of protective genotypes (NPGs) were significantly associated with better survival in a dose-response manner (ptrend <0.001). Compared with the model with sole clinical factors, the addition of protective genotypes to the predict models significantly increased the AUC, i.e., from 72.72% to 75.13% (p = 0.002) and from 72.04% to 74.76 (p = 0.004) for 3-year and 5-year OS, respectively. The expression quantitative trait loci (eQTL) analysis further revealed that the rs4868192 C allele was associated with lower mRNA expression levels of NEURL1B in the whole blood (p = 1.71 × 10-3), while the rs1990975 T allele was correlated with higher mRNA expression levels of FCER2 in the whole blood and normal liver tissues (p = 3.51 × 10-5 and 0.033, respectively). CONCLUSIONS: Three potentially functional SNPs of NEURL1B, CNTN1 and FCER2 may serve as potential prognostic biomarkers for HBV-related HCC.

Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

Laparoscopic prostatectomy with complete urethral reconstruction for sexual active BPH patients.

OBJECTIVE: To describe our technique of transvesical laparoscopic simple prostatectomy (LSP) plus complete urethral reconstruction(CUR). MATERIAL AND METHODS: From May 2019 to May 2021, 28 BPH patients with prostate volumes > 80 ml and the requirement to preserve the ejaculatory function (EF) received LSP plus CUR. Baseline demographics, pathology data, perioperative and postoperative complications, and functional outcomes were assessed. Data were analyzed with the Wilcoxon test. RESULTS: The median prostate volume was 106 ml. All patients successfully underwent LSP with no intraoperative complications or conversions to open surgery. The median operative time was 146 min. A total of five Clavien-Dindo Grade1-2 postoperative complications were noted, including infection, prolonged urine leakage and cardiac arrhythmia. No patient reported postoperative urgent or stress urinary incontinence. Functional outcomes at one-year follow-up demonstrated significant improvement from baseline with median IPSS and Qmax (p both < 0.001). Compared with baseline, no significant difference was observed in IIEF and MSHQ-EjD-SF at 6 and 12 months postoperatively. CONCLUSIONS: Our data support transperitoneal-transvesical LSP plus CUR as a safe and effective surgical technique for treating BPH with large prostate adenoma, regardless of the volume of the median lobe, especially for patients requiring to preserve antegrade ejaculation.

Comparison of RLP and PCNL for large pelvis calculi with CKD.

OBJECTIVES: To compare the effect and safety of retroperitoneal laparoscopic pyelolithotomy (RLP) and percutaneous nephrolithotomy (PCNL) for large pelvis calculi with chronic kidney disease (CKD). MATERIAL AND METHODS: Between June 2017 and July 2021, 62 patients with CKD and large renal pelvis calculi (>4 cm2) were treated with RLP. Another 62 patients receiving PCNL served as controls. The perioperative parameters were compared. All patients were followed up for at least 6 months with the stone-free rate and the recovery of renal function evaluated. RESULTS: Significantly longer operation time (101.47 ± 9.25 vs 62.55 ± 7.54 min), less drop in hemoglobin level (0.90 ± 0.38 vs 2.13 ± 0.80 g/dl), staged operations (0% vs 12.9%), postoperative fever (3.23% vs 16.13%) and delayed bowel movement (3.23% vs 14.52), and shorter hospitalization time (3.90 ± 1.66 vs 4.72 ± 1.80 days) were observed in the RLP group (p < 0.05). The stone-free rates were 100% in the RLP group and 88.7% in the PCNL group at the 3-months follow-up (p < 0.05). The serum creatinine level was significantly lower in the RLP group at 24 h (2.81 ± 1.18 vs 3.00 ± 1.15 mg/dl) and 1 week (2.08 ± 1.13 vs 2.34 ± 1.01 mg/dl) postoperatively (p < 0.05). CONCLUSIONS: Although associated with a longer operation time, RLP is a safer and more efficient surgical option for CKD patients with large pelvic stones than PCNL.

Potentially functional genetic variants in ferroptosis-related CREB3 and GALNT14 genes predict survival of hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Ferroptosis is a known crucial player in the development of cancers. However, the effect of single nucleotide polymorphisms (SNPs) in ferroptosis-related genes on survival in hepatitis B virus (HBV)-related hepatocellular carcinoma (HBV-HCC) patients remains unknown. METHODS: We used two-stage multivariable Cox proportional hazards regression analyses to estimate the associations between 48,774 SNPs in 480 ferroptosis-related genes and overall survival (OS) of 866 HBV-HCC patients. RESULTS: We identified that two potentially functional SNPs (CREB3 rs10814274 C > T and GALNT14 rs17010547 T > C) were significantly independently associated with the OS of HBV-HCC patients (CT + TT verse CC, hazards ratio (HR) = 0.77, 95% confidence interval (CI) = 0.67-0.89, p < 0.001 for rs10814274 and TC + CC verse TT, HR = 0.66, 95% CI = 0.53-0.82, p < 0.001 for rs17010547, respectively). Additional joint assessment of protective genotypes of these two SNPs showed that patients with 1-2 protective genotypes had a significantly better OS compared with those carrying 0 protective genotypes (HR = 0.56, 95% CI = 0.45-0.70, p < 0.001). Moreover, the expression quantitative trait loci (eQTL) analysis revealed that the survival-associated SNP rs10814274 T allele was significantly correlated with reduced CREB3 transcript levels in both normal liver tissues and whole blood cells, while the GALNT14 rs17010547 C allele had a significant correlation with increased GALNT14 transcript levels in whole blood cells. CONCLUSION: These results suggest that genetic variants of CREB3 and GALNT14 may affect the survival of HBV-HCC patients, likely via transcriptional regulation of respective genes. However, further studies are required to confirm these findings.

Potentially Functional Genetic Variants in the NRF2 Signaling Pathway Genes are Associated With HBV-related Hepatocellular Carcinoma Survival.

The nuclear factor E2-related factor 2 (NRF2) signaling pathway is one of the most important cell defense pathways. However, it is unclear whether genetic variants in NRF2 signaling pathway genes are associated with the survival of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). In the present study, we utilized a new hypothesis-driven approach based on biological pathways to investigate the associations between 17919 single nucleotide polymorphisms (SNPs) in 137 NRF2 signaling pathway genes and the overall survival (OS) of 866 patients with HBV-related HCC. As a result, two independent SNPs with potential biological function were identified to be significantly associated with HBV-related HCC OS: [SLC2A9 rs28643326 T>C: hazard ratio (HR) = 0.74, 95% confidence interval (95% CI) = 0.62-0.89, P < 0.001 and SLC5A10 rs2472711 G>T: HR = 0.81, 95% CI = 0.71-0.93, P = 0.003, respectively]. The expression quantitative trait loci (eQTL) analysis further revealed that the rs28643326 C allele was significantly associated with increased levels of SLC2A9 mRNA expression (P < 0.001), and higher mRNA expression levels of SLC2A9 in adjacent normal liver tissues were associated with better survival. Although the association between the rs2472711 T allele and the mRNA expression of SLC5A10 was not statistically significant (P = 0.200), the fact that rs2472711 is located at the DNase I hypersensitivity site and is a marker for promoter and enhancer histones also suggests that it may have the function of regulating its corresponding gene expression. In conclusion, genetic variants of NRF2 signaling pathway genes may serve as potential prognostic biomarkers for HBV-related HCC and also provide a solid basis for further mechanistic exploration.

Quantitative assessment of translocator protein (TSPO) in the non-human primate brain and clinical translation of [18F]LW223 as a TSPO-targeted PET radioligand.

OBJECTIVES: Translocator protein 18 kDa (TSPO) positron emission tomography (PET) can be harnessed for the non-invasive detection of macrophage-driven inflammation. [18F]LW223, a newly reported TSPO PET tracer which was insensitive to rs6971 polymorphism, showed favorable performance characteristics in a recent imaging study involving a rat myocardial infarction model. To enable quantitative neuroimaging with [18F]LW223, we conducted kinetic analysis in the non-human primate (NHP) brain. Further, we sought to assess the utility of [18F]LW223-based TSPO imaging in a first-in-human study. METHODS: Radiosynthesis of [18F]LW223 was accomplished on an automated module, whereas molar activities, stability in formulation, lipophilicity and unbound free fraction (fu) of the probe were measured. Brain penetration and target specificity of [18F]LW223 in NHPs were corroborated by PET-MR imaging under baseline and pre-blocking conditions using the validated TSPO inhibitor, (R)-PK11195, at doses ranging from 5 to 10 mg/kg. Kinetic modeling was performed using one-tissue compartment model (1TCM), two-tissue compartment model (2TCM) and Logan graphical analyses, using dynamic PET data acquisition, arterial blood collection and metabolic stability testing. Clinical PET scans were performed in two healthy volunteers (HVs). Regional brain standard uptake value ratio (SUVr) was assessed for different time intervals. RESULTS: [18F]LW223 was synthesized in non-decay corrected radiochemical yields (n.d.c. RCYs) of 33.3 ± 6.5% with molar activities ranging from 1.8 ± 0.7 Ci/µmol (n = 11). [18F]LW223 was stable in formulation for up to 4 h and LogD7.4 of 2.31 ± 0.13 (n = 6) and fu of 5.80 ± 1.42% (n = 6) were determined. [18F]LW223 exhibited good brain penetration in NHPs, with a peak SUV value of ca. 1.79 in the whole brain. Pre-treatment with (R)-PK11195 substantially accelerated the washout and attenuated the area under the time-activity curve, indicating in vivo specificity of [18F]LW223 towards TSPO. Kinetic modeling demonstrated that 2TCM was the most suitable model for [18F]LW223-based neuroimaging. Global transfer rate constants (K1) and total volumes of distribution (VT) were found to be 0.10 ± 0.01 mL/cm3/min and 2.30 ± 0.17 mL/cm3, respectively. Dynamic PET data analyses across distinct time windows revealed that the VT values were relatively stable after 60 min post-injection. In a preliminary clinical study with two healthy volunteers, [18F]LW223 exhibited good brain uptake and considerable tracer retention across all analyzed brain regions. Of note, an excellent correlation between SUVr with VT was obtained when assessing the time interval from 20 to 40 min post tracer injection (SUVr(20-40 min), R2 = 0.94, p < 0.0001), suggesting this time window may be suitable to estimate specific binding to TSPO in human brain. CONCLUSION: Our findings indicate that [18F]LW223 is suitable for quantitative TSPO-targeted PET imaging in higher species. Employing state-of-the-art kinetic modeling, we found that [18F]LW223 was effective in mapping TSPO throughout the NHP brain, with best model fits obtained from 2TCM and Logan graphical analyses. Overall, our results indicate that [18F]LW223 exhibits favorable tracer performance characteristics in higher species, and this novel imaging tool may hold promise to provide effective neuroinflammation imaging in patients with neurological disease.

Nonenzymatic function of DPP4 promotes diabetes-associated cognitive dysfunction through IGF-2R/PKA/SP1/ERp29/IP3R2 pathway-mediated impairment of Treg function and M1 microglia polarization.

BACKGROUND: Impairment of regulatory T (Treg) cells function is implicated in the pathogenesis of immune imbalance-mediated cognitive impairment. A complete understanding of whether and how this imbalance affect cognitive function in type 2 diabetes is lacking, and the driver affecting this imbalance remains unknown. METHODS: We examined the impact of enzymatic and non-enzymatic function of DPP4 on Treg cell impairment, microglia polarization and diabetes-associated cognitive defects and identified its underlying mechanism in type 2 diabetic patients with cognitive impairment and in db/db mice. RESULTS: We report that DPP4 binds to IGF2-R on Treg cell surface and activates PKA/SP1 signaling, which upregulate ERp29 expression and promote its binding to IP3R2, thereby inhibiting IP3R2 degradation and promoting mitochondria-associated ER membrane formation and mitochondria calcium overload in Tregs. This, in turn, impairs Tregs function and polarizes microglia toward a pro-inflammatory phenotype in the hippocampus and finally leads to neuroinflammation and cognitive impairment in type 2 diabetes. Importantly, inhibiting DPP4 enzymatic activity in type 2 diabetic patients or mutating DPP4 enzymatic active site in db/db mice did not reverse these changes. However, IGF-2R knockdown or blockade ameliorated these effects both in vivo and in vitro. CONCLUSION: These findings highlight the nonenzymatic role of DPP4 in impairing Tregs function, which may facilitate the design of novel immunotherapies for diabetes-associated cognitive impairment.

Associations between genetic variants in sphingolipid metabolism pathway genes and hepatitis B virus-related hepatocellular carcinoma survival.

Background: Although the sphingolipid metabolism pathway is known to play a significant role in tumor progression, there have been few studies on how genetic variants in the sphingolipid metabolism pathway genes affect the survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Methods: We utilized available genotyping data to conduct multivariate Cox proportional hazards regression model analysis, examining the associations of 12,188 single nucleotide polymorphisms (SNPs) in 86 sphingolipid metabolism pathway genes on the survival of 866 HBV-HCC patients, and the model was also used in additive interaction analysis. We used bioinformatics functional prediction and expression quantitative trait locus (eQTL) analysis to explore the potential functions of SNPs and to evaluate the association of SNPs with the corresponding mRNA expression, respectively. We also used the online database TIMER2.0 (http://timer.comp-genomics.org/) to analyze the relationship between the corresponding mRNA expression levels and immune cell infiltration. Results: Our study found that GBA2 rs1570247 G>A was significantly associated with elevated survival of HBV-HCC patients [(hazards ratio (HR)=0.74, 95% confidence interval (CI)=0.64-0.86, P<0.001)]. And on an additive scale, a synergistic effect was observed between the GG genotype of rs1570247 and advanced BCLC stage. Among HBV-HCC patients with advanced BCLC stage, those carrying the GBA2 rs1570247 GG genotype exhibited a significantly elevated risk of mortality (HR=3.32, 95%CI=2.45-4.50). Further functional prediction and eQTL analysis revealed that rs1570247 were located in the 5' untranslated region of the GBA2, the A allele of SNP rs1570247 was associated with higher mRNA expression levels of GBA2 in normal liver tissues (P=0.009). Moreover, we observed a positive correlation between GBA2 mRNA expression and the infiltration level of B lymphocytes cell (R=0.331, P<0.001), while a negative correlation was noted between GBA2 mRNA expression and the infiltration level of macrophage M2 in HCC (R=-0.383, P<0.001). Conclusion: Our findings suggest that GBA2 rs1570247 G>A in sphingolipid metabolism pathway may be a key factor for survival of HBV-HCC patients by regulating the expression of corresponding genes and affecting the infiltration level of immune cells.

Discovery of a highly specific 18F-labeled PET ligand for phosphodiesterase 10A enabled by novel spirocyclic iodonium ylide radiofluorination.

As a member of cyclic nucleotide phosphodiesterase (PDE) enzyme family, PDE10A is in charge of the degradation of cyclic adenosine (cAMP) and guanosine monophosphates (cGMP). While PDE10A is primarily expressed in the medium spiny neurons of the striatum, it has been implicated in a variety of neurological disorders. Indeed, inhibition of PDE10A has proven to be of potential use for the treatment of central nervous system (CNS) pathologies caused by dysfunction of the basal ganglia-of which the striatum constitutes the largest component. A PDE10A-targeted positron emission tomography (PET) radioligand would enable a better assessment of the pathophysiologic role of PDE10A, as well as confirm the relationship between target occupancy and administrated dose of a given drug candidate, thus accelerating the development of effective PDE10A inhibitors. In this study, we designed and synthesized a novel 18F-aryl PDE10A PET radioligand, codenamed [18F]P10A-1910 ([18F]9), in high radiochemical yield and molar activity via spirocyclic iodonium ylide-mediated radiofluorination. [18F]9 possessed good in vitro binding affinity (IC50 = 2.1 nmol/L) and selectivity towards PDE10A. Further, [18F]9 exhibited reasonable lipophilicity (logD = 3.50) and brain permeability (P app > 10 × 10-6 cm/s in MDCK-MDR1 cells). PET imaging studies of [18F]9 revealed high striatal uptake and excellent in vivo specificity with reversible tracer kinetics. Preclinical studies in rodents revealed an improved plasma and brain stability of [18F]9 when compared to the current reference standard for PDE10A-targeted PET, [18F]MNI659. Further, dose-response experiments with a series of escalating doses of PDE10A inhibitor 1 in rhesus monkey brains confirmed the utility of [18F]9 for evaluating target occupancy in vivo in higher species. In conclusion, our results indicated that [18F]9 is a promising PDE10A PET radioligand for clinical translation.

Toxicity assessments and transcriptional effects of monofunctionalized Pt(II) complex under dark and light irradiation condition in Caenorhabditis elegans.

In vivo toxicity of aromatic ring (BODIPY, 1,3,5,7,8-pentamethyl dipyrrin borondifluoride) attached monofunctional Pt(II) complexes mCBP {[cis-Pt(NH3)2Cl] 8-(para-pyridine-methylene),1,3,5,7-tetramethyl dipyrrin borondifluoride}+ Nitrate- and dCBP {[cis-Pt(NH3)2Cl]28-(1,3-pyrimidine-5-methylene),1,3,5,7-tetramethyl dipyrrin borondifluoride}2+ diNitrate2- were tested in Caenorhabditis elegans (C. elegans). dCBP showed promising reactive oxygen ROS (reactive oxygen species) generating capability. This complex resulted reduction of lifespan, body length and egg laying rate under dark and light irradiation in both N2 (wild-type, cisplatin resistant) and ok938 (asna-1, cisplatin sensitive) C. elegans. Expressional change of several key cancer related pathway (JNK (c-Jun N-terminal kinase) and Wnt/ß-catenin (Wingless/Integrated/ß-catenin)) related genes (for instance, jnk-1, wrm-1 and gst-4) were confirmed by RNA sequencing experiments. These transcriptional alternations could explain physiological parameters change in nematode and partially revealed how both Pt(II) based complexes influence cancer related pathways. Furthermore, these associated genes exhibited the function of apoptosis, reduced chemoresistance of cancer cells and most of those expressional changes were linked to extended survival of cancer patients.

Cancer-associated fibroblasts support bone tropic metastasis by acting as coordinators between the tumor microenvironment and bone matrix in breast cancer.

Bone is a common site of metastasis for various types of cancer cells, including breast cancer, and the consequent skeleton-related events observed in patients are severe and often fatal. Currently, it is widely accepted that cancer-associated fibroblasts (CAFs) confer a metastasis-promoting property to breast cancer cells. Furthermore, clinical observations suggest that CAFs mediate the bone tropism of metastatic breast cancer cells. Therefore, a deeper understanding of the mechanism by which CAFs are involved in the bone-tropic metastasis of breast cancer can facilitate the study of the novel and effective therapeutic drugs for the corresponding targets. In this review, we focused on the coordinator role of CAFs in remolding breast cancer cells and remodeling the bone marrow during metastasis. We discussed the potential roles of the CXCL12/CXCR4 axis, the CAFs-CSCs reinforcing loop, and exosomes in this malignant process. In summary, in agreement with Paget's theory, CAFs play a pivotal role in bone colonization by breast cancer cells by providing a "fertile soil" for the "selected seeds" by influencing tumor-intrinsic characteristics and microenvironment (ME).

Marriage of black phosphorus and Cu2+ as effective photothermal agents for PET-guided combination cancer therapy.

The use of photothermal agents (PTAs) in cancer photothermal therapy (PTT) has shown promising results in clinical studies. The rapid degradation of PTAs may address safety concerns but usually limits the photothermal stability required for efficacious treatment. Conversely, PTAs with high photothermal stability usually degrade slowly. The solutions that address the balance between the high photothermal stability and rapid degradation of PTAs are rare. Here, we report that the inherent Cu2+-capturing ability of black phosphorus (BP) can accelerate the degradation of BP, while also enhancing photothermal stability. The incorporation of Cu2+ into BP@Cu nanostructures further enables chemodynamic therapy (CDT)-enhanced PTT. Moreover, by employing 64Cu2+, positron emission tomography (PET) imaging can be achieved for in vivo real-time and quantitative tracking. Therefore, our study not only introduces an "ideal" PTA that bypasses the limitations of PTAs, but also provides the proof-of-concept application of BP-based materials in PET-guided, CDT-enhanced combination cancer therapy.

Single-nucleotide polymorphism rs17548629 in RIPK1 gene may be associated with lung cancer in a young and middle-aged Han Chinese population.

BACKGROUND: Genetic biomarkers of lung cancer (LC) susceptibility may provide a basis for treatment and prevention. This study analyzed an association between SNPs (single nucleotide polymorphisms) in the complementary region of the 3'-UTR (3' untranslated region) of microRNAs of the gene RIPK1 (receptor-interacting serine/threonine-protein kinase 1) and LC among an adult Han Chinese population aged younger than 60 years. Also explored the effect of regulation of the RIPK1 gene via rs17548629 and microRNA-1197 on the occurrence of LC. METHODS: RIPK1 variants (rs17548629, rs77736895) were determined in a population of 571 adults (younger than 60 years) with LC, and 609 gender- and age-matched healthy individuals. Bioinformatics methods predicted the microRNAs bound to rs17548629. Dual luciferase reporter assay was performed to confirm the presence of both rs17548629 and the predicted microRNA. RESULTS: A mutation (T) of rs17548629 was associated with an increased risk for LC in this population under the codominant and recessive genetic models. The risk of lung adenocarcinoma in rs17548629 mutant carriers was 1.769-fold higher than that of the wildtype. In vitro, the luciferase activity of co-transfected mutant psiCHECK2-RIPK1 and microRNA-1197 mimics was less than that of the group transfected with microRNA-1197 mimics only. Factorial analysis indicated interactions between microRNA-1197 mimics and genotypes of rs17548629. CONCLUSION: A mutation (T) of rs17548629 may increase the risk of LC/lung adenocarcinoma in adult Han populations younger than 60 years. When carrying the T allele, rs17548629 may be the target of hsa-miR-1197. This mutation may affect transcriptional level of the RIPK1, thereby promoting the occurrence of LC.

Granulocyte colony-stimulating factor and stromal cell-derived factor-1 combination therapy: A more effective treatment for cerebral ischemic stroke.

BACKGROUND: Drugs that promote angiogenesis include statins, recombinant human granulocyte colony-stimulating factor, and stromal cell-derived factor-1. Low doses of atorvastatin could significantly increase the vascular expressions of endothelial growth factor, and the number of peripheral blood endothelial progenitor cells (EPCs), thus improving angiogenesis and local blood flow. G-CSF is an EPC-mobilization agent used in ischemia studies for targeting angiogenesis after cerebral ischemia via EPCs. In previous clinical trials, consistent conclusions have not been reached about the effectiveness of G-CSF on ischemic stroke. Therefore, the therapeutic effect of G-CSF and its combination with other medicines need further experimental verification. It is known that atorvastatin, rhG-CSF, and SDF-1 are considered the most promising neuroprotective candidates, but a comprehensive comparison of their effects is lacking. AIMS: To compare the effects of atorvastatin, stromal cell-derived factor-1, and recombinant human granulocyte colony-stimulating factor on ischemic stroke. METHODS: Adult male Sprague-Dawley rats were randomly allocated to three groups: normal, sham-operated, and middle cerebral artery occlusion operated. Middle cerebral artery occlusion operated rats were further allocated into saline, atorvastatin, recombinant human granulocyte colony-stimulating factor, and recombinant human granulocyte colony-stimulating factor + stromal cell-derived factor-1 groups. Neurological function evaluation, cerebral infarction and the blood-brain barrier integrity analysis, identification of angiogenic factors, assessment of angiogenesis, expression of growth-associated protein-43, neuroglobin, glial cell-derived neurotrophic factor, and cleaved caspase 3, were performed. RESULTS: Compared with atorvastatin or recombinant human granulocyte colony-stimulating factor alone, recombinant human granulocyte colony-stimulating factor + stromal cell-derived factor-1 treatment improved neurological performance, reduced cerebral infarction and blood-brain barrier disruption after stroke, and increased the content of stromal cell-derived factor-1, vascular endothelial growth factor, monocyte chemotactic protein 1, and basic fibroblast growth factor in peripheral blood. In addition, recombinant human granulocyte colony-stimulating factor + stromal cell-derived factor-1 promoted greater angiogenesis than atorvastatin or recombinant human granulocyte colony-stimulating factor alone and increased the expression of growth-associated protein-43, neuroglobin, and glial cell-derived neurotrophic factor, while decreasing the levels of cleaved caspase 3 in the brain after ischemic stroke. CONCLUSIONS: Combination therapy with recombinant human granulocyte colony-stimulating factor and stromal cell-derived factor-1 is more effective than atorvastatin or recombinant human granulocyte colony-stimulating factor alone in protecting against stroke-induced damage and could be an optimal therapeutic strategy for stroke.

Spontaneous reactivation of a site-specifically placed transgene independent of copy number or DNA methylation.

As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.

Protease-activated receptor-1 (PAR1) promotes epithelial-endothelial transition through Twist1 in hepatocellular carcinoma.

BACKGROUND: Tumor cells transfer into endothelial cells by epithelial-endothelial transition (EET), which is characterized by vasculagenic mimicry (VM) in morphology. VM can change tumor microcirculation, progression, and metastasis. However, the molecular mechanisms of endothelial-like transition remain unclear. EET is a subtype of epithelial-mesenchymal transition (EMT). Twist1, a transcriptional regulatory factor of EMT, is an important factor that induces EET in hepatocellular carcinoma(HCC), but the upstream signal of Twist1 is unclear. METHODS: Expression plasmids, Ca mobilization, and three-dimensional cultures were evaluated. Western blot assay, reporter gene assay, and immunofluorescence staining were conducted. A murine xenograft model was established. Analyses of immunohistochemistry, patient samples, and complementary DNA (cDNA) microarrays were also performed. RESULTS: This study demonstrated that protease-activated receptor-1 (PAR1) can increase the expression of endothelial markers and enhance VM formation by upregulating Twist1 both in vitro and in vivo through thrombin binding. Thrombin not only activates PAR1 but also promotes PAR1 internalization in a time-dependent manner. Clinical pathological analysis further confirms that PAR1 expression is directly correlated with the endothelial marker expression, VM formation, and metastasis and indicates poor survival rate of patients with tumors. CONCLUSION: PAR1 promotes EET through Twist1 in HCC.

Twist1 confers multidrug resistance in colon cancer through upregulation of ATP-binding cassette transporters.

Multidrug resistance is a major problem in colon cancer treatment. However, its molecular mechanisms remain unclear. Recently, the epithelial-mesenchymal transition (EMT) in anticancer drug resistance has attracted increasing attention. This study investigated whether vincristine treatment induces EMT and promotes multidrug resistance in colon cancer. The result showed that vincristine treatment increases the expression of several ATP-binding cassette transporters in invasive human colon adenocarcinoma cell line (HCT-8). Vincristine-resistant HCT-8 cells (HCT-8/V) acquire a mesenchymal phenotype, and thus its migratory and invasive ability are increased both in vitro and in vivo. The master transcriptional factors of EMT, especially Twist1, were significantly increased in the HCT-8/V cell line. Moreover, the ectopic expression of Twist1 increased the chemoresistance of HCT-8 cells to vincristine and increased the expression levels and promoter activities of ABCB1 and ABCC1. Furthermore, Twist1 silencing reverses the EMT phenotype, enhances the chemosensitivity of HCT-8/ V cells to anticancer agents in vitro and in vivo, and downregulates the expression of ABCB1 and ABCC1. Twist1-mediated promotion of ABCB1 and ABCC1 expression levels plays an important role in the drug resistance of colon cancer cells.

Testing Einstein's Equivalence Principle With Fast Radio Bursts.

The accuracy of Einstein's equivalence principle (EEP) can be tested with the observed time delays between correlated particles or photons that are emitted from astronomical sources. Assuming as a lower limit that the time delays are caused mainly by the gravitational potential of the Milky Way, we prove that fast radio bursts (FRBs) of cosmological origin can be used to constrain the EEP with high accuracy. Taking FRB 110220 and two possible FRB/gamma-ray burst (GRB) association systems (FRB/GRB 101011A and FRB/GRB 100704A) as examples, we obtain a strict upper limit on the differences of the parametrized post-Newtonian parameter γ values as low as [γ(1.23 GHz)-γ(1.45 GHz)]<4.36×10(-9). This provides the most stringent limit up to date on the EEP through the relative differential variations of the γ parameter at radio energies, improving by 1 to 2 orders of magnitude the previous results at other energies based on supernova 1987A and GRBs.