[Main characteristics and practical discussions of national drug standards for 196 kinds of Chinese medicine formula granules].

As China is implementing the policy of "Announcement on Ending the Pilot Work of Chinese medicine formula gra-nules", the standard of Chinese medicine formula granules has gradually become the focus of industry development. Up to now, 196 national drug standards for Chinese medicine formula granules have been published by China, which guaranteed the production quality of Chinese medicine formula granules. However, there are still several challenges such as the rational application of national drug standards and the enrichment and improvement of varieties. The basic content of the issued national drug standards for Chinese medicine formula granules was analyzed and compared with the quality standard provisions of the corresponding decoction pieces in the Chinese Pharmacopoeia(2020 edition) in this paper. This paper discussed the main characteristics of paste-forming rate of each medicinal raw materials, "quantity-quality" transformation, equivalent ratio, and so on, and clarified the characteristics of the national standard for Chinese medicine formula granules. This paper provided references for achieving the unified quality control and meeting the overall quality requirements of Chinese medicine formula granules.

[Clinical Characteristics of Acute Myeloid Leukemia Patients with RUNX1 Gene Mutation].

OBJECTIVE: To investigate the incidence of Runt-related transcription factor 1 (RUNX1) gene and its associated gene mutations in patients with acute myeloid leukemia (AML), and analyze its clinical characteristics and prognosis. METHODS: The genomic DNA-PCR method was used to detect the exon of RUNX1 gene, and the gene mutations were analyzed by genetic sequencing. NPM1, DNMT3A, FLT3-ITD, IDH1/2, K/N-RAS, CEPBA, TET2, and WT1 co-mutations were also detected. Patients were followed up to determine efficacy and prognosis. RESULTS: Among 171 patients, the RUNX1 gene mutation was detected in 17 cases, and the mutation rate was 9.9%. The type of RUNX1 gene mutation was 9 missense mutations, 4 frameshift mutations, and 4 nonsense mutations. The peripheral blood leukocyte count of the patients in mutation group was 3 (1-101) ×109/L, which was significantly lower than those in the non-mutation group [26 (1-298)×109/L] (P=0.002), while the platelet count was 79 (22-166)×109/L, which was higher than 50 (8-351)×109/L in the non-mutation group (P=0.010), and the proportion of bone marrow blasts of the patients in the mutation group was 37 (0-72)%, which was lower than 53 (0-98)% in the non-mutation group (P=0.020). The RUNX1 mutation rate in M0 type was 55.6%, and in M4 type was 13.6%, which was significantly higher than other FAB subtypes (P=0.003). There was no significant difference in age, sex, hemoglobin concentration, and counts of peripheral blood mononuclear cells (P>0.05). The prognosis of cytogenetics in the patients in the middle and high-risk groups was 88.1% and 89.7%, which were significantly higher than that in the low-risk group, and the difference showed statistically significant (P=0.018). There was no significantly relationship between RUNX1 and specific karyotype abnormalities, including Trisomy 8, Del (7q), t (8; 21), and Inv (16) (P>0.05). There was a significant relationship between RUNX1 gene mutation and IDH1/2, N/K-RAS gene mutation (P<0.01). The complete response rate (CR) of the patients with chemotherapy in the RUNX1 mutation group(37.5%) was significantly lower than 79.4% in the non-mutated group (P=0.001). The overall survival (OS) of the patients in RUNX1 gene mutation group was lower than that in non-mutation group (P<0.05). CONCLUSION: AML patients with RUNX1 gene mutation shows unique clinical and biological characteristics, RUNX1 mutation can be regarded as a molecular marker of poor prognosis in AML patients.

Exploring prognostic indicators in the pathological images of hepatocellular carcinoma based on deep learning.

OBJECTIVE: Tumour pathology contains rich information, including tissue structure and cell morphology, that reflects disease progression and patient survival. However, phenotypic information is subtle and complex, making the discovery of prognostic indicators from pathological images challenging. DESIGN: An interpretable, weakly supervised deep learning framework incorporating prior knowledge was proposed to analyse hepatocellular carcinoma (HCC) and explore new prognostic phenotypes on pathological whole-slide images (WSIs) from the Zhongshan cohort of 1125 HCC patients (2451 WSIs) and TCGA cohort of 320 HCC patients (320 WSIs). A 'tumour risk score (TRS)' was established to evaluate patient outcomes, and then risk activation mapping (RAM) was applied to visualise the pathological phenotypes of TRS. The multi-omics data of The Cancer Genome Atlas(TCGA) HCC were used to assess the potential pathogenesis underlying TRS. RESULTS: Survival analysis revealed that TRS was an independent prognosticator in both the Zhongshan cohort (p<0.0001) and TCGA cohort (p=0.0003). The predictive ability of TRS was superior to and independent of clinical staging systems, and TRS could evenly stratify patients into up to five groups with significantly different prognoses. Notably, sinusoidal capillarisation, prominent nucleoli and karyotheca, the nucleus/cytoplasm ratio and infiltrating inflammatory cells were identified as the main underlying features of TRS. The multi-omics data of TCGA HCC hint at the relevance of TRS to tumour immune infiltration and genetic alterations such as the FAT3 and RYR2 mutations. CONCLUSION: Our deep learning framework is an effective and labour-saving method for decoding pathological images, providing a valuable means for HCC risk stratification and precise patient treatment.

Heterogeneous immunogenomic features and distinct escape mechanisms in multifocal hepatocellular carcinoma.

BACKGROUND & AIMS: The presence of multifocal tumors, developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO), is a distinct feature of hepatocellular carcinoma (HCC). Immunogenomic characterization of multifocal HCC is important for understanding immune escape in different lesions and developing immunotherapy. METHODS: We combined whole-exome/transcriptome sequencing, multiplex immunostaining, immunopeptidomes, T cell receptor (TCR) sequencing and bioinformatic analyses of 47 tumors from 15 patients with HCC and multifocal lesions. RESULTS: IM and MO demonstrated distinct clonal architecture, mutational spectrum and genetic susceptibility. The immune microenvironment also displayed spatiotemporal heterogeneity, such as less T cell and more M2 macrophage infiltration in IM and higher expression of inhibitory immune checkpoints in MO. Similar to mutational profiles, shared neoantigens and TCR repertoires among tumors from the same patients were abundant in IM but scarce in MO. Combining neoantigen prediction and immunopeptidomes identified T cell-specific neoepitopes and achieved a high verification rate in vitro. Immunoediting mainly occurred in MO but not IM, due to the relatively low immune infiltration. Loss of heterozygosity of human leukocyte antigen (HLA) alleles, identified in 17% of multifocal HCC, hampered the ability of major histocompatibility complex to present neoantigens, especially in IM. An integrated analysis of Immunoscore, immunoediting, TCR clonality and HLA loss of heterozygosity in each tumor could stratify patients into 2 groups based on whether they have a high or low risk of recurrence (p = 0.038). CONCLUSION: Our study comprehensively characterized the genetic structure, neoepitope landscape, T cell profile and immunoediting status that collectively shape tumor evolution and could be used to optimize personalized immunotherapies for multifocal HCC. LAY SUMMARY: Immunogenomic features of multifocal hepatocellular carcinoma (HCC) are important for understanding immune-escape mechanisms and developing more effective immunotherapy. Herein, comprehensive immunogenomic characterization showed that diverse genomic structures within multifocal HCC would leave footprints on the immune landscape. Only a few tumors were under the control of immunosurveillance, while others evaded the immune system through multiple mechanisms that led to poor prognosis. Our study revealed heterogeneous immunogenomic landscapes and immune-constrained tumor evolution, the understanding of which could be used to optimize personalized immunotherapies for multifocal HCC.

Activated and Exhausted MAIT Cells Foster Disease Progression and Indicate Poor Outcome in Hepatocellular Carcinoma.

PURPOSE: Innate immunity is an indispensable arm of tumor immune surveillance, and the liver is an organ with a predominance of innate immunity, where mucosal-associated invariant T (MAIT) cells are enriched. However, little is known about the phenotype, functions, and immunomodulatory role of MAIT cells in hepatocellular carcinoma (HCC).Experimental Design: The distribution, phenotype, and function of MAIT cells in patients with HCC were evaluated by both flow cytometry (FCM) and in vitro bioassays. Transcriptomic analysis of MAIT cells was also performed. Prognostic significance of tumor-infiltrating MAIT cells was validated in four independent cohorts of patients with HCC. RESULTS: Despite their fewer densities in HCC tumor than normal liver, MAIT cells were significantly enriched in the HCC microenvironment compared with other mucosa-associated organs. Tumor-derived MAIT cells displayed a typical CCR7-CD45RA-CD45RO+CD95+ effector memory phenotype with lower costimulatory and effector capabilities. Tumor-educated MAIT cells significantly upregulated inhibitory molecules like PD-1, CTLA-4, TIM-3, secreted significantly less IFNγ and IL17, and produced minimal granzyme B and perforin while shifting to produce tumor-promoting cytokines like IL8. Transcriptome sequencing confirmed that tumor-derived MAIT cells were reprogrammed toward a tumor-promoting direction by downregulating genes enriched in pathways of cytokine secretion and cytolysis effector function like NFKB1 and STAT5B and by upregulating genes like IL8, CXCL12, and HAVCR2 (TIM-3). High infiltration of MAIT cells in HCC significantly correlated with an unfavorable clinical outcome, revealed by FCM, qRT-PCR, and multiplex IHC analyses, respectively. CONCLUSIONS: HCC-infiltrating MAIT cells were functionally impaired and even reprogrammed to shift away from antitumor immunity and toward a tumor-promoting direction.See related commentary by Carbone, p. 3199.

CCL15 Recruits Suppressive Monocytes to Facilitate Immune Escape and Disease Progression in Hepatocellular Carcinoma.

Chemokines play a key role in orchestrating the recruitment and positioning of myeloid cells within the tumor microenvironment. However, the tropism regulation and functions of these cells in hepatocellular carcinoma (HCC) are not completely understood. Herein, by scrutinizing the expression of all chemokines in HCC cell lines and tissues, we found that CCL15 was the most abundantly expressed chemokine in human HCC. Further analyses showed that CCL15 expression was regulated by genetic, epigenetic, and microenvironmental factors, and negatively correlated with patient clinical outcome. In addition to promoting tumor invasion in an autocrine manner, CCL15 specifically recruited CCR1+ cells toward HCC invasive margin, approximately 80% of which were CD14+ monocytes. Clinically, a high density of marginal CCR1+ CD14+ monocytes positively correlated with CCL15 expression and was an independent index for dismal survival. Functionally, these tumor-educated monocytes directly accelerated tumor invasion and metastasis through bursting various pro-tumor factors and activating signal transducer and activator of transcription 1/3, extracellular signal-regulated kinase 1/2, and v-akt murine thymoma viral oncogene homolog signaling in HCC cells. Meanwhile, tumor-derived CCR1+ CD14+ monocytes expressed significantly higher levels of programmed cell death-ligand 1, B7-H3, and T-cell immunoglobulin domain and mucin domain-3 that may lead to immune suppression. Transcriptome sequencing confirmed that tumor-infiltrating CCR1+ CD14+ monocytes were reprogrammed to upregulate immune checkpoints, immune tolerogenic metabolic enzymes (indoleamine and arginase), inflammatory/pro-angiogenic cytokines, matrix remodeling proteases, and inflammatory chemokines. Orthotopic animal models confirmed that CCL15-CCR1 axis forested an inflammatory microenvironment enriched with CCR1+ monocytes and led to increased metastatic potential of HCC cells. Conclusion: A complex tumor-promoting inflammatory microenvironment was shaped by CCL15-CCR1 axis in human HCC. Blockade of CCL15-CCR1 axis in HCC could be an effective anticancer therapy.

Clinical significance of PD-1/PD-Ls gene amplification and overexpression in patients with hepatocellular carcinoma.

Background: The remarkable clinical activity of PD-1 antibody in advanced hepatocellular carcinoma (HCC) highlights the importance of PD-1/PD-L1-mediated immune escape as therapeutic target in HCC. However, the frequency and prognostic significance of PD-Ls genetic alterations in HCC remain unknown. Methods: Fluorescence in situ hybridization were used to determine PD-Ls genetic alterations, and qPCR data coupled with immunofluorescence were used to measure the mRNA and protein levels of PD-Ls. Clinical relevance and prognostic value of 9p24.1 genetic alterations were investigated on tissue microarray containing three independent cohorts of 578 HCC patients. The results were further validated in an independent cohort of 442 HCC patients from The Cancer Genome Atlas (TCGA) database. Results: In total, 7.1%-15.0% for amplification and 15.8%-31.3% for polysomy of 9p24.1 were revealed in three cohorts of HCC patients, similar to the objective response rate of PD-1 antibody in HCC. Patients with 9p24.1 genetic alterations significantly and independently correlated with unfavorable outcomes than those without. FISH and qPCR data coupled with immunofluorescence revealed that genetic alterations of 9p24.1 robustly contributed to PD-L1 and PD-L2 upregulation. In addition, increased expression of PD-L1 instead of PD-L2 also predicted poor survival by multivariate analyses. Meanwhile, high infiltration of PD-1+ immune cells also indicated dismal survival in HCC. Conclusions: Amplification or higher expression of PD-L1 significantly and independently correlated with unfavorable survival in HCC patients, authenticating the PD-1/PD-L1 axis as rational immunotherapeutic targets for HCC.

Spatial and temporal clonal evolution of intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second-most lethal primary liver cancer. Little is known about intratumoral heterogeneity (ITH) and its impact on ICC progression. We aimed to investigate the ITH of ICC in the hope of helping to develop new therapeutic strategies. METHODS: We obtained 69 spatially distinct regions from six operable ICCs. Patient-derived primary cancer cells (PDPCs) were established for each region, followed by whole-exome sequencing (WES) and multi-level validation. RESULTS: We observed widespread ITH for both somatic mutations and clonal architecture, shaped by multiple mechanisms, like clonal "illusion", parallel evolution and chromosome instability. A median of 60.3% of mutations were heterogeneous, among which 85% of the driver mutations were located on the branches of tumor phylogenetic trees. Many truncal and clonal driver mutations occurred in tumor suppressor genes, such as TP53, SMARCB1 and PBRM1 that are involved in DNA repair and chromatin-remodeling. Genome doubling occurred in most cases (5/6) after the accumulation of truncal mutations and was shared by all intratumoral sub-regions. In all cases, ongoing chromosomal instability is evident throughout the evolutionary trajectory of ICC. The recurrence of ICC1239 provided evidence to support the polyclonal metastatic seeding in ICC. The change of mutation landscape and internal diversity among subclones during metastasis, such as the loss of chemoresistance mediator, can be used for new treatment strategies. Targeted therapy against truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, was developed in 5/6 patients. CONCLUSIONS: Integrated investigations of spatial ITH and clonal evolution may provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ICC. LAY SUMMARY: We applied multiregional whole-exome sequencing to investigate the evolution of intrahepatic cholangiocarcinoma (ICC). The results revealed that many factors, such as parallel evolution and chromosome instability, may participate and promote the branch diversity of ICC. Interestingly, in one patient with primary and recurrent metastatic tumors, we found evidence of polyclonal metastatic seeding, indicating that symbiotic communities of multiple clones existed and were maintained during metastasis. More realistically, some truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, could be promising treatment targets in patients with ICC.

FOXP3 Is a HCC suppressor gene and Acts through regulating the TGF-ß/Smad2/3 signaling pathway.

BACKGROUND: FOXP3 has been discovered to be expressed in tumor cells and participate in the regulation of tumor behavior. Herein, we investigated the clinical relevance and biological significance of FOXP3 expression in human hepatocellular carcinoma (HCC). METHODS: Expression profile of FOXP3 was analyzed using real-time RT-PCR, western blotting and immunofluorescence on HCC cell lines, and immunostaing of a tissue microarray containing of 240 primary HCC samples. The potential regulatory roles of FOXP3 were dissected by an integrated approach, combining biochemical assays, analysis of patient survival, genetic manipulation of HCC cell lines, mouse xenograft tumor models and chromatin immunoprecipitation (ChIP) sequencing. RESULTS: FOXP3 was constitutively expressed in HCC cells with the existence of splice variants (especially exon 3 and 4 deleted, Δ3,4-FOXP3). High expression of FOXP3 significantly correlated with low serum α-fetoprotein (AFP) level, absence of vascular invasion and early TNM stage. Survival analyses revealed that increased FOXP3 expression was significantly associated with better survival and reduced recurrence, and served as an independent prognosticator for HCC patients. Furthermore, FOXP3 could potently suppress the proliferation and invasion of HCC cells in vitro and reduce tumor growth in vivo. However, Δ3,4-FOXP3 showed a significant reduction in the tumor-inhibiting effect. The inhibition of FOXP3 on HCC aggressiveness was acted probably by enhancing the TGF-ß/Smad2/3 signaling pathway. CONCLUSION: Our findings suggest that FOXP3 suppresses tumor progression in HCC via TGF-ß/Smad2/3 signaling pathway, highlighting the role of FOXP3 as a prognostic factor and novel target for an optimal therapy against this fatal malignancy.

Telomere length variation in tumor cells and cancer-associated fibroblasts: potential biomarker for hepatocellular carcinoma.

The role of telomere dysfunction and aberrant telomerase activities in hepatocellular carcinoma (HCC) has been overlooked for many years. This study aimed to delineate the variation and prognostic value of telomere length in HCC. Telomere-specific fluorescence in situ hybridization (FISH) and qPCR were used to evaluate telomere length in HCC cell lines, tumor tissues, and isolated non-tumor cells within the tumor. Significant telomere attrition was found in tumor cells and cancer-associated fibroblasts (CAFs) compared to their normal counterparts, but not in intratumor leukocytes or bile duct epithelial cells. Clinical relevance and prognostic value of telomere length were investigated on tissue microarrays of 257 surgically treated HCC patients. Reduced intensity of telomere signals in tumor cells or CAFs correlated with larger tumor size and the presence of vascular invasion (p < 0.05). Shortened telomeres in tumor cells or CAFs associated with reduced survival and increased recurrence, and were identified as independent prognosticators for HCC patients (p < 0.05). These findings were validated in an independent HCC cohort of 371 HCC patients from The Cancer Genome Atlas (TCGA) database, confirming telomere attrition and its prognostic value in HCC. We also showed that telomerase reverse transcriptase promoter (TERTp) mutation correlated with telomere shortening in HCC. Telomere variation in tumor cells and non-tumor cells within the tumor microenvironment of HCC was a valuable prognostic biomarker for this fatal malignancy. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Protein tyrosine phosphatase PTP4A1 promotes proliferation and epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma via the PI3K/AKT pathway.

The protein tyrosine phosphatase PTP4A1 is a key molecule that activates tyrosine phosphorylation, which is important for cancer progression and metastasis. However, the clinical implications and biological function of PTP4A1 in intrahepatic cholangiocarcinoma (ICC) remains unknown. Here, we showed that PTP4A1 was frequently overexpressed in ICC versus adjacent non-tumor tissues. This overexpression significantly correlated with aggressive tumor characteristics like the presence of lymph node metastasis and advanced tumor stages. Survival analysis further indicated that high PTP4A1 expression was significantly and independently associated with worse survival and increased recurrence in ICC patients. Moreover, through forced overexpression and knock-down of PTPT4A1, we demonstrated that PTP4A1 could significantly promote ICC cells proliferation, colony formation, migration, and invasion in vitro, and markedly enhance tumor progression in vivo. Mechanistically, PTP4A1 was involved in PI3K/AKT signaling and its downstream molecules, such as phosphorylation level of GSK3ß and up-regulation of CyclinD1, in ICC cells to promote proliferation. Importantly, PTP4A1 induced ICC cells invasion was through activating PI3K/AKT signaling controlled epithelial-mesenchymal transition (EMT) process by up-regulating Zeb1 and Snail. Thus, PTP4A1 may serve as a potential oncogene that was a valuable prognostic biomarker and therapeutic target for ICC.

Efficacy of losartan for improving insulin resistance and vascular remodeling in hemodialysis patients.

Insulin resistance and vascular remodeling are prevalent and predict cardiovascular mortality in hemodialysis patients. Angiotensin II (Ang II) may be involved in both pathogenesis. In the present study, we investigated the effects of the Ang II receptor blocker losartan on insulin resistance, arterial stiffness, and carotid artery structure in hemodialysis patients. Seventy-two hemodialysis patients were randomly assigned to receive either losartan 50 mg qd (n = 36) or ß-blocker bisoprolol 5 mg qd (n = 36). At the start and at month 12, ambulatory blood pressure (BP) monitoring, aortic pulse wave velocity (PWV) measurements, and carotid artery ultrasound were performed, and homeostasis model assessment index of insulin resistance (HOMA-IR) was determined. During the study period, bioimpedance method was used to evaluate volume status every 3 months. Home-monitored BPs were measured at least monthly. Ambulatory BP decreased significantly and similarly by either losartan or bisoprolol. Decreases in PWVs in losartan group at the end of month 12 were significantly greater than changes in PWV in bisoprolol group (0.9 ± 0.3 vs. 0.4 ± 0.5 m/s, P = 0.021). Common carotid artery intima-media cross-sectional area decreased significantly only in patients treated with losartan (20.3 ± 4.9 vs. 19.1 ± 5.1 mm(2) , P = 0.001), and HOMA-IR was also reduced in losartan group only (1.9 ± 1.0 vs. 1.7 ± 0.8, P = 0.003). Multiple regression analysis showed significant correlations between changes in PWV and changes in HOMA-IR. With comparable BP-lowering efficacy, losartan achieved better improvement in insulin sensitivity, arterial stiffness, and carotid artery hypertrophy in hemodialysis patients. The regression of arterial stiffness may be in part through attenuation in insulin resistance.

Increasing Dialysis Sodium Removal on Arterial Stiffness and Left Ventricular Hypertrophy in Hemodialysis Patients.

OBJECTIVE: The aim of the present study was to examine the effects of a mild increase in dialysis sodium removal on cardiovascular system in hypertensive hemodialysis (HD) patients. METHODS: Sixty four HD patients with pre-HD plasma sodium level higher than 138mmol/l, were randomly assigned into 2 groups. The dialysate sodium was reduced from 138mmol/l to 136mmol/l in the intervention group, while remained at 138mmol/l in the control group. During the study course, home systolic blood pressure (BP) target of 140mmHg was used in all patients, and bioimpedance measurements to guide ultrafiltration were performed monthly. 44-hour ambulatory BP, aortic pulse wave velocity (PWV), left ventricular mass index (LVMI), pre-HD plasma sodium concentration, interdialytic weight gain, and dietary sodium intake, were measured. RESULTS: Better BP control was achieved by 2 groups, with no significant differences. However, less annual averages of antihypertensives were used in the intervention group. The PWV values significantly decreased from 11.8±2.4 to 10.9±2.6m/s in the intervention group (P<0.001), and from 11.6±2.5 to 11.1±2.2m/s in the control group (P=0.012). LVMI regressed from 151±19 to 139±16 g/m2 (P<0.001) in the intervention group only. In addition, values for interdialytic weight gain and pre-HD plasma sodium decreased in the intervention group only. There were no significant differences in annual averages of dietary sodium intake and the frequency of adverse events between the 2 groups. CONCLUSIONS: Increasing dialysis sodium removal was associated with improvements in arterial stiffness, left ventricular hypertrophy, and better BP control in hypertensive HD patients.

[Effect of Cynomorium songaricum polysaccharide on telomere of lung cancer A549 cells].

To study the effect of Cynomorium songaricum polysaccharide (CSRP) on A549 cells telomere of human non-small cell lung cancer, the mice were intragastric administrated with CSRP (0.08 g•kg⁻¹) once daily for 4 days. Then their serum was taken for preparing CSRP drug serum. A549 cells were treated by the drug serum, and the effect of drug serum with different concentrations and different treating time on the proliferation of non-small cell lung cancer A549 cells was determined by MTT test. After treating for 48 hours by the drug serum of different concentrations, the telomere length of the cells was determined by fluorescence quantitative polymerase chain reaction (qPCR); the mRNA expression of telomerase reverse transcriptase (TERT) was determined by RT-qPCR; the cells apoptosis was determined by TUNEL assay. The results demonstrated that CSRP of various concentrations could inhibit the proliferation of the lung cancer A549 cells significantly, and the inhibition effect was strongest at 48 hours with the concentration of 6.0 mL•L⁻¹. At 48 h, that CSRP of the concentrations from 1.5 to 12.0 mL•L⁻¹ could significantly shorten the telomere length of A549 cells, and the effect was strongest with the concentration of 1.5 mg•L⁻¹. CSRP of various concentrations could significantly inhibit the mRNA expression of TERT in A549 cells, and the inhibition effect was stronger when the concentration was ≥6.0 mL•L⁻¹. CSRP of various concentrations could promote A549 cells apoptosis, and the effect was stronger when the concentration was ≥6.0 mL•L⁻¹. In conclusion, CSRP has the anti-cancer effect, and the action mechanism may be associated with inhibiting TERT mRNA expression, shortening telomere length, inhibiting cells proliferation and promoting cells apoptosis.

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor-induced epithelial-mesenchymal transition.

UNLABELLED: Hepatocellular carcinoma (HCC) is the third-most lethal cancer worldwide. Understanding the molecular pathogenesis of HCC recurrence and metastasis is the key to improve patients' prognosis. In this study, we report that protein tyrosine phosphatase receptor S (PTPRS) is significantly down-regulated in nearly 80% of HCCs, and its expression negatively correlates with aggressive pathological features, such as larger tumor size and advanced stage. In addition, PTPRS deficiency is independently associated with shorter survival and increased recurrence in patients, although 16.7% of HCCs show intratumor heterogeneous expression of PTPRS. Restoration of wild-type, but not mutant, PTPRS expression significantly inhibits HCC cell migration and invasion in vitro as well as lung metastasis in vivo, whereas knockdown of its expression significantly promotes invasion and metastasis. Notably, PTPRS-regulated HCC invasiveness is accompanied by typical changes of epithelial-mesenchymal transition (EMT). Moreover, PTPRS forms a complex with epithermal growth factor receptor (EGFR) and regulates its tyrosine residues' phosphorylation. Ectopic expression of EGFR reverses the metastasis-inhibiting effects of PTPRS, whereas silencing of EGFR or inhibiting phosphorylation of key molecules in EGFR downstream pathways reinhibits EMT and metastasis caused by PTPRS down-regulation. Meanwhile, promoter hypermethylation of PTPRS is frequently detected in HCC samples and cell lines. Treatment with a demethylation agent, 5-aza-2'-deoxycytidine, recovers PTPRS expression in a dose-dependent manner. CONCLUSIONS: Epigenetic inactivation of PTPRS may increase phosphorylation and activity of EGFR signaling to promote EMT and metastasis in HCC.

Free radical scavenging activity and neuroprotective potentials of D138, one Cu(II)/Zn(II) Schiff-base complex derived from N,N'-bis(2-hydroxynaphthylmethylidene)-1,3-propanediamine.

There is increasing evidence that free radicals play an important role in neuronal damages induced by diabetes mellitus or cerebral ischemia insults. Antioxidants with free radical scavenging activities have been shown to be beneficial and neuroprotective for these pathological conditions. Here, we report free radical scavenging activity and neuroprotective potential of D138, one copper(II)/zinc(II) Schiff-base complex derived from N,N'-2(2-hydroxynaphthylmethylidene)-1,3-propanediamine. The data from three in vitro assays, 2,2-diphenyl-1-picrylhydrazyl assay, nitro blue tetrazolium assay and hydroxyl radical scavenging assay, indicated that D138 presented a potent free radical scavenging activity. The neuroprotective and antioxidative effects of D138 were further evaluated in vivo using bilateral common carotid artery occlusion (BCCAO) mouse model and streptozotocin (STZ) diabetic mouse model. Our results indicated that treatment of D138 significantly ameliorated the hippocampal neuronal damage and the oxidative stress levels in these animal models. Moreover, D138 also reversed the behavioral deficiencies induced by BCCAO or STZ, as assessed by Y-maze test and fear conditioning test. In conclusion, all these findings support that D138 exerts free radical scavenging and neuroprotective activities and has the potentials to be a potent therapeutic candidate for brain oxidative damage induced by cerebral ischemia or diabetes mellitus.

Effects of increasing diffusive sodium removal on blood pressure control in hemodialysis patients with optimal dry weight.

BACKGROUND: Sodium, apart from volume, may have an independent effect on blood pressure (BP) regulation. METHODS: Sixteen hypertensive hemodialysis patients were enrolled, who have achieved their dry weight assessed by bioimpedance methods, with pre-dialysis plasma sodium levels slightly higher than the facility dialysate sodium concentration 138 mmol/l. After a 1-month period of dialysis with standard dialysate sodium concentration of 138 mmol/l, the patients were followed up for a 4-month period with dialysate sodium set at 136 mmol/l. RESULTS: Along with lowering dialysate sodium, there were significant decreases (-10 mm Hg and -6 mm Hg) in 44-hour ambulatory systolic and diastolic BP at 4 months. Interdialytic weight gain adjusted to the estimated dry weight mildly but significantly decreased (4.81 ± 1.51 vs. 4.36 ± 1.37%, p = 0.047). The post-dialysis volume parameters remained constant throughout the study period. CONCLUSION: In selected hypertensive hemodialysis patients with optimal dry weight, increasing diffusive sodium removal resulted in significant BP decrease. It was probably due to a volume-independent effect.

Rapid cytotoxicity of antimicrobial peptide tempoprin-1CEa in breast cancer cells through membrane destruction and intracellular calcium mechanism.

Temporin-1CEa is an antimicrobial peptide isolated from the skin secretions of the Chinese brown frog (Rana chensinensis). We have previously reported the rapid and broad-spectrum anticancer activity of temporin-1CEa in vitro. However, the detailed mechanisms for temporin-1CEa-induced cancer cell death are still weakly understood. In the present study, the mechanisms of temporin-1CEa-induced rapid cytotoxicity on two human breast cancer cell lines, MDA-MB-231 and MCF-7, were investigated. The MTT assay and the LDH leakage assay indicated that one-hour of incubation with temporin-1CEa led to cytotoxicity in a dose-dependent manner. The morphological observation using electronic microscopes suggested that one-hour exposure of temporin-1CEa resulted in profound morphological changes in both MDA-MB-231 and MCF-7 cells. The membrane-disrupting property of temporin-1CEa was further characterized by induction of cell-surface exposure of phosphatidylserine, elevation of plasma membrane permeability and rapid depolarization of transmembrane potential. Moreover, temporin-1CEa evoked intracellular calcium ion and reactive oxygen species (ROS) elevations as well as collapse of mitochondrial membrane potential (Δφm). In summary, the present study indicates that temporin-1CEa triggers rapid cell death in breast cancer cells. This rapid cytotoxic activity might be mediated by both membrane destruction and intracellular calcium mechanism.

Low-dose Cd induces hepatic gene hypermethylation, along with the persistent reduction of cell death and increase of cell proliferation in rats and mice.

BACKGROUND: Cadmium (Cd) is classified as a human carcinogen probably associated with epigenetic changes. DNA methylation is one of epigenetic mechanisms by which cells control gene expression. Therefore, the present study genome-widely screened the methylation-altered genes in the liver of rats previously exposed to low-dose Cd. METHODOLOGY PRINCIPAL FINDINGS: Rats were exposed to Cd at 20 nmol/kg every other day for 4 weeks and gene methylation was analyzed at the 48(th) week with methylated DNA immunoprecipitation-CpG island microarray. Among the 1629 altered genes, there were 675 genes whose promoter CpG islands (CGIs) were hypermethylated, 899 genes whose promoter CGIs were hypomethylated, and 55 genes whose promoter CGIs were mixed with hyper- and hypo-methylation. Caspase-8 gene promoter CGIs and TNF gene promoter CGIs were hypermethylated and hypomethylated, respectively, along with a low apoptosis rate in Cd-treated rat livers. To link the aberrant methylation of caspase-8 and TNF genes to the low apoptosis induced by low-dose Cd, mice were given chronic exposure to low-dose Cd with and without methylation inhibitor (5-aza-2'-deoxyctidene, 5-aza). At the 48(th) week after Cd exposure, livers from Cd-treated mice displayed the increased caspase-8 CGI methylation and decreased caspase-8 protein expression, along with significant increases in cell proliferation and overexpression of TGF-ß1 and cytokeratin 8/18 (the latter is a new marker of mouse liver preneoplastic lesions), all which were prevented by 5-aza treatment. CONCLUSION/SIGNIFICANCE: These results suggest that Cd-induced global gene hypermethylation, most likely caspase-8 gene promoter hypermethylation that down-regulated its expression, leading to the decreased hepatic apoptosis and increased preneoplastic lesions.

Application of labeled radioimmunoimaging tracing in detecting pulmonary embolism in rabbits after bone cement perfusion and relevant treatment effects.

BACKGROUND: During the process of bone cement joint replacement, some patients show a series of complications, such as a sudden drop in blood pressure or dyspnea. The cause of the complication is considered to be due to emboli caused by the femur prosthesis insertion. The purpose of the present study was to detect the pulmonary embolism in rabbits after bone cement perfusion by radioimmunoimaging, and to explore its protective measures. METHODS: Forty rabbits, 2.5 - 3.0 kg weight, were randomly assigned to four groups, with ten rabbits in each group. Group I (no intervention): Bone cement perfusion was done after medullary cavity reaming and pressurizing. Group II (epinephrine hydrochloride intervention): The medullary cavity was rinsed with a 1:10 000 normal saline-diluted epinephrine hydrochloride solution followed by bone cement perfusion after medullary cavity reaming and pressurizing. Group III (fibrin sealant intervention): The medullary cavity was precoated with fibrin sealant followed by bone cement perfusion after medullary cavity reaming and pressurizing. Group IV (blank control group): The medullary cavity was not perfused with bone cement after reaming. In each group, the rabbits underwent femoral head resection and medullary cavity reaming. Before bone cement perfusion, 2 ml of developing tracer was injected through the ear vein. Radionuclide imaging was performed at 60, 120, and 180 minutes after bone cement perfusion, and the pulmonary radioactivity in vivo was measured. The rabbits were immediately sacrificed, and the pulmonary tissue was removed and its radioactivity was measured in vitro. Pulmonary tissue was then fixed and the pulmonary embolism and the associated pathological changes were observed. RESULTS: The pulmonary radioactivity in vivo was measured at 60, 120, and 180 minutes after bone cement perfusion. The radioactivities of the four groups were 11.67 ± 2.16, 14.59 ± 2.92 and 18.43 ± 4.83 in group I; 8.37 ± 3.05, 10.35 ± 2.24 and 11.48 ± 2.96 in group II; 3.91 ± 1.19, 5.53 ± 2.95 and 7.25 ± 1.26 in group III; 1.04 ± 0.35, 1.14 ± 0.87 and 1.43 ± 0.97 in group IV. The radioactivities of groups I, II, III at 60, 120 and 180 minutes were significantly higher than group IV (P < 0.05). The pulmonary embolism could be detected. Pretreatment with epinephrine hydrochloride and fibrin sealant significantly decreased the pulmonary radioactivity in group II and group III, but it was still higher than in the group IV. CONCLUSIONS: Radioimmunoimaging is an alternative method for the dynamic observation of rabbit pulmonary embolism after bone cement perfusion. Radioimmunoimaging is the optional way to evaluate the effect of pretreatment with epinephrine hydrochloride or fibrin sealant on pulmonary embolism after bone cement perfusion.