Reference intervals and method sensitivity for electrocardiology, hemodynamics, and body temperature parameters in healthy cynomolgus monkeys.

In nonclinical studies, electrocardiograms (ECG) of cynomolgus monkey are recorded intermittently by external leads in manually restrained animals (snapshot recording) or continuously by jacketed external telemetry (JET) or implanted radiotelemetry transmitter in freely moving animals. With the implanted device, blood pressure and core body temperature can be monitored simultaneously. Despite the frequent use of cynomolgus monkeys in nonclinical safety pharmacology testing, few reference data are available for this species, comparisons of the ECG recording methods are limited, and power analyses are seldom conducted. In this study, pretreatment data were recorded from 406, 663, and 131 healthy experimentally naïve monkeys using the snapshot, JET, and implantable method, respectively, from 2019 to 2021. Reference intervals were determined for ECG, blood pressure, and body temperature parameters. Diurnal effects were observed in these parameters, with the exception of QRS and pulse pressure. The QRS, QT, and heart rate-corrected QTc intervals, as well as blood pressure, had a weak positive relationship with age and/or body weight. There were no sex differences in these parameters, and the country of origin only had minimal influences. Compared to telemetry, snapshot ECG data had shorter RR, PR, and QT intervals and longer QRS interval. The JET and implanted telemetry ECG data were comparable. Effect size analysis was conducted to estimate the method sensitivity for each parameter in common non-clinical study design scenarios. Snapshot recording, JET, and implanted telemetry were sensitive to detect 7-15 milliseconds of changes in QTc intervals in standard study designs, indicating these are powerful methods for assessment of QT prolongation in vivo.

A Nanoarchitectonic Approach Enables Triple Modal Synergistic Therapies To Enhance Antitumor Effects.

Improvement of antitumor effects relies on the development of biocompatible nanomaterials and combination of various therapies to produce synergistic effects and avoid resistance. In this work, we developed GBD-Fe, a nanoformulation that effectively integrated chemotherapy (CT), chemodynamic therapy (CDT), and photothermal therapy (PTT). GBD-Fe used gold nanorods as photothermal agents and encapsulated doxorubicin to amplify Fe3+-guided CDT effects by producing H2O2 and reducing the intracellular glutathione levels. In vitro and in vivo experiments were conducted to demonstrate the enhanced accumulation and antitumor effects of this tripronged therapy under magnetic resonance imaging (MRI) guidance. This tripronged approach of CT/CDT/PTT effectively induced tumor cytotoxicity and inhibited tumor growth in tumor-bearing mice and therefore represents a promising strategy to effectively treat tumors.

Knockdown of DNA methyltransferase 1 reduces DNA methylation and alters expression patterns of cardiac genes in embryonic cardiomyocytes.

We previously found that DNA methyltransferase 3a (DNMT3a) plays an important role in regulating embryonic cardiomyocyte gene expression, morphology, and function. In this study, we investigated the role of the most abundant DNMT in mammalian cells, DNMT1, in these processes. It is known that DNMT1 is essential for embryonic development, during which it is involved in regulating cardiomyocyte DNA methylation and gene expression. We used siRNA to knock down DNMT1 expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining and multielectrode array were, respectively, utilized to evaluate cardiomyocyte growth and electrophysiology. RNA sequencing (RNA-Seq) and multiplex bisulfite sequencing were, respectively, performed to examine gene expression and promoter methylation. At 72 h post-transfection, reduction of DNMT1 expression decreased the number and increased the size of embryonic cardiomyocytes. Beat frequency and the amplitude of field action potentials were decreased by DNMT1 siRNA. RNA-Seq analysis identified 801 up-regulated genes and 494 down-regulated genes in the DNMT1 knockdown cells when compared to controls. Pathway analysis of the differentially expressed genes revealed pathways that were associated with cell death and survival, cell morphology, cardiac function, and cardiac disease. Alternative splicing analysis identified 929 differentially expressed exons, including 583 up-regulated exons and 308 down-regulated exons. Moreover, decreased methylation levels were found in the promoters of cardiac genes Myh6, Myh7, Myh7b, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb, mef2c, mef2d, Camta2, Cdkn1A, and Cdkn1C. Of these 13 genes, 6 (Myh6, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb) and 1 (Cdkn1C) had increased or decreased gene expression, respectively. Altogether, these data show that DNMT1 is important in embryonic cardiomyocytes by regulating DNA methylation, gene expression, gene splicing, and cell function.

The Expression of Three Negative Co-Stimulatory B7 Family Molecules in Small Cell Lung Cancer and Their Effect on Prognosis.

BACKGROUND: In recent years, immune checkpoint inhibitors have shown significant effects in a variety of solid tumors. However, due to the low incidence of small cell lung cancer (SCLC) and its unclear mechanism, immune checkpoints in SCLC have not been fully studied. METHODS: We evaluated the expression of PD-L1, B7-H3, and B7-H4 in 115 SCLC tissue specimens using immunohistochemistry. The clinical data of patients with SCLC were retrospectively reviewed to investigate three negative co-stimulatory B7 family molecules' ability to affect the prognosis of SCLC. RESULTS: Among the SCLC patients with complete follow-up data (n = 107), sixty-nine (64.49%) expressed moderate to high B7-H3 levels, which correlated positively with tumor sizes (P < 0.001). Eighty (74.77%) patients expressed moderate to high B7-H4 levels, which correlated positively with metastases (P = 0.049). The positive expression of B7-H3 and B7-H4 correlated significantly with shortened overall survival (OS) (B7-H3, P = 0.006; B7-H4, P = 0.019). PD-L1 was positively expressed only in 13.08% of cancer tissues, and there was no significant correlation with prognosis. The Cox proportional hazards regression showed that B7-H3 was an independent prognostic indicator of OS (P = 0.028; HR = 2.125 [95% CI = 0.985-4.462]). CONCLUSIONS: Our results suggest that B7-H3 has a negative predictive effect on SCLC. This outcome provides a theoretical basis for the subsequent research on immune checkpoint inhibitors targeting B7-H3.

Research on the circadian clock gene HNF4a in different malignant tumors.

Background: The circadian rhythm is produced by multiple feedback loops formed by the core clock genes after transcription and translation, thus regulating various metabolic and physiological functions of the human body. We have shown previously that the abnormal expression of 14 clock genes is related closely to the occurrence and development of different malignant tumors, and these genes may play an anti-cancer or pro-cancer role in different tumors. HNF4a has many typical properties of clock proteins involved in the clock gene negative feedback loop regulation process. We need to explore the function of HNF4a as a circadian clock gene in malignant tumors further. Methods: We used The Cancer Genome Atlas (TCGA) database to download the clinicopathological information of twenty malignant tumors and the corresponding RNA-seq data. The HNF4a RNA-seq data standardized by R language and clinical information were integrated to reveal the relationship between HNF4a and prognosis of patients. Results: Analysis of TCGA data showed that the prognosis of HNF4a was significantly different in BLCA, KIRC, LUSC, and READ. High HNF4a expression is correlated with good prognosis in BLCA, KIRC, and READ but poor prognosis in LUSC. However, HNF4a was associated with the stages, T stages, and lymph node status only in BLCA. Conclusions: HNF4a plays different roles in different malignancies, and the abnormal expression of HNF4a has a great correlation with the biological characteristics of BLCA. The low expression of HNF4a could be a reference index for the metastasis, recurrence, and prognosis of BLCA.

Prognosis of primary hepatic lymphoma: A US population-based analysis.

OBJECTIVE: Primary hepatic lymphoma (PHL) is a rare malignancy with lesions confined to the liver. It is characterized by a large number of monomorphic, medium-sized lymphocytic infiltrates in the hepatic sinusoid. Due to the rarity of this malignancy, our current understanding of PHL is limited. METHODS: We collected incidence, mortality, and clinical data of PHL patients diagnosed between 1975 and 2016 using the Surveillance, Epidemiology, and End Results (SEER) database. The annual percentage changes (APCs) and prognoses were analyzed using the Joinpoint and R package. RESULTS: Among the 1,372 patients, white males were prevalent, and the most common histological subtype was diffuse large B-cell lymphoma (DLBCL). The incidence and mortality rate of PHL was 0.075/100,000 person-years and 0.055/100,000 person-years, respectively. The annual incidence rate of PHL increased significantly, with an APC of 2.74% (P < 0.001). The 3- and 5-year overall survival (OS) rates of patients with PHL were 43.553% and 39.242%, respectively. The 3- and 5-year relative survival (RS) rates were 46.925% and 45.300%, respectively. Multivariate Cox regression analysis revealed that older age, black, DLBCL, and advanced-stage disease were independent predictors of unfavorable OS and RS. The C-index and receiver operating characteristic (ROC) analysis confirmed the prognostic value of the nomograms established in this study. CONCLUSION: The nomogram established in this study is a robust tool to predict the prognosis of PHL patients.

Alpha-Fetoprotein Regulates the Expression of Immune-Related Proteins through the NF-κB (P65) Pathway in Hepatocellular Carcinoma Cells.

BACKGROUND: The prognosis of patients with hepatocellular carcinoma (HCC) is poor, with 60% to 70% of patients developing recurrence and metastasis within five years of radical resection. Alpha-fetoprotein (AFP) plays a significant role in predicting the recurrence and metastasis of HCC after surgery. However, its role in modulating tumor immunity has not been investigated. Our objective was to examine the effect of AFP on the expression of B7 family and activation of the NF-κB (P65) pathway in HCC. METHODS: We generated human hepatoma SMMC-7721 cell lines with or without recombinant AFP transfection (AFPup and control groups). Colony formation assay, Transwell invasion assay, and wound healing assay were used to detect the function of AFP. Liver cancer xenografts were made in BALB/c nude male mice (N = 6 per group). After 28 days of inoculation, the expression of immune genes in the HCC tissues, including PD-L (B7-H1), B7-H3, B7-H4, and P65, was evaluated by quantitative real-time PCR (qPCR) and western blot. In addition, immunofluorescence was used to determine the subcellular localization of the P65 protein, a key factor in the NF-κB pathway. An online HCC patients' dataset was also used to detect the connection between AFP and P65. RESULTS: Overexpression of AFP could enhance proliferation, invasion, and migration of HCC cells. Both qPCR and western blot results demonstrated that the expressions of PD-L1, B7-H4, and P65 were significantly higher in the AFP group compared to the controls (P < 0.05). Immunofluorescence results indicated that the majority of the P65 protein was located in the cytoplasm in the control group but was translocated to the nucleus in the AFPup group. The Spearman correlation coefficient confirms that AFP has a positive correlation with P65 in HCC patients (R = 0.33, P=0.05). CONCLUSION: AFP could enhance proliferation, invasion, and migration in HCC cells. The upregulation of AFP would increase the PD-L1 and B7-H4 mRNA and protein expression in HCC tissues through the upregulation and activation of the P65 protein.

Optimized CyberKnife Lung Treatment: Effect of Fractionated Tracking Volume Change on Tracking Results.

OBJECTIVES: To explore the impact of volume change in the fractionated tracking of stereotactic radiotherapy on the results of synchronous, respiratory tracking algorithm using CyberKnife. METHODS: A total of 38 lung tumor patients receiving stereotactic radiotherapy at our center from March 2018 to October 2019 were counted. Photoshop CS4 image processing software was used to obtain the pixels and the average value of brightness of the tracking volume in the image and calculate the grayscale within the contour of the tracking volume on the real-time X-ray image. At the same time, parameters of the synchronous respiratory tracking algorithm of the fractional CyberKnife were extracted for comparison between the volume of image-guided image tracking and the number of fractions during stereotactic radiotherapy. We also analyzed the relationship between fraction tumor location and characteristics and the calculated results of synchronous respiratory tracking by CyberKnife. RESULTS: There were no significant differences between the first four fractions (p > 0.05) for left lung lesions and no significant differences between the first five fractions for right lung lesions (p ≥ 0.05). For peripheral lung cancer, longer fractional treatment led to greater variation in grayscale (G-A: >4 fractions p < 0.05), while for central lung cancer, longer fractional treatment led to greater variation in parameters of the synchronous respiratory tracking algorithm (Uncertainty A and Uncertainty B: >4 fractions p < 0.05). There was a significant correlation between radiotherapy-graded tumor density and relevant parameters, and the correlation was strong (>0.7, p < 0.05). CONCLUSION: With the increase of treatment fractions, the gray value in the patient tracking volume decreased. Patients of >4 fractions were advised to reevaluate with simulated CT and replan. For tumors with small diameter and low density, the imaging changes of volume should be closely followed during treatment. For left lung and central lung cancer, carefully select the synchronous tracking treatment with 2-view.

Research on circadian clock genes in non-small-cell lung carcinoma.

Circadian clock genes have become a hot topic in cancer research in recent years, and more and more studies are showing that clock genes are involved in regulating cell proliferation cycle and apoptosis of malignant tumors, neuroendocrine and immune function, and other processes. Lung cancer is a malignant tumor with increasing incidence worldwide. The pathogenesis of lung cancer is extremely complicated and includes genetic factors, living environment, and smoking, and the occurrence of lung cancer is related to the regulation of many oncogenes and tumor suppressor genes. But there are few studies on clock genes in lung cancer. Studies on clock genes may help to better understand the mechanism of lung cancer development for an improved treatment. The expressions of all 14 kinds of clock genes in adenocarcinoma (ADC) and squamous cell carcinoma (SCC), two main kinds of non-small-cell lung cancer (NSCLC), were studied based on integration and analysis of data from The Cancer Genome Atlas (TCGA) to show the association between clock gene expression and prognosis of cancer patients. Analysis of TCGA data indicated that overexpression of Cry2, BMAL1, and RORA with underexpression of Timeless and NPAS2 was associated with a favorable prognosis of ADC, and the expression of NPAS2 was associated with the time of patient survival. Additionally, the expression of Cry2 was related to TNM stage. In SCC, high expression of DEC1 was correlated with poor overall survival in patients and the expression of Timeless was associated with the time of patient survival. In NSCLC, circadian clock genes constitute cancer circadian rhythm by interacting with each other, showing that asynchrony with normal tissues, which collectively controlling the occurrence and development of NSCLC.

Research on circadian clock genes in common abdominal malignant tumors.

Circadian rhythm describes the 24-h oscillation in physiology and behavior of living organisms and presents a timing controller for life activity. Studies in recent years have reported that the abnormal expression of clock genes is closely related to the development of common abdominal malignant tumors. The expression of the 14 kinds of clock genes in 6 abdominal malignant tumors from Cancer Genome Atlas (TCGA) data was integrated and analyzed using R and Perl programming languages to show the association between clock gene expression and prognosis of cancer patients. Analysis of TCGA data indicated that the overexpression of Per1-3, Cry2, CLOCK, NR1D2 and RORA with underexpression of Timeless and NPAS2 was associated with a favorable prognosis in kidney cancer. In liver cancer, high expressions of Cry2 and RORA were correlated with prolonged overall survival (OS) in patients, while high expressions of NPAS2 and Timeless were correlated with a poor survival. High expression of CLOCK was positively correlated with OS in colon cancer patients. High expression of Cry2 and low expression of DEC1 were associated with a favorable prognosis in pancreatic cancer patients, respectively. Most of these clock-genes expressions were closely related to the clinical stage and degree of tumor differentiation of patients. Aberrant clock gene expression is related to the biological characteristics of abdominal malignant tumors, which likely has a causal role in cancer development and survival.

Gene expression profile and molecular pathway datasets resulting from benzo(a)pyrene exposure in the liver and testis of adult tilapia.

Benzo(a)pyrene (BaP), the prototype of polycyclic aromatic hydrocarbons, is known to exhibits genotoxic and carcinogenic effects promoting molecular impacts. The dataset presented here is associated with the research article paper entitled "Transcriptome Analysis Reveals Novel Insights Into the Response of Low-dose Benzo(a)pyrene Exposure in Male Tilapia". In this article, we presented a transcriptomic characterization of male tilapia exposure to BaP in the short term. This data provides an extended analysis of changes in the gene expression and identification of pathways in the liver and testis of male tilapia exposure to BaP. We used gene set enrichment analysis (GSEA) and sub-network enrichment analysis (SNEA) to identify gene networks and pathways associated with molecular adverse effects of BaP exposure. The data indicates that target pathways related to promoting carcinogenesis such as DNA repair and DNA replication were affected as well as other crucial biological processes. Moreover, to determine whether some of the key reported genes of DNA damage are affected by BaP exposure, Quantitative PCR (qPCR) was performed. Gene set categories and sub-networks are provided and the corresponding signature differences from BaP exposure are listed. The information in these datasets may contribute to understanding the potential carcinogenesis mechanism of action from low BaP exposure.

Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia.

Despite a wide number of toxicological studies that describe benzo[a]pyrene (BaP) effects, the metabolic mechanisms that underlie these effects in fish are largely unknown. Of great concern is the presence of BaP in aquatic systems, especially those in close proximity to human activity leading to consumption of potentially contaminated foods. BaP is a known carcinogen and it has been reported to have adverse effects on the survival, development and reproduction of fish. The purpose of this study was to investigate if a low dose of BaP can alter genes and key metabolic pathways in the liver and testis in male adult tilapia, and whether these could be associated with biological endpoints disruption. We used both high-throughput RNA-Sequencing to assess whole genome gene expression following repeated intraperitoneal injections of 3 mg/kg of BaP (every 6 days for 26 days) and morphometric endpoints as indicators of general health. Condition factor (K) along with hepatosomatic and gonadosomatic indices (morphometric parameters) were significantly lower in BaP-treated fish than in controls. BaP exposure induced important changes in the gene expression pattern in liver and testis as revealed by both Pathway and Gene Ontology (GO) analyses. Alterations that were shared by both tissues included arachidonic acid metabolism, androgen receptor to prostate-specific antigen signaling, and insulin-associated effects on lipogenesis. The most salient liver-specific effects included: biological processes involved in detoxification, IL6-associated insulin resistance, mTOR hyperactivation, mitotic cytokinesis, spindle pole and microtubule binding. BaP effects that were confined to the testis included: immune system functions, inflammatory response, estrogen and androgen metabolic pathways. Taken together, gene expression and morphometric end point data indicate that the reproductive success of adult male tilapia could be compromised as a result of BaP exposure. These results constitute new insights on the mechanism of action of low dose BaP in a non-model organism (tilapia).

Peritumoral overexpression of ZBP-89 is associated with unfavorable disease-free survival rates in patients with hepatocellular carcinoma following hepatectomy.

Previous studies have revealed that the peritumoral environment has a profound influence on tumor initiation and progression. Zinc-binding protein-89 (ZBP-89) has been observed to be involved with tumor development, recurrence, and metastasis. High intratumoral expression of ZBP-89 has been associated with improved prognosis in several tumor types. However, the prognostic values of peritumoral expression of ZBP-89 remain to be elucidated in patients with hepatocellular carcinoma (HCC) following curative resection. In the present study, peritumoral ZBP-89 expression was examined using immunohistochemistry in 102 HCC patients who had received curative hepatectomy. Expression of ZBP-89 protein was positive in 66.3% of the peritumoral samples from 102 HCC patients. HCC patients with high peritumoral ZBP-89 expression exhibited significantly shorter disease-free survival (DFS) times (P=0.012) than those patients with low peritumoral ZBP-89 expression. Additionally, high ZBP-89 expression in peritumoral HCC tissue was positively associated with the presence of liver cirrhosis. Univariate and multivariate Cox proportional hazard regression analyses demonstrated that albumin levels ≤35 g/l, multiple tumors, tumor sizes ≥5 cm, and macroscopic vascular invasion may serve as independent prognostic factors for overall survival (OS) [hazard ratio (HR)=2.031; P=0.014] in patients with HCC. The multivariate Cox regression model identified that high ZBP-89 expression, multiple tumors and macroscopic vascular invasion were independent prognostic factors for shorter DFS durations. High expression of ZBP-89 in peritumoral HCC tissues was associated with a shorter DFS in HCC patients following curative hepatectomy. Additionally, high ZBP-89 expression in peritumoral HCC tissue was positively associated with the presence of liver cirrhosis in HCC patients, indicating that cirrhosis accompanied by high ZBP-89 expression may be a contributing factor to the poor prognosis of patients with HCC. Therefore, peritumoral ZBP-89 expression may be a good prognostic marker to predict DFS time in HCC patients following curative hepatectomy and may provide novel insights into the molecular mechanisms of HCC initiation.

Hepatitis B virus upregulates GP73 expression by activating the HIF-2α signaling pathway.

Golgi Protein 73 (GP73) is a newly identified diagnostic and prognostic marker for liver cancer. GP73 is highly expressed in liver cancer tissues, however, the mechanism of its overexpression in tumors remains unknown. In the present study, the effect of hepatitis B virus (HBV) on GP73 expression was investigated in HepG2 cells, which are negative for HBV, and in HepG2.2.12 cells, which are integrated with HBV, using reverse transcription-quantitative polymerase chain reaction and western blot analysis. In addition, the cells were transfected with plasmid constructs overexpressing hepatitis B virus protein X (HBx), hypoxia-inducible factor (HIF)-1α, or HIF-2α in order to examine their roles in GP73 expression. The results demonstrated that HBV upregulated the expression of GP73 and HIF-2α in liver cancer cells. HIF-2α induced the expression of GP73 in HepG2 cells and was positively correlated with GP73 expression in liver cancer tissues. By contrast, HBx and HIF-1α did not induce GP73 expression in liver cancer cells. In summary, HBV may upregulate the expression of GP73 by activating the HIF-2α signaling pathway. The present results may illuminate the mechanism by which GP73 is overexpressed in liver cancer tissues.

Low heme oxygenase-1 expression promotes gastric cancer cell apoptosis, inhibits proliferation and invasion, and correlates with increased overall survival in gastric cancer patients.

Heme oxygenase-1 (HO-1) plays a key role in anti-oxidation, anti-apoptosis, and anti-proliferation in various types of cancers. However, the relationship between HO-1 expression and gastric cancer development remains largely unknown. In this study, the protein expression of HO-1 in human gastric cancer was measured by immunohistochemistry on paraffin sections of 89 paired gastric carcinoma tissues and adjacent non-cancer tissues. The correlation of HO-1 expression with 5-year overall survival rate was estimated. The effects of decreased HO-1 expression by two strands of small interfered RNAs (siRNAs) on cell apoptosis, proliferation, and invasion of gastric cancer cell lines were examined by flow cytometry, the MTT assay, and the cell migration assay, respectively. High expression of HO-1 was detected in 11.2% (10/89) of gastric carcinoma tissues, compared with 1.1% (1/89) in matched adjacent normal tissues, and correlated with a decreased survival rate in gastric cancer patients. There were no significant correlations between HO-1 expression and clinical characteristics. Downregulation of HO-1 expression using two strands of siRNAs promoted apoptosis and inhibited the proliferation and invasion of two gastric cancer cell lines, SGC7901 and MKN-28 cells. This study demonstrated that HO-1 plays a vital role in the development of gastric cancer and may serve as a therapeutic target of this type of cancer.

Positive Expression of Programmed Death Ligand 1 in Peritumoral Liver Tissue is Associated with Poor Survival after Curative Resection of Hepatocellular Carcinoma.

BACKGROUND: Recurrence or metastasis of hepatocellular carcinoma (HCC) is mainly intrahepatic after curative resection, demonstrating that the peritumoral environment is important but often neglected. Programmed death ligand 1 (PD-L1) in intratumoral liver tissues is a poor prognosis factor whose impact is removed after curative resection. However, PD-L1 expression remains in the peritumoral liver tissues and its distribution and prognostic value are still not clear. METHODS: We assessed the expression of PD-L1 by immunohistochemistry in peritumoral liver tissues from 90 HCC patients who underwent curative hepatectomy. The results were validated in an independent cohort of additional 90 HCC patients. RESULTS: We found PD-L1 positive expression in 31.11% (28/90) of peritumoral tissues. Peritumoral PD-L1 expression was associated with a significantly worse overall survival (OS) (P=.000) and disease-free survival (DFS) (P=.001) compared to the negative expression group. Additionally, peritumoral PD-L1 positivity significantly correlated with vascular invasion and a lower albumin level (≤35 g/L). Univariate and multivariate Cox regression models both revealed peritumoral PD-L1 as an independent prognostic factor for OS (HR=2.853, P=.002) and DFS (HR=2.362, P=.003). The prognostic value of PD-L1 positivity was validated in the independent data set. CONCLUSIONS: Our data suggest PD-L1 expression in peritumoral hepatocytes is an independent prognostic factor for OS and DFS. This implies that future anti-cancer therapy should target not only residual tumor cells but also the "soil" for promoting tumor growth. Peritumoral PD-L1 could be a good target for adjuvant therapy after hepatectomy.

Reversal of paclitaxel resistance in human ovarian cancer cells with redox-responsive micelles consisting of α-tocopheryl succinate-based polyphosphoester copolymers.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major obstacle in achieving the therapeutic benefits of paclitaxel (PTX) in the treatment of human ovarian carcinoma. This study is aimed to develop an efficient PTX drug delivery approach to overcome MDR. Redox-responsive micelles consisting of amphiphilic polymers containing disulfide linkages, ie, poly (phosphate ester)-SS-D-α-tocopheryl succinate (POPEA-SS-TOS, PSST) were prepared. PTX-loaded PSST micelles (PTX/PSST-M) designed to display synergistic functions, including reversible inhibition of P-gp, intracellular redox-sensitive release and potent anticancer activities. The average size of PTX/PSST-M was 68.1±4.9 nm. The encapsulated PTX was released quickly through redox-triggered dissociation of micelles. The inhibition of P-gp activity and enhanced cellular accumulation of the PSST micelles were validated. PTX/PSST-M showed significantly increased cytotoxicity against PTX-resistant human ovarian cancer A2780/PTX cells: when the cells were treated with PTX/PSST-M for 48 h, the equivalent IC50 value of PTX was reduced from 61.51 to 0.49 µmol/L. The enhanced cytotoxic effects of PTX/PSST-M against A2780/PTX cells were attributed to their synergistic effects on reducing the mitochondrial transmembrane potential, ATP depletion, ROS production, and activation of apoptotic pathways. Furthermore, PTX/PSST-M significantly increased cell apoptosis/necrosis and cell cycle arrest at the G2/M phase in A2780/PTX cells. These results demonstrate that the redox-responsive PSST micelles inhibit P-gp activity and have a good potential to effectively reverse PTX resistance in human ovarian carcinoma cells by activating intrinsic apoptotic pathways.

Bumetanide increases Cl--dependent short-circuit current in late distal colon: Evidence for the presence of active electrogenic Cl- absorption.

Mammalian colonic epithelia consist of cells that are capable of both absorbing and secreting Cl-. The present studies employing Ussing chamber technique identified two opposing short-circuit current (Isc) responses to basolateral bumetanide in rat distal colon. Apart from the transepithelial Cl--secretory Isc in early distal colon that was inhibited by bumetanide, bumetanide also stimulated Isc in late distal colon that had not previously been identified. Since bumetanide inhibits basolateral Na+-K+-2Cl- cotransporter (NKCC) in crypt cells and basolateral K+-Cl- cotransporter (KCC) in surface epithelium, we proposed this stimulatory Isc could represent a KCC-mediated Cl- absorptive current. In support of this hypothesis, ion substitution experiments established Cl- dependency of this absorptive Isc and transport inhibitor studies demonstrated the involvement of an apical Cl- conductance. Current distribution and RNA sequencing analyses revealed that this Cl- absorptive Isc is closely associated with epithelial Na+ channel (ENaC) but is not dependent on ENaC activity. Thus, inhibition of ENaC by 10 µM amiloride or benzamil neither altered the direction nor its activity. Physiological studies suggested that this Cl- absorptive Isc senses dietary Cl- content; thus when dietary Cl- was low, Cl- absorptive Isc was up-regulated. In contrast, when dietary Cl- was increased, Cl- absorptive Isc was down-regulated. We conclude that an active Cl- extrusion mechanism exists in ENaC-expressing late distal colon and likely operates in parallel with ENaC to facilitate NaCl absorption.

Rapid quantification of metabolic intermediates in blood by liquid chromatography-tandem mass spectrometry to investigate congenital lactic acidosis.

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been established to quantify metabolic intermediates, including lactate (Lac), pyruvate (Pyr), acetoacetate (ACAC) and 3-hydroxybutyrate (3-HB) in blood. Samples were deproteinized with methanol-acetonitrile solution, and analytes were separated on an adamantyl group-bonded reverse phase column and detected in multiple reaction monitoring mode. Total analysis time was 4 min per sample. Method validation results displayed that limits of quantification were 10.0 µmol L-1 for Lac and Pyr, and 5.0 µmol L-1for ACAC and 3-HB. The within- and between-run coefficients of variation were in the range of 1.2-6.4% for all analytes. The recoveries were ranged from 95.6 to 111.5%. The reference values of analytes were determined for the pediatric population. Duo to instability of Lac, Pyr and ACAC in vitro, a comprehensive stability assay was performed to determine optimal conditions for sample collection, pretreatment and storage. Results showed that precipitation of protein in blood at bedside combined with low storage temperature could effectively preserve the integrity of Lac, Pyr and 3-HB, but the precipitated protein accelerated degradation of ACAC. Isolation of supernatant fluid slowed degradation of ACAC. Supernatant samples could store at -20 °C for 10 days. The use of plasma or serum to determine these intermediates was not recommended. In this study, 450 samples from patients were analyzed, and 7 patients were diagnosed as congenital lactic acidosis. With the advantages of rapid, accurate and reliable, this method is very suitable for congenital lactic acidosis screening and researches related to energy metabolism.

In Utero Caffeine Exposure Induces Transgenerational Effects on the Adult Heart.

Each year millions of pregnant woman are exposed to caffeine, which acts to antagonize adenosine action. The long-term consequences of this exposure on the developing fetus are largely unknown, although in animal models we have found adverse effects on cardiac function. To assess if these effects are transmitted transgenerationally, we exposed pregnant mice to caffeine equivalent to 2-4 cups of coffee at two embryonic stages. Embryos (F1 generation) exposed to caffeine early from embryonic (E) day 6.5-9.5 developed a phenotype similar to dilated cardiomyopathy by 1 year of age. Embryos exposed to caffeine later (E10.5-13.5) were not affected. We next examined the F2 generation and F3 generation of mice exposed to caffeine from E10.5-13.5, as this coincides with germ cell development. These F2 generation adult mice developed a cardiac phenotype similar to hypertrophic cardiomyopathy. The F3 generation exhibited morphological changes in adult hearts, including increased mass. This report shows that in utero caffeine exposure has long-term effects into adulthood and that prenatal caffeine exposure can exert adverse transgenerational effects on adult cardiac function.