Druggable site near the upper vestibule determines the high affinity and P2X3 homotrimer selectivity of sivopixant/S-600918 and its analogue DDTPA.

BACKGROUND AND PURPOSE: The P2X3 receptor, a trimeric ionotropic purinergic receptor, has emerged as a potential therapeutic target for refractory chronic cough (RCC). Nevertheless, gefapixant/AF-219, the only marketed P2X3 receptor antagonist, might lead taste disorders by modulating the human P2X2/3 (hP2X2/3) heterotrimer. Hence, in RCC drug development, compounds exhibiting strong affinity for the hP2X3 homotrimer and a weak affinity for the hP2X2/3 heterotrimer hold promise. An example of such a molecule is sivopixant/S-600918, a clinical Phase II RCC candidate with a reduced incidence of taste disturbance compared to gefapixant. Sivopixant and its analogue, (3-(4-([3-chloro-4-isopropoxyphenyl]amino)-3-(4-methylbenzyl)-2,6-dioxo-3,6-dihydro-1,3,5-triazin-1(2H)-yl)propanoic acid (DDTPA), exhibit both high affinity and high selectivity for hP2X3 homotrimers, compared with hP2X2/3 heterotrimers. The mechanism underlying the druggable site and its high selectivity remains unclear. EXPERIMENTAL APPROACH: To analyse mechanisms that distinguish this drug candidate from other inhibitors of the P2X3 receptors we used a combination of chimera construction, site covalent occupation, metadynamics, mutagenesis and whole-cell recording. KEY RESULTS: The high affinity and selectivity of sivopixant/DDTPA for hP2X3 receptors was determined by the tri-symmetric site located close to the upper vestibule. Substitution of only four amino acids inside the upper body domain of hP2X2 with those of hP2X3, enabled the hP2X2/3 heterotrimer to exhibit a similar level of apparent affinity for sivopixant/DDTPA as the hP2X3 homotrimer. CONCLUSION AND IMPLICATIONS: From the receptor-ligand recognition perspective, we have elucidated the molecular basis of novel RCC clinical candidates' cough-suppressing properties and reduced side effects, offering a promising approach to the discovery of novel drugs that specifically target P2X3 receptors.

[Study on the Mechanism of Multi-Drug Resistance of Agaricus Blazei Extract FA-2-b-ß Mediated Wnt Signaling Pathway to Reverse Acute T Lymphoblastic Leukemia].

OBJECTIVE: To investigate the mechanism of drug reversing resistance of Agaricus blazei extract FA-2-b-ß on T cell acute lymphoblastic leukemia (T-ALL) cell lines. METHODS: Cell proliferation was detected by CCK-8 assay; the apoptosis, cell cycle mitochondrial membrane potential, and intracellular rhodamine accumulation were detected by flow cytometry, and apoptosis-related gene and protein expression were detected by qPCR and Western blot; the membrane surface protein MDR1 was observed by immunofluorescence microscopy. RESULTS: Different concentrations of FA-2-b-ß significantly inhibited proliferation and induced apoptosis of CCRF-CEM and CEM/C1 (P<0.05), and CCRF-CEM cell cycle were arrested at S phase, and CEM/C1 cells were arrested at G0/G1 phase. Western blot and qPCR results show that FA-2-b-ß inhibited ABCB1、ABCG2、CTNNB、MYC and BCL-2 expression, but upregulated Bax expression. In addition, FA-2-b-ß reversed the resistance characteristics of CEM/C1 drug-resistance cells, which decreased mitochondrial membrane potential, and significantly increased the intracellular rhodamine accumulation, and weakening of the expression of the membrane surface protein MDR1. With the Wnt/ß-catenin inhibitor (ICG001), the process was further intensified. CONCLUSION: Agaricus Blazei Extract FA-2-b-ß inhibits cell proliferation, promotes apoptosis, regulates the cell cycle, reduces mitochondrial energy supply, and down-regulate MDR1 expression to reverse the resistance of CEM/C1, which all suggest it is through regulating the Wnt signaling pathway in T-ALL.

A Novel Aldisine Derivative Exhibits Potential Antitumor Effects by Targeting JAK/STAT3 Signaling.

The JAK/STAT3 signaling pathway is aberrantly hyperactivated in many cancers, promoting cell proliferation, survival, invasiveness, and metastasis. Thus, inhibitors targeting JAK/STAT3 have enormous potential for cancer treatment. Herein, we modified aldisine derivatives by introducing the isothiouronium group, which can improve the antitumor activity of the compounds. We performed a high-throughput screen of 3157 compounds and identified compounds 11a, 11b, and 11c, which contain a pyrrole [2,3-c] azepine structure linked to an isothiouronium group through different lengths of carbon alkyl chains and significantly inhibited JAK/STAT3 activities. Further results showed that compound 11c exhibited the optimal antiproliferative activity and was a pan-JAKs inhibitor capable of inhibiting constitutive and IL-6-induced STAT3 activation. In addition, compound 11c influenced STAT3 downstream gene expression (Bcl-xl, C-Myc, and Cyclin D1) and induced the apoptosis of A549 and DU145 cells in a dose-dependent manner. The antitumor effects of 11c were further demonstrated in an in vivo subcutaneous tumor xenograft experiment with DU145 cells. Taken together, we designed and synthesized a novel small molecule JAKs inhibitor targeting the JAK/STAT3 signaling pathway, which has predicted therapeutic potential for JAK/STAT3 overactivated cancer treatment.

Integrated Biochip-Electronic System with Single-Atom Nanozyme for in Vivo Analysis of Nitric Oxide.

Nitric oxide (NO) exhibits a crucial role in various versatile and distinct physiological functions. Hence, its real-time sensing is highly important. Herein, we developed an integrated nanoelectronic system comprising a cobalt single-atom nanozyme (Co-SAE) chip array sensor and an electronic signal processing module (INDCo-SAE) for both in vitro and in vivo multichannel qualifying of NO in normal and tumor-bearing mice. The high atomic utilization and catalytic activity of Co-SAE endowed an ultrawide linear range for NO varying from 36 to 4.1 × 105 nM with a low detection limit of 12 nM. Combining in situ attenuated total reflectance surface enhanced infrared spectroscopy (ATR-SEIRAS) measurements and density function calculation revealed the activating mechanism of Co-SAE toward NO. The NO adsorption on an active Co atom forms *NO, followed by the reaction between *NO and OH-, which could help design relevant nanozymes. Further, we investigated the NO-producing behaviors of various organs of both normal and tumor-bearing mice using the proposed device. We also evaluated the NO yield produced by the wounded mouse using the designed device and found it to be approximately 15 times that of the normal mouse. This study bridges the technical gap between a biosensor and an integrated system for molecular analysis in vitro and in vivo. The as-fabricated integrated wireless nanoelectronic system with multiple test channels significantly improved the detection efficiency, which can be widely used in designing other portable sensing devices with multiplexed analysis capability.

Risk Factors for Thirty-Day Readmission Following Lumbar Surgery: A Meta-Analysis.

OBJECTIVE: Thirty-day readmission is one of the common complications after lumbar surgery. More 30-day readmission increases the total hospitalization, economic burden, and physical pain of patients, delays the progress of postoperative rehabilitation, and even lead to die. Therefore, it is necessary to analyze the risk factors of 30-day readmission following lumbar surgery. METHODS: We searched for all the clinical trials published from the establishment of the database to May 1, 2022 through the Cochrane Library, Web of Science, Embase, and PubMed. Data including age, American Society of Anesthesiology physical status class, preoperative hematocrit (Hct), diabetes mellitus (DM), current smoker, chronic obstructive pulmonary disease (COPD), length of hospital stay (LHS), operation time, and surgical site infection (SSI) were extracted. We used Review Manager 5.4 for data analysis. RESULTS: Six studies with 30,989 participants were eligible for this meta-analysis. The analysis revealed that there were statistically significant differences in the age (95% confidence interval [CI]: -3.35-2.90, P < 0.001), preoperative Hct (95% CI: 0.75-1.33, P < 0.001), DM (95% CI: 0.56-0.74, P < 0.001), COPD (95% CI: 0.38-0.58, P < 0.001), operation time (95% CI: -35.54-16.18, P < 0.001), LHS (95% CI: -0.54-0.50, P < 0.001), and SSI (95% CI: 0.02-0.03, P < 0.001) between no readmission and readmission groups. In terms of the American Society of Anesthesiology physical status class and current smoker, there was no significant effect on the 30-day readmission (P = 0.16 and P = 0.35 respectively). CONCLUSIONS: Age, preoperative Hct, DM, COPD, operation time, LHS, and SSI are the danger factors of 30-day readmission following lumbar surgery.

Analysis of N6-Methyladenosine RNA Methylation Regulators in Diagnosis and Distinct Molecular Subtypes of Ankylosing Spondylitis.

The most frequent internal modification in eukaryotic mRNA is N6-methyladenosine (m6A). However, what we know about the m6A regulators in Ankylosing spondylitis (AS) is still limited. In our study, eight distinct m6A regulators were selected utilizing Differentially Expressed Gene (DEG) analysis of the Gene Expression Omnibus GSE73754 dataset for making comparisons between AS (Ankylosing spondylitis) and non-AS patients. The random forest model and the nomogram model were used to screen the eight candidate m6A regulators and evaluate their prediction accuracy for the occurrence of AS. Furthermore, based on the selected m6A regulators, the AS patients were divided into two subgroups, and we applied principal component analysis algorithms to calculate their m6A score and evaluate the m6A patterns. Our findings revealed that patients in cluster A were linked to activated CD4 T cell immunity and activated CD8 T cell immunity. With its major contributions in the area of immunology, our research in m6A patterns may benefit the future diagnosis and treatment strategies of AS.

[Spatial Distribution, Source Apportionment, and Ecological Risk Assessment of Soil Heavy Metals in Jianghugongmi Producing Area, Shandong Province].

Taking the Jianghugongmi producing area as the research object, As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn in the soil of the study area were sampled and determined. The correlation of heavy metals was discussed using the multivariate statistical method, the spatial distribution interpolation analysis of heavy metals was carried out using ArcGIS 10.2, the quantitative source analysis of heavy metal pollution was carried out using the enrichment factor (EF) and PMF methods, and the potential ecological risk was evaluated. The results showed that the contents of the soil heavy metals As, Cd, Cu, Hg, Pb, and Zn were lower than the screening values specified in the standard for soil pollution risk control of agricultural land (GB 15618-2018), and the soil ecological environment risk was low; the maximum values of Cr and Ni exceeded the risk screening values, but the risk was low. The main distribution range of pH in the study area was 6.05-6.69, which was suitable for rice growth. The Mohe River indicated the spatial distribution of pH and heavy metals, which was closely related to the supergene geochemical characteristics of the elements. However, Hg and Cd showed different spatial distribution characteristics under human influence. Hg was distributed in the middle and high value distribution area along the west side of the river, and the spatial distribution of Cd was significantly different from north to south. The quantitative source analysis results based on the EF method and PMF showed that the main sources of heavy metals in the study area were agricultural sources, mixed sources, coal sources, and natural sources. The contribution rates of various sources accounted for 24.2%, 35.4%, 9.5%, and 30.9%, respectively. The medium strong ecological risk points of Hg in the study area were distributed along the west side of the Mohe River, whereas the moderate potential ecological risk points of Cd were concentrated in the cultivated land on both sides of the Mohe River, and the potential ecological risk index (Er) of the other elements was<40. Cd and Hg were the main potential ecological risk elements in the study area, whereas Cd was still the main potential pollution element in the cultivated land soil in the study area.

Prediction of post-transplant graft survival by different definitions of early allograft dysfunction.

BACKGROUND: The efficacy of early allograft dysfunction (EAD) definitions in predicting post-transplant graft survival in a Chinese population is still unclear. METHODS: A total of 607 orthotopic liver transplants (OLT) have been included in the current study. Model accuracy was evaluated using receiver operating characteristic (ROC) analysis. Risk factors for EAD was evaluated using univariable analysis and multivariable logistic regression model. RESULTS: The 3-, 6-, and 12-month patient/graft survival were 91.6%/91.4%, 91.1%/90%, and 87.5%/87.3%, respectively. MELDPOD5 had a superior discrimination of 3-month graft survival (C statistic, 0.83), compared with MEAF (C statistic, 0.77) and Olthoff criteria (C statistic, 0.72). Multivariate analysis of risk factors for EAD defined by MELDPOD5, showed that donor body mass index (P=0.001), donor risk index (P=0.006), intraoperative use of packed red blood cells (P=0.001), hypertension of recipient (P=0.004), and preoperative total bilirubin (P<0.001) were independent risk factors. CONCLUSIONS: The results suggest that MLEDPOD5 is a better criterion of EAD for the Chinese population, which might serve as a surrogate end-point for graft survival in clinical study.

Single-Atom Ruthenium Biomimetic Enzyme for Simultaneous Electrochemical Detection of Dopamine and Uric Acid.

Single-atom catalysts have attracted numerous attention due to the high utilization of metallic atoms, abundant active sites, and highly catalytic activities. Herein, a single-atom ruthenium biomimetic enzyme (Ru-Ala-C3N4) is prepared by dispersing Ru atoms on a carbon nitride support for the simultaneous electrochemical detection of dopamine (DA) and uric acid (UA), which are coexisting important biological molecules involving in many physiological and pathological aspects. The morphology and elemental states of the single-atom Ru catalyst are studied by transmission electron microscopy, energy dispersive X-ray elemental mapping, high-angle annular dark field-scanning transmission electron microscopy, and high-resolution X-ray photoelectron spectroscopy. Results show that Ru atoms atomically disperse throughout the C3N4 support by Ru-N chemical bonds. The electrochemical characterizations indicate that the Ru-Ala-C3N4 biosensor can simultaneously detect the oxidation of DA and UA with a separation of peak potential of 180 mV with high sensitivity and excellent selectivity. The calibration curves for DA and UA range from 0.06 to 490 and 0.5 to 2135 µM with detection limits of 20 and 170 nM, respectively. Moreover, the biosensor was applied to detect DA and UA in real biological serum samples using the standard addition method with satisfactory results.

Metabolic engineering of Yarrowia lipolytica for improving squalene production.

The aim of this present research is to enhance the squalene production in Yarrowia lipolytica using pathway engineering and bioprocess engineering. Firstly, to improve the production of squalene, the endogenous HMG-CoA reductase (HMG1) was overexpressed in Y. lipolytica to yield 208.88 mg/L squalene. Secondly, the HMG1 and diacylglycerol acyltranferase (DGA1) were co-overexpressed, the derived recombinant Y. lipolytica SQ-1 strain produced 439.14 mg/L of squalene. Thirdly, by optimizing the fermentation medium, the improved titer of squalene with 514.34 mg/L was obtained by the engineered strain SQ-1 grown on YPD-80 medium. Finally, by optimizing the addition concentrations of acetate, citrate and terbinafine, the 731.18 mg/L squalene was produced in the engineered strain SQ-1 with the addition of 0.5 mg/L terbinafine. This work describes the highest reported squalene titer in Y. lipolytica to date. This study will provide the foundation for further engineering Y. lipolytica capable of cost-efficiently producing squalene.

[Inhibitory Effects of Agaricus Blazei Murrill (FA-2-b-ß) on Proliferation of Chronic Myeloid Leukemia Cells in vivo and Its Mechanism].

OBJECTIVE: To investigated the anti-tumor in vivo effect and mechanism of the acid RNA protein complex (FA-2-b-ß) of Agaricus blazei Murrill extract. METHODS: CCK-8 method was used to detected the inhibitory effect of FA-2-b-ß on proliferation of primary CML cells from newly diagnosed CML patients, the CML mouse model was established by trail-venous injection of primary CML cells, and the survival time, blood cell count and body weight were observed, the immunoflouresence and immunehistochemistry analysis, RT-qPCR, Western bolt were used to detemine the expression of caspase-3 signal pathway-related apoptosis genes and proteins. RESULTS: The experiments in vitro showed that the proliferative inhibitory rate in drug-treated group increased with concentration- and time-dependent manner (r24=0.9092, r48=0.9442, r72=0.9546), the inter group comparison showed the statistical difference of results. The experiments in vitro showed that the survival time prolonged, blood cell count increased and body weight recovered in FA-2-b-ß-treated group and imatinib-treated group, despite the WBC count is still high. The RT-qPCR and Western blot showed that the expression of BAX and caspase-3 gene and protein were up-regulated, the expression of BCL-2, cytochroime C, caspase-8, caspase-9 and BCL-ABL gene and protein were down-regulated. CONCLUSION: The FA-2-b-ß can induce apoptosis of primary CML cells and prolong the survival time of CML model mouse, which may be related with the caspase-3 signal pathway related genes and proteins.

[Primary Mechanisms of CD34+CD38---KG1a Leukemia Stem Cells Apoptosis Induced by FA-2-b-ß Extracted from Qinba Selenium- Mushroo].

OBJECTIVE: To investigate the apoptosis of CD34+CD38--KG1a leukemia stem cells induced by Qinba selenium-mushroom extract(FA-2-b-ß), and its related mechanism. METHODS: CD34+CD38---KG1a cells were isolated from KG1a cell line by magnetic activated cell sorting. The proliferation ability of KG1a stem cells treatd by various concentration of FA-2-b-ß(1.2-2.4 mg/ml) in vitro for 24 and 48 hours were tested by cell counting Kit-8(CCK8). Flow cytometry was used to detect the apoptosis rate of KG1a stem cells in each group after treated by FA-2-b-ß in vitro. Expression of BAX,BCL-2,Casepase-3 and Cyclin D1 protein were detected by Western blot. RESULTS: The proportion of CD34+CD38---KG1a stem cells was (95.35±2.63）% after immunomagnetic isolation. The proliferation of KG1a stem cells was inhibited significantly by FA-2-b-ß, which shows a time- and dose-dependent manner (24 h,r=0.943; 48 h,r=0.976). Flow cytometry shows that with the increasing of drug concentration, the apoptosis was also increased, when KG1a stem cells was treated by FA-2-b-ß for 24 h. Western blot indicated that the expression of apoptosis-related protein BAX and Casepase-3 were up-regulated, the expression of BCL-2 and Cyclin D1 were down-regulated. CONCLUSION: FA-2-b-ß can regulate proliferation and apoptosis KG1a stem cells, the involved mechanism may be related with the activation of mitochondrial-mediated apoptotic pathway.

Pathological process of liver sinusoidal endothelial cells in liver diseases.

Cirrhosis develops from liver fibrosis and is the severe pathological stage of all chronic liver injury. Cirrhosis caused by hepatitis B virus and hepatitis C virus infection is especially common. Liver fibrosis and cirrhosis involve excess production of extracellular matrix, which is closely related to liver sinusoidal endothelial cells (LSECs). Damaged LSECs can synthesize transforming growth factor-beta and platelet-derived growth factor, which activate hepatic stellate cells and facilitate the synthesis of extracellular matrix. Herein, we highlight the angiogenic cytokines of LSECs related to liver fibrosis and cirrhosis at different stages and focus on the formation and development of liver fibrosis and cirrhosis. Inhibition of LSEC angiogenesis and antiangiogenic therapy are described in detail. Targeting LSECs has high therapeutic potential for liver diseases. Further understanding of the mechanism of action will provide stronger evidence for the development of anti-LSEC drugs and new directions for diagnosis and treatment of liver diseases.

MiR-486-5p negatively regulates oncogenic NEK2 in hepatocellular carcinoma.

NEK2 is a member of the NIMA-related family of serine/threonine centrosomal kinases. We analyzed the relationship between differential expression of NEK2 and hepatocellular carcinoma (HCC) patient outcomes after liver transplants. We also studied the microRNAs that affect NEK2 expression. Analysis of multiple microarrays in the Oncomine database revealed that NEK2 expression was higher in HCC tissues than adjacent normal liver tissues. High NEK2 expression correlated with tumor size, pathological grade and macro- and microvascular invasion. Consequently, patients exhibiting high NEK2 expression had poorer prognosis. This was corroborated by our multivariate analysis that showed NEK2 to be an independent prognostic factor for HCC patient survival. Further, high NEK2 expression promoted proliferation, colony formation, migration and invasion of HCC cell lines. Tumor xenograft data from Balb/c nude mice demonstrated that HCC cells with high NEK2 expression formed larger tumors than those with low NEK2 expression. Finally, we showed that miR-486-5p suppressed NEK2 by directly binding to its transcript 3'UTR. We also demonstrated an inverse relationship between miR-486-5p and NEK2 expression in HCC patients. These findings suggest miR-486-5p negatively regulates NEK2, which is a critical prognostic indicator of HCC patient survival after liver transplantation.

A simplified multivisceral transplantation procedure for patients with combined end-stage liver disease and type 2 diabetes mellitus.

In liver transplant patients with type 2 diabetes mellitus (DM), the disease worsens after transplantation because of longterm use of diabetogenic immunosuppressive drugs, making management of those patients a great challenge. The objective of our study was to evaluate the safety and efficacy of a simplified multivisceral transplantation (SMT) procedure for the treatment of patients with end-stage liver disease and concurrent type 2 DM. Forty-four patients who had pretransplant type 2 DM were included. A total of 23 patients received SMT, and 21 patients received orthotopic liver transplantation (OLT). Patient and graft survivals, complications, diabetic control, and quality of life (QOL) were retrospectively analyzed in both groups. The 1-, 3-, and 5-year cumulative patient and graft survival rates were 91.5%, 75.4%, and 75.4% in the SMT group and were 94.4%, 64.4%, and 64.4% in the OLT group, respectively (P = 0.70). Interestingly, 95.7% (22/23) of patients achieved complete remission from DM after SMT compared with 16.7% (3/18) of patients after OLT. The occurrence of biliary complication was significantly higher in the OLT group than that in the SMT group (23.8% versus 0.0%; P = 0.01). Moreover, better QOL was observed in the SMT group than that in the OLT group. In conclusion, the SMT procedure we described here is a safe and viable option for patients with end-stage live disease and concurrent type 2 DM. This SMT procedure offers excellent transplant outcomes and QOL. Liver Transplantation 23 1161-1170 2017 AASLD.

Prognostic value of combined preoperative fibrinogen and neutrophil-lymphocyte ratio in patients with hepatocellular carcinoma after liver transplantation.

OBJECTIVES: Elevated plasma fibrinogen (Fib) correlated with patient's prognosis in several solid tumors. However, few studies have illuminated the relationship between preoperative Fib and prognosis of HCC after liver transplantation. We aimed to clarify the prognostic value of Fib and whether the prognostic accuracy can be enhanced by the combination of Fib and neutrophil-lymphocyte ratio (NLR). RESULTS: Fib was correlated with Child-pugh stage, alpha-fetoprotein (AFP), size of largest tumor, macro- and micro-vascular invasion. Univariate analysis showed preoperative Fib, AFP, NLR, size of largest tumor, tumor number, macro- and micro- vascular invasion were significantly associated with disease-free survival (DFS) and overall survival (OS) in HCC patients with liver transplantation. After multivariate analysis, only Fib and macro-vascular invasion were independently correlated with DFS and OS. Survival analysis showed that preoperative Fib > 2.345 g/L predicted poor prognosis of patients HCC after liver transplantation. Preoperative Fib showed prognostic value in various subgroups of HCC. Furthermore, the predictive range was expanded by the combination of Fib and NLR. MATERIALS AND METHODS: Data were collected retrospectively from 130 HCC patients who underwent liver transplantation. Preoperative Fib, NLR and clinicopathologic variables were analyzed. The survival analysis was performed by the Kaplan-Meier method, and compared by the log-rank test. Univariate and multivariate analyses were performed to identify the prognostic factors for DFS and OS. CONCLUSIONS: Preoperative Fib is an independent effective predictor of prognosis for HCC patients, higher levels of Fib predict poorer outcomes and the combination of Fib and NLR enlarges the prognostic accuracy of testing.

Prognostic value of combined preoperative lactate dehydrogenase and alkaline phosphatase levels in patients with resectable pancreatic ductal adenocarcinoma.

Serum enzymes, including lactate dehydrogenase (LDH) and alkaline phosphatase (ALP), have recently been reported to play important roles in tumor growth. Increases in LDH and ALP have been confirmed to predict poor prognosis in patients with various cancers. However, their prognostic value in pancreatic cancer has not been well studied. Therefore, we reviewed the preoperative data on LDH and ALP in 185 pancreatic ductal adenocarcinoma (PDAC) patients who underwent surgery between July 2005 and December 2010 to explore the prognostic value of these markers. The cutoff points were determined based on the upper limit of their normal values. The Chi-square test was used to analyze the relationships between LDH/ALP and clinical characteristics. Univariate and multivariate analyses were performed to identify the predictive value of the above factors for disease-free survival (DFS) and overall survival (OS). We found that elevation of LDH was related to carbohydrate antigen 19-9 (CA19-9), lymph node involvement, tumor size, TNM, distant metastasis, and recurrence. Additionally, ALP was correlated to perineural invasion. After multivariate analysis, LDH and ALP were identified as independent prognostic factors for DFS and OS, and elevation of LDH/ALP was correlated with poor DFS and OS. Notably, there was a positive correlation between LDH and ALP. The predictive power of LDH combined with ALP was more sensitive than that of either one alone. Therefore, we conclude that the preoperative LDH and ALP values are prognostic factors for PADC, and the prognostic accuracy of testing can be enhanced by the combination of LDH and ALP.

miR-26a regulates mouse hepatocyte proliferation via directly targeting the 3' untranslated region of CCND2 and CCNE2.

BACKGROUND: The deficiency of liver regeneration needs to be addressed in the fields of liver surgery, split liver transplantation and living donor liver transplantation. Researches of microRNAs would broaden our understandings on the mechanisms of various diseases. Our previous research confirmed that miR-26a regulated liver regeneration in mice; however, the relationship between miR-26a and its target, directly or indirectly, remains unclear. Therefore, the present study further investigated the mechanism of miR-26a in regulating mouse hepatocyte proliferation. METHODS: An established mouse liver cell line, Nctc-1469, was transfected with Ad5-miR-26a-EGFP, Ad5-anti-miR-26a-EGFP or Ad5-EGFP vector. Cell proliferation was assessed by MTS, cell apoptosis and cell cycle by flow cytometry, and gene expression by Western blotting and quantitative real-time PCR. Dual-luciferase reporter assays were used to test targets of miR-26a. RESULTS: Compared with the Ad5-EGFP group, Ad5-anti-miR-26a-EGFP down-regulated miR-26a and increased proliferation of hepatocytes, with more cells entering the G1 phase of cell cycle (82.70%+/-1.45% vs 75.80%+/-3.92%), and decreased apoptosis (5.50%+/-0.35% vs 6.73%+/-0.42%). CCND2 and CCNE2 were the direct targeted genes of miR-26a. miR-26a down-regulation up-regulated CCND2 and CCNE2 expressions and down-regulated p53 expression in Nctc-1469 cells. On the contrary, miR-26a over-expression showed the opposite results. CONCLUSIONS: miR-26a regulated mouse hepatocyte proliferation by directly targeting the 3' untranslated regions of cyclin D2/cyclin E2; miR-26a also regulated p53-mediated apoptosis. Our data suggested that miR-26a may be a promising regulator in liver regeneration.

[Study on safety of Tibetan medicine zuotai and preliminary study on clinical safety of its compound dangzuo].

Zuotai (gTso thal) is a typical representative of Tibetan medicines containing heavy metals, but there is still lack of modem safety evaluation data so far. In this study, acute toxicity test, sub-acute toxicity test, one-time administration mercury distribution experiment, long-term mercury accumulative toxicity experiment and preliminary study on clinical safety of Compound Dangzuo were conducted in the hope of obtain the medicinal safety data of Zuotai. In the acute toxicity test, half of KM mice given the lethal dose of Zuotai were not died or poisoned, and LD50 was not found. The maximum tolerated dose of Zuotai was 80 g x kg(-1). In the subacute toxicity test, Zuotai could reduce ALT, AST, Crea levels in serums under low dose (13.34 mg x kg(-1) x d(-1)) and medium dose (53.36 mg x kg(-1) x d(-1)), with significant difference under low dose, and increase the levels of ALT, AST, MDA, Crea in serums under high dose (2 000 mg x kg(-1) x d(-1)); besides, the levels of BUN and GSH in serums reduced with the increase in dose of Zuotai, indicating a significant dose-effect relationship. In the one-time administration distribution experiment, the content of mercury in rat kidney, liver and lung increased after the one-time administration with Zuotai, with a significant dose-dependent relationship in kidney. In the long-term mercury accumulative toxicity experiment, KM mice were administered with equivalent doses of Zuotai for 4.5 months and then stopped drug administration for 1.5 months. Since the 2.5th month, they showed significant mercury accumulation in kidney, which gradually reduced after drug withdrawal, without significant change in mercury content in liver, spleen and brain and ALT, AST, TBIL, BUN and Crea in serum. At the 4.5th month after drug administration, KM mice showed slight structural changes in kidney, liver and spleen tissues, and gradually recovered to normal after drug withdrawal. Besides, no significant difference in weight gain was found between the Zuotai group and the control group. According to the findings of the clinical safety study of Dangzuo, after subjects administered Dangzuo under clinical dose for one month, their serum biochemical indicators, blood routine indicators and urine routine indicators showed no significant adverse change. This study proved that traditional Tibetan medicine Zuotai was slightly toxic, with a better safety in clinical combined administration and no adverse effects on bodies under the clinical dose and clinical medication cycle. However, long-term high-dose administration of Zuotai may have a certain effect on kidney.

Pathological characteristics of liver allografts from donation after brain death followed by cardiac death in pigs.

Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25-30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P<0.05). And there was no significant difference between DBD group and DBCD group (P>0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent.