Clinical significance of upregulated Rho GTPase activating protein 12 causing resistance to tyrosine kinase inhibitors in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is a major health challenge with high incidence and poor survival rates in China.Systemic therapies,particularly tyrosine kinase inhibitors(TKIs),are the first-line treatment for advanced HCC,but resistance is common.The Rho GTPase family member Rho GTPase activating protein 12(ARHGAP12),which regulates cell adhesion and invasion,is a potential therapeutic target for overcoming TKI resistance in HCC.However,no studies on the expression of ARHGAP12 in HCC and its role in resistance to TKIs have been reported.AIM To unveil the expression of ARHGAP12 in HCC,its role in TKI resistance and its potential associated pathways.METHODS This study used single-cell RNA sequencing(scRNA-seq)to evaluate ARHGAP12 mRNA levels and explored its mechanisms through enrichment analysis.CellChat was used to investigate focal adhesion(FA)pathway regulation.We integrated bulk RNA data(RNA-seq and microarray),immunohistochemistry and proteomics to analyze ARHGAP12 mRNA and protein levels,correlating with clinical outcomes.We assessed ARHGAP12 expression in TKI-resistant HCC,integrated conventional HCC to explore its mechanism,identified intersecting FA pathway genes with scRNA-seq data and evaluated its response to TKI and immunotherapy.RESULTS ARHGAP12 mRNA was found to be highly expressed in malignant hepatocytes and to regulate FA.In malignant hepatocytes in high-score FA groups,MDK-[integrin alpha 6(ITGA6)+integrinβ-1(ITGB1)]showed specificity in ligand-receptor interactions.ARHGAP12 mRNA and protein were upregulated in bulk RNA,immunohistochemistry and proteomics,and higher expression was associated with a worse prognosis.ARHGAP12 was also found to be a TKI resistance gene that regulated the FA pathway.ITGB1 was identified as a crossover gene in the FA pathway in both scRNA-seq and bulk RNA.High expression of ARHGAP12 was associated with adverse reactions to sorafenib,cabozantinib and regorafenib,but not to immunotherapy.CONCLUSION ARHGAP12 expression is elevated in HCC and TKI-resistant HCC,and its regulatory role in FA may underlie the TKI-resistant phenotype.

Activated Protein C Resistance in Patients with Pre-Eclampsia in Lagos, Nigeria

Background: Preeclampsia is reported to complicate 2% - 8% of pregnancies globally and is an important cause of maternal and perinatal morbidity and mortality. The aetiology and pathogenesis are still poorly understood and substantial improvement has not been made in the prediction, prevention and treatment of the disease. Objective: To compare the frequency of activated protein C resistance (APC-R) in patients with pre-eclampsia to that of normotensive pregnant women and to determine the correlation between activated protein ratio (APC-ratio) and the severity of pre-eclampsia. Methodology: A cross-sectional study was carried out in 100 pre-eclamptic patients and 100 normotensive pregnant controls. The APC-ratio was determined using the modified activated partial thromboplastin time. Study participants with APC-ratio of less than 2.0 were defined as having APC-R. Data was analyzed using SPSS version 22.0. Results: Mean APC-ratio was significantly lower in pre-eclamptics (2.89 ± 1.70) compared to normotensive pregnant women (3.57 ± 1.06) (p = 0.0008) and the levels were also higher in mild (2.95 ± 1.15) compared to severe pre-eclamptics (2.62 ± 1.14). The frequency of APC-R was 26% among women with pre-eclampsia compared to 4% among normotensive controls (p = 0.000). Among 100 pre-eclamptic women 7 (21.2%) out of 33 with mild pre–eclampsia had APC-R, while 19 (28.4%) out of 67 with severe pre-eclampsia had APC-R. APC-ratio had a significant negative correlation with mean arterial blood pressure (r = −0.324;p = 0.000) and proteinuria (r = −0.379;p = 0.000) among study participants. Conclusion: The frequency of activated protein c resistance is significantly higher in pre-eclamptics compared to normotensive pregnant women and this is more pronounced in those with severe pre-eclampsia compared with those with mild disease. APC-R may therefore be used as a marker of severity in the disease.

Developmental Lead Exposure Alters the Distribution of Protein Kinase C Activity in the Rat Hippocampus

Chronic low-level lead (Pb) exposure in children is known to cause a deficit in learning and memory. In vitro studies have demonstrated that Pb altered protein kinase C (PKC) activityt Especially, hippocampal PKC has been correlated with performance in several learning tasks. The effects of Pb exposure on hippocampal PKC were investigated during development at various postnatal ages: postnatal day (PN) 7, 14, 28, and 56. Two-tenth % Pb acetate was administered to pregnant and lactating dams and then administered to weanling rats in drinking water. PKC activity was measured in both membrane and cytosolic fractions from the hippocampi of the controls and Pb-exposed animals. Pb-induced increase in PKC activity in the cytosolic fraction was obsereved in the PN56 rats. In contrast, PKC activity was decreased by Pb at PN7 in the membrane fraction. Furthermore, a significant decrease in the ratio of membrane to cytosolic PKC activity which is representative of PKC distribution was observed in the PN28 and PN56 Pb-exposed rats relative to the same-age controls. This study indicates that chronic Pb exposure during development influences hippocampal PKC activity and distribution. These changes may be involved in the subclinical neurotoxicity of chronic Pb exposure in young children.

ASSOCIATION OF EXPRESSION OF P53 PROTEIN WITH CELL PROLIFERATIVE ACTIVITY AND PROGNOSIS IN COLORECTAL CANCER

The expression of p53 protein in 66 cases ofcolorectal cancer and its relationship to cell porliferativeactivity, lymph node metastasis as well as prognosis wereinvestigated by means of AB-PAP immunohistochcmicaltechnique- The results showed that 62.1/o of colorectalcancer was positive of p53 expression. The cellproliferative activity and the frequency of lymph nodemetastasis in p53- positive tumors were significantlyhigher than those in p53-negative tumors (P<0.05). Thesurvival rate in patients with p53- positive tumors wassignificantly poorer than those with p53- negative tumors(P<0.05). Our results suggest that the abnormalexpression of p53 and cell proliferation associated withmutations are involved in both human carcinogenesis andlymph node metastasis of colorectal cancer. Examinationof p53 expression is of value in understanding the degreeof malignancy, and evaluating prognosis of the disease.

Preparation and analysis of immunocompetence of recombinant fusion protein of the immunodominant region in chlamydial protease-like activity factor from Chlamydophila pneumoniae and its application in serodiagnosis

To clone the gene coding the immunodominant region in the chlamydial protease-like activity factor （CPAF） from Chlaroydophila pneumoniae, to analyze immunoeoropetenee of the expressed protein, and to evaluate its value in serodiagnosis, the CPAF immunodominant region gene was amplified, ligated into a pGEX6p-2 vector, and then the expressed recombinant protein was purified with glutathione Stransferase （GST） agarose gel FF after renaturation, then identified by SDS-PAGE and Western blot. A new indirect ELISA was developed with the purified protein as coating antigen. The immunogenicity of the recombinant protein was evaluated by immunization to New Zealand rabbits, and its immunoreactivity was analyzed by reacting with anti-C, pneumoniae antibody. 300 clinical sera samples were respectively detected by mieroimmunofluorescenee （MIF） as reference method and the indirect ELISA, and the difference between the two methods was analyzed. Cross-reactivity against Chlamydia trachomatis was investigated with the indirect ELISA to detect anti-C, trachomatis positive antisera. The results indicated that a 51.3 kDa recombinant protein was obtained. Western blot assay proved that the recombinant protein could merely specifically react with human anti- C. pneurnoniae antisera. The titers of the specific IgG antibodies in the immunized New Zealand rabbits were above 1 ： 16 000. Anti- C. pneumoniae IgG positive and negative reference sera were detected with the indirect ELISA, and the concordance rate of negative and positive results were both 100% （40/40）. The sensitivity and specificity of the indirect ELISA in comparison with MIF were 93.8% （45/48） and 100% （252/252） separately by detecting 300 clinical sera samples, and the concordance rate between the two methods was 99.0%. No cross reaction against C. trachomatis was found with the indirect ELISA to detect anti-C, trachomatis positive antisera. In conclusion, the prepared recombinant protein of the CPAF immunodominant region shows excellent immunocompetence and can be used to develop a new indirect ELISA as a method to detect anti-C, pneumoniae antibody for diagnosis of C. pneumoniae infection.

Protein Kinase Activity Changes in the Aging Brain

The aging process in mammals is correlated with changes in psychomotor performance, cognitive function, and ability to adapt to stress (Montgomery et al., 1982; Lorens et al., 1990). These changes may be related to alterations in neuronal tissue that occur during the aging process. The normal aging process may be conceived of as the neuronal cell’s increasing inability to maintain normal cellular function which ultimately results in a number of morphological and biochemical changes. Morphologically, there is a loss of neuronal cells with increasing age (Brizzee and

Influence of sodium fluoride on alkaline phosphatase activity and bone gla protein synthesis in human yellow ligament cells in vitro

Objective To investigate the influence of sodium fluoride(NaF)on alkaline phosphatase(ALP)activity and bone gla protein(BGP)synthesis in yellow ligament cells from different surgical simples in vitro.Methods The human ligament cells

Establishment of Double-antigen Sandwich Time-resolved Fluorescence Immunoassay for Detection of Pest des Petits Ruminants Virus

[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.

Cantharidin and Its Analogues:Anticancer and Ser/Thr Protein Phosphatase Inhibitory Activities

This paper mainly describes the anticancer activities and Ser/Thr protein phosphatase inhibitory activities of cantharidin and its analogues.

Expression of fibroblast activation protein in human pancreatic adenocarcinoma and its clinicopathological significance

AIM: To examine fibroblast activation protein (FAP) expression in pancreatic ductal adenocarcinoma (PDAC) and to analyze its relationship with the clinicopathology of PDAC. METHODS: FAP expression was examined in 134 PDAC specimens by immunohistochemistry, and in four pancreatic cancer cell lines (SW1990, Miapaca-2, AsPC-1 and BxPC-3) by Western blotting assay. We also analyzed the association between FAP expression in PDAC cells and the clinicopathology of PDAC patients. RESULTS: The results showed that the FAP was expressed in both stromal fibroblast cells (98/134, 73.1%) and carcinoma cells (102/134, 76.1%). All 4 pancreatic cancer cell lines expressed FAP protein at different levels. Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its88-kDa seprase subunit were identif ied. Higher FAP expression in carcinoma cells was associated with tumor size (P < 0.001), fi brotic focus (P = 0.003), perineural invasion (P = 0.009) and worse clinical outcome (P = 0.0085). CONCLUSION: FAP is highly expressed in carcinoma cells and f ibroblasts in PDAC tissues, and its expression is associated with desmoplasia and worse prognosis.

Ornithine decarboxylase, mitogen-activated protein kinase and matrix metalloproteinase-2 expressions in human colon tumors

AIM: To investigate the expressions of omithine decarboxylase (ODC), MMP-2, and Erk, and their relationship in human colon tumors.METHODS: ODC activity, MMP-2 expression, and mitogenactivated protein (MAP) kinase activity (Erk phosphorylation) were determined in 58 surgically removed human colon tumors and their adjacent normal tissues, using [1-14C]-ornithine as a substrate, ELISA assay, and Western blotting, respectively.RESULTS: ODC activity, MMP-2 expression, and Erk phosphorylation were significantly elevated in colon tumors, compared to those in adjacent normal tissues. A significant correlation was observed between ODC activities and MMP-2 levels.CONCLUSION: This is the first report showing a significant correlation between ODC activities and MMP-2 levels in human colon tumors. As MMP-2 is involved in cancer invasion and metastasis, and colon cancer overexpresses ODC, suppression of ODC expression may be a rational approach to treat colon cancer which overexpresses ODC.

Caffeic acid phenethyl ester up-regulates antioxidant levels in hepatic stellate cell line T6 via an Nrf2-mediated mitogen activated protein kinases pathway

AIM To investigate the antioxidant effect of caffeic acid phenethyl ester (CAPE) in hepatic stellate cell-T6 (HSC-T6) cells cultured in vitro and the potential mechanisms. METHODS HSC-T6 cells were cultured in vitro and treated with various concentrations of CAPE for 24, 48 and 72 h, respectively. Cell proliferation was investigated using the MTT assay, and cell ultrastructural alterations were observed by transmission electron microscopy. Flow cytometry was employed to investigate the effects of CAPE on apoptosis and the levels of reactive oxygen species in HSC-T6 cells cultured in vitro. An enzyme immunoassay instrument was used to evaluate antioxidant enzyme expression. The effect on alpha-smooth muscle actin was shown using immunofluorescence. Gene and protein levels of Nrf2, related factors, and mitogen activated protein kinases (MAPKs), in HSC-T6 cells were detected using RT-PCR and Western blot, respectively. RESULTS CAPE inhibited the proliferation and activation of HSC-T6 cells cultured in vitro. CAPE increased the antioxidant levels and the translocation of Nrf2 from the cytoplasm to the nucleus in HSC-T6 cells. Moreover, the phosphorylation of MAPKs in cells decreased in response to CAPE. Interestingly, CAPE-induced oxidative stress in the cells was significantly attenuated by pretreatment with MAPKs inhibitors. CONCLUSION CAPE inhibits cell proliferation and up-regulates the antioxidant levels in HSC-T6 cells partly through the Nrf2-MAPKs signaling pathway.

Recurrent thrombotic occlusion of a transjugular intrahepatic portosystemic stent-shunt due to activated protein C resistance

The transjugular intrahepatic portosystemic stent-shunt (TIPS) has successfully been used in the management of refractory variceal bleeding and ascites in patients with portal hypertension. Major drawbacks are the induction of hepatic encephalopathy and shunt dysfunction. We present a 59-year-old woman with alcoholic liver cirrhosis who received a TIPS because of recurrent bleeding from esophageal varices. Stent occlusion occurred 4 mo after placement of the TIPS. Laboratory testing revealed resistance to activated protein C (APC). Combination therapy with low-dose enoxaparin and clopidogrel could not prevent her recurrent stent occlusion. Finally, therapy with high-dose enoxaparin was sufficient to prevent further shunt complications up to now (follow-up period of 1 year). In conclusion, early occlusion of a TIPS warrants testing for thrombophilia. If risk factors are confirmed,anticoagulation should be intensified. There are currently no evidence-based recommendations regarding the best available anticoagulant therapy and surveillance protocol for patients with TIPS.

Neuroprotective mechanisms of rutin for spinal cord injury through anti-oxidation and anti-inflammation and inhibition of p38 mitogen activated protein kinase pathway

Rutin has anti-inflammatory, antioxidant, anti-viral, anti-tumor and immune regulatory effects. However, the neuroprotective effects of rutin in spinal cord injury are unknown. The p38 mitogen activated protein kinase （p38 MAPK） pathway is the most important member of the MAPK family that controls inflammation. We assumed that the mechanism of rutin in the repair of spinal cord injury is associated with the inhibition of p38 MAPK pathway. Allen’s method was used to establish a rat model of spinal cord injury. The rat model was intraperitoneally injected with rutin （30 mg/kg） for 3 days. After treatment with rutin, Basso, Beattie and Bresnahan locomotor function scores increased. Water content, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 levels, p38 MAPK protein expression and caspase-3 and -9 activities in T8–9 spinal cord decreased. Oxidative stress related markers superoxide dismutase and glutathione peroxidase levels increased in peripheral blood. Rutin exerts neuroprotective effect through anti-oxidation, anti-inflammation, anti-apoptosis and inhibition of p38 MAPK pathway.

Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

AIM： To construct a live attenuated Salmonella typhimurium （S. typhimurium） strain harboring the H pylori neutrophil activating protein （HP-NAP） gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity. METHODS： By genetic engineering methods, the genomic DNA of Hpylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction （PCR） and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments. RESULTS： A 435 bp product was cloned using high homology with HP-NAP gene in GenBank （more than 98%）. With identification by PCR and restriction enzyme digestion, a recomoinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with Hpyloril whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 （pIRES-NAP） also induced a specific immune response. CONCLUSION： The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against Hpylori infection.

Helicobacter pylori neutrophil activating protein as target for new drugs against H.pylori inflammation

Helicobacter pylori(H.pylori) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer.With-in this work we present the implication of C-terminal region of H.pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evi-dence that the C-terminal region of H.pylori activating protein is indispensable for neutrophil adhesion to endothelial cells,a step necessary to H.pylori inflammation.In addition we show that arabino galactan proteins derived from chios mastic gum,the natural resin of the plant Pistacia lentiscus var.Chia inhibit neutrophil activation in vitro.

Insulin-like growth factor binding protein-5 influences pancreatic cancer cell growth

AIM： To investigate the functional significance of insulin-like growth factor binding protein-5 （IGFBP-5） overexpression in pancreatic cancer （PaC）.METHODS： The effects of IGFBP-5 on cell growth were assessed by stable transfection of BxPC-3 and PANC-1 cell lines and measuring cell number and DNA synthesis. Alterations in the cell cycle were assessed by flow cytometry and immunoblot analyses. Changes in cell survival and signal transduction were evaluated after mitogen and phosphatidylinositol activated protein kinase 3-kinase （PI3K） inhibitor treatment.RESULTS： After serum deprivation, IGFBP-5 expression increased both cell number and DNA synthesis in BxPC-3 cells, but reduced cell number in PANC-1 cells. Consistent with this observation, cell cycle analysis of IGFBP-5-expressing cells revealed accelerated cell cycle progression in BxPC-3 and G2/M arrest of PANC-1 cells. Signal transduction analysis revealed that Akt activation was increased in BxPC-3, but reduced in PANC-1 cells that express IGFBP-5. Inhibition of PI3K with LY294002 suppressed extracellular signal-regulated kinase-1 and -2 （ERK1/2） activation in BxPC-3, but enhanced ERK1/2 activation in PANC-1 cells that express IGFBP-5. When MEK1/2 was blocked, Akt activation remained elevated in IGFBP-5 expressing PaC cells; however, inhibition of PI3K or MEK1/2 abrogated IGFBP-5-mediated cell survival.CONCLUSION： These results indicate that IGFBP-5 expression affects the cell cycle and survival signal pathways and thus it may be an important mediator of PaC cell growth.

Protective Effects of Activated Protein C on Neurovascular Unit in a Rat Model of Intrauterine Infection-Induced Neonatal White Matter Injury

Summary： Activated protein C （APC）, a natural anticoagulant, has been reported to exert direct vascu- loprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. In- traperitoneal injection of 300 ~tg/kg lipopolysaccharide （LPS） was administered consecutively to preg- nant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infec- tion-induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats imme- diately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eo- sin （H＆E）. Immunohistochemistry was used to evaluate myelin basic protein （MBP） expression in the periventricular white matter region. Blood-brain barrier （BBB） permeability and brain water content ~were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 （PAR1）. Typical pathological changes of white matter injury were ob- served in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely acti- vated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein ex- pression levels were both down-regulated. Our results suggested that APC exerted neuroprotective ef- fects on multiple components of the neurovascular unit in neonatal rats with intrauterine infec- tion-induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

Liver myofibroblasts activate protein C and respond to activated protein C

AIM:To study the protein C activation system in human liver myofibroblasts,and the effects of activated protein C(APC)on these cells.METHODS:Human liver myofibroblasts were obtained by outgrowth.Expression of protease activated receptor 1(PAR-1),endothelial protein C receptor(EPCR) and thrombomodulin(TM)was analyzed by flow cytometry.Extracellular signal-regulated kinase(ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies.Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction(RT-PCR).Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS:Primary cultures of human liver myofibroblasts expressed EPCR on their surface,together with PAR-1 and TM.This receptor system was functional since exposure of myofibroblasts to APC inducedERK1/2 phosphorylation in a dose-and time-dependent manner.Furthermore,APC significantly upregulated the expression of collagen mRNA,as shown by real-time RT-PCR.Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059.Finally,using a cell-based colorimetric assay,we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin.CONCLUSION:These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.