DNA demethylation and tri-methylation of H3K4 at the TACSTD2 promoter are complementary players for TROP2 regulation in colorectal cancer cells.

TROP2 is a powerful cancer driver in colorectal cancer cells. Divergent epigenetic regulation mechanisms for the corresponding TACSTD2 gene exist such as miRNAs or DNA methylation. However, the role of TACSTD2 promoter hypermethylation in colorectal cancer has not been investigated yet. In this study, TROP2 expression strongly correlated with promoter methylation in different colorectal tumor cell lines. Treatment with 5-Azacytidine, a DNMT1 inhibitor, led to demethylation of the TACSTD2 promoter accompanied by an increase in TROP2 protein expression. TROP2 expression correlated with promoter methylation in vivo in human colon tumor tissue, thereby verifying promoter methylation as an important factor in the regulation of TROP2 expression in colorectal cancer. When performing a ChIP-Seq analysis in HCT116 and HT29 cells, we found that TACSTD2 promoter demethylation was accompanied by tri-methylation of H3K4. In silico analysis of GSE156613 data set confirmed that a higher binding of histone mark H3K4me3 around the TACSTD2 promoter was found in TACSTD2 high expressing tumors of colon cancer patients compared to the corresponding adjacent tumor tissue. Moreover, the link between TROP2 and the H3K4me3 code was even evident in tumors showing high intratumoral heterogeneity for TROP2 staining. Our data provide novel evidence for promoter demethylation and simultaneous gains of the active histone mark H3K4me3 across CpG-rich sequences, both being complementary mechanisms in the transcriptional regulation of TACSTD2 in colon cancer. The functional consequences of TROP2 loss in colorectal cancer needs to be further investigated.

An integrated study of glutamine alleviates enteritis induced by glycinin in hybrid groupers using transcriptomics, proteomics and microRNA analyses.

Glutamine has been used to improve intestinal development and immunity in fish. We previously found that dietary glutamine enhances growth and alleviates enteritis in juvenile hybrid groupers (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂). This study aimed to further reveal the protective role of glutamine on glycinin-induced enteritis by integrating transcriptome, proteome, and microRNA analyses. Three isonitrogenous and isolipidic trial diets were formulated: a diet containing 10% glycinin (11S group), 10% glycinin diet supplemented with 2% alanine-glutamine (Gln group), and a diet containing neither glycinin nor alanine-glutamine (fishmeal, FM group). Each experimental diet was fed to triplicate hybrid grouper groups for 8 weeks. The analysis of intestinal transcriptomic and proteomics revealed a total of 570 differentially expressed genes (DEGs) and 169 differentially expressed proteins (DEPs) in the 11S and FM comparison group. Similarly, a total of 626 DEGs and 165 DEPs were identified in the Gln and 11S comparison group. Integration of transcriptome and proteome showed that 117 DEGs showed consistent expression patterns at both the transcriptional and translational levels in the Gln and 11S comparison group. These DEGs showed significant enrichment in pathways associated with intestinal epithelial barrier function, such as extracellular matrix (ECM)-receptor interaction, tight junction, and cell adhesion molecules (P < 0.05). Further, the expression levels of genes (myosin-11, cortactin, tenascin, major histocompatibility complex class I and II) related to these pathways above were significantly upregulated at both the transcriptional and translational levels (P < 0.05). The microRNA results showed that the expression levels of miR-212 (target genes colla1 and colla2) and miR-18a-5p (target gene colla1) in fish fed Gln group were significantly lower compared to the 11S group fish (P < 0.05). In conclusion, ECM-receptor interaction, tight junction, and cell adhesion molecules pathways play a key role in glutamine alleviation of hybrid grouper enteritis induced by high-dose glycinin, in which miRNAs and target mRNAs/proteins participated cooperatively. Our findings provide valuable insights into the RNAs and protein profiles, contributing to a deeper understanding of the underlying mechanism for fish enteritis.

Anti-Inflammatory Activity of Ferula assafoetida Oleo-Gum-Resin (Asafoetida) against TNF-α-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs).

Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 µg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 µg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.

Rs1h-/y exon 3-del rat model of X-linked retinoschisis with early onset and rapid phenotype is rescued by RS1 supplementation.

Animal models of X-linked juvenile retinoschisis (XLRS) are valuable tools for understanding basic biochemical function of retinoschisin (RS1) protein and to investigate outcomes of preclinical efficacy and toxicity studies. In order to work with an eye larger than mouse, we generated and characterized an Rs1h-/y knockout rat model created by removing exon 3. This rat model expresses no normal RS1 protein. The model shares features of an early onset and more severe phenotype of human XLRS. The morphologic pathology includes schisis cavities at postnatal day 15 (p15), photoreceptors that are misplaced into the subretinal space and OPL, and a reduction of photoreceptor cell numbers by p21. By 6 mo age only 1-3 rows of photoreceptors nuclei remain, and the inner/outer segment layers and the OPL shows major changes. Electroretinogram recordings show functional loss with considerable reduction of both the a-wave and b-wave by p28, indicating early age loss and dysfunction of photoreceptors. The ratio of b-/a-wave amplitudes indicates impaired synaptic transmission to bipolar cells in addition. Supplementing the Rs1h-/y exon3-del retina with normal human RS1 protein using AAV8-RS1 delivery improved the retinal structure. This Rs1h-/y rat model provides a further tool to explore underlying mechanisms of XLRS pathology and to evaluate therapeutic intervention for the XLRS condition.

Curcumin by activation of adenosine A2A receptor stimulates protein kinase a and potentiates inhibitory effect of cangrelor on platelets.

Curcumin is a natural polyphenol derived from the turmeric plant (Curcuma longa) which exhibits numerous beneficial effects on different cell types. Inhibition of platelet activation by curcumin is well known, however molecular mechanisms of its action on platelets are not fully defined. In this study, we used laser diffraction method for analysis of platelet aggregation and Western blot for analysis of intracellular signaling mechanisms of curcumin effects on platelets. We identified two new molecular mechanisms involved in the inhibitory effects of curcumin on platelet activation. Firstly, curcumin by activation of adenosine A2A receptor stimulated protein kinase A activation and phosphorylation of Vasodilator-stimulated phosphoprotein. Secondly, we demonstrated that curcumin even at low doses, which did not inhibit platelet aggregation, potentiated inhibitory effect of ADP receptor P2Y12 antagonist cangrelor which partly could be explained by activation of adenosine A2A receptor.

CRISP2, CATSPER1 and PATE1 Expression in Human Asthenozoospermic Semen.

The etiology of human asthenozoospermia is multifactorial. The need to unveil molecular mechanisms underlying this state of infertility is, thus, impelling. Circular RNAs (circRNAs) are involved in microRNA (miRNA) inhibition by a sponge activity to protect mRNA targets. All together they form the competitive endogenous RNA network (ceRNET). Recently, we have identified differentially expressed circRNAs (DE-circRNAs) in normozoospermic and asthenozoospermic patients, associated with high-quality (A-spermatozoa) and low-quality (B-spermatozoa) sperm. Here, we carried out a differential analysis of CRISP2, CATSPER1 and PATE1 mRNA expression in good quality (A-spermatozoa) and low quality (B-spermatozoa) sperm fractions collected from both normozoospermic volunteers and asthenozoospermic patients. These sperm fractions are usually separated on the basis of morphology and motility parameters by a density gradient centrifugation. B-spermatozoa showed low levels of mRNAs. Thus, we identified the possible ceRNET responsible for regulating their expression by focusing on circTRIM2, circEPS15 and circRERE. With the idea that motility perturbations could be rooted in quantitative changes of transcripts in sperm, we evaluated circRNA and mRNA modulation in A-spermatozoa and B-spermatozoa after an oral amino acid supplementation known to improve sperm motility. The profiles of CRISP2, CATSPER1 and PATE1 proteins in the same fractions of sperm well matched with the transcript levels. Our data may strengthen the role of circRNAs in asthenozoospermia and shed light on the molecular pathways linked to sperm motility regulation.

Xanthorrhizol Suppresses Vascular Endothelial Growth Factor-Induced Angiogenesis by Modulating Akt/eNOS Signaling and the NF-[Formula: see text]B-Dependent Expression of Cell Adhesion Molecules.

Angiogenesis plays a crucial role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation and migration are critical steps in tumor angiogenesis. Here, we investigated the anti-angiogenic activity of xanthorrhizol, a sesquiterpenoid isolated from the Indonesian medicinal plant Curcuma xanthorrhiza. Xanthorrhizol at noncytotoxic concentrations inhibited the proliferation, migration, and formation of capillary-like tubes in VEGF-treated human umbilical vein endothelial cells (HUVECs). Xanthorrhizol inhibited the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) and the expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin in VEGF-treated HUVECs. The expression and transcriptional activity of NF-[Formula: see text]B were downregulated by xanthorrhizol in VEGF-treated HUVECs. Furthermore, xanthorrhizol significantly inhibited VEGF-induced angiogenesis in the chorioallantoic membrane of fertilized eggs and Matrigel plugs subcutaneously injected into mice. Xanthorrhizol inhibited tumor volume and tumor-derived angiogenesis in mice inoculated with breast cancer cells. The in vitro and in vivo anti-angiogenic activities of xanthorrhizol were as potent as those of curcumin, a well-known anticancer agent derived from C. longa. Taken together, xanthorrhizol inhibits VEGF-induced angiogenesis of endothelial cells by blocking the activation of the PI3K/Akt/eNOS axis and subsequent upregulation of adhesion molecules induced by the transcriptional activation of NF-[Formula: see text]B. Xanthorrhizol is a promising anti-angiogenic agent and can serve as a beneficial agent to enhance anticancer treatments.

The FLA4-FEI Pathway: A Unique and Mysterious Signaling Module Related to Cell Wall Structure and Stress Signaling.

Cell wall integrity control in plants involves multiple signaling modules that are mostly defined by genetic interactions. The putative co-receptors FEI1 and FEI2 and the extracellular glycoprotein FLA4 present the core components of a signaling pathway that acts in response to environmental conditions and insults to cell wall structure to modulate the balance of various growth regulators and, ultimately, to regulate the performance of the primary cell wall. Although the previously established genetic interactions are presently not matched by intermolecular binding studies, numerous receptor-like molecules that were identified in genome-wide interaction studies potentially contribute to the signaling machinery around the FLA4-FEI core. Apart from its function throughout the model plant Arabidopsis thaliana for the homeostasis of growth and stress responses, the FLA4-FEI pathway might support important agronomic traits in crop plants.

PNN and KCNQ1OT1 Can Predict the Efficacy of Adjuvant Fluoropyrimidine-Based Chemotherapy in Colorectal Cancer Patients.

The benefit of adjuvant chemotherapy in the early stages of colorectal cancer (CRC) is still disappointing and the prediction of treatment outcome quite difficult. Recently, through a transcriptomic approach, we evidenced a role of PNN and KCNQ1OT1 gene expression in predicting response to fluoropyrimidine-based adjuvant chemotherapy in stage III CRC patients. Thus, the aim of this study was to validate in an independent cohort of stages IIIII CRC patients our previous findings. PNN and KCNQ1OT1 mRNA expression levels were evaluated in 74 formalin-fixed paraffin-embedded tumor and matched normal mucosa samples obtained by stages IIIII CRC patients treated with fluoropyrimidine-based adjuvant chemotherapy. PININ, the protein encoded by PNN, was immunohistochemically evaluated in 15 tumor and corresponding normal mucosa samples, selected on the basis of a low, medium, or high mRNA expression tumor/mucosa ratio. PNN and KCNQ1OT1 mRNA mean expression levels were significantly higher in tumor compared with normal tissues. Patients with high PNN or KCNQ1OT1 tumor mRNA levels according to ROC-based cutoffs showed a shorter disease-free survival (DFS) compared with patients with low tumor mRNA gene expression. Also, patients with tumor mRNA expression values of both genes below the identified cutoffs had a significantly longer DFS compared with patients with the expression of one or both genes above the cutoffs. In a representative large cohort of stages IIIII CRC untreated patients retrieved from GEO datasets, no difference in DFS was observed between patients with high and low PNN or KCNQ1OT1 gene expression levels. These data confirm our previous findings and underscore the relevance of PNN and KCNQ1OT1 expression in predicting DFS in early stages of CRC treated with fluoropyrimidine-based adjuvant chemotherapy. If further validated in a prospective case series, both biomarkers could be used to identify patients who benefit from this treatment and to offer alternative chemotherapy regimens to potential unresponsive patients. In relation to the suggested biological role of PNN and KCNQ1OT1 in CRC, they might also be exploited as potential therapeutic targets.

Phosphatidylcholine Ameliorates LPS-Induced Systemic Inflammation and Cognitive Impairments via Mediating the Gut-Brain Axis Balance.

Systemic inflammation will cause an imbalance in the steady state of the gut-brain axis. Phosphatidylcholine (PC) is a phospholipid found in egg yolk that has anti-inflammatory and antioxidant properties. The present research proved that PC supplementation (60 mg/kg body weight) for 35 days prevented inflammatory responses and behavioral disturbances in lipopolysaccharide (LPS)-induced mice. PC could regulate the expression of neurotrophic factors and synaptic proteins, which effectively alleviated the nerve damage and synaptic dysfunction caused by LPS. In addition, PC supplementation ameliorated gut barrier damage, altered gut genes, and improved gut health by modulating the cell adhesion molecule (CAM) pathway. Furthermore, PC remodeled the gut microbiome structure in the mice of the LPS group by increasing the relative abundance of Rikenellaceae and Lachnospiraceae. PC also increased short-chain fatty acid (SCFA) production in LPS-induced mice, which in turn ameliorated brain inflammatory responses. In conclusion, PC supplementation may be a nutritional strategy for the prevention of systemic inflammation via the gut-brain axis.

Methyl-donor supplementation prevents intestinal colonization by Adherent-Invasive E. coli in a mouse model of Crohn's disease.

Deficiencies in methyl-donor molecules (folate, B12 vitamin), DNA methylation alteration and high prevalence of Adherent-Invasive Escherichia coli (AIEC) are frequently observed in Crohn's disease (CD) patients. AIEC bacteria adhere to the enterocytes through abnormally expressed carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) glycoprotein on host cells. This work aims at studying the relationship between methyl-donor molecules and AIEC-induced intestinal inflammatory response. CEABAC10 mice, a mouse model of CD, were fed a control or Methyl-donor Supplemented diet (MS diet). CEACAM6 promoter was hypermethylated in intestinal epithelial cells from mice fed an MS diet, which was associated with a significant decrease in CEACAM6 expression. Transcriptomic analysis revealed increased expression of anti-microbial peptides, increase in HSP70 gene family expression and a decreased expression of inflammatory marker Calprotectin upon MS diet, associated to a lower ability of AIEC bacteria to colonize gut mucosa. We observed in a cohort of CD patients that serum folate concentration was inversely correlated to Crohn's disease endoscopic index of severity and to fecal inflammatory markers. This study demonstrates that methyl-donor supplementation through the diet induces a specific intestinal micro-environment limiting pathobiont colonization of the gut. Clinicians may wish to consider methyl-donor supplementation for methyl-donor deficient CD patients.

Supplementation of endogenous Ahr ligands reverses insulin resistance and associated inflammation in an insulin-dependent diabetic mouse model.

Aryl-hydrocarbon receptor (Ahr) plays an important role in the regulation of intestinal homeostasis. Diabetes is characterized by vascular complications and intestinal dysfunction. We aimed at understanding the relationship between intestinal defense impairment and inflammation in diabetes and effects of Ahr ligands on diabetes-induced insulin resistance, endovascular inflammation, and intercellular adhesion molecule (ICAM) and flavin mono-oxygenase (FMO3) expression. Effects of Ahr ligands, such as tryptophan (Trp) and indole-3-carbinol (I3C) on intestinal barrier and inflammation of Ins2Akita mice were examined. Myeloid differentiation primary response 88 (MYD88) is the adaptor for inflammatory signaling pathways. Ins2Akita-MyD88-/- mice were used to study the role of MyD88. Ins2Akita mice demonstrated decreased Ahr and regenerating islet-derived 3-ß (Reg3ß) expression, and increased Klebsiella pneumoniae translocation. Ins2Akita mice demonstrated increased inducible nitric oxide synthase (iNOS) expression of intestine; ICAM, iNOS, interleukin 1 beta (IL-1ß), and FMO3 expression of liver; and ICAM, iNOS, and FMO3 expression in aorta. Trp and I3C decreased diabetes-induced translocation and increased Ahr and Reg3ß expression of intestine. Ahr ligands reduced diabetes-induced ICAM and FMO3 expression in liver and aorta; IL-6, tumor necrosis factor alpha (TNF-α), and iNOS expression in Kupffer cells; plasma IL-6 and TNF-α levels; dipeptidyl peptidase (DPP4) activity; and insulin insensitivity. Ins2Akita-MyD88-/- mice demonstrated decreased expression of p-NF-κB of liver and ICAM of aorta compared with Ins2Akita mice. Altogether, our data suggest that diabetes induces ICAM and FMO3 expression through the decrease in intestinal defense and MyD88. Ahr ligands reverse diabetes-induced intestinal defense impairment, insulin insensitivity, FMO3/ICAM expression, and systemic inflammation.

Role of some natural anti-oxidants in the down regulation of Kim, VCAM1, Cystatin C protein expression in lead acetate-induced acute kidney injury.

BACKGROUND: Lead is a dangerous systemic toxicant and can provoke life-threatening renal injury. The plan of this study was to evaluate the potential impact of curcumin (CRMN) and L-ascorbic acid (L-ascb) alone or together to counteract lead acetate (Pb-acetate)-induced renal damage in rats and to find out the underlying mechanisms of action of these nutraceuticals. METHODS: Pb-acetate (100 mg/kg/day, i.p.) was injected in male rats along with L-ascb (250 mg/kg/day) and/or CRMN (200 mg/kg/day) orally for 7 days. RESULTS: Pb-acetate administration increased serum urea, creatinine and uric acid. Renal tissue showed a marked depletion in reduced glutathione level and superoxide dismutase activity and elevation in nitric oxide and malondialdehyde levels. Serum C-reactive protein and IL-1ß levels were elevated. Up-regulation of the expression of kidney injury molecule, vascular adhesion molecule-1 and Cystatin C were noticed after Pb-acetate administration. DNA fragmentation was also increased in renal tissues. Histopathological examination revealed a destructed partial layer of Bowman's capsule, proximal and distal convoluted tubules. Treatment with the aforementioned antioxidants ameliorated most of the altered measured biomarker levels. CONCLUSION: Interestingly, the combination of L-ascb and CRMN showed the superlative protective effect against Pb-acetate-induced nephrotoxicity.

Ziziphus spina-christi Leaf Extract Suppressed Mercury Chloride-Induced Nephrotoxicity via Nrf2-Antioxidant Pathway Activation and Inhibition of Inflammatory and Apoptotic Signaling.

Exposure to heavy metals, including mercury chloride (HgCl2), is associated with severe health problems. This study was designed to investigate HgCl2-induced nephrotoxicity and evaluate the protective role of Ziziphus spina-christi leaf extract (ZSCLE). Four randomly selected groups containing seven rats were used. For a period of 28 days, the control group was administered 0.9% saline solution; the second group was administered 300 mg/kg ZSCLE; the third group was administered 0.4 mg/kg HgCl2 dissolved in 0.9% physiological saline solution; and the fourth group was administered an oral supplement of 300 mg/kg ZSCLE one hour after HgCl2 administration. HgCl2 intoxication resulted in Hg accumulation in renal tissue; decreases in body weight, kidney index, and glutathione content and superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities; increases in creatinine, urea, Kim-1 expression, lipid peroxidation, and nitric oxide production; suppression of the Nrf2-antioxidant response pathway; upregulation of Il1ß, Tnfα, and Nos2; and potentiation of proapoptotic activity. ZSCLE exerted beneficial effects against mercury-induced renal toxicity and significantly reversed these alterations to near normal values. These effects resulted from its chelation and antioxidant, anti-inflammatory, and antiapoptotic activities. ZSCLE may prevent or minimize the pathological changes induced by mercury in the kidney.

Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target.

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6-/- cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.

Photoimmunotherapy targeting biliary-pancreatic cancer with humanized anti-TROP2 antibody.

Photoimmunotherapy (PIT) is a new type of tumor-specific treatment utilizing monoclonal antibody (mAb)-photosensitizer conjugates and near-infrared (NIR) light irradiation. One potential PIT target, the type I transmembrane protein TROP2, is expressed at high levels in many cancers, including pancreatic carcinoma (PC) and cholangiocarcinoma (CC), in which its expression is correlated with poor prognosis and tumor aggressiveness. In this study, we assessed the efficacy of PIT utilizing newly developed humanized anti-TROP2 mAb conjugated to the photosensitizer IR700 (TROP2-IR700) for PC and CC. Immunohistochemistry on PC and CC tissue microarrays confirmed that TROP2 is overexpressed in about half of PC and CC specimens. Using cultured PC and CC cells, TROP2-IR700 localized TROP2-specific and target-specific cell killing was observed after NIR light irradiation. In addition, TROP2-IR700 was localized to mouse xenograft tumors expressing TROP2 after intravenous injection. PC and CC xenograft tumor growth was significantly inhibited by TROP2-targeted PIT relative to controls. The efficacy of TROP2-targeted PIT in vitro and against xenografted tumors in vivo suggests promise as a therapy for human PC and CC, both of which currently have dismal prognoses and limited therapeutic options.

Antithrombotic Effect of Artemisia princeps Pampanini Extracts in Vitro and in FeCl3 -Induced Thrombosis Rats.

Extracts of several plants possess antithrombotic effects. Herein, we examined the antithrombotic effects of different extracts of Artemisia princeps Pampanini prepared using distilled water, hot distilled water, 70% ethanol, or subcritical water. The antithrombotic effects were determined using a co-culture system consisting of tumor necrosis factor-alpha (TNF-α)-treated EA.hy926 cells and THP-1 cells. In addition, the coagulation time of plasma collected from healthy volunteers was evaluated in terms of the prothrombin time and activated partial thromboplastin time. A carotid arterial thrombosis model was induced by ferric chloride in Sprague Dawley rats. The rats were treated with either sterile water or three different doses of the subcritical water extract for 2 weeks. The thrombus weight, gene expression of cell adhesion molecules, and histological characteristics were assessed. The results of in vitro studies revealed a significant inhibition in the adhesion of monocytes to EA.hy926 cells stimulated by TNF-α in the subcritical water extract-treated group. We also observed considerable suppression of the occlusion and mRNA expression of cell adhesion molecules in the in vivo experiments. This study suggests that Artemisia princeps Pampanini may have the potential to improve blood coagulation.

Genome-wide enriched pathway analysis of acute post-radiotherapy pain in breast cancer patients: a prospective cohort study.

BACKGROUND: Adjuvant radiotherapy (RT) can increase the risk of developing pain; however, the molecular mechanisms of RT-related pain remain unclear. The current study aimed to identify susceptibility loci and enriched pathways for clinically relevant acute post-RT pain, defined as having moderate to severe pain (pain score ≥ 4) at the completion of RT. METHODS: We conducted a genome-wide association study (GWAS) with 1,344,832 single-nucleotide polymorphisms (SNPs), a gene-based analysis using PLINK set-based tests of 19,621 genes, and a functional enrichment analysis of a gene list of 875 genes with p < 0.05 using NIH DAVID functional annotation module with KEGG pathways and GO terms (n = 380) among 1112 breast cancer patients. RESULTS: About 29% of patients reported acute post-RT pain. None of SNPs nor genes reached genome-wide significant level. Four SNPs showed suggestive associations with post-RT pain; rs16970540 in RFFL or near the LIG3 gene (p = 1.7 × 10-6), rs4584690, and rs7335912 in ABCC4/MPR4 gene (p = 5.5 × 10-6 and p = 7.8 × 10-6, respectively), and rs73633565 in EGFL6 gene (p = 8.1 × 10-6). Gene-based analysis suggested the potential involvement of neurotransmitters, olfactory receptors, and cytochrome P450 in post-RT pain, whereas functional analysis showed glucuronidation (FDR-adjusted p value = 9.46 × 10-7) and olfactory receptor activities (FDR-adjusted p value = 0.032) as the most significantly enriched biological features. CONCLUSIONS: This is the first GWAS suggesting that post-RT pain is a complex polygenic trait influenced by many biological processes and functions such as glucuronidation and olfactory receptor activities. If validated in larger populations, the results can provide biological targets for pain management to improve cancer patients' quality of life. Additionally, these genes can be further tested as predictive biomarkers for personalized pain management.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

Pinin protects astrocytes from cell death after acute ischemic stroke via maintenance of mitochondrial anti-apoptotic and bioenergetics functions.

BACKGROUND: Stroke is the second most common cause of deaths worldwide. After an ischemic stroke, the proliferated reactive astrocytes in the peri-infarct areas play a beneficial role in neuronal survival. As such, astrocytes have gradually become a target for neuroprotection in stroke. The present study assessed the hypothesis that Pinin (Pnn), originally identified as a nuclear and desmosome-associated protein and is now known to possess anti-apoptotic capacity, protects astrocytes from cell death after ischemic stroke and delineated the underlying mechanisms. METHODS: In in vivo experiments, adult male Sprague-Dawley rats (12-week old) were used to induce acute focal cerebral ischemia employing the middle cerebral artery occlusion (MCAO) method. In in vitro experiments, postnatal day 1 (P1) Sprague-Dawley rat pups were used to prepare cultures of primary astrocytes. Oxygen-glucose deprivation (OGD) and re-oxygenation (OGD/R) procedures were employed to mimic the hypoxic-ischemic condition of stroke in those astrocytes. RESULTS: We found in the peri-infarct area of the ipsilateral cortex and striatum in Sprague-Dawley rats after transient MCAO an increase in Pnn expression that correlated positively with the time-course of infarction as detected by T2-weighted imaging and triphenyltetrazolium chloride staining, augmented number of reactive astrocytes that double-labelled with Pnn as determined by immunofluorescence, and enhanced cytotoxic edema as revealed by diffusion weighted imaging; but mirrored the decreased cleaved caspase-3 as measured by western blot. In an OGD and OGD/R model using primary cultured astrocytes, treatment with Pnn siRNA doubled the chance of surviving astrocytes to manifest cell death as revealed by flow cytometry, and blunted activated ERK signaling, reduced Bcl-2 expression and augmented cleaved caspase 3 detected by western blot in the normoxia, OGD or OGD/R group. Gene-knockdown of Pnn also impeded the reversal from decline in cell viability, elevation in lactate dehydrogenase leakage and decrease in ATP production in the OGD/R group. CONCLUSION: We conclude that the endogenous Pnn participates in neuroprotection after acute ischemic stroke by preserving the viability of astrocytes that survived the ischemic challenge via maintenance of mitochondrial anti-apoptotic and bioenergetics functions.