Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated.
4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status.
Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP.
Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

OBJECTIVE: To explore the molecular mechanism of circ_0000326 regulating proliferation, invasion and migration of oral squamous cell carcinoma HSC3 cells.
METHODS: Cancerous tissue and adjacent tissue specimens of 45 patients with oral squamous cell carcinoma (OSCC) admitted to the Second People\'s Hospital of Hefei from March 2020 to June 2021 were collected. qRT-PCR was used to detect the expression levels of circ_0000326 and miR-567. CCK-8, plate clone formation test, scratch test and Transwell test were used to detect cell proliferation, clone formation, migration and invasion. Dual luciferase reporter experiment was used to detect the targeting relationship between circ_0000326 and miR-567. Western blot was used to quantify E-cadherin and N-cadherin protein. SPSS 21.0 software package was used for statistical analysis.
RESULTS: circ_0000326 expression was 4.01±0.29 in OSCC and 1.00±0.13 in paracancerous tissues, while miR-567 expression was 0.28±0.03 and 1.00±0.10, respectively, with significant differences. Compared with the si-NC group, the cell viability and the number of cell clones in the si-circ_0000326 group were significantly decreased(P＜0.05). Compared with the si-NC group, the number of invasive cells and scratch healing rate in the si-circ_0000326 group were significantly decreased (P＜0.05), the expression level of E-cadherin protein was significantly increased (P＜0.05), and the expression level of N-cadherin protein was significantly decreased(P＜0.05). Additionally, circ_0000326 targeted miR-567. miR-567 expression was 1.00±0.00 in pcDNA group, 0.44±0.04 in pcDNA-circ_0000326 group, 0.99±0.06 in si-NC group, and 2.92±0.25 in si-circ_0000326 group with significant differences. Compared with miR-NC group, the cell viability, scratch healing rate, the number of cell clones and the number of invasive cells of miR-567 group were decreased, while E-cadherin protein level was increased(P＜0.05). Compared with si-circ_0000326+anti-miR-NC group, the cell viability, scratch healing rate, N-cadherin protein level, the number of cell clones and the number of invasive cells of si-circ_0000326+anti-miR-567 group were increased(P＜0.05), while E-cadherin protein level was decreased(P＜0.05).
CONCLUSIONS: Interference with the expression of circ_0000326 could reduce the ability of OSCC cell proliferation, colony formation, migration and invasion by promoting the expression of miR-567.

BACKGROUND: Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood.
METHODS: Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail.
RESULTS: CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect.
CONCLUSIONS: These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.

Cadherin-17 (CDH17) is a membranous cell adhesion protein predominantly expressed in intestinal epithelial cells. CDH17 is therefore considered a possible diagnostic and therapeutic target. This study was to comprehensively determine the expression of CDH17 in cancer and to further assess the diagnostic utility of CDH17 immunohistochemistry (IHC). A tissue microarray containing 14,948 interpretable samples from 150 different tumor types and subtypes as well as 76 different normal tissue types was analyzed by IHC. In normal tissues, a membranous CDH17 staining was predominantly seen in the epithelium of the intestine and pancreatic excretory ducts. In tumors, 53 of 150 analyzed categories showed CDH17 positivity including 26 categories with at least one strongly positive case. CDH17 positivity was most common in epithelial and neuroendocrine colorectal neoplasms (50.0%-100%), other gastrointestinal adenocarcinomas (42.7%-61.6%), mucinous ovarian cancer (61.1%), pancreatic acinar cell carcinoma (28.6%), cervical adenocarcinoma (52.6%), bilio-pancreatic adenocarcinomas (40.5-69.8%), and other neuroendocrine neoplasms (5.6%-100%). OnIy 9.9% of 182 pulmonary adenocarcinomas were CDH17 positive. In colorectal adenocarcinomas, reduced CDH17 staining was linked to high pT (p = 0.0147), nodal metastasis (p = 0.0041), V1 (p = 0.0025), L1 (p = 0.0054), location in the right colon (p = 0.0033), and microsatellite instability (p < 0.0001). The CDH17 expression level was unrelated to tumor phenotype in gastric and pancreatic cancer. In summary, our comprehensive overview on CDH17 expression in human tumors identified various tumor entities that might often benefit from anti-CDH17 therapies and suggest utility of CDH17 IHC for the distinction of metastatic gastrointestinal or bilio-pancreatic adenocarcinomas (often positive) from primary pulmonary adenocarcinomas (mostly negative).

This study aimed to use bioinformatics approaches for predicting the anticancer mechanisms of curcumin on triple-negative breast cancer (TNBC) and to verify these predictions through in vitro experiments. Initially, the Cell Counting Kit-8 (CCK8) assay was employed to rigorously investigate the influence of curcumin on the proliferative capacity of TNBC cells. Subsequently, flow cytometry was employed to meticulously assess the impact of curcumin on cellular apoptosis and the cell cycle regulation. Transwell assays were employed to meticulously evaluate the effect of curcumin on the motility of TNBC cells. RNA sequencing was conducted, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of differentially expressed genes, aiming to elucidate the potential anticancer mechanisms underlying curcumin\'s effects. To thoroughly elucidate the interactions among multiple proteins, we constructed a protein-protein interaction (PPI) network. Finally, the expression levels of several key proteins, including fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6, were assessed using the western blot. The CCK8 assay results showed that curcumin significantly inhibited the proliferation of Hs578T and MDA-MB-231 cells. Flow cytometry results showed that curcumin induced apoptosis in these cells and arrested the cell cycle at the G2/M phase. Additionally, Transwell assay results showed that curcumin effectively reduced the motility of Hs578T and MDA-MB-231 cells. Enrichment analysis of RNA sequencing data showed that the mechanism of action of curcumin was significantly associated with signaling pathways such as pathways in cancer, focal adhesion, and PI3K-Akt signaling pathways. Subsequently, we constructed a protein-protein interaction network to elucidate the interactions among multiple proteins. Finally, Western blotting analysis showed that curcumin significantly decreased the expression levels of key proteins including Fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6. Curcumin exhibits its therapeutic potential in TNBC by modulating multiple signaling pathways. It may inhibit the epithelial-mesenchymal transition process by downregulating the expression of proteins involved in the mTOR and PI3K-Akt signaling pathways, thereby suppressing the motility of TNBC cells. These findings provide experimental evidence for considering curcumin as a potential therapeutic strategy in the treatment of TNBC.

OBJECTIVE: According to the literature, high levels of salivary soluble E-cadherin may be lined to advanced stage and poor prognosis in cancers. This research aimed at comparing salivary and serum levels of soluble E-cadherin in cases with esophageal, gastric, and colorectal cancers.
METHODS: This case-control research was performed on 68 patients with gastrointestinal cancers and 57 healthy volunteers as a control group to measure salivary and serum levels of soluble E-cadherin using ELISA kits.
RESULTS: The results showed a statistically significant difference between salivary and serum levels of soluble E-cadherin in the case group, as opposed to the control group (P = 0.031). The salivary level of soluble E-cadherin in the control group was 2.138 ng/mL on average lower than that that in the case group. The median salivary level of soluble E-cadherin among cases with lymph node involvement was significantly higher compared with its median level in the control group (P = 0.049). Soluble E-cadherin salivary level and other variables, such as metastasis and cancer stage, were directly correlated, but it was not statistically significant.
CONCLUSIONS: According to the findings, the salivary level of soluble E-cadherin among cases with gastric cancer could be considered as a prognostic marker, but the diagnostic value of saliva could not be judged in esophageal and colorectal cancers because of small sample size, and more investigations are required for a definite conclusion.

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin cytoskeleton and substrate to study their role in permeability. Our studies show that mechanochemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tricellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force due to disturbed flow that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer extracellular matrix. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechanochemical model to investigate the variation in the permeability of endothelial monolayer due to multiple loads acting simultaneously.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a primary podocytopathy characterized by primary podocyte detection and high proteinuria. The search for biomarkers and factors associated with the progression of this disease is an important task nowdays.
OBJECTIVE: To assess the proteomic profile of urine in patients with FSGS and to isolate urinary biomarkers of podocytopathies.
METHODS: The study included 41 patients diagnosed with chronic glomerulonephritis, 27 men and 14 women. According to the morphological study, 28 patients were diagnosed with FSGS, 9 with steroid-sensitive nephrotic syndrome and 14 with steroid-resistant nephrotic syndrome. The comparison group included 13 patients with membranous nephropathy. The study of the urinary proteome was carried out by targeted liquid chromatography-mass spectrometry using multiple reaction monitoring with synthetic stable isotope labelled peptide standards.
RESULTS: The main differences in the protein profile of urine were found in the subgroups of steroid-sensitive (SS) and steroid-resistant (SR) FSGS. In the FSGS SR group, at the onset of the disease, there was a high concentration of proteins reflecting damage to the glomerular filter (apo-lipoprotein A-IV, orosomucoid, cadherin, hemopexin, vitronectin), as well as proteins associated with tubulo-interstitial inflammation and accumulation of extracellular matrix (retinol- and vitamin D-binding proteins, kininogen-1, lumican and neurophilin-2). Compared with the membranous nephropathy group, FSGS patients had significantly higher urinary concentrations of carnosinase, orosomucoid, cadherin-13, tenascin X, osteopontin, and zinc-alpha-2-glycoprotein.
CONCLUSIONS: Thus, in patients with SR FSGS, the proteomic profile of urine includes more proteins at elevated concentrations, which reflects severe damage to various parts of the nephron compared with patients with SS FSGS and membranous nephropathy.
Обоснование. Фокальный сегментарный гломерулосклероз (ФСГС) относится к первичным подоцитопатиям, которые характеризуются первичным повреждением подоцитов и высокой протеинурией. Поиск биомаркеров и факторов, участвующих в прогрессировании этого заболевания почек, является актуальной задачей в настоящее время. Цель. Оценить протеомный профиль мочи у больных с ФСГС и выделить мочевые биомаркеры подоцитопатий. Материалы и методы. В исследование включен 41 пациент с диагнозом хронического гломерулонефрита – 27 мужчин и 14 женщин. По данным морфологического исследования у 28 пациентов диагностирован ФСГС, у 9 – со стероид-чувствительным нефротическим синдромом и 14 – со стероид-резистентным (СР) нефротическим синдромом. В группу сравнения вошли 13 больных с мембранозной нефропатией. Исследование протеома мочи проводилось методом таргетной хромато-масс-спектрометрии в режиме мониторинга множественных реакций с использованием синтетических изотопно-меченых пептидных стандартов. Результаты. Наибольшие различия по белковому составу мочи выявлены в подгруппах стероид-чувствительного и СР ФСГС. В группе СР ФСГС в дебюте заболевания отмечалось высокое содержание белков, отражающих повреждение гломерулярного фильтра (аполипопротеин А-IV, орозомукоид, кадгерин, гемопексин, витронектин), а также белков, связанных с тубуло-интерстициальным воспалением и накоплением экстрацеллюлярного матрикса (ретинол- и витамин-D-связывающие белки, кининоген-1, люмикан и нейрофилин-2). По сравнению с группой мембранозной нефропатии у больных с ФСГС отмечена достоверно более высокая концентрация в моче карнозиназы, орозомукоида, кадгерина-13, тенасцина X, остеопонтина, цинк-α-2-гликопротеина. Заключение. Таким образом, у больных со СР ФСГС протеомный профиль мочи включает большее количество белков в повышенных концентрациях, что отражает тяжелое повреждение различных отделов нефрона по сравнению с больными со стероид-чувствительным ФСГС и мембранозной нефропатией.

OBJECTIVE: Deoxynivalenol (DON), namely vomitoxin, is one of the most prevalent fungal toxins in cereal crops worldwide. However, the underlying toxic mechanisms of DON remain largely unknown.
METHODS: DON exposure-caused changes in the murine plasma metabolome and gut microbiome were investigated by an LC-MS/MS-based nontargeted metabolomics approach and sequencing of 16S rRNA in fecal samples, respectively. Cellular models were then used to validate the findings from the metabolomics study.
RESULTS: DON exposure increased intestinal barrier permeability evidenced by its-mediated decrease in colonic Claudin 5 and E-cadherin, as well as increases in colonic Ifn-γ, Cxcl9, Cxcl10, and Cxcr3. Furthermore, DON exposure resulted in a significant increase in murine plasma levels of deoxycholic acid (DCA). Also, DON exposure led to gut microbiota dysbiosis, which was associated with DON exposure-caused increase in plasma DCA. In addition, we found not only DON but also DCA dose-dependently caused a significant increase in the levels of IFN-γ, CXCL9, CXCL10, and/or CXCR3, as well as a significant decrease in the expression levels of Claudin 5 and/or E-cadherin in the human colonic epithelial cells (NCM460).
CONCLUSIONS: DON-mediated increase in DCA contributes to DON-caused intestinal injury. DCA may be a potential therapeutic target for DON enterotoxicity.

OBJECTIVE: To investigate the seizure course of PCDH19 clustering epilepsy (PCDH19-CE) in a cohort of female children in China.
METHODS: This ambidirectional cohort study examined 113 female patients with PCDH19-CE through multicentre collaboration. Prognostic factors for seizure freedom were evaluated by multivariate Cox regression analysis.
RESULTS: The median seizure course period from seizure onset was 6 years 6 months. Of 113 patients, 78% and 56% experienced seizure freedom for at least 1 year and at least 2 years respectively. In patients younger than 5 years (n = 30), 5 to 10 years (n = 52), and older than 10 years (n = 31), 57%, 81%, and 94% experienced at least 1 year of seizure freedom, and 32%, 52%, and 84% experienced at least 2 years of seizure freedom, respectively. However, 58% (65 out of 113) relapsed at least once after more than 1 year of seizure freedom without trigger exposure (40%) or because of common triggers, including fever (43%) and antiseizure medication (ASM) reduction (29%). There was an 84% risk of seizure relapse after ASM reduction attempts. The likelihood of seizure freedom decreased with early age at seizure onset and developmental delay.
CONCLUSIONS: Patients with PCDH19-CE exhibit increasing seizure freedom with age, but there is a risk of relapse. ASM reduction in children younger than 10 years old requires caution. Patients with early seizure onset and developmental delay have a reduced chance of seizure freedom.
CONCLUSIONS: The seizure freedom rate in PCDH19 clustering epilepsy gradually increases with age. The disease course is characterized by relapsing-remitting seizures. Antiseizure medication reduction requires caution for patients younger than 10 years of age. Patients with early seizure onset and developmental delay are less likely to achieve seizure freedom.