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The molecular mechanisms that drive muscle adaptations after eccentric exercise training are multifaceted and likely impacted by age. Previous studies have reported that many genes and proteins respond differently in young and older muscles following training. Keratin 18 (Krt18), a cytoskeletal protein involved in force transduction and organization, was found to be upregulated after muscles performed repeated bouts of eccentric contractions, with higher levels observed in young muscle compared with older muscle. Therefore, the purpose of this study was to determine if Krt18 mediates skeletal muscle adaptations following eccentric exercise training. The anterior crural muscles of Krt18 knockout (KO) and wild-type (WT) mice were subjected to either a single bout or repeated bouts of eccentric contractions, with isometric torque assessed across the initial and final bouts. Functionally, Krt18 KO and WT mice did not differ prior to performing any eccentric contractions ( P ≥ 0.100). Muscle strength (tetanic isometric torques) and the ability to adapt to eccentric exercise training were also consistent across strains at all time points ( P ≥ 0.169). Stated differently, immediate strength deficits and the recovery of strength following a single bout or multiple bouts of eccentric contractions were similar between Krt18 KO and WT mice. In summary, the absence of Krt18 does not impede the muscle's ability to adapt to repeated eccentric contractions, suggesting it is not essential for exercise-induced remodeling. NEW & NOTEWORTHY The molecular processes that underlie the changes in skeletal muscle following eccentric exercise training are complex and involve multiple factors. Our findings indicate that Krt18 may not play a significant role in muscle adaptations following eccentric exercise training, likely due to its low expression in skeletal muscle. These results underscore the complexity of the molecular mechanisms that contribute to muscle plasticity and highlight the need for further research in this area.

A complete understanding of neural crest cell mechanodynamics during ocular development will provide insight into postnatal neural crest cell contributions to ophthalmic abnormalities in adult tissues and inform regenerative strategies toward injury repair. Herein, single-cell RNA sequencing in zebrafish during early eye development revealed keratin intermediate filament genes krt8 and krt18a.1 as additional factors expressed during anterior segment development. In situ hybridization and immunofluorescence microscopy confirmed krt8 and krt18a.1 expression in the early neural plate border and migrating cranial neural crest cells. Morpholino oligonucleotide (MO)-mediated knockdown of K8 and K18a.1 markedly disrupted the migration of neural crest cell subpopulations and decreased neural crest cell marker gene expression in the craniofacial region and eye at 48 h postfertilization (hpf), resulting in severe phenotypic defects reminiscent of neurocristopathies. Interestingly, the expression of K18a.1, but not K8, is regulated by retinoic acid (RA) during early-stage development. Further, both keratin proteins were detected during postnatal corneal regeneration in adult zebrafish. Altogether, we demonstrated that both K8 and K18a.1 contribute to the early development and postnatal repair of neural crest cell-derived ocular tissues.

Hypertrophic cardiomyopathy (HCM) is a heart muscle disease associated with an increased risk for sudden cardiac death (SCD). Cytokeratin 18-based proteins, such as M30 and M65 antigens, are known cell-death biomarkers. M30 antigen is released from cells during apoptosis, and M65 antigen is released during cell death from any cause, such as apoptosis or necrosis. We aimed to study the expression of M30 and M65 antigens in peripheral blood obtained by 46 HCM patients and compare with 27 age- and sex-matched patients without HCM. We also investigated the CK18 expression in myocardium from postmortem HCM hearts. M30 and M65 antigens were significantly increased in the HCM vs. non-HCM group (Μ30: 338 ± 197 U/uL vs. 206 ± 166 U/uL, p = 0.003; M65: 428 ± 224 U/uL vs. 246 ± 214 U/uL, p = 0.001), and HCM patients with a higher expression of these markers (M30: 417 ± 208 vs. 271 ± 162 U/uL, p = 0.011; M65: 518 ± 242 vs. 351 ± 178 U/uL, p = 0.011) had a higher risk for SCD. In HCM, both apoptosis and necrosis are increased, but particularly necrosis (M30/M65 ratio: 0.75 ± 0.09 vs. 0.85 ± 0.02, p < 0.001). CK18 is expressed in the HCM myocardium (1.767 ± 0.412 vs. 0.537 ± 0.383, % of area, p = 0.0058). Therefore, M30 and M65 antigens may be novel biomarkers in HCM.

Liver resection (LR) is the primary treatment for hepatic tumors, yet posthepatectomy liver failure (PHLF) remains a significant concern. While the precise etiology of PHLF remains elusive, dysregulated inflammatory processes are pivotal. Therefore, we explored the theragnostic potential of extracellular high-mobility-group-box protein 1 (HMGB1), a key damage-associated molecular pattern (DAMP) released by hepatocytes, in liver recovery post LR in patients and animal models. Plasma from 96 LR patients and liver tissues from a subset of 24 LR patients were analyzed for HMGB1 levels, and associations with PHLF and liver injury markers were assessed. In a murine LR model, the HMGB1 inhibitor glycyrrhizin, was administered to assess its impact on liver regeneration. Furthermore, plasma levels of keratin-18 (K18) and cleaved cytokeratin-18 (ccK18) were quantified to assess suitability as predictive biomarkers for PHLF. Patients experiencing PHLF exhibited elevated levels of intrahepatic and circulating HMGB1, correlating with markers of liver injury. In a murine LR model, inhibition of HMGB1 improved liver function, reduced steatosis, enhanced regeneration and decreased hepatic cell death. Elevated levels of hepatic cell death markers K18 and ccK18 were detected in patients with PHLF and correlations with levels of circulating HMGB1 was observed. Our study underscores the therapeutic and predictive potential of HMGB1 in PHLF mitigation. Elevated HMGB1, K18, and ccK18 levels correlate with patient outcomes, highlighting their predictive significance. Targeting HMGB1 enhances liver regeneration in murine LR models, emphasizing its role in potential intervention and prediction strategies for liver surgery.

Early embryonic mortality resulting from insufficient interaction between the embryo and the uterus leads to the failure of pregnancy in livestock animals. Thus, it is imperative to comprehend the multifaceted process of implantation at molecular levels, which requires synchronized feto-maternal interaction. The in-vitro models serve as valuable tools to investigate the specific stages of implantation. The present study was undertaken to develop a simple method to isolate and culture the primary buffalo endometrial epithelial cells (pBuEECs), followed by proteome profiling of the proliferating cells. Collagenase I was used to separate uterine epithelial cells (UECs) from the ipsilateral uterine horn, and then the cells were separated using a cell strainer. After being seeded on culture plates, UECs developed colonies with characteristic epithelial shape and expressed important markers such as cytokeratin 18 (KRT18), progesterone receptor (PGR), β-estrogen receptor (ESR1), and leukemia inhibitory factor (LIF), which were confirmed by PCR. The purity of epithelial cells was assessed using cytokeratin 18 immunostaining, which indicated approximately 99% purity in cultured cells. The proteome profiling of pBuEECs via high-throughput tandem mass spectrometry (MS), identified a total of 3383 proteins. Bioinformatics analysis revealed enrichment in various biological processes, including cellular processes, metabolic processes, biological regulation, localization, signaling, and developmental processes. Moreover, the KEGG pathway analysis highlighted associations with the ribosome, proteosome, oxidative phosphorylation, spliceosome, and cytoskeleton regulation pathways. In conclusion, these well characterized cells offer valuable in-vitro model to enhance the understanding of implantation and uterine pathophysiology in livestock animals, particularly buffaloes., Competing Interests: Declaration of Competing Interest The authors declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this research article., (Copyright © 2024. Published by Elsevier B.V.)

Objective: Hepatic fibrosis is a severe liver condition characterized by abnormal fibroblast activity, excessive extracellular matrix deposition, inflammation, and structural alterations. Methotrexate (MTX), a pharmaceutical agent widely used for its therapeutic properties, is known to induce hepatotoxicity. However, the precise mechanisms underlying MTX-induced liver injury remain elusive. This study investigates the therapeutic potential of Adipose-Derived Mesenchymal Stem Cells (ADMSCs) in alleviating MTX-induced liver injury in a rat model., Materials and Methods: Thirty male Wistar albino rats were employed in this study. Liver injury was induced in twenty rats by a single MTX dose, while ten rats constituted the control group. The MTX group was further subdivided into two cohorts, one receiving ADMSC treatment and the other saline solution. The treatment duration was 14 days. ADMSCs, isolated from adipose tissue, were characterized by CD13, CD29, and CD105 markers. Biomarker analysis, histopathological evaluations, and various measurements were conducted to assess ADMSCs' therapeutic efficacy., Results: MTX administration significantly increased Transforming Growth Factor-β (TGF-β), Platelet-Derived Growth Factor (PDGF), Plasma Cytokeratin 18, Plasma Malondialdehyde (MDA), and Liver MDA levels, with histopathological liver damage. ADMSC treatment notably lowered TGF-β, PDGF, Plasma Cytokeratin 18, Plasma MDA, and Liver MDA levels, accompanied by reduced liver damage observed histologically. Liver Enzyme ALT levels were also reduced in the MTX and ADMSC groups compared to the MTX and Saline groups., Conclusions: ADMSCs exhibit significant potential in ameliorating MTX-induced liver injury, with notable anti-oxidative and anti-apoptotic properties. These findings suggest that ADMSCs may effectively mitigate oxidative stress and inflammation associated with MTX-induced liver damage. Further research is essential to investigate the clinical application of ADMSCs in liver disease management and uncover the underlying therapeutic mechanisms.

Female infertility is a worldwide concern that impacts the quality of life and well-being of affected couples. Failure of embryo implantation is a major cause of early pregnancy loss and is precisely regulated by a programmed molecular mechanism. Recent studies have shown that proper trophoblast adhesion and invasion are essential for embryo implantation. However, the potential regulatory mechanism involved in trophoblast adhesion and invasion has yet to be fully elucidated. KRT18 has been reported to play a critical role in early embryonic development, but its physiological function in embryo implantation remains unclear. In the present study, we revealed that KRT18 was highly expressed in trophoblast cells and that knockdown of KRT18 in mouse embryos inhibited embryo adhesion and implantation. In vitro experiments further showed that silencing KRT18 disturbed trophoblast migration and invasion. More importantly, we provide evidence that KRT18 directly binds to and stabilizes cell surface E-cadherin in trophoblast cells through microscale thermophoresis (MST) analysis and molecular biology experiments. In brief, our data reveal that KRT18, which is highly expressed in trophoblast cells, plays an important role in the regulation of trophoblast invasion and adhesion during embryo implantation by directly binding to E-cadherin., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Visceral obesity is linked to the progression of fatty liver to nonalcoholic steatohepatitis (NASH). Cytokeratin-18 (CK18) epitopes M30 (CK18M30) and M65 (CK18M65) represent accurate markers for detecting NASH. The aim of this study was to evaluate the association of CK18M30 and CK18M65 levels with anthropometric and metabolic characteristics, liver stiffness, and liver indices of steatosis and fibrosis in a cohort of subjects with visceral obesity; in this cross-sectional study, transient elastography (TE-Fibroscan ® ), anthropometric measurements, metabolic parameters, High Sensitivity C-Reactive Protein (hsCRP), and CK18M30 and CK18M65 levels (Apoptosense ELISA, PEVIVA, Germany) were evaluated. Fatty Liver Index (FLI), Fibrosis 4 (FIB-4), and Aspartate transaminase (AST)-platelet ratio index (APRI) were calculated; among 48 subjects, 47.2% presented metabolic syndrome, 93.8% hepatic steatosis, 60.4% high liver stiffness, and 14.6% hypertransminasemia, while FIB-4 and APRI were normal. CK18M30 and CK18M65 levels were significantly correlated with waist circumference, AST, ALT, HoMA-IR, liver stiffness, and APRI ( p < 0.001). Subjects with CK18 fragments above the median values showed significantly higher waist circumference, HbA1c, AST, ALT, HoMA-IR, FLI, and APRI compared to those with values below the median; CK18M30 and CK18M65 levels correlated well with anthropometric and metabolic characteristics, representing good biomarkers for early identification of NASH in subjects with visceral obesity.

Context: Although type 2 diabetes (T2D) is a risk factor for liver fibrosis in nonalcoholic fatty liver disease (NAFLD), the specific contribution of insulin resistance (IR) relative to other factors is unknown., Objective: Assess the impact on liver fibrosis in NAFLD of adipose tissue (adipose tissue insulin resistance index [adipo-IR]) and liver (Homeostatic Model Assessment of Insulin Resistance [HOMA-IR]) IR in people with T2D and NAFLD., Design: Participants were screened by elastography in the outpatient clinics for hepatic steatosis and fibrosis, including routine metabolites, cytokeratin-18 (a marker of hepatocyte apoptosis/steatohepatitis), and HOMA-IR/adipo-IR., Setting: University ambulatory care practice., Participants: A total of 483 participants with T2D., Intervention: Screening for steatosis and fibrosis with elastography., Main Outcome Measures: Liver steatosis (controlled attenuation parameter), fibrosis (liver stiffness measurement), and measurements of IR (adipo-IR, HOMA-IR) and fibrosis (cytokeratin-18)., Results: Clinically significant liver fibrosis (stage F ≥ 2 = liver stiffness measurement ≥8.0 kPa) was found in 11%, having more features of the metabolic syndrome, lower adiponectin, and higher aspartate aminotransferase (AST), alanine aminotransferase, liver fat, and cytokeratin-18 (P < 0.05-0.01). In multivariable analysis including just clinical variables (model 1), obesity (body mass index [BMI]) had the strongest association with fibrosis (odds ratio, 2.56; CI, 1.87-3.50; P < 0.01). When metabolic measurements and cytokeratin-18 were included (model 2), only BMI, AST, and liver fat remained significant. When fibrosis stage was adjusted for BMI, AST, and steatosis (model 3), only Adipo-IR remained strongly associated with fibrosis (OR, 1.51; CI, 1.05-2.16; P = 0.03), but not BMI, hepatic IR, or steatosis., Conclusions: These findings pinpoint to the central role of dysfunctional, insulin-resistant adipose tissue to advanced fibrosis in T2D, beyond simply BMI or steatosis. The clinical implication is that targeting adipose tissue should be the priority of treatment in NAFLD., Competing Interests: Conflict of Interest K.C. has received research support as principal investigator from the University of Florida from the National Institutes of Health, Cirius, Echosens, Inventiva, Novartis, Novo Nordisk, Poxel, and Zydus. K.C. is also a consultant for Allergan, Altimmune, Arrowhead, AstraZeneca, BMS, Boehringer Ingelheim, Coherus, Eli Lilly, Fractyl, Hanmi, Genentech, Gilead, Intercept, Janssen, Pfizer, Prosciento, Madrigal, and Novo Nordisk. All other authors have no potential conflicts of interest relevant to this article., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Memory impairment is one of the neuropsychological effects of hypobaric hypoxia (HH), which can be associated with programmed cell death, such as apoptosis and ferroptosis. Emerging evidence indicates crosstalk between apoptosis and ferroptosis, while the crosstalk between HH-induced apoptosis and ferroptosis in the hippocampus has not been clarified. Here, microarray profiles were extracted to analyze the differentially expressed genes with and without HH exposure, and keratin 18 (Krt18) was found to be a potential gene related to both apoptosis and ferroptosis. Then, we conducted morphological observations that showed that apoptosis and ferroptosis coexisted in the rat hippocampus after HH exposure. Combined with the real-time q-PCR analysis, the mRNA expression of Krt18 decreased significantly after HH exposure for 1 day and 3 days, and Mapk10 (JNK3) was upregulated at the corresponding time points. After exposure for 7 days, Krt18 and JNK3 showed no significant change. In conclusion, Krt18 may regulate apoptosis and ferroptosis simultaneously, possibly via the JNK signaling pathway, which might provide a potential central target for apoptosis and ferroptosis in hippocampal injury after HH exposure., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cytoskeletal proteins not only define the shape of cells, but also have critical roles in their proliferation, migration and motility, as well as in the establishment and maintenance of tissue organization and integrity. Furthermore, these proteins influence the physiological processes of the male reproductive system and are found in the structure of some cells. This study aimed to determine differences between the pre- and post-pubertal periods for the localization and distribution of actin, desmin, vimentin and cytokeratin-18 in the testes, epididymides and ductus deferentes of Persian and Turkish Angora and Van cats, using immunohistochemistry. The study material was grouped as belonging to the pre-pubertal and post-pubertal periods. The tissue samples of both groups were subjected to routine histological processing and embedded in paraffin. Serial sections cut from the paraffin-embedded tissue blocks were immunohistochemically stained with the indirect streptavidin-biotin complex method. Immunohistochemical findings demonstrated that there was no difference between the pre- and post-pubertal periods for the staining intensity and distribution of the proteins actin, vimentin, desmin and cytokeratin-18 in Persian and Turkish Angora and Van cats. On the other hand, differences were detected between the pre- and post-pubertal periods for the cellular expression and localization of these proteins in the testes, epididymides and ductus deferentes. Thus, the study results suggest that, based on the expression of actin, desmin, vimentin and cytokeratin-18 in the testes, epididymides and ductus deferentes during both periods, these molecular factors could have a contributory role in the development of the male reproductive system and the regulation of its physiological processes., Competing Interests: Declaration of competing interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Endogenous Jaagsiekte sheep retrovirus envelope protein (enJSRV-Env) plays an important role in trophoblast cell fusion in sheep. However, the underlying mechanism remains unclear., Methods: Primary endometrial luminal epithelial cells (LECs) were isolated from the sheep uterus and cocultured with sheep trophoblast cells (STCs). Giemsa staining was conducted to count multinucleated cells in the coculture system. Gain- and loss-of-function assays were performed to explore the role of enJSRV-Env in trophoblast cell fusion in the coculture system. Co-immunoprecipitation and mass spectrometry were carried out to identify the interacting partner of enJSRV-Env in the cocultures. Western blot analysis were conducted to determine the activation of protein kinase A (PKA)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling., Results: Primary LECs were identified by the expression of epithelial marker cytokeratin 18. Overexpression of enJSRV-Env promoted the formation of multinucleated cells in the coculture system. enJSRV-Env activated and physically interacted with PKA, along with the activation of MEK/ERK1/2 signaling. PKA inhibition completely reversed enJSRV-Env-induced MEK/ERK1/2 activation, and ERK1/2 inhibition abolished enJSRV-Env-induced formation of multinucleated cells in the coculture system., Conclusion: enJSRV-Env promotes trophoblast cell fusion in the sheep placenta by activating PKA/MEK/ERK1/2 signaling. This finding reveals a novel mechanism underlying the contribution of enJSRV-Env to trophoblast cell fusion during placental morphogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The Apoptosis Stimulating Protein of p53 2 (ASPP2) is a heterozygous insufficient tumor suppressor; however, its molecular mechanism(s) in tumor suppression is not completely understood. ASPP2 plays an essential role in cell growth, as shown by liver hepatocellular carcinoma (LIHC) RNA-seq assay using the Cancer Genome Atlas (TCGA) and High-Throughput-PCR assay using ASPP2 knockdown cells. These observations were further confirmed by in vivo and in vitro experiments. Mechanistically, N-terminus ASPP2 interacted with Keratin 18 (k18) in vivo and in vitro. Interestingly, the RDIVpSGP motif of ASPP2 associates with 14-3-3 and promotes ASPP2/k18/14-3-3 ternary-complex formation which promotes MEK/ERK signal activation by impairing 14-3-3 and BRAF association. Additionally, ASPP2-rAd injection promotes paclitaxel-suppressed tumor growth by suppressing cell proliferation in the BALB/c nude mice model. ASPP2 and k18 were preferentially downregulated in Hepatocellular Carcinoma (HCC), which predicted poor prognosis in HCC patients. Overall, these findings suggested that ASPP2 promoted BRAF/MEK/ERK signal activation by promoting the formation of an ASPP2/k18/14-3-3 ternary complex via the RDIVpSGP motif at the N terminus. Moreover, this study provides novel insights into the molecular mechanism of tumor suppression in HCC patients., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Explanted livers from patients with inherited metabolic liver diseases possess the potential to be a cell source of good-quality hepatocytes for hepatocyte transplantation (HT). This study evaluated the therapeutic effects of domino HT using hepatocytes isolated from explanted human livers for acute liver failure (ALF)., Methods: Isolated hepatocytes were evaluated for viability and function and then transplanted into D-galactosamine/lipopolysaccharide-induced ALF mice via splenic injection. The survival rate was analyzed by the Kaplan-Meier method and log-rank test. Liver function was evaluated by serum biochemical parameters, and inflammatory cytokine levels were measured by ELISA. The pathological changes in the liver tissues were assessed by hematoxylin-eosin staining. Hepatocyte apoptosis was investigated by TUNEL, and hepatocyte apoptosis-related proteins were detected by western blot. The localization of human hepatocytes in the injured mouse livers was detected by immunohistochemical analyses., Results: Hepatocytes were successfully isolated from explanted livers of 10 pediatric patients with various liver-based metabolic disorders, with an average viability of 85.3% ± 13.0% and average yield of 9.2 × 10 6  ± 3.4 × 10 6 cells/g. Isolated hepatocytes had an excellent ability to secret albumin, produce urea, uptake indocyanine green, storage glycogen, and express alpha 1 antitrypsin, albumin, cytokeratin 18, and CYP3A4. Domino HT significantly reduced mortality, decreased serum levels of alanine aminotransferase and aspartate aminotransferase, and improved the pathological damage. Moreover, transplanted hepatocytes inhibited interleukin-6 and tumor necrosis factor-α levels. Domino HT also ameliorates hepatocyte apoptosis, as evidenced by decreased TUNEL positive cells. Positive staining for human albumin suggested the localization of human hepatocytes in ALF mice livers., Conclusion: Explanted livers from patients with inheritable metabolic disorders can serve as a viable cell source for cell-based therapies. Domino HT using hepatocytes with certain metabolic defects has the potential to be a novel therapeutic strategy for ALF., (© 2022. The Author(s).)

The endometrium undergoes profound changes in tissue architecture and composition, both during the menstrual cycle as well as in the context of pregnancy. Dynamic remodeling processes of the endometrial extracellular matrix (ECM) are a major element of endometrial homeostasis, including changes across the menstrual cycle. A critical element of this tissue microenvironment is the endometrial basement membrane, a specialized layer of proteins that separates the endometrial epithelium from the underlying endometrial ECM. Bioengineering models of the endometrial microenvironment that present an appropriate endometrial ECM and basement membrane may provide an improved environment to study endometrial epithelial cell (EEC) function. Here, we exploit a tiered approach using two-dimensional high-throughput microarrays and three-dimensional gelatin hydrogels to define patterns of EEC attachment and cytokeratin 18 (CK18) expression in response to combinations of endometrial basement membrane proteins. We identify combinations (collagen IV + tenascin C; collagen I + collagen III; hyaluronic acid + tenascin C; collagen V; collagen V + hyaluronic acid; collagen III; and collagen I) that facilitate increased EEC attachment, increased CK18 intensity, or both. We also identify significant EEC mediated remodeling of the methacrylamide-functionalized gelatin matrix environment via analysis of nascent protein deposition. Together, we report efforts to tailor the localization of basement membrane-associated proteins and proteoglycans in order to investigate tissue-engineered models of the endometrial microenvironment.

There is currently a controversial and heated debate about the safety and ethical aspects of fluoride (F) used for human consumption. Thus, this study assessed the effects of prenatal and postnatal F exposure of rats on the salivary glands of their offspring. Pregnant rats were exposed to 0, 10, or 50 mg F/L from the drinking water, from the first day of gestation until offspring weaning (42 days). The offspring rats were euthanized for the collection of the parotid (PA) and submandibular (SM) glands, to assess the oxidative biochemistry and to perform morphometric and immunohistochemical analyses. F exposure was associated with a decrease in the antioxidant competence of PA in the 10 mg F/L group, contrasting with the increase observed in the 50 mg F/L group. On the other hand, the antioxidant competence of the SM glands was decreased at both concentrations. Moreover, both 10 and 50 mg F/L groups showed lower anti-α-smooth muscle actin immunostaining area in SM, while exposure to 50 mg F/L was associated with changes in gland morphometry by increasing the duct area in both glands. These findings demonstrate a greater susceptibility of the SM glands of the offspring to F at high concentration in comparison to PA, reinforcing the need to adhere to the optimum F levels recommended by the regulatory agencies. Such findings must be interpreted with caution, especially considering their translational meaning.

Long non-coding RNAs (lncRNAs) play a crucial role in gastric cancer (GC) progression. And understanding the role of N6-methyladenosine (m6A) in tumorigenesis is an emerging field in cancer research. Here, we identified a novel oncogene, lncRNA LINC02253, in GC. LINC02253 expression was found to be significantly increased in GC. And LINC02253 expression was closely correlated with tumor size, lymph node metastasis and TNM stage of GC. Besides, GC patients with higher LINC02253 expression had worse 5-year overall survival. Additionally, LINC02253 promoted GC cell growth, migration and invasion both in vitro and in vivo. Mechanistically, we determined that LINC02253 increased KRT18 expression through enhancing the stability of KRT18 mRNA. Furthermore, LINC02253 increased m6A modification of KRT18 mRNA to stabilize KRT18 mRNA by recruiting m6A writer METTL3. And, rescue experiments revealed that KRT18 mediated the effects of LINC02253 on growth, migration and invasion of GC cells through activating MAPK/ERK signaling pathway. In conclusion, we demonstrates that oncogenic lncRNA LINC02253 positively regulates GC growth and metastasis via increasing METTL3-mediated mRNA stability of KRT18, extending the understanding of GC pathogenesis regulated by lncRNAs., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: The mechanism by which SARS-CoV-2 triggers cell damage and necrosis are yet to be fully elucidated. We sought to quantify epithelial cell death in patients with COVID-19, with an estimation of relative contributions of apoptosis and necrosis., Methods: Blood samples were collected prospectively from adult patients presenting to the emergency department. Circulating levels of caspase-cleaved (apoptosis) and total cytokeratin 18 (CK-18) (total cell death) were determined using M30 and M65 enzyme assays, respectively. Intact CK-18 (necrosis) was estimated by subtracting M30 levels from M65., Results: A total of 52 COVID-19 patients and 27 matched sick controls (with respiratory symptoms not due to COVID-19) were enrolled. Compared with sick controls, COVID-19 patients had higher levels of M65 (p = 0.046, total cell death) and M30 (p = 0.0079, apoptosis). Hospitalised COVID-19 patients had higher levels of M65 (p= 0.014) and intact CK-18 (p= 0.004, necrosis) than discharged patients. Intensive care unit (ICU)-admitted COVID-19 patients had higher levels of M65 (p= 0.004), M30 (p= 0.004) and intact CK-18 (p= 0.033) than hospitalised non-ICU admitted patients. In multivariable logistic regression, elevated levels of M65, M30 and intact CK-18 were associated with increased odds of ICU admission (OR=22.05, p=0.014, OR=19.71, p=0.012 and OR=14.12, p=0.016, respectively)., Conclusion: Necrosis appears to be the main driver of hospitalisation, whereas apoptosis and necrosis appear to drive ICU admission. Elevated levels CK-18 levels are independent predictors of severe disease, and could be useful for risk stratification of COVID-19 patients and in assessment of therapeutic efficacy in early-phase COVID-19 clinical trials., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Aims: Ballooned hepatocytes represent liver cell degeneration and are histological hallmarks in the diagnosis of non-alcoholic steatohepatitis, a severe form of non-alcoholic fatty liver disease. However, the identification of ballooned hepatocytes is often difficult, especially in the clinical setting of patients with other chronic liver diseases. In this study, we investigated the utility of immunostaining for positive sonic hedgehog (SHh) protein and negative Keratin 8/18 (K8/18) expression on ballooned hepatocytes., Methods and Results: Immunohistochemistry for SHh and K8/18 was evaluated independently by two experienced liver pathologists in non-tumorous liver tissue from 100 cases of resected hepatocellular carcinoma of various aetiology. The degree of hepatocyte ballooning was scored as follows: 0, none; 1, few; 2, many ballooned hepatocytes. These evaluations were performed using routine haematoxylin and eosin (H&E) staining, followed by immunostaining for SHh or K8/18. Using SHh or K8/18 immunostaining combined with H&E staining, the score of ballooned hepatocytes was upgraded in 20 and 19 cases, and downgraded in none and 2 cases, respectively. The percentage of observed agreement for ballooned hepatocytes scoring was 85% and 92%, and the weighted kappa value was 0.806 and 0.893 with SHh or K8/18 immunohistochemistry. Considering the immunohistochemistry results, background liver disease diagnosis was changed in 15 out of 100 cases (15%) evaluated., Conclusions: SHh and K8/18 immunohistochemistry are useful in detecting ballooned hepatocytes, regardless of background liver disease, and improving pathological diagnosis accuracy., (© 2022 John Wiley & Sons Ltd.)

Background: Due to its extremely high prevalence and severity, non-alcoholic fatty liver disease (NALFD) is a serious health and economic concern worldwide. Developing effective methods of diagnosis and therapy demands a deeper understanding of its molecular basis. One of the strategies in such an endeavor is the analysis of alterations in the morphology of liver cells. Such alterations, widely reported in NAFLD patients and disease models, are related to the cytoskeleton. Therefore, the fate of the cytoskeleton components is useful to uncover the molecular basis of NAFLD, to further design innovative approaches for its diagnosis and therapy., Main Findings: Several cytoskeleton proteins are up-regulated in liver cells of NAFLD patients. Under pathological conditions, keratin 18 is released from hepatocytes and its detection in the blood emerges as a non-invasive diagnosis tool. α-Smooth muscle actin is up-regulated in hepatic stellate cells and its down-regulation has been widely tested as a potential NALFD therapeutic approach. Other cytoskeleton proteins, such as vimentin, are also up-regulated., Conclusions: NAFLD progression involves alterations in expression levels of proteins that build the liver cytoskeleton or associate with it. These findings provide a timely opportunity of developing novel approaches for NAFLD diagnosis and therapy., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model’s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2–infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.

Background: To investigate the predictive values of cytokeratin 18 for liver fibrosis in hepatitis C virus (HCV) infected patients with type 2 diabetes mellitus (T2DM)., Methods: 252 HCV-infected patients with T2DM between January 2012 and August 2017 were retrospectively reviewed. Pearson/spearman correlation analysis was used to detect the correlation in the entire cohort. Multivariate linear regression was used to identify independent predictors and logistic regression was for establishing models. Combination models that incorporated CK18 and other methods (i.e. transient elastography, aspartate transaminase-to-platelet ratio index (APRI) and fibrosis-4 index (FIB-4)] were developed in a training cohort of 132 patients. Performance of models was evaluated through discrimination ability and clinical benefits. An internal validation was conducted in 120 consecutive patients., Results: CK18 was found significantly associated with fibrosis scores (r = 0.452, P < .001). CK18 and albumin were confirmed as independent predictors for fibrosis. For predicting significant fibrosis in the validation cohort, the observed AUC values of APRI + CK18 (AUC 0.83) and FIB-4 + CK18 (AUC 0.84) were higher than those of APRI (AUC 0.61) and FIB-4 (AUC 0.65). For predicting advanced fibrosis and cirrhosis, the AUC values of FIB-4 + CK18 (AUC 0.74 and 0.77, respectively) were significantly higher than those of FIB-4 (AUC 0.61 of both). Decision curve analysis confirmed the more clinical benefits can be provided by being combined with CK18., Conclusions: CK18 is an independent predictor of liver fibrosis for HCV-infected patients with T2DM. Noninvasive methods incorporate CK18 and other biomarker indices can have better performance for diagnosing fibrosis and help clinical decision-making., (© 2021. The Author(s).)

The inspection of variations in the proteomic aspects conspire the biomarker discovery in diagnostics of peculiar diseases. Recent developments in high-throughput proteomic techniques have provided leverage in the discovery of biomarkers during the etiology of various diseases. We identified potential biomarkers by utilizing proteomics, bioinformatics and gene expression studies. Meticulous assessment of collagen and hydroxyproline levels along with the glycogen and protein carbonyl levels exhibited deterioration in the N' - Nitrosodiethylamine (NDEA) administered rat livers and subsequent salubrious effect of pomegranate juice. The immunohistochemical inspection of iNOS and nitrite estimation indicated the peccant fibrotic alterations. 2D proteome profiling and MALDI-TOF MS/MS furthered the significant biomarkers to be analyzed for the gene ontology by PANTHER, cluster analysis by DAVID and network simulation by STRING 10.0. Several genes found relevant after MALDI analysis were evaluated by real-time PCR (RTPCR). Our data revealed CYP2b15, HSP70, TRFE, HPT, Il1rl2, Ric8a, Krt18, Hsp90b1 and iNOS as novel biomarkers for the mechanism of pomegranate against liver fibrosis. It can be inferred that NDEA-induced liver fibrosis actuates various biological pathways by the identified biomarkers and pomegranate juice modifies them., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Keratin 8 and keratin 18 (K8/K18) are intermediate filament proteins that form the obligate heteropolymers in hepatocytes and protect the liver against toxins. The mechanisms of protection include the regulation of signaling pathway associated with cell survival. Previous studies show K8/K18 binding with Akt, which is a well-known protein kinase involved in the cell survival signaling pathway. However, the role of K8/K18 in the Akt signaling pathway is unclear. In this study, we found that K8/K18-Akt binding is downregulated by K8/K18 phosphorylation, specifically phosphorylation of K18 ser7/34/53 residues, whereas the binding is upregulated by K8 gly-62-cys mutation. K8/K18 expression in cultured cell system tends to enhance the stability of the Akt protein. A comparison of the Akt signaling pathway in a mouse system with liver damage shows that the pathway is downregulated in K18-null mice compared with nontransgenic mice. K18-null mice with Fas-induced liver damage show enhanced apoptosis combined with the downregulation of the Akt signaling pathway, i.e., lower phosphorylation levels of GSK3β and NFκB, which are the downstream signaling factors in the Akt signaling pathway, in K18-null mice compared with the control mice. Our study indicates that K8/K18 expression protects mice from liver damage by participating in enhancing the Akt signaling pathway.

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 β-cells is dependent on the presence and levels of K18. K18-knockout (K18 ‒/‒ ) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8 ‒/‒ islets. K7, akin to F-actin, is concentrated at the apical vertex of β-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8 ‒/‒ β-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse β-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.

Although hepatocellular carcinoma (HCC) is developed with various etiologies, protection of hepatocytes seems basically essential to prevent the incidence of HCC. Keratin 8 and keratin 18 (K8/K18) are cytoskeletal intermediate filament proteins that are expressed in hepatocytes. They maintain the cell shape and protect cells under stress conditions. Their protective roles in liver damage have been described in studies of mouse models, and K8/K18 mutation frequency in liver patients. Interestingly, K8/K18 bind to signaling proteins such as transcription factors and protein kinases involved in HCC development. Since K8/K18 are abundant cytoskeletal proteins, K8/K18 binding with the signaling factors can alter the availability of the factors. Herein, we discuss the potential roles of K8/K18 in HCC development.

The cervix undergoes extensive remodeling throughout pregnancy and parturition. This process involves both ECM collagen degradation and cellular remodeling, which includes cell proliferation, transition and migration. Progesterone (P4) has been used clinically to delay cervical ripening and prevent preterm birth (PTB). However, the mechanisms by which progesterone affects cell transition and the migration of cervical epithelial and stromal cells are not yet fully known. In this study, we documented the role of a gestational level of P4 in the cellular transition (epithelial-mesenchymal transition [EMT] and mesenchymal-epithelial transition [MET]), cell migration, and inflammatory responses of endocervical epithelial cells (EEC) and cervical stromal cells (CSC). EEC and CSC were treated with LPS and P4 for 6 days. The epithelial:mesenchymal ratio (regular microscopy and cell shape index analysis), shift in intermediate filaments (immunofluorescence microscopy and western blot analyses for cytokeratin [CK]-18 and vimentin), adhesion molecules and transcription factors (western blot analyses for E-cadherin, N-cadherin and SNAIL), were used to determine growth characteristics and EMT and MET changes in EEC and CSC under the indicated conditions. To test cell remodeling, scratch assays followed by cellular analyses as mentioned above were performed. Inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor α [TNFα]) and matrix metallopeptidase 9 (MMP9) were measured by ELISA. LPS promoted EMT (decreased cell shape index, decreased CK-18 and E-cadherin, increased vimentin, N-cadherin, and SNAIL), and increased IL-6 and MMP9 production by EEC. A gestational level of P4 prevented LPS-induced EMT in EEC and exhibited anti-inflammatory effect in both EEC and CSC. LPS slowed down wound healing in CSC but P4 treatment prevented the negative impact of LPS in CSC wound healing. These results may explain the cellular mechanisms by which P4 helps to stabilize the cervical epithelial barrier and preserve the mechanical and tensile strength of the cervical stromal layer, which are important in normal cervical remodeling processes during pregnancy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: Thin endometrium is a common problem encountered in the field of assisted reproductive technology. We explored the effects of platelet-rich fibrin in a thin endometrium rat model., Materials and Methods: Twenty Sprague-Dawley rats were randomly divided into 2 groups. For the thin endometrium group, endometria of left uteri were injected with ethanol. For the experimental group, platelet-rich fibrin was sutured onto the left uteri of endometria injected with ethanol. Right uteri were kept as the normal (control) group. Two weeks after platelet-rich fibrin transplant, uteri were sampled for histology, immunohistochemistry, Western blot, and real-time reverse transcription-polymerase chain reaction., Results: Endometrium thicknesses in normal, thin endometrium, and experimental groups were 632.2 ± 38.28, 434.80 ± 41.37, and 603.0 ± 40.93 μm, respectively. Endometrium thickness in the experi-mental group was significantly increased versus the thin endometrium group (P = .011). Immunohistochemical examination showed that expression levels of cytokeratin 18, vimentin, and leukemia inhibitory factor in the experimental group were significantly higher versus the thin endometrium group (P < .001, P < .006, and P = .001, respectively). In Western blot analysis, cytokeratin 18, integrin β3, leukemia inhibitory factor, and vimentin protein expressions were slightly higher in the experimental and normal groups versus the thin endometrium group. Real-time reverse transcription-polymerase chain reaction showed significantly higher octamer-binding transcription factor 4 mRNA levels in the experimental group versus the thin endometrium group (P < .001). Interleukin 6 and matrix metalloproteinase 9 mRNA levels were significantly upregulated in the experimental group versus the thin endometrium group (P= .004 and P < .001, respectively). Interleukin 1β mRNA expression was significantly lower in the experimental group versus the thin endometrium group (P < .007)., Conclusions: Application of platelet-rich fibrin has a therapeutic effect on thin endometrium in rats. Our results provide new insight on clinical treatment of thin endometrium.

Corneal injuries are among the leading causes of blindness and vision impairment. Trauma, infectious keratitis, thermal and chemical (acids and alkali burn) injuries may lead to irreversible corneal scarring, neovascularization, conjunctivalization, and limbal stem cell deficiency. Bilateral blindness constitutes 12% of total global blindness and corneal transplantation remains a stand-alone treatment modality for the majority of end-stage corneal diseases. However, global shortage of donor corneas, the potential risk of graft rejection, and severe side effects arising from long-term use of immunosuppressive medications, demands alternative therapeutic approaches. Umbilical cord-derived mesenchymal stem cells can be isolated in large numbers using a relatively less invasive procedure. However, their role in injury induced corneal repair is largely unexplored. Here, we isolated, cultured and characterized mesenchymal stem cells from human umbilical cord, and studied the expression of mesenchymal (CD73, CD90, CD105, and CD34), ocular surface and epithelial (PAX6, WNT7A, and CK-8/18) lineage markers through immunofluorescence. The cultured human limbal and corneal epithelial cells were used as controls. Scratch assay was used to study the corneal epithelial repair potential of umbilical cord-derived mesenchymal stem cells, in vitro. The in vitro cultured umbilical cord-derived mesenchymal stem cells were plastic adherent, showed trilineage differentiation and expressed: mesenchymal markers CD90, CD105, CD73; epithelial marker CK-8/18, and ocular lineage developmental markers PAX6 and WNT-7A. Our findings suggest that umbilical cord-derived mesenchymal stem cells promote repair of the injured corneal epithelium by stimulating the proliferation of corneal epithelial cells, in vitro. They may serve as a potential non-ocular source of stem cells for treating injury induced bilateral corneal diseases.

Background: Cytokeratin 18 (CK18) is an intermediate filament protein of the cytokeratin acidic type I group and is primarily expressed in single-layered or "simple" epithelial tissues and carcinomas of different origin., Methods: To systematically determine CK18 expression in normal and cancerous tissues, 11,952 tumor samples from 115 different tumor types and subtypes (including carcinomas, mesenchymal and biphasic tumors) as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format., Results: CK18 was expressed in normal epithelial cells of most organs but absent in normal squamous epithelium. At least an occasional weak CK18 positivity was seen in 90 of 115 (78.3%) tumor types. Wide-spread CK18 positivity was seen in 37 (31.9%) of tumor entities, including adenocarcinomas of the lung, prostate, colon and pancreas as well as ovarian cancer. Tumor categories with variable CK18 immunostaining included cancer types arising from CK18 positive precursor cells but show CK18 downregulation in a fraction of cases, tumor types arising from CK18 negative precursor cells occasionally exhibiting CK18 neo-expression, tumors derived from normal tissues with variable CK18 expression, and tumors with a mixed differentiation. CK18 downregulation was for example seen in renal cell cancers and breast cancers, whereas CK18 neo-expression was found in squamous cell carcinomas of various origins. Down-regulation of CK18 in invasive breast carcinomas of no special type and clear cell renal cell carcinomas (ccRCC) was related to adverse tumor features in both tumors (p ≤ 0.0001) and poor patient prognosis in ccRCC (p = 0.0088). Up-regulation of CK18 in squamous cell carcinomas was linked to high grade and lymph node metastasis (p < 0.05). In summary, CK18 is consistently expressed in various epithelial cancers, especially adenocarcinomas., Conclusions: Down-regulation or loss of CK18 expression in cancers arising from CK18 positive tissues as well as CK18 neo-expression in cancers originating from CK18 negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop., Competing Interests: C.Y.L. is an inventor of intellectual property licensed to Syros Pharmaceuticals, and is the Vice President of Biology and a shareholder of Kronos Bio, Inc. S.L.S. is a shareholder and serves on the Board of Directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics and Decibel Therapeutics; is a shareholder and advises Kojin Therapeutics, Kisbee Therapeutics, Exo Therapeutics, and Eikonizo Therapeutics; serves on the Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma Foundation, Biogen, Inc., and F-Prime Capital Partners and the Board of Advisers of the Genomics Institute of the Novartis Research Foundation; and is a Novartis Faculty Scholar. M.J.W. is an employee and equity holder of Kojin Therapeutics. P.A.C. is an advisor to Pfizer, Inc. and nference, Inc. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. P.W. is a current employee of The Institute of Cancer Research, which has a Rewards to Inventors scheme and has a commercial interest in development of inhibitors of the WNT pathway, CDK8/19, and other CDKs, with intellectual property licensed to Merck and Cyclacel Pharmaceuticals. P.W. is also a consultant for Astex Pharmaceuticals, CV6 Therapeutics, Nextechinvest, Nuevolution, Black Diamond, and STORM Therapeutics and holds equity in Chroma Therapeutics, Nextech, Black Diamond, and STORM. B.N. is an inventor on patent applications related to the dTAG system (WO/2017/024318, WO/2017/024319, WO/2018/148443, and WO/2018/148440). N.S.G. is a Scientific Founder, member of the Scientific Advisory Board (SAB), and equity holder in C4 Therapeutics, Syros, Soltego (board member), B2S, Allorion, and Inception. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voroni, Arbella, Deerfield, and Sanofi., (© 2020 The Authors.)

During puberty, the mammary gland undergoes an intense growth, dependent on the interplay between the Epidermal Growth Factor Receptor (EGFR) in the stroma and different mammary epithelial receptors. We hypothesize that EGFR expressed in the mammary epithelium also has a role in puberty and the epithelial cells can self-sustain by EGFR-mediated autocrine signaling. We adopted mammary cell lines from different species, as in vitro model for the epithelium, and we observed that EGFR-signaling positively affects their survival and proliferation. Once deprived of external growth factors, mammary cells still showed strong Erk 1/2 phosphorylation, abolished upon EGFR inhibition, coupled with a further reduction in survival and proliferation. Based on gene expression analysis, three EGFR-ligands (AREG, EREG and HBEGF) are likely to mediate this autocrine signaling. In conclusion, internal EGFR-activating signals sustain mammary epithelial cell proliferation and survival in vitro., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The establishment and culture of bovine omasal epithelial cells (BOECs) in vitro is a valuable tool for the study of the physiological function, nutrient absorption, and transport mechanisms of the omasum in dairy cows. This paper proposes a method for the culture of primary BOECs. Trypsin digestion was used to subculture the BOECs, which were passaged for 20 generations in vitro, and showed typical epithelial-like characteristics and a cobblestone morphology. The primary BOECs had a fast growth phase (between days 4 and 5) and were validated by their slight β-galactosidase and visible cytokerat in 18 expression. In addition, RT-PCR results demonstrated that the monocarboxylate transporter 1 (MCT1), Na+/H+exchanger 1 (NHE1), and Na+/H+ exchanger 3 (NHE3) were expressed in the isolated primary BOECs. In conclusion, this primary BOEC isolation and culture model is a promising method for the study of nutrient absorption and regulation, as well as the immune regulation of epithelial cell transport in vitro.

To determine the role of 3, 3', 5-triiodo-L thyroxine (T3) in the differentiation of Sertoli cells (SCs) and the factors influencing maturity via the Wilms' tumor 1 (WT1)/non-canonical Wnt signaling pathway, high purity SCs were isolated from newborn calves' testes and cultured in vitro. The SCs were stimulated with T3, and co-treated with short interference (si) RNA to knockdown endogenous WT1 and non-canonical Wnt signalling inhibitor Wnt-c59. Our results suggested that the addition of different concentrations (0, 25, 50, and 100 nM) of T3 in the culture medium changed the expression of KRT-18 (SCs immature marker) and accelerated the differentiation of SCs. T3 (100 nM) treatment induced up-regulated expression of WT1 over time (p < 0.05), while the expression of polarity proteins (Par3, Par6b, and E-cadherin) and Wnt4 were affected to varying degrees (p < 0.05). SCs were treated simultaneously with T3 + Wnt-c59 and T3 + WT1 siRNA, and the results showed that T3 could affect the expression of polarity proteins via WT1/non-canonical Wnt signaling pathway. These data put together indicate that T3 plays a dependent role in the induction of bovine SCs differentiation via WT1/non-canonical Wnt signaling pathway in vitro. This study proposes for the first time that WT1 is a major target for T3., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Serine-52 (Ser52) is the major physiologic site of keratin 18 (K18) phosphorylation. Here, we report that serine-52 phosphorylated K18 (phospho-Ser52 K18) accumulated on centrosomes in a cell cycle-dependent manner. Moreover, we found that phospho-Ser52 K18 was located at the proximal end of the mother centriole. Transfection with the K18 Ser52 → Ala (K18 S52A) mutant prevented centriole localization of phospho-Ser52 K18 and resulted in separation of the mother-daughter centrioles. Inhibition of microtubule polymerization led to the disappearance of aggregated phospho-Ser52 K18 on the centrosome; removal of inhibitors resulted in reaccumulation of phospho-Ser52 K18 in microtubule-organizing centers. Transfection with a K18 S52A mutant inhibited microtubule nucleation. These results reveal a cell cycle-dependent change in centrosome localization of phospho-Ser52 k18 and strongly suggest that the phosphorylation status of Ser52 K18 of mother centrioles plays a critical role in maintaining a tight engagement between mother and daughter centrioles and also contributes to microtubule nucleation.

Leukamenin E is a natural ent -kaurane diterpenoid isolated from Isodon racemosa (Hemsl) Hara that has been found to be a novel and potential keratin filament inhibitor, but its underlying mechanisms remain largely unknown. Here, we show that leukamenin E induces keratin filaments (KFs) depolymerization, largely independently of microfilament (MFs) and microtubules (MTs) in well-spread cells and inhibition of KFs assembly in spreading cells. These effects are accompanied by keratin phosphorylation at K8-Ser73/Ser431 and K18-Ser52 via the by extracellular signal-regulated kinases (ERK) pathway in primary liver carcinoma cells (PLC) and human umbilical vein endothelial cells (HUVECs). Moreover, leukamenin E increases soluble pK8-Ser73/Ser431, pK18-Ser52, and pan-keratin in the cytoplasmic supernatant by immunofluorescence imaging and Western blotting assay. Accordingly, leukamenin E inhibits the spreading and migration of cells. We propose that leukamenin E-induced keratin phosphorylation may interfere with the initiation of KFs assembly and block the formation of a new KFs network, leading to the inhibition of cell spreading. Leukamenin E is a potential target drug for inhibition of KFs assembly.

Objective: To identify the chaperone of polypyrimidine tractor-binding protein-associated splicing factor (PSF) in myeloid leukemia cells, and to explore the mechanism and redistributive pattern to cell surface of PSF in chemo-sensitive HL60 cells and resistant HL60/DOX cells., Methods: The eukaryotic expression vector of PSF was transfected with liposomes transiently, then flow cytometry was used to detect the expression level of PSF on the cell surface 24 h, 48 h and 72 h after vector transfections. We constructed a chimeric expression vector, streptavidin binding peptide (SBP)-PSF, meanwhile this vector was transfected and made SBP-PSF fusion protein overexpress. In addition, we used streptavidin magnetic beads to precipitate the cellular chaperonin of PSF and then identified its chaperonin by mass spectrometry (MS). Lentiviral vectors containing cytokeratin18 (K18) interference sequences were transfected into 293T cells to prepare lentivirus. HL60 and HL60/DOX cells were infected with lentivirus to obtain stable interfering K18 cell lines. Next, flow cytometry was used to test the membrane relocation level of PSF. Together, these methods confirmed the similar or different mechanisms of the PSF redistributing to membrane synergistically mediated by K18 in HL60 and HL60/DOX cells., Results: The expression of membrane relocated PSF was detected every day for three days (at the end of 24 h, 48 h and 72 h) after transient overexpression. The expressing rate of PSF on the cell surface was 22.4%±3.5%, 37.9%±6.0%, 58.3%±8.8%, respectively in sensitive HL60 cells, while that was 4.7%±0.5%, 3.9%±0.6%, 2.9%±0.6% , respectively in resistant HL60/DOX cells. The difference of expressing rate on each day was significant, P<0.01. We identified K18 detected by co-immunoprecipitation and mass spectrum assay which was the cellular chaperone of PSF. We found that K18 knockdown decreased the PSF expression level which redistributed on cell surface from 48.9%±5.4% to 6.2%±1.0% in sensitive HL60 cells, and from 9.11%±1.2% to 2.21%±0.51% in resistant HL60/DOX cells, respectively., Conclusion: K18 is the intracellular chaperonin of PSF. The interaction of PSF and K18 mediates its redistribution to cell membrane in sensitive cells. While in resistant cells, PSF failed to relocate at the cell surface and accumulated in cells, which mediated resistance to chemotherapeutics.

Aims: To investigate the role of IL-28B and CK-18 M30 in the diagnosis of non-alcoholic steatohepatitis (NASH) in rats., Methods: The rat NASH model was constructed by high-fat diet feeding and confirmed by liver tissue pathology analysis. The CK-18 M30, IL-28B, liver function and blood lipid were detected. The pathology of liver tissues was observed by H&E staining. The ROC curves of liver pathological scores, IL-28B, and CK-18 M30 were plotted and the sensitivity/specificity of each index was calculated., Results: The liver tissue pathology at the end of the 12th week met NASH diagnostic criteria. The liver pathological scores of NASH model were higher than those of the control group (P < 0.05). Compared with the control group, the body weight of rats in the NASH group was lower (P < 0.05) and the liver index was higher (P < 0.05). Moreover, the serum levels of alanine aminotransferase, triglyceride, low-density lipoprotein, total cholesterol and CK-18 M30 in the NASH group were higher (P < 0.05) and positively correlated with liver pathological scores (P < 0.05), but IL-28B in serum and liver tissue was lower (P < 0.05) and negatively correlated with liver pathological scores (P < 0.05). According to the ROC curves, the sensitivity/specificity of each index was shown as following: liver pathological scores (1.000/1.000), IL-28B of liver tissue (1.000/0.857), serum CK-18 M30 plus IL-28B (0.857/1.000), serum IL-28B (0.857/0.857) and serum CK-18 M30 (1.000/0.857)., Conclusions: IL-28B level is related to the pathological changes of livers in SD rats during the development of NASH. The combination of serum CK-18 M30 level and serum IL-28B level may be a promising non-invasive detection method for the diagnosis of NASH., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier GmbH.)

Objective: HIV-infected patients are at increased risk of nonalcoholic steatohepatitis (NASH). Vitamin E is recommended for treatment of NASH in the general population. However, its safety and efficacy among HIV-infected patients remain unknown., Design: Single-centre, phase IV, open-label, single arm clinical trial., Methods: HIV mono-infected patients without significant alcohol intake or viral hepatitis coinfection were included. The diagnosis of NASH was based on the co-existence of fatty liver, diagnosed by controlled attenuation parameter (CAP) at least 248 dB/m and significant hepatocyte apoptosis, defined by the serum biomarker cytokeratin 18 (CK-18) greater than 130.5 U/L. Participants were treated with 800 IU daily of oral vitamin E (alpha-tocopherol) for 24 weeks, and followed for an additional 24 weeks postdiscontinuation. Generalized linear mixed effects models were used to evaluate changes in alanine aminotransferase (ALT), CAP and CK-18 at the completion of treatment and end of follow-up, controlling for pretreatment trends., Results: A total of 27 patients were included. Four (15%) had a pretreatment liver biopsy, which confirmed the diagnosis of NASH in all cases. Compared with baseline, 24 weeks of vitamin E treatment improved ALT [-27 units/l; 95% confidence interval (CI) -37 to -17], CAP scores (-22 dB/m; 95% CI -42 to -1) and CK-18 (-123 units/l; 95% CI -201 to -46). Conversely, there was no change in BMI. No serious adverse event was reported and no patient was lost to follow-up., Conclusion: In this first clinical trial, we showed that vitamin E is an effective and well tolerated treatment for NASH in HIV-infected patients.

Purpose: Paget's disease (PD) of the breast is an uncommon disease of the nipple usually accompanied by an underlying carcinoma, often HER2 + , and accounting for 0.5-5% of all breast cancer. To date, histogenesis of PD of the breast remains controversial, as two theories-transformation and epidermotropic-have been proposed to explain this disease. Currently, animal models recapitulating PD of the nipple have not been described., Methods: HER2-enriched DT13 breast cancer cells were injected into the mammary fat pad of NOD scid gamma null (NSG) female mice. Immunohistochemical staining and pathological studies were performed on tumor samples, and diagnosis of PD of the nipple was confirmed by expression of proteins characteristic of Paget cells (epidermal growth factor 2 (HER2), androgen receptor (AR), cytokeratin 7 (CK7), cytokeratin 8/18 (CK8/18), and mucin 1 (MUC1)). In addition, DT13 cells grown in 2D culture and in soft agar assays were sensitive to in vitro treatment with pharmacological inhibitors targeting Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways., Results: Mice developed tumors and nipple lesions that were detected exclusively on the tumor-bearing mammary fat pad. Tumor cells were positive for proteins characteristic of Paget cells. In vitro, DT13 cells were sensitive to inhibition of Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways., Conclusions: Our results suggest that injection of HER2 + DT13 cells into the mammary fat pad of NSG mice recapitulates critical aspects of the pathophysiology of PD of the nipple, supporting the epidermotropic theory as the more likely to explain the histogenesis of this disease.

We examined a 10-year-old cow in which about half of the liver was displaced by malignant tissue consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Cytokeratin (CK) 18 and 7 were expressed in the latter. Metastasis was present in the hepatic, pancreaticoduodenal and mediastinal lymph nodes, where malignant cells had hepatocellular features, but more pleomorphic and atypical than in the primary lesion. Areas composed solely of CC cells or less-differentiated HCC cells were observed. In contrast, well-differentiated HCC cells were almost always admixed with the other two types, and may have had the ability to transform into CC cells and to dedifferentiate into less-differentiated cells. This report suggests that CK18 is an excellent marker for biliary differentiation in cattle.

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18 -knockout ( Krt18 -KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18 -null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

The histomorphological changes occurring in the Dama dama reticulum during prenatal development have been investigated. Twenty-five Dama dama embryos were used, from the first stages of prenatal life until birth. Differentiation of the reticulum was observed at 23% gestation. By 25% gestation the reticular wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Primary reticular crests were visible at 38% gestation. Secondary reticular crests were observed at 61% gestation. Neuroendocrine cells were detected by synaptophysin (SYP) at 35% gestation, in the lamina propria-submucosa, tunica muscularis, and serosa. Epithelial Cytokeratin-18 (CK-18) cells were observed at 35% gestation extended throughout the epithelial layers. The glial cells (vimentin -VIM- and glial fibrillary acidic protein-GFAP-markers) were discerned at 25% and 43% gestation, respectively, in myenteric and submucosal plexuses, lamina propria, muscularis mucosae, tunica muscularis, and perivascular connective tissue. The neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) markers were immunodetected at 75% and 80 gestation, respectively, in the lamina propria-submucosa, muscularis mucosae, tunica muscularis, serosa, and myenteric plexuses. The prenatal development of the fallow deer reticular mucosa evidenced a considerable precocity similar to that previously reported in goat and red deer., (© 2020 Japanese Society of Animal Science.)

The aim of this study was to investigate the effect of synbiotic and probiotic supplementation on serum vascular dysfunction and necrosis markers in hemodialysis (HD) patients. In this randomized, double-blind, placebo-controlled trial, 75 HD patients were randomly assigned to either the synbiotic or probiotic or placebo group. The patients in the synbiotic group received 15 g of prebiotics and 5 g probiotic powder containing Lactobacillus acidophilus strain T16 (IBRC-M10785), Bifidobacterium bifidum strain BIA-6, Bifidobacterium lactis strain BIA-6, Bifidobacterium longum strain LAF-5 (2.7 × 10 7  CFU/g each) in sachets (n = 25), whereas the probiotic group received 5 g probiotics same to the first group with 15 g of maltodextrin powder in sachets (n = 25) and the placebo group received 20 g of maltodextrin powder in sachets (n = 25) for 12 weeks. At baseline and the end of the study, serum concentrations of soluble intercellular adhesion molecule type 1 (sICAM-1), soluble vascular cell adhesion molecule type 1 (sVCAM-1), cytokeratin 18 (CK-18) as the necrosis marker, uric acid, and phosphate levels were measured. Feces also were collected for microbiota colony counting. Serum ICAM-1 level reduced significantly in the synbiotic group after the intervention period (P = 0.02), and this reduction was significantly different in the synbiotic group in comparison to the placebo group (P = 0.03). Serum levels of VCAM-1 and CK-18 were not significantly different between the groups. However, the reduction in serum levels of VCAM-1 in the synbiotic group was significantly higher in comparison to the placebo group (P = 0.01). Multivariate linear regression analysis revealed that ∆ phosphate was the sole independent determinant of ∆ICAM-1 (P = 0 < 001). The study indicated that synbiotic supplementation reduced serum ICAM-1 level, which is a risk factor for cardiovascular diseases in HD patients, but has no effect on the necrosis marker. Trial registration: www.irct.ir (IRCT2017041233393N1).

Despite considerable recent insight into the molecular phenotypes and type 2 innate immune functions of tuft cells in rodents, there is sparse knowledge about the region-specific presence and molecular phenotypes of tuft cells in the human digestive tract. Here, we traced cholinergic tuft cells throughout the human alimentary tract with immunohistochemistry and deciphered their region-specific distribution and biomolecule coexistence patterns. While absent from the human stomach, cholinergic tuft cells localized to villi and crypts in the small and large intestines. In the biliary tract, they were present in the epithelium of extra-hepatic peribiliary glands, but not observed in the epithelia of the gall bladder and the common duct of the biliary tract. In the pancreas, solitary cholinergic tuft cells were frequently observed in the epithelia of small and medium-size intra- and inter-lobular ducts, while they were absent from acinar cells and from the main pancreatic duct. Double immunofluorescence revealed co-expression of choline acetyltransferase with structural (cytokeratin 18, villin, advillin) tuft cell markers and eicosanoid signaling (cyclooxygenase 1, hematopoietic prostaglandin D synthase, 5-lipoxygenase activating protein) biomolecules. Our results indicate that region-specific cholinergic signaling of tuft cells plays a role in mucosal immunity in health and disease, especially in infection and cancer.

Chronic lung allograft dysfunction (CLAD) remains the leading cause of late death after lung transplantation. Epithelial injury is thought to be a key event in the pathogenesis of CLAD. M30 and M65 are fragments of cytokeratin-18 released specifically during epithelial cell apoptosis and total cell death, respectively. We investigated whether M30 and M65 levels in bronchoalveolar lavage (BAL) correlate with CLAD subtypes: restrictive allograft syndrome (RAS) versus bronchiolitis obliterans syndrome (BOS). BALs were obtained from 26 patients with established CLAD (10 RAS, 16 BOS) and 19 long-term CLAD-free controls. Samples with concurrent infection or acute rejection were excluded. Protein levels were measured by ELISA. Variables were compared using Kruskal-Wallis, Mann-Whitney U test and Chi-squared tests. Association of M30 and M65 levels with post-CLAD survival was assessed using a Cox PH models. M65 levels were significantly higher in RAS compared to BOS and long-term CLAD-free controls and correlated with worse post-CLAD survival. Lung epithelial cell death is enhanced in patients with RAS. Detection of BAL M65 may be used to differentiate CLAD subtypes and as a prognostic marker in patients with established CLAD. Understanding the role of epithelial cell death in CLAD pathogenesis may help identify new therapeutic targets to improve outcome., (© 2019 Steunstichting ESOT.)

Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory-Denk bodies found in patients with various liver diseases. However, the functions of other post-translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site-specific mutation assay combined with nano-liquid chromatography-tandem mass spectrometry, we identified K8-Lys108 and K18-Lys187/426 as acetylation sites, and K8-Arg47 and K18-Arg55 as methylation sites. Keratin mutation (Arg-to-Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease-associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18-del65-72 and K18-I150V variants increased. Notably, the highly acetylated/methylated K18-I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease-associated variants regulate keratin protein stability. These findings extend our understanding of how disease-associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.-Jang, K.-H., Yoon, H.-N., Lee, J., Yi, H., Park, S.-Y., Lee, S.-Y., Lim, Y., Lee, H.-J., Cho, J.-W., Paik, Y.-K., Hancock, W. S., Ku, N.-O. Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.

The amnion membrane that lines the human intrauterine cavity is composed of amnion epithelial cells (AECs) connected to an extracellular matrix containing amnion mesenchymal cells (AMCs) through a basement membrane. Cellular interactions and transitions are mechanisms that facilitate membrane remodeling to maintain its integrity. Dysregulation of cellular remodeling, primarily mediated by oxidative stress (OS), is often associated with preterm birth. However, the mechanisms that maintain membrane homeostasis remain unclear. To understand these mechanisms, we developed an amnion membrane organ-on-chip (AM-OOC) and tested the interactive and transition properties of primary human AECs and AMCs under normal and OS conditions. AM-OOC contained 2 chambers connected by type IV collagen-coated microchannels, allowing independent culture conditions that permitted cellular migration and interactions. Cells grown either independently or coculture were exposed to OS inducing cigarette smoke extract, antioxidant N -acetyl-l-cysteine (NAC), or both. When grown independently, AECs transitioned to AMCs and migrated, whereas AMCs migrated without transition. OS caused AECs' transition but prevented migration, whereas AMCs' migration was unhindered. Coculture of cells facilitated transition, migration, and eventual integration in the contiguous population. OS cotreatment in both chambers facilitated AECs' transition, prevented migration, and increased inflammation, a process that was prevented by NAC. AM-OOC recapitulated cellular mechanisms observed in utero and enabled experimental manipulation of cells to determine their roles during pregnancy and parturition.-Richardson, L., Jeong, S., Kim, S., Han, A., Menon, R. Amnion membrane organ-on-chip: an innovative approach to study cellular interactions.