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The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.

During pulmonary mucormycosis, inhaled sporangiospores adhere to, germinate, and invade airway epithelial cells to establish infection. We provide evidence that HIF1α plays dual roles in airway epithelial cells during Mucorales infection. We observed an increase in HIF1α protein accumulation and increased expression of many known HIF1α-responsive genes during in vitro infection, indicating that HIF1α signaling is activated by Mucorales infection. Inhibition of HIF1α signaling led to a substantial decrease in the ability of R. delemar to invade cultured airway epithelial cells. Transcriptome analysis revealed that R. delemar infection induces the expression of many pro-inflammatory genes whose expression was significantly reduced by HIF1α inhibition. Importantly, pharmacological inhibition of HIF1α increased survival in a mouse model of pulmonary mucormycosis without reducing fungal burden. These results suggest that HIF1α plays two opposing roles during mucormycosis: one that facilitates the ability of Mucorales to invade the host cells and one that facilitates the ability of the host to mount an innate immune response.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p

Acute lung injury (ALI) is characterized by respiratory failure resulting from the disruption of the epithelial and endothelial barriers as well as immune system. In this study, we evaluated the therapeutic potential of airway epithelial cell-derived extracellular vesicles (EVs) in maintaining lung homeostasis. We isolated human bronchial epithelial cell-derived EVs (HBEC-EVs), which endogenously express various immune-related surface markers and investigated their immunomodulatory potential in ALI. In ALI cellular models, HBEC-EVs demonstrated immunosuppressive effects by reducing the secretion of proinflammatory cytokines in both THP-1 macrophages and HBECs. Mechanistically, these effects were partially ascribed to nine of the top 10 miRNAs enriched in HBEC-EVs, governing toll-like receptor-NF-κB signaling pathways. Proteomic analysis revealed the presence of proteins in HBEC-EVs involved in WNT and NF-κB signaling pathways, pivotal in inflammation regulation. ANXA1, a constituent of HBEC-EVs, interacts with formyl peptide receptor (FPR)2, eliciting anti-inflammatory responses by suppressing NF-κB signaling in inflamed epithelium, including type II alveolar epithelial cells. In a mouse model of ALI, intratracheal administration of HBEC-EVs reduced lung injury, inflammatory cell infiltration, and cytokine levels. Collectively, these findings suggest the therapeutic potential of HBEC-EVs, through their miRNAs and ANXA1 cargo, in mitigating lung injury and inflammation in ALI patients.

UNLABELLED: Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1-sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1. SIGNIFICANCE: This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.

PURPOSE: Identifying molecular and immune features to guide immune checkpoint inhibitor (ICI)-based regimens remains an unmet clinical need. EXPERIMENTAL DESIGN: Tissue and longitudinal blood specimens from phase III trial S1400I in patients with metastatic squamous non-small cell carcinoma (SqNSCLC) treated with nivolumab monotherapy (nivo) or nivolumab plus ipilimumab (nivo+ipi) were subjected to multi-omics analyses including multiplex immunofluorescence (mIF), nCounter PanCancer Immune Profiling Panel, whole-exome sequencing, and Olink. RESULTS: Higher immune scores from immune gene expression profiling or immune cell infiltration by mIF were associated with response to ICIs and improved survival, except regulatory T cells, which were associated with worse overall survival (OS) for patients receiving nivo+ipi. Immune cell density and closer proximity of CD8+GZB+ T cells to malignant cells were associated with superior progression-free survival and OS. The cold immune landscape of NSCLC was associated with a higher level of chromosomal copy-number variation (CNV) burden. Patients with LRP1B-mutant tumors had a shorter survival than patients with LRP1B-wild-type tumors. Olink assays revealed soluble proteins such as LAMP3 increased in responders while IL6 and CXCL13 increased in nonresponders. Upregulation of serum CXCL13, MMP12, CSF-1, and IL8 were associated with worse survival before radiologic progression. CONCLUSIONS: The frequency, distribution, and clustering of immune cells relative to malignant ones can impact ICI efficacy in patients with SqNSCLC. High CNV burden may contribute to the cold immune microenvironment. Soluble inflammation/immune-related proteins in the blood have the potential to monitor therapeutic benefit from ICI treatment in patients with SqNSCLC.

Endurance exercise can disturb intestinal epithelial integrity, leading to increased systemic indicators of cell injury, hyperpermeability, and pathogenic translocation. However, the interaction between exercise, diet, and gastrointestinal disturbance still warrants exploration. This study examined whether a 6-day dietary intervention influenced perturbations to intestinal epithelial disruption in response to a 25-km race walk. Twenty-eight male race walkers adhered to a high carbohydrate (CHO)/energy diet (65% CHO, energy availability = 40 kcal·kg FFM−1·day−1) for 6 days prior to a Baseline 25-km race walk. Athletes were then split into three subgroups: high CHO/energy diet (n = 10); low-CHO, high-fat diet (LCHF: n = 8; <50 g/day CHO, energy availability = 40 kcal·kg FFM−1·day−1); and low energy availability (n = 10; 65% CHO, energy availability = 15 kcal·kg FFM−1·day−1) for a further 6-day dietary intervention period prior to a second 25-km race walk (Adaptation). During both trials, venous blood was collected pre-, post-, and 1 hr postexercise and analyzed for markers of intestinal epithelial disruption. Intestinal fatty acid-binding protein concentration was significantly higher (twofold increase) in response to exercise during Adaptation compared to Baseline in the LCHF group (p =.001). Similar findings were observed for soluble CD14 (p <.001) and lipopolysaccharide-binding protein (p =.003), where postexercise concentrations were higher (53% and 36%, respectively) during Adaptation than Baseline in LCHF. No differences in high CHO/energy diet or low energy availability were apparent for any blood markers assessed (p >.05). A short-term LCHF diet increased intestinal epithelial cell injury in response to a 25-km race walk. No effect of low energy availability on gastrointestinal injury or symptoms was observed. [ABSTRACT FROM AUTHOR]

Frankincense is produced by Boswellia trees, which can be found throughout the Middle East and parts of Africa and Asia. Boswellia serrata extract has been shown to have anti-cancer, anti-inflammatory, and antimicrobial effects. Periodontitis is an oral chronic inflammatory disease that affects nearly half of the US population. We investigated the antimicrobial effects of B. serrata extract on two oral pathogens associated with periodontitis. Using the minimum inhibitory concentration and crystal violet staining methods, we demonstrated that Porphyromonas gingivalis growth and biofilm formation were impaired by treatment with B. serrata extracts. However, the effects on Fusobacterium nucleatum growth and biofilm formation were not significant. Using quantification of colony-forming units and microscopy techniques, we also showed that concentrations of B. serrata that were not toxic for host cells decreased intracellular P. gingivalis infection in human gingival epithelial cells. Our results show antimicrobial activity of a natural product extracted from Boswellia trees (B. serrata) against periodontopathogens. Thus, B. serrata has the potential for preventing and/or treating periodontal diseases. Future studies will identify the molecular components of B. serrata extracts responsible for the beneficial effects.

Different exogenous electric fields (EF) can guide cell migration, disrupt proliferation, and program cell development. Studies have shown that many of these processes were initiated at the cell membrane, but the mechanism has been unclear, especially for conventionally non-excitable cells. In this study, we focus on the electrostatic aspects of EF coupling with the cell membrane by eliminating Faradaic processes using dielectric-coated microelectrodes. Our data unveil a distinctive biphasic response of the ERK signaling pathway of epithelial cells (MCF10A) to alternate current (AC) EF. The ERK signal exhibits both inhibition and activation phases, with the former triggered by a lower threshold of AC EF, featuring a swifter peaking time and briefer refractory periods than the later-occurring activation phase, induced at a higher threshold. Interestingly, the biphasic ERK responses are sensitive to the waveform and timing of EF stimulation pulses, depicting the characteristics of electrostatic and dissipative interactions. Blocker tests and correlated changes of active Ras on the cell membrane with ERK signals indicated that both EGFR and Ras were involved in the rich ERK dynamics induced by EF. We propose that the frequency-dependent dielectric relaxation process could be an important mechanism to couple EF energy to the cell membrane region and modulate membrane protein-initiated signaling pathways, which can be further explored to precisely control cell behavior and fate with high temporal and spatial resolution.

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells.

A core pathophysiologic feature underlying many respiratory diseases is multiciliated cell dysfunction, leading to inadequate mucociliary clearance. Due to the prevalence and highly variable etiology of mucociliary dysfunction in respiratory diseases, it is critical to understand the mechanisms controlling multiciliogenesis that may be targeted to restore functional mucociliary clearance. Multicilin, in a complex with E2F4, is necessary and sufficient to drive multiciliogenesis in airway epithelia, however this does not apply to all cell types, nor does it occur evenly across all cells in the same cell population. In this study we further investigated how co-factors regulate the ability of Multicilin to drive multiciliogenesis. Combining data in mouse embryonic fibroblasts and human bronchial epithelial cells, we identify RBL2 as a repressor of the transcriptional activity of Multicilin. Knockdown of RBL2 in submerged cultures or phosphorylation of RBL2 in response to apical air exposure, in the presence of Multicilin, allows multiciliogenesis to progress. These data demonstrate a dynamic interaction between RBL2 and Multicilin that regulates the capacity of cells to differentiate and multiciliate. Identification of this mechanism has important implications for facilitating MCC differentiation in diseases with impaired mucociliary clearance.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we use integrative single-cell sequencing (scRNA-seq and scATAC-seq) on insectivorous (Eptesicus fuscus; big brown bat) and frugivorous (Artibeus jamaicensis; Jamaican fruit bat) bat kidneys and pancreases and identify key cell population, gene expression and regulatory differences associated with the Jamaican fruit bat that also relate to human disease, particularly diabetes. We find a decrease in loop of Henle and an increase in collecting duct cells, and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the Jamaican fruit bat kidney. The Jamaican fruit bat pancreas shows an increase in endocrine and a decrease in exocrine cells, and differences in genes and regulatory elements involved in insulin regulation. We also find that these frugivorous bats share several molecular characteristics with human diabetes. Combined, our work provides insights from a frugivorous mammal that could be leveraged for therapeutic purposes.

Oral epitheliolysis or mucosal shedding is an infrequently described phenomenon characterized by epithelial desquamation, revealing mucosa of normal color and texture underneath. The condition has a predilection for middle-aged females and primarily affects nonkeratinized oral tissues. Although some cases are idiopathic, certain oral hygiene products have been implicated as the culprit, and resolution of the condition documented with their discontinuation. Severity of desquamation and symptoms vary with frequency and duration of contact with the irritant and its concentration. A case is presented of a dramatic case of shedding oral mucosa in an elderly female, which appeared to be caused by habitual chewing of an aspirincontaining over-the-counter analgesic. [ABSTRACT FROM AUTHOR]

The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF. • The presence of oviductal epithelial cells during embryo in vitro culture enhanced porcine blastocyst formation. • The oviductal epithelial cells during embryo culture decrease ROS levels in porcine in vitro produced cleaved embryos. • The porcine oviductal epithelial cells expressed OVGP-1 during in vitro culture. [ABSTRACT FROM AUTHOR]

The vocal fold is an architecturally complex organ comprising a heterogeneous mixture of various layers of individual epithelial and mesenchymal cell lineages. Here we performed single‐cell RNA sequencing profiling of 5836 cells from the vocal folds of adult Sprague–Dawley rats. Combined with immunostaining, we generated a spatial and transcriptional map of the vocal fold cells and characterized the subpopulations of epithelial cells, mesenchymal cells, endothelial cells, and immune cells. We also identified a novel epithelial‐to‐mesenchymal transition‐associated epithelial cell subset that was mainly found in the basal epithelial layers. We further confirmed that this subset acts as intermediate cells with similar genetic features to epithelial‐to‐mesenchymal transition in head and neck squamous cell carcinoma. Finally, we present the complex intracellular communication network involved homeostasis using CellChat analysis. These studies define the cellular and molecular framework of the biology and pathology of the VF mucosa and reveal the functional importance of developmental pathways in pathological states in cancer. [ABSTRACT FROM AUTHOR]

The alveolar surface of the lung is lined by an epithelium consisting of type I (AECI) and type II alveolar epithelial cells (AECII). This epithelium is covered by a liquid alveolar lining layer (ALL). Besides intra‐alveolar surfactant, ALL also contains the alveolar epithelial glycocalyx on the apical side of AECI and AECII. To better understand the alveolar epithelial glycocalyx, its ultrastructural visualization by transmission electron microscopy is required. The aim of this study was to systematically re‐evaluate routine cytochemical methods for visualization of the alveolar epithelial glycocalyx and specifically its glycan components. For this purpose, we used chemical fixation by vascular perfusion with aldehydes as a common routine approach in mice. After fixation, staining is needed for glycocalyx visualization. Cytochemical staining agents such as alcian blue, ruthenium red, and lanthanum nitrate were compared. In addition, SNL (Sambucus nigra lectin) and UEA1 (Ulex europaeus agglutinin I) were used for sialic acid and fucose‐specific labeling. Alcian blue showed the strongest staining, with cloud‐like structures, whereas ruthenium red appeared as thread‐like structures. On the other hand, lanthanum nitrate did not stain the alveolar epithelial glycocalyx. For specific sialic acid and fucose labeling, both lectins presented a specific signal. In conclusion, these methods can be used routinely for assessing ultrastructural changes of the alveolar epithelial glycocalyx in experimental in vivo models under different physiological and pathological conditions. In addition, cytochemical staining by tissue massage and post‐embedding lectin labeling after vascular perfusion support 3R (reduction, refinement, replacement) principles of animal welfare. [ABSTRACT FROM AUTHOR]

Cisplatin-induced renal tubular injury largely restricts the wide-spread usage of cisplatin in the treatment of malignancies. Identifying the key signaling pathways that regulate cisplatin-induced renal tubular injury is thus clinically important. PARVB, a focal adhesion protein, plays a crucial role in tumorigenesis. However, the function of PARVB in kidney disease is largely unknown. To investigate whether and how PARVB contributes to cisplatin-induced renal tubular injury, a mouse model (PARVB cKO) was generated in which PARVB gene was specifically deleted from proximal tubular epithelial cells using the Cre-LoxP system. In this study, we found depletion of PARVB in proximal tubular epithelial cells significantly attenuates cisplatin-induced renal tubular injury, including tubular cell death and inflammation. Mechanistically, PARVB associates with transforming growth factor-β-activated kinase 1 (TAK1), a central regulator of cell survival and inflammation that is critically involved in mediating cisplatin-induced renal tubular injury. Depletion of PARVB promotes cisplatin-induced TAK1 degradation, inhibits TAK1 downstream signaling, and ultimately alleviates cisplatin-induced tubular cell damage. Restoration of PARVB or TAK1 in PARVB-deficient cells aggravates cisplatin-induced tubular cell injury. Finally, we demonstrated that PARVB regulates TAK1 protein expression through an E3 ligase ITCH-dependent pathway. PARVB prevents ITCH association with TAK1 to block its ubiquitination. Our study reveals that PARVB deficiency protects against cisplatin-induced tubular injury through regulation of TAK1 signaling and indicates targeting this pathway may provide a novel therapeutic strategy to alleviate cisplatin-induced kidney damage. [ABSTRACT FROM AUTHOR]

This study describes a complex human in vitro model for evaluating anti‐inflammatory drug response in the alveoli that may contribute to the reduction of animal testing in the pre‐clinical stage of drug development. The model is based on the human alveolar epithelial cell line Arlo co‐cultured with macrophages differentiated from the THP‐1 cell line, creating a physiological biological microenvironment. To mimic the three‐dimensional architecture and dynamic expansion and relaxation of the air‐blood‐barrier, they are grown on a stretchable microphysiological lung‐on‐chip. For validating the in vitro model, three different protocols have been developed to demonstrate the clinically established anti‐inflammatory effect of glucocorticoids to reduce certain inflammatory markers after different pro‐inflammatory stimuli: (1) an inflammation caused by bacterial LPS (lipopolysaccharides) to simulate an LPS‐induced acute lung injury measured best with cytokine IL‐6 release; (2) an inflammation caused by LPS at ALI (air‐liquid interface) to investigate aerosolized anti‐inflammatory treatment, measured with chemokine IL‐8 release; and (3) an inflammation with a combination of human inflammatory cytokines TNFα and IFNγ to simulate a critical cytokine storm leading to epithelial barrier disruption, where the eventual weakening or protection of the epithelial barrier can be measured. In all cases, the presence of macrophages appeared to be crucial to mediating inflammatory changes in the alveolar epithelium. LPS induction led to inflammatory changes independently of stretch conditions. Dynamic stretch, emulating breathing‐like mechanics, was essential for in vitro modeling of the clinically relevant outcome of epithelial barrier disruption upon TNFα/IFNγ‐induced inflammation. [ABSTRACT FROM AUTHOR]

PurposeMethodsResultsConclusionThis study aimed to evaluate the tomographic, biomechanical, and in vivo confocal microscopic (IVCM) effects of chronic gout disease on human cornea.This prospective study included 60 eyes of 30 participants with chronic gout disease and 60 eyes of 30 healthy controls. Corneal thickness, keratometry (K) readings, and corneal aberrations were measured with Sirius 3 D corneal tomography system (Sirius, CSO, Italy). Corneal biomechanical properties (corneal hysteresis [CH], corneal resistance factor [CRF], and intraocular pressure [IOP] parameters) were assessed with an ocular response analyzer (ORA, Reichert Ophthalmic Instruments). The number and morphology of corneal endothelial cells and the number of basal epithelial cells were evaluated with IVCM (Confoscan 4.0). Tear breakup time (TBUT) was also evaluated.The mean diagnosis time of the patients with gout was 91.2 ± 69.6 months (12–300 month). Among corneal tomography measurements, K readings were similar between the two groups, while total and higher-order aberrations(coma, trefoil,s pherical, and quadrafoil) were significantly higher in the gout group. In the evaluation of biomechanical measurements, the CH value was significantly lower and the corneal-compensated IOP value was significantly higher in the gout group (p = 0.02, p = 0.01, respectively). The two groups did not significantly differ regarding the CRF or Goldmann IOP (p = 0.61, p = 0.15, respectively). Among the IVCM parameters, the number of corneal basal epithelial cells and the percentage of corneal endothelial hexagonality were significantly lower in the gout group, but no significant difference was detected in terms of the number of endothelial cells or polymegathism (p = 0.02, p < 0.001, p = 0.18, p = 0.59, respectively). While TBUT was significantly lower in the gout group (p < 0.001).This study showed that chronic gout disease increases the corneal aberrations and decreases the basal epithelial cell count, hexagonality ratio of endothelial cell and corneal biomechanics. [ABSTRACT FROM AUTHOR]

Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this, we used the Drosophila ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this 3D structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type–specific basal geometries starts with patterning of the basal extracellular matrix, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We find the Dystroglycan receptor complex (DGC) is required for this patterning by promoting localized Laminin accumulation at the basal surface of cells. Moreover, our results reveal that localized accumulation of Laminin and the DGC are required for directing Integrin adhesion. This induces cell basal geometry remodeling by anchoring the basal surface of cells to the extracellular matrix at specific, Laminin-rich locations. We propose that patterning of a basal extracellular matrix by generating discrete Laminin domains can direct Integrin adhesion to induce cell shape remodeling in epithelial morphogenesis. Cell shape remodeling is a main driver of epithelial tissue morphogenesis, but how cell basal geometry is regulated remains unclear. This work identifies a novel morphogenetic pathway involving laminin and integrin adhesion that coordinates the remodelling of epithelial cell shape by patterning their basement membrane. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and was the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while the exons encoding p14ARF remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines. [ABSTRACT FROM AUTHOR]

Background: This study aimed to identify the roles of L-tryptophan (Trp) and its rate-limiting enzymes on the receptivity of bovine endometrial epithelial cells. Real-time PCR was conducted to analyze the differential expression of genes between different groups of bovine endometrial epithelial cells. Western blot was performed to detect Cyclooxygenase-2 (COX2) expression after treatment with Trp or kynurenine (the main metabolites of Trp). The kynurenine assay was used to examine if Trp or prostaglandin E2 (PGE2) can increase the production of kynurenine in the bovine endometrial epithelial cells. Results: Trp significantly stimulates insulin growth factor binding protein 1 (IGFBP1) expression, a common endometrial marker of conceptus elongation and uterus receptivity for ruminants. When bovine endometrial epithelial cells are treated with Trp, tryptophan hydroxylase-1 remains unchanged, but tryptophan 2,3-dioxygenase 2 (TDO2) is significantly increased, suggesting tryptophan is mainly metabolized through the kynurenine pathway. Kynurenine significantly stimulates IGFBP1 expression. Furthermore, Trp and kynurenine significantly increase the expression of aryl hydrocarbon receptor (AHR). CH223191, an AHR inhibitor, abrogates the induction of Trp and kynurenine on IGFBP1. PGE2 significantly induces the expression of TDO2, AHR, and IGFBP1. Conclusions: The regulation between Trp / kynurenine and PGE2 may be crucial for the receptivity of the bovine uterus. [ABSTRACT FROM AUTHOR]

BamA, an Omp85 superfamily member, is universally conserved and essential for cell viability. Using anti‐Oma87 antibodies, we focus on understanding the effect of Oma87 of Acinetobacter baumannii on pathogenicity. Oma87 was expressed, purified, and used to induce anti‐Oma87 antibodies in BALB/c mice. Acute toxicity of the protein was evaluated in mice. HeLa cells were infected with both live and killed A. baumannii 19606 and a clinical isolate. The effects of anti‐Oma87 sera on A. baumannii adherence, internalization, and proliferation in HeLa cells were studied. The roles of microfilaments and microtubules in A. baumannii invasion were demonstrated by Actin disruption. Reduced bacterial population and biofilm formation were noted. The ability of A. baumannii to provoke autophagy through Oma87 induction leads to incomplete autophagy and potentially facilitates bacterial replication. Actin‐mediated uptake, attachment, and invasion demonstrated A. baumannii survival and multiplication within vacuoles in the host cell. The findings underscore the potential of Oma87 as a therapeutic intervention target in infections caused by A. baumannii. This comprehensive analysis contributes valuable information for understanding the virulence mechanisms of A. baumannii, potentially guiding future strategies to combat infections caused by this pathogen. [ABSTRACT FROM AUTHOR]

Background and Aim: Increased oxidative stress within the airways is associated to epithelial damage and amplification of inflammatory responses that in turn contribute to Chronic Obstructive Pulmonary Disease (COPD) progression. This study was aimed to identify whether a new formulation of N-acetylcisteine (NAC), carnitine, curcumin and B2 vitamin could counteract oxidative stress and downstream pro-inflammatory events promoted by cigarette smoke extract (CSE) exposure in primary bronchial epithelial cells (PBEC), both submerged/ undifferentiated (S-PBEC) and cultured at the air-liquid interface (ALI-PBEC). Methods: PBEC were exposed to CSE with/without the new formulation or NAC alone and ROS production, IL-8 and IL-6 gene expression and protein release were evaluated. Results: CSE increased ROS, IL-8 and IL-6 gene expression and protein release and the new formulation counteracted these effects. NAC alone was not effective on IL-8 and IL-6 release. The effects of a similar nutraceutical formulation were evaluated in COPD patients treated for six months. The results showed that the treatment reduced the concentration of IL-8 in nasal wash and improved quality of life. Conclusion: The tested formulation, exerting antioxidant and anti-inflammatory effects, can preserve airway epithelial homeostasis and improve clinical symptoms in COPD. [ABSTRACT FROM AUTHOR]

α -1 antitrypsin (A1AT) is a 52 kDa acute-phase glycoprotein belonging to the serine protease inhibitor superfamily (SERPIN). It is primarily synthesized by hepatocytes and to a lesser extent by monocytes, macrophages, intestinal epithelial cells, and bronchial epithelial cells. A1AT is encoded by SERPINA1 locus, also known as PI locus, highly polymorphic with at least 100 allelic variants described and responsible for different A1AT serum levels and function. A1AT inhibits a variety of serine proteinases, but its main target is represented by Neutrophil Elastase (NE). However, recent attention has been directed towards its immune-regulatory and homeostatic activities. A1AT exerts immune-regulatory effects on different cell types involved in innate and adaptive immunity. Additionally, it plays a role in metal and lipid metabolism, contributing to homeostasis. An adequate comprehension of these mechanisms could support the use of A1AT augmentation therapy in many disorders characterized by a chronic immune response. The aim of this review is to provide an up-to-date understanding of the molecular mechanisms and regulatory pathways responsible for immune-regulatory and homeostatic activities of A1AT. This knowledge aims to support the use of A1AT in therapeutic applications. Furthermore, the review summarizes the current state of knowledge regarding the application of A1AT in clinical and laboratory settings human and animal models. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer (CC) is the fourthmost commonmalignancy among women globally and serves as the main cause of cancer-related deaths among women in developing countries. The early symptoms of CC are often not apparent, with diagnoses typically made at advanced stages, which lead to poor clinical prognoses. In recent years, numerous studies have shown that there is a close relationship betweenmast cells (MCs) and tumor development. However, research on the role MCs played in CC is still very limited at that time. Thus, the study conducted a single-cellmulti-omics analysis on human CC cells, aiming to explore the mechanisms by which MCs interact with the tumor microenvironment in CC. The goal was to provide a scientific basis for the prevention, diagnosis, and treatment of CC, with the hope of improving patients’ prognoses and quality of life. Method: The present study acquired single-cell RNA sequencing data from ten CC tumor samples in the ArrayExpress database. Slingshot and AUCcell were utilized to infer and assess the differentiation trajectory and cell plasticity of MCs subpopulations. Differential expression analysis of MCs subpopulations in CC was performed, employing Gene Ontology, gene set enrichment analysis, and gene set variation analysis. CellChat software package was applied to predict cell communication between MCs subpopulations and CC cells. Cellular functional experiments validated the functionality of TNFRSF12A in HeLa and Caski cell lines. Additionally, a risk scoring model was constructed to evaluate the differences in clinical features, prognosis, immune infiltration, immune checkpoint, and functional enrichment across various risk scores. Copy number variation levels were computed using inference of copy number variations. Result: The obtained 93,524 high-quality cells were classified into ten cell types, including T_NK cells, endothelial cells, fibroblasts, smooth muscle cells, epithelial cells, B cells, plasma cells, MCs, neutrophils, and myeloid cells. Furthermore, a total of 1,392 MCs were subdivided into seven subpopulations: C0 CTSG+ MCs, C1 CALR+ MCs, C2 ALOX5+ MCs, C3 ANXA2+ MCs, C4 MGP+ MCs, C5 IL32+ MCs, and C6 ADGRL4+ MCs. Notably, the C2 subpopulation showed close associations with tumor-related MCs, with Slingshot results indicating that C2 subpopulation resided at the intermediate-to-late stage of differentiation, potentially representing a crucial transition point in the benign-to-malignant transformation of CC. CNVscore and bulk analysis results further confirmed the transforming state of the C2 subpopulation. CellChat analysis revealed TNFRSF12A as a key receptor involved in the actions of C2 ALOX5+ MCs. Moreover, in vitro experiments indicated that downregulating the TNFRSF12A gene may partially inhibit the development of CC. Additionally, a prognosis model and immune infiltration analysis based on the marker genes of the C2 subpopulation provided valuable guidance for patient prognosis and clinical intervention strategies. Conclusions: We first identified the transformative tumor-associated MCs subpopulation C2 ALOX5+ MCs within CC, which was at a critical stage of tumor differentiation and impacted the progression of CC. In vitro experiments confirmed the inhibitory effect of knocking down the TNFRSF12A gene on the development of CC. The prognostic model constructed based on the C2 ALOX5 +MCs subset demonstrated excellent predictive value. These findings offer a fresh perspective for clinical decision-making in CC. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) technology has emerged as a powerful tool for dissecting cellular heterogeneity and understanding the intricate biology of diseases, including cancer. Endometrial cancer (EC) stands out as the most prevalent gynecological malignancy in Europe and the second most diagnosed worldwide, yet its cellular complexity remains poorly understood. In this review, we explore the contributions of scRNA-seq studies to shed light on the tumor cells and cellular landscape of EC. We discuss the diverse tumoral and microenvironmental populations identified through scRNA-seq, highlighting the implications for understanding disease progression. Furthermore, we address potential limitations inherent in scRNA-seq studies, such as technical biases and sample size constraints, emphasizing the need for larger-scale research encompassing a broader spectrum of EC histological subtypes. Notably, a significant proportion of scRNA-seq analyses have focused on primary endometrioid carcinoma tumors, underscoring the need to incorporate additional histological and aggressive types to comprehensively capture the heterogeneity of EC. By critically evaluating the current state of scRNA-seq research in EC, this review underscores the importance of advancing towards more comprehensive studies to accelerate our understanding of this complex disease. [ABSTRACT FROM AUTHOR]

Background: Gonadotrophin-releasing hormone (GnRH) administration significantly decreases the pregnancy rate of recipient ewes after embryo transfer, possibly because GnRH affects endometrial epithelial cell function. Therefore, this study investigated the effect of GnRH on endometrial epithelial cells. Methods: Transcriptome sequencing was used to determine the regulatory effect of GnRH on the ewe endometrium, and the S100A4 gene, which showed altered transcription, was screened as a candidate regulator of this effect. Endometrial epithelial cells were further isolated, the S100A4 protein was immunoprecipitated, and host proteins that interacted with S100A4 were identified by mass spectrometry. We further verified the effects of S100A4 and GNAI2 on the proliferation of endometrial epithelial cells via overexpression/ knockdown experiments and subsequent CCK-8 and EdU assays. The effect of S100A4 deletion in endometrial cells on reproduction was verified in mice with S100A4 knockout. Results: Our results showed that S100A4 gene transcription in endometrial cells was significantly inhibited after GnRH administration. GNAI2 was identified as a downstream interacting protein of S100A4, and S100A4 was confirmed to activate the MAPK signaling pathway to promote cell proliferation by targeting GNAI2. Conclusion: GnRH can suppress the expression of S100A4 in the endometrium, consequently inhibiting the proliferation of endometrial cells through the S100A4/GNAI2/MAPK signaling pathway. These findings suggest a potential explanation for the limited efficacy of GnRH in promoting embryo implantation. [ABSTRACT FROM AUTHOR]

Introduction: The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route.Methods: Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rβ that constitute the bovIFN-λ3 receptor.Results: Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rβ subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rβ subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells.Conclusion: Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells. [ABSTRACT FROM AUTHOR]

Background: Worldwide, ulcerative colitis (UC) is becoming increasingly fast growing. Ginsenoside Rh2 has been reported to alleviate UC. However, the latent biological mechanism of Rh2 in the treatment of UC remains uncertain. In this study, the goal was to determine the therapeutic effect of Rh2 on dextran sulfate sodium (DSS)-induced UC. Methods: A DSS-induced UC mouse model was established and divided into 7 groups for Rh2 gavage and/or miR-125a-5p lentivirus injection (n = 10 per group). Colonic specimens were collected for phenotypic and pathological analysis. miR-125a-5p and specific protein 1 (SP1) expression, inflammation-related factors IL-6 and IL-10, and apoptosis were detected in mice. Human normal colon epithelial cell line NCM460 was treated with H2O2 and ferric chloride hexahydrate to construct an in vitro cell model of colitis and induce ferroptosis. Independent sample t-test was used to compare cell proliferation, cell entry, apoptosis, and oxidative stress between the two groups. One way analysis of variance combined with the least significant difference t test was used for comparison between groups. Multiple time points were compared by repeated measurement analysis of variance. Results: DSS-induced UC mice had significantly decreased body weight, increased disease activity index, decreased colon length, and decreased miR-125a-5p expression (all P < 0.05). In the DSS-induced mouse model, the expression of miR-125a-5p rebounded and ferroptosis was inhibited after Rh2 treatment (all P < 0.05). Inhibition of miR-125a-5p or upregulation of SP1 expression counteracted the protective effects of Rh2 on UC mice and ferroptosis cell models (all P < 0.05). Conclusions: Rh2 mitigated DSS-induced colitis in mice and restrained ferroptosis by targeting miR-125a-5p. Downregulating miR-125a-5p or elevating SP1 could counteract the protective impacts of Rh2 on ferroptotic cells. The findings convey that Rh2 has a latent application value in the treatment of UC. Highlights: Rh2 mitigates DSS-induced ferroptosis in mice; MiR-125a-5p represses DSS-induced ferroptosis in mice. Rh2 has protective impacts on ferroptotic cells. MiR-125a-5p targets SP1; Repression of miR-125a-5p or elevation of SP1 can counteract the protective effects of Rh2 on ferroptotic cells. [ABSTRACT FROM AUTHOR]

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70 kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health. [ABSTRACT FROM AUTHOR]

Objective: It has been shown that clonal mutations occur in hematopoietic stem cells with advancing age and increase the risk of death due to atherosclerotic vascular diseases, similarly to myeloproliferative neoplasms. Endothelial cells (ECs) and hematopoietic stem cells develop from common stem cells called hemangioblasts in the early embryonic period. However, the presence of hemangioblasts in the postnatal period is controversial. In this study, JAK2 gene variants were examined in patients with atherosclerotic carotid disease and without any hematological malignancies. Materials and Methods: Ten consecutive patients (8 men and 2 women) with symptomatic atherosclerotic carotid stenosis were included in this study. ECs (CD31+CD45-) were separated from tissue samples taken by carotid endarterectomy. JAK2 variants were examined in ECs, peripheral blood mononuclear cells, and oral epithelial cells of the patients with next-generation sequencing. Results: The median age of the patients was 74 (range: 58-80) years and the median body mass index value was 24.44 (range: 18.42- 30.85) kg/m2. Smoking history was present in 50%, hypertension in 80%, diabetes in 70%, and ischemic heart disease in 70% of the cases. The JAK2V617F mutation was detected in the peripheral blood mononuclear cells of 3 of the 10 patients, and 2 patients also had the JAK2V617F mutation in their ECs. The JAK2V617F mutation was not found in the oral epithelial cells of any of the patients. Conclusion: In this study, for the first time in the literature, we showed that the JAK2V617F mutation was found somatically in both peripheral blood cells and ECs in patients with atherosclerosis. This finding may support that ECs and hematopoietic cells originate from a common clone or that somatic mutations can be transmitted to ECs by other mechanisms. Examining the molecular and functional changes caused by the JAK2V617F mutation in ECs may help open a new avenue for treating atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: To investigate the involvement of LINC02605 in the progression of paediatric Mycoplasma pneumoniae pneumonia (MPP). Methods: One hundred and thirty‐two children with MPP (90 simple MPP and 42 MPP + diarrhoea) were enrolled, and their plasma was collected for detection of LINC026505 expression. CCK‐8 kit and commercial apoptosis kit were introduced to determine cell growth and apoptosis. In silico prediction analyses were conducted to predict the downstream miRNA for LINC02605, following verification by dual luciferase reporter assay. The lipid‐associated membrane proteins (LAMPs) were used to treat A549 and Coca‐2 cells. Results: LIN02605 was highly expressed in the MPP, especially in MPP complicated with diarrhoea. LINC02605 downregulation in A549 cells correlated with significant suppression of cell apoptosis rate and growth inhibition rate in vitro. Introduction of miR‐539‐5p inhibited luciferase activity in a reporter system containing the wild‐type LINC02605 and CXCL1. After stimulation with LAMPs, overexpression of LINC02605 and CXCL1 and inhibition of miR‐539‐5p were found. miR‐539‐5p and CXCL1 knockdown resulted in a rescue effect on the LINC02605‐inhibited cell apoptosis. LAMPs induced IL‐1β in intestinal epithelial cells and IL‐1β induced LINC02605 expression in A549 cells. Conclusions: LINC02605 was upregulated in MPP and miR‐539‐5p was a target for LINC02605. LINC02605 may be involved in the crosstalk between the gastrointestinal tract and the respiratory tract. [ABSTRACT FROM AUTHOR]

Objective: PM2.5 is closed linked to asthma exacerbation. The Notch1 pathway acts as an important pathway, ultimately inducing T-helper cells that express GATA3 and its corresponding Th2 cytokines. The regulatory effects of miR-139-5p on the Notch1 pathway have been indicated in cancer. However, studies on miR-139-5p have not applied asthma-related models. The role of miR-139-5p and its regulatory effects on the Notch1-GATA3 pathway in asthma exacerbation induced by acute PM2.5 exposure has not been elucidated. We hypothesize that acute PM2.5 exposure induces asthma exacerbation by regulating the expression of miR-139-5p and activating the Notch1-GATA3 pathway. Methods: We first employed Diseased Human Bronchial Epithelial Cells-Asthma cells to establish an in vitro model of acute exposure to PM2.5, and explored the relationship between the different concentrations and durations of acute PM2.5 exposure and the activation of Notch1-GATA3 pathway. We investigated the protein and mRNA expression changes of Notch1, upstream Jagged1, downstream GATA3, as well as the regulatory effect of miR-139-5p involved in it. Results: The miR-139-5p expression increased within 24 h of PM2.5 exposure. However, if PM2.5 exposure was sustained, miR-139-5p expression turned to decrease, accompanied by upregulations of the mRNA and protein expression of Notch1-GATA3 pathway. Overexpression of miR-139-5p blocked Notch1-GATA3 pathway activation induced by acute PM2.5 exposure. Conclusion: Acute PM2.5 exposure can activate Notch1-GATA3 pathway in asthma bronchial epithelial cells model, which might be involved in PM2.5-induced asthma exacerbation. miR-139-5p has a potential protective role of inhibiting PM2.5-induced asthma airway inflammation by targeting Notch1. [ABSTRACT FROM AUTHOR]

Development of respiratory tissue constructs is challenging due to the complex structure of native respiratory tissue and the unique biomechanical conditions induced by breathing. While studies have shown that the inclusion of biomechanical stimulus mimicking physiological conditions greatly benefits the development of engineered tissues, to our knowledge no studies investigating the influence of biomechanical stimulus on the development of respiratory tissue models produced through three-dimensional (3D) bioprinting have been reported. This paper presents a study on the utilization of a novel breath-mimicking ventilated incubator to impart biomechanical stimulus during the culture of 3D respiratory bioprinted constructs. Constructs were bioprinted using an alginate/collagen hydrogel containing human primary pulmonary fibroblasts with further seeding of human primary bronchial epithelial cells. Biomechanical stimulus was then applied via a novel ventilated incubator capable of mimicking the pressure and airflow conditions of multiple breathing conditions: standard incubation, shallow breathing, normal breathing, and heavy breathing, over a two-week time period. At time points between 1 and 14 days, constructs were characterized in terms of mechanical properties, cell proliferation, and morphology. The results illustrated that incubation conditions mimicking normal and heavy breathing led to greater and more continuous cell proliferation and further indicated a more physiologically relevant respiratory tissue model. [ABSTRACT FROM AUTHOR]

Elevated levels of NEFA caused by negative energy balance in transition cows induce cellular dyshomeostasis. Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) can maintain cellular homeostasis and act as a critical regulator of stress responses besides functioning in the ubiquitin-like system. The objective of this study was to elucidate the UFL1 working mechanism on promoting cellular adaptations in bovine mammary epithelial cells (BMECs) in response to NEFA challenge, with an emphasis on the ER and mitochondrial function. The results showed that exogenous NEFA and UFL1 depletion resulted in the disorder of ER and mitochondrial homeostasis and the damage of BMEC integrity, overexpression of UFL1 effectively alleviated the NEFA-induced cellular dyshomeostasis. Mechanistically, our study found that UFL1 had a strong interaction with IRE1α and could modulate the IRE1α/XBP1 pathway of unfolded protein response in NEFA-stimulated BMECs, thereby contributing to the modulation of cellular homeostasis. These findings imply that targeting UFL1 may be a therapeutic alternative to relieve NEB-induced metabolic changes in perinatal dairy cows. [Display omitted] • NEFA and UFL1 depletion resulted in the disorder of ER and mitochondrial homeostasis. • UFL1 overexpression effectively alleviated the NEFA-induced cellular dyshomeostasis. • UFL1 could interact with IRE1α and modulate the IRE1α/XBP1 pathway of UPR in BMECs. • IRE1α functioned as a positive regulator in MAMs homeostasis of NEFA-induced BMECs. [ABSTRACT FROM AUTHOR]

There are several in vitro systems that enable evaluation of the absorption direction, but there are few quantitative systems that enable easy evaluation of the excretion direction. Enteroids, organoids derived from intestine, have been frozen and passaged for various research. But it is not clear how the freezing and passaging affect the expression and function of transporters. We investigated the effects of passage and cryopreservation of enteroids. We focused on P-gp (P-glycoprotein) and compared the transfer rates of rhodamine 123 (Rh123) into the lumen of enteroids with and without a P-gp inhibitor. mRNA expression levels did not change significantly before and after passage and cryopreservation. Accumulation of Rh123 in the lumen of enteroids was observed. With some P-gp inhibitors, excretion of Rh123 into the lumen of enteroids was inhibited and the nonexcreted Rh123 accumulated in enteroids epithelial cells. The transfer rate of Rh123 into the lumen of enteroids with a P-gp inhibitor was significantly decreased compared to that of without a P-gp inhibitor. Before and after passage and cryopreservation, the transfer rate was almost the same as that of primary cultured enteroids. We succeeded in easily evaluating whether a component is a substrate of P-gp using enteroids. [Display omitted] • mRNA expression level of efflux intestinal transporter was not changed before and after passage and number of culture days. • This method allows semi-quantitative evaluation of efflux transporters function regardless of the shapes of enteroids. • Our method enables to easily evaluate whether a component is a substrate of P-gp using enteroids. [ABSTRACT FROM AUTHOR]

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size‐based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan‐A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan‐A‐positive, 3/9 PNL2‐positive, and 8/9 S100‐positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative‐stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p <.05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours. [ABSTRACT FROM AUTHOR]

Severe burn patients frequently suffer from 1,25-Dihydroxyvitamin D3 (1,25-[OH]2-D3) deficiency. In this study, we investigated the effect of 1,25-[OH]2-D3 on early mortality post severe burn and potential underlying mechanisms. Our results indicate that 1,25-[OH]2-D3 significantly reduced early mortality in mice post severe burn injury. A decrease in serum lipopolysaccharide levels and an increase in serum superoxide dismutase activity were found after administration of 1,25-[OH]2-D3. Furthermore, 1,25-[OH]2-D3 demonstrated protective effects on both intestinal and lung histology and ameliorated lung inflammation. Its anti-inflammatory effect was further confirmed in airway epithelial cells. In conclusion, our study provides evidence that 1,25-[OH]2-D3 has a significant impact on the reduction of early mortality post severe burn injury, possibly through its ability to alleviate endotoxemia, oxidative stress, and inflammation. Our findings highlight the potential of 1,25-[OH]2-D3 to protect the intestinal mucosal barrier in the early stage following major burn injury and opens up new avenues for clinical application of 1,25-[OH]2-D3 in burn patients. [Display omitted] ● 1,25-dihydroxyvitamin D3 significantly reduced early mortality in mice post severe burn injury.1,25-[OH]2-D3 markedly reduced early mortality in mice post severe burn injury. ● A decrease in serum lipopolysaccharide levels and an increase in serum superoxide dismutase activity were found after administration of 1,25-dihydroxyvitamin D3. 1,25-[OH]2-D3 markedly reduced early mortality in mice post severe burn injury. ● 1,25-dihydroxyvitamin D3 demonstrated protective effects on both intestinal and lung histology and ameliorated lung inflammation. 1,25-[OH]2-D3 had protective effects on both intestinal and lung histology and ameliorated lung inflammation. [ABSTRACT FROM AUTHOR]

This research was designed to elucidate the anti-inflammatory impacts of liquiritin on lipopolysaccharide (LPS)-activated human corneal epithelial cells (HCECs). The Cell Counting kit-8 (CCK-8) assay was adopted to assess cell viability. The enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α. Transcriptome analysis was conducted to identify the genes that exhibited differential expression between different treatment. The model group included cells treated with LPS (10 µg/mL), the treatment group comprised cells treated with liquiritin (80 µM) and LPS (10 µg/mL), and the control group consisted of untreated cells. To further validate the expression levels of the selected genes, including CSF2, CXCL1, CXCL2, CXCL8, IL1A, IL1B, IL24, IL6, and LTB, quantitative real-time PCR was performed. The expression of proteins related to the Akt/NF-κB signaling pathway was assessed through western blot analysis. NF-κB nuclear translocation was evaluated through immunofluorescence staining. The secretion of IL-6, IL-8, and TNF-α in LPS-induced HCECs was significantly downregulated by liquiritin. Based on the transcriptome analysis, the mRNA expression of pro-inflammatory cytokines, namely IL-6, IL-8, IL-1β, IL-24, TNF-α, and IL-1α was overproduced by LPS stimulation, and suppressed after liquiritin treatment. Furthermore, the Western blot results revealed a remarkable reduction in the phosphorylation degrees of NF-κB p65, IκB, and Akt upon treatment with liquiritin. Additionally, immunofluorescence analysis confirmed liquiritin's inhibition of LPS-induced p65 nuclear translocation. Collectively, these findings imply that liquiritin suppresses the expression of proinflammatory cytokines, and the anti-inflammatory impacts of liquiritin may be caused by its repression of the Akt/NF-κB signaling pathway in LPS-induced HCECs. These data indicate that liquiritin could provide a potential therapeutic application for inflammation-associated corneal diseases. [ABSTRACT FROM AUTHOR]

To explore the correlation of endoplasmic reticulum stress (ERS) and oxidative stress (OS), and the protective effect of Sestrin2 (SESN2) on human lens epithelial cells (HLECs). Tunicamycin (TM) was used to induce ERS in HLECs. 4-Phenylbutyric acid (4-PBA) was used to inhibit ERS. Eupatilin applied to HLECs as SESN2 agonist. SESN2 expression was knocked down via si-RNA in HLECs. The morphological changes of HLECs were observed by microscope. ER-tracker to evaluate ERS, ROS production assay to measure ROS, flow cytometry to calculate cell apoptosis rate. Immunofluorescence to observe Nrf2 translocation, and effects of TM or EUP on SESN2. Western blot and qPCR were used to evaluate the expression of GRP78, PERK, ATF4, CHOP, Nrf2, and SESN2 expression in HLECs with different treatment groups. ERS can elevate the expression of ROS and Nrf2 to induce OS. Upregulation of SESN2 was observed in ERS-mediate OS. Overexpression of SESN2 can reduce the overexpression of ERS-related protein GRP78, PERK, ATF4, proapoptotic protein CHOP, OS-related protein Nrf2, as well as ROS, and alleviate ERS injury at the same time. Whereas knockdown of SESN2 can upregulate the expression of GRP78, PERK, ATF4, CHOP, Nrf2, ROS, and deteriorate ERS damage. ERS can induce OS, they form a vicious cycle to induce apoptosis in HLECs, which may contribute to cataract formation. SESN2 could protect HLECs against the apoptosis by regulating the vicious cycle between ERS and OS. [ABSTRACT FROM AUTHOR]

Workers in the iron casting industries are exposed to various chemicals, especially graphite in furnace process. This study aims to investigate the toxic effects of graphite particles on human lung cells. Particle characteristics were confirmed by electron microscope and light scattering. Cell viability and oxidative stress markers were measured. The expression of oxidative repair genes, namely OGG1, MTH1, and ITPA, was evaluated. The average particle size was determined to be 172.1 ± 11.96 nm. The median inhibition concentration (IC50) of graphite particles was 46.75 µg/mL. Notably, 25 and 50 µg/mL concentrations resulted in significant GSH depletion and MDA production. The high concentration of graphite particles (200 µg/mL) led to OGG1 suppression and increased MTH1 expression. Based on these findings, graphite exposure may induce toxicity in human lung cells by increasing oxidative stress. Further research is necessary to fully understand the mechanisms underlying graphite toxicity. [ABSTRACT FROM AUTHOR]

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild‐type (WT) and αF2M mice. Patch‐clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride‐sensitive short‐circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single‐channel activity was significantly lower in kidney tubules from Na+‐restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+‐restricted αF2Mvs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. Key points: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole‐animal physiology remain to be addressed.We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice).We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction.ENaC function at the organ level was preserved in salt‐restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis.These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo. [ABSTRACT FROM AUTHOR]

Intraductal papillary mucinous neoplasms (IPMNs) are potential precursor lesions of pancreatic cancer. We assessed the efficacy of screening for KRAS proto‐oncogene, GTPase (KRAS), and GNAS complex locus (GNAS) mutations in cell‐free DNA (cfDNA)—using digital droplet polymerase chain reaction (ddPCR) and circulating epithelial cell (CEC) detection—as biomarkers for risk stratification in IPMN patients. We prospectively collected plasma samples from 25 resected patients at risk of malignant progression, and 23 under clinical surveillance. Our findings revealed KRAS mutations in 10.4% and GNAS mutations in 18.8% of the overall cohort. Among resected IPMN patients, KRAS and GNAS mutation detection rates were 16.0% and 32.0%, respectively, whereas both rates were 4.0% in conservatively managed IPMN. GNAS mutations in cfDNA were significantly more prevalent in resected IPMN (P = 0.024) compared with IPMN under surveillance. No CECs were detected. The absence of KRAS and GNAS mutations could be a reliable marker for branch duct IPMN without worrisome features. The emergence of GNAS mutations could prompt enhanced imaging surveillance. Neither the presence of established worrisome features nor GNAS or KRAS mutations appear effective in identifying high‐grade dysplasia among IPMN patients. [ABSTRACT FROM AUTHOR]

Aging is a gradual, inevitable physiologic process. The organ aging is related to the persistence of chronic inflammation, but the understanding of inflammatory state during renal aging is lacking currently. Single-cell transcriptome sequencing was performed on aging mouse kidney to reveal the molecular phenotype and composition changes of different cell types. In the early stage of aging, immune cells such as T, B cells and mononuclear macrophages increased in kidney. The molecular state of T cells in aging kidney changed and polarized. Among them, we identified a group of GZMK + CD8 + T cells with high expression of Eomes , Pdcd1 and Ifng and a group of Il17a + T cells with high expression of Il17a and Il23r. Moreover, the cytokines and inflammations can aggravate tissue damage eventually. Furthermore, we found the interaction between different types of epithelial cells and T cells increased during the renal aging. These results identify the changes of T cells in the early stage of aging kidney and suggest that GZMK+CD8+ T cells might be a potential target to ameliorate age-associated dysfunctions of kidney(Graphical Abstract). [Display omitted] • Kidney undergoes the most severe structural and functional deterioration with aging. • Organ aging is related to chronic inflammation, and persist inflammation hinders intrinsic cell repair. • Immune cells increased in the early stage of kidney aging. • The molecular state of T cells in aging kidney changed and polarized. • The crosstalk between different types of epithelial cells and immune cells increased. [ABSTRACT FROM AUTHOR]

Epithelial cell networks imply a packing geometry characterized by various cell shapes and distributions in terms of number of cell neighbors and areas. Despite such simple characteristics describing cell sheets, the formation of bubble‐like cells during the morphogenesis of epithelial tissues remains poorly understood. This study proposes a topological mathematical model of morphogenesis in a squamous epithelial. We introduce a new potential that takes into account not only the elasticity of cell perimeter and area but also the elasticity of their internal angles. Additionally, we incorporate an integral equation for chemical signaling, allowing us to consider chemo‐mechanical cell interactions. In addition to the listed factors, the model takes into account essential processes in real epithelial, such as cell proliferation and intercalation. The presented mathematical model has yielded novel insights into the packing of epithelial sheets. It has been found that there are two main states: one consists of cells of the same size, and the other consists of "bubble" cells. An example is provided of the possibility of accounting for chemo‐mechanical interactions in a multicellular environment. The introduction of a parameter determining the flexibility of cell shapes enables the modeling of more complex cell behaviors, such as considering change of cell phenotype. The developed mathematical model of morphogenesis of squamous epithelium allows progress in understanding the processes of formation of cell networks. The results obtained from mathematical modeling are of significant importance for understanding the mechanisms of morphogenesis and development of epithelial tissues. Additionally, the obtained results can be applied in developing methods to influence morphogenetic processes in medical applications. [ABSTRACT FROM AUTHOR]