Search

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.

Primary cultures of proximal tubule cells are widely used to model the behavior of kidney epithelial cells in vitro. However, de-differentiation of primary cells upon culture has been observed and appreciated for decades, yet the mechanisms driving this phenomenon remain poorly understood. This confounds the interpretation of experiments using primary kidney epithelial cells and prevents their use to engineer functional kidney tissue ex vivo. In this report, we measure the dynamics of cell-state transformations in early primary culture of mouse proximal tubules to identify key pathways and processes that correlate with and may drive de-differentiation. Our data show that the loss of proximal-tubule-specific genes is rapid, uniform, and sustained even after confluent, polarized epithelial monolayers develop. This de-differentiation occurs uniformly across many common culture condition variations. Changes in early culture were strongly associated with the loss of HNF4A. Exogenous re-expression of HNF4A can promote expression of a subset of proximal tubule genes in a de-differentiated proximal tubule cell line. Using genetically labeled proximal tubule cells, we show that selective pressures very early in culture influence which cells grow to confluence. Together, these data indicate that the loss of in vivo function in proximal tubule cultures occurs very early and suggest that the sustained loss of HNF4A is a key regulatory event mediating this change. [ABSTRACT FROM AUTHOR]

Skin-resident regulatory T cells (Tregs) play an irreplaceable role in orchestrating cutaneous immune homeostasis and repair, including the promotion of hair regeneration via the Notch signaling ligand Jagged-1 (Jag1). While skin Tregs are indispensable for facilitating tissue repair post-wounding, it remains unknown if Jag1-expressing skin Tregs impact wound healing. Using a tamoxifen inducible Foxp3creERT2Jag1fl/fl model, we show that loss of functional Jag1 in Tregs significantly delays the rate of full-thickness wound closure. Unlike in hair regeneration, skin Tregs do not utilize Jag1 to impact epithelial stem cells during wound healing. Instead, mice with Treg-specific Jag1 ablation exhibit a significant reduction in Ly6G + neutrophil accumulation at the wound site. However, during both homeostasis and wound healing, the loss of Jag1 in Tregs does not impact the overall abundance or activation profile of immune cell targets in the skin, such as CD4+ and CD8+ T cells, or pro-inflammatory macrophages. This collectively suggests that skin Tregs may utilize Jag1-Notch signalling to co-ordinate innate cell recruitment under conditions of injury but not homeostasis. Overall, our study demonstrates the importance of Jag1 expression in Tregs to facilitate adequate wound repair in the skin. [ABSTRACT FROM AUTHOR]

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female. [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]

Bronchopulmonary dysplasia (BPD) is the most common sequela of prematurity and is characterized by alveolar simplification and lung angiogenesis failure. The aim of this study was to explore the immune signatures of BPD. Differentially expressed gene analysis and immune infiltration analysis were conducted to identify key immune cell types and related genes by using the mRNA-seq dataset GSE25286. The expression patterns of key genes were validated in the scRNA-seq dataset GSE209664 and in experiments. The cell–cell crosstalk of key immune cells was explored with CellChat. We found that differentially expressed genes between BPD mice and controls were mostly enriched in leukocyte migration and M1 macrophages were highly enriched in BPD lungs. Hub genes (Cybb, Papss2, F7 and Fpr2) were validated at the single-cell level, among which the downregulation of Cybb was most closely related to macrophage infiltration. The reduced mRNA and protein levels of Cybb were further validated in animal experiments. Colocalization analysis of Cybb and macrophage markers demonstrated a significant decrease of Cybb in M1 macrophages. Cell–cell crosstalk found that alveolar epithelial cells interacted actively with macrophages through MIF-(CD74 + CD44) signalling. In conclusion, M1 macrophages played important roles in promoting BPD-like lung injury, which was correlated with a specific reduction of Cybb in macrophages and the potential activation of MIF signalling. [ABSTRACT FROM AUTHOR]

Tibetan pigs are a unique swine strain adapted to the hypoxic environment of the plateau regions in China. The unique mechanisms underlying the adaption by Tibetan pigs, however, are still elusive. Only few studies have investigated hypoxia-associated molecular regulation in the lung tissues of animals living in the plateau region of China. Our previous study reported that ssc-miR-101-3p expression significantly differed in the lung tissues of Tibetan pigs at different altitudes, suggesting that ssc-miR-101-3p plays an important role in the adaptation of Tibetan pigs to high altitude. To understand the underlying molecular mechanism, in this study, the target genes of ssc-miR-101-3p and their functions were analyzed via various methods including qRT-PCR and GO and KEGG pathway enrichment analyses. The action of ssc-miR-101-3p was investigated by culturing alveolar type-II epithelial cells (ATII) of Tibetan pigs under hypoxic conditions and transfecting ATII cells with vectors overexpressing or inhibiting ssc-miR-101-3p. Overexpression of ssc-miR-101-3p significantly increased the proliferation of ATII cells and decreased the expression of inflammatory and apoptotic factors. The target genes of ssc-miR-101-3p were significantly enriched in FOXO and PI3K-AKT signaling pathways required to mitigate lung injury. Further, FOXO3 was identified as a direct target of ssc-miR-101-3p. Interestingly, ssc-miR-101-3p overexpression reversed the damaging effect of FOXO3 in the ATII cells. In conclusion, ssc-miR-101-3p targeting FOXO3 could inhibit hypoxia-induced apoptosis and inflammatory response in ATII cells of Tibetan pigs. These results provided new insights into the molecular mechanisms elucidating the response of lung tissues of Tibetan pigs to hypoxic stress. [ABSTRACT FROM AUTHOR]

Urate nephropathy, a common complication of hyperuricemia, has garnered increasing attention worldwide. However, the exact pathogenesis of this condition remains unclear. Currently, inflammation is widely accepted as the key factor in urate nephropathy. Therefore, the aim of this study was to elucidate the interaction of lincRNA-p21/AIF-1/CMPK2/NLRP3 via exosomes in urate nephropathy. This study evaluated the effect of lincRNA-p21/AIF-1/CMPK2/NLRP3 using clinical data collected from patients with urate nephropathy and human renal tubular epithelial cells (HK2) cultured with different concentrations of urate. In clinical research section, the level of lincRNA-p21/AIF-1 in exosomes of urine in patients with hyperuricemia or urate nephropathy was found to be increased, particularly in patients with urate nephropathy. In vitro study section, the level of exosomes, inflammation, autophagy, and apoptosis was increased in HK2 cells induced by urate. Additionally, the expression of lincRNA-p21, AIF-1, CMPK2, and NLRP3 was upregulated in exosomes and HK2 cells. Furthermore, manipulating the activity of lincRNA-p21, AIF-1, CMPK2, and NLRP3 through overexpression or interference vectors regulated the level of inflammation, autophagy, and apoptosis in HK2 cells. In conclusion, the pathway of lincRNA-p21/AIF-1/CMPK2/NLRP3 contributed to inflammation, autophagy, and apoptosis of human renal tubular epithelial cell induced by urate via exosomes. Additionally, the specific exosomes in urine might serve as novel biomarkers for urate nephropathy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cells are effective against hematological cancers, but are less effective against solid tumors such as non-small cell lung cancer (NSCLC). One of the reasons is that only a few cell surface targets specific for NSCLC cells have been identified. Here, we report that CD98 heavy chain (hc) protein is overexpressed on the surface of NSCLC cells and is a potential target for CAR T cells against NSCLC. Screening of over 10,000 mAb clones raised against NSCLC cell lines showed that mAb H2A011 bound to NSCLC cells but not normal lung epithelial cells. H2A011 recognized CD98hc. Although CAR T cells derived from H2A011 could not be established presumably due to the high level of H2A011 reactivity in activated T cells, those derived from the anti-CD98hc mAb R8H283, which had been shown to lack reactivity with CD98hc glycoforms expressed on normal hematopoietic cells and some normal tissues, were successfully developed. R8H283 specifically reacted with NSCLC cells in six of 15 patients. R8H283-derived CAR T cells exerted significant anti-tumor effects in a xenograft NSCLC model in vivo. These results suggest that R8H283 CAR T cells may become a new therapeutic tool for NSCLC, although careful testing for off-tumor reactivity should be performed in the future. [ABSTRACT FROM AUTHOR]

Epithelial–Mesenchymal Transition (EMT) of retinal pigment epithelial (RPE) cells is recognized as pivotal in various retinal diseases. Previous studies have suggested a reciprocal regulation between reactive oxygen species (ROS) and EMT, though the involvement of peroxidized lipids or the effects of reducing them has remained unclear. The present study disclosed that EMT of ARPE-19 cells induced by TGF-β2 and TNF-α involves increased lipid peroxidation, and Ferrostatin-1 (Fer-1), a lipophilic antioxidative agent, successfully inhibited the increase in lipid peroxidation. Fer-1 suppressed the formation of EMT-associated fibrotic deposits, while EMT induction or Fer-1 treatment did not influence the cell viability or proliferation. Functionally, Fer-1 impeded EMT-driven cell migration and reduction in transepithelial electrical resistance. It demonstrated regulatory prowess by downregulating the mesenchymal marker fibronectin, upregulating the epithelial marker ZO-1, and inhibiting the EMT-associated transcriptional factor ZEB1. Additionally, VEGF, a major pathogenic cytokine in various retinal diseases, is also upregulated during EMT, and Fer-1 significantly mitigated the effect. The present study disclosed the involvement of lipid peroxidation in EMT of RPE cells, and suggests the suppression of lipid peroxidation may be a potential therapeutic target in retinal diseases in which EMT is implicated. [ABSTRACT FROM AUTHOR]

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases. [ABSTRACT FROM AUTHOR]

Sepsis-induced acute lung injury (SALI) poses a significant threat with high incidence and mortality rates. Ginsenoside Rg1 (GRg1), derived from Ginseng in traditional Chinese medicine, has been found to reduce inflammation and protect lung epithelial cells against tissue damage. However, the specific roles and mechanisms by which GRg1 mitigates SALI have yet to be fully elucidated. In this context, we employed a relevant SALI mouse model, alongside network pharmacology, molecular docking, and molecular dynamics simulation to pinpoint GRg1's action targets, complemented by in vitro assays to explore the underlying mechanisms. Our research shows that GRg1 alleviates CLP-induced SALI, decreasing lung tissue damage and levels of serum proinflammatory factor IL-6, TNF-α, and IL-1β, also enhancing the survival rate of CLP mice. A total of 116 common targets between GRg1 and ALI, with specific core targets including AKT1, VEGFA, SRC, IGF1, ESR1, STAT3, and ALB. Further in vitro experiments assessed GRg1's intervention effects on MLE-12 cells exposed to LPS, with qRT-PCR analysis and molecular dynamics simulations confirming AKT1 as the key target with the favorable binding activity for GRg1. Western blot results indicated that GRg1 increased the Bcl-2/Bax protein expression ratio to reduce apoptosis and decreased the high expression of cleaved caspase-3 in LPS-induced MLE-12 cells. More results showed significant increases in the phosphorylation of PI3K and AKT1. Flow cytometric analysis using PI and Annexin-V assays further verified that GRg1 decreased the apoptosis rate in LPS-stimulated MLE-12 cells (from 14.85 to 6.54%, p < 0.05). The employment of the AKT1 inhibitor LY294002 confirmed these trends, indicating that AKT1's inhibition negates GRg1's protective effects on LPS-stimulated MLE-12 cells. In conclusion, our research highlights GRg1's potential as an effective adjunct therapy for SALI, primarily by inhibiting apoptosis in alveolar epithelial cells and reducing pro-inflammatory cytokine secretion, thus significantly enhancing the survival rates of CLP mice. These beneficial effects are mediated through targeting AKT1 and activating the PI3K-AKT pathway. [ABSTRACT FROM AUTHOR]

This study explores the application of the RIP3-caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay's capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay's adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research. [ABSTRACT FROM AUTHOR]

This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson's staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1's 3′-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions. [ABSTRACT FROM AUTHOR]

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more. [ABSTRACT FROM AUTHOR]

Among the myriad of nanoparticles, silica nanoparticles (SiO2NPs) have gained significant attention since they are extensively produced and used across several kinds of industries. Because of its widespread usage, there has been increasing concern about the potential health effects. This study aims to evaluate the effects of SiO2NPs on Interleukin-6 (IL-6) gene expression in human lung epithelial cell lines (A549). In this study, A549 cells were exposed to SiO2NPs at concentrations of 0, 1, 10, 50, 100, and 200 µg/mL for 24 and 48 h. The IL-6 gene expression was assessed using Real-Time RT-PCR. Additionally, the impact of SiO2NPs on the viability of A549 cells was determined by MTT assay. Statistical analysis was performed using GraphPad Prism software 8.0. MTT assay results indicated a concentration-dependent impact on cell survival. After 24 h, survival decreased from 80 to 68% (1–100 µg/mL), rising to 77% at higher concentrations. After 48 h, survival dropped from 97 to 80%, decreasing to 90% at higher concentrations. RT-PCR showed a dose–response relationship in cellular toxicity up to 10 µg/mL. At higher concentrations, there was increased IL-6 gene expression, mitigating SiO2NP-induced cytotoxic effects. The study shows that the viability and proliferation of A549 cells are impacted by different SiO2NPs concentrations. There may be a potential correlation between IL-6 gene expression reduction and a mechanism linked to cellular toxicity. However, at higher concentrations, an unknown mechanism increases IL-6 gene expression, reducing SiO2NPs' cytotoxic effects. These effects are concentration-dependent and not influenced by exposure times. Further investigation is recommended to determine this mechanism's nature and implications, particularly in cancer research. [ABSTRACT FROM AUTHOR]

Vaginitis, a prevalent gynecological condition in women, is mainly caused by an imbalance in the vaginal micro-ecology. The two most common types of vaginitis are vaginal bacteriosis and vulvovaginal candidiasis, triggered by the virulent Gardnerella vaginalis and Candida albicans, respectively. In this study, a strain capable of inhibiting G. vaginalis and C. albicans was screened from vaginal secretions and identified as Lactobacillus gasseri based on 16S rRNA sequences. The strain, named L. gasseri VHProbi E09, could inhibit the growth of G. vaginalis and C. albicans under co-culture conditions by 99.07% ± 0.26% and 99.95% ± 0.01%, respectively. In addition, it could significantly inhibit the adhesion of these pathogens to vaginal epithelial cells. The strain further showed the ability to inhibit the enteropathogenic bacteria Escherichia coli and Salmonella enteritidis, to tolerate artificial gastric and intestinal fluids and to adhere to intestinal Caco-2 cells. These results suggest that L. gasseri VHProbi E09 holds promise for clinical trials and animal studies whether administered orally or directly into the vagina. Whole-genome analysis also revealed a genome consisting of 1752 genes for L. gasseri VHProbi E09, with subsequent analyses identifying seven genes related to adhesion and three genes related to bacteriocins. These adhesion- and bacteriocin-related genes provide a theoretical basis for understanding the mechanism of bacterial inhibition of the strain. The research conducted in this study suggests that L. gasseri VHProbi E09 may be considered as a potential probiotic, and further research can delve deeper into its efficacy as an agent which can restore a healthy vaginal ecosystem. [ABSTRACT FROM AUTHOR]

Cigarette smoke (CS) is one of the leading causes of pulmonary diseases and can induce lung secretome alteration. CS exposure-induced damages to human pulmonary epithelial cells and microvascular endothelial cells have been extensively demonstrated; however, the effects of the secretome of lung epithelial cells exposed to CS extracts (CSE) on lung microvascular endothelial cells are not fully understood. In this study, we aimed to determine the effects of the secretome of lung epithelial cells exposed to CSE on lung microvascular endothelial cells. Human lung epithelial cells, A549, were exposed to CSE, and the secretome was collected. Human lung microvascular endothelial cells, HULEC-5a, were used to evaluate the effect of the secretome of A549 exposed to CSE. Secretome profile, endothelial cell death, inflammation, and permeability markers were determined. CSE altered the secretome expression of A549 cells, and secretome derived from CSE-exposed A549 cells caused respiratory endothelial cell death, inflammation, and moderately enhanced endothelial permeability. This study demonstrates the potential role of cellular interaction between endothelial and epithelial cells during exposure to CSE and provides novel therapeutic targets or beneficial biomarkers using secretome analysis for CSE-related respiratory diseases. [ABSTRACT FROM AUTHOR]

Human milk (HM) components affect immune cell toll-like receptor 4 (TLR4) signaling. However, studies examining the immunomodulatory impacts of HM on TLR4 signaling in intestinal epithelial cells (IECs) are limited. This study utilized both a TLR4 reporter cell line and a Caco-2 IEC model to examine the effects of HM on lipopolysaccharide (LPS)-induced TLR4 activation and cytokine responses, respectively. Additionally, we performed fast protein liquid chromatography and mass spectrometry to identify a HM component that contributes to the effect of HM on LPS/TLR4 signaling. HM enhances LPS-induced TLR4 signaling as well as LPS-induced IEC gene expression of pro-inflammatory cytokines and negative regulators of NF-κB. Human serum albumin (HSA) present in HM contributes to these effects. HSA within HM synergizes with LPS to induce IEC gene expression of pro-inflammatory cytokines and negative regulators of NF-κB. Altogether, this study provides mechanistic evidence behind the immunomodulatory function of HM on IECs, which may contribute to an enhanced immune response in breast-fed neonates. [ABSTRACT FROM AUTHOR]

Abstract Epithelial tissue forms and maintains a critical barrier function in the body. A novel culture design aimed at promoting uniform maturation of epithelial cells using liquid materials is described. Culturing Madin–Darby canine kidney (MDCK) cells at the liquid–liquid interface yielded reduced migration and stimulated active cell growth. Similar to solid–liquid interfaces, cells cultured on a fibronectin-coated liquid–liquid interface exhibited active migration and growth, ultimately reaching a confluent state. These cells exhibited reduced stress fiber formation and adopted a cobblestone-like shape, which led to their even distribution in the culture vessel. To inhibit stress fiber formation and apoptosis, the exposure of cells on liquid–liquid interfaces to Y27632, a specific inhibitor of the Rho-associated protein kinase (ROCK), facilitated tight junction formation (frequency of ZO-2-positive cells, F Z = 0.73). In Y27632-exposed cells on the liquid–liquid interface, the value obtained by subtracting the standard deviation of the ratio of nucleus densities in each region that compartmentalized a culture vessel from 1, denoted as H LN, was 0.93 ± 0.01, indicated even cell distribution in the culture vessel at t = 72 h. The behavior of epithelial cells on liquid–liquid interfaces contributes to the promotion of their uniform maturation.

The relationship between the use of tobacco products and SARS-CoV-2 infection is poorly understood and controversial. Few studies have examined the effect of electronic cigarettes (ECs) on SARS-CoV-2 infection. We tested the hypothesis that EC fluids and aerosols with nicotine promote SARS-COV-2 infection by increasing viral entry into human respiratory epithelial cells. Responses of BEAS-2B cells to JUUL aerosols or their individual constituents were compared using three exposure platforms: submerged culture, air-liquid-interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex system, which produces authentic heated EC aerosols. In general, nicotine and nicotine + propylene glycol/vegetable glycerin aerosols increased ACE2 (angiotensin converting enzyme 2) levels, the SARS-CoV-2 receptor; and increased the activity of TMPRSS2 (transmembrane serine protease 2), an enzyme essential for viral entry. Lentivirus pseudoparticles with spike protein were used to test viral penetration. Exposure to nicotine, EC fluids, or aerosols altered the infection machinery and increased viral entry into cells. While most data were in good agreement across the three exposure platforms, cells were more responsive to treatments when exposed at the ALI in the Cultex system, even though the exposures were brief and intermittent. While both nicotine and JUUL aerosols increased SARS-CoV-2 infection, JUUL significantly decreased the effect of nicotine alone. These data support the idea that vaping can increase the likelihood of contracting COVID-19 and that e-liquid composition may modulate this effect.

Squamous cell lung cancer maintains its growth through elevated glucose consumption, but selective glucose consumption inhibitors are lacking. Here, we discovered using a high-throughput screen new compounds that block glucose consumption in three squamous cell lung cancer cell lines and identified 79 compounds that block glucose consumption in one or more of these cell lines. Based on its ability to block glucose consumption in all three cell lines, pacritinib, an inhibitor of FMS Related Receptor Tyrosine Kinase 3 (FLT3) and Janus Kinase 2 (JAK2), was further studied. Pacritinib decreased glucose consumption in squamous cell lung cancer cells in cell culture and in vivo without affecting glucose consumption in healthy tissues. Pacritinib blocked hexokinase activity, and Hexokinase 1 and 2 mRNA and protein expression. Overexpression of Hexokinase 1 blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Overexpression of FLT3 but not JAK2 significantly increased glucose consumption and blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Additional FLT3 inhibitors blocked glucose consumption in squamous cell lung cancer cells. Our study identifies FLT3 inhibitors as a new class of inhibitors that can block glucose consumption in squamous cell lung cancer.

The observed sex disparity in bladder cancer (BlCa) argues that androgen receptor (AR) signaling has a role in these malignancies. BlCas express full-length AR (FL-AR), constitutively active AR splice variants, including AR-v19, or both, and their depletion limits BlCa viability. However, the mechanistic basis of AR-dependence is unknown. Here, we depleted FL-AR, AR-v19, or all AR forms (T-AR), and performed RNA-seq studies to uncover that different AR forms govern distinct but partially overlapping transcriptional programs. Overlapping alterations include a decrease in mTOR and an increase of hypoxia regulated transcripts accompanied by a decline in oxygen consumption rate (OCR). Queries of BlCa databases revealed a significant negative correlation between AR expression and multiple hypoxia-associated transcripts arguing that this regulatory mechanism is a feature of high-grade malignancies. Our analysis of a 1600-compound library identified niclosamide as a strong ATPase inhibitor that reduces OCR in BlCa cells, decreased cell viability and induced apoptosis in a dose and time dependent manner. These results suggest that BlCa cells hijack AR signaling to enhance metabolic activity, promoting cell proliferation and survival; hence targeting this AR downstream vulnerability presents an attractive strategy to limit BlCa.

Aplacophoran molluscs are shell-less and have a worm-like body which is covered by biomineralized sclerites. We investigated sclerite crystallography and the sclerite mosaic of the Solenogastres species Dorymenia sarsii, Anamenia gorgonophila, and Simrothiella margaritacea with electron-backscattered-diffraction (EBSD), laser-confocal-microscopy and FE-SEM imaging. The soft tissue of the molluscs is covered by spicule-shaped, aragonitic sclerites. These are sub-parallel to the soft body of the organism. We find, for all three species, that individual sclerites are untwinned aragonite single crystals. For individual sclerites, aragonite c-axis is parallel to the morphological, long axis of the sclerite. Aragonite a- and b-axes are perpendicular to sclerite aragonite c-axis. For the scleritomes of the investigated species we find different sclerite and aragonite crystal arrangement patterns. For the A. gorgonophila scleritome, sclerite assembly is disordered such that sclerites with their morphological, long axis (always the aragonite c-axis) are pointing in many different directions, being, more or less, tangential to cuticle surface. For D. sarsii, the sclerite axes (equal to aragonite c-axes) show a stronger tendency to parallel arrangement, while for S. margaritacea, sclerite and aragonite organization is strongly structured into sequential rows of orthogonally alternating sclerite directions. The different arrangements are well reflected in the structured orientational distributions of aragonite a-, b-, c-axes across the EBSD-mapped parts of the scleritomes. We discuss that morphological and crystallographic preferred orientation (texture) is not generated by competitive growth selection (the crystals are not in contact), but is determined by templating on organic matter of the sclerite-secreting epithelial cells and associated papillae. [ABSTRACT FROM AUTHOR]

Allergic conjunctivitis (AC) is the most common form of allergic eye disease and an increasingly prevalent condition. Topical eye drop treatments are the usual approach for managing AC, although their impact on the ocular surface is not frequently investigated. The aim of this study was to perform a comparative physicochemical characterization, and in vitro biological evaluations in primary conjunctival and corneal epithelial cells of the new multidose preservative-free bilastine 0.6% and main commercially available eye drops. MTT assay was used to measure cell viability; oxidative stress was analyzed with a ROS-sensitive probe; and apoptosis was evaluated monitoring caspase 3/7 activation. Differences in pH value, osmolarity, viscosity and phosphate levels were identified. Among all formulations, bilastine exhibited pH, osmolarity and viscosity values closer to tear film (7.4, 300 mOsm/l and ~ 1.5–10 mPa·s, respectively), and was the only phosphates-free solution. Single-dose ketotifen did not induce ROS production, and single-dose azelastine and bilastine only induced a mild increase. Bilastine and single-dose ketotifen and azelastine showed high survival rates attributable to the absence of preservative in its formulation, not inducing caspase-3/7-mediated apoptosis after 24 h. Our findings support the use of the new bilastine 0.6% for treating patients with AC to preserve and maintain the integrity of the ocular surface. [ABSTRACT FROM AUTHOR]

To analyze the clinical characteristics of mucinous obstruction diagnosed by dacryoendoscopy and compared the cytological changes with membranous obstruction using a modified liquid-based thin prep cytology method. A retrospective chart review was conducted on 53 eyes of 51 patients with mucus obstruction based on dacryoendoscopic findings from January 2022 to October 2022. Liquid-based thin-prep cytology was performed by irrigating the inside of the nasolacrimal drainage system with saline during dacryoendoscopy-guided silicone tube intubation. Pathological findings were analyzed through a comparison of mucinous obstruction with membranous obstruction as determined by dacryoendoscopic findings. The modified liquid-based thin prep cytology technique had a higher cytology detection rate across all cases. Mucinous obstruction exhibited a significantly higher number of successful canalicular irrigation test cases compared to membranous obstruction. In mucinous obstruction, epithelial squamous cells were more frequently detected in pre-sac obstruction, whereas columnar epithelial cells were predominant in post-sac obstruction. Inflammatory cells showed a stronger correlation with primary change and post-sac obstruction. Bacterial colonies were observed exclusively in cases of mucinous obstruction. The use of a modified liquid-based thin prep cytology method enables the examination of histopathological changes in the lacrimal passage in primary acquired nasolacrimal duct obstruction (PANDO), particularly in cases of mucinous obstruction, without the need for invasive biopsies. These findings enhance the understanding of the etiopathogenesis of mucinous obstruction, complementing knowledge of membranous obstruction in PANDO. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is known to elicit a robust immune response. This study aimed to investigate the impact of LPS on the transcriptome of human nasal epithelial cells (HNEpC). HNEpC were cultured and stimulated with LPS (1 μg/mL) or an equivalent amount of normal culture medium. Subsequently, total RNA was extracted, purified, and sequenced using next-generation RNA sequencing technology. Differentially expressed genes (DEGs) were identified and subjected to functional enrichment analysis. A protein–protein interaction (PPI) network of DEGs was constructed, followed by Ingenuity Pathway Analysis (IPA) to identify molecular pathways influenced by LPS exposure on HNEpC. Validation of key genes was performed using quantitative real-time PCR (qRT-PCR). A total of 97 DEGs, comprising 48 up-regulated genes and 49 down-regulated genes, were identified. Results from functional enrichment analysis, PPI, and IPA indicated that DEGs were predominantly enriched in chemokine-related signaling pathways. Subsequent qRT-PCR validation demonstrated significant upregulation of key genes in these pathways in LPS-treated HNEpC compared to control cells. In conclusion, LPS intervention profoundly altered the transcriptome of HNEpC, potentially exacerbating inflammatory responses through the activation of chemokine-related signaling pathways. [ABSTRACT FROM AUTHOR]

TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the etiological agent of Chagas disease, invades many cell types affecting numerous host-signalling pathways. During the T. cruzi infection, we demonstrated modulations in the host RNA polymerase II activity with the downregulation of ribonucleoproteins affecting host transcription and splicing machinery. These alterations could be a result of the initial damage to the host DNA caused by the presence of the parasite, however, the mechanisms are not well understood. Herein, we examined whether infection by T. cruzi coincided with enhanced DNA damage in the host cell. We studied the engagement of the DNA damage response (DDR) pathways at the different time points (0–24 h post-infection, hpi) by T. cruzi in LLC-MK2 cells. In response to double-strand breaks (DSB), maximum phosphorylation of the histone variant H2AX is observed at 2hpi and promotes recruitment of the DDR p53-binding protein (53BP1). During T. cruzi infection, Ataxia-telangiectasia mutated protein (ATM) and DNA-PK protein kinases remained active in a time-dependent manner and played roles in regulating the host response to DSB. The host DNA lesions caused by the infection are likely orchestrated by the non-homologous end joining (NHEJ) pathway to maintain the host genome integrity. [ABSTRACT FROM AUTHOR]

Abnormal mitochondria have been observed in bronchial- and alveolar epithelial cells of patients with chronic obstructive pulmonary disease (COPD). However, it is unknown if alterations in the molecular pathways regulating mitochondrial turnover (mitochondrial biogenesis vs mitophagy) are involved. Therefore, in this study, the abundance of key molecules controlling mitochondrial turnover were assessed in peripheral lung tissue from non-COPD patients (n = 6) and COPD patients (n = 11; GOLDII n = 4/11; GOLDIV n = 7/11) and in both undifferentiated and differentiated human primary bronchial epithelial cells (PBEC) from non-COPD patients and COPD patients (n = 4–7 patients/group). We observed significantly decreased transcript levels of key molecules controlling mitochondrial biogenesis (PPARGC1B, PPRC1, PPARD) in peripheral lung tissue from severe COPD patients. Interestingly, mRNA levels of the transcription factor TFAM (mitochondrial biogenesis) and BNIP3L (mitophagy) were increased in these patients. In general, these alterations were not recapitulated in undifferentiated and differentiated PBECs with the exception of decreased PPARGC1B expression in both PBEC models. Although these findings provide valuable insight in these pathways in bronchial epithelial cells and peripheral lung tissue of COPD patients, whether or not these alterations contribute to COPD pathogenesis, underlie changes in mitochondrial function or may represent compensatory mechanisms remains to be established. [ABSTRACT FROM AUTHOR]

Parabens (PBs) are a class of preservatives commonly used in cosmetics and pharmaceuticals. Studies have shown that these compounds may act as endocrine disruptors, affecting thyroxine levels in humans. PBs with longer chain substituents, such as butylparaben (BuP), are less prone to complete biotransformation and are therefore more likely to accumulate in the body. In this study, the effect of high-dose exposure to BuP on thyroid microstructure, ultrastructure, and function was investigated in rats. 50 mg/kg bw per day of BuP was injected subcutaneously into the neck of rats for 4 weeks. Rat thyroid weight, microstructure, and ultrastructure were determined, and the levels of thyroid sodium/iodide symporter (NIS), serum thyroid hormones, and thyroid autoantibodies were measured. The human thyroid cell line was used to study the mechanism of BuP on thyroid epithelial cells. The weight of the thyroid gland of BuP-exposed rats was increased, the structure of the thyroid follicles was irregular and damaged, the mitochondria and rough endoplasmic reticulum were swollen and damaged, and the microvilli at the tip of the epithelium were reduced and disappeared. Serum total T3, total T4, free T3, and free T4 were decreased in BuP-exposed rats, and TSH, peroxidase antibody, and thyroglobulin antibody were increased. In vitro, BuP decreased the level of NIS in thyroid epithelial cells, inhibited proliferation and viability, and induced apoptosis in a dose-dependent manner. This study demonstrated that high-dose exposure to BuP induced structural, ultrastructural, and functional impairment to the thyroid gland of rats, which may be one of the factors leading to hypothyroidism. [ABSTRACT FROM AUTHOR]

Cigarette smoke (CS) exposure is known to cause injury to respiratory tract epithelial cells and is a contributing factor in the development of chronic obstructive pulmonary disease and lung cancer. Electronic cigarettes (e-cigarettes) are gaining popularity as a potential substitute for conventional cigarettes due to their potential for aiding smoking cessation. However, the safety of e-cigarettes remains uncertain, and scientific evidence on this topic is still limited. In this study, we aimed to investigate the effects of CS and e-cigarette smoke (ECS) of different flavors on human lung bronchial epithelial cells. Real-time smoke exposure was carried out using an air–liquid interface system, and cell viability was assessed. RNA-Seq transcriptome analysis was performed to compare the differences between CS and ECS. The transcriptome analysis revealed a significantly higher number of differentially expressed genes in CS than in ECS. Moreover, the impact of mint-flavored e-cigarettes on cells was found to be greater than that of tobacco-flavored e-cigarettes, as evidenced by the greater number of differentially expressed genes. These findings provide a reference for future safety research on traditional cigarettes and e-cigarettes, particularly those of different flavors. The use of omics-scale methodologies has improved our ability to understand the biological effects of CS and ECS on human respiratory tract epithelial cells, which can aid in the development of novel approaches for smoking cessation and lung disease prevention. [ABSTRACT FROM AUTHOR]

Dry eye syndrome (DES) is a complex ocular condition characterized by an unstable tear film and inadequate tear production, leading to tissue damage. Despite its common occurrence, there is currently no comprehensive in vitro model that accurately reproduce the cellular characteristics of DES. Here we modified a corneal epithelium-on-a-chip (CEpOC) model to recapitulate DES by subjecting HCE-T human corneal epithelial cells to an air–liquid (AL) interface stimulus. We then assessed the effects of AL stimulation both in the presence and absence of diclofenac (DCF), non-steroidal anti-inflammatory drug. Transcriptomic analysis revealed distinct gene expression changes in response to AL and AL_DCF, affecting pathways related to development, epithelial structure, inflammation, and extracellular matrix remodeling. Both treatments upregulated PIEZO2, linked to corneal damage signaling, while downregulating OCLN, involved in cell–cell junctions. They increased the expression of inflammatory genes (e.g., IL-6) and reduced mucin production genes (e.g., MUC16), reflecting dry eye characteristics. Metabolomic analysis showed increased secretion of metabolites associated with cell damage and inflammation (e.g., methyl-2-oxovaleric acid, 3-methyl-2-oxobutanoic acid, lauroyl-carnitine) in response to AL and even more with AL_DCF, indicating a shift in cellular metabolism. This study showcases the potential use of AL stimulus within the CEpOC to induce cellular characteristics relevant to DES. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma (LUAD) is one of the sole causes of death in lung cancer patients. This study combined with single-cell RNA-seq analysis to identify tumor stem-related prognostic models to predict the prognosis of lung adenocarcinoma, chemotherapy agents, and immunotherapy efficacy. mRNA expression-based stemness index (mRNAsi) was determined by One Class Linear Regression (OCLR). Differentially expressed genes (DEGs) were detected by limma package. Single-cell RNA-seq analysis in GSE123902 dataset was performed using Seurat package. Weighted Co-Expression Network Analysis (WGCNA) was built by rms package. Cell differentiation ability was determined by CytoTRACE. Cell communication analysis was performed by CellCall and CellChat package. Prognosis model was constructed by 10 machine learning and 101 combinations. Drug predictive analysis was conducted by pRRophetic package. Immune microenvironment landscape was determined by ESTIMATE, MCP-Counter, ssGSEA analysis. Tumor samples have higher mRNAsi, and the high mRNAsi group presents a worse prognosis. Turquoise module was highly correlated with mRNAsi in TCGA-LUAD dataset. scRNA analysis showed that 22 epithelial cell clusters were obtained, and higher CSCs malignant epithelial cells have more complex cellular communication with other cells and presented dedifferentiation phenomenon. Cellular senescence and Hippo signaling pathway are the major difference pathways between high- and low CSCs malignant epithelial cells. The pseudo-temporal analysis shows that cluster1, 2, high CSC epithelial cells, are concentrated at the end of the differentiation trajectory. Finally, 13 genes were obtained by intersecting genes in turquoise module, Top200 genes in hdWGCNA, DEGs in high- and low- mRNAsi group as well as DEGs in tumor samples vs. normal group. Among 101 prognostic models, average c-index (0.71) was highest in CoxBoost + RSF model. The high-risk group samples had immunosuppressive status, higher tumor malignancy and low benefit from immunotherapy. This work found that malignant tumors and malignant epithelial cells have high CSC characteristics, and identified a model that could predict the prognosis, immune microenvironment, and immunotherapy of LUAD, based on CSC-related genes. These results provided reference value for the clinical diagnosis and treatment of LUAD. [ABSTRACT FROM AUTHOR]

Epithelial–mesenchymal transition (EMT) is the process by which epithelial cells acquire mesenchymal characteristics. This process induces cell migration and invasion, which are closely related to cancer metastasis and malignancy. EMT consists of various intermediate states that express both epithelial and mesenchymal traits, called partial EMT. Recently, several studies have focused on the roles of voltage-gated potassium (Kv) channels associated with EMT in cancer cell migration and invasion. In this study, we demonstrate the relationship between Kv3.4 and EMT and confirm the effects of cell migration and invasion. With TGF-β treatment, EMT was induced and Kv3.4 was also increased in A549 cells, human lung carcinoma cells. The knockdown of Kv3.4 blocked the EMT progression reducing cell migration and invasion. However, the Kv3.4 overexpressed cells acquired mesenchymal characteristics and increased cell migration and invasion. The overexpression of Kv3.4 also has a synergistic effect with TGF-β in promoting cell migration. Therefore, we conclude that Kv3.4 regulates cancer migration and invasion through TGF-β-induced EMT and these results provide insights into the understanding of cancer metastasis. [ABSTRACT FROM AUTHOR]

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the nasal mucosa, and epithelial–mesenchymal transition (EMT) is thought to be an essential process in the pathogenesis of CRSwNP. However, the mechanisms of epithelial and fibroblastic changes at the single-cell level are unclear. In this study, we investigated the epithelial cell, fibroblast, and key gene alterations in the development of CRSwNP. We revealed major cell types involved in CRSwNP and nasal mucosal inflammation formation, then mapped epithelial and fibroblast subpopulations. We showed that the apical and glandular epithelial cells and the ADGRB3+ and POSTN+ fibroblasts were the key cell subtypes in the progression of CRSwNP. Pseudotime and cell cycle analysis identified dynamic changes between epithelial cells and fibroblasts during its development. WFDC2 and CCL26 were identified as the key marker genes involved in the development of CRSwNP and were validated by IHC staining, which may provide a potential novel target for future CRSwNP therapy. ScRNA-seq data provided insights into the cellular landscape and the relationship between epithelial cells and fibroblasts in the progression of CRSwNP. WFDC2 and CCL26 were identified as the key genes involved in the development of CRSwNP and may be the potential markers for gene therapy. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) remains a high mortality rate with dramatic lung inflammation and alveolar epithelial cell death. Although fatty acid β-oxidation (FAO) impairment has been implicated in the pathogenesis of ALI, whether Carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme for FAO, plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. Accordingly, we focused on exploring the effect of CPT1A in the context of ALI and the underlying mechanisms. We found that overexpression of CPT1A (AAV-CPT1A) effectively alleviated lung injury by reduction of lung wet-to-dry ratio, inflammatory cell infiltration, and protein levels in the BALF of ALI mice. Meanwhile, AAV-CPT1A significantly lessened histopathological changes and several cytokines' secretions. In contrast, blocking CPT1A with etomoxir augmented inflammatory responses and lung injury in ALI mice. Furthermore, we found that overexpression of CPT1A with lentivirus reduced the apoptosis rates of alveolar epithelial cells and the expression of apoptosis-related proteins induced by LPS in MLE12 cells, while etomoxir increased the apoptosis of MLE12 cells. Overexpression of CPT1A prevented the drop in bioenergetics, palmitate oxidation, and ATP levels. In conclusion, the results rendered CPT1A worthy of further development into a pharmaceutical drug for the treatment of ALI. [ABSTRACT FROM AUTHOR]

Boron is an essential trace element with roles in growth, development, and physiological functions; however, its mechanism of action is still unclear. In this study, the regulatory roles of the PI3K/Akt signaling pathway on boron-induced changes in barrier function, proliferation, and apoptosis in rat intestinal epithelial cells were evaluated. Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein expression levels of PCNA were higher, while the proportions of cells in the G0/G1 and S phases, apoptosis rate, and caspase-3 mRNA and protein expression levels were lower in cells treated with 0.8 mmol/L boron than in control IEC-6 cells (P < 0.01 or P < 0.05). However, 40 mmol/L boron decreased ZO-1 and Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein levels of PCNA and increased the apoptosis rate and caspase-3 mRNA expression (P < 0.01 or P < 0.05). After specifically blocking PI3K and Akt signals (using LY294002 and MK-2206 2HCL), 0.8 mmol/L boron had no effects on Occludin, PCNA level, apoptosis rates, and caspase-3 levels (P < 0.05); however, the proliferation rate and PCNA levels decreased significantly (P < 0.01 or P < 0.05). The addition of 40 mmol/L boron did not affect ZO-1 and Occludin levels and did not affect the apoptosis rate or PCNA and caspase-3 levels. These results suggested that the PI3K/Akt signaling pathway mediates the effects of low-dose boron on IEC-6 cells. [ABSTRACT FROM AUTHOR]

To investigate whether aldosterone (ALD) and hydrocortisone (HC) change the gene expression of SLC7A5, which encodes the large neutral amino acid transporter small subunit 1 (LAT1), and the transport activity of LAT1 in the retinal pigment epithelium (RPE) in vitro. ARPE-19 cells were grown to confluence. After withdrawing the serum, ALD or HC was added with several doses and incubated, and SLC7A5 gene expression was measured. The influx and efflux transport of sodium fluorescein (Na-F) were evaluated using the Transwell culture system. SLC7A5 gene expression was upregulated by ALD and downregulated by HC in a dose-dependent manner. Both ALD and HC significantly increased the influx and efflux Na-F transport of RPE cells at a dose that did not change the expression of SLC7A5. JPH203, a specific inhibitor of LAT1, significantly reduced accelerated Na-F transport. Both ALD and HC increased the gene expression of zonula occludin-1 (ZO-1) although they did not change the immunoreactivity of ZO-1 in RPE cells. LAT1 may play an important role in increasing Na-F transport associated with ALD and HC administration. A specific LAT1 inhibitor may effectively regulate the increased material transport of RPE induced by ALD and HC. [ABSTRACT FROM AUTHOR]

The effect of MDANP effects on ER stress signalling not well known or elucidated. Endoplasmic reticulum (ER) stress plays a critical role in necrotizing enterocolitis (NEC) pathogenesis through the PERK-eIF2ɑ-QRICH1 axis. The present study aimed to explore the protective effects of MDANP in NEC development. Firstly, a function screening was designed to identify the candidate peptides in human milk, and then the identified peptides were validated in NEC patients. In vivo, NEC was induced in mice pups and divided into four groups: (1) control group, (2) NEC group, (3) MDANP + NEC group, and (4) NS + NEC group. In vitro, lentivirus-mediated QRICH1 silencing, was used to transfect NCM460 cell lines, then stimulated with LPS. After LPS stimulation, cells were treated with chemically synthesized MDANP, and the essential proteins in the QRICH1 signalling pathway in cells were tested and compared. After the small-scale screening, a peptide (SKSKKFRRPDIQYPDATDED) named MDANP was determined as the principal peptide. Its protective effect against NEC through inhibiting the expression of ERS key proteins and impeding the intestinal cells' apoptosis was observed in the animal models. Furthermore, the inhibitive effect of MDANP on apoptosis of intestinal epithelial cells through modulating the PERK-eIF2ɑ-QRICH1 ERS pathway was also confirmed in vitro. Taken together, our data suggest that MDANP effectively ameliorates apoptosis in NEC through attenuating PERK-eIF2ɑ-QRICH1. [ABSTRACT FROM AUTHOR]

Epithelial cells undergoing EMT experience significant alterations at transcriptional and morphological levels. However, changes in the cytoskeleton, especially cytoskeleton dynamics are poorly described. Addressing the question we induced EMT in three cell lines (MCF-7, HaCaT and A-549) and analyzed morphological and cytoskeletal changes there using immunostaining and life cell imaging of cells transfected with microtubule and focal adhesion markers. In all studied cell lines, cell area after EMT increased, MCF-7 and A-549 cells became elongated, while HaCaT cells kept the aspect ratio the same. We next analyzed three components of the cytoskeleton: microtubules, stress fibers and focal adhesions. The following changes were observed after EMT in cultured cells: (i) Organization of microtubules becomes more radial; and the growth rate of microtubule plus ends was accelerated; (ii) Actin stress fibers become co-aligned forming the longitudinal cell axis; and (iii) Focal adhesions had decreased area in all cancer cell lines studied and became more numerous in HaCaT cells. We conclude that among dynamic components of the cytoskeleton, the most significant changes during EMT happen in the regulation of microtubules. [ABSTRACT FROM AUTHOR]

The small intestinal mucosa constitutes a physical barrier separating the gut lumen from sterile internal tissues. Junctional complexes between cells regulate transport across the barrier, preventing water loss and the entry of noxious molecules or pathogens. Inflammatory diseases in cattle disrupt this barrier; nonetheless, mechanisms of barrier disruption in cattle are poorly understood. We investigated the direct effects of three inflammatory cytokines, TNFα, IFNγ, and IL-18, on the bovine intestinal barrier utilizing intestinal organoids. Flux of fluorescein isothiocyanate (FITC)-labeled dextran was used to investigate barrier permeability. Immunocytochemistry and transmission electron microscopy were used to investigate junctional morphology, specifically tortuosity and length/width, respectively. Immunocytochemistry and flow cytometry was used to investigate cellular turnover via proliferation and apoptosis. Our study shows that 24-h cytokine treatment with TNFα or IFNγ significantly increased dextran permeability and tight junctional tortuosity, and reduced cellular proliferation. TNFα reduced the percentage of G2/M phase cells, and IFNγ treatment increased cell apoptotic rate. IL-18 did not directly induce significant changes to barrier permeability or cellular turnover. Our study concludes that the inflammatory cytokines, TNFα and IFNγ, directly induce intestinal epithelial barrier dysfunction and alter the tight junctional morphology and rate of cellular turnover in bovine intestinal epithelial cells.

Breast cancer is a heterogenous disease that can be classified into multiple subtypes including the most aggressive basal-like and triple-negative subtypes. Understanding the heterogeneity within the normal mammary basal epithelial cells holds the key to inform us about basal-like cancer cell differentiation dynamics as well as potential cells of origin. Although it is known that the mammary basal compartment contains small pools of stem cells that fuel normal tissue morphogenesis and regeneration, a comprehensive yet focused analysis of the transcriptional makeup of the basal cells is lacking. We used single-cell RNA-sequencing and multiplexed RNA in-situ hybridization to characterize mammary basal cell heterogeneity. We used bioinformatic and computational pipelines to characterize the molecular features as well as predict differentiation dynamics and cell-cell communications of the newly identified basal cell states. We used genetic cell labeling to map the in vivo fates of cells in one of these states. We identified four major distinct transcriptional states within the mammary basal cells that exhibit gene expression signatures suggestive of different functional activity and metabolic preference. Our in vivo labeling and ex vivo organoid culture data suggest that one of these states, marked by Egr2 expression, represents a dynamic transcriptional state that all basal cells transit through during pubertal mammary morphogenesis. Our study provides a systematic approach to understanding the molecular heterogeneity of mammary basal cells and identifies previously unknown dynamics of basal cell transcriptional states.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes severe inflammation in various infectious diseases, such as bloodstream infections, respiratory and urinary tract infections, which leads to high mortality. Polydatin (PD), an active ingredient of Yinhuapinggan granule, has attracted worldwide attention for its powerful antioxidant, anti-inflammatory, antitumor, and antibacterial capacity. However, very little is known about the effect of PD on CRKP. In this research, we evaluated the inhibitory effects of PD on both the bacterial level and the bacterial-cell co-culture level on anti-biofilm and efflux pumps and the other was the inhibitory effect on apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) after CRKP induction. Additionally, we validated the mechanism of action by qRT-PCR and western blot in human lung epithelial cells. Firstly, PD was observed to have an inhibitory effect on the biofilm of CRKP and the efflux pump AcrAB-TolC. Mechanically, CRKP not only inhibited the activation of Nuclear Factor erythroid 2-Related Factor 2 (Nrf-2) but also increased the level of ROS in cells. These results showed that PD could inhibit ROS and activate Nrf-2 production. Together, our research demonstrated that PD inhibited bacterial biofilm formation and efflux pump AcrAB—TolC expression and inhibited CRKP-induced cell damage by regulating ROS and Nrf-2-regulated antioxidant pathways. [ABSTRACT FROM AUTHOR]

In this study, we aimed to identify molecular markers associated with type II alveolar epithelial cell injury in acute lung injury (ALI) models using bioinformatics methods. The objective was to provide new insights for the diagnosis and treatment of ALI/ARDS. We downloaded RNA SEQ datasets (GSE109913, GSE179418, and GSE119123) from the Gene Expression Omnibus (GEO) and used R language package to screen differentially expressed genes (DEGs). DEGs were annotated using Gene Ontology (GO), and their pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG). DEGs were imported into the STRING database and analyzed using Cytoscape software to determine the protein network of DEGs and calculate the top 10 nodes for the hub genes. Finally, potential therapeutic drugs for the hub genes were predicted using the DGIdb database. We identified 78 DEGs, including 70 up-regulated genes and 8 down-regulated genes. GO analysis revealed that the DEGs were mainly involved in biological processes such as granulocyte migration, response to bacterial-derived molecules, and cytokine-mediated signaling pathways. Additionally, they had cytokine activity, chemokine activity, and receptor ligand activity, and functioned in related receptor binding, CXCR chemokine receptor binding, G protein-coupled receptor binding, and other molecular functions. KEGG analysis indicated that the DEGs were mainly involved in TNF signaling pathway, IL-17 signaling pathway, NF-κB signal pathway, chemokine signal pathway, cytokine-cytokine receptor interaction signal pathway, and others. We identified eight hub genes, including IRF7, IFIT1, IFIT3, PSMB8, PSMB9, BST2, OASL2, and ZBP1, which were all up-regulated genes. We identified several hub genes of type II alveolar epithelial cells in ALI mouse models using bioinformatics analysis. These results provide new targets for understanding and treating of ALI. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) is a major cause of illness and death but has no effective therapy. The heat-labile enterotoxin LT is a significant virulence factor produced by ETEC. The heat-labile enterotoxin-B (LT-B) subunit may enter host cells by binding to monosialotetrahexosylganglioside-a (GM1a), a monosialoganglioside found on the plasma membrane surface of animal epithelial cells. This research was conducted to develop conformationally comparable peptides to the carbohydrate epitope of GM1a for the treatment of ETEC. We used the LT-B subunit to select LT-B-binding peptides that structurally resemble GM1a. The ganglioside microarray and docking simulations were used to identify three GM1a ganglioside-binding domain (GBD) peptides based on LT-B recognition. Peptides had an inhibiting effect on the binding of LT-B to GM1a. The binding capacity, functional inhibitory activity, and in vitro effects of the GBD peptides were evaluated using HCT-8 cells, a human intestinal epithelial cell line, to evaluate the feasibility of deploying GBD peptides to combat bacterial infections. KILSYTESMAGKREMVIIT was the most efficient peptide in inhibiting cellular absorption of LT-B in cells. Our findings offer compelling evidence that GM1a GBD-like peptides might act as new therapeutics to inhibit LT-B binding to epithelial cells and avoid the subsequent physiological consequences of LT. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis onco-BCG bacilli used in immunotherapy of bladder cancer are candidates for training of immune cells towards microbial pathogens. Increasing antibiotic resistance of gastric pathogen Helicobacter pylori (Hp) prompts the search for new anti-Hp and immunomodulatory formulations. Colonization of gastric mucosa by Hp through mucin 5 AC (MUC5AC) ligands could potentially be a therapeutic target. The aim of this study was to examine the ability of onco-BCG mycobacteria to reduce Hp adhesion to gastric epithelial cells using Cavia porcellus model. Animals were inoculated per os with 0.85% NaCl, Hp alone, onco-BCG alone or with onco-BCG and Hp. After 7/28 days Mucin5AC and Hp binding to gastric epithelium were assessed in gastric tissue specimens by staining with anti-Mucin5AC and anti-Hp antibodies, respectively, both fluorescently labeled. Primary gastric epithelial cells were treated ex vivo with live Hp or Hp surface antigens (glycine extract or lipopolysaccharide) alone or with onco-BCG. In such cells MUC5AC and Hp binding were determined as above. Mycobacteria reduced the amount of MUC5AC animals infected with Hp and in gastric epithelial cells pulsed in vitro with Hp components. Decrease of MUC5AC driven in cell cultures in vitro and in gastric tissue exposed ex vivo to mycobacteria was related to diminished adhesion of H. pylori bacilli. Vaccine mycobacteria by diminishing the amount of MUC5AC in gastric epithelial cells may reduce Hp adhesion. [ABSTRACT FROM AUTHOR]

Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences. [ABSTRACT FROM AUTHOR]

Besides metabolic homeostasis regulation, adipokines are recently emerged as important players in regulating immunity and inflammation. Helminth infection has known to modulate circulating adipokine secretion; however, the regulation and function of adipokines in response to helminth infection is still unclear. Here, we investigated the regulation and function of adiponectin during T. spiralis infection. While there was no change in circulating level of adiponectin, we found an increased adiponectin, but not leptin expression in the small intestine. Interestingly, the intestinal adiponectin expression was strongly associated with the expression of epithelial cell-derived cytokines IL-25, IL-33, and TSLP following infection. Indeed, mice deficiency of IL-25 receptor exhibited no intestinal adiponectin induction upon helminth infection. Interestingly, IL-25-induced adiponectin modulated intestinal epithelial cell responses by enhancing occludin and CCL17 expression. Using LPS-induced intestinal epithelial barrier dysfunctions in a Caco-2 cell monolayer model, adiponectin pretreatment enhanced a Transepithelial electrical resistance (TEER) and occludin expression. More importantly, adiponectin pretreatment of Caco2 cells prevented T. spiralis larval invasion in vitro and its administration during infection enhanced intestinal IL-13 secretion and worm expulsion in vivo. Altogether, our data suggest that intestinal adiponectin expression induced by helminth infection through the regulation of IL-25 promotes worm clearance and intestinal barrier function. [ABSTRACT FROM AUTHOR]