Invasion Of Epithelial Cells Research Articles

A phenotypic screening approach to target p60AmotL2-expressing invasive cancer cells

BackgroundTumor cells have the ability to invade and form small clusters that protrude into adjacent tissues, a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro, p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction.MethodsUsing epithelial cells transfected with inducible p60AmotL2, we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo, drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models, both in 2D and in 3D, and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells.ResultsWe identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly, these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models, as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells.ConclusionsBET inhibitors specifically target p60AmotL2-expressing invasive cancer cells, likely by exploiting differences in chromatin accessibility, leading to cell death. Additionally, our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells.

Effects of silencing hsa_circ_0015326 on proliferation, migration, invasion, and apoptosis of epithelial ovarian cancer cells.

Although the treatment of ovarian cancer has made great progress, there are still many patients who are not timely detected and given targeted therapy due to unknown pathogenesis. Recent studies have found that hsa_circ_0015326 is upregulated in ovarian cancer and is involved in the proliferation, invasion, and migration of ovarian cancer cells. However, whether hsa_circ_0015326 can be used as a new target of ovarian cancer needs further investigation. Therefore, the effect of hsa_circ_0015326 on epithelial ovarian cancer was investigated in this study. At first, si-hsa_circ_0015326 lentivirus was transfected into epithelial ovarian cancer cells. Then real-time fluorescence quantitative PCR (qRT-PCR) was used to detect hsa_circ_0015326 level. The proliferation of ovarian cancer cells was detected by CCK-8 assay. The horizontal and vertical migration abilities of the cells were detected by wound-healing assay and Transwell assay, respectively. Transwell assay was also used to determine the invasion rate. As for the apoptosis rate, it was assessed by flow cytometry. As a result, the expression level of hsa_circ_0015326 in A2780 and SKOV3 was found to be higher than that in IOSE-80. However, after transfecting si-hsa_circ_0015326 and si-NC into the cells, the proliferation, migration, and invasion abilities of A2780 and SKOV3 cells in the si-hsa_circ_0015326 group were significantly reduced in comparison to those in the si-NC and mock groups, while their apoptosis rates were elevated. Collectively, silencing hsa_circ_0015326 bears the capability of inhibiting the proliferation, migration, and invasion of ovarian cancer cells while increasing apoptosis rate. It can be concluded that hsa_circ_0015326 promotes the malignant biological activities of epithelial ovarian cancer cells.

Epithelial ingrowth in descemet membrane endothelial keratoplasty associated with vitreous loss

BackgroundEpithelial ingrowth is a rare but potentially sight-threatening complication caused by the invasion of corneal or conjunctival epithelial cells into the eye during ocular surgeries. DMEK is emerging as a widely used surgery for endothelial keratoplasty with its improved safety profile. We describe a case of epithelial ingrowth in the graft-host interface after uneventful DMEK associated with vitreous prolapse in the anterior chamber.Case presentationAn 81-year-old female with Fuchs endothelial dystrophy underwent DMEK for corneal decompensation following cataract surgery. During the DMEK procedure, vitreous prolapse was observed around the intraocular lens (IOL). Her early postoperative course was unremarkable, but a dense paracentral interface opacity was observed during the 3-month follow-up. The area of epithelial ingrowth was imaged with optical coherence tomography (OCT) as a uniform nodule with a discrete increase in interface hyperreflectivity. A low-energy YAG laser was applied to remove the opacity. She maintained good vision and clear cornea without reoccurrence after treatment.ConclusionsWe propose that, in addition to the introduction of epithelial cells during surgery, vitreous retention in the anterior chamber may be a risk factor by providing a scaffold that potentially aggravates epithelial ingrowth in DMEK. Our case demonstrated that early YAG intervention may disrupt interface epithelial cell growth, and the transmitted laser energy may fragment the scaffold vitreous noninvasively.

Salmonella manipulates macrophage migration via SteC-mediated myosin light chain activation to penetrate the gut-vascular barrier

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.

Abstract 6849: Characterizing a discernible stromal fibroblastic gene signature in the shift from high-grade prostatic intraepithelial neoplasia to aggressive prostate cancer

Abstract Background: High-grade prostatic intraepithelial neoplasia (HGPIN) is a preneoplastic lesion that precedes the development of both indolent and aggressive variants of prostate cancer (PCa). However, the specific stromal molecular mechanisms underlying the progression of HGPIN and their contribution to epithelial cancer cell motility and invasiveness remain poorly understood. This study aims to elucidate the driver genomic alterations involved in shaping the perineoplastic stroma of HGPIN, with the goal of unraveling their role in promoting cancer invasion, and metastasis. Methods: We performed gene expression profiling on laser capture microdissected perineoplastic stroma of HGPIN and PCa in tissue samples from 25 PCa cases, including 12 low-grade (Gleason 6) and 13 high-grade (Gleason >/= 8) cases. We used the standard moderated t-test from the Bioconductor limma package to assess the statistical significance of differential gene expression between perineoplastic stroma high grade PCa and perineoplastic stroma HGPIN, as well as between perineoplastic stroma low grade PCa and HGPIN. The -log10 p-values represents the level of statistical significance for each gene, while the log2 fold change represents the magnitude of gene expression difference between the two comparison groups. Additionally, we computed Gene Set Enrichment Analysis (GSEA) scores using the Bioconductor package and discerned the leading-edge genes among the pathways. This study is supported by the NIH-NCI-T32 program for next-generation pathologists at our institution. Results: In the gene expression analysis comparing High Grade PCa and HGPIN, several genes, including POSTN, BGN, GREM1, C1QA, THBS4, COL1A1, COL1A2, SFRP4, RNU6-847P, FCGR2P, and TOMM22, exhibited the highest level of both biological and statistical significance. GSEA revealed that the multi-invasive pathway had the highest enrichment score (>2). Notably, the top overexpressed genes in this pathway exhibited a fibroblastic signature (in bold), including POSTN, BGN, GREM1, FBN1, COL1A2, COL3A1, COL5A2, ASPN, SPARC, VCAN, COMP, INHBA, THBS2, and SFRP4. The presence of this signature indicates the invasiveness of the disease. Importantly, these genes were not associated with the progression to low grade PCa, indicating their exclusive association with the aggressive variant. Conclusion: To our knowledge, this study is the first to explore the stroma's role in HGPIN-to-aggressive PCa progression. It highlights a key stromal pathway enriched with fibroblastic gene signature. These findings offer potential biomarkers for targeted therapies and improved care through HGPIN patient stratification. Citation Format: Mohammad K. Alexanderani, Lucio Queiroz, Mohamed Omar, Claudio Zanettini, Hubert Pakula, Luigi Marchionni, Massimo Loda. Characterizing a discernible stromal fibroblastic gene signature in the shift from high-grade prostatic intraepithelial neoplasia to aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6849.

Listeria monocytogenes: the silent assassin.

Listeriosis is a foodborne infection in humans caused by Listeria monocytogenes. Consumption of contaminated food can lead to severe infection in vulnerable patients, that can be fatal. Clinical manifestations include sepsis and meningitis, and in pregnancy-associated infection, miscarriage and stillbirth. Diagnosis is confirmed by culture and identification of the pathogen from blood, cerebrospinal fluid, vaginal swab, placenta or amniotic fluid. Treatment regimens recommend amoxicillin, ampicillin or an aminoglycoside. Virulence factors mediate bacterial adhesion and invasion of gut epithelial cells. Other factors mediate biofilm formation and tolerance to low temperatures and high salt concentrations facilitating persistence and survival in the environment.

Mitochondrial carrier homolog 2 increases malignant phenotype of human gastric epithelial cells and promotes proliferation, invasion, and migration of gastric cancer cells.

The precise role of mitochondrial carrier homolog 2 (MTCH2) in promoting malignancy in gastric mucosal cells and its involvement in gastric cancer cell metastasis have not been fully elucidated. To determine the role of MTCH2 in gastric cancer. We collected 65 samples of poorly differentiated gastric cancer tissue and adjacent tissues, constructed MTCH2-overexpressing and MTCH2-knockdown cell models, and evaluated the proliferation, migration, and invasion of human gastric epithelial cells (GES-1) and human gastric cancer cells (AGS) cells. The mitochondrial membrane potential (MMP), mitochondrial permeability transformation pore (mPTP) and ATP fluorescence probe were used to detect mitochondrial function. Mitochondrial function and ATP synthase protein levels were detected via Western blotting. The expression of MTCH2 and ATP2A2 in gastric cancer tissues was significantly greater than that in adjacent tissues. Overexpression of MTCH2 promoted colony formation, invasion, migration, MMP expression and ATP production in GES-1 and AGS cells while upregulating ATP2A2 expression and inhibiting cell apoptosis; knockdown of MTCH2 had the opposite effect, promoting overactivation of the mPTP and promoting apoptosis. MTCH2 can increase the malignant phenotype of GES-1 cells and promote the proliferation, invasion, and migration of gastric cancer cells by regulating mitochondrial function, providing a basis for targeted therapy for gastric cancer cells.

Helicobacter Pylori and Sociodemographic Characteristics Distribution

Introduction and Aim: Helicobacter pylori is a gram-negative bacterium that affects up to half of the world's population, particularly in poorer nations. It is the leading cause of chronic gastritis, ulcers, gastric lymphoma, and gastric cancer, with more frequent cases in children and adolescents. Transmission can occur through fecal-oral, gastro-oral, oral-oral, or sexual means. The infection is caused by H. pylori's urease activity, invasion of host gastric epithelial cells, and adherence to host cell receptors. Early treatment can enhance gastric protection. Materials and Methods: Between November 2022 and July 2023, 103 samples were collected from patients at Mihrabani Surgical Hospital in Erbil City to identify Helicobacter pylori infection. Blood samples and stool samples were used to test for antibodies. Results: The study found a relatively equal gender representation in the patient and control groups, with 48.48% of men in the patient group and 48.64% in the control group. However, there are significant differences in age distribution, with G3 of age (<35) having the highest prevalence of H.pylori (48.48%). Additionally, 62.12% of patients are rural residents, while 97.29% of the control group are urban residents. Finally, 56.07% of patients are married, indicating a significant difference. Conclusion: The study found equal gender representation in patient and control groups but significant age differences, rural residency, and marital status, with G3 under 35 having the highest H.pylori prevalence.

Nanobody-mediated neutralization of candidalysin prevents epithelial damage and inflammatory responses that drive vulvovaginal candidiasis pathogenesis.

Candida albicans can cause mucosal infections in humans. This includes oropharyngeal candidiasis, which is commonly observed in human immunodeficiency virus infected patients, and vulvovaginal candidiasis (VVC), which is the most frequent manifestation of candidiasis. Epithelial cell invasion by C. albicans hyphae is accompanied by the secretion of candidalysin, a peptide toxin that causes epithelial cell cytotoxicity. During vaginal infections, candidalysin-driven tissue damage triggers epithelial signaling pathways, leading to hyperinflammatory responses and immunopathology, a hallmark of VVC. Therefore, we proposed blocking candidalysin activity using nanobodies to reduce epithelial damage and inflammation as a therapeutic strategy for VVC. Anti-candidalysin nanobodies were confirmed to localize around epithelial-invading C. albicans hyphae, even within the invasion pocket where candidalysin is secreted. The nanobodies reduced candidalysin-induced damage to epithelial cells and downstream proinflammatory responses. Accordingly, the nanobodies also decreased neutrophil activation and recruitment. In silico mathematical modeling enabled the quantification of epithelial damage caused by candidalysin under various nanobody dosing strategies. Thus, nanobody-mediated neutralization of candidalysin offers a novel therapeutic approach to block immunopathogenic events during VVC and alleviate symptoms.IMPORTANCEWorldwide, vaginal infections caused by Candida albicans (VVC) annually affect millions of women, with symptoms significantly impacting quality of life. Current treatments are based on anti-fungals and probiotics that target the fungus. However, in some cases, infections are recurrent, called recurrent VVC, which often fails to respond to treatment. Vaginal mucosal tissue damage caused by the C. albicans peptide toxin candidalysin is a key driver in the induction of hyperinflammatory responses that fail to clear the infection and contribute to immunopathology and disease severity. In this pre-clinical evaluation, we show that nanobody-mediated candidalysin neutralization reduces tissue damage and thereby limits inflammation. Implementation of candidalysin-neutralizing nanobodies may prove an attractive strategy to alleviate symptoms in complicated VVC cases.

Epithelial Cell Infection Analyses with Shigella.

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

PEtN-Modified O-Antigen Enhances Shigella Pathogenesis by Promoting Epithelial Cell Invasion and Inhibiting Complement Binding.

Shigellosis poses an ongoing global public health threat. The presence and length of the O-antigen in lipopolysaccharide play critical roles in Shigella pathogenesis. The plasmid-mediated opt gene encodes a phosphoethanolamine (PEtN) transferase that catalyzes the addition of PEtN to the O-antigen of Shigella flexneri serotype X and Y strains, converting them into serotype Xv and Yv strains, respectively. Since 2002, these modified strains have become prevalent in China. Here we demonstrate that PEtN-mediated O-antigen modification in S. flexneri increase the severity of corneal infection in guinea pigs without any adaptive cost. This heightened virulence is associated with epithelial cell adhesion and invasion, as well as an enhanced inflammatory response of macrophage. Notably, PEtN addition allow S. flexneri to attenuate the binding of complement C3 and better resist phagocytosis, potentially contributing to the retention of S. flexneri in the host environment.

Extracellular vesicles from Staphylococcus aureus promote the pathogenicity of Pseudomonas aeruginosa

Co-infections with Staphylococcus aureus and Pseudomonas aeruginosa are common in patients with chronic wounds, but little is known about their synergistic effect mediated by extracellular vesicles (EVs). In this study, we investigated the effect of EVs derived from S. aureus (SaEVs) on the pathogenicity of P. aeruginosa. By using lipophilic dye, we could confirm the fusion between SaEV and P. aeruginosa membranes. However, SaEVs did not alter the growth and antibiotic susceptible pattern of P. aeruginosa. Differential proteomic analysis between SaEV-treated and non-treated P. aeruginosa was performed, and the results revealed that lipopolysaccharide (LPS) biosynthesis protein in P. aeruginosa significantly increased after SaEV-treatment. Regarding this result, we also found that SaEVs promoted LPS production, biofilm formation, and expression of polysaccharide polymerization-related genes in P. aeruginosa. Furthermore, invasion of epithelial cells by SaEV-pretreated P. aeruginosa was enhanced. On the other hand, uptake of P. aeruginosa by RAW 264.7 macrophages was impaired after pretreatment P. aeruginosa with SaEVs. Proteomic analysis SaEVs revealed that SaEVs contain the proteins involving in host cell colonization, inhibition of host immune response, anti-phagocytosis of the macrophages, and protein translocation and iron uptake of S. aureus. In conclusion, SaEVs serve as a mediator that promote P. aeruginosa pathogenicity by enhancing LPS biosynthesis, biofilm formation, epithelial cell invasion, and macrophage uptake impairment.

Cirsilineol inhibits the proliferation and migration of endometriotic cells

Purpose: To determine the effect of cirsilineol on the proliferation and migration of endometriotic cells.
 Methods: Human endometriotic epithelial cells 12Z were treated with 5, 10 and 20 μM cirsilineol, respectively. Cell proliferation was evaluated using BrdU incorporation assay whereas wound healing assay was used to investigate cell migration, and cell invasion was assessed by Transwell assay. Cell cycle was investigated using flow cytometry while protein expression and phosphorylation levels of p65 and IκBα were evaluated by Western blot.
 Results: Treatment with cirsilineol at either 10 μM or 20 μM resulted in a significant reduction in cell proliferation and cell cycle arrest at the G2/M phase (p < 0.05). Moreover, cirsilineol weakened cell migration and invasion in 12Z cells, but did not alter the expression of p65. However, it significantly downregulated the phosphorylation of p65 and increased the expression of IκBα, while attenuating the phosphorylation level of IκBα (p < 0.05). Furthermore, 5 μM cirsilineol did not show any significant effects on these cellular activities, suggesting the activity of cirsilineol in 12Z cells is dose-dependent.
 Conclusion: Cirsilineol inhibits cell proliferation, induces cell cycle arrest, suppresses migration and invasion of endometriotic epithelial cells by regulating NF-κB pathway. Thus, cirsilineol might be a potential therapeutic candidate for endometriosis treatmen

A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

Galactomannan inhibits Trichinella spiralis invasion of intestinal epithelium cells and enhances antibody-dependent cellular cytotoxicity related killing of larvae by driving macrophage polarization.

Previous studies have shown that recombinant Trichinella spiralis galectin (rTsgal) is characterized by a carbohydrate recognition domain sequence motif binding to beta-galactoside, and that rTsgal promotes larval invasion of intestinal epithelial cells. Galactomannan is an immunostimulatory polysaccharide composed of a mannan backbone with galactose residues. The aim of this study was to investigate whether galactomannan inhibits larval intrusion of intestinal epithelial cells and enhances antibody-dependent cellular cytotoxicity (ADCC), killing newborn larvae by polarizing macrophages to the M1 phenotype. The results showed that galactomannan specially binds to rTsgal, and abrogated rTsgal facilitation of larval invasion of intestinal epithelial cells. The results of qPCR, Western blotting, and flow cytometry showed that galactomannan and rTsgal activated macrophage M1 polarization, as demonstrated by high expression of iNOS (M1 marker) and M1 related genes (IL-1β, IL-6, and TNF-α), and increased CD86+ macrophages. Galactomannan and rTsgal also increased NO production. The killing ability of macrophage-mediated ADCC on larvae was also significantly enhanced in galactomannan- and rTsgal-treated macrophages. The results demonstrated that Tsgal may be considered a potential vaccine target molecule against T. spiralis invasion, and galactomannan may be a novel adjuvant therapeutic agent and potential vaccine adjuvant against T. spiralis infection.

The RNA chaperone Hfq has a multifaceted role in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qβ replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E.ictaluri by creating an hfq mutant (EiΔhfq) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella. The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, EiΔhfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, EiΔhfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. EiΔhfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the EiΔhfq vaccination completely protected catfish against subsequent EiWT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri, affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.

MicroRNA transcriptome analysis reveals the immune regulatory mechanism of Crassostrea hongkongesis against Vibrio harveyi infection

MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate target-genes expression and play crucial roles in post-transcriptional regulation and immune system regulation. The Hong Kong oyster (Crassostrea hongkongesis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen host interaction. Vibrio harveyi, a Gram negative luminescent marine bacterium, is widely distributed in coastal water environments and can cause large-scale death of C. hongkongesis. However, little in formation is available on the immune regulatory mechanisms of C. hongkongesis infected with V. harveyi. Therefore, we performed microRNA transcriptome analysis for elucidating the immunoregulation mechanism of C. hongkongesis infected with V. harveyi. The results show that a total of 308468208 clean reads and 288371159 clean tags were obtained. 222 differentially expressed miRNAs were identified. A total of 388 target genes that were differentially expressed and negatively correlated with miRNA expression were predicted by 222 DEmiRs. GO enrichment analysis of 388 DETGs showed that they were mainly enriched in the immune-related term of membrane-bounded vesicle, endocytic vesicle lumen, antigen processing and presentation of exogenous peptide antigen via MHC class I, antigen processing and presentation of peptide antigen via MHC class I, and other immune-related term. KEGG enrichment analysis showed that DETGs were mainly enriched in the Complement and coagulation cascades, Herpes simplex virus 1 infection, Bacterial invasion of epithelial cells, Antigen processing and presentation and NOD-like receptor signaling pathway. The 16 key DEmiRs and their target genes form a regulatory network for seven immune-related pathways. These results suggest that V. harveyi infection induces a complex miRNA response with wide-ranging effects on immune gene expression in the C. hongkongesis. This study explored the immune response of C. hongkongesis to V. harveyi infection at the level of miRNAs, which provides new ideas for the healthy culture and selective breeding of C. hongkongesis.

Oral aged garlic (Allium sativum) alleviates ulcerative colitis in mice by improving gut homeostasis.

Aged garlic, obtained by heating raw garlic (Allium sativum) under high temperature and controlled humidity for a period, possesses a wide range of bioactivities, but its role in ulcerative colitis and its mechanism are not fully elucidated. We investigated the bioactive constituents in aged garlic (AG) and explored the effect of oral AG delivery on DSS-induced murine colitis. The results revealed that the aging process up-regulated anti-oxidative, anti-inflammatory and anti-microbial compounds such as dihydrocaffeic acid, 5-acetylsalicylic acid, verticine, S-allyl-L-cysteine and D-fucose. Oral AG obviously alleviated colitis, reducing colon damage and enhancing anti-oxidative and anti-inflammatory effects. Escherichia coli and Streptococcus equinus dramatically were enriched in the colon of mice with colitis that were strongly associated with Parkinson's disease, bacterial invasion of epithelial cells, aerobactin biosynthesis, and heme biosynthesis, but a distinct AG-mediated alteration in the colon due to increasing abundance of Akkermansia muciniphila, Lactobacillus sp. L-YJ, Bifidobacterium breve, Blautia wexlerae, Desulfomicrobium sp. P100A, and Lentilactobacillus hilgardii was observed. Next, we demonstrated that colonic microbiome reconstruction by oral AG significantly increased the production of short-chain fatty acids such as acetic acid, propionic acid, isobutyric acid, and isovaleric acid. This study provides the first data indicating that oral AG ameliorates colonic inflammation in a gut microbiota-dependent manner. Our findings provide novel insights into the AG-mediated remission of colitis and AG as a functional food for modulating the gut microbiota to prevent and treat colitis.

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive “contact-IAMs” involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.

LncRNA HOTAIR down-expression inhibits the invasion and tumorigenicity of epithelial ovarian cancer cells by suppressing TGF-β1 and ZEB1

BackgroundEpithelial ovarian cancer (EOC) is a pathological type with a higher mortality rate among gynecological cancers today. Long-chain noncoding RNAs (lncRNAs) can regulate the transcription and expression of cellular genes. However, the downstream molecules regulated by lncRNA HOTAIR have not been well studied. The effects of downregulated lncRNA HOTAIR on EOC invasiveness and tumorigenicity in nude mice, along with TGF- β1 and ZEB1 in epithelial ovarian cancer cells, need to be investigated in further research.ResultsRT-qPCR was used to detect lncRNA HOTAIR and TGF-β1 and ZEB1 mRNA expression in EOC SKOV3 cells. The expression of lncRNA HOTAIR in SKOV3 cells transfected with the recombinant shHOTAIR interference plasmid was significantly lower than that of the negative control. Compared with the negative control, the matrix gel invasion ability of shHOTAIR SKOV3 cells in vitro and their tumorigenicity in nude mice were significantly reduced. Moreover, compared with the control, the expression of ZEB1 protein in shHOTAIR-SKOV3 xenograft tumors was significantly reduced. Downregulation of lncRNA HOTAIR expression significantly reduced TGF-β1 and ZEB1 mRNA expression, but increased the expression of E-cadherin mRNA. In summary, downregulated lncRNA HOTAIR in EOC SKOV3 cells transfected with shHOTAIR can inhibit TGF-β1, reduce ZEB1, increase E-cadherin, and significantly reduce the invasiveness and tumorigenicity of ovarian epithelial cancer SKOV3 cells.ConclusionsThese results suggest that the lncRNA HOTAIR may be an effective target for the treatment of human EOC.