Computational insights into allosteric inhibition of focal adhesion kinase: A combined pharmacophore modeling and molecular dynamics approach.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that modulates integrin and growth factor signaling pathways and is implicated in cancer cell migration, proliferation, and survival. Over the past decade various, FAK kinase, FERM, and FAT domain inhibitors have been reported and a few kinase domain inhibitors are under clinical consideration. However, few of them were identified as multikinase inhibitors. In kinase drug design selectivity is always a point of concern, to improve selectivity allosteric inhibitor development is the best choice. The current research utilized a pharmacophore modeling (PM) approach to identify novel allosteric inhibitors of FAK. The all-available allosteric inhibitor bound 3D structures with PDB ids 4EBV, 4EBW, and 4I4F were utilized for the pharmacophore modeling. The validated PM models were utilized to map a database of 770,550 compounds prepared from ZINC, EXIMED, SPECS, ASINEX, and InterBioScreen, aiming to identify potential allosteric inhibitors. The obtained compounds from screening step were forwarded to molecular docking (MD) for the prediction of binding orientation inside the allosteric site and the results were evaluated with the known FAK allosteric inhibitor (REF). Finally, 14 FAK-inhibitor complexes were selected from the docking study and were studied under molecular dynamics simulations (MDS) for 500 ns. The complexes were ranked according to binding free energy (BFE) and those demonstrated higher affinity for allosteric site of FAK than REF inhibitors were selected. The selected complexes were further analyzed for intermolecular interactions and finally, three potential allosteric inhibitor candidates for the inhibition of FAK protein were identified. We believe that identified scaffolds may help in drug development against FAK as an anticancer agent.

Functional Analysis of Membrane-Associated Scaffolding Tight Junction (TJ) Proteins in Tumorigenic Characteristics of B16-F10 Mouse Melanoma Cells.

Tight junction (TJ) proteins (Tjps), Tjp1 and Tjp2, are tight junction-associated scaffold proteins that bind to the transmembrane proteins of tight junctions and the underlying cytoskeleton. In this study, we first analyzed the tumorigenic characteristics of B16-F10 melanoma cells, including cell proliferation, migration, invasion, metastatic potential, and the expression patterns of related proteins, after the CRISPR-Cas9-mediated knockout (KO) of Tjp genes. The proliferation of Tjp1 and Tjp2 KO cells significantly increased in vitro. Other tumorigenic characteristics, including migration and invasion, were significantly enhanced in Tjp1 and Tjp2 KO cells. Zonula occludens (ZO)-associated protein Claudin-1 (CLDN-1), which is a major component of tight junctions and functions in controlling cell-to-cell adhesion, was decreased in Tjp KO cells. Additionally, Tjp KO significantly stimulated tumor growth and metastasis in an in vivo mouse model. We performed a transcriptome analysis using next-generation sequencing (NGS) to elucidate the key genes involved in the mechanisms of action of Tjp1 and Tjp2. Among the various genes affected by Tjp KO-, cell cycle-, cell migration-, angiogenesis-, and cell-cell adhesion-related genes were significantly altered. In particular, we found that the Ninjurin-1 (Ninj1) and Catenin alpha-1 (Ctnna1) genes, which are known to play fundamental roles in Tjps, were significantly downregulated in Tjp KO cells. In summary, tumorigenic characteristics, including cell proliferation, migration, invasion, tumor growth, and metastatic potential, were significantly increased in Tjp1 and Tjp2 KO cells, and the knockout of Tjp genes significantly affected the expression of related proteins.

Revolutionizing Drug Targeting Strategies: Integrating Artificial Intelligence and Structure-Based Methods in PROTAC Development.

PROteolysis TArgeting Chimera (PROTAC) is an emerging technology in chemical biology and drug discovery. This technique facilitates the complete removal of the target proteins that are "undruggable" or challenging to target through chemical molecules via the Ubiquitin-Proteasome System (UPS). PROTACs have been widely explored and outperformed not only in cancer but also in other diseases. During the past few decades, several academic institutes and pharma companies have poured more efforts into PROTAC-related technologies, setting the stage for several major degrader trial readouts in clinical phases. Despite their promising results, the formation of robust ternary orientation, off-target activity, poor permeability, and binding affinity are some of the limitations that hinder their development. Recent advancements in computational technologies have facilitated progress in the development of PROTACs. Researchers have been able to utilize these technologies to explore a wider range of E3 ligases and optimize linkers, thereby gaining a better understanding of the effectiveness and safety of PROTACs in clinical settings. In this review, we briefly explore the computational strategies reported to date for the formation of PROTAC components and discuss the key challenges and opportunities for further research in this area.

Anticancer Effects of 6-Gingerol through Downregulating Iron Transport and PD-L1 Expression in Non-Small Cell Lung Cancer Cells.

Iron homeostasis is considered a key factor in human metabolism, and abrogation in the system could create adverse effects, including cancer. Moreover, 6-gingerol is a widely used bioactive phenolic compound with anticancer activity, and studies on its exact mechanisms on non-small cell lung cancer (NSCLC) cells are still undergoing. This study aimed to find the mechanism of cell death induction by 6-gingerol in NSCLC cells. Western blotting, real-time polymerase chain reaction, and flow cytometry were used for molecular signaling studies, and invasion and tumorsphere formation assay were also used with comet assay for cellular processes. Our results show that 6-gingerol inhibited cancer cell proliferation and induced DNA damage response, cell cycle arrest, and apoptosis in NSCLC cells, and cell death induction was found to be the mitochondrial-dependent intrinsic apoptosis pathway. The role of iron homeostasis in the cell death induction of 6-gingerol was also investigated, and iron metabolism played a vital role in the anticancer ability of 6-gingerol by downregulating EGFR/JAK2/STAT5b signaling or upregulating p53 and downregulating PD-L1 expression. Also, 6-gingerol induced miR-34a and miR-200c expression, which may indicate regulation of PD-L1 expression by 6-gingerol. These results suggest that 6-gingerol could be a candidate drug against NSCLC cells and that 6-gingerol could play a vital role in cancer immunotherapy.

Inhibition of Urban Particulate Matter-Induced Airway Inflammation by RIPK3 through the Regulation of Tight Junction Protein Production.

Urban particulate matter (UPM) is a high-hazard cause of various diseases in humans, including in the respiratory tract, skin, heart, and even brain. Unfortunately, there is no established treatment for the damage caused by UPM in the respiratory epithelium. In addition, although RIPK3 is known to induce necroptosis, its intracellular role as a negative regulator in human lungs and bronchial epithelia remains unclear. Here, the endogenous expression of RIPK3 was significantly decreased 6 h after exposure to UPM. In RIPK3-ovexpressed cells, RIPK3 was not moved to the cytoplasm from the nucleus. Interestingly, the overexpression of RIPK3 dramatically decreased TEER and F-actin formation. Its overexpression also decreased the expression of genes for pro-inflammatory cytokines (IL-6 and IL-8) and tight junctions (ZO-1, -2, -3, E-cadherin, and claudin) during UPM-induced airway inflammation. Importantly, overexpression of RIPK3 inhibited the UPM-induced ROS production by inhibiting the activation of iNOS and eNOS and by regulating mitochondrial fission processing. In addition, UPM-induced activation of the iκB and NF-κB signaling pathways was dramatically decreased by RIPK3, and the expression of pro-inflammatory cytokines was decreased by inhibiting the iκB signaling pathway. Our data indicated that RIPK3 is essential for the UPM-induced inflammatory microenvironment to maintain homeostasis. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for UPM-induced pulmonary inflammation.

PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCß3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

ß-Asarone Alleviates High-Glucose-Induced Oxidative Damage via Inhibition of ROS Generation and Inactivation of the NF-κB/NLRP3 Inflammasome Pathway in Human Retinal Pigment Epithelial Cells.

Diabetic retinopathy (DR) is the leading cause of vision loss and a major complication of diabetes. Hyperglycemia-induced accumulation of reactive oxygen species (ROS) is an important risk factor for DR. ß-asarone, a major component of volatile oil extracted from Acori graminei Rhizoma, exerts antioxidant effects; however, its efficacy in DR remains unknown. In this study, we investigated whether ß-asarone inhibits high-glucose (HG)-induced oxidative damage in human retinal pigment epithelial (RPE) ARPE-19 cells. We found that ß-asarone significantly alleviated cytotoxicity, apoptosis, and DNA damage in HG-treated ARPE-19 cells via scavenging of ROS generation. ß-Asarone also significantly attenuated the excessive accumulation of lactate dehydrogenase and mitochondrial ROS by increasing the manganese superoxide dismutase and glutathione activities. HG conditions markedly increased the release of interleukin (IL)-1ß and IL-18 and upregulated their protein expression and activation of the nuclear factor-kappa B (NF-κB) signaling pathway, whereas ß-asarone reversed these effects. Moreover, expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome multiprotein complex molecules, including thioredoxin-interacting protein, NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain, and cysteinyl aspartate-specific proteinase-1, were increased in ARPE-19 cells under HG conditions. However, their expression levels remained similar to those in the control group in the presence of ß-asarone. Therefore, ß-asarone protects RPE cells from HG-induced injury by blocking ROS generation and NF-κB/NLRP3 inflammasome activation, indicating its potential as a therapeutic agent for DR treatment.

GNAQ-Regulated ZO-1 and ZO-2 Act as Tumor Suppressors by Modulating EMT Potential and Tumor-Repressive Microenvironment in Lung Cancer.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in the development and progression of lung cancer by promoting its invasiveness and metastasis. Using integrative analyses of the public lung cancer database, we found that the expression levels of the tight junction proteins, zonula occluden (ZO)-1 and ZO-2, were lower in lung cancer tissues, including both lung adenocarcinoma and lung squamous cell carcinoma than in normal lung tissues analyzed using The Cancer Genome Atlas (TCGA). Although the ectopic expression or knockdown of ZO-1 and ZO-2 did not affect the growth of lung cancer cells, they significantly regulated cell migration and invasion. When M0 macrophages were co-cultured with ZO-1 or ZO-2 knockdown Calu-1 cells, M2-like polarization was efficiently induced. Conversely, co-culture of M0 THP-1 cells with A549 cells stably expressing ZO-1 or ZO-2 significantly reduced M2 differentiation. We also identified G protein subunit alpha q (GNAQ) as a potential ZO-1- and ZO-2-specific activator through analysis of correlated genes with the TCGA lung cancer database. Our results suggest that the GNAQ-ZO-1/2 axis may play a tumor-suppressive role in lung cancer development and progression and highlight ZO-1 and ZO-2 as key EMT- and tumor microenvironment-suppressive proteins. These findings provide new insights for the development of targeted therapies for lung cancer.

Nrf2-mediated activation of HO-1 is required in the blocking effect of compound K, a ginseng saponin metabolite, against oxidative stress damage in ARPE-19 human retinal pigment epithelial cells.

Background: The beneficial effects of compound K (CK) on different chronic diseases have been shown to be at least related to antioxidant action. Nevertheless, since its antioxidant activity in human retinal pigment epithelial (RPE) cells is still unknown, here we investigated whether CK alleviates oxidative stress-stimulated damage in RPE ARPE-19 cells. Methods: The cytoprotective consequence of CK in hydrogen peroxide (H2O2)-treated cells was evaluated by cell viability, DNA damage, and apoptosis assays. Fluorescence analysis and immunoblotting were performed to investigate the inhibitory action of CK on reactive oxygen species (ROS) production and mitochondrial dysfunction. Results: H2O2-promoted cytotoxicity, oxidative stress, DNA damage, mitochondrial impairment, and apoptosis were significantly attenuated by CK in ARPE-19 cells. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation level and its shuttling to the nucleus were increased, which was correlated with upregulated activation of heme oxygenase-1 (HO-1). However, zinc protoporphyrin, a blocker of HO-1, significantly abrogated the preventive action of CK in H2O2-treated ARPE-19 cells. Conclusion: This study indicates that activation of Nrf2/HO-1 signaling by CK plays an important role in rescuing ARPE-19 cells from oxidative cellular damage.

Inhibition of Pseudomonas aeruginosa LPS-Induced airway inflammation by RIPK3 in human airway.

Although the physiological function of receptor-interacting protein kinase (RIPK) 3 has emerged as a critical mediator of programmed necrosis/necroptosis, the intracellular role it plays as an attenuator in human lungs and human bronchial epithelia remains unclear. Here, we show that the expression of RIPK3 dramatically decreased in the inflamed tissues of human lungs, and moved from the nucleus to the cytoplasm. The overexpression of RIPK3 dramatically increased F-actin formation and decreased the expression of genes for pro-inflammatory cytokines (IL-6 and IL-1ß), but not siRNA-RIPK3. Interestingly, whereas RIPK3 was bound to histone 1b without LPS stimulation, the interaction between them was disrupted after 15 min of LPS treatment. Histone methylation could not maintain the binding of RIPK3 and activated movement towards the cytoplasm. In the cytoplasm, overexpressed RIPK3 continuously attenuated pro-inflammatory cytokine gene expression by inhibiting NF-κB activation, preventing the progression of inflammation during Pseudomonas aeruginosa infection. Our data indicated that RIPK3 is critical for the regulation of the LPS-induced inflammatory microenvironment. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for bacterial infection-induced pulmonary inflammation.

Induction of apoptosis through inactivation of ROS-dependent PI3K/Akt signaling pathway by platycodin D in human bladder urothelial carcinoma cells.

Platycodin D (PD) is a triterpenoid saponin, a major bioactive constituent of the roots of Platycodon grandiflorum, which is well known for possessing various pharmacological properties. However, the anti-cancer mechanism of PD in bladder cancer cells remains poorly understood. In the current study, we investigated the effect of PD on the growth of human bladder urothelial carcinoma cells. PD treatment significantly reduced the cell survival of bladder cancer cells associated with induction of apoptosis and DNA damage. PD inhibited the expression of inhibitor of apoptosis family members, activated caspases, and induced cleavage of poly (ADP-ribose) polymerase. PD also increased the release of cytochrome c into the cytoplasm by disrupting the mitochondrial membrane potential while upregulating the expression ratio of Bax to Bcl-2. The PD-mediated anti-proliferative effect was significantly inhibited by pre-treatment with a pancaspase inhibitor, but not by an inhibitor of necroptosis. Moreover, PD suppressed the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and the apoptosis-inducing effect of PD was further enhanced by a PI3K inhibitor. In addition, PD increased the accumulation of reactive oxygen species (ROS), whereas N-acetyl cysteine (NAC), an ROS inhibitor, significantly attenuated the growth inhibition and inactivation of the PI3K/Akt/mTOR signaling caused by PD. Furthermore, NAC significantly suppressed apoptosis, DNA damage, and decreased cell viability induced by PD treatment. Collectively, our findings indicated that PD blocked the growth of bladder urothelial carcinoma cells by inducing ROS-mediated inactivation of the PI3K/Akt/mTOR signaling.

Protection against Oxidative Stress-Induced Apoptosis by Fermented Sea Tangle (Laminaria japonica Aresch) in Osteoblastic MC3T3-E1 Cells through Activation of Nrf2 Signaling Pathway.

The purpose of the present study was to explore the efficacy of fermented extract of sea tangle (Laminaria japonica Aresch, FST) with Lactobacillus brevis on DNA damage and apoptosis in hydrogen peroxide (H2O2)-stimulated osteoblastic MC3T3-E1 cells and clarify related signaling pathways. Our results showed that exposure to FST significantly improved cell viability, inhibited apoptosis, and suppressed the generation of reactive oxygen species (ROS) in H2O2-stimulated cells. In addition, H2O2 triggered DNA damage in MC3T3-E1 cells was markedly attenuated by FST pretreatment. Moreover, H2O2-induced mitochondrial dysfunctions associated with apoptotic events, including loss of mitochondrial membrane potential (MMP), decreased Bcl-2/Bcl-2 associated x-protein (Bax) ratio, and cytosolic release of cytochrome c, were reduced in the presence of FST. FST also diminished H2O2-induced activation of caspase-3, which was associated with the ability of FST to protect the degradation of poly (ADP-ribose) polymerase. Furthermore, FST notably enhanced nuclear translocation and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) in the presence of H2O2 with concomitant upregulation of heme oxygenase-1 (HO-1) expression. However, artificial blockade of this pathway by the HO-1 inhibitor, zinc protoporphyrin IX, greatly abolished the protective effect of FST against H2O2-induced MC3T3-E1 cell injury. Taken together, these results demonstrate that FST could protect MC3T3-E1 cells from H2O2-induced damage by maintaining mitochondrial function while eliminating ROS along with activation of the Nrf2/HO-1 antioxidant pathway.

Expression profiles of human endogenous retrovirus (HERV)-K and HERV-R Env proteins in various cancers.

The vertebrate genome contains an endogenous retrovirus that has been inherited from the past millions of years. Although approximately 8% of human chromosomal DNA consists of sequences derived from human endogenous retrovirus (HERV) fragments, most of the HERVs are currently inactive and noninfectious due to recombination, deletions, and mutations after insertion into the host genome. Several studies suggested that Human endogenous retroviruses (HERVs) factors are significantly related to certain cancers. However, only limited studies have been conducted to analyze the expression of HERV derived elements at protein levels in certain cancers. Herein, we analyzed the expression profiles of HERV-K envelope (Env) and HERV-R Env proteins in eleven different kinds of cancer tissues. Furthermore, the expression patterns of both protein and correlation with various clinical data in each tissue were analyzed. The expressions of both HERV-K Env and HERV-R Env protein were identified to be significantly high in most of the tumors compared with normal surrounding tissues. Correlations between HERV Env expressions and clinical investigations varied depending on the HERV types and cancers. Overall expression patterns of HERV-K Env and HERV-R Env proteins were different in every individual but a similar pattern of expressions was observed in the same individual. These results demonstrate the expression profiles of HERV-K and HERV-R Env proteins in various cancer tissues and provide a good reference for the association of endogenous retroviral Env proteins in the progression of various cancers. Furthermore, the results elucidate the relationship between HERV-Env expression and the clinical significance of certain cancers. [BMB Reports 2021; 54(7): 368-373].

Urban Aerosol Particulate Matter Promotes Necrosis and Autophagy via Reactive Oxygen Species-Mediated Cellular Disorders that are Accompanied by Cell Cycle Arrest in Retinal Pigment Epithelial Cells.

Urban particulate matter (UPM) is recognized as a grave public health problem worldwide. Although a few studies have linked UPM to ocular surface diseases, few studies have reported on retinal dysfunction. Thus, the aim of the present study was to evaluate the influence of UPM on the retina and identify the main mechanism of UPM toxicity. In this study, we found that UPM significantly induced cytotoxicity with morphological changes in ARPE-19 human retinal pigment epithelial (RPE) cells and increased necrosis and autophagy but not apoptosis. Furthermore, UPM significantly increased G2/M arrest and simultaneously induced alterations in cell cycle regulators. In addition, DNA damage and mitochondrial dysfunction were remarkably enhanced by UPM. However, the pretreatment with the potent reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) effectively suppressed UPM-mediated cytotoxicity, necrosis, autophagy, and cell cycle arrest. Moreover, NAC markedly restored UPM-induced DNA damage and mitochondrial dysfunction. Meanwhile, UPM increased the expression of mitophagy-regulated proteins, but NAC had no effect on mitophagy. Taken together, although further studies are needed to identify the role of mitophagy in UPM-induced RPE injury, the present study provides the first evidence that ROS-mediated cellular damage through necrosis and autophagy is one of the mechanisms of UPM-induced retinal disorders.

The PDZ motif peptide of ZO-1 attenuates Pseudomonas aeruginosa LPS-induced airway inflammation.

Pseudomonas aeruginosa is known to play a role in many human diseases. Therefore, examining the negative control mechanisms of tight junction protein ZO-1 on the exotoxin LPS of P. aeruginosa-induced diseases could be critical in the development of novel therapeutics. We found that ZO-1 expression dramatically decreased in inflammatory human lung tissues. Interestingly, PDZ1 deletion of the PDZ domain in the ZO-1 protein dramatically decreased LPS-induced F-actin formation and increased the expression of genes for pro-inflammatory cytokines, but not PDZ2 and PDZ3 of the ZO-1 protein. We also found that the consensus PDZ peptide (based on PDZ1) of ZO-1 down-regulates the expression of pro-inflammatory cytokine genes and F-actin formation; in contrast, the GG24,25AA mutant PDZ peptide cannot control these genes. LPS activates IL-8 secretion extracellularly in a time-dependent manner, while the secretion is inhibited by PDZ peptide. Whereas increased IL-8 secretion by LPS activates the CXCR2 receptor, overexpressed RGS12 negatively regulates LPS-induced CXCR2/IL-8 signaling. The PDZ peptide also decreases LPS-induced inflammatory cell populations, pro-inflammatory cytokine gene expression, and TEER in bronchoalveolar lavage fluid and cultured alveolar macrophages. Collectively, we suggest that the PDZ peptide may be a potential therapeutic for bacteria-induced respiratory diseases.

Down-regulation of diesel particulate matter-induced airway inflammation by the PDZ motif peptide of ZO-1.

Although diesel airborne particulate matter (PM2.5) has been known to play a role in many human diseases, there is no direct evidence that therapeutic drugs or proteins can diminish PM2.5-induced diseases. Nevertheless, studies examining the negative control mechanisms of PM2.5-induced diseases are critical to develop novel therapeutic medications. In this study, the consensus PDZ peptide of ZO-1 inhibited PM2.5-induced inflammatory cell infiltration, pro-inflammatory cytokine gene expression, and TEER in bronchoalveolar lavage (BAL) fluid and AM cells. Our data indicated that the PDZ domain in ZO-1 is critical for regulation of the PM2.5-induced inflammatory microenvironment. Therefore, the PDZ peptide may be a potential therapeutic candidate during PM-induced respiratory diseases.

Diesel particulate matter2.5 promotes epithelial-mesenchymal transition of human retinal pigment epithelial cells via generation of reactive oxygen species.

Although several studies have linked PM2.5 (particulate matter with a diameter less than 2.5 µm) to ocular surface diseases such as keratitis and conjunctivitis, very few studies have previously addressed its effect on the retina. Therefore, the aim of this study was to evaluate the effect of PM2.5 on epithelial-mesenchymal transition (EMT), a process involved in disorders of the retinal pigment epithelial (RPE) on APRE-19 cells. PM2.5 changed the phenotype of RPE cells from epithelial to fibroblast-like mesenchymal, and increased cell migration. Exposure to PM2.5 markedly increased the expression of mesenchymal markers, but reduced the levels of epithelial markers. Moreover, PM2.5 promoted the phosphorylation of MAPKs and the expression of transforming growth factor-ß (TGF-ß)-mediated nuclear transcriptional factors. However, these PM2.5-mediated changes were completely reversed by LY2109761, a small molecule inhibitor of the TGF-ß receptor type I/II kinases, and N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger. Interestingly, NAC, but not LY2109761, effectively restored the PM2.5-induced mitochondrial defects, including increased ROS, decreased mitochondrial activity, and mitochondrial membrane potential disruption. Collectively, our findings indicate that the TGF-ß/Smad/ERK/p38 MAPK signaling pathway is activated downstream of cellular ROS during PM2.5-induced EMT. The present study provides the first evidence that EMT of RPE may be one of the mechanisms of PM2.5-induced retinal dysfunction.

Specific bacteriophage of Bordetella bronchiseptica regulates B. bronchiseptica-induced microRNA expression profiles to decrease inflammation in swine nasal turbinate cells.

BACKGROUND: Respiratory diseases in pigs are the main health concerns for swine producers. Similar to the diseases in human and other animals, respiratory diseases are primary related to morbidity and are the result of infection with bacteria, viruses, or both. B. bronchiseptica causes serious respiratory diseases in the swine airway track. However, the B. bronchiseptica-specific bacteriophage has diverse advantages such as decreasing antibiotic overuse and possible therapeutic potential against bacteria. OBJECTIVE: The objects of this study were to investigate the therapeutic effect of specific B. bronchiseptica bacteriophages and to identify genes related to bacteriophage signaling utilizing RNA microarrays in swine nasal turbinate cells. METHODS: Bor-BRP-1 phages were applied 24 h prior to B.bronchiseptica infection (1 × 107 cfu/ml) at several concentrations of bacterial infection. Cells were incubated to detect cytokines and 24 h to detect mucin production. And real-time quantitative PCR was performed to examine related genes expression. To determine the change of total gene expression based on B.bronchiseptica and Bor-BRP-1 treatment, we performed RNA sequencing experiments. RESULTS: The results showed that B. bronchiseptica induced increased expression of several inflammatory genes such as IL-1ß, IL-6, and Muc1 in a dose-dependent manner. However, Bor-BRP-1 induced reduction of gene expression compared to the B. bronchiseptica induction group. In addition, microarrays detected Bor-BRP-1-altered inflammatory gene expression against B. bronchiseptica, reducing B. bronchiseptica-induced airway inflammation in swine epithelial cells. CONCLUSION: These results suggest that the specific bacteriophage has a therapeutic potential to defend against B. bronchiseptica infection by altering inflammatory gene expression profiles.

Pasteurella multocida specific bacteriophage suppresses P. multocida-induced inflammation: identification of genes related to bacteriophage signaling by Pasteurella multocida-infected swine nasal turbinate cells.

BACKGROUND: Although Pasteurella multocida is highly prevalent pathogen in animals and plays an important role in swine respiratory diseases, only a few studies on the use of bacteriophages specific to Pasteurella multocida disease have been reported. OBJECTIVE: The object of this study was to investigate the therapeutic effect of specific P. multocida bacteriophages and to identify genes related to bacteriophage signaling utilizing RNA microarrays in swine nasal turbinate cells. METHODS: Pas-MUP-1 phages were applied 24 h prior to P. multocida infection (1 × 107 cfu/ml) at several concentrations of bacterial infection. Cells were incubated to detect cytokines and 24 h to detect mucin production. And real-time quantitative PCR was performed to examine related genes expression. To determine the change of total gene expression based on P. multocida and Pas-MUP-1 treatment, we performed RNA sequencing experiments. RESULTS: We found that P. multocida-infected PT-K75 cells show increased gene expression of IL-1ß, IL-6, and Muc1 in a dose-dependent manner. Interestingly, these genes resulted in decreased expression in P. multocida pretreated with the P. multocida-specific Pas-MUP-1 bacteriophage. RNA sequencing analysis revealed that bacteriophage administration regulated genes associated with immune and inflammatory responses, and the regulated genes were dramatically concentrated in the cytokine/chemokine-based signaling pathways. Pas-MUP-1 treatment was shown to regulate P. multocida induced gene expression in the bacteria. CONCLUSION: These results suggest the specific bacteriophage has therapeutic potential as an alternative to antibiotic treatment to defend against P. multocida infection by altering inflammatory gene expression profiles.

Exercise training reduces the risk of opportunistic infections after acute exercise and improves cytokine antigen recognition.

In general, acute exercise is thought to inhibit immune function and increase the risk of opportunistic infections, but there is some opposition to this due to a lack of quantitative evaluation. Therefore, we quantified the effect of exercise on immune function and observed the interaction between antigens and cytokines using an intramuscular infection with Trichinella spiralis (T. spiralis), a common parasitic infection model. C57BL/6 mice were used for a non-infection experiment and an infection (Inf) experiment. Each experiment was divided further into three groups: one control (CON) group, and an exercise pre-infection (PIE)-only group and exercise-sustained (ES) group, each of which was subjected to exercise for 7 weeks. All animals in the infection experiment were infected with T. spiralis 30 min after acute exercise. After infection, the ES and Inf-ES groups continued exercise for 7 additional weeks. The number of T. spiralis nurse cells remaining in skeletal muscles was fewer in the infected exercise groups compared with the infected control. Expression of interleukin-6 (IL-6) and interleukin-10 (IL-10) was higher in the Inf-CON group and transforming growth factor beta (TGF-ß) expression was lower in the Inf-CON group than in the CON group, as measured by RT-PCR. In the infection experiment, only IL-10 had significant differences between the groups. Immunofluorescence revealed that most cytokines were specifically expressed around the antigenic nurse cells following exercise. In conclusion, exercise training does not increase the risk of opportunistic infections even after acute exercise, but rather reduces it. These results may be due to antigen-specific immune responses.