Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress.

BACKGROUND: Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. RESULTS: At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10 mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. CONCLUSION: Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.

Bigger problems from smaller colonies: emergence of antibiotic-tolerant small colony variants of Mycobacterium avium complex in MAC-pulmonary disease patients.

BACKGROUND: Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria that includes Mycobacterium avium and Mycobacterium intracellulare. MAC pulmonary disease (MAC-PD) poses a threat to immunocompromised individuals and those with structural pulmonary diseases worldwide. The standard treatment regimen for MAC-PD includes a macrolide in combination with rifampicin and ethambutol. However, the treatment failure and disease recurrence rates after successful treatment remain high. RESULTS: In the present study, we investigated the unique characteristics of small colony variants (SCVs) isolated from patients with MAC-PD. Furthermore, revertant (RVT) phenotype, emerged from the SCVs after prolonged incubation on 7H10 agar. We observed that SCVs exhibited slower growth rates than wild-type (WT) strains but had higher minimum inhibitory concentrations (MICs) against multiple antibiotics. However, some antibiotics showed low MICs for the WT, SCVs, and RVT phenotypes. Additionally, the genotypes were identical among SCVs, WT, and RVT. Based on the MIC data, we conducted time-kill kinetic experiments using various antibiotic combinations. The response to antibiotics varied among the phenotypes, with RVT being the most susceptible, WT showing intermediate susceptibility, and SCVs displaying the lowest susceptibility. CONCLUSIONS: In conclusion, the emergence of the SCVs phenotype represents a survival strategy adopted by MAC to adapt to hostile environments and persist during infection within the host. Additionally, combining the current drugs in the treatment regimen with additional drugs that promote the conversion of SCVs to RVT may offer a promising strategy to improve the clinical outcomes of patients with refractory MAC-PD.

Subcellular localization and ER-mediated cytotoxic function of α1A and α1ACT in spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a polyglutamine (polyQ) disease, which is caused by the elongation of CAG repeats encoding polyQ in the CACNA1A gene. The CACNA1A gene encodes two proteins, namely, α1A (a subunit of the plasma membrane calcium channel), which is translated in its entire length, and α1ACT, which is translated from the second cistron, and both proteins have a polyQ tract. The α1A-polyQ and α1ACT-polyQ proteins with an elongated polyQ stretch have been reported to form aggregates in cells and induce neuronal cell death, but the subcellular localization of these proteins and their cytotoxic properties remain unclear. In this study, we first analyzed SCA6 model mice and found that α1A-polyQlong localized mainly to the Golgi apparatus, whereas a portion of α1ACT-polyQlong localized to the nucleus. Analysis using Neuro2a cells also showed similar subcellular localizations of these proteins, and a proportion of both proteins localized to the endoplasmic reticulum (ER). Cytotoxic studies demonstrated that both proteins induce both the ER stress response and apoptosis, indicating that they are able to induce ER stress-induced apoptosis.

Regulation of Benzo[a]pyrene-Induced Hepatic Lipid Accumulation through CYP1B1-Induced mTOR-Mediated Lipophagy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by lipid accumulation within the liver. The pathogenesis underlying its development is poorly understood. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon and a group 1 carcinogen. The aryl hydrocarbon receptor activation by B[a]P induces cytochrome P450 (CYP) enzymes, contributing to hepatic lipid accumulation. However, the molecular mechanism through which the B[a]P-mediated induction of CYP enzymes causes hepatic lipid accumulation is unknown. This research was conducted to elucidate the role of CYP1B1 in regulating B[a]P-induced lipid accumulation within hepatocytes. B[a]P increased hepatic lipid accumulation, which was mitigated by CYP1B1 knockdown. An increase in the mammalian target of rapamycin (mTOR) by B[a]P was specifically reduced by CYP1B1 knockdown. The reduction of mTOR increased the expression of autophagic flux-related genes and promoted phagolysosome formation. Both the expression and translocation of TFE3, a central regulator of lipophagy, were induced, along with the expression of lipophagy-related genes. Conversely, enhanced mTOR activity reduced TFE3 expression and translocation, which reduced the expression of lipophagy-related genes, diminished phagolysosome production, and increased lipid accumulation. Our results indicate that B[a]P-induced hepatic lipid accumulation is caused by CYP1B1-induced mTOR and the reduction of lipophagy, thereby introducing novel targets and mechanisms to provide insights for understanding B[a]P-induced MASLD.

In Silico-Based Design of a Hybrid Peptide with Antimicrobial Activity against Multidrug-Resistant Pseudomonas aeruginosa Using a Spider Toxin Peptide.

The escalating prevalence of antibiotic-resistant bacteria poses an immediate and grave threat to public health. Antimicrobial peptides (AMPs) have gained significant attention as a promising alternative to conventional antibiotics. Animal venom comprises a diverse array of bioactive compounds, which can be a rich source for identifying new functional peptides. In this study, we identified a toxin peptide, Lycotoxin-Pa1a (Lytx-Pa1a), from the transcriptome of the Pardosa astrigera spider venom gland. To enhance its functional properties, we employed an in silico approach to design a novel hybrid peptide, KFH-Pa1a, by predicting antibacterial and cytotoxic functionalities and incorporating the amino-terminal Cu(II)- and Ni(II) (ATCUN)-binding motif. KFH-Pa1a demonstrated markedly superior antimicrobial efficacy against pathogens, including multidrug-resistant (MDR) Pseudomonas aeruginosa, compared to Lytx-Pa1a. Notably, KFH-Pa1a exerted several distinct mechanisms, including the disruption of the bacterial cytoplasmic membrane, the generation of intracellular ROS, and the cleavage and inhibition of bacterial DNA. Additionally, the hybrid peptide showed synergistic activity when combined with conventional antibiotics. Our research not only identified a novel toxin peptide from spider venom but demonstrated in silico-based design of hybrid AMP with strong antimicrobial activity that can contribute to combating MDR pathogens, broadening the utilization of biological resources by incorporating computational approaches.

The Identification of a Novel Spider Toxin Peptide, Lycotoxin-Pa2a, with Antibacterial and Anti-Inflammatory Activities.

With the increasing challenge of controlling infectious diseases due to the emergence of antibiotic-resistant strains, the importance of discovering new antimicrobial agents is rapidly increasing. Animal venoms contain a variety of functional peptides, making them a promising platform for pharmaceutical development. In this study, a novel toxin peptide with antibacterial and anti-inflammatory activities was discovered from the spider venom gland transcriptome by implementing computational approaches. Lycotoxin-Pa2a (Lytx-Pa2a) showed homology to known-spider toxin, where functional prediction indicated the potential of both antibacterial and anti-inflammatory peptides without hemolytic activity. The colony-forming assay and minimum inhibitory concentration test showed that Lytx-Pa2a exhibited comparable or stronger antibacterial activity against pathogenic strains than melittin. Following mechanistic studies revealed that Lytx-Pa2a disrupts both cytoplasmic and outer membranes of bacteria while simultaneously inducing the accumulation of reactive oxygen species. The peptide exerted no significant toxicity when treated to human primary cells, murine macrophages, and bovine red blood cells. Moreover, Lytx-Pa2a alleviated lipopolysaccharide-induced inflammation in mouse macrophages by suppressing the expression of inflammatory mediators. These findings not only suggested that Lytx-Pa2a with dual activity can be utilized as a new antimicrobial agent for infectious diseases but also demonstrated the implementation of in silico methods for discovering a novel functional peptide, which may enhance the future utilization of biological resources.

Curcumin Disrupts a Positive Feedback Loop between ADMSCs and Cancer Cells in the Breast Tumor Microenvironment via the CXCL12/CXCR4 Axis.

Adipose tissue has a significant impact on breast cancer initiation and progression owing to its substantial proportion in the breast. Adipose-derived mesenchymal stem cells (ADMSCs) are major players in the breast tumor microenvironment (TME) as they interact with cancer cells. The intricate interaction between ADMSCs and cancer cells not only drives the differentiation of ADMSCs into cancer-associated fibroblasts (CAFs) but also the metastasis of cancer cells, which is attributed to the CXCL12/CXCR4 axis. We investigated the effects of curcumin, a flavonoid known for CXCL12/CXCR4 axis inhibition, on breast TME by analyzing whether it can disrupt the ADMSC-cancer positive loop. Using MCF7 breast cancer cell-derived conditioned medium (MCF7-CM), we induced ADMSC transformation and verified that curcumin diminished the phenotypic change, inhibiting CAF marker expression. Additionally, curcumin suppressed the CXCL12/CXCR4 axis and its downstream signaling both in ADMSCs and MCF7 cells. The CM from ADMSCs, whose ADMSC-to-CAF transformation was repressed by the curcumin treatment, inhibited the positive feedback loop between ADMSCs and MCF7 as well as epithelial-mesenchymal transition in MCF7. Our study showed that curcumin is a potent anti-cancer agent that can remodel the breast TME, thereby restricting the ADMSC-cancer positive feedback loop associated with the CXCL12/CXCR4 axis.

Development of droplet digital PCR-based detection of bacterial pathogens in prosthetic joint infection: a preliminary study using a synthesized model plasmid.

Periprosthetic joint infection (PJI) can be diagnosed to characterize the microorganisms constituting a biofilm, which is an essential procedure for proper treatment. The gold standard method for detecting and identifying the causative microorganism is culture of microorganisms from patients-derived sample.; however, this method takes a long time and has low sensitivity. To compensate for these limitations, identification methods based on real-time PCR (RT-PCR) have been widely used. However, RT-PCR also has limitations, including low sensitivity and the requirement of a standard curve for quantification. Therefore, to prevent significant proliferation of pathogenic bacteria, it is important to detect a limited number of infectious bacteria during early stages of PJI. In the present study, we developed droplet digital PCR-based detection of bacterial pathogens in PJI. And we evaluated the analytical performance of the assay using a model plasmid, based on the 16S ribosomal DNA sequence of target bacteria commonly found in PJI. We also prepared genomic DNA extracted from E. coli, S. aureus, and S. epidermidis to test whether ddPCR provides better sensitivity and quantification of the target sequences. ddPCR detected 400 attograms of target DNA, which was more than 10 times less than that detected by real-time PCR using synthesized plasmid. In addition, ddPCR detected target regions from genomic DNA of 50 femtograms for E. coli, 70 femtograms for S. epidermidis, and 90 femtograms for S. aureus. The results indicate that ddPCR has the potential to decrease the microbial detection limit and provide precise detection, signifying its effectiveness for early PJI.

Generation of a human embryonic stem cell reporter line, TMEM119-EGFP, for the visualisation of in vitro differentiated human microglia.

Transmembrane protein 119 (TMEM119) is a recently identified microglia marker that is not expressed by other immune cells. Using CRISPR/Cas9 technology, we introduced enhanced green fluorescence protein (EGFP), into the H9 WA-09 human embryonic stem cell line, directly before the TMEM119 stop codon. Sanger sequencing confirmed successful insertion of the EGFP sequence. The newly created cell line expressed a normal morphology and karyotype, several pluripotency markers, and the ability to differentiate into all three germ layers. H9-TMEM119-EGFP can be used to provide a deeper understanding of the role of TMEM119 in microglia by monitoring its expression under different experimental conditions.

In silico identification of novel antimicrobial peptides from the venom gland transcriptome of the spider Argiope bruennichi (Scopoli, 1772).

As the emergence and prevalence of antibiotic-resistant strains have resulted in a global crisis, there is an urgent need for new antimicrobial agents. Antimicrobial peptides (AMPs) exhibit inhibitory activity against a wide spectrum of pathogens and can be utilized as an alternative to conventional antibiotics. In this study, two novel AMPs were identified from the venom transcriptome of the spider Argiope bruennichi (Scopoli, 1772) using in silico methods, and their antimicrobial activity was experimentally validated. Aranetoxin-Ab2a (AATX-Ab2a) and Aranetoxin-Ab3a (AATX-Ab3a) were identified by homology analysis and were predicted to have high levels of antimicrobial activity based on in silico analysis. Both peptides were found to have antibacterial effect against Gram-positive and -negative strains, and, in particular, showed significant inhibitory activity against multidrug-resistant Pseudomonas aeruginosa isolates. In addition, AATX-Ab2a and AATX-Ab3a inhibited animal and vegetable fungal strains, while showing low toxicity to normal human cells. The antimicrobial activity of the peptides was attributed to the increased permeability of microbial membranes. The study described the discovery of novel antibiotic candidates, AATX-Ab2a and AATX-Ab3a, using the spider venom gland transcriptome, and validated an in silico-based method for identifying functional substances from biological resources.

Anti-heat shock protein 10 IgG in chronic spontaneous urticaria: Relation with miRNA-101-5p and platelet-activating factor.

BACKGROUND: Anti-heat shock protein (HSP) autoantibodies are detected in autoimmune diseases. We sought to ascertain whether anti-HSP10 IgG is present in patients with CSU and to elucidate the role of HSP10 in CSU pathogenesis. METHOD: Using a human proteome microarray, six potential autoantibodies had higher expression in 10 CSU samples compared with 10 normal controls (NCs). Among them, HSP10 IgG autoantibody was quantified by immune dot-blot assay in sera from 86 CSU patients and 44 NCs. The serum levels of HSP10 and microRNA-101-5p were measured in CSU patients and NCs. The effects of HSP10 and miR-101-5p on mast cell degranulation in response to IgE, compound 48/80, and platelet-activating factor (PAF) were investigated. RESULTS: CSU patients had higher IgG positivity to HSP10 (40.7% vs. 11.4%, p = .001), lower serum HSP10 levels (5.8 ± 3.6 vs. 12.2 ± 6.6 pg/mL, p < .001) than in NCs, and their urticaria severity was associated with anti-HSP10 IgG positivity, while HSP10 levels were related to urticaria control status. MiR-101-5p was increased in CSU patients. PAF enhanced IL4 production in PBMCs from CSU patients. IL-4 upregulated miR-101-5p and reduced HSP10 expression in keratinocytes. Transfection of miR-101-5p reduced HSP10 expression in keratinocytes. MiR-101-5p promoted PAF-induced mast cell degranulation, while HSP10 specifically prevented it. CONCLUSION: A new autoantibody, anti-HSP10 IgG was detected in CSU patients, which showed a significant correlation with UAS7 scores. A decreased serum HSP10 level was associated with upregulation of miR-101-5p due to increased IL-4 and PAF in CSU patients. Modulation of miR-101-5p and HSP10 may be a novel therapeutic approach for CSU.

A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting.

Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous Mycobacterium strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for Mycobacterium intracellulare, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of M. intracellulare ATCC 13950 in silico, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different M. intracellulare strains using an in silico test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 M. intracellulare clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95-98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in M. intracellulare strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous M. intracellulare.

Chronic Exposure to TDI Induces Cell Migration and Invasion via TGF-ß1 Signal Transduction.

Toluene diisocyanate (TDI) is commonly used in manufacturing, and it is highly reactive and causes respiratory damage. This study aims to identify the mechanism of tumorigenesis in bronchial epithelial cells induced by chronic TDI exposure. In addition, transcriptome analysis results confirmed that TDI increases transforming growth factor-beta 1 (TGF-ß1) expression and regulates genes associated with cancerous characteristics in bronchial cells. Our chronically TDI-exposed model exhibited elongated spindle-like morphology, a mesenchymal characteristic. Epithelial-mesenchymal transition (EMT) was evaluated following chronic TDI exposure, and EMT biomarkers increased concentration-dependently. Furthermore, our results indicated diminished cell adhesion molecules and intensified cell migration and invasion. In order to investigate the cellular regulatory mechanisms resulting from chronic TDI exposure, we focused on TGF-ß1, a key factor regulated by TDI exposure. As predicted, TGF-ß1 was significantly up-regulated and secreted in chronically TDI-exposed cells. In addition, SMAD2/3 was also activated considerably as it is the direct target of TGF-ß1 and TGF-ß1 receptors. Inhibiting TGF-ß1 signaling through blocking of the TGF-ß receptor attenuated EMT and cell migration in chronically TDI-exposed cells. Our results corroborate that chronic TDI exposure upregulates TGF-ß1 secretion, activates TGF-ß1 signal transduction, and leads to EMT and other cancer properties.

Comparative genetic characterization of CMY-2-type beta-lactamase producing pathogenic Escherichia coli isolated from humans and pigs suffering from diarrhea in Korea.

BACKGROUND: Pathogenic Escherichia coli are an important cause of bacterial infections in both humans and pigs and many of antimicrobials are used for the treatment of E. coli infection. The objective of this study was to investigate the characteristics and relationship between humans and pigs regarding third-generation cephalosporin resistance and CMY-2-producing E. coli in Korea. RESULTS: All 103 third-generation cephalosporin-resistant E. coli isolates showed multidrug resistance. Also, except for ß-lactam/ß-lactamase inhibitor combinations, all antimicrobials resistant rates were higher in pigs than in humans. A total of 36 isolates (humans: five isolates; pigs: 31 isolates) were positive for the CMY-2-encoding genes and thirty-two (88.9%) isolates detected class 1 integrons with 10 different gene cassette arrangements, and only 1 isolate detected a class 2 integron. The most common virulence genes in pigs were LT (71.0%), F18 (51.6%), and STb (51.6%), while stx2 (80.0%) was the most frequently detected gene in humans. Stx2 gene was also detected in pigs (6.5%). Interestingly, 36 CMY-2-producing E. coli isolates showed a high diversity of sequence types (ST), and ST88 was present in E. coli from both pigs (11 isolates) and humans (one isolate). CONCLUSION: Our findings suggest that a critical need for comprehensive surveillance of third-generation cephalosporin resistance is necessary to preserve the usefulness of third-generation cephalosporins in both humans and pigs.

Antigenic Determinant of Helicobacter pylori FlaA for Developing Serological Diagnostic Methods in Children.

The early diagnosis of Helicobacter pylori infection is important for gastric cancer prevention and treatment. Although endoscopic biopsy is widely used for H. pylori diagnosis, an accurate biopsy cannot be performed until a lesion becomes clear, especially in pediatric patients. Therefore, it is necessary to develop convenient and accurate methods for early diagnosis. FlaA, an essential factor for H. pylori survival, shows high antigenicity and can be used as a diagnostic marker. We attempted to identify effective antigens containing epitopes of high diagnostic value in FlaA. Full-sized FlaA was divided into several fragments and cloned, and its antigenicity was investigated using Western blotting. The FlaA fragment of 1345-1395 bp had strong immunogenicity. ELISA was performed with serum samples from children by using the 1345-1395 bp recombinant antigen fragment. IgG reactivity showed 90.0% sensitivity and 90.5% specificity, and IgM reactivity showed 100% sensitivity and specificity. The FlaA fragment of 1345-1395 bp discovered in the present study has antigenicity and is of high value as a candidate antigen for serological diagnosis. The FlaA 1345-1395 bp epitope can be used as a diagnostic marker for H. pylori infection, thereby controlling various gastric diseases such as gastric cancer and peptic ulcers caused by H. pylori.

Modulating macrophage function to reinforce host innate resistance against Mycobacterium avium complex infection.

Mycobacterium avium complex (MAC) is the main causative agent of infectious diseases in humans among nontuberculous mycobacteria (NTM) that are ubiquitous organisms found in environmental media such as soil as well as in domestic and natural waters. MAC is a primary causative agent of NTM-lung disease that threaten immunocompromised or structural lung disease patients. The incidence and the prevalence of M. tuberculosis infection have been reduced, while MAC infections and mortality rates have increased, making it a cause of global health concern. The emergence of drug resistance and the side effects of long-term drug use have led to a poor outcome of treatment regimens against MAC infections. Therefore, the development of host-directed therapy (HDT) has recently gained interest, aiming to accelerate mycobacterial clearance and reversing lung damage by employing the immune system using a novel adjuvant strategy to improve the clinical outcome of MAC infection. Therefore, in this review, we discuss the innate immune responses that contribute to MAC infection focusing on macrophages, chief innate immune cells, and host susceptibility factors in patients. We also discuss potential HDTs that can act on the signaling pathway of macrophages, thereby contributing to antimycobacterial activity as a part of the innate immune response during MAC infection. Furthermore, this review provides new insights into MAC infection control that modulates and enhances macrophage function, promoting host antimicrobial activity in response to potential HDTs and thus presenting a deeper understanding of the interactions between macrophages and MACs during infection.

De Novo Design of AC-P19M, a Novel Anticancer Peptide with Apoptotic Effects on Lung Cancer Cells and Anti-Angiogenic Activity.

Despite the current developments in cancer therapeutics, efforts to excavate new anticancer agents continue rigorously due to obstacles, such as side effects and drug resistance. Anticancer peptides (ACPs) can be utilized to treat cancer because of their effectiveness on a variety of molecular targets, along with high selectivity and specificity for cancer cells. In the present study, a novel ACP was de novo designed using in silico methods, and its functionality and molecular mechanisms of action were explored. AC-P19M was discovered through functional prediction and sequence modification based on peptide sequences currently available in the database. The peptide exhibited anticancer activity against lung cancer cells, A549 and H460, by disrupting cellular membranes and inducing apoptosis while showing low toxicity towards normal and red blood cells. In addition, the peptide inhibited the migration and invasion of lung cancer cells and reversed epithelial-mesenchymal transition. Moreover, AC-P19M showed anti-angiogenic activity through the inhibition of vascular endothelial growth factor receptor 2 signaling. Our findings suggest that AC-P19M is a novel ACP that directly or indirectly targets cancer cells, demonstrating the potential development of an anticancer agent and providing insights into the discovery of functional substances based on an in silico approach.

Quercetin and Isorhamnetin Reduce Benzo[a]pyrene-Induced Genotoxicity by Inducing RAD51 Expression through Downregulation of miR-34a.

Benzo[a]pyrene (B[a]P) is metabolized in the liver into highly reactive mutagenic and genotoxic metabolites, which induce carcinogenesis. The mutagenic factors, including B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and reactive oxygen species, generated during B[a]P metabolism can cause DNA damage, such as BPDE-DNA adducts, 8-oxo-dG, and double-strand breaks (DSBs). In this study, we mechanistically investigated the effects of quercetin and its major metabolite isorhamnetin on the repair of B[a]P-induced DNA DSBs. Whole-transcriptome analysis showed that quercetin and isorhamnetin each modulate the expression levels of genes involved in DNA repair, especially those in homologous recombination. RAD51 was identified as a key gene whose expression level was decreased in B[a]P-treated cells and increased by quercetin or isorhamnetin treatment. Furthermore, the number of γH2AX foci induced by B[a]P was significantly decreased by quercetin or isorhamnetin, whereas RAD51 mRNA and protein levels were increased. Additionally, among the five microRNAs (miRs) known to downregulate RAD51, miR-34a level was significantly downregulated by quercetin or isorhamnetin. The protective effect of quercetin or isorhamnetin was lower in cells transfected with a miR-34a mimic than in non-transfected cells, and the B[a]P-induced DNA DSBs remained unrepaired. Our results show that quercetin and isorhamnetin each upregulates RAD51 by downregulating miR-34a and thereby suppresses B[a]P-induced DNA damage.

Immunomodulatory Effect and Bone Homeostasis Regulation in Osteoblasts Differentiated from hADMSCs via the PD-1/PD-L1 Axis.

Human mesenchymal stem cells (hMSCs) are promising candidates for stem cell therapy and are known to secrete programmed death-1 (PD-1) ligand 1 (PD-L1) regulating T cell-mediated immunosuppression. Given the limitations of current stem cell therapy approaches, improvements in immunomodulatory capacity and stem cell differentiation efficacy are needed. In this study, we propose novel strategies to overcome the challenges that remain in hMSC-mediated bone regeneration. We found that PD-1 is highly expressed in osteoblasts, and the PD-1/PD-L1 axis mediated the decreased proinflammatory cytokine expressions in differentiated osteoblasts cocultured with human adipose derived mesenchymal stem cells (hADMSCs). Moreover, the decrease was attenuated by PD-1/PD-L1 pathway inhibition. Osteogenic properties including osteogenic gene expression and calcium deposits were increased in osteoblasts cocultured with hADMSCs compared with those that were monocultured. Osteoblasts treated with PD-L1 and exosomes from hADMSCs also exhibited enhanced osteogenic properties, including calcium deposits and osteogenic gene expression. In our cocultured system that mimics the physiological conditions of the bone matrix, the PD-1/PD-L1 axis mediated the increased expression of osteogenic genes, thereby enhancing the osteogenic properties, while the calcium deposits of osteoblasts were maintained. Our results provide the therapeutic potentials and novel roles of the PD-1/PD-L1 axis in bone matrix for modulating the bone properties and immunosuppressive potentials that can aid in the prevention of bone diseases via maintaining bone homeostasis.