Histone Deacetylase Inhibitors Restore the Odontogenic Differentiation Potential of Dental Pulp Stem Cells under Hyperglycemic Conditions.

OBJECTIVE: Complications arising from diabetes can result in stem cell dysfunction, impairing their ability to undergo differentiation into various cellular lineages. The present study evaluated the effect of histone deacetylase inhibitors, Valproic acid and Trichostatin A, on the odontogenic differentiation potential of dental pulp stem cells under hyperglycemic conditions. METHODS: Streptozotocin (STZ) induced diabetes mellitus in 12 male Wistar rats. Dental parameters were examined using micro-computed tomography. The odontogenic potential of human pulp stem cells exposed to 30 mM glucose was assessed through alkaline phosphatase assays, examination of gene expression for dentin matrix protein 1 and dentin sialoprotein using real-time PCR, and alizarin red staining for calcium deposition. RESULTS: Along with reduced dentin thickness and root length in diabetic rats, the results revealed a significant increase in histone deacetylase 3 and 2 gene expressions in isolated diabetic pulp tissues compared to the control groups. The gene expression of odontogenic-related markers and alkaline phosphatase activity in human cultured pulp stem cells under hyperglycemic conditions significantly decreased. Adding Valproic acid and Trichostatin A restored the odontogenic differentiation markers, including calcium deposition, gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and alkaline phosphatase activity. CONCLUSION: The data suggests that hyperglycemic conditions negatively impact the odontogenic potential of pulp mesenchymal stem cells. However, histone deacetylase inhibitors improve the impaired odontogenic differentiation capacity. This study implies that histone deacetylases may represent a potential therapeutic target for enhancing the regenerative mineralization of pulp cells in diabetic patients.

In Vitro and Ex Vivo Investigation of the Antibacterial Effects of Methylene Blue against Methicillin-Resistant Staphylococcus aureus.

Methylene blue (MB) is a water-soluble dye that has a number of medical applications. Methicillin-resistant Staphylococcus aureus (MRSA) was selected as a subject for research due to the numerous serious clinical diseases it might cause and because there is a significant global resistance challenge. Our main goal was to determine and analyze the antibacterial effects of MB against S. aureus both in vitro and ex vivo to enhance treatment options. A total of 104 MRSA isolates recovered from various clinical specimens were included in this study. Minimum inhibitory concentration (MIC) values of MB against MRSA isolates were determined by the agar dilution method. One randomly selected MRSA isolate and a methicillin-susceptible S. aureus strain (S. aureus ATCC 25923) were employed for further evaluation of the antibacterial effects of MB in in vitro and ex vivo time-kill assays. A disc diffusion method-based MB + antibiotic synergy assay was performed to analyze the subinhibitory effects of MB on ten isolates. MICs of MB against 104 MRSA isolates, detected by the agar dilution method, ranged between 16 and 64 µg/mL. MB concentrations of 4 and 16 µg/mL showed a bactericidal effect at 24 h in the ex vivo time-kill assays and in vitro time-kill assays, respectively. We observed a significant synergy between cefoxitin and methylene blue at a concentration of 1-2 µg/mL in two (20%) test isolates. Employing MB, which has well-defined pharmacokinetics, bioavailability, and safety profiles, for the treatment of MRSA infections and nasal decolonization could be a good strategy.

ARID3A and ARID3B exert direct regulatory control over the long non-coding RNAs (lncRNAs) MALAT1 and NORAD within the context of non-small cell lung cancer (NSCLC).

Lung cancer, known for its high mortality rates and poor prognosis, remains one of the most prevalent cancer types. Early detection and effective treatment methods are crucial for improving survival rates. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases. Long non-coding RNAs (lncRNAs), which play vital roles in various biological processes, have been implicated in the development of cancer and can impact key therapeutic targets in different cancer types. In NSCLC, the dysregulation of specific lncRNAs, such as MALAT1 and NORAD, has been associated with neoplastic initiation, progression, metastasis, tumor angiogenesis, chemoresistance, and genomic instability. Both MALAT1 and NORAD directly regulate the expression of the transcription factor E2F1, thereby influencing cell cycle progression. Additionally, MALAT1 has been reported to affect the expression of p53 target genes, leading to cell cycle progression through the repression of p53 promoter activity. NORAD, on the other hand, is indirectly regulated by p53. The AT-rich interaction domain (ARID) family of DNA-binding proteins, particularly ARID3A and ARID3B, are involved in various biological processes such as cell proliferation, differentiation, and development. They also play significant roles in E2F-dependent transcription and are transcriptional targets of p53. The intricate balance between promoting cellular proliferation through the pRB-E2F pathway and inducing growth arrest through the p53 pathway underscores the crucial regulatory role of ARID3A, ARID3B, and their interaction with lncRNAs MALAT1 and NORAD. In this study, we aimed to investigate the potential interactive and functional connections among ARID3A, ARID3B, MALAT1, and NORAD in NSCLC, considering their involvement in the pRB-E2F and p53 pathways. Our findings strongly suggest that ARID3A and ARID3B play a regulatory role in controlling MALAT1 and NORAD in NSCLC. Specifically, our study demonstrates that the activities of MALAT1 and NORAD were markedly increased upon the overexpression of ARID3A and ARID3B. Therefore, we can conclude that ARID3A and ARID3B likely contribute significantly to the oncogenic functions of MALAT1 and NORAD in NSCLC. Consequently, targeting ARID3A and ARID3B could hold promise as a therapeutic approach in NSCLC, given their direct control over the expression of MALAT1 and NORAD.

The role of miR-223 in breast cancer; an integrated analysis.

BACKGROUND: Breast cancer (BRCA) is the most common and leading cause of cancer-related death in women. MicroRNAs (miRNAs) are short non-coding RNA fragments that play a role in regulating gene expression including the cancer-related pathways. Although dysregulation of miR-223 has been demonstrated in recent studies to have prognostic value in various cancers, its diagnostic and prognostic role in BRCA remains unknown. METHODS: The expression and the prognostic value of miR-223 were evaluated using the TCGA data and verified by qRT-PCR. Subsequently, potential oncogenic targets of miR-223 were identified by using three different miRNA target prediction tools and the GEPIA database. In addition to these databases, protein-protein interaction network, molecular functions, prognostic value, and the expression level of miR-223 targets were included by using several other bioinformatics tools and databases; such as, UALCAN, GeneMANIA and Metascape. RESULTS: The bioinformatic results demonstrated that miR-223 downregulated in BRCA and associated with poor prognosis of patients. In vitro experiments validated that miR-223 significantly downregulated in BRCA cells, MCF-7, SK-BR3, MDA-MB-231 and HCC1500, compared to normal breast cell line hTERT-HME1. Furthermore, ANLN, DYNLT1, LRRC59, SLC12A8 and TPM3 genes were identified as the potential oncogenic target genes of miR-223 based on their expression and prognosis in BRCA. Additionally, protein-protein interaction network of these target genes was mainly enriched in dynein intermediate chain binding, cell division, regulation of cell cycle process, and positive regulation of cellular component biogenesis. CONCLUSIONS: The results suggests that miR-223 and its targets, ANLN, DYNLT1, LRRC59, SLC12A8 and TPM3, might be reliable potential prognostic biomarkers in BRCA patients.

RNAi-mediated siRNA sequences to combat the COVID-19 pandemic with the inhibition of SARS-CoV2.

The outbreak of the COVID-19 pandemic has cost five million lives to date, and was caused by a positive-sense RNA virus named SARS-CoV2. The lack of drugs specific to SARS-CoV2, leads us to search for an effective and specific therapeutic approach. Small interfering RNA (siRNA) is able to activate the RNA interference (RNAi) pathway to silence the specific targeted gene and inhibit the viral replication, and it has not yet attracted enough attention as a SARS-CoV2 antiviral agent. It could be a potential weapon to combat this pandemic until the completion of full scale, effective mass vaccination. For this study, specific siRNAs were designed using a web-based bioinformatics tool (siDirect2.0) against 14 target sequences. These might have a high probability of silencing the essential proteins of SARS-CoV2. such as: 3CLpro/Mpro/nsp5, nsp7, Rd-Rp/nsp12, ZD, NTPase/HEL or nsp13, PLpro/nsp3, envelope protein (E), spike glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), ORF8, ORF3a, nsp2, and its respective 5' and 3'-UTR. Among these potential drug targets, the majority of them contain highly conserved sequences; the rest are chosen on the basis of their role in viral replication and survival. The traditional vaccine development technology using SARS-CoV2 protein takes 6-8 months; meanwhile the virus undergoes several mutations in the candidate protein chosen for vaccine development. By the time the protein-based vaccine reaches the market, the virus would have undergone several mutations, such that the antibodies against the viral sequence may not be effective in restricting the newly mutated viruses. However, siRNA technology can make sequences based on real time viral mutation status. This has the potential for suppressing SARS-CoV2 viral replication, through RNAi technology.

Adipose-Derived Mesenchymal Stem Cell Transplantation in Chemotherapy-Induced Premature Ovarian Insufficiency: the Role of Connexin and Pannexin.

In women undergoing chemotherapy, it is inevitable that infertility risk will increase because of impaired reproductive functions. Premature ovarian insufficiency (POI), which occurs as a devastating result of chemotherapy, is the complete depletion or dysfunction of ovarian follicles. Adipose-derived mesenchymal stem cells (ADMSCs) transplantation is among the alternative treatment methods for POI, which currently do not have an effective treatment method. Apoptosis of granulosa cells in POI is seen as the main mechanism of the disease. It is also reported that in addition to molecules directly associated with apoptosis, connexins, and pannexins are also potential effector molecules in apoptosis. The roles of these molecules in POI, which are known to play a role in many important mechanisms in the ovary, are unknown. In this study, it was aimed to analyze the expressions of Connexin43 and Pannexin1, which are thought to be effective in the formation of POI, and to show the relationship between the antiapoptotic effects of ADMSCs transplantation and these molecules in POI. For this purpose, Caspase3, Connexin43, Pannexin1 proteins, and mRNA expressions were analyzed by immunohistochemistry and RT-qPCR, and AMH levels were measured by ELISA. It was determined that Pannexin1, Caspase3 proteins, and mRNA levels increased in the POI, while Pannexin1 and Caspase3 expressions decreased in the ADMSCs treated group. While Connexin43 level decreased in POI, Connexin43 protein and mRNA levels increased in ADMSCs group. Consequently, this study demonstrated for the first time that Connexin43 and Pannexin1 were associated with apoptosis in POI. In addition, it was revealed that ADMSCs transplantation could produce antiapoptotic effects by modulating these molecules.

ARID3A regulates autophagy related gene BECN1 expression and inhibits proliferation of osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor which has unclear pathobiology. Hence, enlightening the exact molecular mechanism underlying osteosarcoma progression is crucial for developing new treatment strategies. One member of the ARID family of DNA binding proteins is ARID3A that is implicated in osteosarcoma pathogenesis. ARID3A could bind E2F1 and regulate the transcription of E2F1 targets. At the same time, BECN1 is a well-characterized autophagy regulator gene that is a direct target of E2F1. The present study aimed to investigate the effect of ARID3A on the expression of BECN1 in osteosarcoma cells. First, we determined gene expression levels of ARID3A, BECN1, and E2F1 in U-2 OS by qPCR and confirmed with online datasets from GEO database. In addition, the prognostic value of these genes was also evaluated from Kaplan-Meier plotter database. Next, ARID3A was overexpressed and silenced in order to investigate the effect of ARID3A on BECN1 expression and proliferation of U-2 OS cells. Our results demonstrated that BECN1 was negatively correlated with E2F1 and positively correlated with ARID3A based on initial expression and prognostic effect in OS. Overexpression of ARID3A upregulated BECN1 while silenced ARID3A downregulated BECN1 expression in U-2 OS cells. Additionally, silencing of ARID3A promoted colony formation and proliferation, whereas overexpression of ARID3A suppressed colony formation and proliferation of U-2 OS cells. Taken together, these results indicate that ARID3A could function as tumor suppressor and affect the expression level of BECN1 in U-2 OS cells.

The relationship of 4-vinylcyclohexene diepoxide toxicity with cell death, oxidative stress, and gap junctions in female rat ovaries.

PURPOSE: It was aimed to investigate the damage caused by VCD toxicity in the ovary, which women working in the industrial field are frequently exposed to, and to show the relationship between gap junction protein, oxidative stress, and apoptosis, which is thought to be effective in the emergence of this damage. METHODS: Rats were divided into three groups as control, sham, and VCD. Histological stainings were performed for histopathological evaluations in ovary. Serum AMH level was measured with the ELISA. Then, iNOS, caspase 3, connexin 43 protein, and mRNA expression levels were analyzed by immunohistochemistry and RT-qPCR methods. RESULTS: As a result of the analyses, different amounts of degenerations such as hemorrhage, vacuolization, and fibrosis were observed in the ovary. VCD group AMH level decreased compared to control. In VCD group, iNOS and caspase 3 expressions increased, while connexin 43 expression decreased. CONCLUSIONS: It was shown that VCD caused damage to all ovarian tissue. Also, it was revealed for the first time that VCD triggered apoptosis by increasing oxidative stress in the ovary and suppressed connexin 43 which was also effective in the survival of granulosa cells. The devastating effect of exposure to occupational chemicals such as VCD on fertility was demonstrated in this study.

Distinct and overlapping roles of ARID3A and ARID3B in regulating E2F­dependent transcription via direct binding to E2F target genes.

The AT­rich interacting domain (ARID) family of DNA­binding proteins is involved in various biological processes, including the regulation of gene expression during cell proliferation, differentiation and development. ARID3A and ARID3B are involved in chromatin remodeling and can bind to E2F1 and retinoblastoma tumor suppressor protein (RB), respectively. However, their role in regulating E2F target gene expression remains poorly understood. E2F transcription factors are critical regulators of cell cycle progression and are modulated by RB. Herein, putative ARID3­binding sites (BSs) in E2F target genes were identified, including Cdc2, cyclin E1 and p107, and it was found that ARID3A and ARID3B bound to these BSs in living cells. The mutation of ARID3 BSs reduced Cdc2 promoter activity, while ARID3A and ARID3B overexpression increased the promoter activity, depending on both ARID3 and E2F BSs. ARID3B knockdown blocked the transcription of Cdc2, cyclin E1 and p107 in normal human dermal fibroblasts (NHDFs), whereas the effects of ARID3A knockdown varied depending on the target genes. ARID3B overexpression, but not that of ARID3A, upregulated the transcription of E2F target genes, and activated cyclin E1 transcription and induced cell death with E2F1 assistance. Finally, ARID3A and ARID3B knockdown attenuated the cell cycle progression of NHDFs and T98G cells, and suppressed tumor cell growth. On the whole, these results indicate that ARID3A and ARID3B play distinct and overlapping roles in E2F­dependent transcription by directly binding to the E2F target genes. The present study provides novel insight into the mechanisms underlying the E2F dysregulation caused by ARID3A and ARID3B overexpression, which may have a significant influence on the progression of tumorigenesis.

Epigenetic alterations in aging tooth and the reprogramming potential.

Tooth compartments and associated supportive tissues exhibit significant alterations during aging, leading to their impaired functioning. Aging not only affects the structure and function of dental tissue but also reduces its capacity to maintain physiological homeostasis and the healing process. Decreased cementocyte viability; diminished regenerative potential of stem cells residing in the pulp, alveolar bone and periodontal ligament; and impaired osteogenic and odontogenic differentiation capacity of progenitor cells are among the cellular impacts associated with oral aging. Various physiological and pathological phenomena are regulated by the epigenome, and hence, changes in epigenetic markers due to external stimuli have been reported in aging oral tissues and are considered a possible molecular mechanism underlying dental aging. The role of nutri-epigenetics in aging has emerged as an attractive research area. Thus far, various nutrients and bioactive compounds have been identified to have a modulatory effect on the epigenetic machinery, showing a promising response in dental aging. The human microbiota is another key player in aging and can be a target for anti-aging interventions in dental tissue. Considering the reversible characteristics of epigenetic markers and the potential for environmental factors to manipulate the epigenome, to minimize the deteriorative effects of aging, it is important to evaluate the linkage between external stimuli and their effects in terms of age-related epigenetic modifications.

Long noncoding RNA ERICD interacts with ARID3A via E2F1 and regulates migration and proliferation of osteosarcoma cells.

Long noncoding RNA (lncRNA) dysregulation is known to be taking part in majority of cancers, including osteosarcoma. In one of our previous studies, we showed that lncRNA MEG3 is being regulated by microRNA-664a (miR-664a) suppresses the migratory potential of osteosarcoma cells (U-2OS). We now report a novel lncRNA, namely, ERICD, which is linked to the transcription factor AT-rich interaction domain 3A (ARID3A) in U-2OS cells. We show that ARID3A binds to ERICD and indirectly interacts with each other via the E2F transcription factor 1 (E2F1). Furthermore, small interfering RNA (siRNA)-mediated knockdown of ERICD inhibited cell migration, formation of colonies, and proliferation in U-2OS cells. Overexpression of ARID3A inhibited cell migration, colony formation, and proliferation, whereas siRNA-mediated knockdown of ARID3A promoted cell migration, colony formation, and proliferation. Our findings indicate that ARID3A and lncRNA ERICD have plausible tumor suppressive and oncogenic functions, respectively, in osteosarcoma. Our data demonstrate the converse interaction between ARID3A and lncRNA ERICD that target DNA-binding proteins and dysregulation of their expression through E2F1 augments osteosarcoma progression. The cell rescue experiment also indicated E2F1 to be involved in the regulation of ARID3A and ERICD.

Effects of Moringa oleifera Lam Extract (MOLE) in the heat shock protein 70 expression and germ cell apoptosis on experimentally induced cryptorchid testes of rats.

Moringa oleifera (Moringaceae) is a plant known for having high antioxidant potency, anticancer, hepatoprotective, cardioprotective etc. and many more activities. Besides these, Moringaceae has the potential for attenuating the male sexual dysfunction. Reactive oxygen species/ROS were increased in cryptorchidism and therefore cause infertility by damaging sperm DNA and germ cell apoptosis. There was an increase in heat shock proteins (HSP) in cells, which is affected by heat shock. In the present study, the antioxidant effects of two different doses of M. oleifera Lam Extract (MOLE) on experimentally induced cryptorchid testes of rats was investigated. Forty two male rats (16 days old) were divided into four groups: a normal control group, a cryptorchidism-induced control group and two cryptorchidism-induced groups treated orally with either 400 or 800 mg/kg MOLE for 2 weeks. Our study showed that there were ruptures from interstitial spaces, separation of the germ cells from basal membrane, falling of the germ cells into the lumen, perivascular fibrosis, oedema, increased level of HSP70, apoptosis, malondialdehyde (MDA) and decrease in the level of superoxide dismutase (SOD) after the cryptorchidism. We found that pathological damages, oxidative stress, expression of the HSP70 and germ cell apoptosis were decreased in treated groups with MOLE. In brief, we can say that aqueous extract of M. oleifera reduces the oxidative stress in a unilateral cryptorchidism induced rats, and it might attenuate histopathological damages, HSP expression and germ cell apoptosis.

Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E.

In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.

[Role of ARID3A in E2F target gene expression and cell growth].

UNLABELLED: ARID3A is a member of the AT-rich interaction domain (ARID) family of DNA-binding proteins. ARID3A was isolated as proteins binding to E2F1, and stimulates transcription mediated by the E2F transcription factor that plays a central role in regulating cell cycle progression. However, the function of ARID3A in E2F-target-gene expression has not been fully understood. METHODS: Gene-silencing and overexpression experiments were carried out using siRNA and recombinant adenoviruses, respectively. E2F responsive gene expression was measured by RT-PCR. Effects of ARID3A silencing on DNA synthesis and cell growth were determined by EdU incorporation and colony formation assay, respectively. RESULTS: siRNA mediated gene silencing of ARID3A blocked the transcription of E2F-target genes, such as E2F1, p107, CDC2 and CDC6 in normal human dermal fibroblasts (NHDFs). Although adenoviral-mediated overexpression of ARID3A did not up-regulate the transcription of these E2F-target genes in quiescent NHDFs, E2F1 overexpression was unable to overcome the blockade of CDC6 expression by ARID3A silencing. Furthermore, ARID3A silencing attenuated S phase entry of NHDFs, and suppressed growth of human tumor cell lines. CONCLUSION: These results indicate that ARID3A plays an important role for E2F-mediated transcriptional activation and cell growth.