XTT Assay Research Articles

Comprehensive Evaluation of Decellularized Dental Pulp as a New Biomaterial for Regeneration of the Pulp-Dentin Complex

Objective: To develop a detergent-enzymatic method and evaluate the quality of a decellularized pulp scaffold for regenerative endodontics.Materials and methods: Biomaterial and mesenchymal stem cells (MSCs) were derived from dental pulp that was obtained following third molar extraction indicated for orthodontic reasons in patients aged 14-18 years. The detergent-enzymatic method enabled to obtain a decellularized scaffold from pulp samples. The proliferative activity and viability of dental pulp-derived MSCs were assessed using trypan blue staining and XTT assay. To assess tissue response, Wistar rats underwent subcutaneous implantation of native and decellularized dental pulp. Explanted samples were stained with hematoxylin-eosin on days 7 and 14.Results: The detergent-enzymatic treatment of the dental pulp demonstrated the absence of nuclear material, whereas the histoarchitecture of the dental pulp was disturbed. The DNA content in the sample of the decellularized scaffold was 22.79 ± 2.1 ng/mg of tissue; the amount of DNA in the native sample was 78.5 ± 5.4 ng/mg of tissue. According to XTT assay results, no cytotoxicity of the decellularized scaffold against MSCs was found. Biopsy specimens of the rats with implanted decellularized dental pulp were characterized by no signs of inflammation.Conclusions: The study results will enable to create a biomaterial that can be the base of a tissue-engineered structure of the dental pulp and be used for the regeneration of the pulp-dentin complex.

Nanoparticle-Mediated Delivery of Deferasirox: A Promising Strategy Against Invasive Aspergillosis

Background: Invasive aspergillosis (IA) is a deadly fungal lung infection. Antifungal resistance and treatment side effects are major concerns. Iron chelators are vital for IA management, but systemic use can cause side effects. We developed nanoparticles (NPs) to selectively deliver the iron chelator deferasirox (DFX) for IA treatment. Methods: DFX was encapsulated in poly(lactic-co-glycolic acid) (PLGA) NPs using a single emulsion solvent evaporation method. The NPs were characterized by light scattering and electron microscopy. DFX loading efficiency and release were assessed spectrophotometrically. Toxicity was evaluated using SRB, luciferase, and XTT assays. Therapeutic efficacy was tested in an IA mouse model, assessing fungal burden by qPCR and biodistribution via imaging. Results: DFX-NPs had a size of ~50 nm and a charge of ~−30 mV, with a loading efficiency of ~80%. Release kinetics showed DFX release via diffusion and bioerosion. The EC50 of DFX-NPs was significantly lower (p < 0.001) than the free drug, and they were significantly less toxic (p < 0.0001) in mammalian cell cultures. In vivo, NP treatment significantly reduced Af burden (p < 0.05). Conclusion: The designed DFX-NPs effectively target and kill Af with minimal toxicity to mammalian cells. The significant in vivo therapeutic efficacy suggests these NPs could be a safe and effective treatment for IA.

The Role of the Bone Morphogenetic Protein Antagonist Noggin in Nucleus Pulposus Intervertebral Disc Cells.

Low back pain (LBP) is a significant global health issue, contributing to disability and socioeconomic burdens worldwide. The degeneration of the human intervertebral disc (IVD) is a critical factor in the pathogenesis of LBP. Recent studies have emphasized the significance of a specific set of genes and extracellular matrix (ECM) in IVD health. In particular, Noggin has emerged as a critical gene due to its high expression levels in healthy nucleus pulposus cells (NPCs) observed in our previous research. In this study, it was hypothesized that decreased Noggin expression in NPCs is associated with IVD degeneration and contributes to LBP development. A lentivirus-mediated RNAi was applied to knock down Noggin expression in primary NPCs from six human donors. The NPCs after transduction were evaluated through cell viability analysis, XTT assay, and cell apoptosis analyses. After two weeks, a colony formation assay was used to examine the anchor-independent growth ability of transduced cells. At the transcript level, anabolic and catabolic markers were quantified using RT-qPCR. The results demonstrated that lentivirus-mediated downregulation of Noggin significantly inhibited cell proliferation, reduced cell viability, and suppressed colony formation, while inducing apoptosis in human NPCs in vitro. Notably, it disrupted cellular anabolic processes and promoted catabolic activity in human NPCs post-transduction. Our findings indicated that the degeneration of human IVD is possibly related to decreased Noggin expression in NPCs. This research provides valuable insights into the role of Noggin in IVD homeostasis and its implications in LBP treatment.

Electric Stimulation at 448 kHz Modulates Proliferation and Differentiation of Follicle Dermal Papilla Cells

Dermal papilla cells (DPCs) regulate the hair cycle and play important roles in hair growth and regeneration. Alopecia is a pathology caused by a deregulation in the hair cycle phases. Currently, the use of physical therapies such as radiofrequency (RF) as an alternative to pharmacological treatment is increasing. Electrical stimulation by capacitive resistive electrical transfer (CRET) is one of these therapies. The objective of the present study was to analyze the effect of RF-CRET currents on DPCs. Cells were treated with subthermal 448 kHz CRET currents with two different types of signals: standard (CRET-STD) or modulated (CRET-MOD). Viability (XTT Assay), proliferation (Ki67 and ERK1/2), apoptosis (p53 and caspase 3), differentiation (β-catenin and α-SMA), and anagen markers (versican and PPARγ) were analyzed by immunofluorescence and immunoblot. CRET caused effects on the proliferation and survival of DPCs associated with increases in the expression of p-MAPK-ERK1/2, cyclin D1, and decreases in the expression of p53 and caspase 3. Also, CRET caused significant transient increases in the expression of β-catenin, involved in hair growth, and in the expression of anagen phase markers such as versican and PPARγ related to hair follicle maintenance. The present study highlights the ability of treatment with CRET therapy to cause molecular alterations in DPC involved in hair regeneration.

Enoxaparin Protects C6 Glioma Cells from Glutamate-Induced Cytotoxicity by Reducing Oxidative Stress and Apoptosis.

Recent studies suggest enoxaparin may protect the central nervous system (CNS) from damage. However, its specific effects on glial cells and the underlying mechanisms involving cell death and oxidative stress require further investigation. Therefore, this research investigated enoxaparin's potential to safeguard C6 glioma cells against glutamate-induced cytotoxicity, specifically focusing on its influence on oxidative stress and apoptotic mechanisms. To investigate the neuroprotective effects of enoxaparin against glutamate-induced cytotoxicity in C6 cells, four groups were established: a control group, a group exposed to 10mM glutamate, a group treated with enoxaparin at concentrations ranging from 25 to 200µM, and a group receiving both 10mM glutamate and enoxaparin at concentrations ranging from 25 to 200µM. Cell viability was measured using an XTT assay. To evaluate the effects of enoxaparin on oxidative stress, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using ELISA, along with total antioxidant status (TAS) and total oxidant status (TOS). Apoptosis was evaluated using flow cytometry, and caspase-3 activity, a key marker of apoptosis, was assessed using caspase-3 immunofluorescence staining. Enoxaparin at 50, 100, and 200µM markedly increased cell viability in the enoxaparin + glutamate group. Enoxaparin treatment in the enoxaparin + glutamate group also significantly elevated levels of SOD and TAS, while concurrently decreasing MDA and TOS levels. These changes indicate a reduction in oxidative stress. Enoxaparin treatment further resulted in a significant decline in cleaved caspase-3 levels, a marker of apoptosis. Enoxaparin pre-treatment reduced cell death according to flow cytometry analysis. This study suggests enoxaparin's potential to shield C6 glioma cells from glutamate-induced cell death by mitigating both oxidative stress and apoptotic pathways. More research is needed to confirm this effect.

Noradrenaline Protects Human Microglial Cells (HMC3) Against Apoptosis and DNA Damage Induced by LPS and Aβ1-42 Aggregates In Vitro.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, characterized by the accumulation of amyloid-beta (Aβ) plaques and neuroinflammation. This study investigates the protective effects of noradrenaline (NA) on human microglial cells exposed to lipopolysaccharides (LPS) and Aβ aggregates-major contributors to inflammation and cellular damage in AD. The reduced Aβ aggregation in the HMC3 human microglial cells co-treated with Aβ and NA was confirmed by thioflavin T (ThT) assay, fluorescent ThT staining, and immunocytochemistry (ICC). The significantly increased viability of HMC3 cells after 48 h of incubation with NA at 50 µM, 25 µM, and 10 µM, exposed to IC50 LPS and IC50 Aβ, was confirmed by XTT and LDH assays. Moreover, we found that NA treatment at 25 μM and 50 μM concentrations in HMC3 cells exposed to IC50 LPS or IC50 Aβ results in an increased proliferation of HMC3 cells, their return to normal morphology, decreased levels of DNA damage, reduced caspase-3 activity, decreased expression of pro-apoptotic DDIT3 and BAX, and increased expression of anti-apoptotic BCL-2 genes and proteins, leading to enhanced cell survival, when compared to that of the HMC3 cells treated only with IC50 LPS or IC50 Aβ. Furthermore, we showed that NA induces the degradation of both extracellular and intracellular Aβ deposits and downregulates hypoxia-inducible factor 1α (HIF-1α), which is linked to impaired Aβ clearance and AD progression. These findings indicate that NA holds promise as a therapeutic target to address microglial dysfunction and potentially slow the progression of AD. Its neuroprotective effects, particularly in reducing inflammation and regulating microglial activity, warrant further investigation into its broader role in mitigating neuroinflammation and preserving microglial function in AD.

PHOTOINACTIVATION OF CANDIDA ALBICANS BIOFILM WITH GREEN LASER MEDIATED BY THE PAPAYA LEAF EXTRACT CHLOROPHYLL

This study aims to activate the effectiveness of Photodynamic Inactivation (PDI) as an antibacterial agent by using a green laser and papaya leaf chlorophyll extract to prevent Candida albicans cell death. Papaya leaf extract chlorophyll is known to have potential as a photosensitizer (PS) through its antimicrobial properties and ability to absorb optimal light photons at a wavelength range of 405–680 nm. Activation of chlorophyll molecules with appropriate light produces Reactive Oxygen Species (ROS), which are toxic to pathogenic microbes such as Candida albicans. The research method involves using PDI with a green laser light source and chlorophyll extract on Candida albicans biofilms. Four main treatment groups were applied, negative control (C-), positive controls with 10% (C1+) and 15% chlorophyll (C2+), irradiation for 60, 120, 180, 240, and 300 seconds (L1–L5), and combinations of irradiation with chlorophyll (L1F1–L5F2, where F1 for 10% chlorophyll and F2 for 15% chlorophyll), with measurements performed three times for each treatment. Living Candida albicans cells were detected using the XTT assay staining method. The results showed a significant decrease in activity in all treatment groups. Maximum activity was achieved in the L5F1 and L5F2 treatment groups with inactivation of 80% (p<0.05) and 83% (p<0.05), respectively. This study concludes that high papaya leaf extract chlorophyll concentrations combined with a green laser effectively inhibit Candida albicans biofilm.

Steroid-based hydrazones as anabolic agents: Design, synthesis, computational studies, and biological evaluation in human skeletal muscle cell line

Hydrazone derivatives were synthesized in a three-step process starting from diosgenin and then tested for their anabolic activity. Through in silico analysis, we identified proteins associated with muscle growth to elucidate the plausible mechanisms and interactions between these compounds and the active site amino acids, thereby providing a rationale for the observed biological results. The results of the in vitro assay on the human skeletal muscle cell line showed an increase in the number of viable cells, which was determined using increasing concentrations of each compound through the XTT assay. Additionally, the increase in protein concentration was measured using the Bradford method. The experimental evaluation revealed that all compounds have differences in increased protein/cell percentage compared to the control and testosterone, which suggests a hyperplastic/hypertrophic effect with a normal morphology.

Oncolytic Coxsackievirus B3 Strain PD-H Is Effective Against a Broad Spectrum of Pancreatic Cancer Cell Lines and Induces a Growth Delay in Pancreatic KPC Cell Tumors In Vivo

Pancreatic cancer is one of the deadliest cancers globally, with limited success from existing therapies, including chemotherapies and immunotherapies like checkpoint inhibitors for patients with advanced pancreatic ductal adenocarcinoma (PDAC). A promising new approach is the use of oncolytic viruses (OV), a form of immunotherapy that has been demonstrated clinical effectiveness in various cancers. Here we investigated the potential of the oncolytic coxsackievirus B3 strain (CVB3) PD-H as a new treatment for pancreatic cancer. In vitro, PD-H exhibited robust replication, as measured by plaque assays, and potent lytic activity, as assessed by XTT assays, in most pancreatic tumor cell lines, outperforming two other coxsackievirus strains tested, H3N-375/1TS and CVA21. Thus, H3N-375/1TS showed efficient replication and lytic efficiency in distinctly fewer tumor cell lines, while most tumor cells were resistant to CVA21. The oncolytic efficiency of the three OV largely correlated with mRNA expression levels of viral receptors and their ability to induce apoptosis, as measured by cleaved caspase 3/7 activity in the tumor cells. In a syngeneic mouse model with subcutaneous pancreatic tumors, intratumoral administration of PD-H significantly inhibited tumor growth but did not completely stop tumor progression. Importantly, no virus-related side effects were observed. Although pancreatic tumors respond to PD-H treatment, its therapeutic efficacy is limited. Combining PD-H with other treatments, such as those aiming at reducing the desmoplastic stroma which impedes viral infection and spread within the tumor, may enhance its efficacy.

Aucubin Induces Apoptotic Cell Death Through Caspase-dependent Pathways in Human Osteosarcoma MG-63 Cells

Background Osteosarcomas (OSs) are predominantly generated by mesenchymal cells, which commonly develop in the distal and upper regions of the bones. OS has high mortality rates and treatment failures, mostly due to the presence of lung metastases. Purpose The present work aimed to scrutinize the anticancer potentials of aucubin against OS human osteosarcoma (MG-63) cells. Methods The proliferation of the control and aucubin-treated MG-63 cells was studied by an XTT assay. Various fluorescent staining assays were done to assess the endogenous reactive oxygen species (ROS) accumulation and apoptosis in the aucubin-treated MG-63 cells. The caspase-3, -8, and -9 activities were investigated using the assay kits. Results The XTT assay results confirmed that the aucubin treatment considerably reduced the MG-63 cells. The findings of the various fluorescent staining assays evidenced that the aucubin treatment remarkably promoted endogenous ROS accumulation and apoptosis in the OS cells. The caspase activities were also improved in the OS cells after the aucubin treatment. Conclusion The results of this work show that aucubin treatments inhibit cell proliferation and trigger apoptosis in MG-63 cells, suggesting its potential as a promising anticancer agent. These findings will facilitate the future development of aucubin as a talented anticancer agent to treat OS.

Cytotoxicity assessment of eluates from vacuum-forming thermoplastics.

This study aimed to evaluate possible cytotoxic effects of thermoplastic materials commonly used for occlusal splints and orthodontic appliances. Seven thermoplastics were included: three variants of the Essix sheets (C+, Plus, and Tray Rite; Dentsply Sirona), three thermoplastics (Bleach Heavy, Splint, and X-Heavy; Cavex Holland) and Invisalign (Align Technology). Cylindrical specimens (n = 24; 10mm diameter) were incubated in cell culture medium for 24h and 14 days. After incubation, the medium was collected, serially diluted, and dosed to primary human gingival fibroblasts in triplicate. Medium processed like the samples was used as negative control. Cell viability was evaluated by XTT and LDH assay to assess metabolic activity and membrane integrity, respectively. Next, cell cycle was assessed with flow cytometry after exposing HGFs to undiluted extracts. The 24-hour and 14-day extracts did not evoke cytotoxicity after 24-hour incubation. No significant differences in cell viability (one-way ANOVA, p > 0.05 ) in the XTT and LDH assays or in cell cycle distribution between the different materials (two-way ANOVA, p > 0.05 ). The thermoplastics tested in the study showed no evident in-vitro cytotoxic effects. Further investigation should focus on determining which compounds are released from thermoplastic materials and assessing potential toxicity related to exposure to these compounds. Our study adds to the growing body of evidence on the biocompatibility of dental thermoplastics. This can aid clinical decision-making, as thermoplastics are expected to be safe to use in terms of cytotoxicity.

Radiofrequency Currents Modulate Inflammatory Processes in Keratinocytes.

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes.

Alkaloid-rich extract of jujube seed regenerate the antiproliferative effect of paclitaxel on paclitaxel-resistant MDA-MB-231 breast cancer cell line in 2D and 3D cultures

IntroductionCancer is the second leading cause of death globally, and among all types of cancer, triple-negative breast cancer (TNBC) has one of the worst prognoses. The repeated use of chemotherapeutic drugs often leads to drug resistance, complicating treatment outcomes. This study aimed to investigate the effect of jujube alkaloid-rich extract on drug sensitivity and its antitumor effects on paclitaxel-resistant MDA-MB-231 cells in vitro. Materials and methodsThis study utilized the MDA-MB-231 breast cancer cell line and its paclitaxel-resistant variant. Cell viability was measured using the XTT assay, while apoptosis was detected through Annexin-PI assays. Various concentrations of jujube extract, alone and combined with paclitaxel, were assessed for synergistic effects in 2D and 3D culture models. ResultsThe presence of alkaloids in the jujube seed extract was confirmed via the Dragendorff test, with 0.395 g of alkaloids present in 5 g of jujube seed powder. A combination of paclitaxel and jujube extract exhibited dose-dependent cytotoxic effects on paclitaxel-resistant MDA-MB-231 cells. The combination of jujube extract and paclitaxel significantly decreased IC50 from 541.3 μg/ml to 124.3 μg/ml, and the IC20 decreased from 92.1 μg/ml to 23.9 μg/ml when combined with paclitaxel in 2D culture model. The cytotoxicity of the combination was reduced in the 3D culture model. Apoptosis rates were 0.75% in the control group, 13.89% in the alkaloid-rich extract group, 2.59% in the paclitaxel group, and 42.90% in the combination group. ConclusionCombining alkaloid-rich jujube seed extract with paclitaxel regenerates the cytotoxicity and apoptotic ability of paclitaxel in the paclitaxel-resistant MDA-MB-231 breast cancer cells in both 2D and 3D culture models. This suggests that such combinations might be a viable strategy to overcome drug resistance in TNBC treatments.

In vitro anticancer efficacy of Calendula Officinalis extract-loaded chitosan nanoparticles against gastric and colon cancer cells

Objective This study assessed the anticancer activities of Calendula officinalis-loaded chitosan nanoparticles in gastric and colon cancer cells compared with fibroblast cells and examined the balance between ROS and antioxidants. Methods Considering this information, we synthesized Calendula officinalis-loaded chitosan nanoparticles (CO-CSNPs) via the ionic gelation method. Their characterizations were carried out with ZetaSizer, UV-Vis, FTIR and SEM devices including size, morphology and surface zeta potential analysis, loading capacity, encapsulation efficiency, in vitro drug release, and chemical interactions. The anticancer activities of CO, CSNPs, and CO-CSNPs were tested against AGS, Caco-2, and normal NIH-3T3 cells using an XTT assay. The anticancer effects were evaluated using DAPI staining, scratch assay, reactive oxygen species (ROS) detection and the CUPRAC method on cellular and non-cellular processes that promote anticancer mechanisms. Results Results showed that CO and CO-CNPs exhibited anticancer activity against AGS and Caco-2. Further, the formulation of CO with CSNPs enhanced the anticancer activity of CO while having no cytotoxicity on NIH-3T3. DAPI staining, scratch assay, ROS, and CUPRAC method confirmed the anticancer activity of CO and CO-CSNPs, which resulted in a reduction in the number of apoptotic cells, inhibited migration, triggered apoptotic pathway via ROS, and higher antioxidant activity. Conclusions The results of the study indicate that CO-CSNPs are a promising therapeutic formulation for gastric and colon cancer treatment. We consider that this study will lead to the investigation of molecular mechanisms of CO-CSNPs in cancer treatment and their investigation in clinical studies.

Preparation and characterization of chitosan nanoparticles with extracts of Rheum ribes, evaluation of biological activities of extracts and extract loaded nanoparticles

The biological activities of different parts of the Rheum ribes plant were evaluated comparatively. Extracts showing strong biological activity were identified and it was determined which of the extract-loaded nanoparticles showed stronger activity. Cytotoxic activity of R. ribes extracts was calculated on glial (C6) and fibroblast (NIH 3T3) cells using XTT assay. Spectrophotometry was used to evaluate the impact of these compounds on the enzyme activities of human carbonic anhydrase I and II (hCA I and hCA II). The findings showed that chitosan NPs with extracts loaded on them have a lower IC50 value and more cytotoxic activity in C6 cells than chitosan NPs with only extracts. R. ribes young shoots ultrasonic methanol extract (RYU) was shown to have the strongest antiproliferative efficacy against C6 cells. Results showed that RYU and ultrasonic methanol extract of R. ribes radix (RRU) were determined as the best carbonic anhydrase inhibitors. According to results of particle size, encapsulation efficiency, and release studies of chitosan NPs, it has been observed that they are suitable for application. At a concentration of 10 µg/mL, it was found that none of the R. ribes extracts exhibited cytotoxic action toward the NIH 3T3 cell line. According to results of particle size, encapsulation efficiency, and release studies of chitosan NPs, it has been observed that they are suitable for application. It was observed that none of the extracts of R. ribes at a concentration of 10 µg/mL showed cytotoxic activity in the NIH 3T3 cell line.

Isolation and comparative genotoxicity screening of trichokonins VI and VIII on CHO-K1 cells

Peptaibols are fungal peptides that exhibit efficacy against pathogen microorganisms. Trichokonin VI (TK-VI) and trichokonin VIII (TK-VIII) are known peptaibols isolated from the endolichenic fungi Hypocrea sp. Previous investigations reported that trichokonin VI presents antiproliferative effects on tumor cells. This study is pioneering in elucidating the genotoxic effects of TK-VI and TK-VIII, contributing to the thorough assessment of their safety as potential therapeutic agents. The present investigation aimed to evaluate the genotoxicity of TK-VI and TK-VIII on CHO-K1 cells. Cytotoxicity was evaluated using the XTT assay and clonogenic survival assays, followed by evaluation of DNA damage using the comet assay and micronucleus test conducted in vitro. The XTT assay results indicated IC50 values of 10.30 µM and 9.89 µM for TK-VI and TK-VIII, respectively. The clonogenic survival assay indicated that concentrations of 10 µM or higher completely inhibited the cell colony formation. In the comet assay, both TK-VI and TK-VIII increased the DNA damage score and the frequency of comet nuclei in all tested concentrations. In the micronucleus assay, TK-VI and TK-VIII at 10 µM increased the frequency of MN in CHO-K1 cells. Both TK-VI and TK-VIII exhibited genotoxic effects. Our findings underscore the importance of considering the genotoxicological safety of peptaibols, particularly when assessing their potential for other biological activities.

MoMo30 Binds to SARS-CoV-2 Spike Variants and Blocks Infection by SARS-CoV-2 Pseudovirus.

MoMo30 is an antiviral protein isolated from aqueous extracts of Momordica balsamina L. (Senegalese bitter melon). Previously, we demonstrated MoMo30's antiviral activity against HIV-1. Here, we explore whether MoMo30 has antiviral activity against the COVID-19 virus, SARS-CoV-2. MLV particles pseudotyped with the SARS-CoV-2 Spike glycoprotein and a Luciferase reporter gene (SARS2-PsV) were developed from a three-way co-transfection of HEK293-T17 cells. MoMo30's inhibition of SARS2-PsV infection was measured using a luciferase assay and its cytotoxicity using an XTT assay. Additionally, MoMo30's interactions with the variants and domains of Spike were determined by ELISA. We show that MoMo30 inhibits SARS2-PsV infection. We also report evidence of the direct interaction of MoMo30 and SARS-CoV-2 Spike from WH-1, Alpha, Delta, and Omicron variants. Furthermore, MoMo30 interacts with both the S1 and S2 domains of Spike but not the receptor binding domain (RBD), suggesting that MoMo30 inhibits SARS-CoV-2 infection by inhibiting fusion of the virus and the host cell via interactions with Spike.

Dysregulation of lncRNA TFAP2A-AS1 is involved in the pathogenesis of pulpitis by the regulation of microRNA-32-5p.

This study was designed to evaluate TFAP2A-AS1 expression in the dental pulp of teeth with or without pulpitis and to determine the function of TFAP2A-AS1 in pulp cells. GSE92681 was analyzed to filter out differentially expressed lncRNAs. Pulp samples from teeth with pulpitis and healthy teeth (control) were examined using real-time (RT) quantitative polymerase chain reaction (qPCR). Human dental pulp stem cells (hDPSCs) were cultured in a specific medium for osteogenic induction, or treated with lipopolysaccharide (LPS) to simulate inflammation. The viability and apoptosis of human DPSCs (hDPSCs) were determined by XTT assay and apoptosis detection kit. Inflammation was induced by LPS and assessed by measuring the expression and release of inflammatory cytokines after TFAP2A-AS1 knockdown. Osteogenic differentiation of hDPSCs was investigated by determining expression levels of osteogenic markers and alkaline phosphatase (ALP) activity after TFAP2A-AS1 overexpression. The downstream microRNA (miRNA) was predicted. Dual-luciferase reporter was used to confirm the binding between miR-32-5p and TFAP2A-AS1. The expression of TFAP2A-AS1 was evaluated in inflamed pulp using RT-qPCR. TFAP2A-AS1 had a discriminatory ability for healthy individuals and patients with pulpitis. The expression of TFAP2A-AS1 decreased upon the osteogenic differentiation of hDPSCs, and increased upon the LPS induction. TFAP2A-AS1 can reverse the osteogenic differentiation of hDPSCs, as evidenced by decreased levels of dentine sialophosphoprotein, dentin matrix protein-1, and ALP activity. TFAP2A-AS1 knockdown can promote cell proliferation of hDPSCs and relieve LPS-induced inflammation, as evidenced by decreased levels of TNF-α, IL-1β, and IL-6. miR-32-5p was identified as a downstream miRNA of TFAP2A-AS1. This study demonstrated the expression and potential function of TFAP2A-AS1 in the human dental pulp. TFAP2A-AS1 can inhibit odontogenic differentiation but promote inflammation in pulp cells.

Antiviral Activity of Graphene Oxide-Silver Nanocomposites Against Murine Betacoronavirus.

The high infectivity of coronaviruses has led to increased interest in developing new strategies to prevent virus spread. Silver nanoparticles (AgNPs) and graphene oxide (GO) have attracted much attention in the antiviral field. We investigated the potential antiviral activity of GO and AgNPs combined in the nanocomposite GO-Ag against murine betacoronavirus MHV using an in vitro model. GO, AgNPs, and GO-Ag characterization (size distribution, zeta potential, TEM visualization, FT-IR, and EDX analysis) and XTT assay were performed. The antiviral activity of GO-Ag nanocomposites was evaluated by RT-qPCR and TCID50 assays. The results were compared with free AgNPs and pure GO. Cell growth and morphology of MHV-infected hepatocytes treated with GO-Ag composites were analyzed by JuLI™Br. Immunofluorescence was used to visualize the cell receptor used by MHV. Ultrastructural SEM analysis was performed to examine cell morphology after MHV infection and GO-Ag composite treatment. A significant reduction in virus titer was observed for all nanocomposites tested, ranging from 3.2 to 7.3 log10 TCID50. The highest titer reduction was obtained for GO 5 µg/mL - Ag 25 µg/mL in the post-treatment method. These results were confirmed by RT-qPCR analysis. The results indicate that GO-Ag nanocomposites exhibited better antiviral activity compared to AgNPs and GO. Moreover, the attachment of AgNPs to the GO flake platform reduced their cytotoxicity. In addition, the GO-Ag composite modulates the distribution of the Ceacam1 cell receptor and can modulate cell morphology. Graphene oxide sheets act as a stabilizing agent, inhibiting the accumulation of AgNPs and reducing their cellular toxicity. The GO-Ag composite can physically bind and inhibit murine betacoronavirus from entering cells. Furthermore, the constant presence of GO-Ag can inhibit MHV replication and significantly limit its extracellular release. In conclusion, GO-Ag shows promise as an antiviral coating on solid surfaces to minimize virus transmission and spread.

Biocompatible melanin from the marine black yeast Hortaea werneckii R23 with antioxidant and photoprotection property.

Pigments from diverse sources have a great deal of interest due to its multifaceted applications. Hence, this study reports the physicochemical and functional characterization of the black pigment melanin from the marine black yeast Hortaea werneckii R23. In the present study, Hortaea werneckii R23, produced a black pigment in the yeast biomass. The pigment was extracted from the harvested yeast biomass and followed by pigment purification, characterization and identification was done. Physicochemical characterization of the pigment showed acid precipitation, alkali solubilization, insolubility in most organic solvents and water. The black pigment was confirmed as melanin based on ultraviolet-visible spectroscopy, Fourier-transform infrared, and Nuclear magnetic resonance spectroscopy analyses. Furthermore, the analyses of the elemental composition indicated that the pigment possessed a moderately high percentage of nitrogen and also detectable proportion of sulfur. All these Physicochemical properties indicated that H. werneckii melanin (HwM) mostly consisted of eumelanin. HwM exhibited strong antioxidant potential as reactive oxygen species (ROS) scavenger by in vitro DPPH (1,1-diphenyl-2-picryl hydrazyl) and ABTS (2,2-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid) radical scavenging assay, and lipid peroxidation assay. The photoprotectant role of HwM on UV-irradiated human epithelial cells (HEp-2) revealed its potential effect in photoprotection. In addition, cytotoxicity study by XTT and SRB assay confirmed its biocompatibility with HEp-2 cells. From these findings, it is evident that the HwM from the marine black yeast possesses strong antioxidant and photoprotectant activity, moreover, it is biocompatible to human epithelial cells. So HwM could be used as a protective agent against oxidative stress associated disorders in an environment-friendly perspective.