Cytotoxic Concentrations Research Articles

Antiviral Activity of Flavonoids from Bauhinia holophylla Leaves against Zika virus

Zika virus (ZIKV) is involved in the etiology of serious nervous system pathologies. Currently, there are no specific and effective vaccines or antiviral drugs to prevent the diseases caused by ZIKV. This study aimed to assess the activity of flavonoids present in crude hydroethanolic extract (CHE) and fractions obtained from B. holophylla leaves against ZIKV. O-glycosylated flavonoids were characterized by high-performance liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS). The cytotoxic concentration and the effective concentration for 50% of the cells (CC50 and EC50, respectively) were determined, and the selectivity index (SI) was calculated. Molecular networks were constructed based on the chemical composition of the samples and global antiviral activity data using the Global Natural Products Social Molecular Networking (GNPS) platform. Protein–ligand docking was performed in the NS2B-NS3 protease, NS3 helicase, and NS5 methyltransferase of the ZIKV. CHE showed greater antiviral activity at a multiplicity of infection (MOI) of 1.0, with an EC50 of 11.93 µg/mL, SI = 13.38, and reduced cytopathic effects. Molecular networks indicated that O-glycosylated flavonoids are responsible for the activity against ZIKV, being quercetin-O-deoxyhexoside more selective and effective. Molecular docking confirmed the inhibitory activity of quercetin-O-deoxyhexoside, which showed an affinity for the tested targets, especially for NS2B-NS3 protease. The results showed that B. holophylla has flavonoids with potential for future therapeutic applications against ZIKV.

Microalgae extracts modulates the immune response in Trypanosoma cruzi-infected human cells

Chagas disease (CD) is caused by the hemoflagellate protozoan Trypanosoma cruzi. The control of the infection depends of the innate and acquired immune response of host. Moreover, CD plays a significant role in the immune response, and, in this context, microalgae can be an interesting alternative due to its immunomodulatory and trypanocidal effects. This study aimed to evaluate, in vitro, immunomodulatory potentials of the aqueous extracts of Chlorella vulgaris and Tetradesmus obliquus. Both microalgae extracts (ME) were obtained by sonication, and the selectivity index (SI) was determined by assays of inhibitory concentration (IC50) in T. cruzi trypomastigotes cells; as well as the cytotoxic concentrations (CC50) in human peripheral mononuclear cells (PBMC). The immune response was evaluated in T. cruzi-infected PBMC using the IC50 value. ME led to inhibition of T. cruzi trypomastigotes after 24 h of treatment, in which the IC50 values were 112.1 µg/ml to C. vulgaris and 15.8 µg ml−1 to T. obliquus. On the other hand, C. vulgaris did not affect the viability of PBMCs in concentrations up to 1000 µg ml−1, while T. obliquus was non-toxic to PBMCs in concentrations up to 253.44 µg ml−1. In addition, T. obliquus displayed a higher SI against T. cruzi (SI = 16.8), when compared with C. vulgaris (SI = 8.9). C. vulgaris decreased the levels of IFN, indicating a reduction of the inflammatory process; while T. obliquus displayed an interesting immunomodulatory effect, since discretely increased the levels of TNF and stimulated the production of the anti-inflammatory cytokine IL-10. This study confirms that ME are effective against T. cruzi trypomastigotes, and may able to control the parasitemia and preventing the progress of CD while regulating the inflammatory process.

High Efficacy of Green Synthesized Silver Nanoparticles for Treatment of Toxoplasma Gondii Infection Through Their Immunomodulatory, Anti-Inflammatory, and Antioxidant Potency.

The present experimental survey designed to green synthesis, characterization, as well as in vitro and in vivo anti-Toxplasma gondii activity of silver nanoparticles (SLN) green synthesized by Lupinus arcticus extract. SLN were green synthesized based on the reducing by L. arcticus extract through the precipitation technique. In vitro lethal effects of SLN on T. gondii tachyzoites, infectivity rate, parasites inside of the human macrophage cells (THP-1 cells), nitric oxide (NO) triggering, and iNOS and interferon gamma (IFN-γ) expression genes were evaluated. In vivo, after establishment of toxoplasmosis in BALB/c mice via T. gondii ME49 strain, mice received SLN at 10 and 20mg/kg/day alone and combined to pyrimethamine at 5mg/kg for 14 days. SLN exhibited a spherical form with a size ranging from 25 to 90nm. The 50% inhibitory concentration (IC50) value of SLN and pyrimethamine against tachyzoites was 29.1 and 25.7µg/mL, respectively. While, the 50% cytotoxic concentration (CC50) value of SLN and pyrimethamine against THP-1 cells was 412.3µg/mL and 269.5µg/mL, respectively. SLN in combined with pyrimethamine obviously (p < 0.05) decreased the number and size of the T. gondii cysts in the infected mice. The level of NO, iNOS and IFN-γ genes was obviously (p < 0.001) upregulated. SLN obviously (p < 0.05) decreased the liver level of oxidative stress and increased the level of antioxidant factors. The findings displayed the promising beneficial effects of SLN mainly in combination with current synthetic drugs against latent T. gondii infection in mice. But we need more experiments to approve these findings, clarifying all possible mechanisms, and its efficiency in clinical phases.

Radiolabeled 15-mer peptide internalization is mediated by megalin (LRP2 receptor) in a CRISPR/Cas9-based LRP2 knockout human kidney cell model.

Megalin (LRP2 receptor) mediates the endocytosis of radiolabeled peptides into proximal tubular kidney cells, which may cause nephrotoxicity due to the accumulation of a radioactive tracer. The study aimed to develop a cellular model of human kidney HK2 cells with LRP2 knockout (KO) using CRISPR/Cas9 technique. This model was employed for the determination of the megalin-mediated accumulation of 68Ga- and 99mTc-labeled 15-mer peptide developed to target the vascular endothelial growth factor (VEGF) receptor in oncology radiodiagnostics. The gene editing in the LRP2 KO model was verified by testing two well-known megalin ligands when higher viability of KO cells was observed after gentamicin treatment at cytotoxic concentrations and lower FITC-albumin internalization by the KO cells was detected in accumulation studies. Fluorescent-activated cell sorting was used to separate genetically modified LRP2 KO cell subpopulations. Moreover, flow cytometry with a specific antibody against megalin confirmed LRP2 knockout. The verified KO model identified both 68Ga- and 99mTc-radiolabeled 15-mer peptides as megalin ligands in accumulation studies. We found that both radiolabeled 15-mers enter LRP2 KO HK2 cells to a lesser extent compared to parent cells. Differences in megalin-mediated cellular uptake depending on the radiolabeling were not observed. Using biomolecular docking, the interaction site of the 15-mer with megalin was also described. The CRISPR/Cas9 knockout of LRP2 in human kidney HK2 cells is an effective approach for the determination of radiopeptide internalization mediated by megalin. This in vitro method provided direct molecular evidence for the cellular uptake of radiolabeled anti-VEGFR 15-mer peptides via megalin.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Anti-Leishmanial Effects of a Novel Biocompatible Non-Invasive Nanofibers Containing Royal Jelly and Propolis against Iranian Strain of Leishmania major (MRHO/IR/75/ER): an In-Vitro Study.

Current medications especially the pentavalent antimonial compounds have been used as the first line treatment of cutaneous leishmaniasis (CL), but they have limitations due to serious side effects such as drug resistance, cardio and nephrotoxicity, and high costs. Hence, the demand to find more usable drugs is evident. Synthesis and development of natural, effective, biocompatible, and harmless compounds against Leishmania major is the principal priority of this study. By electrospinning method, a new type of nanofiber were synthesized from royal jelly and propolis with different ratios. Nanofibers were characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscopy (TEM), Thermogravimetric Analysis (TGA), Contact angle, and Fourier-transform infrared spectroscopy (FTIR). The Half-maximal inhibitory concentration (IC50), Half-maximal effective concentration (EC50) and the 50% cytotoxic concentration (CC50) for different concentrations of nanofibers were determined using quantitative calorimetric methods. Inductively coupled plasma-optical emission spectrometry (ICP-OES) and flow cytometry were performed as complementary tests. The results showed that the proposed formulas provide a new achievement that, despite the significant killing activity on L. major, has negligible cytotoxicity on the host cells. Royal jelly nanofibers have significantly shown the best 72 hours results (IC50= 35 μg/ml and EC50=16.4 μg/ml) and the least cytotoxicity. This study presents a great challenge to introduce a new low-cost treatment method for CL, accelerate wound healing, and reduce scarring with minimal side effects and biocompatible materials. Royal jelly and propolis nanofibers significantly inhibit the growth of L. major in-vitro.

Microwave-assisted ultrafine silver nanoparticle synthesis using Mitragyna speciosa for antimalarial applications

Abstract This study investigates the antiplasmodial activity of ultrafine silver nanoparticles (AgNPs, 2–5 nm) synthesized using a green approach involving the Mitragyna speciosa extract and emphasizing the microwave-assisted irradiation technique. Various synthesis parameters were optimized, resulting in the successful production of spherical AgNPs, which exhibited a characteristic surface plasmon resonance peak at around 440 nm. The synthesized AgNPs demonstrated high stability, indicated by a zeta potential value of −28 mV. The antimalarial efficacy of the microwave-assisted AgNPs against the P. falciparum strain was evaluated, demonstrating a half-maximum inhibitory concentration (IC50) value of 1.56 µg·mL−1. Further enhancement in the antimalarial performance was observed when the AgNPs were conjugated with chloroquine (CQ), a traditional antimalarial drug, achieving an impressive IC50 value of 24 ng·mL−1. Additionally, all formulations exhibited low toxicity, with a cytotoxic concentration (CC50) exceeding 800 µg·mL−1 in Vero cells. Complementing these experimental findings, specific computational studies offered insights into the interactions between silver atoms and bioactive compounds in M. speciosa, as well as shedding light on the dynamics of CQ functionalization. These experimental and computational findings emphasize the potential of a sustainable, low-toxicity, and cost-effective AgNP synthesis process, showcasing significant promise in advancing green nanotechnology for the development of effective antimalarial medications.

Molecular mechanisms underlying Tao-Hong-Si-Wu decoction treating hyperpigmentation based on network pharmacology, Mendelian randomization analysis, and experimental verification

Context Hyperpigmentation, a common skin condition marked by excessive melanin production, currently has limited effective treatment options. Objective This study explores the effects of Tao-Hong-Si-Wu decoction (THSWD) on hyperpigmentation and to elucidate the underlying mechanisms. Materials and methods We employed network pharmacology, Mendelian randomization, and molecular docking to identify THSWD’s hub targets and mechanisms against hyperpigmentation. The Cell Counting Kit-8 (CCK-8) assay determined suitable THSWD treatment concentrations for PIG1 cells. These cells were exposed to graded concentrations of THSWD-containing serum (2.5%, 5%, 10%, 15%, 20%, 30%, 40%, and 50%) and treated with α-MSH (100 nM) to induce an in vitro hyperpigmentation model. Assessments included melanin content, tyrosinase activity, and Western blotting. Results ALB, IL6, and MAPK3 emerged as primary targets, while quercetin, apigenin, and luteolin were the core active ingredients. The CCK-8 assay indicated that concentrations between 2.5% and 20% were suitable for PIG1 cells, with a 50% cytotoxicity concentration (CC50) of 32.14%. THSWD treatment significantly reduced melanin content and tyrosinase activity in α-MSH-induced PIG1 cells, along with downregulating MC1R and MITF expression. THSWD increased ALB and p-MAPK3/MAPK3 levels and decreased IL6 expression in the model cells. Discussion and conclusion THSWD mitigates hyperpigmentation by targeting ALB, IL6, and MAPK3. This study paves the way for clinical applications of THSWD as a novel treatment for hyperpigmentation and offers new targeted therapeutic strategies.

Novel synthesis of new triazine sulfonamides with antitumor, anti-microbial and anti-SARS-CoV-2 activities

Novel approach for synthesizing triazine sulfonamide derivatives is accomplished via reacting the sulfaguanidine derivatives with N-cyanodithioiminocarbonate. Further reaction of the novel triazine sulfonamide analogues with various secondary amines and anilines generated various substituted triazine sulfonamide analogues of promising broad-spectrum activities including anti-microbial, anti-tumor, and anti-viral properties. The in vitro anti-proliferative activities of most of the novel compounds were evaluated on the NCI-60 cell line panel. The antifungal and antibacterial activities of the compounds were also estimated. The anti-viral activity against SARS CoV-2 virus was performed using MTT cytotoxicity assay to evaluate the half-maximal cytotoxic concentration (CC50) and inhibitory concentration 50 (IC50) of a representative compound from the novel triazine sulfonamide category. Compound 3a demonstrated potent antiviral activity against SARS-CoV-2 with IC50 = 2.378 µM as compared to the activity of the antiviral drug remdesivir (IC50 = 10.11 µM). Our results indicate that, upon optimization, these new triazine sulfonamides could potentially serve as novel antiviral drugs.

The effect of ginger extract on expression of Gli1 and Patched-1 genes involved in breast cancer signaling pathway under in vitro condition

Introduction and Aim: Breast cancer ranks among the leading causes of death in women worldwide. Ginger has shown potential efficacy against certain cancer types, surpassing conventional therapies such as chemotherapy and radiation. However, its molecular mechanisms remain less understood. Materials and Methods: In this study, MCF-7 cancer cells were cultured and treated with various concentrations of aqueous ginger extract (20, 30, 45, 65 microgram/mL) for 12, 48, and 72 hours. The effects were assessed through gene expression analysis and cell vitality assays (MTT). Results: The cell vitality test revealed a direct correlation between cytotoxicity and extract concentration. Concentrations exceeding 30 microgram/mL exhibited significant cell death (IC50 of 104.03 microgram/mL). Gene expression analysis demonstrated an increase in Patched-1 and a decrease in Gli1 expression with rising extract concentrations. The maximum Patched-1 expression occurred at 65 microgram/mL after 72 hours. Patched-1 (oncogene) and Gli1 (tumor suppressor) are pivotal genes in the hedgehog (Hh) signalling pathway associated with breast cancer. Conclusion: It appears that ginger compounds play a substantial role in regulating cancer progression by influencing key components of the hedgehog signalling pathway.

Brincidofovir Effectively Inhibits Proliferation of Pseudorabies Virus by Disrupting Viral Replication.

Pseudorabies is an acute and febrile infectious disease caused by pseudorabies virus (PRV), a member of the family Herpesviridae. Currently, PRV is predominantly endemoepidemic and has caused significant economic losses among domestic pigs. Other animals have been proven to be susceptible to PRV, with a mortality rate of 100%. In addition, 30 human cases of PRV infection have been reported in China since 2017, and all patients have shown severe neurological symptoms and eventually died or developed various neurological sequelae. In these cases, broad-spectrum anti-herpesvirus drugs and integrated treatments were mostly applied. However, the inhibitory effect of the commonly used anti-herpesvirus drugs (e.g., acyclovir, etc.) against PRV were evaluated and found to be limited in this study. It is therefore urgent and important to develop drugs that are clinically effective against PRV infection. Here, we constructed a high-throughput method for screening antiviral drugs based on fluorescence-tagged PRV strains and multi-modal microplate readers that detect fluorescence intensity to account for virus proliferation. A total of 2104 small molecule drugs approved by the U.S. Food and Drug Administration (FDA) were studied and validated by applying this screening model, and 104 drugs providing more than 75% inhibition of fluorescence intensity were selected. Furthermore, 10 drugs that could significantly inhibit PRV proliferation in vitro were strictly identified based on their cytopathic effects, virus titer, and viral gene expression, etc. Based on the determined 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50), the selectivity index (SI) was calculated to be 26.3-3937.2 for these 10 drugs, indicating excellent drugability. The antiviral effects of the 10 drugs were then assessed in a mouse model. It was found that 10 mg/kg brincidofovir administered continuously for 5 days provided 100% protection in mice challenged with lethal doses of the human-origin PRV strain hSD-1/2019. Brincidofovir significantly attenuated symptoms and pathological changes in infected mice. Additionally, time-of-addition experiments confirmed that brincidofovir inhibited the proliferation of PRV mainly by interfering with the viral replication stage. Therefore, this study confirms that brincidofovir can significantly inhibit PRV both in vitro and in vivo and is expected to be an effective drug candidate for the clinical treatment of PRV infections.

Antiproliferative and immunomodulative potential of Citrullus colocynthis and its bioactive compounds in human lymphocytes and lung cells

Ethnopharmacological relevanceCitrullus colocynthis (L.) Schrad is a member of the Cucurbitaceae plant family which has been used in traditional medicine for the treatment of lung diseases such as asthma and bronchitis. Aim of the studyThe study was conducted to investigate antiproliferative and immunomodulating effects of C. colocynthis and isolated cucurbitacins on human T lymphocytes and lung epithelial cells in order to evaluate their potential in the treatment of airway diseases. Materials and methodsDifferent concentrations of an ethanolic extract of C. colocynthis fruits and cucurbitacins B (CuB), E (CuE) and E-glucopyranoside (CuE-Glu) were analysed for their cytotoxicity and immunomodulatory potential on Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors and on the epithelial lung cancer cell line A549. Viability and proliferation were tested using WST1 and CFSE assays. Flow cytometric analysis of AnnexinV/PI staining was used to investigate cell death through apoptosis/necrosis. Effects on regulatory mechanisms of T lymphocytes, such as CD69 and CD25 marker activation, cytokine production of the cytokines interleukin 2 (IL2), tumor necrosis factor α (TNFα) and interferon γ (IFNy) were also analysed via flow cytometry. Influences on the activator protein 1 (AP1), nuclear factor of activated T-cells (NFAT) or nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NFκB) pathways were analysed in the Jurkat reporter cell line. Cytokine secretion in A549 cells stimulated with virus-like particles was analysed using the bead-based Legendplex™ assay. ResultsNon-toxic concentrations of C. colocynthis and CuE-Glu showed dose-dependent effects on viability and proliferation in both T lymphocytes and A549 cells. The extracts inhibited lymphocyte activation and suppressed T cell effector functions, which was also shown by lower production of cytokines IL2, TNFα and IFNy. A dose dependent inhibition of the pathways NFκB, NFAT and AP1 in Jurkat cells could be observed. In A549 cells, especially CuE and CuE-Glu showed inhibitory effects on cytokine production following a simulated viral infection. Unglycosylated cucurbitacins were more effective in suppressing the immune function in lymphocytes than glycosylated cucurbitacins, however this activity is limited to cytotoxic concentrations. ConclusionIn our study we could confirm the immunmodulating effect of C. colocynthis and cucurbitacins B, E and E-glucopyranoside in vitro by suppression of different pathways of inflammation and T cell proliferation. Activity in a lung cell model using a virus-like stimulation shows promise for further research regarding cucurbitacins in airway diseases.

A phenotypic screen of the Global Health Priority Box identifies an insecticide with anthelmintic activity

BackgroundInfection with parasitic nematodes (helminths), particularly those of the order Strongylida (such as Haemonchus contortus), can cause significant and burdensome diseases in humans and animals. Widespread drug (anthelmintic) resistance in livestock parasites, the absence of vaccines against most of these nematodes, and a lack of new and effective chemical entities on the commercial market demands the discovery of new anthelmintics. In the present study, we searched the Global Health Priority Box (Medicines for Malaria Venture) for new candidates for anthelmintic development.MethodsWe employed a whole-organism, motility-based phenotypic screening assay to identify compounds from the Global Health Priority Box with activity against larvae of the model parasite H. contortus, and the free-living comparator nematode Caenorhabditis elegans. Hit compounds were further validated via dose–response assays, with lead candidates then assessed for nematocidal activity against H. contortus adult worms, and additionally, for cytotoxic and mitotoxic effects on human hepatoma (HepG2) cells.ResultsThe primary screen against H. contortus and C. elegans revealed or reidentified 16 hit compounds; further validation established MMV1794206, otherwise known as ‘flufenerim’, as a significant inhibitor of H. contortus larval motility (half-maximal inhibitory concentration [IC50] = 18 μM) and development (IC50 = 1.2 μM), H. contortus adult female motility (100% after 12 h of incubation) and C. elegans larval motility (IC50 = 0.22 μM). Further testing on a mammalian cell line (human hepatoma HepG2 cells), however, identified flufenerim to be both cytotoxic (half-maximal cytotoxic concentration [CC50] < 0.7 μM) and mitotoxic (half-maximal mitotoxic concentration [MC50] < 0.7 μM).ConclusionsThe in vitro efficacy of MMV1794206 against the most pathogenic stages of H. contortus, as well as the free-living C. elegans, suggests the potential for development as a broad-spectrum anthelmintic compound; however, the high toxicity towards mammalian cells presents a significant hindrance. Further work should seek to establish the protein–drug interactions of MMV1794206 in a nematode model, to unravel the mechanism of action, in addition to an advanced structure–activity relationship investigation to optimise anthelmintic activity and eliminate mammalian cell toxicity.Graphical

Silica Microparticles from Sugarcane By-Products as an Encapsulation System for Retinoids Aimed at Topical Sustained Release.

The encapsulation of retinol within silica microparticles has emerged as a promising opportunity in the realm of cosmetic and pharmaceutical formulations, driven by the need to reinforce the photoprotection and oxidation stability of retinol. This work examines the process of encapsulating retinol into silica microparticles. The association efficiency, microparticle size, molecular structure, morphology, oxidation, and release profile, as well as biocompatibility and skin sensitization, were evaluated. Results showed that 0.03% of retinol and 9% of emulsifier leads to an association efficiency higher than 99% and a particle size with an average of 5.2 µm. FTIR results indicate that there is an association of retinol with the silica microparticles, and some may be on the surface. Microscopy indicates that when association happens, there is less aggregation of the particles. Oxidation occurs in two different phases, the first related to the retinol on the surface and the second to the associated retinol. In addition, a burst release of up to 3 h (30% free retinol, 17% associated retinol) was observed, as well as a sustained release of 44% of retinol up to 24 h. Encapsulation allowed an increase in the minimal skin cytotoxic concentrations of retinol from 0.04 μg/mL to 1.25 mg/mL without skin sensitization. Overall, retinol is protected when associated with silica microparticles, being safe to use in cosmetics and dermatology.

Formulation of category-2 hypoglycemic Improved Traditional Medicines from selected Cameroonian medicinal plants: &lt;i&gt;Mangifera indica, Persea americana&lt;/i&gt; and &lt;i&gt;Ageratum conyzoides&lt;/i&gt;

Diabetes mellitus is the fourth leading cause of death worldwide, and constitutes a major public health crisis. Management of the adverse condition relies mostly on synthetic drugs such as metformin, glibenclamide and insulin. However, treatment with synthetic drugs is challenged by side effects and high cost especially to patients in low-income countries like Cameroon. This study set out to formulate Improved Traditional Medicines (ITMs) in the form of capsules from lyophilized aqueous leaf extracts of Ageratum conyzoides, Mangifera indica and Persea americana, in combination, coded as ITM-1, and leaf extract of Mangifera indica alone, referred to as ITM-2. Phytochemical profiling of individual extracts was carried out using standard methods, while their antioxidant activity was evaluated in vitro by the 2,2-diphenyl-1-picryhadrazyl reducing (DPPH) assay and the Ferric (Iron) reducing antioxidant power assay (FRAP). Capsule formulation was guided by findings from prior Physicochemical and pharmacotechnical analysis of individual extracts and their combination. Safety studies were carried out both in vitro (cytotoxicity testing in Vero cells); and in vivo (acute toxicity tests using the mouse model). Preliminary evaluation of the antidiabetic potential of the formulated ITMs was achieved through determination of their acute hypoglycemic and antihyperglycemic properties in Wistar rats (Oral Glucose Tolerance Test). Major classes of phytochemicals detected were alkaloids, phenols, tannins, flavonoids, anthocyanins, tri-terpenes and anthraquinones. The DPPH and FRAP assays showed dose-dependent antioxidant activity for ITM-1 and ITM-2. Both ITMs showed no toxic effects, be it in vitro, (Cytotoxic Concentration 50% - CC50&gt;1000 µg/mL in cells) or in vivo. At the dose of 25 mg/Kg, both ITMs exhibited significant hypoglycemic effects in Wistar rats. The ITM-2 capsules completely suppressed post-prandial glucose peak in rats as compared to the negative control (distilled water), ITM-1 capsule was able to cause restoration of the glucose levels to normal levels after 120 minutes

ANTIVIRAL ACTIVITY OF SAUSSUREA LAPPA ETHANOL EXTRACT AGAINST SARS-COV-2: IN VITRO STUDY

Objective: The study aims to investigate the antiviral activity of S. lappa against Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) in vitro. Methods: The extracts are obtained by ultrasonic-assisted extraction (UAE) with a 96% ethanol solvent. Thin-layer chromatography (TLC) uses n-hexane: ethyl acetate and chloroform: methanol) as mobile phases. The staining outcome is subsequently examined using UV visualizers with a wavelength of 366 nm. To assess the antiviral activity of Vero E6 cells, extracts were employed at doses of 25, 50, 75, and 100 µg/ml, with remdesivir serving as the positive control. Supernatants were collected on days 1, 2, 3, and 6 for qRT-PCR testing with target genes E and ORF1ab. Time-addition experiments were conducted to determine how the extract works as antiviral. Protein expression was tested with Western blots with antibodies S and N SARS-CoV-2. Results: TLC identifies terpenoid chemicals present in the ethanol extract of S. lappa. The ethanol extract of S. lappa exhibited antiviral effects against SARS-CoV-2, with an inhibitory concentration 50 (IC50) of 40 µg/ml, a cytotoxic concentration 50 (CC50) of 131.4 µg/ml, and a selectivity index of 3.51. The extract can potentially impact the entry-post-entry phase of SARS-CoV-2 infection in Vero cells. The immunoblotting results demonstrated a reduction in the expression of S and N proteins in the treatment group compared to the negative control. Conclusion: S. lappa ethanol extract has antiviral activity against SARS-CoV-2 based on an in vitro study.

Molecular targets of cisplatin in HeLa cells explored through competitive activity-based protein profiling strategy

Cisplatin is widely used as anticancer drugs, and DNA is considered as the main target. Considering its high affinity towards cysteines and the important role of cystine containing proteins, we applied a competitive activity-based protein profiling strategy to identify protein cysteines that bind with cisplatin in HeLa cells. Living cells were treated with cisplatin at cytotoxic concentrations, then the protein was extracted. After labeling with desthiobiotin iodoacetamide (DBIA) probe, protein was precipitated, digested and isotopically labeled, subsequently the peptides were combined, and the biotinylated cysteine-containing peptides were enriched and quantified by LC-MS/MS. A total of 3571 peptides which originated from 1871 proteins were identified using the DBIA probe. Among them, 46 proteins were screened as targets, including proteins that have been identified as binding proteins by previous study. A novel cisplatin target, calpain-1 (CAPN1), was identified and validated as binding with cisplatin in vitro.

The SMAC mimetic GDC-0152 is a direct ABCB1-ATPase activity modulator and BIRC5 expression suppressor in cancer cells

Upregulation of the multidrug efflux pump ABCB1/MDR1 (P-gp) and the anti-apoptotic protein BIRC5/Survivin promotes multidrug resistance in various human cancers. GDC-0152 is a DIABLO/SMAC mimetic currently being tested in patients with solid tumors. However, it is still unclear whether GDC-0152 is therapeutically applicable for patients with ABCB1-overexpressing multidrug-resistant tumors, and the molecular mechanism of action of GDC-0152 in cancer cells is still incompletely understood. In this study, we found that the potency of GDC-0152 is unaffected by the expression of ABCB1 in cancer cells. Interestingly, through in silico and in vitro analysis, we discovered that GDC-0152 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multidrug efflux activity at sub-cytotoxic concentrations (i.e., 0.25×IC50 or less). Further investigation revealed that GDC-0152 also decreases BIRC5 expression, induces mitophagy, and lowers intracellular ATP levels in cancer cells at low cytotoxic concentrations (i.e., 0.5×IC50). Co-treatment with GDC-0152 restored the sensitivity to the known ABCB1 substrates, including paclitaxel, vincristine, and YM155 in ABCB1-expressing multidrug-resistant cancer cells, and it also restored the sensitivity to tamoxifen in BIRC5-overexpressing tamoxifen-resistant breast cancer cells in vitro. Moreover, co-treatment with GDC-0152 restored and potentiated the anticancer effects of paclitaxel in ABCB1 and BIRC5 co-expressing xenograft tumors in vivo. In conclusion, GDC-0152 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide essential information to physicians for designing a more patient-specific GDC-0152 clinical trial program in the future.

Antibody functionalized capacitance sensor for label-free and real-time detection of bacteria and antibiotic susceptibility

The effective management of infectious diseases and the growing concern of antibiotic resistance necessitates accurate and targeted therapies, highlighting the importance of antibiotic susceptibility testing. This study aimed to develop a real-time impedimetric biosensor for identifying and monitoring bacterial growth and antibiotic susceptibility. The biosensor employed a gold 8-channel disk-shaped microelectrode array with specific antibodies as bio-recognition elements. This setup was allowed for the analysis of bacterial samples, including Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus. These microorganisms were successfully cultured and detected within 1 h of incubation even with a minimal bacterial concentration of 10 CFU/ml. Overall, the developed biosensor array exhibits promising capabilities for monitoring S. aureus, B. cereus and M. luteus, showcasing an excellent linear response ranging from 10 to 104 CFU/ml with a detection limit of 0.95, 1.22 and 1.04 CFU/mL respectively. Moreover, real-time monitoring of antibiotic susceptibility was facilitated by changes in capacitance, which dropped when bacteria were exposed to antibiotic doses higher than their minimum inhibitory concentration (MIC), indicating suppressed bacterial growth. The capacitance measurements enabled determination of half-maximal cytotoxic concentrations (CC50) values for each bacteria-antibiotic pair. As a proof-of-concept application, the developed sensor array was employed as a sensing platform for the real time detection of bacteria in milk samples, which ensured the reliability of the sensor for in-field detection of foodborne pathogens and rapid antimicrobial susceptibility tests (ASTs).

Determination of Non-Cytotoxic Concentrations of Purine Analogues on Different Types of In Vitro-Incubated Embryonic Cells: A Pilot Study

Different concentrations of the methylxanthine/purine analogues aminophylline and 61-tartrat, were tested on in vitro-incubated embryonic avian cells from duck line DEC 99, as well as of mammalian embryonic cells from bovine line EBTr and mouse fibroblast line 3T3. In all cases, the C\(C_{50}\)/ml cell suspension, presenting the cytotoxic concentration, in which were observed 50\% death or changes, was determined. On the avian embryonic cells, the determined values of the methylxantine/purine analogues were 2.4x \(10^{-6}\) M/L about the aminophylline and 2.1x \(10^{-6}\) M/L about the 61-tartrat. The assessed C\(C_{50}\)/mL of the same compounds on the mammalian cells were 2.4x \(10^{-5}\) M/L about the aminophylline and 2.1x \(10^{-5}\) M/L about the 61-tartrat, respectively. The embryonic mammalian cells were more resistant to both substances than the embryonic avian cells. On the other hand, the assessed values of both C\(C_{50}\)/ml and maximal non-toxic concentration (MNC - in which no cellular mortality or other changes can be detected) of each one of the two methylxantine/purine analogues were very near about EBTr and 3T3 mammalian cell lines, and the mouse cells, which are proved as widely used experimental in vitro-model, showed some advantages in both cases. In this way, the current data suggest a possibility about application of methylxantine/purine derivatives about genetic and/or epigenetic reparations, on DNA- and mRNA-levels of low differentiated mammalian cells.