MTT Reduction Research Articles

Overexpression of mTOR in Leukocytes from ALS8 Patients.

The mutated VAPBP56S (vesicle B associated membrane protein - P56S) protein has been described in a Brazilian family and classified as Amyotrophic Lateral Sclerosis type 8 (ALS8). We aimed to study altered biochemical and immunological parameters in cells from ALS8 patients to identify possible biomarkers or therapeutic targets. Wild-type VAPB, VAPBP56S, mTOR, proinflammatory cytokines, and oxidant/reducing levels in serum, leucocytes, and cellular lysate from ALS8 patients and health Controls were performed by ELISA, fluorimetry, and spectrophotometry. Our results showed similar levels of mutant and wild-type VAPB in serum and intracellular lysate (p > 0.05) when ALS8 patients and Controls were compared. IL-1β, IL-6, and IL-18 levels in patients and Controls showed no difference, suggesting an absence of peripheral inflammation (p > 0.05). Oxidative metabolic response, assessed by mitochondrial ROS production, and reductive response by MTT reduction, were higher in the ALS8 group compared to Controls (p < 0.05), although not characterizing typical oxidative stress in ALS8 patients. Total mTOR levels (phosphorylated or non-phosphorylated) of ALS8 patients were significantly lower in serum and higher in intracellular lysate than the mean equivalents in Controls (p < 0.05). A similar result was observed when we quantified the phosphorylated protein (p < 0.05). We demonstrate the possibility of using these biochemical and immunological parameters as potential therapeutic targets or biomarkers. Furthermore, by hypothesis, we suggest a hormetic response in which both VAPB forms could coexist in different proportions throughout life. The mutated VAPBP56S production would increase with aging and predominate over the wild-type VAPB levels, determining the onset of symptoms and aggravating the disease.

Disruption of mitochondrial bioenergetics and calcium homeostasis by phytanic acid in the heart: Potential relevance for the cardiomyopathy in Refsum disease

Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10–30 μM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50–100 μM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.

Applications of comet and MTT assays in studying Dunaliella algae species

Chloroplasts and unicellular algae genomes, are the most important and vital intracellular organelles which determine the cells physiological performance. Using Comet and MTT assay technics in algal studies, will provide researchers prominent features about both their photosynthesis and genomes. Using two unicellular algal groups, Dunaliella salina (orange) and Dunaliella viridis (green), collected from a hypersaline lake (Maharloo lake) located east of Shiraz, Iran, MTT and Comet assays along with their trouble shootings were used to study these two algae. Results showed that, MTT was reduced due to the presence of oxidoreductive enzymes both in photosynthetic reaction centers and in electron transport chains. It was concluded that, MTT reduction in unicellular algal cells, mostly reflects the chloroplasts activity. The rates of light absorbance at λ = 570 nm due to MTT reduction for 100 000 D. salina cells/well (R2 = 0.9457) as well as for 160 000 D. viridis cells/well (R2=9838) were the same as equal to 1. Due to the presence of beta-carotene in D. salina cells, lower number of cell was required to give the same absorbance as compared to D. viridis. In continuation of this study, non-physiological apoptosis cells death caused by Cis‑platinum drug was investigated in these two algal species. Using Comet assay, the DNA damage was determined in both positive and negative control groups. The tail averages in positive control group and in negative control group, were 14.9 % and 0.73 % respectively. Unicellular aquatic algae that lack cell wall, are suitable models to be used in such studies. We would like to recommend the application of MTT and Comet assays technics due to their high sensitivities for revealing damages done to both photosynthetic system and to DNA which will be effective tools for monitoring the biological and non-biological environmental hazards.

Allophylus africanus Stem Bark Extract Modulates the Mitochondrial Apoptotic Pathway in Human Stomach Cancer Cells.

The present work aimed to detail the mechanisms elicited by Allophylus africanus P. Beauv. stem bark extract in human stomach cancer cells and to identify the bioactives underlying the cytotoxicity. MTT reduction and LDH leakage assays allowed characterizing the cytotoxic effects in AGS cells, which were further detailed by morphological analysis using phalloidin and Hoechst 33258. Proapoptotic mechanisms were elucidated through a mitochondrial membrane potential assay and by assessing the impact upon the activity of caspase-9 and -3. The extract displayed selective cytotoxicity against AGS cells. The absence of plasma membrane permeabilization, along with apoptotic body formation, suggested that pro-apoptotic effects triggered cell death. Intrinsic apoptosis pathway activation was verified, as mitochondrial membrane potential decrease and activation of caspase-9 and -3 were observed. HPLC-DAD profiling enabled the identification of two apigenin-di-C-glycosides, vicenin-2 (1) and apigenin-6-C-hexoside-8-C-pentoside (3), as well as three mono-C-glycosides-O-glycosylated derivatives, apigenin-7-O-hexoside-8-C-hexoside (2), apigenin-8-C-(2-rhamnosyl)hexoside (4) and apigenin-6-C-(2-rhamnosyl)hexoside (5). Isovitexin-2″-O-rhamnoside (5) is the main constituent, accounting for nearly 40% of the total quantifiable flavonoid content. Our results allowed us to establish the relationship between the presence of vicenin-2 and other apigenin derivatives with the contribution to the cytotoxic effects on the presented AGS cells. Our findings attest the anticancer potential of A. africanus stem bark against gastric adenocarcinoma, calling for studies to develop herbal-based products and/or the use of apigenin derivatives in chemotherapeutic drug development.

Protection and Disinfection Activities of Oregano and Thyme Essential Oils Encapsulated in Poly(ε-caprolactone) Nanocapsules.

The biocolonization of building materials by microorganisms is one of the main causes of their degradation. Fungi and bacteria products can have an undesirable impact on human health. The protection and disinfection of sandstone and wood materials are of great interest. In this study, we evaluated the protection and disinfection activity of oregano and thyme essential oils encapsulated in poly(ε-caprolactone) nanocapsules (Or-NCs, Th-NCs) against four types of environmental microorganisms: Pleurotus eryngii, Purpureocillium lilacinum (fungal strains), Pseudomonas vancouverensis, and Flavobacterium sp. (bacterial strains). The surfaces of sandstone and whitewood samples were inoculated with these microorganisms before or after applying Or-NCs and Th-NCs. The concentration-dependent effect of Or-NCs and Th-NCs on biofilm viability was determined by the MTT reduction assay. The results showed that Or-NCs and Th-NCs possess effective disinfection and anti-biofilm activity. Diffuse reflectivity measurements revealed no visible color changes of the materials after the application of the nanoencapsulated essential oils.

Influence of menthol on biofilm formation, ergosterol content, and cell surface hydrophobicity of Candida glabrata.

Resistance to synthetic antifungals has become one of the leading public health challenges around the world. Accordingly, novel antifungal products like naturally occurring molecules can be one of the potential ways to reach efficient curative approaches to control candidiasis. This work evaluated the effect of menthol on cell surface hydrophobicity (CSH), biofilm formation, growth and ergosterol content of Candida glabrata (C. glabrata), a yeast with a high resistance against antifungal agents. Disc diffusion method (susceptibility to synthetic antifungals), broth micro-dilution method (Susceptibility to menthol), MTT reduction assay (biofilm formation), HPLC technique (ergosterol content) and adherence to n-hexadecane (CSH) were employed to determine the influence of menthol against C. glabrata isolates. The MIC range of menthol versus C. glabrata was 1250-5000µg/mL (mean±SD: 3375 ± 1375µg/mL). The mean rate of C. glabrata biofilm formation was decreased up to 97.67, 81.15, 71.21, 63.72, 47.53, 26.31 and 0.051% at 625, 1250, 2500, 5000, 10000, 20000 and 40000µg/mL concentrations, respectively. The percentages of CSH were significantly in groups treated with MIC/2 (17.51±5.52%) and MIC/4 (26±5.87%) concentrations of menthol. Also, the percentage changes in membrane ergosterol were 15.97, 45.34 and 73.40% at 0.125, 0.25 and 0.5mg/mL concentrations of menthol, respectively, in comparison with untreated control. The results showed the menthol impact versus sessile and planktonic C. glabrata cells, and the interference with ergosterol content, CSH, and biofilm formation, which made it a potent natural antifungal.

Biobran/MGN-3, an Arabinoxylan Rice Bran, Protects against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An In Vitro and In Silico Study.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), poses a serious global public health threat for which there is currently no satisfactory treatment. This study examines the efficacy of Biobran/MGN-3 against SARS-CoV-2. Biobran is an arabinoxylan rice bran that has been shown to significantly inhibit the related influenza virus in geriatric subjects. Here, Biobran's anti-SARS-CoV-2 activity was assessed using MTT and plaque reduction assays, RT-PCR, ELISA techniques, and measurements of SARS-CoV-2-related gene expression and protein levels. For Vero E6 cells infected with SARS-CoV-2, Biobran reduced the viral load by 91.9% at a dose of 100 μg/mL, it reduced viral counts (PFU/mL) by 90.6% at 50 μg/mL, and it exhibited a significant selectivity index (EC50/IC50) of 22.5. In addition, Biobran at 10 μg/mL inhibited papain-like proteinase (PLpro) by 87% and ACE2 SARS-CoV-2 S-protein RBD by 90.5%, and it significantly suppressed SARS-CoV-2 gene expression, down-regulating E-gene and RdRp gene expression by 93% each at a dose of 50 μg/mL and inhibiting the E-protein by 91.3%. An in silico docking study was also performed to examine the protein-protein interaction (PPI) between SARS-CoV-2 RBD and DC-SIGN as well as between serine carboxypeptidase and papain-like protease PLpro. Serine carboxypeptidase, an active ingredient in Biobran, was found to interfere with the binding of SARS-CoV-2 to its receptor DC-SIGN on Vero cells, thus preventing the cell entry of SARS-CoV-2. In addition, it impairs the viral replication cycle by binding to PLpro. We conclude that Biobran possesses potent antiviral activity against SARS-CoV-2 in vitro and suggest that Biobran may be able to prevent SARS-CoV-2 infection. This warrants further investigation in clinical trials.

Silver Nanoparticles Phytofabricated through Azadirachta indica: Anticancer, Apoptotic, and Wound-Healing Properties.

Silver nanoparticles (AgNPs) have unlocked numerous novel disciplines in nanobiotechnological protocols due to their larger surface area-to-volume ratios, which are attributed to the marked reactivity of nanosilver, and due to their extremely small size, which enables AgNPs to enter cells, interact with organelles, and yield distinct biological effects. AgNPs are capable of bypassing immune cells, staying in the system for longer periods and with a higher distribution, reaching target tissues at higher concentrations, avoiding diffusion to adjacent tissues, releasing therapeutic agents or drugs for specific stimuli to achieve a longer duration at a specific rate, and yielding desired effects. The phytofabrication of AgNPs is a cost-effective, one-step, environmentally friendly, and easy method that harnesses sustainable resources and naturally available components of plant extracts (PEs). In addition, it processes various catalytic activities for the degradation of various organic pollutants. For the phytofabrication of AgNPs, plant products can be used in a multifunctional manner as a reducing agent, a stabilizing agent, and a functionalizing agent. In addition, they can be used to curtail the requirements for any additional stabilizing agents and to help the reaction stages subside. Azadirachta indica, a very common and prominent medicinal plant grown throughout the Indian subcontinent, possesses free radical scavenging and other pharmaceutical properties via the regulation of proinflammatory enzymes, such as COX and TOX. It also demonstrates anticancer activities through cell-signaling pathways, modulating tumor-suppressing genes such as p53 and pTEN, transcriptional factors, angiogenesis, and apoptosis via bcl2 and bax. In addition, it possesses antibacterial activities. Phytofabricated AgNPs have been applied in the areas of drug delivery, bioimaging, biosensing, cancer treatment, cosmetics, and cell biology. Such pharmaceutical and biological activities of phytofabricated AgNPs are attributed to more than 300 phytochemicals found in Azadirachta indica, and are especially abundant in flavonoids, polyphenols, diterpenoids, triterpenoids, limonoids, tannins, coumarin, nimbolide, azadirachtin, azadirone, azadiradione, and gedunin. Parts of Azadirachta indica, including the leaves in various forms, have been used for wound healing or as a repellent. This study was aimed at examining previously biosynthesized (from Azadirachta indica) AgNPs for anticancer, wound-healing, and antimicrobial actions (through MTT reduction assay, scratch assay, and microbroth dilution methods, respectively). Additionally, apoptosis in cancer cells and the antibiofilm capabilities of AgNPs were examined through caspase-3 expression, dentine block, and crystal violet methods. We found that biogenic silver nanoparticles are capable of inducing cytotoxicity in HCT-116 colon carcinoma cells (IC50 of 744.23 µg/mL, R2: 0.94), but are ineffective against MCF-7 breast cancer cells (IC50 &gt;&gt; 1000 µg/mL, R2: 0.86). AgNPs (IC50 value) induced a significant increase in caspase-3 expression (a 1.5-fold increase) in HCT-116, as compared with control cells. FITC-MFI was 1936 in HCT-116-treated cells, as compared to being 4551 in cisplatin and 1297 in untreated cells. AgNPs (6.26 µg/mL and 62.5 µg/mL) induced the cellular migration (40.2% and 33.23%, respectively) of V79 Chinese hamster lung fibroblasts; however, the improvement in wound healing was not significant as it was for the controls. AgNPs (MIC of 10 µg/mL) were very effective against MDR Enterococcus faecalis in the planktonic mode as well as in the biofilm mode. AgNPs (10 µg/mL and 320 µg/mL) reduced the E. faecalis biofilm by &gt;50% and &gt;80%, respectively. Natural products, such as Syzygium aromaticum (clove) oil (MIC of 312.5 µg/mL) and eugenol (MIC of 625 µg/mL), showed significant antimicrobial effects against A. indica. Our findings indicate that A. indica-functionalized AgNPs are effective against cancer cells and can induce apoptosis in HCT-116 colon carcinoma cells; however, the anticancer properties of AgNPs can also be upgraded through active targeting (functionalized with enzymes, antibiotics, photosensitizers, or antibodies) in immunotherapy, photothermal therapy, and photodynamic therapy. Our findings also suggest that functionalized AgNPs could be pivotal in the development of a novel, non-cytotoxic, biocompatible therapeutic agent for infected chronic wounds, ulcers, and skin lesions involving MDR pathogens via their incorporation into scaffolds, composites, patches, microgels, or formulations for microneedles, dressings, bandages, gels, or other drug-delivery systems.

Evaluation of MTT reducers and strongly colored substances in the Short Time Exposure test method for assessing eye irritation potential.

The Short Time Exposure (STE) test evaluates eye irritation potential using a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assays may underpredict results for some substances that directly reduce MTT (i.e., MTT reducers) or interfere with absorbance because of their strong color (i.e., strongly colored substances). Based on previous research, we selected 25 substances as MTT reducers. Of these, 13 were expected to be MTT reducers at 5% dilution (5% MTT reducers) of the STE test condition. These 13 substances were then tested to determine whether the results were interfered from direct MTT reduction. Those 5% MTT reducers that were classified as irritants based on in vivo data were identified as irritants by the STE test. In addition, the low cell viability results at 5% dilution suggested that direct MTT reduction had not occurred. Next, the remaining 5% MTT reducers that were classified as non-irritants based on in vivo data were identified as non-irritants by the STE test. We then examined two strongly colored substances. One was classified as an irritant based on in vivo data and was confirmed as an irritant by the STE test. The other was classified as a non-irritant by the STE test. This was further evaluated using a medium that did not contain MTT; the result indicated that it was a non-irritant correctly. In conclusion, the STE test is useful for evaluating eye irritation potential without the drawback of underprediction for MTT reducers and strongly colored substances.

Heparin prevents the cytotoxic activity of Bothrops jararacussu and Apis mellifera venoms in renal cells

Envenomation by Bothrops snakes and Apis mellifera bee may imply systemic disorders which affect well-perfused organs such as kidneys, a process that can lead to acute renal failure. Nevertheless, there is scarce information regarding a direct renal cell effect and the putative antagonism by antivenoms. Here the cytotoxic effect of B. jararacussu and A. mellifera venoms was evaluated in the renal proximal tubule cell line LLC-PK1, as well as the antagonism of this effect by heparin. B. jararacussu venom showed significant cytotoxicity as assessed by LDH release and MTT reduction, with a sharp decline of the cell number after 180 min (>90% at 50 μg/mL). A. mellifera venom produced a much faster and potent cytotoxic activity, conferring almost no viable cells after 15 min at 25 μg/mL. Phase contrast microscopy revealed that while B. jararacussu venom induced a progressive loss of cell adhesion and detachment, A. mellifera venom promoted a rapid plasma membrane disruption and nuclear condensation suggestive of necrotic cell death. Pre-incubation of both venoms with heparin for 30 min significantly reduced cytotoxicity. Our results demonstrate direct toxicity of B. jararacussu and A. mellifera venoms toward renal cells but with distinct kinetics and cell pattern, suggesting different mechanisms of action. In addition, the antagonistic, cytoprotective effect of heparin ascribes such compound as a promising drug for preventing renal failure from envenomation.

Silver Nanoparticles and Glycyrrhiza glabra (Licorice) Root Extract as Modifying Agents of Hydrogels Designed as Innovative Dressings.

The interest in the application of plant extracts as modifiers of polymers intended for biomedical purposes is constantly increasing. The therapeutical properties of the licorice root, including its anti-inflammatory and antibacterial activity, make this plant particularly promising. The same applies to silver nanoparticles showing antibacterial properties. Thus the main purpose of the research was to design hydrogel dressings containing both licorice root extract and nanosilver so as to obtain a system promoting wound regeneration processes by preventing infection and inflammation within the wound. The first step included the preparation of the plant extract via the solid-liquid extraction using the Soxhlet extractor and the synthesis of silver nanoparticles by the chemical reduction of silver ions using a sodium borohydride as a reducing agent. Subsequently, hydrogels were synthesized via photopolymerization and subjected to studies aiming at characterizing their sorption properties, surface morphology via scanning electron microscopy, and their impact on simulated physiological liquids supported by defining these liquids' influence on hydrogels' structures by FT-IR spectroscopy. Next, the tensile strength of hydrogels and their percentage elongation were determined. Performed studies also allowed for determining the hydrogels' wettability and free surface energies. Finally, the cytotoxicity of hydrogels towards L929 murine fibroblasts via the MTT reduction assay was also verified. It was demonstrated that developed materials showed stability in simulated physiological liquids. Moreover, hydrogels were characterized by high elasticity (percentage elongation within the range of 24-29%), and their surfaces were hydrophilic (wetting angles below 90°). Hydrogels containing both licorice extract and nanosilver showed smooth and homogeneous surfaces. Importantly, cytotoxic properties towards L929 murine fibroblasts were excluded; thus, developed materials seem to have great potential for application as innovative dressings.

Repellent active ingredients encapsulated in polymeric nanoparticles: potential alternative formulations to control arboviruses

Dengue, yellow fever, Chinkungunya, Zika virus, and West Nile fever have infected millions and killed a considerable number of humans since their emergence. These arboviruses are transmitted by mosquito bites and topical chemical repellents are the most commonly used method to protect against vector arthropod species. This study aimed to develop a new generation of repellent formulations to promote improved arboviruses transmission control. A repellent system based on polycaprolactone (PCL)-polymeric nanoparticles was developed for the dual encapsulation of IR3535 and geraniol and further incorporation into a thermosensitive hydrogel. The physicochemical and morphological parameters of the prepared formulations were evaluated by dynamic light scattering (DLS), nano tracking analysis (NTA), atomic force microscopy (AFM). In vitro release mechanisms and permeation performance were evaluated before and after nanoparticles incorporation into the hydrogels. FTIR analysis was performed to evaluate the effect of formulation epidermal contact. Potential cytotoxicity was evaluated using the MTT reduction test and disc diffusion methods. The nanoparticle formulations were stable over 120days with encapsulation efficiency (EE) of 60% and 99% for IR3535 and geraniol, respectively. AFM analysis revealed a spherical nanoparticle morphology. After 24h, 7 ± 0.1% and 83 ± 2% of the GRL and IR3535, respectively, were released while the same formulation incorporated in poloxamer 407 hydrogel released 11 ± 0.9% and 29 ± 3% of the loaded GRL and IR3535, respectively. GRL permeation from PCL nanoparticles and PCL nanoparticles in the hydrogel showed similar profiles, while IR3535 permeation was modulated by formulation compositions. Differences in IR3535 permeated amounts were higher for PCL nanoparticles in the hydrogels (36.9 ± 1.1mg/cm2) compared to the IR3535-PCL nanoparticles (29.2 ± 1.5mg/cm2). However, both active permeation concentrations were low at 24h, indicating that the formulations (PCL nanoparticles and PCL in hydrogel) controlled the bioactive percutaneous absorption. Minor changes in the stratum corneum (SC) caused by interaction with the formulations may not represent a consumer safety risk. The cytotoxicity results presented herein indicate the carrier systems based on poly-epsilon caprolactone (PCL) exhibited a reduced toxic effect when compared to emulsions, opening perspectives for these systems to be used as a tool to prolong protection times with lower active repellent concentrations.

Development of Chitosan/Gelatin-Based Hydrogels Incorporated with Albumin Particles.

The research subject of this paper are natural polymer-based hydrogels modified with albumin particles. The proteins were obtained via the salt-induced precipitation method, and next characterized using dynamic light scattering (DLS), UV-Vis spectroscopy and FT-IR spectroscopy. The most favorable composition showing monodispersity and particles with a size lower than 40 nm was selected for modification of hydrogels. Such systems were obtained via the photopolymerization performed under the influence of UV radiation using diacrylate poly(ethylene glycol) as a crosslinking agent and 2-hydroxy-2-methylpropiophenone as a photoinitiator. Next, the hydrogels' swelling ability, mechanical properties, wettability and surface morphology were characterized. Moreover, FT-IR spectroscopy, incubation studies in simulated physiological liquids, pro-inflammatory activity analysis and MTT reduction assay with L929 murine fibroblasts were performed. The release profiles of proteins from hydrogels were also verified. Materials modified with proteins showed higher swelling ability, increased flexibility even by 50% and increased surface hydrophilicity. Hydrogels' contact angles were within the range 62-69° while the tensile strength of albumin-containing hydrogels was approx. 0.11 MPa. Furthermore, the possibility of the effective release of protein particles from hydrogels in acidic environment (approximately 70%) was determined. Incubation studies showed hydrogels' stability and lack of their degradation in tested media. The viability of fibroblasts was 89.54% for unmodified hydrogel, and approx. 92.73% for albumin-modified hydrogel, and such an increase indicated the positive impact of the albumin on murine fibroblast proliferation.

Zinc Oxide Nanoparticles Induce Toxicity in H9c2 Rat Cardiomyoblasts.

Zinc oxide nanoparticles (ZnO NPs) are widely used in the cosmetic industry. They are nano-optical and nano-electrical devices, and their antimicrobial properties are applied in food packaging and medicine. ZnO NPs penetrate the body through inhalation, oral, and dermal exposure and spread through circulation to various systems and organs. Since the cardiovascular system is one of the most vulnerable systems, in this work, we studied ZnO NPs toxicity in H9c2 rat cardiomyoblasts. Cardiac cells were exposed to different concentrations of ZnO NPs, and then the morphology, proliferation, viability, mitochondrial membrane potential (ΔΨm), redox state, and protein expression were measured. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining showed strong morphological damage. ZnO NPs were not observed inside cells, suggesting that Zn2+ ions were internalized, causing the damage. ZnO NPs strongly inhibited cell proliferation and MTT reduction at 10 and 20 μg/cm2 after 72 h of treatment. ZnO NPs at 20 μg/cm2 elevated DCF fluorescence, indicating alterations in the cellular redox state associated with changes in ΔΨm and cell death. ZnO NPs also reduced the intracellular expression of troponin I and atrial natriuretic peptide. ZnO NPs are toxic for cardiac cells; therefore, consumption of products containing them could cause heart damage and the development of cardiovascular diseases.

“The Effects of Silver Nanoparticle Shape on Protein Adsorption and Neural Stem Cell Viability”

Silver nanoparticles (AgNPs) are important and widely used as antimicrobials and nanodrug carriers. The increased use of AgNPs in consumer products has raised concerns about nanosafety; for instance, AgNPs may be inhaled and translocated to the brain via olfactory neural stem cells/progenitors. While the biological effects of nanoparticle size have been widely investigated, there are little data on the effects of particle shape on cellular phenotype. Therefore, here we investigated the interactions between AgNP spheres, rods, cubes, and triangles and human plasma proteins as well as their effects on the viability of NE-4C neural stem cells. Nanoparticles were synthesized by wet chemistry methods and characterized by UV-vis spectroscopy, dynamic light scattering, zeta potential measurement, transmission electron microscopy, nanoparticle tracking analysis, and differential centrifugal sedimentation. NE-4C cell viability was assessed using the MTT reduction assay, and the cellular uptake of differently shaped nanoparticles was monitored by electron microscopy. All 50 nm (in at least one dimension) AgNPs exerted toxic effects, with rods and cubes displaying greater toxicity than spheres and triangles. These cellular and physicochemical results indicate that edges on the AgNPs increase toxicity, presumably due to enhanced ion dissolution from the edges.

Autophagy (but not metabolism) is a key event in mitoxantrone-induced cytotoxicity in differentiated AC16 cardiac cells.

Mitoxantrone (MTX) is an antineoplastic agent used to treat advanced breast cancer, prostate cancer, acute leukemia, lymphoma and multiple sclerosis. Although it is known to cause cumulative dose-related cardiotoxicity, the underlying mechanisms are still poorly understood. This study aims to compare the cardiotoxicity of MTX and its' pharmacologically active metabolite naphthoquinoxaline (NAPHT) in an in vitro cardiac model, human-differentiated AC16 cells, and determine the role of metabolism in the cardiotoxic effects. Concentration-dependent cytotoxicity was observed after MTX exposure, affecting mitochondrial function and lysosome uptake. On the other hand, the metabolite NAPHT only caused concentration-dependent cytotoxicity in the MTT reduction assay. When assessing the effect of different inhibitors/inducers of metabolism, it was observed that metyrapone (a cytochrome P450 inhibitor) and phenobarbital (a cytochrome P450 inducer) slightly increased MTX cytotoxicity, while 1-aminobenzotriazole (a suicide cytochrome P450 inhibitor) decreased fairly the MTX-triggered cytotoxicity in differentiated AC16 cells. When focusing in autophagy, the mTOR inhibitor rapamycin and the autophagy inhibitor 3-methyladenine exacerbated the cytotoxicity caused by MTX and NAPHT, while the autophagy blocker, chloroquine, partially reduced the cytotoxicity of MTX. In addition, we observed a decrease in p62, beclin-1, and ATG5 levels and an increase in LC3-II levels in MTX-incubated cells. In conclusion, in our in vitro model, neither metabolism nor exogenously given NAPHT are major contributors to MTX toxicity as seen by the residual influence of metabolism modulators used on the observed cytotoxicity and by NAPHT's low cytotoxicity profile. Conversely, autophagy is involved in MTX-induced cytotoxicity and MTX seems to act as an autophagy inducer, possibly through p62/LC3-II involvement.

Comparative efficacy of coconut water diluent with different semen extenders for cryopreservation of Nili Ravi buffalo bull semen

In this study, efforts were made to investigate fresh semen parameters and to select a suitable extender for buffalo semen cryopreservation. Experiment I, fresh undiluted seminal parameters were determined while Experiment II, efficacy comparison of coconut water extender (CWE) with tris citric acid extender (TCAE) and skimmed milk extender (SME) was made. Four bulls single ejaculate was collected weekly for 5 and 10 weeks for Experiment I and II, respectively however osmotic pressure replicates were 16 and 6 for semen and seminal plasma, respectively. In Experiment I, each bull spermatozoa concentration, motility (%), semen volume and pooled semen percentage NAR, PMI, viability and MTT (3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction rate was checked. In Experiment II, pooled semen added to extenders and then equilibrated (4oC) and filled to obtain 20×106 spermatozoa/0.5 ml straws before plunging in liquid nitrogen. Percentage thawed spermatozoa viability, normal acrosomal ridge (NAR), motility, DNA damage, plasma membrane integrity (PMI) and lipid peroxidation (nM) recorded. In Experiment I, seminal parameters as spermatozoa concentration (1226.43±71.48 million/mL), semen volume (2.84±0.14 mL), viability (90.05±0.71%), motility (77.13±0.71%), PMI (86.23±0.34%), NAR (94.67±0.30%) and MTT reduction rate (0.290±0.06) while osmotic pressure of seminal plasma (294.83±3.87 mOsm/kg) and semen (290.87±2.58 mOsm/kg) was recorded. In Experiment II, TCAE higher (P&lt;0.05) sperm motility noted compared to CWE and SME whereas percentage viability, NAR, PMI, DNA damage was non-significant. Lipid peroxidation compared to SME and TCAE was higher (P&lt;0.05) in CWE. In conclusion, based on sperm motility and lipid per oxidation, TCAE was more efficient for cryopreservation of buffalo semen.

P131 Optimization of MTT assay protocol in Pythium insidiosum zoospores incubated together with neutrophils

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesThe tetrazolium-based MTT assay is widely used to assess cell viability. This colorimetric assay measures cell viability through the enzymatic reduction of MTT, a yellow water-soluble tetrazolium dye, to purple-colored formazan crystals. The zoospores of Pythium insidiosum are the infective form causing pythiosis. There remains a lack of standardized protocol for P. insidiosum zoospore viability assay. Here, we optimized the MTT assay to assess the activity of neutrophils on zoospores.MethodsNeutrophils and zoospores were incubated for 2 h, 4 h, and overnight at 37°C. Neutrophils were lysed in water (pH = 11), MTT was added to each well and incubated at 37°C for 2 h. Then, SDS-HCl solution was added to each well, and absorbance at 570 nm was measured using a microplate reader. The percentage of viable zoospores was then determined.ResultsThe viable zoospores metabolized MTT to insoluble purple formazan product. The zoospores germinated into hyphae when incubated for ˃2 h. Because of the rapid germination of zoospores into hyphae, reliable results would be obtained with an optimum incubation time of 2 h for the zoospores.ConclusionThe simplicity and low cost of the assay make it a valuable tool for zoospore viability studies, with a focus on immunology and immunotherapy.

P012 Antibiofilm activity of staphylococcal peptide derivative(s) against Candida auris biofilms in vitro and in an animal model of catheter-associated infection

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectives Candida auris has emerged as a major multidrug-resistant nosocomial pathogen worldwide. The organism exhibits a persistent, colonizing phenotype, usually associated with biofilm formation on hospital surfaces, medical equipment, and indwelling medical devices. Biofilm formation by C. auris can further aggravate the infection acquisition and outcome, owing to the intrinsic resistance of biofilms to disinfectants, antiseptics, and antifungal drugs. The present study aimed to evaluate the preventive and therapeutic efficacy of select peptide derivative(s) from staphylococci against C. auris biofilms in vitro and in a mouse model of C. auris catheter-associated infection.MethodsThree potentially antimicrobial, staphylococcal alpha-helical amphipathic peptides (19-23 amino acids) were evaluated for antifungal and antibiofilm activity against clinical isolates of C. auris. The antifungal activity against C. auris planktonic cells was tested by broth microdilution assay according to the method of the Clinical and Laboratory Standards Institute. Biofilm assays were performed in 96 well, flat-bottomed microtiter plates in RPMI-1640, and the effect of the test agents on biofilm formation (MBPC, minimum biofilm preventive concentration) as well as pre-formed biofilms (MBIC, minimum biofilm inhibitory concentration) was determined by subjecting to a two-fold dilution range of the test agents (0.03-64 μm) for 24 h at 37°C, followed by XTT dye reduction test. Field-Emission Scanning Electron Microscopy was performed to observe the effect of peptides on biofilm morphology. The cytotoxicity of these compounds was elucidated on HeLa, HEK-293, and Raw 264.7 murine macrophages by MTT reduction test. The synergistic effect of the selected peptide with representative antifungal drugs belonging to three different classes (amphotericin B, caspofungin, and voriconazole) was tested by fractional inhibitory concentration assays. In vivo activity of the selected peptide was determined in a murine model of subcutaneous C. auris catheter-associated infection, alone and in combination with amphotericin B and caspofungin.ResultsBased on the antimicrobial activity, antibiofilm activity, and cytotoxicity data, the 19 amino acid, alpha helical staphylococcal peptide derivative (charge, +3; hydrophobicity, 0.634, and hydrophobic moment, 0.623) exhibited promising activity against C. auris biofilms. The peptide was particularly effective in preventing C. auris biofilm formation, with a median MBPC50 of 1 μm. It demonstrated synergistic activity with amphotericin B (FIC index, 0.3) as well as caspofungin (FIC index, 0.18), and an additive effect with voriconazole (FIC index, 0.71). When combined with 0.125 μm of the peptide derivative, nearly 4-to-8-fold lower amount of the drug was required to achieve results comparable to the drug-only controls. Furthermore, nearly 99% reduction in biofilm formation was noted at clinically-achievable trough levels of amphotericin B or caspofungin in combination with this peptide derivative. Similar results were noted in vivo, with nearly 99% reduction in biofilm formation of C. auris in catheter lumen using combination therapy with 0.125 μm of the peptide.ConclusionThe present study demonstrates that a 19 amino acid, alpha helical staphylococcal peptide derivative exhibits promising antibiofilm activity against C. auris, particularly in preventing biofilm formation, in vitro and in a murine model of subcutaneous catheter-associated infection.

Phytochemical Analysis and Biological Activity of Salvia compressa Vent.

Salvia extracts have various biological activities and are rich sources of bioactive metabolites. We identified five phytochemicals from S. compressa extract and assessed its biological properties. The plant's shoots were extracted using dichloromethane, and the constituents were isolated using column chromatography. High-resolution NMR spectroscopy characterized the chemical structures of the compounds (1 - 5). The cytotoxic effect of the extract was examined against MCF-7 cells by MTT reduction assay, while cisplatin was tested as a reference cytotoxic compound. The antibacterial activity was assessed using nutrient broth micro-dilution (NBMD), and chloramphenicol was used as the positive control. Citrostadienol (1), β-sitosterol (2), two glyceride esters of linolenic, linoleic, and palmitic acids (3, 4), and geraniol (5) were isolated from S. compressa for the first time. The extract showed activity against MCF-7 breast cancer cells and reduced cell viability to 68.2 ± 13.1% compared to the control drug at the concentration of 50 µg/mL, while it was not active against seven test bacteria. The anti-complementary activity of the isolated steroids suggests S. compressa for future anti-inflammatory research.