Search results for: In vitro cytotoxicity

Authors: Martyna Chotomska, Aleksandra Studzinska, Marta Lisowska, Justyna Szubert, Aleksandra Tabis, Jacek Bania, Arkadiusz Miazek

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Objectives: Assessing antigen-specific T cell responses is of utmost importance for the pre-clinical testing of prototype vaccines against intracellular pathogens and tumor antigens. Mainly two types of in vitro assays are used for this purpose 1) enzyme-linked immunospot (ELISpot) and 2) intracellular cytokine staining (ICS). Both are time-consuming, relatively expensive, and require manual dexterity. Here, we assess if a straightforward detection of luciferase activity in blood samples of transgenic reporter mice expressing a secreted Lucia luciferase under the transcriptional control of IFN-γ promoter parallels the sensitivity of IFNγ ELISpot assay. Methods: IFN-γ-LUCIA mouse strain carrying multiple copies of Lucia luciferase transgene under the transcriptional control of IFNγ minimal promoter were generated by pronuclear injection of linear DNA. The specificity of transgene expression and mobilization was assessed in vitro using transgenic splenocytes exposed to various mitogens. The IFN-γ-LUCIA mice were immunized with 50mg of ovalbumin (OVA) emulsified in incomplete Freund’s adjuvant three times every two weeks by subcutaneous injections. Blood samples were collected before and five days after each immunization. Luciferase activity was assessed in blood serum. Peripheral blood mononuclear cells were separated and assessed for frequencies of OVA-specific IFNγ-secreting T cells. Results: We show that in vitro cultured splenocytes of IFN-γ-LUCIA mice respond by 2 and 3 fold increase in secreted luciferase activity to T cell mitogens concanavalin A and phorbol myristate acetate, respectively but fail to respond to B cell-stimulating E.coli lipopolysaccharide. Immunization of IFN-γ-LUCIA mice with OVA leads to over 4 fold increase in luciferase activity in blood serum five days post-immunization with a barely detectable increase in OVA-specific, IFNγ-secreting T cells by ELISpot. Second and third immunizations, further increase the luciferase activity and coincidently also increase the frequencies of OVA-specific T cells by ELISpot. Conclusions: We conclude that minimally invasive monitoring of luciferase secretions in blood serum of IFN-γ-LUCIA mice constitutes a sensitive method for evaluating primary and memory Th1 responses to protein antigens. As such, this method may complement existing methods for rapid immunogenicity assessment of prototype vaccines.

Keywords: ELISpot, immunogenicity, interferon-gamma, reporter mice, vaccines

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Authors: Ashleigh Williams

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Micro- and nanoplastics’ (MNLPs) omnipresence and ecological accumulation is evident when surveying recent environmental impact studies. For example, in 2014 it was estimated that at least 52.3 trillion plastic microparticles are floating at sea, and scientists have even found plastics present remote Arctic ice and snow (5,6). Plastics have even found their way into precipitation, with more than 1000 tons of microplastic rain precipitating onto the Western United States in 2020. Even more recent studies evaluating the chemical safety of reusable plastic bottles found that hundreds of chemicals leached into the control liquid in the bottle (ddH2O, ph = 7) during a 24-hour time period. A consequence of the increased abundance in plastic waste in the air, land, and water every year is the bioaccumulation of MNLPs in ecosystems and trophic niches of the animal food chain, which could potentially cause increased direct and indirect exposure of humans to MNLPs via inhalation, ingestion, and dermal contact. Though the detrimental, toxic effects of MNLPs have been established in marine biota, much less is known about the potentially hazardous health effects of chronic MNLP ingestion in humans. Recent data indicate that long-term exposure to MNLPs could cause possible inflammatory and dysbiotic effects. However, toxicity seems to be largely dose-, as well as size-dependent. In addition, the transcytotic uptake of MNLPs through the intestinal epithelia in humans remain relatively unknown. To this point, the goal of the current study was to investigate the mechanisms of micro- and nanoplastic uptake and transcytosis of Polystyrene (PE) in human stem-cell derived, physiologically relevant in vitro intestinal model systems, and to compare the relative effect of particle size (30 nm, 100 nm, 500 nm and 1 µm), and concentration (0 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL) on polystyrene MNLP uptake, transcytosis and intestinal epithelial model integrity. Observational and quantitative data obtained from confocal microscopy, immunostaining, transepithelial electrical resistance (TEER) measurements, cryosectioning, and ELISA cytokine assays of the proinflammatory cytokines Interleukin-6 and Interleukin-8 were used to evaluate the localization and transcytosis of polystyrene MNPs and its impact on epithelial integrity in human-derived intestinal in vitro model systems. The effect of Microfold (M) cell induction on polystyrene micro- and nanoparticle (MNP) uptake, transcytosis, and potential inflammation was also assessed and compared to samples grown under standard conditions. Microfold (M) cells, link the human intestinal system to the immune system and are the primary cells in the epithelium responsible for sampling and transporting foreign matter of interest from the lumen of the gut to underlying immune cells. Given the uptake capabilities of Microfold cells to interact both specifically and nonspecific to abiotic and biotic materials, it was expected that M- cell induced in vitro samples would have increased binding, localization, and potentially transcytosis of Polystyrene MNLPs across the epithelial barrier. Experimental results of this study would not only help in the evaluation of the plastic toxicity, but would allow for more detailed modeling of gut inflammation and the intestinal immune system.

Keywords: nanoplastics, enteroids, intestinal barrier, tissue engineering, microfold (M) cells

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Authors: Rajinikanth Siddalingam, Poonguzhali Subramanian

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The present study aims to prepare and evaluate the self-nano emulsifying drug delivery (SNEDDS) system to enhance the dissolution rate of a poorly soluble drug dutasteride. The formulation was prepared using capryol PGMC, Cremophor EL, and polyethylene glycol (PEG) 400 as oil, surfactant and co-surfactant, respectively. The pseudo-ternary phase diagrams with presence and absence of drug were plotted to find out the nano emulsification range and also to evaluate the effect of dutasteride on the emulsification behavior of the phases. Prepared SNEDDS formulations were evaluated for its particle size distribution, nano emulsifying properties, robustness to dilution, self-emulsification time, turbidity measurement, drug content and in-vitro dissolution. The optimized formulations are further evaluated for heating cooling cycle, centrifugation studies, freeze-thaw cycling, particle size distribution and zeta potential were carried out to confirm the stability of the formed SNEDDS formulations. The particle size, zeta potential and polydispersity index of the optimized formulation found to be 35.45 nm, -15.45 and 0.19, respectively. The in vitro results are revealed that the prepared formulation enhanced the dissolution rate of dutasteride significantly as compared with pure drug. The in vivo studies in was conducted using rats and the results are revealed that SNEDDS formulation has enhanced the bioavailability of dutasteride drug significantly as compared with raw drug. Based the results, it was concluded that the dutasteride-loaded SNEDDS shows potential to enhance the dissolution of dutasteride, thus improving the bioavailability and therapeutic effects.

Keywords: self-emulsifying drug delivery system, dutasteride, enhancement of bioavailability, dissolution enhancement

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Authors: Jae Bok Heo, Yun Mi Lee, Hee Rang Yun

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Several GTPases are required for ribosome biogenesis and assembly. We recently characterized rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), belonging to the YlqF/YawG family of GTPases, as playing a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating the function of OsNug2, yeast two-hybrid screens were carried out using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a potential interacting protein candidate. In vitro pull down and bimolecular fluorescence complementation assays confirmed the interaction between OsNug2 and OsSTK1, and like green fluorescent protein-tagged OsNug2, green fluorescent protein-tagged OsSTK1 was targeted to the nucleus of Arabidopsis protoplasts. OsSTK1 was not found to affect the GTP-binding activity of OsNug2; however, when recombinant OsSTK1 was included in OsNug2 assay reaction mixtures, OsSTK1 increased the GTPase activity of OsNug2. To test whether OsSTK1 phosphorylates OsNug2 in vitro, a kinase assay was performed. OsSTK1 was found to have weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Yeast complementation testing resulted in a GAL::OsNug2(S209N) mutant-harboring yeast strain exhibiting a growth-defective phenotype on galactose medium at 39°C, divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of mutant OsNug2(S209N) was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings reported here indicate that OsSTK1 functions as a positive regulator protein of OsNug2 by enhancing the GTPase activity of OsNug2, and that the phosphorylation of serine 209 of OsNug2 is essential for the complete function of OsNug2 in ribosome biogenesis.

Keywords: OsSTK1, OsNug2, GTPase activity, GTP binding activity, phosphorylation

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Authors: R. Razavizadeh, F. Adabavazeh, M. Rezaee Chermahini

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Salinity is one of the most widespread agricultural problems in arid and semi-arid areas that limits the plant growth and crop productivity. In this study, the salt stress effects on protein, reducing sugar, proline contents and antioxidant enzymes activities of Carum copticum L. under in vitro conditions were studied. Seeds of C. copticum were cultured in Murashige and Skoog (MS) medium containing 0, 25, 50, 100 and 150 mM NaCl and calli were cultured in MS medium containing 1 μM 2, 4-dichlorophenoxyacetic acid, 4 μM benzyl amino purine and different levels of NaCl (0, 25, 50, 100 and 150 mM). After NaCl treatment for 28 days, the proline and reducing sugar contents of shoots, roots and calli increased significantly in relation to the severity of the salt stress. The highest amount of proline and carbohydrate were observed at 150 and 100 mM NaCl, respectively. The reducing sugar accumulation in shoots was the highest as compared to roots, whereas, proline contents did not show any significant difference in roots and shoots under salt stress. The results showed significant reduction of protein contents in seedlings and calli. Based on these results, proteins extracted from the shoots, roots and calli of C. copticum treated with 150 mM NaCl showed the lowest contents. The positive relationships were observed between activity of antioxidant enzymes and the increase in stress levels. Catalase, ascorbate peroxidase and superoxide dismutase activity increased significantly under salt concentrations in comparison to the control. These results suggest that the accumulation of proline and sugars, and activation of antioxidant enzymes play adaptive roles in the adaptation of seedlings and callus of C. copticum to saline conditions.

Keywords: antioxidant enzymes, Carum copticum, organic solutes, salt stress

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Authors: Koo-Yeon Kim, Eun-Hye Kim, Tae-Il Son

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Epidermal Growth Factor (EGF, Mw=6,045) has been reported to have high efficiency of wound repair and anti-wrinkle effect. However, the half-life of EGF in the body is too short to exert the biological activity effectively when applied in free form. Growth Factors can be stabilized by immobilization with carbohydrates from thermal and proteolytic degradation. Low molecular weight chitosan (LMCS) and its derivate prepared by hydrogen peroxide has high solubility. LM6A6DC was successfully prepared as a reactive carbohydrate for the stabilization of EGF by the reactions of LMCS with alkalization, tosylation, azidation and reduction. The structure of LM6A6DC was confirmed by FT-IR, 1H NMR and elementary analysis. For enhancing the stability of free EGF, EGF was attached with LM6A6DC by using water-soluble carbodiimide. EGF-LM6A6DC conjugates did not show any cytotoxicity on the Normal Human Dermal Fibroblast(NHDF) 3T3 proliferation at least under 100 ㎍/㎖. In the result, it was considered that LM6A6DC is suitable to immobilize of growth factor.

Keywords: epidermal growth factor (EGF), low-molecular-weight chitosan, immobilization

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Authors: Hajji Lobna, Chattaoui Mayssa, Regaieg Hajer, M'Hamdi-Boughalleb Naima, Rhouma Ali, Horrigue-Raouani Najet

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In this study, three local isolates of Trichoderma (Tr1: T. viride, Tr2: T. harzianum and Tr3: T. asperellum) were isolated and evaluated for their biocontrol effectiveness under in vitro conditions and in greenhouse. In vitro bioassay revealed a biopotential control against Fusarium oxysporum f. sp. radicis lycopersici and Meloidogyne javanica (RKN) separately. All species of Trichoderma exhibited biocontrol performance and (Tr1) Trichoderma viride was the most efficient. In fact, growth rate inhibition of Fusarium oxysporum f. sp. radicis lycopersici (FORL) was reached 75.5% with Tr1. Parasitism rate of root-knot nematode was 60% for juveniles and 75% for eggs with the same one. Pots experiment results showed that Tr1 and Tr2, compared to chemical treatment, enhanced the plant growth and exhibited better antagonism against root-knot nematode and root-rot fungi separated or combined. All Trichoderma isolates revealed a bioprotection potential against Fusarium oxysporum f. sp. radicis lycopersici. When pathogen fungi inoculated alone, Fusarium wilt index and browning vascular rate were reduced significantly with Tr1 (0.91, 2.38%) and Tr2 (1.5, 5.5%), respectively. In the case of combined infection with Fusarium and nematode, the same isolate of Trichoderma Tr1 and Tr2 decreased Fusarium wilt index at 1.1 and 0.83 and reduced the browning vascular rate at 6.5% and 6%, respectively. Similarly, the isolate Tr1 and Tr2 caused maximum inhibition of nematode multiplication. Multiplication rate was declined at 4% with both isolates either tomato infected by nematode separately or concomitantly with Fusarium. The chemical treatment was moderate in activity against Meloidogyne javanica and Fusarium oxysporum f. sp. radicis lycopersici alone and combined.

Keywords: trichoderma spp., meloidogyne javanica, Fusarium oxysporum f.sp.radicis lycopersici, biocontrol

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Authors: Teodora Mocan, Lucian Mocan, Cornel Iancu, Flaviu A. Tabaran, Bartos Dana, Matea Cristian

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Antimicrobial resistance, which threatens the efficacy of the existing antibiotics represents a worldwide public health issue. At the current time, vancomycin is the only responsive treatment although has significant cytotoxicity, is partially effective and it is poorly retained by infected tissues. From a clinical point of view, attractive alternative approaches for treating such Meticillin-Resistant Staphylococcus Aureus (MRSA) strains would be using agents that cause physical damage to the bacteria. Modular nanopharmaceuticals systems are being designed to address all of these multifunctional capabilities for the ideal bacterial treatment, with the ability to mix and match appropriate functions. Here we present a novel method of selective laser photothermal ablation of MRSA bacteria mediated by gold nanoparticles bound to PBP antibody against PBP protein located on the MRSA surface.

Keywords: MRSA, laser, nanoparticle, antibody

Procedia PDF Downloads 257

Authors: Georgia Saxami, Evangelia N. Kerezoudi, Evdokia K. Mitsou, Marigoula Vlassopoulou, Georgios Zervakis, Adamantini Kyriacou

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Autism Spectrum Disorder (ASD) is a complex group of developmental disorders of the brain, characterized by social and communication dysfunctions, stereotypes and repetitive behaviors. The potential interaction between gut microbiota (GM) and autism has not been fully elucidated. Children with autism often suffer gastrointestinal dysfunctions, while alterations or dysbiosis of GM have also been observed. Treatment with dietary components has been postulated to regulate GM and improve gastrointestinal symptoms, but there is a lack of evidence for such approaches in autism, especially for prebiotics. This study assessed the effects of Pleurotus eryngii mushroom (candidate prebiotic) and inulin (known prebiotic compound) on gut microbial composition, using faecal samples from autistic children in an in vitro batch culture fermentation system. Selected members of GM were enumerated at baseline (0 h) and after 24 h fermentation by quantitative PCR. After 24 h fermentation, inulin and P. eryngii mushroom induced a significant increase in total bacteria and Faecalibacterium prausnitzii compared to the negative control (gut microbiota of each autistic donor with no carbohydrate source), whereas both treatments induced a significant increase in levels of total bacteria, Bifidobacterium spp. and Prevotella spp. compared to baseline (t=0h) (p for all <0.05). Furthermore, this study evaluated the impact of fermentation supernatants (FSs), derived from P. eryngii mushroom or inulin, on the expression levels of tight junctions’ genes (zonulin-1, occludin and claudin-1) in Caco-2 cells stimulated by bacterial lipopolysaccharides (LPS). Pre-incubation of Caco-2 cells with FS from P. eryngii mushroom led to a significant increase in the expression levels of zonulin-1, occludin and claudin-1 genes compared to the untreated cells, the cells that were subjected to LPS and the cells that were challenged with FS from negative control (p for all <0.05). In addition, incubation with FS from P. eryngii mushroom led to the highest mean expression values for zonulin-1 and claudin-1 genes, which differed significantly compared to inulin (p for all <0.05). Overall, this research highlighted the beneficial in vitro effects of P. eryngii mushroom on the composition of GM of autistic children after 24 h of fermentation. Also, our data highlighted the potential preventive effect of P. eryngii FSs against dysregulation of the intestinal barrier, through upregulation of tight junctions’ genes associated with the integrity and function of the intestinal barrier. This research has been financed by "Supporting Researchers with Emphasis on Young Researchers - Round B", Operational Program "Human Resource Development, Education and Lifelong Learning."

Keywords: gut microbiota, intestinal barrier, autism spectrum disorders, Pleurotus Eryngii

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Authors: Abdu Zakari, Said Jibrin, Fatope Majekodunmi Oladeji, Mohammed Hassan Shagga, Andrew Sule

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From the Stems of Bark of Echinaceae angustifolia DC three known triterpenes 3a,5,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9-yl acetate (lupeol acetate), 4,4,6a,6b,8a,10,11,14b,octamethyl1,1,2,3,4,4a,5,6,6a,6b,7,8,8a, 9,10, 11,12,12a,14,14a,14b-icosahydropicen-3-yl acetate (derivative of β-amyrin and 9- hydroxy-1-isopropenyl-5a,5b,8,8,11a-pentamethyl-icosahydro-cyclopenta[a]chrysene- 3a-carboxylic acid (betulinic acid), labelled as Ea-7-38, Ea-9-10 and Ea-12-85) were isolated and characterized. All isolates were tested for their cytotoxicities against Artemia salina (brine shrimp larvae). Compound Ea-12-85 exhibited potent cytotoxic activity against the Artemia salina, Ea-7-38, Ea-9-10 were found to be non-toxic in the cytotoxicity test. The result of the study has justified the claim of the traditional medicine practitioners in Girei for the treatment of complicated malaria disease using the stem bark of E. angustifolia DC.

Keywords: cytotoxic, terpenes, Echinaceae angustifolia, brine shrimp, artemia salina

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Authors: S. Beekrum, B. Odhav, R. Lalloo, E. A. Amonsou

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Marine microalgae are rich sources of novel and biologically active metabolites; therefore they may be used in the food industry as natural food ingredients and functional foods. They have several biological applications related to health benefits, among others. The aim of the study focused on the screening of phytochemicals from Amphora sp. biomass extracts, and to examine the in vitro antioxidant and antimicrobial potential. Amphora sp. biomass was obtained from CSIR (South Africa) and methanol, hexane and water extracts were prepared. The in vitro antimicrobial effect of extracts were tested against some pathogens (Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans), using the disc diffusion assay. Qualitative analyses of phytochemicals were conducted by chemical tests. The present investigation revealed that all extracts showed relatively strong antibacterial activity against most of the tested bacteria. The highest phenolic content was found in the methanolic extract. Results of the DPPH assay showed that the biomass contained strong antioxidant capacity, 79% in the methanolic extract and 85% in the hexane extract. Extracts have displayed effectively reducing power and superoxide anion radical scavenging activity. Results of this study have highlighted potential antioxidant activity in the methanol and hexane extracts. The results of the phytochemical screening showed the presence of terpenoids and sterols with potential applications as food flavorants and functional foods, respectively. The use of Amphora sp. as a natural antioxidant source and a potential source of antibacterial compounds and phytochemicals in the food industry appears promising and should be investigated further.

Keywords: antioxidants, antimicrobial, microalgae, phytochemicals, cymbella

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Authors: G. Agrawal, R. Agrawal, A. Pich

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The pH and temperature responsive biodegradable poly(N-vinylcaprolactam) (PVCL) based microgels functionalized with itaconic acid (IA) units are prepared via precipitation polymerization for drug delivery applications. Volume phase transition temperature (VPTT) of the obtained microgels is influenced by both IA content and pH of the surrounding medium. The developed microgels can be degraded under acidic conditions due to the presence of hydrazone based crosslinking points inside the microgel network. The microgel particles are able to effectively encapsulate doxorubicin (DOX) drug and exhibit low drug leakage under physiological conditions. At low pH, rapid DOX release is observed due to the changes in electrostatic interactions along with the degradation of particles. The results of the cytotoxicity assay further display that the DOX-loaded microgel exhibit effective antitumor activity against HeLa cells demonstrating their great potential as drug delivery carriers for cancer therapy.

Keywords: degradable, drug delivery, hydrazone linkages, microgels, responsive

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Authors: Simona Moravcova, Jiri Novotny, Zdenka Bendova

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The pineal gland of the vertebrate brain is a circumventricular organ which serves as a major neuroendocrine gland with the primary function of rhythmic secretion of neurohormone melatonin under the control of the hypothalamic suprachiasmatic nucleus (SCN). Soon after the onset of the darkness, the activity of the key rate-limiting enzyme for melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT), raises due to the increased release of norepinephrine from sympathetic neurons terminating on the parenchymal cells where it binds to β-adrenergic receptors. Melatonin codes the length of the night, and it is well recognized for its anti-inflammatory effects. However, to our knowledge, less is known about the effect of the immune system on the melatonin biosynthesis and the precise role of the STAT3 in the signaling pathway leading to the expression of AANAT. Lipopolysaccharide (LPS) is the essential component in the outer surface membrane of gram-negative bacteria and acts as a strong stimulator of natural and innate immunity. STAT3 acts as an important factor in immune response. Here we investigated the effect of LPS on the components of the melatonergic synthetic pathway in the pineal gland. The experiments were performed both in vivo and in vitro. The changes in AANAT activity were determined by radioenzymatic assay. PCR analyses were carried out to detect aa-nat, icer, spi-3 and stat3 gene expression. From our results, it is apparent that the high basal level of phosphorylated forms of STAT3 can be elevated after systemic as well as in vitro administration of LPS. Our experiments have shown that LPS reduces melatonin synthesis, nevertheless, the activity of AANAT was increased. Moreover, the basal level of phosphorylated STAT3 counteracts β-adrenergic receptor-mediated aa-nat gene expression and sustains its own and spi-3 gene expression. In conclusion, LPS can affect immunomodulators such as melatonin in the pineal gland.

Keywords: AANAT, lipopolysaccharide, pineal gland, rat, STAT3

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Authors: Amir Ali, Laura Yael Mendoza

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The application of nanomaterials is becoming the most effective strategy of elicitation to produce a desirable level of plant biomass with complex medicinal compounds. This study was designed to check the influence of phytosynthesized iron nanoparticles (FeNPs) on physical growth characteristics, antioxidant status, and production of steviol glycosides of in vitro grown Stevia rebaudiana. Effect of different concentrations of iron nanoparticles replacement of iron sulfate in MS medium (stock solution) on invitro stevia plant growth following positive control (MS basal medium), negative control (iron sulfate devoid medium), iron sulfate devoid MS medium and supplemented with FeNPs at different concentrations (5.6 mg/L, 11.2 mg/L, 16.8 mg/L, 22.4 mg/L) was evaluated. The iron deficiency leads to a drastic reduction in plant growth. In contrast, applying FeNPs leads to improvement in plant height, leave diameter, improved leave morphology, etc., in a concentration-dependent manner. Furthermore, the stress caused by FeNPs at 16.8 mg/L in cultures produced higher levels of total phenolic content (3.7 ± 0.042 mg/g dry weight: DW) and total flavonoid content (1.9 ± 0.022 mg/g DW and antioxidant activity (78 ± 4.6%). In addition, plants grown in the presence of FeNPs at 22.4 mg/L resulted in higher enzymatic antioxidant activities (SOD = 3.5 ± 0.042 U/mg; POD = 2.6 ± 0.026 U/mg; CAT = 2.8 ± 0.034 U/mg and APx = 3.6 ± 0.043 U/ mg), respectively. Furthermore, exposure to a higher dose of FeNPs (22.4 mg/L) exhibited the maximum amount of stevioside (stevioside: 4.6 ± 0.058 mg/g (DW) and rebaudioside A: 4.9 ± 0.068 mg/g DW) as compared to other doses. The current investigation confirms the effectiveness of FeNPs in growth media. It offers a suitable prospect for commercially desirable production of S. rebaudiana biomass with higher sweet glycosides profiles in vitro.

Keywords: cell culture, stevia, iron nanoparticles, antioxidants

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Authors: Kandi Sridhar, Charles Albert Linton

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Grapes are excellent sources of bioactive compounds, which have been suggested to be responsible for lowering the risk of chronic diseases. Fresh and freeze-dried extracts of Kyoho and Jubilee, commercial grape varieties available in Taiwan and attractive for their quality berries, were investigated for their total phenolics and total flavonoids contents and related dose-dependent antioxidants properties using various in vitro assays. The efficiency of the extraction yield ranged from 7.10 % to 25.53 % (w/w), depending on solvent used. Fresh samples of Kyoho and Jubilee exhibited total polyphenolic contents (351.56 ± 23.08 and 328.67 ± 16.54 µg GAE/mL, respectively), whereas Kyoho freeze-dried methanol: water extracts contains the good levels of total flavonoids (4767.82 ± 22.20 µg QE/mL). Kyoho and Jubilee freeze-dried extracts exhibited the highest total flavonoid contents. There was a weak correlation between total phenolic and flavonoid assays (r= -0.05, R2 = 0.02, p > 0.05). Kyoho fresh and freeze-dried samples showed the DPPH (11.51 – 77.82 %), superoxide scavenging activity (33.61 – 81.95 %), and total antioxidant inhibition (92.01 – 99.28 %), respectively. Total flavonoids were statistically correlated with EC50 DPPH scavenging radicals (r =0.91, p < 0.01), EC50 nitric oxide (r = 0.25, p > 0.05), and EC50 lipid peroxidation radicals (r = 0.38, p > 0.05). These results suggested that the two commercial grape cultivars in Taiwan could be used as a good source of natural antioxidants. Thus, consumption of grapes as a source antioxidant might lower the risk of chronic diseases. Moreover, future studies will investigate and develop phenolic acid profile for the cultivars in Taiwan.

Keywords: antioxidants, EC50 radical scavenging activity, grape cultivars, total phenolics

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Authors: Mayuva Youngsabanant-Areekijseree, Chanikarn Srinark, S. Sengsai, Mayuree Pumipaiboon

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Our project was aimed at morphology of oocytes, follicle cells and follicular fluid proteins study of Large White pig (at local slaughter house in Nakhon Pathom Province). The porcine oocytes and follicular fluid of healthy small follicles (1-2 mm), medium follicles (3-6 mm in diameters) and large follicles (7-8 mm and 10 mm in diameter) were aspirated and collected from the ovary by sterile technique. Then, the oocytes and the follicle cells were separated from the fluid. The oocytes were round shape and surrounded by zona pellucida with numerous layers of cumulus cells. Based on the number of cumulus cell layers surrounding oocytes, the oocytes were classified into 5 types, which were intact-, multi-, partial-cumulus layer oocyte, completely denuded oocyte and degenerative oocyte. The collected oocytes showed high percentages of intact- and multi- cumulus cell layers in the small follicles (53.48%) medium follicles (56.94%) and large follicles (56.52%) which have high potential to develop into mature oocytes in vitro. Proteins from follicular fluid of 3 size follicles were separated by SDS-PAGE and LC/MS/MS. The molecular weight of follicular fluid proteins from the small follicles were 24, 60-65, 79, 110, 140, 160, and > 220 kDa. Meanwhile, the follicular fluid protein from medium and large follicle contained 52, 65, 79, 90, 110, 120, 160, 190 and > 220 kDa. Almost all proteins played important roles in promoting and regulating growth and development of oocytes and ovulation. This finding was an initial tool for in vitro testing and applied biotechnology research. Acknowledgements: The project was funded by a grant from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: follicular fluid protein, LC/MS/MS, porcine oocyte, SDS-PAGE, reproductive biology

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Authors: C. E. Chinma, S. O. Azeez, J. C. Anuonye, O. B. Ocheme, C. M. Yakubu, S. James, E. U. Ohuoba, I. A. Baba

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There is growing interest in the use of rice bran protein in food formulation due to its hypoallergenic protein, high nutritional value and health promoting potentials. For the first time, the amino acid profile, protein digestibility, antioxidant, and functional properties of protein concentrate from some local varieties of rice bran from Nigeria were studied for possible food applications. Protein concentrates were prepared from rice bran and analysed using standard methods. Results showed that protein content of Kwandala, Yardass, Jeep, and Jamila were 69.24%, 69.97%, 68.73%, and 71.62%, respectively while total essential amino acid were 52.71, 53.03, 51.86, and 55.75g/100g protein, respectively. In vitro protein digestibility of protein concentrate from Kwandala, Yardass, Jeep and Jamila were 90.70%, 91.39%, 90.57% and 91.63% respectively. DPPH radical inhibition of protein from Kwandala, Yardass, Jeep, and Jamila were 48.15%, 48.90%, 47.56%, and 53.29%, respectively while ferric reducing ability power were 0.52, 0.55, 0.47 and 0.67mmol TE per gram, respectively. Protein concentrate from Jamila had higher onset (92.57oC) and denaturation temperature (102.13oC), and enthalpy (0.72J/g) than Jeep (91.46oC, 101.76oC, and 0.68J/g, respectively), Kwandala (90.32oC, 100.54oC and 0.57J/g, respectively), and Yardass (88.94oC, 99.45oC, and 0.51J/g, respectively). In vitro digestibility of protein from Kwandala, Yardas, Jeep, and Jamila were 90.70%, 91.39%, 90.57% and 91.63% respectively. Oil absorption capacity of Kwandala, Yardass, Jeep, and Jamila were 3.61, 3.73, 3.40, and 4.23g oil/g sample respectively, while water absorption capacity were 4.19, 4.32, 3.55 and 4.48g water/g sample, respectively. Protein concentrates had low bulk density (0.37-0.43g/ml). Protein concentrate from Jamila rice bran had the highest foam capacity (37.25%), followed by Yardass (34.20%), Kwandala (30.14%) and Jeep (28.90%). Protein concentrates showed low emulsifying and gelling capacities. In conclusion, protein concentrate prepared from these local rice bran varieties could serve as functional ingredients in food formulations and for enriching low protein foods.

Keywords: rice bran protein, amino acid profile, protein digestibility, antioxidant and functional properties

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Authors: Kuan-Hao Tsui, Li-Te Lin, Chia-Jung Li

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The combination of Y-27632 and Vitamin C significantly enhances the quality of aging germ cells by reducing reactive oxygen species (ROS) production, restoring mitochondrial membrane potential balance, and promoting mitochondrial fusion. The age-related decline in oocyte quality contributes to reduced fertility, increased aneuploidy, and diminished embryo quality, with mitochondrial dysfunction in both oocytes and granulosa cells being a key factor in this decline. Experiments on aging germ cells investigated the effects of the Y-27632 and Vitamin C combination. In vivo studies involved aged mice to assess oocyte maturation and ROS accumulation during culture. The assessment included mitochondrial activity, ROS levels, mitochondrial membrane potential, and mitochondrial dynamics. Cellular energy metabolism and ATP production were also measured. The combination treatment effectively addressed mitochondrial dysfunction and regulated cellular energy metabolism, promoting oxygen respiration and increasing ATP production. In aged mice, this supplement treatment enhanced in vitro oocyte maturation and prevented ROS accumulation in aging oocytes during culture. While these findings are promising, further research is needed to explore the long-term effects and potential side effects of the Y-27632 and Vitamin C combination. Additionally, translating these findings to human subjects requires careful consideration. Overall, the study suggests that the Y-27632 and Vitamin C combination could be a promising intervention to mitigate aging-related dysfunction in germ cells, potentially enhancing oocyte quality, particularly in the context of in vitro fertilization.

Keywords: ovarian aging, supplements, ROS, mitochondria

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Authors: Natasa Ilic, Alisa Gruden-Movsesijan, Maja Kosanovic, Sofija Glamoclija, Marina Bekic, Ljiljana Sofronic-Milosavljevic, Sergej Tomic

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As a very promising candidate for modulation of immune response in the sense of biasing the inflammatory towards an anti-inflammatory type of response, Trichinella spiralis infection was shown to successfully alleviate the severity of experimental autoimmune encephalomyelitis, the animal model of human disease multiple sclerosis. This effect is achieved via its excretory-secretory muscle larvae (ES L1) products which affect the maturation status and function of dendritic cells (DCs) by inducing the tolerogenic status of DCs, which leads to the mitigation of the Th1 type of response and the activation of a regulatory type of immune response both in vitro and in vivo. ES L1 alone or via treated DCs successfully mitigated EAE in the same manner as the infection itself. On the other hand, it has been shown that T. spiralis infection slows down the tumour growth and significantly reduces the tumour size in the model of mouse melanoma, while ES L1 possesses a pro-apoptotic and anti-survival effect on melanoma cells in vitro. Hence, although the mechanisms still need to be revealed, T. spiralis infection and its ES L1 products have a bit of controversial potential to modulate both inflammatory diseases and malignancies. The recent discovery of T. spiralis extracellular vesicles (TsEVs) suggested that the induction of complex regulation of the immune response requires simultaneous delivery of different signals in nano-sized packages. This study aimed to explore whether TsEVs bare the similar potential as ES L1 to influence the status of DCs in initiation, progression and regulation of immune response, but also to investigate the effect of both ES L1 and TsEVs on myeloid derived suppressor cells (MDSC) which present the regular tumour tissue environment. TsEVs were enriched from the conditioned medium of T. spiralis muscle larvae by differential centrifugation and used for the treatment of human monocyte-derived DCs and MDSC. On DCs, TsEVs induced low expression of HLA DR and CD40, moderate CD83 and CD86, and increased expression of ILT3 and CCR7 on treated DCs, i.e., they induced tolerogenic DCs. Such DCs possess the capacity to polarize T cell immune response towards regulatory type, with an increased proportion of IL-10 and TGF-β producing cells, similarly to ES L1. These findings indicated that the ability of TsEVs to induce tolerogenic DCs favoring anti-inflammatory responses may be helpful in coping with diseases that involve Th1/Th17-, but also Th2-mediated inflammation. In MDSC in vitro model, although both ES L1 and TsEVs had the same impact on MDSC phenotype i.e., they acted suppressive, ES L1 treated MDSC, unlike TsEVs treated ones, induced T cell response characterized by the increased RoRγT and IFN-γ, while the proportion of regulatory cells was decreased followed by the decrease in IL-10 and TGF-β positive cells proportion within this population. These findings indicate the interesting ability of ES L1 to modulate T cells response via MDSC towards pro-inflamatory type, suggesting that, unlike TsEVs which consistently demonstrate the suppresive effect on inflammatory response, it could be used also for the development of new approaches aimed for the treatment of malignant diseases. Acknowledgment: This work was funded by the Promis project – Nano-MDCS-Thera, Science Fund, Republic of Serbia.

Keywords: dendritic cells, myeloid derived suppressor cells, immunomodulation, Trichinella spiralis

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Authors: Huixin Wei, Shibin Wang, Linan Li, Lei Zhou, Xinhao Tu

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Facial skin is a biomedical material with complex mechanical properties of anisotropy, viscoelasticity, and hyperelasticity. The mechanical properties of facial skin are crucial for a number of applications including facial plastic surgery, animation, dermatology, cosmetic industry, and impact biomechanics. Skin is a complex multi-layered material which can be broadly divided into three main layers, the epidermis, the dermis, and the hypodermis. Collagen fibers account for 75% of the dry weight of dermal tissue, and it is these fibers which are responsible for the mechanical properties of skin. Many research on the anisotropic mechanical properties are mainly concentrated on in vitro, but there is a great difference between in vivo and in vitro for mechanical properties of the skin. In this study, we presented a method to measure the mechanical properties of facial skin in vivo. Digital image correlation (DIC) and indentation tests were used to obtain the experiment data, including the deformation of facial surface and indentation force-displacement curve. Then, the experiment was simulated using a finite element (FE) model. Application of Computed Tomography (CT) and reconstruction techniques obtained the real tissue geometry. A three-dimensional FE model of facial skin, including a bi-layer system, was obtained. As the epidermis is relatively thin, the epidermis and dermis were regarded as one layer and below it was hypodermis in this study. The upper layer was modeled as a Gasser-Ogden-Holzapfel (GOH) model to describe hyperelastic and anisotropic behaviors of the dermis. The under layer was modeled as a linear elastic model. In conclusion, the material properties of two-layer were determined by minimizing the error between the FE data and experimental data.

Keywords: facial skin, indentation test, finite element, digital image correlation, computed tomography

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Authors: Richa Sharma, Uma Nahar, Sadhna Sharma, Indu Verma

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Counteracting the deadly pathogen Mycobacterium tuberculosis (M. tb) effectively is still a global challenge. Scrutinizing alternative weapons like antimicrobial peptides to strengthen existing tuberculosis artillery is urgently required. Considering the antimycobacterial potential of Human Beta Defensin 1 (HBD-1) along with isoniazid, the present study was designed to explore the ability of HBD-1 to act against active and dormant M. tb. HBD-1 was screened in silico using antimicrobial peptide prediction servers to identify its short antimicrobial motif. The activity of both HBD-1 and its selected motif (Pep B) was determined at different concentrations against actively growing M. tb in vitro and ex vivo in monocyte derived macrophages (MDMs). Log phase M. tb was grown along with HBD-1 and Pep B for 7 days. M. tb infected MDMs were treated with HBD-1 and Pep B for 72 hours. Thereafter, colony forming unit (CFU) enumeration was performed to determine activity of both peptides against actively growing in vitro and intracellular M. tb. The dormant M. tb models were prepared by following two approaches and treated with different concentrations of HBD-1 and Pep B. Firstly, 20-22 days old M. tbH37Rv was grown in potassium deficient Sauton media for 35 days. The presence of dormant bacilli was confirmed by Nile red staining. Dormant bacilli were further treated with rifampicin, isoniazid, HBD-1 and its motif for 7 days. The effect of both peptides on latent bacilli was assessed by colony forming units (CFU) and most probable number (MPN) enumeration. Secondly, human PBMC granuloma model was prepared by infecting PBMCs seeded on collagen matrix with M. tb(MOI 0.1) for 10 days. Histopathology was done to confirm granuloma formation. The granuloma thus formed was incubated for 72 hours with rifampicin, HBD-1 and Pep B individually. Difference in bacillary load was determined by CFU enumeration. The minimum inhibitory concentrations of HBD-1 and Pep B restricting growth of mycobacteria in vitro were 2μg/ml and 20μg/ml respectively. The intracellular mycobacterial load was reduced significantly by HBD-1 and Pep B at 1μg/ml and 5μg/ml respectively. Nile red positive bacterial population, high MPN/ low CFU count and tolerance to isoniazid, confirmed the formation of potassium deficienybaseddormancy model. HBD-1 (8μg/ml) showed 96% and 99% killing and Pep B (40μg/ml) lowered dormant bacillary load by 68.89% and 92.49% based on CFU and MPN enumeration respectively. Further, H&E stained aggregates of macrophages and lymphocytes, acid fast bacilli surrounded by cellular aggregates and rifampicin resistance, indicated the formation of human granuloma dormancy model. HBD-1 (8μg/ml) led to 81.3% reduction in CFU whereas its motif Pep B (40μg/ml) showed only 54.66% decrease in bacterial load inside granuloma. Thus, the present study indicated that HBD-1 and its motif are effective antimicrobial players against both actively growing and dormant M. tb. They should be further explored to tap their potential to design a powerful weapon for combating tuberculosis.

Keywords: antimicrobial peptides, dormant, human beta defensin 1, tuberculosis

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Authors: Nandini Nalika, Suhel Parvez

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Nanotechnology has emerged to play a vital role in developing all through the industrial world with an immense production of nanomaterials including nanoparticles (NPs). Many toxicological studies have confirmed that due to unique small size and physico-chemical properties of NPs (1-100nm), they can be potentially hazardous. Metallic NPs of small size have been shown to induce higher levels of cellular oxidative stress and can easily pass through the Blood Brain Barrier (BBB) and significantly accumulate in brain. With the wide applications of titanium dioxide nanoparticles (TNPs) in day-to-day life in form of cosmetics, paints, sterilisation and so on, there is growing concern regarding the deleterious effects of TNPs on central nervous system and mitochondria appear to be important cellular organelles targeted to the pro-oxidative effects of NPs and an important source that contribute significantly for the production of reactive oxygen species after some toxicity or an injury. The aim of our study was to elucidate the effect of TNPs in anatase form with different concentrations (5-50 µg/ml) following with various oxidative stress markers in isolated brain mitochondria as an in vitro model. Oxidative stress was determined by measuring the different oxidative stress markers like lipid peroxidation as well as the protein carbonyl content which was found to be significantly increased. Reduced glutathione content and major glutathione metabolizing enzymes were also modulated signifying the role of glutathione redox cycle in the pathophysiology of TNPs. The study also includes the mitochondrial enzymes (Complex 1, Complex II, complex IV, Complex V ) and the enzymes showed toxicity in a relatively short time due to the effect of TNPs. The study provide a range of concentration that were toxic to the neuronal cells and data pointing to a general toxicity in brain mitochondria by TNPs, therefore, it is in need to consider the proper utilization of NPs in the environment.

Keywords: mitochondria, nanoparticles, brain, in vitro

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Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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Authors: Affiah D. U., Katuri I. P., Emefiene M. E., Amienyo C. A.

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Broccoli (Brassica oleraceae L., var. italica) is one of the most important vegetables that is high in nutrients and bioactive compounds. It easily grown on a wide range of soil types and is adaptable to many different climatic conditions. This study was carried out within Jos North and environs in vitro to evaluate Neem (Azadirachta indica) leaves extract against Albugo candida, the causative agent of white blisters disease of broccoli. Through the survey, prevalence and incidence were accessed and a fluffy white growth symptom on the underside of leaves was also observed on the field. Infected leaves samples were collected from three different farms namely: Farin Gada, Naraguta, and Juth and the organism associated with the disease was isolated. Pathogenicity test carried out revealed the fungal isolate Albugo candida to be responsible for the disease. Antimicrobial susceptibility test was performed using agar well diffusion method to determine the minimum inhibitory concentrations of two extract of Azadirachta indica leaves against the organism. Ethanolic extract had the highest antifungal activities of 3.30±0.21 - 17.61± 0.11 while aqueous extract had the least antifungal activities of 0.00±0.00 - 13.23±0.12. The minimum inhibitory concentration of aqueous was 100 mg/ml while its minimum fungicidal concentration was at 200 mg/ml. For ethanol, the minimum inhibitory concentration was 50 mg/ml while its minimum fungicidal concentration was 100 mg/ml. Plants being less toxic in usage over synthetic or inorganic chemicals makes them easy to handle, easily accessible and renewable. Due to the biosafety of plant extracts and its availability since the plant-based extracts of the two different solvents were found to be effective against the test organism hence, it is recommended for in-depth research to make it readily available for control of other pathogens and pests.

Keywords: antifungal, biocontrol, broccoli, fungi

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Authors: C. Panebianco, K. Adamberg, S. Adamberg, C. Saracino, M. Jaagura, K. Kolk, A. Di Chio, P. Graziano, R. Vilu, V. Pazienza

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Background/Aims: Despite recent advances in treatment options, a modest impact on the outcome of the pancreatic cancer (PC) is observed so far. Short-term fasting cycles have the potential to improve the efficacy of chemotherapy against PC. However, diseased people may refuse to follow the fasting regimen and fasting may worsen the weight loss often occurring in cancer patients. Therefore, alternative approaches are needed. The aim of this study was to assess the effect of Engineered Low glycemic food ELGIF mimicking diet on growth of cancer cell lines in vitro and in an in vivo pancreatic cancer mouse xenograft model. Materials and Methods: BxPC-3, MiaPaca-2 and Panc-1 cells were cultured in control and ELGIF mimicking diet culturing condition to evaluate the tumor growth and proliferation pathways. Pancreatic cancer xenograft mice were subjected to ELGIF to assess the tumor volume and weight as compared to mice fed with control diet. Results: Pancreatic cancer cells cultured in ELGIF mimicking medium showed decreased levels of proliferation as compared to those cultured in the standard medium. Consistently, xenograft pancreatic cancer mice subjected to ELGIF diet displayed a significant decrease in tumor growth. Conclusion: A positive effect of ELGIF diet on proliferation in vitro is associated with the decrease of tumor progression in the in vivo PC xenograft mouse model. These results suggest that engineered dietary interventions could be supportive as synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients.

Keywords: functional food, microbiota, mouse model, pancreatic cancer

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Authors: Chaoran Liu

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Bioactive polysaccharides and polysaccharide-protein complex derived from mushrooms and fungi have a wide range of immunomodulatory activity with low side-effects and have therefore the potential to be developed as an adjuvant in cancer therapies. Mushrooms sclerotium is rich in polysaccharides and the polysaccharides isolated from the sclerotium of Polyporus rhinocerus have shown potent in vivo and in vitro immunomodulatory effects. Macrophages are considered to be an important component of the innate immune response against bacterial infection and cancer. To better understanding the immunomodulatory effects and its underlying mechanisms of sclerotial water-soluble polysaccharides extracted from P. rhinocerus on macrophages, the objectives of this study are to purify the water-soluble novel sclerotial polysaccharides and to characterize the structure and properties as well as to study the detailed molecular mechanisms of the in vitro immunomodulating effects in murine macrophages. The hot water-soluble fraction PRW from the sclerotium of P. rhinocerus was obtained using solvent extraction. PRW was further fractionated by membrane ultrafiltration to a give a fraction (PRW1) with molecular mass less than 50 kDa. PRW1 was characterized to be a polysaccharide-protein complex composed of 45.7% polysaccharide and 44.2% protein. The chemical structure of the carbohydrate moiety of PRW1 was elucidated by GC and FTIR to be mainly beta-D-glucan with trace amount of galactose and mannose. The immunomodulatory effects of PRW1 on murine RAW 264.7 macrophages were demonstrated in terms of the increase in nitric oxide production and cytokine production. Mechanistically, PRW1 initiates ERK phosphorylation to activate macrophages within 15 min and significantly improves the expression level of inducible NOS (iNOS) from 6 h after treatment. In summary, this study indicates that PRW1 is a potent immunomodulatory agent for macrophages and suggests that mushroom sclerotia from Polyporus rhinocerus requires for further investigation in cancer research.

Keywords: Polyporus rhinocerus, mushroom sclerotia, Polysaccharide-Protein Complex, macrophage activation

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Authors: Mohammad Y. Alfaifi

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Anything that poses a challenge or a threat to our well-being is a stress. Understanding the genetic material and cellular response of rats threatened with Repeated swimming stress provides insights that can influence human health. The aim of the present study was to assess the genetical damage and cytological changes caused by exposure of the test organism (Rattus rattus) to forced swimming stress. For this purpose, animals have been submerged in water path 15 minutes daily for 2 weeks. Following that, we performed a micronuclei (MN) test using MNNCE (Micronucleated normocromatic erythrocytes) and MNPCE (Micronucleated polychromatic erythrocytes), NDI (Nuclear division index) and cytological parameters using NDCI (nuclear division cytotoxicity index), necrotic and apoptotic cells in rat's bone marrow samples. Results showed that there was a slightly but not significant increase in the frequency of micronucleated as well as in cytological parameters in bone marrow cells.

Keywords: submergence stress, micronucleus, NDI, NDCI, toxicity, chromosomal aberrations

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Authors: Calvin A. Omolo, Rahul S. Kalhapure, Mahantesh Jadhav, Sanjeev Rambharose, Chunderika Mocktar, Thirumala Govender

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Treatment of infections is compromised by limitations of conventional dosage forms and drug resistance. Nanocarrier system is a strategy to overcome these challenges and improve therapy. Thus, the development of novel materials for drug delivery via nanocarriers is essential. The aim of the study was to synthesize a multi-arm polymer (6-mPEPEA) for enhanced activity of vancomycin (VM) against susceptible and resistant Staphylococcus aureus (MRSA). The synthesis steps of the star polymer followed reported procedures. The synthesized 6-mPEPEA was characterized by FTIR, ¹H and ¹³CNMR and MTT assays. VM loaded micelles were prepared from 6-mPEPEA and characterized for size, polydispersity index (PI) and surface charge (ZP) (Dynamic Light Scattering), morphology by TEM, drug loading (UV Spectrophotometry), drug release (dialysis bag), in vitro and in vivo efficacy against sensitive and resistant S. aureus. 6-mPEPEA was synthesized, and its structure was confirmed. MTT assays confirmed its nontoxic nature with a high cell viability (77%-85%). Unimolecular spherical micelles were prepared. Size, PI, and ZP was 52.48 ± 2.6 nm, 0.103 ± 0.047, -7.3 ± 1.3 mV, respectively and drug loading was 62.24 ± 3.8%. There was a 91% drug release from VCM-6-mPEPEA after 72 hours. In vitro antibacterial test revealed that VM-6-mPEPEA had 8 and 16-fold greater activity against S. aureus and MRSA when compared to bare VM. Further investigations using flow cytometry showed that VM-6-mPEPEA had 99.5% killing rate of MRSA at the MIC concentration. In vivo antibacterial activity revealed that treatment with VM-6-mPEPEA had a 190 and a 15-fold reduction in the MRSA load in untreated and VM treated respectively. These findings confirmed the potential of 6-mPEPEA as a promising bio-degradable nanocarrier for antibiotic delivery to improve treatment of bacterial infections.

Keywords: biosafe, MRSA, nanocarrier, resistance, unimolecular-micelles

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Authors: Hamza Assiry, Gamal A. Mohamed, Sabrin R. M. Ibrahim, Hossam M. Abdallah

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Chemical investigation of the aerial parts of Barleria trispinosa(Forssk.) Vahl. resulted in isolation of four major iridoids that were identified as 6,8-O,O-diacetylshanhiside methyl ester (acetyl barlerin) (1), 8-O-acetylshanzhiside methyl ester (barlerin) (2), shanzhiside methyl ester (3), and 6- ⍺ -L-rhamnopyranosyl-8-O-acetylshanzihiside methyl ester (4). The isolated compounds were confirmed by detailed one and two-dimensional NMR. Isolated compounds were tested for their cytotoxic activity on breast cancer (MCF-7, MDA-MB-231) and colon cancer (LS174T) cell linesusing sulphorhodamine B (SRB) assay. It is noteworthy that compound 1 demonstrated a significant cytotoxic potential towards MDA-MB-231 cell line with IC5016.7 ± 2.7µg / mL compared to doxorubicin whereas compounds 2, showed moderate cytotoxic potential with IC5021.2 ± 1.9µg / mL on MCF-7. The other compounds showed moderate activity on the tested cell lines.

Keywords: acanthaceae, cytotoxicity, metabolites, barleria trispinosa

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Authors: Nurul Hamirah Kamsani, Mohd Hamim Rajikin, Nor Ashikin Mohamed Noor Khan, Sharaniza Abdul Rahim

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Cytoskeletal structures, in particular actin and tubulin, provide a fundamental framework in all cells, including embryos. Under influence of nicotine, the cytoskeletal organization may be subjected to oxidative stress (OS) insult and cause alteration. Tocotrienol-rich fraction (TRF) is proven to enhance fertility better than the other sub-group of Vitamin E, tocopherols (TCPs). The objective of this study was to evaluate the effects of TRF on 1) actin and tubulin of 2- and 8-cell murine embryos and 2) the regulation of reactive oxygen species (ROS)-scavenging enzymes; induced by nicotine. Twenty four female Balb/C were subjected to either subcutaneous (sc) injection of 0.9% NaCl; sc injection of 3.0 mg/kg bw/day nicotine; sc injection of 3.0 mg/kg bw/day nicotine + oral gavage (OG) of 60 mg/kg bw/day TRF; or OG of 60 mg/kg bw/day TRF for 7 consecutive days. After superovulation and mating, animals were euthanized. 2-cell developing embryos were retrieved. 50% of the retrieved embryos were visualized under confocal laser staining microscopy (CLSM) for alterations of actin and tubulin. The remaining amount of embryos was cultured in vitro until 8-cell stage followed by CLSM visualization. Blood plasma was subjected to OS assays. Plasma malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined and analysed accordingly. At both 2- and 8-cell developing stages, actin intensities were significantly reduced in the nicotine group (p<0.001). After the intervention, actin intensity was significantly increased compared to that of the nicotine group (p<0.001). The same trend was seen in tubulin at both cell stages. TRF has minimized the deleterious effects of nicotine in actin and tubulin of both 2- and 8-cell developmental stages during pre-implantation embryonic development in mice in vitro. Levels of endogenous anti-oxidative enzymes were sustained close to control accompanied by decreased levels of OS biomarker.

Keywords: actin, nicotine, pre-implantation embryos, tocotrienol rich fraction, tubulin

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