Search results for: enzyme assay

Authors: K. Suresh, R. Arunkumar

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The objective of this study is to investigate the preparation of gramine nano suspension and protective effect of Gramine on the apoptosis of laryngeal cancer cells cell line (HEp-2 and KB). The growth inhibition rate of Hep-2 and KB cells in vitro were measured by MTT assay and apoptosis by, levels of reactive oxygen species, mitochondrial membrane potential, morphological changes and flowcytometry. Based on the results, we determined the effective doses of gramine as 127.23µm/ml for 24 hr and 119.81 µm/ml for 48hr in hep-2 cell line and 147.58 µm ml for 24 hr and 123.74µm µm/ml for 48hr in KB cell line. cytotoxicity effects of gramine were confirmed by treatment of HEp-2 cell and KB cell with IC50 concentration of gramine resulted in sequences of events marked by the enhance the apoptosis accompanied by loss of cell viability, modulation of reactive oxygen species and cell cycle arrest through the induction of G0/G1 phase arrest on HEp-2 cells. Our study suggests that the nanosuspension of gramine possesses the more cytotoxic effect of cancer cells and a novel candidate for cancer chemoprevention.

Keywords: apoptosis, HEp-2 cell line, KB cell line mitochondria, gramine, nanosuspension

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Authors: Liza Meutia Sari, Gus Permana Subita, Elza Ibrahim Auerkari

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Background: Many herbs have been discovered to be potential sources of anticancer drugs. Biji Pinang or areca nut (Areca catechu Linn.) has a high content of phenolics and flavonoids, and which is related to antioxidant activity. However, data on its effects on oral squamous cell carcinoma is not available. Objectives: Identification of the cytotoxicity and apoptosis activity in HSC-2 and HSC-3. Methods: The areca nut was extracted by ethanol 96%, MTS assay and apoptosis activity with flow cytometry. Results: The extract of areca nut showed higher toxicity on HSC-3 cell compared to HSC-2. The IC₅₀ of HSC-3 was 164.06 μg/ml vs. 629.50 μg/ml in HSC-2. There was an increase in late apoptosis percentage after 24 and 48 hours in HSC-2. There was a significant increase in early apoptosis percentage after 24 hours and late in 48 hours in HSC-3. Conclusion: The antioxidant activity of the extract of areca nut might be associated with the selective cytotoxicity on HSC-2 and HSC-3. Apoptosis is the major cell death mechanism involved. The areca nut may play an important role in anticancer herb medicine.

Keywords: areca nut, cytotoxicity, apoptosis, oral carcinoma

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Authors: Salmin K. Alshalmani, Nada H. Zobi, Ismaeel H. Bozakouk

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Marine organisms are potentially prolific sources of highly bio active secondary metabolites that might represent useful leads in the development of new pharmaceutical agents. The Libyan marine biodiversity including macroalgae remains partially unexplored in term of their potential bio activities. The phytochemical analysis of the alcoholic extracts of some commonly occurring seaweed Cystoseira compressa, enteromorpha intestinals, corallina, and Ulva lactuca and their evaluated for antibacterial activity by well diffusion assay were studied. Four different solvents namely water, ethanol 99 %, methanol 99 %, and methylated spirit 95 % were used for extraction. The phytochemical analysis revealed the presence of carbohydrates, steroids, tannin & phenols, saponins, proteins, and glycosides. The extracts were subjected for study of antibacterial activity. The zone of inhibition ranged between 8 to 16 mm in aqueous extract and up to 16 mm in methanol extract. The maximum activity (16 mm) was recorded from methanol extract of Ulva lactuca against Staphylococcus aureus and, minimum activity (8mm) recorded by Cystoseira compressa against S. aureus.

Keywords: macroalgae, phytochemicals, antibacterial activity, methanolic extract

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Authors: Deokyeong Choe, Sung Hun Youn, Younggon Kim, Chul Soo Shin

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L-Cysteine, a sulfur-containing amino acid, has been often used in the pharmaceutical, cosmetic, food, and feed additive industries. This amino acid has been usually produced by acid-hydrolysis of human hair and poultry feathers. There are many problems, such as avoidance for use of animal hair, low yields, and formation of harmful waste material. As an alternative, the enzymatic conversion of D, L-2-amino-Δ2-thiazoline-4-carboxylic acid (ATC) to L-cysteine has been developed as an environmental-friendly method. However, the substrate solubility was too low to be used in industry. In this study, high concentrations of eutectic substrate solutions were prepared to solve the problem. Eutectic melting occurred at 39°C after mixing ATC and malonic acid at a molar ratio of 1:1. The characteristics of eutectic mixtures were analyzed by FE-SEM, EDS mapping, and XPS. However, since sorbitol, MnSO4, and NaOH should be added as supplements to the substrate mixture for the activation and stabilization of the enzyme, strategies for sequential addition of total five compounds, ATC, malonic acid, sorbitol, MnSO4, and NaOH were established. As a result, eutectic substrate mixtures of 670 mM ATC were successfully formulated. After 6 h of enzymatic reaction, 550 mM L-cysteine was made.

Keywords: D, L-2-amino-Δ2-thiazoline-4-carboxylicacid, enzymatic conversion, eutectic solution, l-cysteine

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Authors: Adrian Garrido Sanchis, Richard Pashley

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The MS2 (ATCC15597-B1) virus was used as a surrogate to estimate the inactivation rates for enteric viruses when using a hot air bubble column evaporator (HBCE) system in the treatment of household wastewater. In this study, we have combined MS2 virus surface charging properties with thermal inactivation rates, using an improved double layer plaque assay technique, in order to assess the efficiency of the HBCE process for virus removal in water. When bubbling a continuous flow of dry air, at 200°C, only heats the aqueous solution in the bubble column to about 50°C. Viruses are not inactivated by this solution temperature, as confirmed separately from water bath heating experiments. Hence, the efficiency of the HBCE process for virus removal in water appeared to be caused entirely by collisions between the hot air bubbles and the virus organisms. This new energy efficient treatment for water reuse applications can reduce the thermal energy required to only 25% (about 113.7 kJ/L) of that required for boiling (about 450 kJ/L).

Keywords: MS2 virus inactivation, water reuse, hot bubble column evaporator, water treatment

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Authors: Deepanwita Deka, Dhruva Kumar Jha

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Endophytes are the microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects. Endophytes are rich source of therapeutic substances like antimicrobial, anticancerous, herbicidal, insecticidal, immunomodulatory compounds. Brucea mollis, commonly known as Quinine in Assam, belonging to the family Simaroubaceae, is a shrub or small tree, recorded as endangered species in North East India by CAMP survey in 2003. It is traditionally being used as antimalarial and antimicrobial agent and has antiplasmodial, cytotoxic, anticancer, diuretic, cardiovascular effect etc. Being endangered and medicinal; this plant may host certain noble endophytes which need to be studied in depth. The aim of the present study was isolation and identification of potent endophytic fungi from Brucea mollis, an endangered medicinal plant, to protect it from extinction due to over use for medicinal purposes. Aseptically collected leaves, barks and roots samples of healthy plants were washed and cut into a total of 648 segments of about 2 cm long and 0.5 cm broad with sterile knife, comprising 216 segments each from leaves, barks and roots. These segments were surface sterilized using ethanol, mercuric chloride (HgCl2) and aqueous solution of sodium hypochlorite (NaClO). Different media viz., Czapeck-Dox-Agar (CDA, Himedia), Potato-Dextrose-Agar (PDA, Himedia), Malt Extract Agar (MEA, Himedia), Sabourad Dextrose Agar (SDA, Himedia), V8 juice agar, nutrient agar and water agar media and media amended with plant extracts were used separately for the isolation of the endophytic fungi. A total of 11 fungal species were recovered from leaf, bark and root tissues of B. mollis. The isolates were screened for antimicrobial, antioxidant and enzymatic activities using certain protocols. Cochliobolus geniculatus was identified as the most dominant species. The mycelia sterilia (creamy white) showing highest inhibitory activity against Candida albicans (MTCC 183) was induced to sporulate using modified PDA media. The isolate was identified as Geosmithia pallida. The internal transcribed spacer of rDNA was sequenced for confirmation of the taxonomic identity of the sterile mycelia (creamy white). The internal transcribed spacer r-DNA sequence was submitted to the NCBI (KU693285) for the first time from India. G. pallida and Penicillium showed highest antioxidant activity among all the isolates. The antioxidant activity of G. pallida and Penicillium didn’t show statistically significant difference (P˃0.05). G. pallida, Cochliobolus geniculatus and P. purpurogenum respectively showed highest cellulase, amylase and protease activities. Thus, endopytic fungal isolates may be used as potential natural resource of pharmaceutical importance. The endophytic fungi, Geosmithia pallida, may be used for synthesis of pharmaceutically important natural products and consequently can replace plants hitherto used for the same purpose. This study suggests that endophytes should be investigated more aggressively to better understand the endophyte biology of B. mollis.

Keywords: Antimicrobial activity, antioxidant activity, Brucea mollis, endophytic fungi, enzyme activity, Geosmithia pallida

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Authors: Forough Ataollahi, Sumit Pramanik, Belinda Pingguan-Murphy, Wan Abu Bakar Bin Wan Abas, Noor Azuan Bin Abu Osman

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Endothelium proliferation is an important process in cardiovascular homeostasis and can be regulated by extracellular environment, as cells can actively sense mechanical environment. In this study, we evaluated endothelial cell proliferation on PDMS/alumina (Al2O3) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 5% and 10% Al2O3 at curing temperature 50˚C for 4 h and then characterized by mechanical, structural and morphological analyses. Higher stiffness was found in the composites compared to the pure PDMS substrate. Cell proliferation of the cultured bovine aortic endothelial cells on substrate materials were evaluated via Resazurin assay and 1, 1’-Dioctadecyl-1, 3, 3, 3’, 3’-Tetramethylindocarbocyanine Perchlorate-Acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The results revealed that stiffer substrates promote more endothelial cells proliferation to the less stiff substrates. Therefore, this study firmly hypothesizes that the stiffness elevates endothelial cells proliferation.

Keywords: stiffness, proliferation, bovine aortic endothelial cells, extra cellular matrix, vascular

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Authors: D. Zujur, J. Moret, D. Rodriguez, L. Cruz, J. Lira, L. Gil, E. Dominguez, J. F. Alvarez-Barreto

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In this work, chitosan (CH) has been used to produce a novel coating for Ti6Al4V, the most widely used alloy in orthopedic implants, so as to improve the biological tissue response at the metallic surface. The Ti6Al4V surface was sandblasted with alumina particles and observed by SEM. Chitosan was chemically modified, via crodiimide chemistry, with lactobionic and 4-azidebenzoic acid to make it soluble at physiological pH and photo-crosslinkable, respectively. The reaction was verified by FTIR, NMR, and UV/vis spectroscopy. Ti6Al4V surfaces were coated with solutions of the modified CH and exposed to UV light, causing the polymer crosslinking, and formation of a hydrogel on the surface. The crosslinking reaction was monitored by FTIR at different exposure times. Coating morphology was observed by SEM. The coating´s cytocompatibility was determined in vitro through the culture of rat bone marrow´s mesenchymal stem cells, using an MTT assay. The results show that the developed coating is cytocompatible, easy to apply and could be used for further studies in the encapsulation of bioactive molecules to improve osteogenic potential at the tissue-implant interface.

Keywords: chitosan, photo-crosslinking, Ti6Al4V, bioactive coating, hydrogel

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Authors: S. Ghalem, M. Mesmoudi, I. Daoudand, H. Allali

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The nitrogen-containing bisphosphonates (N-BPs) are well established as the treatments of choice for disorders of excessive bone resorption, myeloma and bone metastases, and osteoporosis. They inhibit farnesyl pyrophosphate synthase (FFPS), a key enzyme in the mevalonate pathway, resulting in inhibition of the prenylation of small GTP-binding proteins in osteoclasts and disruption of their cytoskeleton, adhesion/spreading, and invasion of cancer cells. A very few examples for synthesis of α-amino bisphosphonates based on several amino acids are known from the literature. In the present work, esters of aminoacid react with ketophsophonate (or their analog acid or acyl) to afford the desired products, α-iminophosphonates. The reaction of imine with dimethyl phosphate in the presence of catalytic amount of I2 give ester of α-aminobisphosphonate as sole product in good yield. Finally, we used computational docking methods to predict how several α-aminobisphosphonates bind to FPPS and how R and X influence. Pamidronate, β-aminobisphosphonate already marketed, was used as reference. These results are of interest since they represent a new and simple way to sythesize α-aminobisphosphonates with a free COOH group increased by R2 functionalisable and opening up the possibility of using the molecular docking to facilitate the design of other, novel FFPS inhibitors.

Keywords: drug research, cancer, α-amino bisphosphonates, molecular docking

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Authors: Meiling Yu, Nadia Rouatbi, Khuloud T. Al-Jamal

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Background: Treatment of neurological disorders with modern medical and surgical approaches remains difficult. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. The treatment of brain diseases with gene therapy requires the gene-editing tool to be delivered efficiently to the central nervous system. In this study, we explored the efficiency of different delivery routes, namely intravenous (i.v.), intra-cranial (i.c.), and intra-nasal (i.n.), to deliver stable nucleic acid-lipid particles (SNALPs) containing gene-editing tools namely Cas9 mRNA and sgRNA encoding for GFP as a reporter protein. We hypothesise that SNALPs can reach the brain and perform gene-editing to different extents depending on the administration route. Intranasal administration (i.n.) offers an attractive and non-invasive way to access the brain circumventing the blood–brain barrier. Successful delivery of gene-editing tools to the brain offers a great opportunity for therapeutic target validation and nucleic acids therapeutics delivery to improve treatment options for a range of neurodegenerative diseases. In this study, we utilised Rosa26-Cas9 knock-in mice, expressing GFP, to study brain distribution and gene-editing efficiency of SNALPs after i.v.; i.c. and i.n. routes of administration. Methods: Single guide RNA (sgRNA) against GFP has been designed and validated by in vitro nuclease assay. SNALPs were formulated and characterised using dynamic light scattering. The encapsulation efficiency of nucleic acids (NA) was measured by RiboGreen™ assay. SNALPs were incubated in serum to assess their ability to protect NA from degradation. Rosa26-Cas9 knock-in mice were i.v., i.n., or i.c. administered with SNALPs to test in vivo gene-editing (GFP knockout) efficiency. SNALPs were given as three doses of 0.64 mg/kg sgGFP following i.v. and i.n. or a single dose of 0.25 mg/kg sgGFP following i.c.. knockout efficiency was assessed after seven days using Sanger Sequencing and Inference of CRISPR Edits (ICE) analysis. In vivo, the biodistribution of DiR labelled SNALPs (SNALPs-DiR) was assessed at 24h post-administration using IVIS Lumina Series III. Results: Serum-stable SNALPs produced were 130-140 nm in diameter with ~90% nucleic acid loading efficiency. SNALPs could reach and stay in the brain for up to 24h following i.v.; i.n. and i.c. administration. Decreasing GFP expression (around 50% after i.v. and i.c. and 20% following i.n.) was confirmed by optical imaging. Despite the small number of mice used, ICE analysis confirmed GFP knockout in mice brains. Additional studies are currently taking place to increase mice numbers. Conclusion: Results confirmed efficient gene knockout achieved by SNALPs in Rosa26-Cas9 knock-in mice expressing GFP following different routes of administrations in the following order i.v.= i.c.> i.n. Each of the administration routes has its pros and cons. The next stages of the project involve assessing gene-editing efficiency in wild-type mice and replacing GFP as a model target with therapeutic target genes implicated in Motor Neuron Disease pathology.

Keywords: CRISPR, nanoparticles, brain diseases, administration routes

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Authors: Piyaporn Buaklang, Narisa Kengtrong Bordeerat

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The use of nanoparticles is increasing worldwide and there are many nanotech-based daily products available in the market. The toxicity of nanoparticles results from their extremely small size which can be transported easily into the blood stream and other organs. We aimed to study the genotoxicity of two nanoparticles, Titanium dioxide (TiO2-NPs) and Zinc oxide (ZnO-NPs), in TK6 cells by micronucleus assay. The cells were tested at 8, 24, and 48 hours after exposed to 0.10, 0.25, 0.50 and 1.00 µg/mL of TiO2-NPs particles size < 25 nm and < 100 nm and to ZnO-NPs at 1, 10, 50, and 100 µg/mL, particles size < 50 nm and < 100 nm. At 24 hours of incubation transmission electron microscope (TEM) revealed that the nanoparticles TiO2-NPs at 1.00 µg/mL and ZnO-NPs at 10 µg/mL were able to be taken into the cells and induced the production of increasing amount of micronucleus in dose-dependent manner. The effect of the two nanoparticles on chromosome aberration indicated that TiO2-NPs and ZnO-NPs are genotoxic. In addition, the toxicity of TiO2-NPs was found to be 10 times more toxic than ZnO-NPs after 24 hours exposure. Analysis showed that the TiO2-NPs induced formation of micronucleus was both time and dose dependent, whereas the genotoxicity of ZnO-NPs was only dose dependent. In conclusion, TiO2-NPs and ZnO-NPs were able to transport through the cells membrane and directly genotoxic to TK6 cells in dose-dependent manner.

Keywords: nanoparticles, genotoxicity, human lymphoblast cells (TK6), micronucleus

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Authors: Rekia. Cherbi, Mokhtar. Saidi, Mohamed. Yousfi, Zhor. Rahmani

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Lawsonia alba (Henna) is widely used in folkloric medicinal for a treatment of various skin diseases such as Eczema (atopic dermatitis), boils and sores. The aim of the present study is to determine the antioxidant activity, total phenolics, flavonoids, and condensed tannins content of extracts from the seeds of Lawsonia. alba grown in Algeria and selected from three different regions (Adrar, Biskra, and Ouargla). Total phenolics content ranged from 68,42 ± 0,54 to 88,31 ± 0,78mg gallic acid equivalents (GAE)/g dry weight, the flavonoids content varied from 1,13 ± 0,0035 to 1,367 ± 0,002mg quercetin equivalents (Q)/ g dry weight and condensed tannins (14,47 ± 0,138 to 25,50 ± 0,076 mg catechin equivalents (CE)/g dry weight). The antioxidant activities of the extracts were evaluated by DPPH assay. The results showed that all extracts from the seeds of Lawsonia. alba seem to be good trappers of radicals, the IC50 values of the extracts ranged between 0,00826 and 0,01 g/l.

Keywords: antioxidant activity, Lawsonia. alba, phenolic compounds, seeds

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Authors: Zhanara B. Suleimenova, Zhazira K. Saduyeva

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Filamentous fungi are major producers of enzymes that have important applications in the food and beverage industries. The overall objective of this research is a strain improvement technology for efficient industrial enzymes production. The new way of filamentous fungi cultivation method has been developed. Such technology prolong producers’ cultivation period up to 60 days and create the opportunity to obtain enzymes repeatedly in every 2-3 days of fungal cultivation. This method is based on immobilizing enzymes producers with solid support in submerged conditions of growth. Immobilizing has a range of advantages: Decreasing the price of the final product, absence of foreign substances, controlled process of enzyme-genesis, ability of various enzymes simultaneous production, etc. Design of proposed technology gives the opportunity to increase the activity of immobilized cells culture filtrate comparing to free cells, growing in periodic culture conditions. Thus, proposed research focuses on new, more versatile, microorganisms capable of squeezing more end-products as well as proposed cultivation technology led to increased enzymatic productivity by several times.

Keywords: filamentous fungi, immobilization, industrial enzymes production, strain improvement

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Authors: Sweta Iyer, Nemeshwaree Behary, Jinping Guan, Guoqiang Chen, Vincent Nierstrasz

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Flavin mononucleotide widely known as 'FMN' is a biobased resource derived from riboflavin. The isoalloxazine ring present in the FMN molecule attributes the photoluminescence phenomenon, whereas FMN molecule in the presence of bacterial luciferase enzyme and co-factors such as NADH, long chain aldehyde leads to bioluminescence reaction. In this study, the FMN molecule was treated on cellulosic textile using chromojet technique and the photoluminescence property was characterized using spectroscopy technique. Further, the FMN was used as a substrate along with enzymes and co-factors to treat the non-woven textile, and the bioluminescence property was explored using luminometer equipment. The investigation revealed photoluminescence property on cellulosic textile, and the emission peak was observed at a wavelength around 530 nm with an average corrected spectral intensity of 10×106 CPS/Microamps. In addition, the measurement of nonwoven textile using bioluminescence reaction system exhibited light intensity measured in the form of relative light units (RLU). The study enabled to explore the use of FMN as both photoluminescent and bioluminescent textile. Further investigation would require for stability study of the same to provide an eco-efficient approach to obtain luminescent textile.

Keywords: flavin mononucleotide, photoluminescence, bioluminescence, luminescent textile

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Authors: Abubakar Umar Birnin-yauri

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Plastic wastes arising from Polyethylene (PE)-based materials are increasingly becoming environmental problem, this is owed to the fact that these PE waste materials will only decompose over hundreds, or even thousands of years, during which they cause serious environmental problems. In this research, Polymer blends prepared from PE, Cashewnut flour (CNF) and Gum Arabic (GA) were studied in order to assay their biodegradation potentials via composting method. Different sample formulations were made i.e., X1= (70% PE, 25% CNF and 5% GA, X2= (70% PE, 20% CNF and 10% GA), X3= (70% PE, 15% CNF and 15% GA), X4 = (70% PE, 10% CNF and 20% GA) and X5 = (70% PE, 5% CNF and 25% GA) respectively. The results obtained showed that X1 recorded weight loss of 9.89% of its original weight after the first 20 days and 37.45% after 100 day, and X2 lost 12.67 % after the first 20 days and 42.56% after 100day, sample X5 experienced the greatest weight lost in the two methods adopted which are 52.9% and 57.89%. Instrumental analysis such as Fourier Transform Infrared Spectroscopy, Thermogravimetric analysis and Scanning electron microscopy were performed on the polymer blends before and after biodegradation. The study revealed that the biodegradation of the polymer blends is influenced by the contents of both the CNF and GA added into the blends.

Keywords: polyethylene, cashewnut, gum Arabic, biodegradation, blend, environment

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Authors: Mansouri Ouarda, Adbdennour Cherif, Boukarma Ziad

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Environmental and biological monitoring are used for the evaluation of exposure to industrial chemicals, and provide a tool for assessing workers’ exposure to chemicals. The organs or tissues where the first biological effects can be observed with increasing amounts of lead toxicity. This study aims at evaluating the effect of the metal element-trace; lead, on the sex hormones in male workers, exposed to this metal on the level of the manufacturing plant of lead accumulators. The results indicate a significant reduction of the testosterone concentration in exposed workers compared to the control. However, the rate of LH was strongly increased at the individuals exposed to Pb. A significant difference concerning the rate of FSH, the hormone Prolactin and cortisol was recorded. The indicators of the lead poisoning indicate a very highly significant increase in the value of Pbs which vary between (142-796 µg/L) among which 50% of the workers present a high lead poisoning and the value of PPZ which vary between (43-554µg/L). The biochemical parameters show a significant increase in the rate of the créatinine, the urea and the acid urique. The hepatic results show no significant differentiation in the rate of TGO and TGP between both groups of study. However the rates of the enzyme phosphatase alkaline, triglyceride, and cholesterol a significant difference were registered.

Keywords: hormons, parameters, physilogical, Pbs, PPZ

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Authors: A. Jamsheera, F. Arjmand, D. K. Mohapatra

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Small molecules binding to specific sites along DNA molecule are considered as potential chemotherapeutic agents. Their role as mediators of key biological functions and their unique intrinsic properties make them particularly attractive therapeutic agents. Keeping in view, novel dipeptide complexes Cu(II)-Val-Pro (1), Zn(II)-Val-Pro (2), Cu(II)-Ala-Pro (3) and Zn(II)-Ala-Pro (4) were synthesized and thoroughly characterized using different spectroscopic techniques including elemental analyses, IR, NMR, ESI–MS and molar conductance measurements. The solution stability study carried out by UV–vis absorption titration over a broad range of pH proved the stability of the complexes in solution. In vitro DNA binding studies of complexes 1–4 carried out employing absorption, fluorescence, circular dichroism and viscometric studies revealed the binding of complexes to DNA via groove binding. UV–vis titrations of 1–4 with mononucleotides of interest viz., 5´-GMP and 5´-TMP were also carried out. The DNA cleavage activity of the complexes 1 and 2 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents and the cleavage mechanism involved a hydrolytic pathway. Furthermore, in vitro antitumor activity of complex 1 was screened against human cancer cell lines of different histological origin.

Keywords: dipeptide Cu(II) and Zn(II) complexes, DNA binding profile, pBR322 DNA cleavage, in vitro anticancer activity

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Authors: Silvia Di Giacomo, Marcello Locatelli, Simone Carradori, Francesco Cacciagrano, Chiara Toniolo, Gabriela Mazzanti, Luisa Mannina, Stefania Cesa, Antonella Di Sotto

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Hyperglycemia represents the main pathogenic factor in the development of diabetes complications and has been found associated with mitochondrial dysfunction and oxidative stress, which in turn increase cell dysfunction. Therefore, counteract oxidative species appears to be a suitable strategy for preventing the hyperglycemia-induce cell damage and support the pharmacotherapy of diabetes and metabolic diseases. Antidiabetic potential of many food sources has been linked to the presence of polyphenolic metabolites, particularly flavonoids such as quercetin and its glycosylated form rutin. In line with this evidence, in the present study, we assayed the potential anti-hyperglycemic activity of an ethyl acetate extract from the peel of Punica granatum L. var. Dente di Cavallo (PGE), a fruit well known to traditional medicine for the beneficial properties of its edible juice. The effect of the extract on the glucidic metabolism has been evaluated by assessing its ability to inhibit α-amylase and α-glucosidase, two digestive enzymes responsible for the hydrolysis of dietary carbohydrates: their inhibition can delay the carbohydrate digestion and reduce glucose absorption, thus representing an important strategy for the management of hyperglycemia. Also, the PGE ability to block the release of advanced glycated end-products (AGEs), whose accumulation is known to be responsible for diabetic vascular complications, was studied. The iron-reducing and chelating activities, which are the primary mechanisms by which AGE inhibitors stop their metal-catalyzed formation, were evaluated as possible antioxidant mechanisms. At last, the phenolic content of PGE was characterized by chromatographic and spectrophotometric methods. Our results displayed the ability of PGE to inhibit α-amylase enzyme with a similar potency to the positive control: the IC₅₀ values were 52.2 (CL 27.7 - 101.2) µg/ml and 35.6 (CL 22.8 - 55.5) µg/ml for acarbose and PGE, respectively. PGE also inhibited the α-glucosidase enzyme with about a 25 higher potency than the positive controls of acarbose and quercetin. Furthermore, the extract exhibited ferrous and ferric ion chelating ability, with a maximum effect of 82.1% and 80.6% at a concentration of 250 µg/ml respectively, and reducing properties, reaching the maximum effect of 80.5% at a concentration of 10 µg/ml. At last, PGE was found able to inhibit the AGE production (maximum inhibition of 82.2% at the concentration of 1000 µg/ml), although with lower potency with respect to the positive control rutin. The phytochemical analysis of PGE displayed the presence of high levels of total polyphenols, tannins, and flavonoids, among which ellagic acid, gallic acid and catechin were identified. Altogether these data highlight the ability of PGE to control the carbohydrate metabolism at different levels, both by inhibiting the metabolic enzymes and by affecting the AGE formation likely by chelating mechanisms. It is also noteworthy that peel from pomegranate, although being a waste of juice production, can be reviewed as a nutraceutical source. In conclusion, present results suggest the possible role of PGE as a remedy for preventing hyperglycemia complications and encourage further in vivo studies.

Keywords: anti-hyperglycemic activity, antioxidant properties, nutraceuticals, polyphenols, pomegranate

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Authors: E. Carraro, Sa. Bonetta, Si. Bonetta, E. Ceretti, G. C. V. Viola, C. Pignata, S. Levorato, T. Salvatori, S. Vannini, V. Romanazzi, A. Carducci, G. Donzelli, T. Schilirò, A. De Donno, T. Grassi, S. Bonizzoni, A. Bonetti, G. Gilli, U. Gelatti

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In 2013, air pollution and particulate matter were classified as carcinogenic to human by the IARC. At present, PM is Europe's most problematic pollutant in terms of harm to health, as reported by European Environmental Agency (EEA) in the EEA Technical Report on Air quality in Europe, 2015. A percentage between 17-30 of the EU urban population lives in areas where the EU air quality 24-hour limit value for PM10 is exceeded. Many studies have found a consistent association between exposure to PM and the incidence and mortality for some chronic diseases (i.e. lung cancer, cardiovascular diseases). Among the mechanisms responsible for these adverse effects, genotoxic damage is of particular concern. Children are a high-risk group in terms of the health effects of air pollution and early exposure during childhood can increase the risk of developing chronic diseases in adulthood. The MAPEC_LIFE (Monitoring Air Pollution Effects on Children for supporting public health policy) is a project founded by EU Life+ Programme (LIFE12 ENV/IT/000614) which intends to evaluate the associations between air pollution and early biological effects in children and to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in children. This work is focused on the micronuclei frequency in child buccal cells in association with airborne PM levels taking into account the influence of other factors associated with the lifestyle of children. The micronucleus test was performed in exfoliated buccal cells of 6–8 years old children from 5 Italian towns with different air pollution levels. Data on air quality during the study period were obtained from the Regional Agency for Environmental Protection. A questionnaire administered to children’s parents was used to obtain details on family socio-economic status, children health condition, exposures to other indoor and outdoor pollutants (i.e. passive smoke) and life-style, with particular reference to eating habits. During the first sampling campaign (winter 2014-15) 1315 children were recruited and sampled for Micronuclei test in buccal cells. In the sampling period the levels of the main pollutants and PM10 were, as expected, higher in the North of Italy (PM10 mean values 62 μg/m3 in Torino and 40 μg/m3 in Brescia) than in the other towns (Pisa, Perugia, Lecce). A higher Micronucleus frequency in buccal cells of children was found in Brescia (0.6/1000 cells) than in the other towns (range 0.3-0.5/1000 cells). The statistical analysis underlines a relation of the micronuclei frequency with PM concentrations, traffic level near child residence, and level of education of parents. The results suggest that, in addition to air pollution exposure, some other factors, related to lifestyle or further exposures, may influence micronucleus frequency and cellular response to air pollutants.

Keywords: air pollution, buccal cells, children, micronucleus cytome assay

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Authors: Maomao Cao

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Objective: To test the performance of ctDNA methylation for the detection of liver cancer. Methods: A total of 1233 individuals have been recruited in 2017. 15 male and 15 female samples (including 10 cases of liver cancer) were randomly selected in the present study. CfDNA was extracted by MagPure Circulating DNA Maxi Kit. The concentration of cfDNA was obtained by Qubit™ dsDNA HS Assay Kit. A pre-constructed predictive model was used to analyze methylation data and to give a predictive score for each cfDNA sample. Individuals with a predictive score greater than or equal to 80 were classified as having liver cancer. CT tests were considered the gold standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the diagnosis of liver cancer were calculated. Results: 9 patients were diagnosed with liver cancer according to the prediction model (with high sensitivity and threshold of 80 points), with scores of 99.2, 91.9, 96.6, 92.4, 91.3, 92.5, 96.8, 91.1, and 92.2, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of ctDNA methylation for the diagnosis of liver cancer were 0.70, 0.90, 0.78, and 0.86, respectively. Conclusions: ctDNA methylation could be an acceptable diagnostic modality for the detection of liver cancer.

Keywords: liver cancer, ctDNA methylation, detection, diagnostic performance

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Authors: Manal E. A Elhalwagy, Nadia Amin Abdulmajeed, Hanan S. Alnahdi, Enas N. Danial

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Baron is herbicide includes (48% glyphosate) widely used in Egypt. The present study assesses the cytotoxic and genotoxic effect of baron on rats liver. Two groups of rats were treated orally with 1/10 LD 50, (275.49 mg kg -1) and 1/40 LD 50, (68.86 mg kg-1) glyphosate for 28 days compared with control group. Serum and liver tissues were taken at 14 and 28 days of treatment. An inhibition in Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were recorded at both treatment periods and reduction in total serum protein (TP) and albumin (ALB). However, non-significant changes in serum acetylcholinesterase (AChE). Elevation in oxidative stress biomarker malondyaldehyde (MDA) and the decline in detoxification biomarker total reduced glutathione (GSH), Glutathione S-transferase (GST) and superoxide dismutase (SOD) in liver tissues led to increase in percentage of DNA damage. Destruction in liver tissue architecture was observed . Although, Baron was classified in the safe category pesticides repeated exposure to small doses has great danger effect.

Keywords: glyphosate, liver toxicity, oxidative stress, DNA damage, commet assay

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Authors: Abdolreza Esmaeilzadeh, Mehri Mirzaei

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Objective: Rheumatic disease is a chronic disease that causes pain, stiffness, swelling and limited motion and function of many joints. RA is the most common form of autoimmune arthritis, affecting more than 1.3 million Americans. Of these, about 75% are women. Materials and Methods: This study was formed due to the misconception about ANA test, which is frequently performed with methods based upon solid phase as ELISA. This experiment was conducted on 430 patients, with clinical symptoms that are likely affected with rheumatic diseases, simultaneously by means of ANA and FANA. Results: 36 cases (8.37%) of patients, despite positive ANA, have demonstrated negative results via Indirect Immunofluorescence Assay (IIFA), (false positive). 116 cases (27%) have demonstrated negative ANA results, by means of the ELISA technique, although they had positive IIFA results. Conclusion: Other advantages of IIFA are antibody titration and specific pattern detection that have the capability of distinguishing positive dsDNA results. According to the restrictions and false negative cases, in patients, IIFA test is highly recommended for these disease's diagnosis.

Keywords: autoimmune disease, IIFA, EIA, rheumatic disease

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Authors: Prajna Paramita Naik, Sujit Kumar Bhutia

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Background: Regardless of the development multimodal treatment strategies, oral squamous cell carcinoma (OSCC) is often associated with a high rate of recurrence, metastasis and chemo- and radio- resistance. The present study inspected the relevance of CD44, ABCB1 and ADAM17 expression as a putative stem cell compartment in oral squamous cell carcinoma (OSCC) and deciphered the role of autophagy in regulating the expression of aforementioned proteins, stemness and chemoresistance. Methods: A retrospective analysis of CD44, ABCB1 and ADAM17 expression with respect to the various clinicopathological factors of sixty OSCC patients were determined via immunohistochemistry. The correlation among CD44, ABCB1 and ADAM17 expression was established. Sphere formation assay, flow cytometry and fluorescence microscopy were conducted to elucidate the stemness and chemoresistance nature of established cisplatin-resistant oral cancer cells (FaDu). The pattern of expression of CD44, ABCB1 and ADAM17 in parental (FaDu-P) and resistant FaDu cells (FaDu-CDDP-R) were investigated through fluorescence microscopy. Western blot analysis of autophagy marker proteins was performed to compare the status of autophagy in parental and resistant FaDu cell. To investigate the role of autophagy in chemoresistance and stemness, sphere formation assay, immunofluorescence and Western blot analysis was performed post transfection with siATG14 and the level of expression of autophagic proteins, mitochondrial protein and stemness-associated proteins were analyzed. The statistical analysis was performed by GraphPad Prism 4.0 software. p-value was defined as follows: not significant (n.s.): p > 0.05;*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001 were considered statistically significant. Results: In OSCC, high CD44, ABCB1 and ADAM17 expression were significantly correlated with higher tumor grades and poor differentiation. However, the expression of these proteins was not related to the age and sex of OSCC patients. Moreover, the expression of CD44, ABCB1 and ADAM17 were positively correlated with each other. In vitro and OSCC tissue double labeling experiment data showed that CD44+ cells were highly associated with ABCB1 and ADAM17 expression. Further, FaDu-CDDP-R cells showed higher sphere forming capacity along with increased fraction of the CD44+ population and β-catenin expression FaDu-CDDP-R cells also showed accelerated expression of CD44, ABCB1 and ADAM17. A comparatively higher autophagic flux was observed in FaDu-CDDP-R against FaDu-P cells. The expression of mitochondrial proteins was noticeably reduced in resistant cells as compared to parental cells indicating the occurrence of autophagy-mediated mitochondrial degradation in oral cancer. Moreover, inhibition of autophagy was coupled with the decreased formation of orospheres suggesting autophagy-mediated stemness in oral cancer. Blockade of autophagy was also found to induce the restoration of mitochondrial proteins in FaDu-CDDP-R cells indicating the involvement of mitophagy in chemoresistance. Furthermore, a reduced expression of CD44, ABCB1 and ADAM17 was also observed in ATG14 deficient cells FaDu-P and FaDu-CDDP-R cells. Conclusion: The CD44+ ⁄ABCB1+ ⁄ADAM17+ expression in OSCC might be associated with chemoresistance and a putative CSC compartment. Further, the present study highlights the contribution of mitophagy in chemoresistance and confirms the potential involvement of autophagic regulation in acquisition of stem-like characteristics in OSCC.

Keywords: ABCB1, ADAM17, autophagy, CD44, chemoresistance, mitophagy, OSCC, stemness

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Authors: Josselyn Mata Calidonio, Arianna I. Maddox, Kimberly Hamad-Schifferli

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Rapid diagnostics are critical infectious disease tools that are designed to detect a known biomarker using antibodies specific to that biomarker. However, a way to detect unknown viruses has not yet been achieved in a paper test format. We describe here a route to make an adaptable paper immunoassay that can detect an unknown biomarker, demonstrating it on SARS-CoV-2 variants. The immunoassay repurposes cross-reactive antibodies raised against the alpha variant. Gold nanoparticles of two different colors conjugated to two different antibodies create a colorimetric signal, and machine learning of the resulting colorimetric pattern is used to train the assay to discriminate between variants of alpha and Omicron BA.5. By using principal component analysis, the colorimetric test patterns can pick up and discriminate an unknown that it has not encountered before, Omicron BA.1. The test has an accuracy of 100% and a potential calculated discriminatory power of 900. We show that it can be used adaptively and that it can be used to pick up emerging variants without the need to raise new antibodies.

Keywords: adaptive immunoassay, detecting unknown viruses, gold nanoparticles, paper immunoassay, repurposing antibodies

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Authors: Sweta Iyer, Nemeshwaree Behary, Vincent Nierstrasz

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Various organisms involve bioluminescence for their particular biological function. The bio-based molecules responsible for bioluminescence vary from one species to another, research has been done to identify the chemistry and different mechanisms involved in light production in living organisms. The light emitting chemical systems such as firefly and bacterial luminous mostly involves enzyme-catalyzed reactions and is widely used for ATP measurement, bioluminescence imaging, environmental biosensors etc. Our strategy is to design bioluminescent textiles using such bioluminescent systems. Hence, a detailed literature work was carried out to study on how to mimic bioluminescence effect seen in nature. Reaction mechanisms in various bioluminescent living organisms were studied and the components or molecules responsible for luminescence were identified. However, the challenge is to obtain the same effect on textiles by immobilizing enzymes responsible for light creation. Another challenge is also to regenerate substrates involved in the reaction system to create a longer lasting illumination in bioluminescent textiles. Natural film-forming polymers were used to immobilize the reactive components including enzymes on textile materials to design a biomimetic luminescent textile.

Keywords: bioluminescence, biomimetic, immobilize, luminescent textile

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Authors: Chijindu Okpalaoka, Charles Chinedu Onuselogu

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COVID-19, humanity's coronavirus disease caused by SARS-CoV-2, was first detected in late 2019 in Wuhan, China. COVID-19 lacked an established conventional pharmaceutical therapy, and as a result, the outbreak quickly became an epidemic affecting the entire World. Only a qPCR assay is reliable for diagnosing COVID-19. Clustered, regularly interspaced short palindromic repeats (CRISPR) technology is being researched for speedy and specific identification of COVID-19, among other therapeutic techniques. Apart from its therapeutic capabilities, the CRISPR technique is being evaluated to develop antiviral therapies; nevertheless, no CRISPR-based medication has been approved for human use to date. Prophylactic antiviral CRISPR in living being cells, a Cas 13-based approach against coronavirus, has been developed. While this method can be evolved into a treatment approach, it may face substantial obstacles in human clinical trials for licensure. This study discussed the potential applications of CRISPR-based techniques for developing a speedy and accurate feasible treatment alternative for the COVID-19 virus.

Keywords: COVID-19, CRISPR technique, Cas13, SARS-CoV-2, prophylactic antiviral

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Authors: Meryem Abi-Ayad, Biagio Arcidiacono, Eusebio Chiefari, Daniela Foti, Mohamed Benyoucef, Antonio Brunetti

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Lipoprotein Lipase (LPL) is a key enzyme for lipid metabolism; its genetic polymorphism can be a candidate for modulating lipids parameters in metabolic syndrome. The objective of the present study was to determine whether lipoproteins lipase polymorphisMetS (LPL-HindIII) could be associated with moderate hypertriglyceridemia (secondary to metabolism syndrome). The polymorphism Hind III (rs320) was assessed by PCR-RFLP in 51 MetS patients and 17 healthy controls from the hospital in Tlemcen. The logistic regression analyses showed no significant association with Hind III genotype and hypertriglyceridemia (TG ≥ 1,5g/l or TG lower treatment) (P=0,455), metabolic syndrome (P=0,455), hypertension (P=0,802) and type 2 diabetes (P=0,144). In terms of plasma biomarkers, although not statistically significant, there was a difference in TG levels (P > 0,05), which was lowest among carriers of the homogenous mutant allele (H-). In this study, there was no association between the rare allele (H-) and disease protection, and between the frequent allele (H+) and disease prevalence (hypertriglyceridemia, metabolic syndrome, hypertension, type 2 diabetes).

Keywords: moderate secondary hypertriglyceridemia, metabolic syndrome, lipids, polymorphism lipoprotein lipase, HindIII(rs320)

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Authors: Reza Sadeghi

Abstract:

In this study, microwave heating was employed for controlling Oryzaephilus mercator. adults infesting stored Iranian dried fig. For this purpose, the dried fig samples were artificially infested with O. mercator and then heated in a microwave oven (2450 MHz) at the power outputs of 450, 720, and 900 W for 10, 20, 30, and 40 s, respectively. Subsequently, changes in the colors of the product samples under the effects of the varied microwave applications were investigated in terms of lightness (ΔL*), redness (Δa*), and yellowness (Δb*) using an image processing technique. The results revealed that both parameters of microwave power and exposure time had significant impacts on the pest mortality rates (p<0.01). In fact, a direct positive relationship was obtained between the mortality rate and microwave irradiation power. Complete mortality was achieved for the pest at the power of 900 W and exposure time of 40 s. The dried fig samples experienced fewer changes in their color parameters. Considering the successful pest control and acceptable changes in the product quality, microwave irradiation can be introduced as an appropriate alternative to chemical fumigants.

Keywords: colorimetric assay, microwave heating, Oryzaephilus mercator, mortality

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Authors: O. J. Jesumoroti, Faridoon, R. Klein, K. A. Iobb, D. Mnkadhla, H. C. Hoppe, P. T. Kaye

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The 1, 3-aryl diketo acids (DKA) based agents represent an important class of HIV integrase (IN) strand transfer inhibitors. In other to study the chelating role of the divalent metal ion in the inhibition of IN strand transfer, we designed and synthesized a series of 2-hydroxy-3-oxo-3-phenyl propionate derivatives with the notion that such compounds could interact with the divalent ion in the active site of IN. The synthetic sequence to the desired compounds involves the concept of Doebner knoevenagel condensation, Fischer esterification and ketohydroxylation using neuclophilic re-oxidant; compounds were characterized by their IR, IHNMR, 13CNMR, HRMS spectroscopic data and melting point determination. Also, molecular docking was employed in this study and it was revealed that there is interaction with the active site of the enzyme. However, there is disparity in the corresponding anti-HIV activity determined by the experimental bioassay. These compounds lack potency at low micromolar concentration when compared to the results of the docking studies. Nevertheless, the results of the study suggest modification of the aryl ring with one or two hydroxyl groups to improve the inhibitory activity.

Keywords: anti-HIV-1 integrase, ketohydroxylation, molecular docking, propionate derivatives

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Authors: Mahdi Rahaie

Abstract:

MicroRNAs are a novel class of small RNAs which regulate gene expression by translational repression or degradation of messenger RNAs. To produce sensitive, simple and cost-effective assays for microRNAs, detection is in urgent demand due to important role of these biomolecules in progression of human disease such as Alzheimer’s, Multiple sclerosis, and some other neurodegenerative diseases. Herein, we report several novel, sensitive and specific microRNA nanobiosensors which were designed based on colorimetric and fluorescence detection of nanoparticles and hybridization chain reaction amplification as an enzyme-free amplification. These new strategies eliminate the need for enzymatic reactions, chemical changes, separation processes and sophisticated equipment whereas less limit of detection with most specify are acceptable. The important features of these methods are high sensitivity and specificity to differentiate between perfectly matched, mismatched and non-complementary target microRNAs and also decent response in the real sample analysis with blood plasma. These nanobiosensors can clinically be used not only for the early detection of neuro diseases but also for every sickness related to miRNAs by direct detection of the plasma microRNAs in real clinical samples, without a need for sample preparation, RNA extraction and/or amplification.

Keywords: hybridization chain reaction, microRNA, nanobiosensor, neurodegenerative diseases

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