Search results for: comet assay

Authors: Fan Gao, Lior Pachter

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The primary tool currently used to pre-process 10X Chromium single-cell ATAC-seq data is Cell Ranger, which can take very long to run on standard datasets. To facilitate rapid pre-processing that enables reproducible workflows, we present a suite of tools called scATAK for pre-processing single-cell ATAC-seq data that is 15 to 18 times faster than Cell Ranger on mouse and human samples. Our tool can also calculate chromatin interaction potential matrices, and generate open chromatin signal and interaction traces for cell groups. We use scATAK tool to explore the chromatin regulatory landscape of a healthy adult human brain and unveil cell-type specific features, and show that it provides a convenient and computational efficient approach for pre-processing single-cell ATAC-seq data.

Keywords: single-cell, ATAC-seq, bioinformatics, open chromatin landscape, chromatin interactome

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Authors: Eun Jung Sohn, Hwan Tae Park

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Autophagy is a cellular response to stress or environment on cell survival. Here, we investigated the role of ectopic expression of miR 200c-3p in autophagy. Ectopic expression of miR 200c-3p increased the expression of IRE1alpha, ATF6 and CHOP by western blot and RT-qPCR. Furthermore, the level of microRNA 200c-3p was enhanced by treatment of TG or overexpression of GRP 78. Also, ectopic expression of miR200c-3p increased the LC3 II expression by western blot and RT-qPCR. Also, we found that western blot assay showed that miR200c-3p inhibitor was blocked the starvation–induced LC3II levels. Furthermore, starvation stress increased the level of miR200c-3p in different kinetics. Ectopic expression of miR200c-3p attenuated LC3II expression in IRE1 siRNA transfected PC3 cells. Here, we first demonstrate that miR200c-3p regulates autophagy via ER stress pathway.

Keywords: Autophagy, ER stress, LC3II, miR200c-3p

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Authors: Hiroshi Nakayama, Yuji Ito

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Synthetic cannabinoids have attracted much public attention recently in Japan. 1-pentyl-3-(1-naphthoyl)-indole (JWH-018), 1-pentyl-2-methyl-3-(1-naphthoyl) indole (JWH-015), 1-(5-fluoropentyl)-3- (1-(2,2,3,3- tetramethylcyclopropyl)) indole (XLR-11) and 1-methyl-3- (1-admantyl) indole (JWH-018 adamantyl analog) are known as synthetic cannabinoids and are also considered dangerous illegal drugs in Japan. It has become necessary to develop sensitive and useful methods for detection of synthetic cannabinoids. We produced two monoclonal antibodies (MAb) against synthetic cannabinoids, named NT1 (IgG1) and NT2 (IgG1), using Hybridoma technology. The cross-reactivity of these produced MAbs was evaluated using a competitive enzyme-linked immunosorbent assay (ELISA). In the results, we found both of these antibodies recognize many kinds of synthetic cannabinoids analog. However, neither of these antibodies recognizes naphtoic acid, 1-methyl-indole and indole known as a raw material of synthetic cannabinoid. Thus, the MAbs produced in this study could be a useful tool for the detection of synthetic cannabinoids.

Keywords: ELISA, monoclonal antibody, sensor, synthetic cannabinoid

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Authors: Hassan Waseem

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Cytomegalovirus (CMV) is ubiquitously distributed viral agent responsible for different clinical manifestations that may vary according to the immunologic status of the patient. CMV can cause morbidity and mortality among fetuses and patients with compromised immune system. A cross-sectional study was carried out in Islamabad to investigate the prevalence and risk factors associated with CMV infection among pregnant women. Blood samples of 172 pregnant women visiting Mother and Child Healthcare, Pakistan Institute of Medical Sciences (PIMS) Islamabad were taken. In present study, serum samples of the women were checked for CMV-specific IgG and IgM antibodies by enzyme linked immunosorbent assay (ELISA). Clinical, obstetrical and socio-demographical characteristics of the women were collected by using structured questionnaires. Out of 172 pregnant women included in the study, 171 (99.4%) were CMV specific IgG positive and 30 (17.4%) were found positive for CMV-IgM antibodies. The CMV has taken an endemic form in Pakistan so, routine screening of CMV among pregnant women is recommended.

Keywords: Cytomegalovirus, blood transfusion, ELISA, seroprevalence

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Authors: Saeeda Nadir Ali, Najma Sultana, Muhammad Saeed Arayne, Amtul Qayoom

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Present study describes a simple and a fast liquid chromatographic method using ultraviolet detector for simultaneous determination of anxiety relief medicine alprazolam with ACE inhibitors i.e; lisinopril, captopril and enalapril employing purospher star C18 (25 cm, 0.46 cm, 5 µm). Separation was achieved within 5 min at ambient temperature via methanol: water (8:2 v/v) with pH adjusted to 2.9, monitoring the detector response at 220 nm. Optimum parameters were set up as per ICH (2006) guidelines. Calibration range was found out to be 0.312-10 µg mL-1 for alprazolam and 0.625-20 µg mL-1 for all the ACE inhibitors with correlation coefficients > 0.998 and detection limits 85, 37, 68 and 32 ng mL-1 for lisinopril, captopril, enalapril and alprazolam respectively. Intra-day, inter-day precision and accuracy of the assay were in acceptable range of 0.05-1.62% RSD and 98.85-100.76% recovery. Method was determined to be robust and effectively useful for the estimation of studied drugs in dosage formulations and human serum without obstruction of excipients or serum components.

Keywords: alprazolam, ACE inhibitors, RP HPLC, serum

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Authors: Ibrahim Alshunaibir

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Infection with Helicobacter pylori is common worldwide but few studies focus on the prevalence and spread of the infection in Saudi Arabia. This study was undertaken to observe the epidemiology of Helicobacter pylori infection in patients suffering from gastrointestinal sign and symptoms in one of the largest hospitals in the capital of Saudi Arabia, Riyadh. Methods: Enzyme-linked immunosorbent assay (ELISA) was undertaken for this study with nearly 6000 samples collected and examined for patients suffering from (dyspeptic) symptoms ranging in their age from 5 to 75 years. Results: The prevalence of helicobacter infection was 67% increasing with age. Female shows higher percentage of H. pylori infection than male. Conclusions: The percentage rate was higher in female than male. This study shows a high percentage of helicobacter infection in Saudi Arabia.

Keywords: Helicobacter pylori, percentage, dyspeptic, Saudi Arabia

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Authors: M. Yahia, N. Chaoui, A. Chaouch, Massinissa Yahia

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Our study was designed to determine the metabolic changes of some biochemical parameters (cholesterol, triglyceride, Iron, uric acid, Urea and folic acid) and highlight their changes in 57 women of the region Batna, during the first trimester of pregnancy. This practical work was done with 27 women with missed miscarriage, compared with 30 control subjects of normal pregnant women. The assay results revealed a highly significant difference (P = 0.0006) between the two groups in serum iron (64.00 vs 93.54) and in the rate of folate (6.70 vs 9.22) (P <0.001) but no difference was found regarding the rate of Ca (9.69 vs 10.20), urea (0.19 vs 0.17), UA (33.96 vs 32.76), CH (1.283 vs 1.431), and TG (0.8852 vs 0.8290). The present study indicates that iron deficiency and folate are associated with missed miscarriage, but no direct pathophysiological link has been determined. Further in-depth studies are needed to determine the exact mechanism by which these deficits lead to a missed miscarriage.

Keywords: biochemical parameters, pregnant women, missed miscarriage, Algeria

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Authors: Atefeh Esmailnejad, Gholamreza Nikbakht Brujeni

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Major histocompatibility complex (MHC) is one of the best characterized genetic regions associated with immune responses and controlling disease resistance in chicken. Association of the MHC with a wide range of immune responses makes it a valuable predictive factor for the disease pathogenesis and outcome. In this study, the association of MHC with cell-mediated immune responses was analyzed in commercial broiler chicken. The tandem repeat LEI0258 was applied to investigate the MHC polymorphism. Cell-mediated immune response was evaluated by peripheral blood lymphocyte proliferation assay using MTT method. Association study revealed a significant influence of MHC alleles on cellular immune responses in this population. Alleles 385 and 448 bp were associated with elevated cell-mediated immunity. Haplotypes associated with improved immune responses could be considered as candidate markers for disease resistance and applied to breeding strategies.

Keywords: MHC, cell-mediated immunity, broiler, chicken

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Authors: Ahmed M. Haddad, Hadeel S. El-Shaal, Gadallah M. Abu-Elreesh

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Some of filamentous fungi can be used for biodiesel production as they are able to accumulate high amounts of intracellular lipids when grown at stress conditions. Aspergillus fumigatus sp. was isolated from Nile delta soil in Egypt. The fungus was primarily screened for its capacity to accumulate lipids using Nile red staining assay. The fungus could accumulate more than 20% of its biomass as lipids when grown at optimized minimal medium. After lipid extraction, we could use fungal cell debris to remove some heavy metals from contaminated waste water. The fungal cell debris could remove Cd, Cr, and Zn with absorption efficiency of 73%, 83.43%, and 69.39% respectively. In conclusion, the Aspergillus fumigatus isolate may be considered as a promising biodiesel producer, and its biomass waste can be further used for bioremediation of wastewater contaminated with heavy metals.

Keywords: biodiesel, bioremediation, fungi, heavy metals, lipids, oleaginous

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Authors: Gulten Yilmaz, A. Beril Tugrul, Mustafa Demir, Dogan Yasar, Bayram Demir, Bulent Buyuk

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A Positron Emission Tomography (PET) is a radioisotope imaging technique that illustrates the organs and the metabolisms of the human body. This technique is based on the simultaneous detection of 511 keV annihilation photons, annihilated as a result of electrons annihilating positrons that radiate from positron-emitting radioisotopes that enter biological active molecules in the body. This study was conducted on ten patients in an effort to conduct patient-related experimental studies. Dosage monitoring for the bladder, which was the organ that received the highest dose during PET applications, was conducted for 24 hours. Assessment based on measuring urination activities after injecting patients was also a part of this study. The MIRD method was used to conduct dosage calculations for results obtained from experimental studies. Results obtained experimentally and theoretically were assessed comparatively.

Keywords: PET/CT, TLD, MIRD, dose measurement, patient doses

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Authors: Sundru Manjulata Devi, Prakash M. Halami

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The immemorial use of fermented foods from vegetables, dairy and other biological sources are of great demand in India because of their health benefits. However, the diversity of Lactobacillus plantarum group (LPG) of vegetable origin has not been revealed yet, particularly with reference to their probiotic functionalities. In the present study, the different species of probiotic Lactobacillus plantarum group (LPG) i.e., L. plantarum subsp. plantarum MTCC 5422 (from fermented cereals), L. plantarum subsp. argentoratensis FG16 (from fermented bamboo shoot) and L. paraplantarum MTCC 9483 (from fermented gundruk) (as characterized by multiplex recA PCR assay) were considered to investigate their relative expression of MUB domains of mub gene (mucin binding protein) by Real time PCR. Initially, the allelic variation in the mub gene was assessed and found to encode three different variants (Type I, II and III). All the three types had 8, 9 and 10 MUB domains respectively (as analysed by Pfam database) and were found to be responsible for adhesion of bacteria to the host intestinal epithelial cells. These domains either get inserted or deleted during speciation or evolutionary events and lead to divergence. The reverse transcriptase qPCR analysis with mubLPF1+R1 primer pair supported variation in amplicon sizes with 300, 500 and 700 bp among different LPG strains. The relative expression of these MUB domains significantly unregulated in the presence of 1% mucin in overnight grown cultures. Simultaneously, the mub gene expressed efficiently by 7 fold in the culture L. paraplantarum MTCC 9483 with 10 MUB domains. An increase in the expression levels for L. plantarum subsp. plantarum MTCC 5422 and L. plantarum subsp. argentoratensis FG16 (MCC 2974) with 9 and 8 repetitive domains was around 4 and 2 fold, respectively. The detection and expression of an integrase (int) gene in the upstream region of mub gene reveals the excision and integration of these repetitive domains. Concurrently, an in vitro adhesion assay to mucin and exclusion of pathogens (such as Listeria monocytogenes and Micrococcus leuteus) was investigated and observed that the L. paraplantarum MTCC 9483 with more adhesion domains has more ability to adhere to mucin and inhibited the growth of pathogens. The production and expression of plantaricin-like bacteriocin (plnNC8 type) in MTCC 9483 suggests the pathogen inhibition. Hence, the expression of MUB domains can act as potential biomarkers in the screening of a novel probiotic LPG strain with adherence property. The present study provides a platform for an easy, rapid, less time consuming, low-cost methodology for the detection of potential probiotic bacteria. It was known that the traditional practices followed in the preparation of fermented bamboo shoots/gundruk/cereals of Indian foods contain different kinds of neutraceuticals for functional food and novel compounds with health promoting factors. In future, a detailed study of these food products can add more nutritive value, consumption and suitable for commercialization.

Keywords: adhesion gene, fermented foods, MUB domains, probiotics

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Authors: Marisa Rio, Sharanya Bola, Richard H. W. Funk, Gerald Gerlach

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Cell migration, wound healing and regeneration are some of the physiological phenomena in which electric fields (EFs) have proven to have an important function. Physiologically, cells experience electrical signals in the form of transmembrane potentials, ion fluxes through protein channels as well as electric fields at their surface. As soon as a wound is created, the disruption of the epithelial layers generates an electric field of ca. 40-200 mV/mm, directing cell migration towards the wound site, starting the healing process. In vitro electrotaxis, experiments have shown cells respond to DC EFs polarizing and migrating towards one of the poles (cathode or anode). A standard electrotaxis experiment consists of an electrotaxis chamber where cells are cultured, a DC power source and agar salt bridges that help delaying toxic products from the electrodes to attain the cell surface. The electric field strengths used in such an experiment are uniform and homogeneous. In contrast, the endogenous electric field strength around a wound tend to be multi-field and non-homogeneous. In this study, we present a custom device that enables electrotaxis experiments in non-homogeneous DC electric fields. Its main feature involves the replacement of conventional metallic electrodes, separated from the electrotaxis channel by agarose gel bridges, through electrolyte-filled microchannels. The connection to the DC source is made by Ag/AgCl electrodes, incased in agarose gel and placed at the end of each microfluidic channel. An SU-8 membrane closes the fluidic channels and simultaneously serves as the single connection from each of them to the central electrotaxis chamber. The electric field distribution and current density were numerically simulated with the steady-state electric conduction module from ANSYS 16.0. Simulation data confirms the application of nonhomogeneous EF of physiological strength. To validate the biocompatibility of the device cellular viability of the photoreceptor-derived 661W cell line was accessed. The cells have not shown any signs of apoptosis, damage or detachment during stimulation. Furthermore, immunofluorescence staining, namely by vinculin and actin labelling, allowed the assessment of adhesion efficiency and orientation of the cytoskeleton, respectively. Cellular motility in the presence and absence of applied DC EFs was verified. The movement of individual cells was tracked for the duration of the experiments, confirming the EF-induced, cathodal-directed motility of the studied cell line. The in vitro monolayer wound assay, or “scratch assay” is a standard protocol to quantitatively access cell migration in vitro. It encompasses the growth of a confluent cell monolayer followed by the mechanic creation of a scratch, representing a wound. Hence, wound dynamics was monitored over time and compared for control and applied the electric field to quantify cellular population motility.

Keywords: DC non-homogeneous electric fields, electrotaxis, microfluidic biochip, wound healing

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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: Nusrat Masood, Vijaya Dubey, Suaib Luqman

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Caspase 3, a member of cysteine-aspartic acid protease family, is an imperative indicator for cell death particularly when substantiating apoptosis. Thus, caspase 3 is an interesting target for the discovery and development of anticancer agent. We adopted a four level assessment of both terpenoids and flavonoids and thus experimentally performed the enzymatic assay in cell free system as well as in cancer cell line which was validated through real time expression and molecular interaction studies. A significant difference was observed with both the class of natural products indicating terpenoids as better activators of caspase 3 compared to flavonoids both in the cell free system as well as in cell lines. The expression analysis, activation constant and binding energy also correlate well with the enzyme activity. Overall, terpenoids had an unswerving effect on caspase 3 in all the tested system while flavonoids indirectly affect enzyme activity.

Keywords: Caspase 3, terpenoids, flavonoids, activation constant, binding energy

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Authors: Azhar Alhasawi, Vasu D. Appanna

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Chitin, the second most abundant bio-polymer in nature after cellulose, composed of β (1→4) linked N-acetylglucosamine (GlcNAc), is a major structural component in the cell walls of fungi and the shells of crustaceans. Chitin and its derivatives are gaining importance of economic value due to its biological activity and its industrial and biomedical applications. There are several methods to hydrolyze chitin to NAG, but they are typically expensive and environmentally unfriendly. Chitinase which catalyzes the breakdown of chitin to NAG has received much attention owing to its various applications in biotechnology. The presented research examines the ability of the versatile soil microbe, Pseudomonas fluorescens grown in chitin medium to produce chitinase and a variety of value-added products under abiotic stress. We have found that with high pH, Pseudomonas fluorescens enable to metabolize chitin more than with neutral pH and the overexpression of chitinase was also increased. P-dimethylaminobenzaldehyde (DMAB) assay for NAG production will be monitored and a combination of sodium dodecyl polyacrylamide gels will be used to monitor the proteomic and metabolomic changes as a result of the abiotic stress. The bioreactor of chitinase will also be utilized.

Keywords: Pseudomonas fluorescens, chitin, DMAB, chitinase

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Authors: Ting-Ying Su, Meng-Shiou Lee, Shyang-Chwen Sheu

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Garlic is used commonly as a seasoning around the world. But some people suffer from allergy to garlic. Garlic may also cause burning of mouth, stomach, and throat. In some Buddhist traditions, consuming garlic is not allowed. The objective of this study is to develop a LAMP based method for detection of garlic in food. We designed specific primers targeted on ITS1-5.8S rRNA-ITS2 sequence of garlic DNA. The LAMP assay was performed using a set of four different primers F3, B3, FIP and BIP at 60˚C in less than 60 mins. Results showed that the primer was not cross-reactive to other commonly used spice including Chinese leek, Chinese onion, green onion, onion, pepper, basil, parsley, pepper and ginger. As low as 2% of garlic DNA could be detected. Garlic still could be detected by developed LAMP after boiled at 100˚C for 80 minutes and autoclaved at 121˚C for 60 minutes. Commercial products labeled with garlic ingredient could be identified by the developed method.

Keywords: garlic, loop-mediated isothermal amplification, processing, DNA

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Authors: Anita Bajpai, Sarah Duan, Ashlee Erskine, Shehzein Khan, Raymond Kramer

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Introduction: This research focuses on exploring the beneficial effects if any, of Vitamin-D in reducing the need for supplemental oxygen among hospitalized COVID-19 patients. Two questions are investigated – Q1)Doeshaving a healthy level of baselineVitamin-D 25-OH (≥ 30ng/ml) help,andQ2) does administering Vitamin-D therapy after-the-factduring inpatient hospitalization help? Methods/Study Design: This is a comprehensive, retrospective, observational study of all inpatients at RUHS from March through December 2020 who tested positive for COVID-19 based on real-time reverse transcriptase–polymerase chain reaction assay of nasal and pharyngeal swabs and rapid assay antigen test. To address Q1, we looked atall N1=182 patients whose baseline plasma Vitamin-D 25-OH was known and who needed supplemental oxygen. Of this, a total of 121 patients had a healthy Vitamin-D level of ≥30 ng/mlwhile the remaining 61 patients had low or borderline (≤ 29.9ng/ml)level. Similarly, for Q2, we looked at a total of N2=893 patients who were given supplemental oxygen, of which713 were not given Vitamin-D and 180 were given Vitamin-D therapy. The numerical value of the maximum amount of oxygen flow rate(dependent variable) administered was recorded for each patient. The mean values and associated standard deviations for each group were calculated. Thesetwo sets of independent data served as the basis for independent, two-sample t-Test statistical analysis. To be accommodative of any reasonable benefitof Vitamin-D, ap-value of 0.10(α< 10%) was set as the cutoff point for statistical significance. Results: Given the large sample sizes, the calculated statistical power for both our studies exceeded the customary norm of 80% or better (β< 0.2). For Q1, the mean value for maximumoxygen flow rate for the group with healthybaseline level of Vitamin-D was 8.6 L/min vs.12.6L/min for those with low or borderline levels, yielding a p-value of 0.07 (p < 0.10) with the conclusion that those with a healthy level of baseline Vitamin-D needed statistically significant lower levels of supplemental oxygen. ForQ2, the mean value for a maximum oxygen flow rate for those not administered Vitamin-Dwas 12.5 L/min vs.12.8L/min for those given Vitamin-D, yielding a p-valueof 0.87 (p > 0.10). We thereforeconcludedthat there was no statistically significant difference in the use of oxygen therapy between those who were or were not administered Vitamin-D after-the-fact in the hospital. Discussion/Conclusion: We found that patients who had healthy levels of Vitamin-D at baseline needed statistically significant lower levels of supplemental oxygen. Vitamin-D is well documented, including in a recent article in the Lancet, for its anti-inflammatory role as an adjuvant in the regulation of cytokines and immune cells. Interestingly, we found no statistically significant advantage for giving Vitamin-D to hospitalized patients. It may be a case of “too little too late”. A randomized clinical trial reported in JAMA also did not find any reduction in hospital stay of patients given Vitamin-D. Such conclusions come with a caveat that any delayed marginal benefits may not have materialized promptly in the presence of a significant inflammatory condition. Since Vitamin-D is a low-cost, low-risk option, it may still be useful on an inpatient basis until more definitive findings are established.

Keywords: COVID-19, vitamin-D, supplemental oxygen, vitamin-D in primary care

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Authors: Asima Shah, F. A. Masoodi, Adil Gani, Bilal Ahmad Ashwar

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The present study was designed to evaluate the effect of γ-rays on the antioxidant and antiproliferative potential of β-glucan isolated from oats. The β-glucan was irradiated with 0, 2, 6, and 10 kGy by gamma ray. The samples were characterized by FT-IR, GPC, and quantitative estimation by Megazyme β-glucan assay kit. The average molecular weight of non-irradiated β-glucan was 199 kDa that decreased to 70 kDa at 10 kGy. Both FT-IR spectrum and chemical analysis revealed that the extracted β-glucan was pure having minor impurities. Antioxidant activity was evaluated by DPPH, lipid peroxidation, reducing power, metal chelating ability and oxidative DNA damage assays. Results revealed that the antioxidant activity of β-glucan increased with the increase in irradiation dose. Irradiated β-glucan also exhibited dose dependent cancer cell growth inhibition with irradiation doses. The study revealed that low molecular weight β-glucan with enhanced antioxidant and antiproliferative activities can be produced by a simple irradiation method.

Keywords: γ-irradiation, antioxidant activity, antiproliferative activity, β-glucan, oats

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Authors: Sajad Haghshenas

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Emerging evidence from clinical, genetic, and animal model studies suggests that the N-methyl-D-aspartate (NMDA) glutamate receptors (NMDAR) may contribute to the pathophysiology and aetiology of neurological and psychiatric disorders and the patients with impaired NMDR receptors experience psychological symptoms. Therefore, we hypothesised that NMDAR receptors play a key role in the development of attention deficit hyperactivity disorder (ADHD). In this comparative analytical study, we utilized western blotting method to assay the expression levels of NMDA subunits NR1 and NR2 in the blood plasma of 50 male individuals diagnosed with ADHD in comparison to 20 healthy controls. The findings from the western blotting analysis provide support for the hypothesis that individuals with ADHD exhibit significantly lower levels of NR1/2 receptors compared to those without the disorder. Further research is needed to explore the potential causal relationship between reduced NR1/NR2 receptor levels and the development of ADHD.

Keywords: expression, glutamate receptors, NR1, NR2, ADHD

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Authors: Yu Pu, Chien-Ping Hsiao, Chien-Chang Huang, Chieh-Lun Cheng

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Free radicals can accelerate aging on human skin by causing lipid oxidation, protein denaturation, and even DNA mutation. Substances with the ability of anti-free radicals can be used as functional components in cosmetic products. Research are attracted to develop new anti-free radical components for cosmetic application. This study was aimed to evaluate the microbial metabolites on free radical scavenging ability. Two microorganisms, PU-01 and PU-02, were isolated from soil of hot spring environment and grew in LB agar at 50°C for 24 h. The suspension was collected by centrifugation at 4800 g for 3 min, The anti-free radical activity was determined by DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging assay. The result showed that the growth medium of PU-01 presented a higher DPPH scavenging effect than that of PU-02. This study presented potential anti-free radical components from microbial metabolites that might be applied in anti-aging cosmetics.

Keywords: anti-ageing, anti-free radical, biotechnology, microorganism

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Authors: Ghazala Yaqub, Zubi Sadiq, Almas Hamid, Saira Iqbal

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This paper is the very first report of three dual action hybrids synthesized by covalent linkage of carbazole based novel antibacterial compounds with efflux pump inhibitors i.e., indole acetic acid/gallic acid. Novel carbazole based antibacterial compounds were prepared first and then these were covalently linked with efflux pump inhibitors which leads to the successful formation of hybrids. All prepared compounds were evaluated for their bacterial cell killing capability against Escherichia coli, Staphylococcus aureus, Pasteurella multocida and Bacillus subtilis. Compound were effective against all tested bacterial strains at different concentrations. But when these compounds were linked with efflux pump inhibitors they showed dramatic enhancement in their bacterial cell killing potential and minimum inhibitory concentration of all hybrids ranges from 7.250 µg/mL to 0.0283 µg/mL.

Keywords: antimicrobial assay, carbazole, dual action hybrids, efflux pump inhibitors

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Authors: Reshma Jolly, Mohammad Shakir, Mohammad Shoeb Khan, Noor E. Iram

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A nanocomposite system integrating hydroxyapatite, chitin and starch (n-HA/CT/ST) has been synthesized via co-precipitation approach at room temperature, addressing the issues of biocompatibility, mechanical strength and cytotoxicity required for Bone tissue engineering. The interactions, crystallite size and surface morphology against n-HA/CT (nano-hydroxyapatite/chitin) nanocomposite have been obtained by correlating and comparing the results of FTIR, SEM, TEM and XRD. The comparative study of the bioactivity of n-HA/CT and n-HA/CT/ST nanocomposites revealed that the incorporation of starch as templating agent improved these properties in n-HA/CT/ST nanocomposite. The rise in thermal stability in n-HA/CT/ST nanocomposite as compared to n-HA/CT has been observed by comparing the TGA results. The comparison of SEM images of both the scaffolds indicated that the addition of ST influenced the surface morphology of n-HA/CT scaffold which appeared to be rougher and porous. The MTT assay on murine fibroblast L929 cells and in-vitro bioactivity of n-HA/CT/ST matrix referred superior non-toxic property of n-HA/CT/ST nanocomposite and higher possibility of osteo-integration in-vivo, respectively.

Keywords: bioactive, chitin, hyroxyapatite, nanocomposite

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Authors: Vijayalakshmi Marella, NageswaraRaoPilli

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This paper describes a simple, rapid and sensitive liquid chromatography / tandem mass spectrometry assay for the determination of montelukast in human plasma using montelukast d6 as an internal standard. Analyte and the internal standard were extracted from 50 µL of human plasma via solid phase extraction technique without evaporation, drying and reconstitution steps. The chromatographic separation was achieved on a C18 column by using a mixture of methanol and 5mM ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 0.8 mL/min. Good linearity results were obtained during the entire course of validation. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze more number of samples in short time, thus increasing the productivity. The proposed method was found to be applicable to clinical studies.

Keywords: Montelukast, tandem mass spectrometry, montelukast d6, FDA guidelines

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Authors: Pierre Moulis, Markus Herderich, Doris Rauhut, Patricia Ballestra

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Nowadays, it is starting to be more frequent to detect wines with mousy off-flavor. The reasons behind this could be the significant decrease in sulphur dioxide, the increase in pH, and the trend for spontaneous fermentation in wine. This off-flavor can be produced by Brettanomyces bruxellensis or some Lactic acid bacteria. So far there is no study working on the influence of the genetic group on the production of these microorganisms. Objectives: The objectives of this research are to increase knowledge and to have a better understanding of the microbiological phenomena related to the production of the mousy off-flavor in the wine. Methodologies: In this research, microorganisms were screened in an N-heterocycle assay medium (this medium contained all known precursors) and the production of mousy compounds was quantified by Stir Bar Sorptive Extraction-Gas Chromatography-Mass Spectrometry (SBSE-GC-MS). Main contributions: Brettanomyces bruxellensis and Oenococcus oeni could produce mousiness at a different amount depending on the strain. But there is no group effect.

Keywords: mousy off-flavor, wine, Brettanomyces bruxellensis, Oenococcus oeni

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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: Alec Chabwinja, Cannan Tawonezvi, Jerneja Vidmar, Constance Chingwaru, Walter Chingwaru

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Objective: To evaluate the potential of commercial fermented / probiotic products available in Zimbabwe or internationally, and strains of Lactobacillus plantarum (L. plantarum) as prophylaxis and therapy against diarrhoeal and sexually transmitted infections. Methods: The antimicrobial potential of cultures of lactobacilli enriched from 4 Zimbabwean commercial food/beverage products, namely Dairibord Lacto sour milk (DLSM), Probrand sour milk (PSM), Kefalos Vuka cheese (KVC) and Chibuku opaque beer (COB); three probiotic products obtainable in Europe and internationally; and four strains of L. plantarum obtained from Balkan traditional cheeses and Zimbabwean foods against clinical strains of Escherichia coli (E. coli) and non-clinical strains of Candida albicans and Rhodotorula spp. was assayed using the well diffusion method. Three commercial Agar diffusion assay and a competitive exclusion assay were carried out on Mueller-Hinton agar. Results: Crude cultures of putative lactobacillus strains obtained from Zimbabwean dairy products (Probrand sour milk, Kefalos Vuka vuka cheese and Chibuku opaque beer) exhibited significantly greater antimicrobial activities against clinical strains of E. coli than strains of L. plantarum isolated from Balkan cheeses (CLP1, CLP2 or CLP3) or crude microbial cultures from commercial paediatric probiotic products (BG, PJ and PL) of a culture of Lactobacillus rhamnosus LGG (p < 0.05). Furthermore, the following has high antifungal activities against the two yeasts: supernatant-free microbial pellet (SFMP) from an extract of M. azedarach leaves (27mm ± 2.5) > cell-free culture supernatants (CFCS) from Maaz Dairy sour milk and Mnandi sour milk (approximately 26mm ± 1.8) > CFCS and SFMP from Amansi hodzeko (25mm ± 1.5) > CFCS from Parinari curatellifolia fruit (24mm ± 1.5), SFMP from P. curatellifolia fruit (24mm ± 1.4) and SFMP from mahewu (20mm ± 1.5). These cultures also showed high tolerance to acidic conditions (~pH4). Conclusions: The putative lactobacilli from four commercial Zimbabwean dairy products (Probrand sour milk, Kefalos Vuka vuka cheese and Chibuku opaque beer), and three strains of L. plantarum from Balkan cheeses (CLP1, CLP2 or CLP3) exhibited high antibacterial activities, while Maaz Dairy sour-, Mnandi sour- and Amansi hodzeko milk products had high antifungal activities. Our selection of Zimbabwean probiotic products has potential for further development into probiotic products for use in the control diarrhea caused by pathogenic strains of E. coli or yeast infections. Studies to characterise the probiotic potential of the live cultures in the products are underway.

Keywords: lactic acid bacteria, Staphylococcus aureus, Streptococcus spp, yeast, inhibition, acid tolerance

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Authors: M. Abdel-Hamid, P. Saporito, R. V. Mateiu, A. Osman, E. Romeih, H. Jenssen

Abstract:

Camel milk whey (CMW) was hydrolyzed with papain from Carica papaya and fractionated by size exclusion chromatography (SEC). The antibacterial and anti-biofilm activity of the CMW, Camel milk whey hydrolysate (CMWH) and the obtained SEC-fractions was assessed against Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (MRSA). SEC-F2 (fraction 2) exhibited antibacterial effectiveness against MRSA and P. aeruginosa with the minimum inhibitory concentration of 0.31 and 0.156 mg/ml, respectively. Furthermore, SEC-F2 significantly decreased biofilm biomass by 71% and 83 % for MRSA and P. aeruginosa in a crystal violet microplate assay. Scanning electron microscopy showed that the SEC-F2 caused changes in the treated bacterial cells. Additionally, LC/MS analysis was used to characterize the peptides of SEC-F2. Two major peptides were detected in SEC-F2 having masses of 414.05 Da and 456.06 Da. In conclusion, this study has demonstrated that hydrolysis of CMW with papain generates small and extremely potent antibacterial and anti-biofilm peptides against both MRSA and P. aeruginosa.

Keywords: camel milk, whey proteins, antibacterial peptide, anti-biofilm

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Authors: Andrés Quiroz, Emilio Hormazabal, Ana Mutis, Fernando Ortega, Manuel Chacón-Fuentes, Leonardo Parra

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Red clover (Trifolium pratense L.) is an important forage species in temperate regions of the world. The main limitation of this species worldwide is a lack of persistence related to the high mortality of plants due to a complex of biotic and abiotic factors, determining a life span of two or three seasons. Because of the importance of red clover in Chile, a red clover breeding program was started at INIA Carillanca Research Center in 1989, with the main objective of improving the survival of plants, forage yield, and persistence. The main selection criteria for selecting new varieties have been based on agronomical parameters and biotic factors. The main biotic factor associated with red clover mortality in Chile is Hylastinus obscurus (Coleoptera: Curculionidae). Both larval and adults feed on the roots, causing weakening and subsequent death of clover plants. Pesticides have not been successful for controlling infestations of this root borer. Therefore, alternative strategies for controlling this pest are a high priority for red clover producers. Currently, the role of semiochemical in the interaction between H. obscurus and red clover plants has been widely studied for our group. Specifically, from the red clover foliage has been identified limonene is eliciting repellency from the root borer. Limonene is generated in the plant from two independent biosynthetic pathways, the mevalonic acid, and deoxyxylulose pathway. Mevalonate pathway enzymes are localized in the cytosol, whereas the deoxyxylulose phosphate pathway enzymes are found in plastids. In summary, limonene can be determinated by enzymatic bioassay using GPP as substrate and by limonene synthase expression. Therefore, the main objective of this work was to study genetic variation of limonene in material provided by INIA´s Red Clover breeding program. Protein extraction was carried out homogenizing 250 mg of leave tissue and suspended in 6 mL of extraction buffer (PEG 1500, PVP-30, 20 mM MgCl2 and antioxidants) and stirred on ice for 20 min. After centrifugation, aliquots of 2.5 mL were desalted on PD-10 columns, resulting in a final volume of 3.5 mL. Protein determination was performed according to Bradford with BSA as a standard. Monoterpene synthase assays were performed with 50 µL of protein extracts transferred into gas-tight 2 mL crimp seal vials after addition of 4 µL MgCl₂ and 41 µL assay buffer. The assay was started by adding 5 µL of a GPP solution. The mixture was incubated for 30 min at 40 °C. Biosynthesized limonene was quantified in a GC equipped with a chiral column and using synthetic R and S-limonene standards. The enzymatic the production of R and S-limonene from different Superqueli-Carillanca genotypes is shown in this work. Preliminary results showed significant differences in limonene content among the genotypes analyzed. These results constitute an important base for selecting genotypes with a high content of this repellent monoterpene towards H. obscurus.

Keywords: head space, limonene enzymatic determination, red clover, Hylastinus obscurus

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Authors: Ana Paula Rosifini Alves Claro, André Luiz Reis Rangel, Nathan Trujillo, Ketul C. Popat

Abstract:

In the present work, in vitro cytotoxicity was evaluated after nanotubes growth on Ti15Mo alloy surface. TiO2 nanotubes were obtained by anodizing technique at room temperature in an electrolyte with 0.25 %NH4F and glycerol at a constant anodic potential of 20 V for 24 hours. The morphology of nanotubes was observed by field emission scanning electron microscopy (FE-SEM; XL 30 FEG, Philips). Crystal structure was analyzed by wide-angle X-ray diffraction. A cell culture model using human fibroblast-like cells was used to study the effect of TiO2 nanotubes growth on the cytotoxicity of the Ti15Mo alloy for 1, 4 and 7 days culture period. The MTT assay was used to evaluate cell viability and cell adhesion was evaluated by scanning electron microscopy. Results show that Ti15Mo alloy with TiO2 nanotubes on surface is nontoxic and exhibit good interaction with surface.

Keywords: titanium alloys, TiO2 nanotubes, cell growth, Ti-15Mo alloy

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Authors: Reza Dehghani Bidgoli

Abstract:

Although Artemisia species are one of the most important medicinal plants, there are a few reports on chemistry or activity of their essential oils because of low amounts of the oils in this genus. In this study, chemical composition of essential oils leaves and stems of Artemisia sieberi and Artemisia aucheri growing wild in Kashan rangelands, central Iran, have been analyzed using GC–MS technique. Analysis revealed 50 identified compounds, representing 96.55% of the oil and 23 identified compounds representing 97.83% of the oil on Artemisia sieberi and Artemisia aucheri respectively. The yield of essential oil extraction is very higher than those of previous reports. In both plants α-thujene is the main component in both of them, with an extra value, 74.42%, in aucheri species. Several compounds (some with significant compositions), were found in these varieties of Artemisia which are not recorded in previous literature. Antioxidant activities of the essential oils were evaluated for the first time in this research work using β-carotene/linoleic acid assay and found to be surprisingly attributed directly to α-pinene contents in them.

Keywords: essential oil, artemisia aucheri, artemisia sieberi, α-thujene, antioxidant activity

Procedia PDF Downloads 424