Search results for: bifunctional enzyme

Authors: Shanakr Bhattarai, V. S. Tiwari, Barak Akabayov

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The plummeting number of infections and death is due to the development of drug-resistant bacteria. In addition, the number of approved antibiotic drugs by the Food and Drug Administration (FDA) is insufficient. Therefore, developing new drugs and finding novel targets for central metabolic pathways in bacteria is urgently needed. One of the promising targets is DNA replication machinery which consists of many essential proteins and enzymes. DnaG primase is an essential enzyme and a central part of the DNA replication machinery. DnaG primase synthesizes short RNA primers that initiate the Okazaki fragments by the lagging strand DNA polymerase. Therefore, it is reasonable to assume that inhibition of primase activity will stall DNA replication and prevent bacterial proliferation. We did the expression and purification of eight different bacterial DnaGs (Mycobacterium tuberculosis(Mtb), Bacillus anthracis (Ba), Mycobacterium smegmatis (Msmeg), Francisella tularencis (Ft), Vibrio cholerae (Vc) and Yersinia pestis (Yp), Staphylococcus aureus(Saureus), Escherichia coli(Ecoli)) followed by the radioactive activity assay. After obtaining the pure and active protein DnaG, we synthesized the inhibitors for them. The inhibitors were divided into five different groups, each containing five molecules, and the cocktail inhibition assay was performed against each DnaGs. The groups of molecules inhibiting the DnaGs were further tested with individual molecules belonging to inhibiting groups. Each molecule showing inhibition was titrated against the corresponding DnaGs to find IC50. We got a molecule(VS167) that acted as broad inhibitors, inhibiting all eight DnaGs. Molecules VS180 and VS186 inhibited seven DnaGs (except Saureus). Similarly, two molecules(VS 173, VS176) inhibited five DnaGs (Mtb, Ba, Ft, Yp, Ecoli). VS261 inhibited four DnaGs (Mtb, Ba, Ft, Vc). MS50 inhibited Ba and Vc DnaGs. And some of the inhibitors inhibited only one DnaGs. Thus we found the broad and specific inhibitors for different bacterial DnaGs, and their Structure-activity analysis(SAR) was done. Further, We tried to explain the similarities among the enzyme DnaGs from different bacteria based on their inhibition pattern.

Keywords: DNA replication, DnaG, okazaki fragments, antibiotic drugs

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Authors: Sanjiv Kumar Soni, Chetna Janveja

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The approach of utilizing zero cost kitchen waste residues for growing suitable strains of fungi for the induction of a cocktail of hydrolytic enzymes and ethanol generation has been validated in the present study with the objective of developing an indigenous biorefinery for low cost bioethanol production with the generation of zero waste. Solid state fermentation has been carried out to evaluate the potential of various steam pretreated kitchen waste residues as substrates for the co-production of multiple carbohydrases including cellulases, hemicellulases, pectinase and amylases by a locally isolated strain of Aspergillus niger C-5. Of all the residues, potato peels induced the maximum yields of all the enzyme components corresponding to 64.0±1.92 IU of CMCase, 17.0±0.54 IU of FPase , 42.8±1.28 IU of β-glucosidase, 990.0±28.90 IU of xylanase, 53.2±2.12 IU of mannanase, 126.0±3.72 IU of pectinase, 31500.0±375.78 IU of α-amylase and 488.8±9.82 IU of glucoamylase/g dry substrate respectively. Saccharification of various kitchen refuse residues using inhouse produced crude enzyme cocktail resulted in the release of 610±10.56, 570±8.89, 435±6.54, 475±4.56, 445±4.27, 385±4.49, 370±6.89, 490±10.45 mg of total reducing sugars/g of dried potato peels, orange peels, pineapple peels, mausami peels, onion peels, banana stalks, pea pods and composite mixture respectively revealing carbohydrate conversion efficiencies in the range of 97.0-99.4%. After fermentation of released hexoses by Saccharomyces cerevisae, ethanol yields ranging from 80-262 mL/ kg of dry residues were obtained. The study has successfully evaluated the valorization of kitchen garbage, a highly biodegradable component in Municipal Solid Waste by using it as a substrate for the in-house co-production of multiple carbohydrases and employing the steam treated residues as a feed stock for bioethanol production. Such valorization of kitchen garbage may reduce the level of Municipal Solid Waste going into land-fills thus lowering the emissions of greenhouse gases. Moreover, the solid residue left after the bioconversion may be used as a biofertilizer for improving the fertility of the soils.

Keywords: kitchen waste, bioethanol, solid waste, bioconversion, waste management

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Authors: Shady S. Hassan, Brijesh K. Tiwari, Gwilym A. Williams, Amit K. Jaiswal

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Waste materials from a wide range of agro-industrial processes may be used as substrates for microbial growth, and subsequently the production of a range of high value products and bioenergy. In addition, utilization of these agro-residues in bioprocesses has the dual advantage of providing alternative substrates, as well as solving their disposal problems. Spent coffee grounds (SCG) are a by-product (45%) of coffee processing. SCG is a lignocellulosic material, which is composed mainly of cellulose, hemicelluloses, and lignin. Thus, a pretreatment process is required to facilitate an efficient enzymatic hydrolysis of such carbohydrates. In this context, microwave pretreatment of lignocellulosic biomass without the addition of harsh chemicals represents a green technology. Moreover, microwave treatment has a high heating efficiency and is easy to implement. Thus, microwave pretreatment of SCG without adding of harsh chemicals investigated as a green technology to enhance enzyme hydrolysis. In the present work, microwave pretreatment experiments were conducted on SCG at varying power levels (100, 250, 440, 600, and 1000 W) for 60 s. By increasing microwave power to a certain level (which vary by varying biomass), reducing sugar increases, then reducing sugar from biomass start to decrease with microwave power increase beyond this level. Microwave pretreatment of SCG at 60s followed by enzymatic hydrolysis resulted in total reducing sugars of 91.6 ± 7.0 mg/g of biomass (at microwave power of 100 w). Fourier transform Infrared Spectroscopy (FTIR) was employed to investigate changes in functional groups of biomass after pretreatment, while high-performance liquid chromatography (HPLC) was employed for determination of glucose. Pretreatment of lignocellulose using microwave was found to be an effective and energy efficient technology to improve saccharification and glucose yield. Energy performance will be evaluated for the microwave pretreatment, and the enzyme hydrolysate will be used as media component substitute for the production of ethanol and other high value products.

Keywords: lignocellulose, microwave, pretreatment, spent coffee grounds

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Authors: Soultana Konstantinidou, Tiziana Schmidt, Elena Landi, Alessandro De Carli, Giovanni Maltinti, Darius Witt, Alicja Dziadosz, Agnieszka Lindstaedt, Michele Lai, Mauro Pistello, Valentina Cappello, Luciana Dente, Chiara Gabellini, Piotr Barski, Vittoria Raffa

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CRISPR/Cas9 technology has gained the interest of researchers in the field of biotechnology for genome editing. Since its discovery as a microbial adaptive immune defense, this system has been widely adopted and is acknowledged for having a variety of applications. However, critical barriers related to safety and delivery are persisting. Here, we propose a new concept of genome engineering, which is based on a nano-formulation of Cas9. The Cas9 enzyme was conjugated to a gold nanoparticle (AuNP-Cas9). The AuNP-Cas9 maintained its cleavage efficiency in vitro, to the same extent as the ribonucleoprotein, including non-conjugated Cas9 enzyme, and showed high gene editing efficiency in vivo in zebrafish embryos. Since CRISPR/Cas9 technology is extensively used in cancer research, melanoma was selected as a validation target. Cell studies were performed in A375 human melanoma cells. Particles per se had no impact on cell metabolism and proliferation. Intriguingly, the AuNP-Cas9 internalized spontaneously in cells and localized as a single particle in the cytoplasm and organelles. More importantly, the AuNP-Cas9 showed a high nuclear localization signal. The AuNP-Cas9, overcoming the delivery difficulties of Cas9, could be used in cellular biology and localization studies. Taking advantage of the plasmonic properties of gold nanoparticles, this technology could potentially be a bio-tool for combining gene editing and photothermal therapy in cancer cells. Further work will be focused on intracellular interactions of the nano-formulation and characterization of the optical properties.

Keywords: CRISPR/Cas9, gene editing, gold nanoparticles, nanotechnology

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Authors: Emma Castiglioni, Nigel Scrutton, Derren Heyes, Alistair Fielding

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By using light as trigger, it is possible to study many biological processes, such as the activity of genes, proteins, and other molecules, with precise spatiotemporal control. Caged compounds, where biologically active molecules are generated from an inert precursor upon laser photolysis, offer the potential to initiate such biological reactions with high temporal resolution. As light acts as the trigger for cleaving the protecting group, the ‘caging’ technique provides a number of advantages as it can be intracellular, rapid and controlled in a quantitative manner. We are developing caging strategies to study the catalytic cycle of a number of enzyme systems, such as nitric oxide synthase and ethanolamine ammonia lyase. These include the use of caged substrates, caged electrons and the possibility of caging the enzyme itself. In addition, we are developing a novel freeze-quench instrument to study these reactions, which combines rapid mixing and flashing capabilities. Reaction intermediates will be trapped at low temperatures and will be analysed by using electron paramagnetic resonance (EPR) spectroscopy to identify the involvement of any radical species during catalysis. EPR techniques typically require relatively long measurement times and very often, low temperatures to fully characterise these short-lived species. Therefore, common rapid mixing techniques, such as stopped-flow or quench-flow are not directly suitable. However, the combination of rapid freeze-quench (RFQ) followed by EPR analysis provides the ideal approach to kinetically trap and spectroscopically characterise these transient radical species. In a typical RFQ experiment, two reagent solutions are delivered to the mixer via two syringes driven by a pneumatic actuator or stepper motor. The new mixed solution is then sprayed into a cryogenic liquid or surface, and the frozen sample is then collected and packed into an EPR tube for analysis. The earliest RFQ instrument consisted of a hydraulic ram unit as a drive unit with direct spraying of the sample into a cryogenic liquid (nitrogen, isopentane or petroleum). Improvements to the RFQ technique have arisen from the design of new mixers in order to reduce both the volume and the mixing time. In addition, the cryogenic isopentane bath has been coupled to a filtering system or replaced by spraying the solution onto a surface that is frozen via thermal conductivity with a cryogenic liquid. In our work, we are developing a novel RFQ instrument which combines the freeze-quench technology with flashing capabilities to enable the studies of both thermally-activated and light-activated biological reactions. This instrument also uses a new rotating plate design based on magnetic couplings and removes the need for mechanical motorised rotation, which can otherwise be problematic at cryogenic temperatures.

Keywords: caged compounds, freeze-quench apparatus, photolysis, radicals

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Authors: Md. Alauddin, Md. Ruhul Amin, Md. Omar Faruque, Muhammad Ali Siddiquee, Zakir Hossain Howlader, Mohammad Asaduzzaman

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Diabetes and hypertension are the major lifestyle related diseases. The α-amylase and angiotensin converting enzymes (ACE) are the key enzymes that regulate diabetes and hypertension. The aim was to develop a drug for the treatment of diabetes and hypertension. The Rice Bran (RB) sample (Oryza sativa; BRRI-Dhan-84) was collected from the Bangladesh Rice Research Institute (BRRI), and rice bran proteins were isolated and hydrolyzed by hydrolyzing enzyme alcalase and trypsin. In vivo experiment suggested that rice bran bioactives has an effect on regulating the expression of several key gluconeogenesis and lipogenesis-regulating genes, such as glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, and fatty acid synthase. The above genes have a connection of regulating the glucose level, lipids profile as well as act as an anti-inflammatory agent. A molecular docking, bioinformatics and in vitro experiments were performed. We found rice bran protein hydrolysates significantly (<0.05) influence the peptide concentration in the case of trypsin, alcalase, and (trypsin + alcalase) digestion. The in vitro analysis found that protein hydrolysate significantly (<0.05) reduced diabetic and hypertension as well as oxidative stress. A molecular docking study showed that the YY and IP peptide have a significantly strong binding affinity to the active site of the ACE enzyme and α-amylase with -7.8Kcal/mol and -6.2Kcal/mol, respectively. The Molecular dynamics (MD) simulation and Swiss ADME data analysis showed that less toxicity risk, good physicochemical properties, pharmacokinetics, and drug-likeness with drug scores 0.45 and 0.55 of YY and IP peptides, respectively. Thus, rice bran bioactive could be a good candidate for the treatment of diabetes and hypertension.

Keywords: anti-hypertensive and anti-hyperglycemic, anti-oxidative, bioinformatics, in vitro study, rice bran proteins and peptides

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Authors: Monisha Elumalai, Joana Guerreiro, Joana Carvalho, Marta Prado

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DNA biosensors popularity has been increasing over the past few years. Traditional analytical techniques tend to require complex steps and expensive equipment however DNA biosensors have the advantage of getting simple, fast and economic. Additionally, the combination of DNA biosensors with nanomaterials offers the opportunity to improve the selectivity, sensitivity and the overall performance of the devices. DNA biosensors are based on oligonucleotides as sensing elements. These oligonucleotides are highly specific to complementary DNA sequences resulting in the hybridization of the strands. DNA biosensors are not only an advantage in the clinical field but also applicable in numerous research areas such as food analysis or environmental control. Zebra Mussels (ZM), Dreissena polymorpha are invasive species responsible for enormous negative impacts on the environment and ecosystems. Generally, the detection of ZM is made when the observation of adult or macroscopic larvae's is made however at this stage is too late to avoid the harmful effects. Therefore, there is a need to develop an analytical tool for the early detection of ZM. Here, we present a portable plasmonic biosensor for the detection of environmental DNA (eDNA) released to the environment from this invasive species. The plasmonic DNA biosensor combines gold nanoparticles, as transducer elements, due to their great optical properties and high sensitivity. The detection strategy is based on the immobilization of a short base pair DNA sequence on the nanoparticles surface followed by specific hybridization in the presence of a complementary target DNA. The hybridization events are tracked by the optical response provided by the nanospheres and their surrounding environment. The identification of the DNA sequences (synthetic target and probes) to detect Zebra mussel were designed by using Geneious software in order to maximize the specificity. Moreover, to increase the optical response enzyme amplification of DNA might be used. The gold nanospheres were synthesized and characterized by UV-visible spectrophotometry and transmission electron microscopy (TEM). The obtained nanospheres present the maximum localized surface plasmon resonance (LSPR) peak position are found to be around 519 nm and a diameter of 17nm. The DNA probes modified with a sulfur group at one end of the sequence were then loaded on the gold nanospheres at different ionic strengths and DNA probe concentrations. The optimal DNA probe loading will be selected based on the stability of the optical signal followed by the hybridization study. Hybridization process leads to either nanoparticle dispersion or aggregation based on the presence or absence of the target DNA. Finally, this detection system will be integrated into an optical sensing platform. Considering that the developed device will be used in the field, it should fulfill the inexpensive and portability requirements. The sensing devices based on specific DNA detection holds great potential and can be exploited for sensing applications in-loco.

Keywords: ZM DNA, DNA probes, nicking enzyme, gold nanoparticles

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Authors: Shama Ranjan Barua, Tofazzal M. Rakib, Mohammad Alamgir Hossain, Tania Ferdushy, Sharmin Chowdhury

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Rotavirus is one of the main etiologies of neonatal diarrhea in bovine calves that causes significant economic loss in Bangladesh. The present study was carried out to investigate the pathology of neonatal enteritis in calves due to bovine rotavirus infection in south-eastern part of Bangladesh. Rotavirus was identified by using ELISA, RT-PCR (Reverse Transcription Polymerase Chain Reaction), real-time RT-PCR. We examined 12 dead calves with history of diarrhea during necropsy. Among 12 dead calves, in gross examination, 6 were found with pathological changes in intestine, 5 calves had congestion of small intestine and rest one had no distinct pathological changes. Intestinal contents and/or faecal samples of all dead calves were collected and examined to confirm the presence of bovine rotavirus A using Enzyme linked immunosorbant assay (ELISA), RT-PCR and real-time RT-PCR. Out 12 samples, 5 (42%) samples revealed presence of bovine rotavirus A in three diagnostic tests. The histopathological changes were found almost exclusively limited in the small intestine. The lesions of rotaviral enteritis ranged from slight to moderate shortening (atrophy) of villi in the jejunum and ileum with necrotic crypts. The villi were blunt and covered by immature epithelial cells. Infected cells, stained with Haematoxylin and Eosin staining method, showed characteristic syncytia and eosinophilc intracytoplasmic inclusion body. The presence of intracytoplasmic inclusion bodies in enterocytes is the indication of viral etiology. The presence of rotavirus in the affected tissues and/or lesions was confirmed by three different immunological and molecular tests. The findings of histopathological changes will be helpful in future diagnosis of rotaviral infection in dead calves.

Keywords: calves, diarrhea, pathology, rotavirus

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Authors: Lintle Mohase, Ninikoe Lebusa, Mpho Stephen Mafa

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Wheat is an economically important cereal crop. However, the changing climatic conditions that intensify drought in production areas, and additional pest infestation, such as the Russian wheat aphid (RWA, Diuraphis noxia), severely hamper its production. Drought and pest management require an additional water supply through irrigation and applying inorganic nutrients (including silicon) as alternative strategies to mitigate the stress effects. Therefore, other approaches are needed to enhance wheat productivity during drought stress and aphid abundance. Two wheat cultivars were raised under greenhouse conditions, exposed to drought stress, and treated with silicon before infestation with the South African RWA biotype 2 (RWASA2). The morphological evaluations showed that severe drought or a combination of drought and infestation significantly reduced the plant height of wheat cultivars. Silicon treatment did not alleviate the growth reduction. The biochemical responses were measured using spectrophotometric assays with specific substrates. An evaluation of the enzyme activities associated with oxidative stress and defence responses indicated that drought stress increased NADPH oxidase activity, while silicon treatment significantly reduced it in drought-stressed and infested plants. At 48 and 72 hours sampling periods, a combination of silicon, drought and infestation treatment significantly increased peroxidase activity compared to drought and infestation treatment. The treatment also increased β-1,3-glucanase activity 72 hours after infestation. In addition, silicon and drought treatment increased glucose but reduced sucrose accumulation. Furthermore, silicon, drought, and infestation treatment combinations reduced the sucrose content. Finally, silicon significantly increased the trehalose content under severe drought and infestation, evident at 48 and 72-hour sampling periods. Our findings shed light on silicon’s ability to induce protective biochemical responses during drought and aphid infestation.

Keywords: drought, enzyme activity, silicon, soluble sugars, Russian wheat aphid, wheat

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Authors: Elisangela Sobreira, Denise Teixeira

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Visceral leishmaniasis is a public health problem in Brazil, it is the main reservoir dog. In the period 2012-2016 78 diagnoses were performed in dogs suspected. Blood samples were collected from the cephalic vein obtaining serum used for the indirect immunofluorescence test and enzyme-linked immunosorbent assay, while it collected a drop of blood for the rapid chromatographic immunoassay. Obtained in 32 dogs positive. The test is important for the control of this disease and is used routinely in the Zoonoses Control Center.

Keywords: Brazil, dogs, Leismaniasis, Zoonoses center

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Authors: Fergal Fouhy, Alan O’Keeffe, Sean Costelloe, Michael Clarkson

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Background: Sarcoidosis is a systemic, non-infectious disease of unknown aetiology, characterized by non-caseating granulomatous inflammation. The lung is most often affected (90%); however, the condition can affect all organs, including the kidneys. There is limited evidence describing the incidence and characteristics of renal involvement in sarcoidosis. Serum angiotensin-converting enzyme (ACE) is a recognised biomarker used in the diagnosis and monitoring of sarcoidosis. Methods: A single-centre, retrospective cohort study of patients presenting to Cork University Hospital (CUH) in 2015 with first-time elevations of serum ACE was performed. This included an initial database review of ACE and other biochemistry results, followed by a medical chart review to confirm the presence or absence of sarcoidosis and management thereof. Acute kidney injury (AKI) was staged using the AKIN criteria, and chronic kidney disease (CKD) was staged using the KDIGO criteria. Follow-up was assessed over five years tracking serum creatinine, serum calcium, and estimated glomerular filtration rates (eGFR). Results: 119 patients were identified as having a first raised serum ACE in 2015. Seventy-nine male patients and forty female patients were identified. The mean age of patients identified was 47 years old. 11% had CKD at baseline. 18% developed an AKI at least once within the next five years. A further 6% developed CKD during this time period. 13% developed hypercalcemia. The patients within the lowest quartile of serums ACE had an incidence of sarcoidosis of 5%. None of this group developed hypercalcemia, 23% developed AKI, and 7% developed CKD. Of the patients with a serum ACE in the highest quartile, almost all had documented diagnoses of sarcoidosis with an incidence of 96%. 3% of this group developed hypercalcemia, 13% AKI and 3% developed CKD. Conclusions: There was an unexpectedly high incidence of AKI in patients who had a raised serum ACE. Not all patients with a raised serum ACE had a confirmed diagnosis of sarcoidosis. There does not appear to be a relationship between increased serum ACE levels and increased incidence of hypercalcaemia, AKI, and CKD. Ideally, all patients should have biopsy-proven sarcoidosis. This is an initial study that should be replicated with larger numbers and including multiple centres.

Keywords: sarcoidosis, acute kidney injury, chronic kidney disease, hypercalcemia

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Authors: K. Yu Vlasova, M. A. Abakumov, H. Wishwarsao, M. Sokolsky, N. V. Nukolova, A. G. Majouga, Y. I. Golovin, N. L. Klyachko, A. V. Kabanov

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Introduction:Nowadays pharmaceutical medicine is aimed to create systems for combined therapy, diagnostic, drug delivery and controlled release of active molecules to target cells. Magnetic nanoparticles (MNPs) are used to achieve this aim. MNPs can be applied in molecular diagnostics, magnetic resonance imaging (T1/T2 contrast agents), drug delivery, hyperthermia and could improve therapeutic effect of drugs. The most common drug containers, containing MNPs, are liposomes, micelles and polymeric molecules bonded to the MNPs surface. Usually superparamagnetic nanoparticles are used (the general diameter is about 5-6 nm) and all effects of high frequency magnetic field (MF) application are based on Neel relaxation resulting in heating of surrounded media. In this work we try to develop a new method to improve drug release from MNPs under super low frequency MF. We suppose that under low frequency MF exposures the Brown’s relaxation dominates and MNPs rotation could occur leading to conformation changes and release of bioactive molecules immobilized on MNPs surface.The aim of this work was to synthesize different systems with active drug (biopolymers coated MNPs nanoclusters with immobilized enzymes and doxorubicin (Dox) loaded magnetic liposomes/micelles) and investigate the effect of super low frequency MF on these drug containers. Methods: We have synthesized MNPs of magnetite with magnetic core diameter 7-12 nm . The MNPs were coated with block-copolymer of polylysine and polyethylene glycol. Superoxide dismutase 1 (SOD1) was electrostatically adsorbed on the surface of the clusters. Liposomes were prepared as follow: MNPs, phosphatidylcholine and cholesterol were dispersed in chloroform, dried to get film and then dispersed in distillated water, sonicated. Dox was added to the solution, pH was adjusted to 7.4 and excess of drug was removed by centrifugation through 3 kDa filters. Results: Polylysine coated MNPs formed nanosized clusters (as observed by TEM) with intensity average diameter of 112±5 nm and zeta potential 12±3 mV. After low frequency AC MF exposure we observed change of immobilized enzyme activity and hydrodynamic size of clusters. We suppose that the biomolecules (enzymes) are released from the MNPs surface followed with additional aggregation of complexes at the MF in medium. Centrifugation of the nanosuspension after AC MF exposures resulted in increase of positive charge of clusters and change in enzyme concentration in comparison with control sample without MF, thus confirming desorption of negatively charged enzyme from the positively charged surface of MNPs. Dox loaded magnetic liposomes had average diameter of 160±8 nm and polydispersity index (PDI) 0.25±0.07. Liposomes were stable in DW and PBS at pH=7.4 at 370C during a week. After MF application (10 min of exposure, 50 Hz, 230 mT) diameter of liposomes raised to 190±10 nm and PDI was 0.38±0.05. We explain this by destroying and/or reorganization of lipid bilayer, that leads to changes in release of drug in comparison with control without MF exposure. Conclusion: A new application of low frequency AC MF for drug delivery and controlled drug release was shown. Investigation was supported by RSF-14-13-00731 grant, K1-2014-022 grant.

Keywords: magnetic nanoparticles, low frequency magnetic field, drug delivery, controlled drug release

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Authors: Ghada Ben Hamad, Zohir Younsi, Hassane Naji, Noureddine Lebaz, Naoual Belouaggadia

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This study deals with a bifunctional micro-encapsulated phase change (MCP) material, capric-stearic acid/graphene oxide-TiO2, which has been successfully developed by in situ hydrolysis and polycondensation of tetrabutyl titanate and modification of graphene oxide (GO) on the TiO2 doped shell. The use of graphene and doped TiO2 is a promising approach to provide photocatalytic activity under visible light and improve the microcapsules physicochemical properties. The morphology and chemical structure of the resulting microcapsule samples were determined by using Fourier transform infrared (FT-IR) spectroscopy, scanning electronic microscope (SEM), and X-ray diffractometer (XRD) methods. The ultraviolet, visible spectrophotometer (UV–vis), the differential scanning calorimeter (DSC) and the thermogravimetric analyzer (TGA) were used to investigate the absorption of visible and ultraviolet (UV), the thermal properties, and thermal stabilities of the microcapsules. Note that, the visible light photocatalytic activity was assessed for the toluene and benzene gaseous removal in a suitable test room. The microcapsules exhibit an interesting spherical morphology and an average diameter of 15 to 25 μm. The addition of graphene can enhance the rigidity of the shell and improve the microcapsules thermal reliability. At the same time, the thermal analysis tests showed that the synthesized microcapsules had a high solar thermal energy-storage and better thermal stability. In addition, the capric-stearic acid microcapsules exhibited high solar photocatalytic activity with respect to atmospheric pollutants under natural sunlight. The fatty acid samples obtained with the GO/TiO2 shell showed great potential for applications of solar energy storage, solar photocatalytic degradation of air pollutants and buildings energy conservation.

Keywords: thermal energy storage, microencapsulation, titanium dioxide, photocatalysis, graphene oxide

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Authors: Amina Aloui, Jalila Ben Salah-Abbès, Abdellah Zinedine, Amar Riba, Noel Durand, Catherine Brabet, Didier Montet, Samir Abbès

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Mycotoxin contamination of food and feed-isa globaconcern, both economically and for public health. Aflatoxin M1 (AFM1) is the principal hydroxylated metabolite of aflatoxin B1. It is frequently found in milk and other dairy products. It is responsible for the development of hepatocellular carcinoma and immunotoxic in humans and animals. The reduction of its bioavailabilitybecomesa great demand in order to protect human and animal health. The use of probiotic bacteria and clay are demonstrated to be able to bind AFM1 in vitro. This study aimed to investigate, in vivo, the activity of two-component mixture: L. rhamnosusGAF06 (LR) and bentonite for reducing the oxidative stress and the histological alterationsinduced by AFM1 in the liver andkidneys. For the experiment, male mice were divided into 7 groups (6 mice/group) and treated, orally, by AFM1, alone or in combination with LR and/or bentonite, for 10 days as follows: group 1 control, group 2 treated with LR alone (2.108 CFU/ml), group 3 treated with bentonite alone (1g/kg), group 4 treated with AFM1 alone (100μg/kg), group 5 co-treated with LR+AFM1, group 6 co-treated with bentonite+AFM1, group 7 co-treated with bentonite+LR+AFM1. At the end of the treatment, the mice were sacrificed, and the livers and kidneys were collected for histological assays. Intracellular antioxidant activities and lipid peroxidation were also studied. The results showed that AFM1causeddamage in liver and kidney tissues, being evidence of hepatotoxicity and nephrotoxicity marked by necrotic cells. It increased the MDA level and decreased the antioxidant enzyme activities (SOD) in both organs. In contrast, the co-treatment with AFM1 plus LR and/or bentonitesignificantly improved the hepatic and renal tissues, regulated kidney, and liver antioxidant enzyme activities. This improvement was more remarkable with the administration of LR-bentonite mixture with AFM1.LR and bentonite alone showed to be safe during the treatment. This mixture can be a promising candidate for future applications in biotechnological processes that aimed to detoxify AFM1in food and feed.

Keywords: aflatoxin M1, bentonite, L. rhamnosus GAF06, oxidative stress, prevention

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Authors: Mahdokht Sadeghvishkaei, Azizah Abdul-Hamid, Amin Ismail, Nazamid Saari

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Hypertension is a common and serious chronic health problem and known as the most important risk factor for development of many diseases such as stroke. Since angiotensin I-converting enzyme (ACE) is the key enzyme involved in blood pressure, one of the well accepted mechanisms to control hypertension is through ACE inhibition. The ACE inhibitory effect of Actinopyga lecanora (stone fish) proteolysate in vitro had been reported. Hence, this study aimed to evaluate the ACE inhibitory potential of Actinopyga lecanora proteolysate in vivo in normotensive rats. Therefore the ACE inhibitory capability of the proteolysate to prevent increasing systolic blood pressure, after inducing hypertension by angiotensin I was examined. The pre-fed rats with the proteolysates at various doses (200, 400, 800 mg/kg body weight) revealed the significant (p ≤ 0.05) suppression effect compared with control groups. Furthermore, different doses of the proteolysate (200, 400, 800 mg/kg body weight) were examined to find its optimum effective dose. Results depicted that 800 mg proteolysate/kg body weight significantly reduced systolic blood pressure without negative effect on normal blood pressure (p ≤ 0.05). Furthermore, Sub-acute toxicity study based on OECD guideline demonstrated the safety of the proteolysate in vivo. The present study indicated that the proteolysate at a dose of 1000 mg/kg daily for 14 days did not cause toxicity signs such as death, changes in activity, or piloerection. Since there are no significant differences between treated groups and control groups, hematological and biochemical analysis confirmed safety of the proteolysate (p > 0.05). In addition, there were no significant differences between organs weights of the treated groups and the control groups. Morphologically, neither histopathological changes, nor gross abnormalities were observed. However, the proteolysate caused significant decrease in body weight in relation to the control groups (p ≤ 0.05) probably due to appetite stimulation by the proteolysate, leading to decreased food consumption in sub-acute group. It is concluded that the proteolysate generated from Actinopyga lecanora possess a significant anti-hypertensive effect and would be potentially used as natural alternative of ACE inhibitors.

Keywords: ACE inhibition, Actinopyga lecanora, anti-hypertensive activity, bioactive peptides, normotensive rats

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Authors: Salman Akrokayan

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Synthetic cDNA (ScDNA) of granulocyte colony-stimulating factor (G-CSF) was constructed using a DNA synthesizer with the aim to increase its expression level. 5' end of the ScDNA of G-CSF coding region was modified by decreasing the GC content without altering the predicted amino acids sequence. The identity of the resulting protein from ScDNA was confirmed by the highly specific enzyme-linked immunosorbent assay. In conclusion, a synthetic G-CSF cDNA in combination with the recombinant DNA protocol offers a rapid and reliable strategy for synthesizing the target protein. However, the commercial utilization of this methodology requires rigorous validation and quality control.

Keywords: synthetic cDNA, recombinant G-CSF, cloning, gene expression

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Authors: Navpreet Kaur, Himkusha Thakur, Nirmal Prabhakar

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The most controversial issue in crop production is the use of Organophosphate insecticides. This is evident in many reports that Organophosphate (OP) insecticides, among the broad range of pesticides are mainly involved in acute and chronic poisoning cases. OPs detection is of crucial importance for health protection, food and environmental safety. In our study, a nanocomposite of poly (3,4 ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs) has been deposited electrochemically onto the surface of fluorine doped tin oxide sheets (FTO) for the analysis of malathion OP. The -COOH functionalization of MWCNTs has been done for the covalent binding with amino groups of AChE enzyme. The use of PEDOT-MWCNT films exhibited an excellent conductivity, enables fast transfer kinetics and provided a favourable biocompatible microenvironment for AChE, for the significant malathion OP detection. The prepared biosensors were characterized by Fourier transform infrared spectrometry (FTIR), Field emission-scanning electron microscopy (FE-SEM) and electrochemical studies. Various optimization studies were done for different parameters including pH (7.5), AChE concentration (50 mU), substrate concentration (0.3 mM) and inhibition time (10 min). Substrate kinetics has been performed and studied for the determination of Michaelis Menten constant. The detection limit for malathion OP was calculated to be 1 fM within the linear range 1 fM to 1 µM. The activity of inhibited AChE enzyme was restored to 98% of its original value by 2-pyridine aldoxime methiodide (2-PAM) (5 mM) treatment for 11 min. The oxime 2-PAM is able to remove malathion from the active site of AChE by means of trans-esterification reaction. The storage stability and reusability of the prepared biosensor is observed to be 30 days and seven times, respectively. The application of the developed biosensor has also been evaluated for spiked lettuce sample. Recoveries of malathion from the spiked lettuce sample ranged between 96-98%. The low detection limit obtained by the developed biosensor made them reliable, sensitive and a low cost process.

Keywords: PEDOT-MWCNT, malathion, organophosphates, acetylcholinesterase, biosensor, oxime (2-PAM)

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Authors: Abdul-Dahiru El-Yuguda, Saka Saheed Baba, Tawa Monilade Adisa, Mustapha Bala Abubakar

Abstract:

Background: Chikungunya (CHIK) virus, a previously anecdotally described arbovirus, is now assuming a worldwide public health burden. The CHIK virus infection is characterized by potentially life threatening and debilitating arthritis in addition to the high fever, arthralgia, myalgia, headache and rash. Method: Three hundred and seventy (370) serum samples were collected from outpatients with febrile illness attending University of Maiduguri Teaching Hospital, Nigeria, and was used to detect for Chikungunya (CHIK) virus IgG and IgM antibodies using the Enzyme Linked Immunosorbent Assays (ELISAs). Result: Out of the 370 sera tested, 39 (10.5%) were positive for presence of CHIK virus antibodies. A total of 24 (6.5%) tested positive for CHIK virus IgM only while none (0.0%) was positive for presence of CHIK virus IgG only and 15 (4.1%) of the serum samples were positive for both IgG and IgM antibodies. A significant difference (p<0.0001) was observed in the distribution of CHIK virus antibodies in relation to gender. The males had prevalence of 8.5% IgM antibodies as against 4.6% observed in females. On the other hand 4.6% of the females were positive for concurrent CHIK virus IgG and IgM antibodies when compared to a prevalence of 3.4% observed in males. Only the age groups ≤ 60 years and the undisclosed age group were positive for presence of CHIK virus IgG and/or IgM antibodies. No significant difference (p>0.05) was observed in the seasonal prevalence of CHIK virus antibodies among the study subjects Analysis of the prevalence of CHIK virus antibodies in relation to clinical presentation (as observed by Clinicians) of the patients revealed that headache and fever were the most frequently encountered ailments. Conclusion: The CHIK virus IgM and concurrent IgM and IgG antibody prevalence rates of 6.5% and 4.1% observed in this study indicates a current infection and the lack of IgG antibody alone observed shows that the infection is not endemic but sporadic. Recommendation: Further studies should be carried to establish the seasonal prevalence of CHIK virus infection vis-à-vis vector dynamics in the study area. A comprehensive study need to be carried out on the molecular characterization of the CHIK virus circulating in Nigeria with a view to developing CHIK virus vaccine.

Keywords: Chikungunya virus, IgM and IgG antibodies, febrile patients, enzyme linked immunosorbent assay

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Authors: Menna Ewida, Dalal Abou El-Ella, Dina Lasheen, Huessin El-Subbagh

Abstract:

Introduction: Vascular Endothelial Growth Factor receptor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis to create new blood vessels. VEGF family binds to three trans-membrane tyrosine kinase receptors,Dihydrofolate reductase (DHFR) is an enzyme of crucial importance in medicinal chemistry. DHFR catalyzes the reduction 7,8 dihydro-folate to tetrahydrofolate and intimately couples with thymidylate synthase which is a pivotal enzyme that catalysis the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) utilizing N5,N10-methylene tetrahydrofolate as a cofactor which functions as the source of the methyl group. Purpose: Novel substituted Thiazole agents were designed as DHFR and VEGF-TK inhibitors with increased synergistic activity and decreased side effects. Methods: Five series of compounds were designed with a rational that mimic the pharmacophoric features present in the reported active compounds that target DHFR & VEGFR. These molecules were docked against Methotrexate & Sorafenib as controls. An in silico ADMET study was also performed to validate the bioavailability of the newly designed compounds. The in silico molecular docking & ADMET study were also applied to the non-classical antifolates for comparison. The interaction energy comparable to that of MTX for DHFRI and Sorafenib for VEGF-TKI activity were recorded. Results: Compound 5 exhibited the highest interaction energy when docked against Sorafenib, While Compound 9 showed the highest interaction energy when docked against MTX with the perfect binding mode. Comparable results were also obtained for the ADMET study. Most of the compounds showed absorption within (95-99) zone which varies according to the type of substituents. Conclusions: The Substituted Thiazole Analogues could be a suitable template for antitumor drugs that possess enhanced bioavailability and act as DHFR and VEGF-TK inhibitors.

Keywords: anti-tumor agents, DHFR, drug design, molecular modeling, VEGFR-TKIs

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Authors: Francesca Lombardi, Francesca Rosaria Augello, Benedetta Cinque, Maria Grazia Cifone, Paola Palumbo

Abstract:

Glioblastoma multiforme (GBM) is a high-grade primary brain tumor refractory to current forms of treatment. The survival benefits of patients with GBM remain unsatisfactory due to the intrinsic or acquired resistance to temozolomide (TMZ), an alkylating agent, used as the first-line chemotherapeutic drug to treat GBM patients. Its cytotoxic effect is visualized by the induction of O6-methylguanine (O6MeG) within DNA. Cyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of GBM, its inhibition shows anticancer activities. In the present study, it was verified if the combination of a COX-2 selective inhibitor, NS398, with TMZ could counteract the TMZ resistance. In particular, the effect of NS398 mixed with TMZ was investigated in the GBM TMZ-resistant cell line, T98G. Cells were pretreated with NS398 (100µM, 24 hours) and then exposed to TMZ alone (200µM), NS398 alone, or both for 72 hours, after which cell growth rate and cycle phases, as well as apoptosis level, were evaluated. Coadministration of NS398 and TMZ caused a significant decrease in cell growth and a progressive increase of dead cells detected by trypan blue staining. Moreover, a significant level of apoptotic cell percentage and alteration of cell cycle phases were observed in T98G treated with TMZ-NS398 combination when compared to untreated cells or TMZ-treated cells. TMZ-resistant tumors, as GBM, express elevated levels of DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). The mixture drastically reduced MGMT expression in the TMZ-resistant cell line T98G, known to express high levels of MGMT basically. Moreover, while TMZ alone did not influence the COX-2 protein expression, the combination successfully reduced it. In conclusion, these results demonstrated that NS398 enhanced the efficacy of TMZ through cell number reduction, apoptosis induction, and decreased MGMT levels, suggesting the ability of drug combination to reduce the chemoresistance. This drug combination deserves attention and could be considered as a promising therapeutic strategy for GBM patients.

Keywords: COX-2, COX-2 inhibitor, glioblastoma, NS398, T98G, temozolomide

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Authors: Mohamed M. Ibrahim, Gaber A. M. Mersal, Salih Al-Juaid, Samir A. El-Shazly

Abstract:

A series of mixed ligand complexes, viz., [Cu(BPy)(Gly)X]Y {X = Cl (1), Y = 0; X = 0, Y = ClO4- (2); X = H2O, Y = NO3- (3); X = H2O, Y = CH3COO- (4); and [Cu(BPy)(Gly)-(H2O)]2(SO4) (5) have been synthesized. Their structures and properties were characterized by elemental analysis, thermal analaysis, IR, UV–vis, and ESR spectroscopy, as well as electrochemical measurements including cyclic voltammetry, electrical molar conductivity, and magnetic moment measurements. Complexes 1 and 2 formed slightly distorted square-pyramidal coordination geometries of CuN3OCl and CuN3O2, respectively in which the N,O-donor glycine and N,N-donor bipyridyl bind at the basal plane with chloride ion or water as the axial ligand. Complex 3 shows square planar CuN3O coordination geometry, which exhibits chemically significant hydrogen bonding interactions besides showing coordination polymer formation. The superoxide dismutase and catalase-like activities of all complexes were tested and were found to be promising candidates as durable electron-transfer catalyst being close to the efficiency of the mimicking enzymes displaying either catalase or tyrosinase activity to serve for complete reactive oxygen species (ROS) detoxification, both with respect to superoxide radicals and related peroxides. The DNA binding interaction with super coiled pGEM-T plasmid DNA was investigated by using spectral (absorption and emission) titration and electrochemical techniques. The results revealed that DNA intercalate with complexes 1 and 2 through the groove binding mode. The calculated intrinsic binding constant (Kb) of 1 and 2 were 4.71 and 2.429 × 105 M−1, respectively. Gel electrophoresis study reveals the fact that both complexes cleave super coiled pGEM-T plasmid DNA to nicked and linear forms in the absence of any additives. On the other hand, the interaction of both complexes with DNA, the quasi-reversible CuII/CuI redox couple slightly improves its reversibility with considerable decrease in current intensity. All the experimental results indicate that the bipyridyl mixed copper(II) complex (1) intercalate more effectively into the DNA base pairs.

Keywords: enzyme mimics, mixed ligand complexes, X-ray structures, antioxidant, DNA-binding, DNA cleavage

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Authors: Zakir Hossain, Arzu Pervin, Halima Jahan, Rabeya Akter, Abdel Omri

Abstract:

Replacement of fish meal with soybean meal is an effective way to relieve the pressure on fish meal as the supply of this feed ingredient is dwindling and certainly is not sustainable in long term at present levels in commercial feeds. This study was designed to determine the effect of fishmeal (FM) replacement with soybean meal (SBM) in diet on growth, digestive enzyme activity, antioxidation and gut histomorphology of tilapia (Oreochromis niloticus). Five diets were formulated where SBM0 contained 100% FM, FM substituted with graded levels of a mix of SBM to replace 25% (SBM25), 50% (SBM50), 75% (SBM75) and 100% (SBM100) of FM. Juvenile tilapia having weight and length of 6.60±0.13 g and 5.42±0.17 cm were randomly divided into five treatment groups having 40 individual each group and fed to visual satiation for 90 days. Diet with SBM was increased significant in body weight gain and specific growth rate in fish compared to the fish fed with SBM100. Fish having the similar weight (74.34±5.41 g) fed the diets SBM50, SBM75 and SBM100 containing higher level of SBM showed significantly longer intestine compared to SBM0. Villus height of stomach and intestine were significantly greater in the fish fed with the diets SBM0, SBM25 and SBM50 compared to SBM100. Muscular thickness was inversely changed with the increasing villus height. Protease activity was increased significantly in stomach, anterior and posterior intestine of fish fed with SBM0 and SBM25 compared to SBM100. In anterior and posterior segment of intestine, significantly higher lipase activity was observed in fish fed with the diets SBM0 and SBM25 compared to diet SBM100. In stomach, amylase activity was also significantly greater in SBM0 compared to SBM100. The antioxidant enzymes including catalase and superoxide dismutase of liver were significantly (P < 0.05) higher in the O. niloticus fed SBM100 compared to the ones fed SBM0. These results suggest that the replacement of FM upto 75% with SBM could be possible considering the growth performances, gut health and activities digestive enzymes and antioxidant enzymes in O. niloticus.

Keywords: soybean meal, fish meal, digestive enzymes, anti-oxidant enzymes

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Authors: Krasimira N. Zhilkova, Mariya K. Kyulavska, Roza P. Mateva

Abstract:

The anionic polymerization of -caprolactam (CL) with bifunctional activators has been extensively studied as an effective and beneficial method of improving chemical and impact resistances, elasticity and other mechanical properties of polyamide (PA6). In presence of activators or macroactivators (MAs) also called polymeric activators (PACs) the anionic polymerization of lactams proceeds rapidly at a temperature range of 130-180C, well below the melting point of PA-6 (220C) permitting thus the direct manufacturing of copolymer product together with desired modifications of polyamide properties. Copolymers of PA6 with an elastic polypropylene glycol (PPG) middle block into main chain were successfully synthesized via activated anionic ring opening polymerization (ROP) of CL. Using novel PACs based on PPG polyols (with differ molecular weight) the anionic ROP of CL was realized and investigated in the presence of a basic initiator sodium salt of CL (NaCL). The PACs were synthesized as N-carbamoyllactam derivatives of hydroxyl terminated PPG functionalized with isophorone diisocyanate [IPh, 5-Isocyanato-1-(isocyanatomethyl)-1,3,3-trimethylcyclohexane] and blocked then with CL units via an addition reaction. The block copolymers were analyzed and proved with 1H-NMR and FT-IR spectroscopy. The influence of the CL/PACs ratio in feed, the length of the PPG segments and polymerization conditions on the kinetics of anionic ROP, on average molecular weight, and on the structure of the obtained block copolymers were investigated. The structure and phase behaviour of the copolymers were explored with differential scanning calorimetry, wide-angle X-ray diffraction, thermogravimetric analysis and dynamic mechanical thermal analysis. The crystallinity dependence of PPG content incorporated into copolymers main backbone was estimate. Additionally, the mechanical properties of the obtained copolymers were studied by notched impact test. From the performed investigation in this study could be concluded that using PPG based PACs at the chosen ROP conditions leads to obtaining well-defined PA6-b-PPG-b-PA6 copolymers with improved impact resistance.

Keywords: anionic ring opening polymerization, caprolactam, polyamide copolymers, polypropylene glycol

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Authors: G. Amariei, K. Boltes, R. Rosal, P. Leton

Abstract:

Wastewater treatment plants (WWTPs) are supposed to hold an important place in the reduction of emerging contaminants, but provide an environment that has potential for the development and/or spread of adaptation, as bacteria are continuously mixed with contaminants at sub-inhibitory concentrations. Reviewing the literature, there are little data available regarding the use of adapted bacteria forming activated sludge community for toxicity assessment, and only individual validations have been performed. Therefore, the aim of this work was to study the toxicity of Triclosan (TCS) and Ibuprofen (IBU), individually and in binary combination, on adapted activated sludge (AS). For this purpose a battery of biomarkers were assessed, involving oxidative stress and cytotoxicity responses: glutation-S-transferase (GST), catalase (CAT) and viable cells with FDA. In addition, we compared the toxic effects on adapted bacteria with unadapted bacteria, from a previous research. Adapted AS comes from three continuous-flow AS laboratory systems; two systems received IBU and TCS, individually; while the other received the binary combination, for 14 days. After adaptation, each bacterial culture condition was exposure to IBU, TCS and the combination, at 12 h. The concentration of IBU and TCS ranged 0.5-4mg/L and 0.012-0.1 mg/L, respectively. Batch toxicity experiments were performed using Oxygraph system (Hansatech), for determining the activity of CAT enzyme based on the quantification of oxygen production rate. Fluorimetric technique was applied as well, using a Fluoroskan Ascent Fl (Thermo) for determining the activity of GST enzyme, using monochlorobimane-GSH as substrate, and to the estimation of viable cell of the sludge, by fluorescence staining using Fluorescein Diacetate (FDA). For IBU adapted sludge, CAT activity it was increased at low concentration of IBU, TCS and mixture. However, increasing the concentration the behavior was different: while IBU tends to stabilize the CAT activity, TCS and the mixture decreased this one. GST activity was significantly increased by TCS and mixture. For IBU, no variations it was observed. For TCS adapted sludge, no significant variations on CAT activity it was observed. GST activity it was significant decreased for all contaminants. For mixture adapted sludge the behaviour of CAT activity it was similar to IBU adapted sludge. GST activity it was decreased at all concentration of IBU. While the presence of TCS and mixture, respectively, increased the GST activity. These findings were consistent with the viability cells evaluation, which clearly showed a variation of sludge viability. Our results suggest that, compared with unadapted bacteria, the adapted bacteria conditions plays a relevant role in the toxicity behaviour towards activated sludge communities.

Keywords: adapted sludge community, mixture, PPCPs, toxicity

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Authors: Neslihan Demirci, Serdar Durdağı

Abstract:

Mycobacterium tuberculosis is still a worldwide disease-causing agent that, according to WHO, led to the death of 1.5 million people from tuberculosis (TB) in 2020. The bacteria reside in macrophages located specifically in the lung. There is a known quadruple drug therapy regimen for TB consisting of isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB). Over the past 60 years, there have been great contributions to treatment options, such as recently approved delamanid (OPC67683) and bedaquiline (TMC207/R207910), targeting mycolic acid and ATP synthesis, respectively. Also, there are natural compounds that can block the tryptophanyl-tRNA synthetase (TrpRS) enzyme, chuangxinmycin, and indolmycin. Yet, already the drug resistance is reported for those agents. In this study, the newly released TrpRS enzyme structure is investigated for potential inhibitor drugs from already synthesized molecules to help the treatment of resistant cases and to propose an alternative drug for the quadruple drug therapy of tuberculosis. Maestro, Schrodinger is used for docking and molecular dynamic simulations. In-house library containing ~8000 compounds among FDA-approved indole-containing compounds, a total of 57 obtained from the ChemBL were used for both ATP and tryptophan binding pocket docking. Best of indole-containing 57 compounds were subjected to hit expansion and compared later with virtual screening workflow (VSW) results. After docking, VSW was done. Glide-XP docking algorithm was chosen. When compared, VSW alone performed better than the hit expansion module. Best scored compounds were kept for ten ns molecular dynamic simulations by Desmond. Further, 100 ns molecular dynamic simulation was performed for elected molecules according to Z-score. The top three MMGBSA-scored compounds were subjected to steered molecular dynamic (SMD) simulations by Gromacs. While SMD simulations are still being conducted, ponesimod (for multiple sclerosis), vilanterol (β₂ adrenoreceptor agonist), and silodosin (for benign prostatic hyperplasia) were found to have a significant affinity for tuberculosis TrpRS, which is the propulsive force for the urge to expand the research with in vitro studies. Interestingly, top-scored ponesimod has been reported to have a side effect that makes the patient prone to upper respiratory tract infections.

Keywords: drug repurposing, molecular dynamics, tryptophanyl-tRNA synthetase, tuberculosis

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Authors: Aruma Baduge Kithma De Silva

Abstract:

Haskap (Lonicera caerulea L.), also known as blue honeysuckle, is a newly commercialized berry crop in Canada. Haskap berries are rich in polyphenols, including, anthocyanins, which are known for potential health-promoting properties. Cyanidin-3-O-glucoside (C3G) is the most abundant anthocyanin of haskap berries. The compound C3G has the ability to reduce the risk of type 2 diabetes (T2D), which has become an increasingly common health issue around the world. The T2D is characterized as a metabolic disorder of hyperglycemia and insulin resistance. It has been demonstrated that C3G has anti-diabetic effects through several ways, including inhibition of dipeptidyl peptidase-4 (DPP-4), reduction of gluconeogenesis, improvement in insulin sensitivity, and inhibition of activities of carbohydrate hydrolyzing enzymes, including α-amylase and α-glucosidase. The goal of this study was to investigate the influence of variety and harvests maturity of haskap on C3G, other fruit quality characteristics and anti-diabetic activities of haskap berries using in vitro studies. The polyphenols present in four commercially grown haskap cultivars, Aurora, Rebecca, Larissa, and Evie harvested at five harvesting dates (H1-H5) apart from 2-3 days, were extracted separately. High-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) analyzes of polyphenols revealed that haskap berries contain predominantly anthocyanins, flavonols, flavan-3-ols, and phenolic acids. The compound C3G was the most prominent anthocyanin, which is available in approximately 79% of total anthocyanin in four cultivars. The Larissa at H5 contained the highest C3G content. The antioxidant capacity of Evie at H5 was greater than other cultivars. Furthermore, Larissa H5 showed the greatest inhibition of carbohydrate hydrolyzing enzymes including alpha-glucosidase and alpha-amylase. In conclusion, the haskap variety and harvesting date influenced the polyphenol composition and biological properties. The variety Larissa, at H5 harvesting date, contained the highest polyphenol content and the ability of inhibition of the carbohydrate hydrolyzing enzyme as well as DPP4 enzyme in order to reduce type 2 diabetes.

Keywords: anthocyanin, Haskap, type 2 diabetes, polyphenol

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Authors: Shameel Pervez, Saad Aziz Durrani, Ibatsam Khokhar

Abstract:

Pectinase is an enzyme that breaks down pectin, a compound responsible for structural integrity of the plant. Pectin is difficult to break down mechanically and the cost is very high, that is why many industries including food industries use pectinase enzyme produced by microbes for pectin breakdown. Apple (Malus domestica) is an important fruit in terms of market value. Every year, millions of apples are wasted due to post-harvest rot caused by fungi. Fungi are natural decomposers of our ecosystem and are infamous for post-harvest rot of apple fruit but at the same time they are prized for their high production of valuable extracellular enzymes such as pectinase. In this study, fungi belonging to different genus were isolated from rotten apples. Rotten samples of apple were picked from different markets of Lahore. After surface sterilization, the rotten parts were cut into small pieces and placed onto MEA media plates for three days. Afterwards, distinct colonies were picked and purified by sub-culturing. The isolates were identified to genus level through the study of basic colony morphology and microscopic features. The isolates were then subjected to screening for pectinase activity on MS media to compare pectinase production and were then subsequently tested for pathogenic activity through wound suspension method to evaluate the pathogenic activity of isolates in comparison with their pectinolytic activity. A total of twelve fungal strains were isolates from rotten apples. They were belonging to genus Penicillium, Alternaria, Paecilomyces and Rhizopus. Upon screening for pectinolytic activity, isolates Pen 1, Pen 4, and Rz showed high pectinolytic activity and were further subjected to DNA isolation and partial sequencing for species identification. The results of partial sequencing were combined with in-depth study of morphological features revealing Pen 1 as Penicillium janthinellum, Pen 4 as Penicillium griseofulvum, and Rz as Rhizopus microsporus. Pathogenic activity of all twelve isolates was evaluated. Penicillium spp. were highly pathogenic and destructive and same was the case with Paecilomyces sp. and Rhizopus sp. However, Alternaria spp. were found to be more consistent in their pathogenic activity, on all types of apples.

Keywords: apple, pectinase, fungal pathogens, penicillium, rhizopus

Procedia PDF Downloads 63

Authors: Navpreet Kaur, Himkusha Thakur, Nirmal Prabhakar

Abstract:

One of the most publicized and controversial issue in crop production is the use of agrichemicals- also known as pesticides. This is evident in many reports that Organophosphate (OP) insecticides, among the broad range of pesticides are mainly involved in acute and chronic poisoning cases. Therefore, detection of OPs is very necessary for health protection, food and environmental safety. In our study, a nanocomposite of poly (3,4 ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs) has been deposited electrochemically onto the surface of fluorine doped tin oxide sheets (FTO) for the analysis of malathion OP. The -COOH functionalization of MWCNTs has been done for the covalent binding with amino groups of AChE enzyme. The use of PEDOT-MWCNT films exhibited an excellent conductivity, enables fast transfer kinetics and provided a favourable biocompatible microenvironment for AChE, for the significant malathion OP detection. The prepared PEDOT-MWCNT/FTO and AChE/PEDOT-MWCNT/FTO nano-biosensors were characterized by Fourier transform infrared spectrometry (FTIR), Field emission-scanning electron microscopy (FE-SEM) and electrochemical studies. Electrochemical studies were done using Cyclic Voltammetry (CV) or Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS). Various optimization studies were done for different parameters including pH (7.5), AChE concentration (50 mU), substrate concentration (0.3 mM) and inhibition time (10 min). The detection limit for malathion OP was calculated to be 1 fM within the linear range 1 fM to 1 µM. The activity of inhibited AChE enzyme was restored to 98% of its original value by 2-pyridine aldoxime methiodide (2-PAM) (5 mM) treatment for 11 min. The oxime 2-PAM is able to remove malathion from the active site of AChE by means of trans-esterification reaction. The storage stability and reusability of the prepared nano-biosensor is observed to be 30 days and seven times, respectively. The application of the developed nano-biosensor has also been evaluated for spiked lettuce sample. Recoveries of malathion from the spiked lettuce sample ranged between 96-98%. The low detection limit obtained by the developed nano-biosensor made them reliable, sensitive and a low cost process.

Keywords: PEDOT-MWCNT, malathion, organophosphates, acetylcholinesterase, nano-biosensor, oxime (2-PAM)

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Authors: Anteneh Wodaje Bayeh, Daniel Manaye Kabtamu, Chen-Hao Wang

Abstract:

As one of the most promising electrochemical energy storage systems, vanadium redox flow batteries (VRFBs) have received increasing attention owing to their attractive features for largescale storage applications. However, their high production cost and relatively low energy efficiency still limit their feasibility. For practical implementation, it is of great interest to improve their efficiency and reduce their cost. One of the key components of VRFBs that can greatly influence the efficiency and final cost is the electrode, which provide the reactions sites for redox couples (VO²⁺/VO₂ + and V²⁺/V³⁺). Carbon-based materials are considered to be the most feasible electrode materials in the VRFB because of their excellent potential in terms of operation range, good permeability, large surface area, and reasonable cost. However, owing to limited electrochemical activity and reversibility and poor wettability due to its hydrophobic properties, the performance of the cell employing carbon-based electrodes remained limited. To address the challenges, we synthesized heteroatom-doped bimetallic oxide grown on the surface of carbon through the one-step approach. When applied to VRFBs, the prepared electrode exhibits significant electrocatalytic effect toward the VO²⁺/VO₂ + and V³⁺/V²⁺ redox reaction compared with that of pristine carbon. It is found that the presence of heteroatom on metal oxide promotes the absorption of vanadium ions. The controlled morphology of bimetallic metal oxide also exposes more active sites for the redox reaction of vanadium ions. Hence, the prepared electrode displays the best electrochemical performance with energy and voltage efficiencies of 74.8% and 78.9%, respectively, which is much higher than those of 59.8% and 63.2% obtained from the pristine carbon at high current density. Moreover, the electrode exhibit durability and stability in an acidic electrolyte during long-term operation for 1000 cycles at the higher current density.

Keywords: VRFB, VO²⁺/VO₂ + and V³⁺/V²⁺ redox couples, graphite felt, heteroatom-doping

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Authors: Ozlem Oral, Emre Taskin, Aysel Yuce, Serap Dokmeci, Devrim Gozuacik

Abstract:

Autophagy is an evolutionary conserved lysosome-dependent catabolic pathway, responsible for the degradation of long-lived proteins, abnormal aggregates and damaged organelles which cannot be degraded by the ubiquitin-proteasome system. Lysosomes degrade the substrates through the activity of lysosomal hydrolases and lysosomal membrane-bound proteins. Mutations in the coding region of these proteins cause malfunctional lysosomes, which contributes to the pathogenesis of lysosomal storage diseases. Gaucher disease is a lysosomal storage disease resulting from the mutation of a lysosomal membrane-associated glycoprotein called glucocerebrosidase and its cofactor saposin C. The disease leads to intracellular accumulation of glucosylceramide and other glycolipids. Because of the essential role of lysosomes in autophagic degradation, Gaucher disease may directly be linked to this pathway. In this study, we investigated the expression of autophagy and/or lysosome-related genes and proteins in fibroblast cells isolated from patients with different mutations. We carried out confocal microscopy analysis and examined autophagic flux by utilizing the differential pH sensitivities of RFP and GFP in mRFP-GFP-LC3 probe. We also evaluated lysosomal pH by active lysosome staining and lysosomal enzyme activity. Beside lysosomes, we also performed proteasomal activity and cell death analysis in patient samples. Our data showed significant attenuation in the expression of key autophagy-related genes and accumulation of their proteins in mutant cells. We found decreased the ability of autophagosomes to fuse with lysosomes, associated with elevated lysosomal pH and reduced lysosomal enzyme activity. Proteasomal degradation and cell death analysis showed reduced proteolytic activity of the proteasome, which consequently leads to increased susceptibility to cell death. Our data indicate that the major degradation pathways are affected by multifunctional lysosomes in mutant patient cells and may underlie in the mechanism of clinical severity of Gaucher patients. (This project is supported by TUBITAK-3501-National Young Researchers Career Development Program, Project No: 112T130).

Keywords: autophagy, Gaucher's disease, glucocerebrosidase, mutant fibroblasts

Procedia PDF Downloads 324