Search results for: enzyme inhibitors

Authors: Neha Sharma, Kanthi K. Kondepudi, Naveen Gupta

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Phytases are phytate specific phosphatases catalyzing the step-wise dephosphorylation of phytate, which acts as an anti-nutritional factor in food due to its strong binding capacity to minerals. In recent years microbial phytases have been explored for improving nutritional quality of food. But the major limitation is acceptability of phytases from these microorganisms. Therefore, efforts are being made to isolate organisms which are generally regarded as safe for human consumption such as Lactic Acid Bacteria (LAB). Phytases from these organisms will have an edge over other phytase sources due to its probiotic attributes. Only few LAB have been reported to give phytase activity that too is generally seen as intracellular. LAB producing extracellular phytase will be more useful as it can degrade phytate more effectively. Moreover, enzyme from such isolate will have application in food processing also. Only few species of Lactobacillus producing extracellular phytase have been reported so far. This study reports the isolation of a probiotic strain of Lactobacillus fermentum spp KA1 which produces extracellular phytase. Conditions for the optimal production of phytase have been optimized and the enzyme production resulted in an approximately 13-fold increase in yield. The phytate degradation potential of extracellular phytase in rice bran has been explored and conditions for optimal degradation were optimized. Under optimal conditions, there was 43.26% release of inorganic phosphate and 6.45% decrease of phytate content.

Keywords: Lactobacillus, phytase, phytate reduction, rice bran

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Authors: Hany M. Abd El-Lateef

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Two novel Schiff bases, 5-bromo-2-[(E)-(pyridin-3-ylimino) methyl] phenol (HBSAP) and 5-bromo-2-[(E)-(quinolin-8-ylimino) methyl] phenol (HBSAQ) have been synthesized. They have been characterized by elemental analysis and spectroscopic techniques (UV–Vis, IR and NMR). Moreover, the molecular structure of HBSAP and HBSAQ compounds are determined by single crystal X-ray diffraction technique. The inhibition activity of HBSAP and HBSAQ for carbon steel in 3.5 %NaCl+0.1 M HCl for both short and long immersion time, at different temperatures (20-50 ºC), was investigated using electrochemistry and surface characterization. The potentiodynamic polarization shows that the inhibitors molecule is more adsorbed on the cathodic sites. Its efficiency increases with increasing inhibitor concentrations (92.8 % at the optimal concentration of 10-3 M for HBSAQ). Adsorption of the inhibitors on the carbon steel surface was found to obey Langmuir’s adsorption isotherm with physical/chemical nature of the adsorption, as it is shown also by scanning electron microscopy. Further, the electronic structural calculations using quantum chemical methods were found to be in a good agreement with the results of the experimental studies.

Keywords: carbon steel, Schiff bases, corrosion inhibition, SEM, electrochemical techniques

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Authors: Aussara Panya, Nunghathai Sawasdee, Mutita Junking, Chatchawan Srisawat, Kiattawee Choowongkomon, Pa-Thai Yenchitsomanus

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Dengue virus (DENV) infection is a global public health problem with approximately 100 million infected cases a year. Presently, there is no approved vaccine or effective drug available; therefore, the development of anti-DENV drug is urgently needed. The clinical reports revealing the positive association between the disease severity and viral titer has been reported previously suggesting that the anti-DENV drug therapy can possibly ameliorate the disease severity. Although several anti-DENV agents showed inhibitory activities against DENV infection, to date none of them accomplishes clinical use in the patients. The surface envelope (E) protein of DENV is critical for the viral entry step, which includes attachment and membrane fusion; thus, the blocking of envelope protein is an attractive strategy for anti-DENV drug development. To search the safe anti-DENV agent, this study aimed to search for novel peptide inhibitors to counter DENV infection through the targeting of E protein using a structure-based in silico design. Two selected strategies has been used including to identify the peptide inhibitor which interfere the membrane fusion process whereby the hydrophobic pocket on the E protein was the target, the destabilization of virion structure organization through the disruption of the interaction between the envelope and membrane proteins, respectively. The molecular docking technique has been used in the first strategy to search for the peptide inhibitors that specifically bind to the hydrophobic pocket. The second strategy, the peptide inhibitor has been designed to mimic the ectodomain portion of membrane protein to disrupt the protein-protein interaction. The designed peptides were tested for the effects on cell viability to measure the toxic to peptide to the cells and their inhibitory assay to inhibit the DENV infection in Vero cells. Furthermore, their antiviral effects on viral replication, intracellular protein level and viral production have been observed by using the qPCR, cell-based flavivirus immunodetection and immunofluorescence assay. None of tested peptides showed the significant effect on cell viability. The small peptide inhibitors achieved from molecular docking, Glu-Phe (EF), effectively inhibited DENV infection in cell culture system. Its most potential effect was observed for DENV2 with a half maximal inhibition concentration (IC50) of 96 μM, but it partially inhibited other serotypes. Treatment of EF at 200 µM on infected cells also significantly reduced the viral genome and protein to 83.47% and 84.15%, respectively, corresponding to the reduction of infected cell numbers. An additional approach was carried out by using peptide mimicking membrane (M) protein, namely MLH40. Treatment of MLH40 caused the reduction of foci formation in four individual DENV serotype (DENV1-4) with IC50 of 24-31 μM. Further characterization suggested that the MLH40 specifically blocked viral attachment to host membrane, and treatment with 100 μM could diminish 80% of viral attachment. In summary, targeting the hydrophobic pocket and M-binding site on the E protein by using the peptide inhibitors could inhibit DENV infection. The results provide proof of-concept for the development of antiviral therapeutic peptide inhibitors to counter DENV infection through the use of a structure-based design targeting conserved viral protein.

Keywords: dengue virus, dengue virus infection, drug design, peptide inhibitor

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Authors: F. Fushai, M.Tekere, M. Masafu, F. Siebrits, A. Kanengoni, F. Nherera

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The effects of dietary fibre source on the histomorphology of the ileal epithelium were examined in growing pigs fed high fibre (242-250 g total dietary fibre kg-1 dry matter) diets fortified with Roxazyme® G2. The control was a standard, low fibre (141 g total dietary fibre kg-1 dry matter) diet formulated from dehulled soybean (Glycine max), maize (Zea Mays) meal and hominy chop. Five fibrous diets were evaluated in which fibre was increased by partial substitution of the grains in the control diet with maize cobs, soybean hulls, barley (Hordeum vulgare L) brewer’s grains, Lucerne (Medicago sativa) hay or wheat (Triticum aestivum) bran. Each diet was duplicated and 220 mg Roxazyme® G2 kg-1 dry mater was added to one of the mixtures. Seventy-two intact Large White X Landrace male pigs of weight 32 ± 5.6 kg pigs were randomly allocated to the diets in a complete randomised design with a 2 (fibre source) X (enzyme) factorial arrangement of treatments. The pigs were fed ad libitum for 10 weeks. Ileal tissue samples were taken at slaughter, at a point 50cm above the ileal-caecal valve. Villi length and area, and crypt depth were measured by computerised image analyses. The villi length: crypt ratio was calculated. The diet and the supplemental enzyme cocktail did not affect (p>0.05) any of the measured parameters. Significant (p=0.016) diet X enzyme interaction was observed for villi length whereby the enzyme reduced the villi length of pigs on the soy-hulls, standard and wheat bran diets, with an opposite effect on pigs on the maize cob, brewer’s grain, Lucerne diets. The results suggested fibre-source dependent changes in the morphology of the ileal epithelium of pigs fed high fibre, maize-soybean diets fortified with Roxazyme® G2.

Keywords: fibre, growing pigs, histomorphology, ileum, Roxazyme® G2

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Authors: Akanksha Bhatnagar, Sandhya Kortegare, Felice Elefant

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Context: Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by progressive cognitive decline and memory loss. The cause of AD is not fully understood, but it is thought to be caused by a combination of genetic, environmental, and lifestyle factors. One of the hallmarks of AD is the loss of neurons in the hippocampus, a brain region that is important for memory and learning. This loss of neurons is thought to be caused by a decrease in histone acetylation, which is a process that regulates gene expression. Research Aim: The research aim of the study was to develop mall molecule compounds that can enhance the activity of Tip60, a histone acetyltransferase that is important for memory and learning. Methodology/Analysis: The researchers used in silico structural modeling and a pharmacophore-based virtual screening approach to design and synthesize small molecule compounds strongly predicted to target and enhance Tip60’s HAT activity. The compounds were then tested in vitro and in vivo to assess their ability to enhance Tip60 activity and rescue cognitive deficits in AD models. Findings: The researchers found that several of the compounds were able to enhance Tip60 activity and rescue cognitive deficits in AD models. The compounds were also developed to cross the blood-brain barrier, which is an important factor for the development of potential AD therapeutics. Theoretical Importance: The findings of this study suggest that Tip60 HAT activators have the potential to be developed as therapeutic agents for AD. The compounds are specific to Tip60, which suggests that they may have fewer side effects than other HDAC inhibitors. Additionally, the compounds are able to cross the blood-brain barrier, which is a major hurdle for the development of AD therapeutics. Data Collection: The study collected data from a variety of sources, including in vitro assays and animal models. The in vitro assays assessed the ability of compounds to enhance Tip60 activity using histone acetyltransferase (HAT) enzyme assays and chromatin immunoprecipitation assays. Animal models were used to assess the ability of the compounds to rescue cognitive deficits in AD models using a variety of behavioral tests, including locomotor ability, sensory learning, and recognition tasks. The human clinical trials will be used to assess the safety and efficacy of the compounds in humans. Questions: The question addressed by this study was whether Tip60 HAT activators could be developed as therapeutic agents for AD. Conclusions: The findings of this study suggest that Tip60 HAT activators have the potential to be developed as therapeutic agents for AD. The compounds are specific to Tip60, which suggests that they may have fewer side effects than other HDAC inhibitors. Additionally, the compounds are able to cross the blood-brain barrier, which is a major hurdle for the development of AD therapeutics. Further research is needed to confirm the safety and efficacy of these compounds in humans.

Keywords: Alzheimer's disease, cognition, neuroepigenetics, drug discovery

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Authors: Yezli-Touiker Samira, Amrani-Kirane Leila, Soltani Mazouni Nadia

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Due to secondary effects of conventional insecticides on the environment, the agrochemical research has resulted in the discovery of novel molecules. That research work will help in the development of a new group of pesticides that may be cheaper and less hazardous to the environment and non-target organisms which is the main desired outcome of the present work. Angiotensin-converting enzyme as a target for the development of novel insect growth regulators. Captopril is an inhibitor of angiotensin converting enzyme (ACE) it was tested in vivo by topical application on reproduction of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). The compound is diluted in acetone and applied topically to newly emerged pupae (10µg/ 2µl). The effects of this molecule was studied,on the biochemistry of ovary (on amounts nucleic acid, proteins, the qualitative analysis of the ovarian proteins and the reproductive potential (duration of the pre-oviposition, duration of the oviposition, number of eggs laid and hatching percentage). Captopril reduces significantly quantity of ovarian proteins and nucleic acid. The electrophoresis profile reveals the absence of tree bands at the treated series. This molecule reduced the duration of the oviposition period, the fecundity and the eggviability.

Keywords: environment, ephestia kuehniella, captopril, reproduction, the agrochemical research

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Authors: Ibrahim M. Labouta, Marwa H. El-Wakil, Hayam M. Ashour, Ahmed M. Hassan, Manal N. Saudi

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The receptor tyrosine kinase c-Met is an attractive target for therapeutic treatment of cancers nowadays. Among the wide variety of heterocycles that have been explored for developing c-Met kinase inhibitors, the 1,2,4-triazines have been rarely investigated, although they are well known in the literature to possess antitumor activities. Herein we describe the design and synthesis of a novel series of 1,2,4-triazine derivatives possessing N-acylarylhydrazone moiety and another series combining the 1,2,4-triazine scaffold to the well-known anticancer drug 6-MP in order to explore their “double-drug” effect. The synthesized compounds were evaluated for their in vitro antitumor activity against three c-Met addicted cancer cell lines (A549, HT-29 and MKN-45). Most compounds showed moderate to excellent antiproliferative activity and four compounds showed potent inhibitory activity more than the reference drug Foretinib against one or more cancer cell lines. The obtained results revealed that the potent compounds are highly selective to A549 (lung adenocarcinoma) cancer cell line. The c-Met kinase inhibitory activity of the potent derivatives is still under investigation. The present study clearly demonstrates that the 1,2,4-triazine core ring exhibits promising antitumor activity with potential c-Met kinase inhibitory activity.

Keywords: 1, 2, 4-triazine, antitumor, c-Met inhibitor, double-drug

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Authors: Munisath Khandoker, Stephan Haefele, Andy Gregory

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Extracellular enzymes play a key role in soil organic carbon (SOC) decomposition and nutrient cycling and are known indicators for soil health; however, it is not understood how these enzymes respond to different land uses and their relationships to other soil properties have not been extensively reviewed. The relationships among the activities of three soil enzymes: β-glucosaminidase (NAG), phosphomonoesterase (PHO) and β-glucosidase (GLU), were examined. The impact of soil organic amendments, soil types and land management on soil enzyme activities were reviewed, and it was hypothesized that soils with increased SOC have increased enzyme activity. Long-term experiments at Rothamsted Research Woburn and Harpenden sites in the UK were used to evaluate how different management practices affect enzyme activity involved in carbon (C) and nitrogen (N) cycling in the soil. Samples were collected from soils with different organic treatments such as straw, farmyard manure (FYM), compost additions, cover crops and permanent grass cover to assess whether SOC can be linked with increased levels of enzymatic activity and what influence, if any, enzymatic activity has on total C and N in the soil. Investigating the interactions of important enzymes with soil characteristics and SOC can help to better understand the health of soils. Studies on long-term experiments with known histories and large datasets can better help with this. SOC tends to decrease during land use changes from natural ecosystems to agricultural systems; therefore, it is imperative that agricultural lands find ways to increase and/or maintain SOC in the soil.

Keywords: biological soil health indicators, extracellular enzymes, soil health, soil, microbiology

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Authors: Azad A. Meerkhan, Alaa Hani Razak, Bayan M. S. Younis

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The efficiency of enzyme-linked immunosorbent assay (ELISA) in sheep infected with Fasciola hepatica was studied. 232 jaundiced sheep among 5208 sheep slaughter in the Duhok abattoir (regardless of the age and gender) between the period of May. 2012 to Oct. 2012 were examined by direct examination (Searching of adult flukes in the bile duct) and by Enzyme-linked immunosorbent assay (ELISA) to detect the prevalence of fascioliasis in the studied population which showed a high observed infection ratio in Sep. 2012 (12.2%) with the high (ELISA) result of infection in May. 2012 (25.36%). Significant differences were found between the two ways in all of the months with the highest difference in May. 2012 and the net deference between the both ways was 6.91%.

Keywords: fascioliasis, Fasciola hepatica, layers, liver fluk, ELISA, direct examination

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Authors: L. Kutateldze, T. Urushadze, R. Khvedelidze, N. Zakariashvili, I. Khokhashvili, T. Sadunishvili

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Microbial cellulase/xylanase have shown their potential application in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing, and agriculture. Extremophilic micromycetes and their enzymes that are resistant to critical values of temperature and pH, and retaining enzyme activity for a long time are of great industrial interest. Among strains of microscopic fungi from the collection of S. Durmishidze Institute of Biochemistry and Biotechnology, strains isolated from different ecological niches of Southern Caucasus-active producers of cellulase/xylanase have been selected by means of screening under deep cultivation conditions. Extremophilic micromycetes and their enzymes that are resistant to critical values of temperature and pH, and retaining enzyme activity for a long time are of great industrial interest. Among strains of microscopic fungi from the collection of S. Durmishidze Institute of Biochemistry and Biotechnology, strains isolated from different ecological niches of Southern Caucasus-active producers of cellulase/xylanase have been selected by means of screening under deep cultivation conditions. Representatives of the genera Aspergillus, Penicillium and Trichoderma are outstanding by relatively high activities of these enzymes. Among the producers were revealed thermophilic strains, representatives of the genus Aspergillus-Aspergillus terreus, Aspergillus versicolor, Aspergillus wentii, also strains of Sporotrichum pulverulentum and Chaetomium thermophile. As a result of optimization of cultivation media and conditions, activities of enzymes produced by the strains have been increased by 4 -189 %. Two strains, active producers of cellulase/xylanase – Penicillium canescence E2 (mesophile) and Aspergillus versicolor Z17 (thermophile) were chosen for further studies. Cellulase/xylanase enzyme preparations from two different genera of microscopic fungi Penicillium canescence E2 and Aspergillus versicolor Z 17 were obtained with activities 220 U/g /1200 U/g and 125 U/g /940 U/g, correspondingly. Main technical characteristics were as follows: the highest enzyme activities were obtained for mesophilic strain Penicillium canescence E2 at 45-500C, while almost the same enzyme activities were fixed for the thermophilic strain Aspergillus versicolor Z 17 at temperature 60-65°C, exceeding the temperature optimum of the mesophile by 150C. Optimum pH of action of the studied cellulase/xylanases from mesophileic and thermophilic strains were similar and equaled to 4.5-5.0 It has been shown that cellulase/xylanase technical preparations from selected strains of Penicillium canescence E2 and Aspergillus versicolor Z17 hydrolyzed cellulose of untreated wheat straw to reducible sugars by 46-52%, and to glucose by 22-27%. However the thermophilic enzyme preparations from the thermophilic A.versicolor strains conducted the process at 600C higher by 100C as compared to mesophlic analogue. Rate of hydrolyses of the pretreated substrate by the same enzyme preparations to reducible sugars and glucose conducted at optimum for their action 60 and 500C was 52-61% and 29-33%, correspondingly. Thus, maximum yield of glucose and reducible sugars form untreated and pretreated wheat straw was achieved at higher temperature (600C) by enzyme preparations from thermophilic strain, which gives advantage for their industrial application.

Keywords: cellulase/xylanase, cellulose hydrolysis, microscopic fungi, thermophilic strain

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Authors: Joana Antunes, Thomas Levée, Barbara Radovani, Anu Suonpää, Paulina Saloranta, Liji Sobhana, Petri Ihalainen

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Lignin is an important component in the exploitation of lignocellulosic biomass. It has been shown that within the next years, the yield of added-value lignin-based chemicals and materials will generate renewable alternatives to oil-based products (e.g. polymeric composites, resins and adhesives) and enhance the economic feasibility of biorefineries. In this paper, a novel technology for lignin valorisation (METNIN™) is presented. METNIN™ is based on the oxidative action of an alkaliphilic enzyme in aqueous alkaline conditions (pH 10-11) at mild temperature (40-50 °C) combined with a cascading membrane operation, yielding a collection of lignin fractions (from oligomeric down to mixture of tri-, di- and monomeric units) with distinct molecular weight distribution, low polydispersity and favourable physicochemical properties. The alkaline process conditions ensure the high processibility of crude lignin in an aqueous environment and the efficiency of the enzyme, yielding better compatibility of lignin towards targeted applications. The application of a selected lignin fraction produced by METNIN™ as a suitable lignopolyol to completely replace a commercial polyol in polyurethane rigid foam formulations is presented as a prototype. Liquid lignopolyols with a high lignin content were prepared by oxypropylation and their full utilization in the polyurethane rigid foam formulation was successfully demonstrated. Moreover, selected technical specifications of different foam demonstrators were determined, including closed cell count, water uptake and compression characteristics. These specifications are within industrial standards for rigid foam applications. The lignin loading in the lignopolyol was a major factor determining the properties of the foam. In addition to polyurethane foam demonstrators, other examples of lignin-based products related to resins and sizing applications will be presented.

Keywords: enzyme, lignin valorisation, polyol, polyurethane foam

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Authors: S. Yezli-Touiker, L. Kirane-Amrani, N. Soltani-Mazouni

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Ephestia kuehniella Zeller Lepidoptera, Pyralidae commonly called Mediterranean flour moth, is serious cosmopolitan pest of stored grain products, particularly flour Month. This species is also a source of allergen that causes asthma and rhinitis. Captopril is an inhibitor of angiotensin converting enzyme (ACE) it was tested in vivo by topical application on development of E. kuehniella. The compound is diluted in acetone and applied topically to newly emerged pupae (10mg/2ml). Report chitin protein of cuticule and ecdysteroid Amounts were determined in vivo. Results show that the captopril does not affect chitin protein of cuticule but traitment with captopril increase the hormonal production, the quantitative analysis reveals the presence of two peaks one at third and another at fifth day.

Keywords: Ephestia kuehniella, cuticule, hormone, captopril

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Authors: Pauline Nyssen, Ange Mouithys-Mickalad, Maryse Hoebeke

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Inflammation is a complex physiological phenomenon involving chemical and enzymatic mechanisms. Polymorphonuclear neutrophil leukocytes (PMNs) play an important role by producing reactive oxygen species (ROS) and releasing myeloperoxidase (MPO), a pro-oxidant enzyme. Released both in the phagolysosome and the extracellular medium, MPO produces during its peroxidase and halogenation cycles oxidant species, including hypochlorous acid, involved in the destruction of pathogen agents, like bacteria or viruses. Inflammatory pathologies, like rheumatoid arthritis, atherosclerosis induce an excessive stimulation of the PMNs and, therefore, an uncontrolled release of ROS and MPO in the extracellular medium, causing severe damages to the surrounding tissues and biomolecules such as proteins, lipids, and DNA. The treatment of chronic inflammatory pathologies remains a challenge. For many years, MPO has been used as a target for the development of effective treatments. Numerous studies have been focused on the design of new drugs presenting more efficient MPO inhibitory properties. However, some designed inhibitors can be toxic. An alternative consists of assessing the potential inhibitory action of clinically-known molecules, having antioxidant activity. Propofol, 2,6-diisopropyl phenol, which is used as an intravenous anesthetic agent, meets these requirements. Besides its anesthetic action employed to induce a sedative state during surgery or in intensive care units, propofol and its injectable form Diprivan indeed present antioxidant properties and act as ROS and free radical scavengers. A study has also evidenced the ability of propofol to inhibit the formation of the neutrophil extracellular traps fibers, which are important to trap pathogen microorganisms during the inflammation process. The aim of this study was to investigate the potential inhibitory action mechanism of propofol and Diprivan on MPO activity. To go into the anti-inflammatory action of propofol in-depth, two of its oxidative derivatives, 2,6-diisopropyl-1,4-p-benzoquinone (PPFQ) and 3,5,3’,5’-tetra isopropyl-(4,4’)-diphenoquinone (PPFDQ), were studied regarding their inhibitory action. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling have evidenced the low anti-catalytic action of propofol. Stopped-flow absorption spectroscopy and direct MPO activity analysis have proved that propofol acts as a reversible MPO inhibitor by interacting as a reductive substrate in the peroxidase cycle and promoting the accumulation of redox compound II. Overall, Diprivan exhibited a weaker inhibitory action than the active molecule propofol. In contrast, PPFQ seemed to bind and obstruct the enzyme active site, preventing the trigger of the MPO oxidant cycles. PPFQ induced a better chlorination cycle inhibition at basic and neutral pH in comparison to propofol. PPFDQ did not show any MPO inhibition activity. The three interest molecules have also demonstrated their inhibition ability on an important step of the inflammation pathway, the PMNs superoxide anions production, thanks to EPR spectroscopy and chemiluminescence. In conclusion, propofol presents an interesting immunomodulatory activity by acting as a reductive substrate in the peroxidase cycle of MPO, slowing down its activity, whereas PPFQ acts more as an anti-catalytic substrate. Although PPFDQ has no impact on MPO, it can act on the inflammation process by inhibiting the superoxide anions production by PMNs.

Keywords: Diprivan, inhibitor, myeloperoxidase, propofol, spectroscopy

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Authors: Elena G. Salina, Vadim A. Makarov

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With the emergence of primary resistance to the current drugs and wide distribution of latent tuberculosis infection, a need for new compounds with a novel mode of action is growing steadily. Copper-mediated innate immunity and antibacterial toxicity propose novel strategies in TB drug discovery and development. Transcriptome of M. tuberculosis was obtained by RNA-seq, intracellular copper content was measured by ISP MS and complexes of 1-hydroxy-2-thiopyridines with copper were detected by HPLC.1-hydroxy-2-thiopyridine derivatives were found to be highly active in vitro against both actively growing and dormant non-culturable M. tuberculosis. Transcriptome response to 1-hydroxy-2-thiopyridines revealed signs of copper toxicity in M. tuberculosis bacilli. Indeed, Cu was found to accumulate inside cells treated with 1-hydroxy-2-thiopyridines. These compounds were found to form stable charged lipophylic complexes with Cu²⁺ ions which transport into mycobacterial cell. Subsequent metabolic destruction of the complex led to transformation of 1-hydroxy-2-thiopyridines into 2-methylmercapto-2-ethoxycarbonylpyridines, which did not possess antitubercular activity and releasing of free Cu²⁺ in the cytoplasm. 1-hydroxy-2-thiopyridines are a potent class of Cu-dependent inhibitors of M. tuberculosis which may control M. tuberculosis infection by impairment of copper homeostasis. Acknowledgment: This work was financially supported by the Ministry of Education and Science of the RussianFederation (Agreement No 14.616.21.0065; unique identifier RFMEFI61616X0065).

Keywords: copper toxicity, drug discovery, M. tuberculosis inhibitors, 2-thiopyridines

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Authors: Kaniz Roksana, Aluthgun Hewage Shaini, Cheng Zhu

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Lead is environmentally hazardous because it may persist for a long time in soil, water, and air, and it can travel large distances when carried by wind or water. Lead is toxic to many different species of organisms and has the potential to disrupt ecosystem stability. Moreover, lead can contaminate crops and livestock, which can then have an adverse effect on human health. This study was conducted to use the enzyme-induced calcium carbonate precipitation (EICP) technique from soybean crude extract urease along coconut fiber derived biochar’s (CFB) to bioremediate lead. To study the desorption rates of heavy metals from the soil, lead (Pb) was added to the soil at load ratios of 50 and 100 mg/kg. There were five separate treatment soil columns created: control sample, only CFB, only EICP, EICP with 2% (w/w) CFB, and EICP with 4% (w/w) CFB. Laboratory scale experiment demonstrates significant lead removal from soil. The amount of CaCO₃ precipitated in the soil was measured using a gravimetric acid digestion test, which related heavy metal desorption to the amount of precipitated calcium carbonate. These findings were validated using a scanning electron microscope (SEM), which revealed calcium carbonate and lead coprecipitation. As a result, the study reveals that the EICP technique, in conjunction with coconut fiber biochar, could be an efficient alternative in the remediation of heavy metal ion-contaminated soils.

Keywords: enzyme induced calcium carbonate precipitation (EICP), coconut fiber derived biochar’s (CFB), bioremediation, heavy metal

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Authors: Hakim Chayeb

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Macrophages (Mo) play an essential role in host defense against pathogens. These inflammatory cells contain a large group of inducible enzymes such as NO synthase (NOS). This study was conducted to characterize experimentally induced inflammation in vivo by lipopolysaccharides (LPS). LPS is an essential component of the outer membrane of Gram-negative bacteria and a potent inducer of macrophage. Except control rats, all rats received different doses of LPS intra-peritoneally. The involvement of inducible NO synthase (iNOS) and constitutive (cNOS ) in the modulation of the inflammatory response was studied by treating the rats with L-NAME (non-selective NOS inhibitor) or aminoguanidine (AG inhibitor of iNOS). Inhibitors were injected 24 hours before LPS administration. The results showed that esterase activity (a marker of macrophage infiltration) which is induced by LPS is reduced by AG, was potentiated by treatment with L-NAME in tissue homogenates of the liver, kidney and spleen. Meanwhile, the concentrations of nitric oxide (NO) induced by LPS were reduced with AG and are completely inhibited with L-NAME in the tissues studied. NO concentrations and plasma transaminase levels have undergone remarkable increases in rats treated with LPS alone. However, the AG significantly reduced these rates. Our results highlighted the role of NO synthase inhibitors in reducing of inflammatory responses that characterize many infectious diseases.

Keywords: aminoguanidine, esterase, LPS, L-NAME, macrophage, nitric oxide

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Authors: Shafaqat Ali, Afzal Hussain, Muhammad Rizwan, Longhua Wu

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Chromium (Cr) is one of the widespread and toxic trace elements present in the agricultural land. Chromium can enter into the food chain mainly through agricultural crops grown on Cr-contaminated soils such as rice (Oryza sativa L.). The current study was done to evaluate the effects of increasing concentrations foliar applied zinc (Zn) chelated with lysine (Zn-lys) (0, 10, 20, and 30 mg L⁻¹) on rice biomass, photosynthesis, oxidative stress, key antioxidant enzyme activities and Cr uptake under increasing levels of Cr in the soil (0, 100, 500 mg kg⁻¹). Cr-induced toxicity reduced the height of plants, biomass, chlorophyll contents, gas exchange parameters, and antioxidant enzyme activities while increased the Cr concentrations and oxidative stress (malondialdehyde, electrolyte leakage, and H₂O₂) in shoots and roots than control plants. Foliar application of Zn-lys increased the plant growth, photosynthesis, Zn concentrations, and enzyme activities in rice seedlings. In addition, Zn-lys reduced the Cr concentrations and oxidative stress compared to the respective Cr treatments alone. The present results indicate that foliar Zn-lys stimulates the antioxidant defense system in rice, increase the rice growth while reduced the Cr concentrations in plants by promoting the Zn uptake and photosynthesis. Taken together, foliar spray of Zn-lys chelate can efficiently be employed for improving plant growth and Zn contents while reducing Cr concentration in rice grown in Cr-contaminated and Zn-deficient soils.

Keywords: antioxidants, chromium, zinc-lysine, oxidative stress, photosynthesis, tolerance

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Authors: Serap Pektas

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Ataxia telangiectasia mutated (ATM) is a serine-threonine kinase, which is the major regulator of the DNA damage response. ATM is activated upon the formation of DNA double-strand breaks (DSBs) in the cells. ATM phosphorylates the proteins involved in apoptotic responses, cell cycle checkpoint control, DNA repair, etc. Tumor protein p53, known as p53 is one of these proteins that phosphorylated by ATM. Phosphorylation of p53 at Ser15 residue leads to p53 stabilization in the cells. Often enzymes activity is affected by hydrogen ion concentration (pH). In order to find the optimal pH range for ATM activity, steady-state kinetic assays were performed at acidic and basic pH ranges. Ser15 phosphorylation of p53 is determined by using ELISA. The results indicated that the phosphorylation rate was better at basic pH range compared with the acidic pH range. This could be due to enzyme stability, or enzyme-substrate interaction is pH dependent.

Keywords: ataxia telangiectasia mutated, DNA double strand breaks, DNA repair, tumor protein p53

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Authors: Mark P. Mayala, Henry Mayala, Khuzeima Khanbhai

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Background: Heart failure has been a rising concern in Tanzania. New drugs have been introduced, including the group of drugs called Angiotensin receptor Neprilysin Inhibitor (ARNI), but due to their high cost, angiotensin-converting enzymes inhibitors (ACEIs) and Angiotensin receptor blockers (ARBs) have been mostly used in Tanzania. However, according to our knowledge, the efficacy comparison of the two groups is yet to be studied in Tanzania. The aim of this study was to compare the efficacy of ACEIs and ARBs among patients with heart failure. Methodology: This was a hospital-based prospective cohort study done at Jakaya Kikwete Cardiac Institution (JKCI), Tanzania, from June to December 2020. Consecutive enrollment was done until fulfilling the inclusion criteria. Clinical details were measured at baseline. We assessed the relationship between ARBs and ACEIs users with N-terminal pro-brain natriuretic peptide (NT pro-BNP) levels at admission and at 1-month follow-up using a chi-square test. A Kaplan-Meier curve was used to estimate the survival time of the two groups. Results: 155 HF patients were enrolled, with a mean age of 48 years, whereby 52.3% were male, and their mean left ventricular ejection fraction (LVEF) was 37.3%. 52 (33.5%) heart failure patients were on ACEIs, 57 (36.8%) on ARBs, and 46 (29.7%) were neither using ACEIs nor ARBs. At least half of the patients did not receive a guideline-directed medical therapy (GDMT), with only 82 (52.9%) receiving a GDMT. A drop in NT pro-BNP levels was observed during admission and at 1-month follow-up on both groups, from 6389.2 pg/ml to 4000.1 pg/ml for ARB users and 5877.7 pg/ml to 1328.2 pg/ml for the ACEIs users. There was no statistical difference between the two groups when estimated by the Kaplan-Meier curve, though more deaths were observed in those who were neither on ACEIs nor ARBs, with a calculated P value of 0.01. Conclusion: This study demonstrates that ACEIs have more efficacy and overall better clinical outcome than ARBs, but this should be taken under the patient-based case, considering the side effects of ACEIs and patients’ adherence.

Keywords: angiotensin converting enzymes inhibitors, angiotensin receptor blockers, guideline direct medical therapy, N-terminal pro-brain natriuretic peptide

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Authors: Ovidiu Vlaicu, Leontina Banica, Dan Otelea, Andrei-Jose Petrescu, Costin-Ioan Popescu

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Hepatitis C Virus (HCV) infects 170 million peoples worldwide. Major advances have been made recently in HCV standard of care with interferon-free therapy being already approved. Despite major progress in HCV therapy, the genotype associated treatment efficacy and toxicity still represent issues to address. To identify endogenous factors involved in different stages of HCV life cycle, we have developed a trans-packaging system for HCV subgenomic replicons lacking core protein gene. Huh7 cells were used to generate a packaging cell line expressing the core protein in an inducible manner. The core packaging cell line was able to trans-complemented various subgenomic replicons to secret infectious trans-complemented HCV particles (HCV-TCP). Further, we constructed subgenomic replicons with foreign epitopes suitable for immunoaffinity purification or fluorescence microscopy studies. We have shown that the insertion has not effects on the efficacy of trans-complementation yielding similar titers to the control subgenomic replicon. This system will be a valuable tool in studying pre- and post-assembly events in HCV life cycle and for the fast identification of HCV assembly inhibitors.

Keywords: assembly inhibitors, core protein, HCV, trans-complementation

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Authors: Mohamed Moussaoui, Mouna Baassi, Soukayna Baammi, Hatim Soufi, Mohammed Salah, Rachid Daoud, Achraf EL Allali, Mohammed Elalaoui Belghiti, Said Belaaouad

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The present study aims to investigate the quantitative structure-activity relationship (QSAR) of a series of Thiazole derivatives reported as anticancer agents (hepatocellular carcinoma), using principally the electronic descriptors calculated by the density functional theory (DFT) method and by applying the multiple linear regression method. The developed model showed good statistical parameters (R²= 0.725, R²ₐ𝒹ⱼ= 0.653, MSE = 0.060, R²ₜₑₛₜ= 0.827, Q²𝒸ᵥ = 0.536). The energy of the highest occupied molecular orbital (EHOMO) orbital, electronic energy (TE), shape coefficient (I), number of rotatable bonds (NROT), and index of refraction (n) were revealed to be the main descriptors influencing the anti-cancer activity. Additional Thiazole derivatives were then designed and their activities and pharmacokinetic properties were predicted using the validated QSAR model. These designed molecules underwent evaluation through molecular docking (MD) and molecular dynamic (MD) simulations, with binding affinity calculated using the MMPBSA script according to a 100 ns simulation trajectory. This process aimed to study both their affinity and stability towards Cyclin-Dependent Kinase 2 (CDK2), a target protein for cancer disease treatment. The research concluded by identifying four CDK2 inhibitors - A1, A3, A5, and A6 - displaying satisfactory pharmacokinetic properties. MDs results indicated that the designed compound A5 remained stable in the active center of the CDK2 protein, suggesting its potential as an effective inhibitor for the treatment of hepatocellular carcinoma. The findings of this study could contribute significantly to the development of effective CDK2 inhibitors.

Keywords: QSAR, ADMET, Thiazole, anticancer, molecular docking, molecular dynamic simulations, MMPBSA calculation

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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: Yutong Wan, John N. Wood

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Introduction: SCN9A encoded Nav1.7 is an ideal therapeutic target with minimal side effects for the pharmaceutical industry because SCN9A variants can cause both human gains of function pain-related mutations and loss of function pain-free mutations. This study reviews the clinical effectiveness of existing Nav1.7 inhibitors, which theoretically should be powerful analgesics. Methods: A systematic review is conducted on the effectiveness of current Nav1.7 blockers undergoing clinical trials. Studies were mainly extracted from PubMed, U.S. National Library of Medicine Clinical Trials, World Health Organization International Clinical Trials Registry, ISRCTN registry platform, and Integrated Research Approval System by NHS. Only studies with full text available and those conducted using double-blinded, placebo controlled, and randomised designs and reporting at least one analgesic measurement were included. Results: Overall, 61 trials were screened, and eight studies covering PF 05089771 (Pfizer), TV 45070 (Teva & Xenon), and BIIB074 (Biogen) met the inclusion criteria. Most studies were excluded because results were not published. All three compounds demonstrated insignificant analgesic effects, and the comparison between PF 05089771 and pregabalin/ibuprofen showed that PF 05089771 was a much weaker analgesic. All three drug candidates only have mild side effects, indicating the potentials for further investigation of Nav1.7 antagonists. Discussion: The failure of current Nav1.7 small molecule inhibitors might attribute to ignorance of the key role of endogenous systems in Nav1.7 null mutants, the lack of selectivity and blocking potency, and central impermeability. The synergistic combination of analgesic drugs, a recent UCL patent, combining a small dose of Nav1.7 blockers and opioids or enkephalinase inhibitors dramatically enhanced the analgesic effects. Conclusion: The current clinical testing Nav1.7 blockers are generally disappointing. However, the newer generation of Nav1.7 targeting analgesics has overcome the major constraints of its predecessors.

Keywords: chronic pain, Nav1.7 blockers, SCN9A, systematic review

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Authors: Hakan Arslan, Deniz Ekinci, Alper Gungor, Gurkan Bilir, Omer Tas, Mehmet Altun

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Drought is one of the major abiotic stresses affecting the agricultural production worldwide. In this study, Leaf samples were taken from the carrot plants grown under drought stress conditions during the harvesting period. The plants were irrigated in three irrigation interval (4, 6 and 8 days) and Irrigation water regime was set up in pots. The changes in activities of antioxidant enzymes such as glutathione reductase (GR), glutathione s-transferase (GST), superoxide dismutase (SOD)) in leaves of black carrot were investigated. The activities of antioxidant enzymes (GR, GST, SOD) were varied significantly with irrigation intervals. The highest value of GR, GST and SOD were determined in the irrigation interval of 6 days. All antioxidant activity values were decreased in 8 days of irrigation interval. As a result of the study, it has been suggested that optimum irrigation intervals for plants can be used in antioxidant enzymes.

Keywords: antioxidant enzyme, carrot, drought, irrigation interval

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Authors: Y. Moldes-Diz, M. Gamallo, G. Eibes, C. Vazquez-Vazquez, G. Feijoo, J. M. Lema, M. T. Moreira

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Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are excellent biocatalysts for biotechnological and environmental applications because of their high activity, selectivity, and specificity. Specifically, these characteristics allow them to perform the oxidation of recalcitrant compounds with simple requirements for the catalysis (presence of molecular oxygen). Nevertheless, the low stability under unfavorable conditions (pH, inactivating agents or temperature) and high production costs still limits their use for practical applications. Immobilization of enzymes has proven particularly valuable to avoid some of the aforementioned drawbacks. Magnetic nanoparticles (MNPs) have received increasing attention as carriers for enzyme immobilization since they can potentially provide an easy recovery of the biocatalyst from the reaction medium under an external magnetic field. In the present work, silica-coated magnetic nanoparticles (Fe3O4@SiO2) were prepared, characterized and used for laccase immobilization by covalent binding. The synthesis of Fe3O4@SiO2 was performed in a two-step procedure: co-precipitation and reverse microemulsion. The influence of immobilization conditions: concentrations of the functionalization agent (3-aminopropyl-triethoxy-silane) and the cross-linker (glutaraldehyde) as well as the influence of pH, T or inactivating agents were evaluated. In general, immobilized laccase showed superior stability compared to that of free enzyme. The reusability of the biocatalyst was demonstrated in successive batch reactions, where enzyme activity was maintained above 65% after 8 cycles of oxidation of the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate).

Keywords: silica-coated magnetic nanoparticles, laccase, immobilization, regeneration

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Authors: Prasanna Belur, P. R. Ashwini, Sampath Charanyaa, I. Regupathi

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Indian oil sardine (Sardinella longiceps) are one of the richest and cheapest sources of n-3 polyunsaturated fatty acids (n-3 PUFA) such as Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA). The health benefits conferred by n-3 PUFA upon consumption, in the prevention and treatment of coronary, neuromuscular, immunological disorders and allergic conditions are well documented. Natural refined Indian Sardine oil generally contain about 25% (w/w) n-3 PUFA along with various unsaturated and saturated fatty acids in the form of mono, di, and triglycerides. Having high concentration of n-3 PUFA content in the glyceride form is most desirable for human consumption to avail maximum health benefits. Thus, enhancing the n-3 PUFA content while retaining it in the glyceride form with green technology is the need of the hour. In this study, refined Indian Sardine oil was subjected to selective hydrolysis by Candida antartica lipase to enhance n-3 PUFA content. The degree of hydrolysis and enhancement of n-3 PUFA content was estimated by determining acid value, Iodine value, EPA and DHA content (by Gas Chromatographic methods after derivitization) before and after hydrolysis. Various reaction parameters such as pH, temperature, enzyme load, lipid to aqueous phase volume ratio and incubation time were optimized by conducting trials with one parameter at a time approach. Incubating enzyme solution with refined sardine oil with a volume ratio of 1:1, at pH 7.0, for 60 minutes at 50 °C, with an enzyme load of 60 mg/ml was found to be optimum. After enzymatic treatment, the oil was subjected to refining to remove free fatty acids and moisture content using previously optimized refining technology. Enzymatic treatment at the optimal conditions resulted in 12.11 % enhancement in Degree of hydrolysis. Iodine number had increased by 9.7 % and n-3 PUFA content was enhanced by 112 % (w/w). Selective enhancement of n-3 PUFA glycerides, eliminating saturated and unsaturated fatty acids from the oil using enzyme is an interesting preposition as this technique is environment-friendly, cost effective and provide natural source of n-3 PUFA rich oil.

Keywords: Candida antartica, lipase, n-3 polyunsaturated fatty acids, sardine oil

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Authors: Anahita Izadyar, My Ni Van, Kayleigh Amber Rodriguez, Ilwoo Seok, Elizabeth E. Hood

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Using a recombinant enzyme derived from corn and a simple modification, we fabricated a facile, fast, and cost-beneficial biosensor to measure glucose. The Nafion/ Plant Produced Mn Peroxidase (PPMP)– glucose oxidase (GOx)- Bovine serum albumin (BSA) /Au electrode showed an excellent amperometric response to detect glucose. This biosensor is capable of responding to a wide range of glucose—20.0 µM−15.0 mM and has a lower detection limit (LOD) of 2.90µM. The reproducibility response using six electrodes is also very substantial and indicates the high capability of this biosensor to detect a wide range of 3.10±0.19µM to 13.2±1.8 mM glucose concentration. Selectivity of this electrode was investigated in an optimized experimental solution contains 10% diet green tea with citrus containing ascorbic acid (AA), and citric acid (CA) in a wide concentration of glucose at 0.02 to 14.0mM with an LOD of 3.10µM. Reproducibility was also investigated using 4 electrodes in this sample and shows notable results in the wide concentration range of 3.35±0.45µM to of 13.0 ± 0.81 mM. We also used other voltammetry methods to evaluate this biosensor. We applied linear sweep voltammetry (LSV) and this technique shows a wide range of 0.10−15.0 mM to detect glucose with a lower detection limit of 19.5µM. The performance and strength of this enzyme biosensor were the simplicity, wide linear ranges, sensitivities, selectivity, and low limits of detection. We expect that the modified biosensor has the potential for monitoring various biofluids.

Keywords: plant-produced manganese peroxidase, enzyme-based biosensors, glucose, modified gold electrode, glucose oxidase

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Authors: Uzma Saqib, Mirza S. Baig

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Myeloid differentiation primary response protein 88 (MYD88) has long been considered a central player in the inflammatory pathway. Recent studies clearly suggest that it is an important therapeutic target in inflammation. On the other hand, a recent study on the interaction between the orphan nuclear receptor (Nur77) and p38α, leading to increased lipopolysaccharide-induced hyperinflammatory response, suggests this binary complex as a therapeutic target. In this study, we have designed inhibitors that can inhibit both MYD88 and Nur77 at the same time. Since both MYD88 and Nur77 are an integral part of the pathways involving lipopolysaccharide-induced activation of NF-κB-mediated inflammation, we tried to target both proteins with the same library in order to retrieve compounds having dual inhibitory properties. To perform this, we developed a homodimeric model of MYD88 and, along with the crystal structure of Nur77, screened a virtual library of compounds from the traditional Chinese medicine database containing ~61,000 compounds. We analyzed the resulting hits for their efficacy for dual binding and probed them for developing a common pharmacophore model that could be used as a prototype to screen compound libraries as well as to guide combinatorial library design to search for ideal dual-target inhibitors. Thus, our study explores the identification of novel leads having dual inhibiting effects due to binding to both MYD88 and Nur77 targets.

Keywords: drug design, Nur77, MYD88, inflammation

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Authors: So Young Kim, Eun-Young Kwon, Bora Choi, Mi Kyeong Yu, Seon Jeong Lee, Myung-Sook Choi

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Lemon (Citrus limon) has various beneficial effect. Eriocitrin (eriodictyol 7-rutinoside) is the main ingredient of lemon fruit and is known to have antioxidative effects. However, there has been little research about the effects of eriocitrin on obesity and regulation of lipid profiles levels. In the present study, we investigated the anti-obesity and lipid-lowering effects of eriocitrin in mice fed high-fat diet (HFD). The 4 week-old male C57BL/6 mice were randomly divided into two groups and were fed HFD (20% fat, w/w) and HFD supplemented with eriocitrin (0.005%, w/w, EC) for 16 weeks. Food intake, body weight and white adipose tissue weight (WAT) were measured and plasma free fatty acid (FFA), apolipoprotein (Apo) B100 level and hepatic enzyme activity were analyzed. No differences were shown between the HFD and EC groups in body weight and food intake. However EC supplementation significantly reduced the weights of epididymal, subcutaneous and total WAT. In addition, the levels of plasma FFA and Apo B100 were significantly decreased in the EC group compared with the HFD group. Moreover, the activities of glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) related to fatty acids synthesis were significantly lower in the EC group than in the HFD group in liver. Therefore, this study indicates that eriocitrin has beneficial effects on adiposity and nonalcholic fatty liver diseases by modulating hepatic lipid-regulating enzyme activities and plasma lipid profile.

Keywords: antiobesity, eriocitrin, high fat diet, lipid lowering

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Authors: Orapan Paecharoenchai, Tanasait Ngawhirunpat, Theerasak Rojanarata, Auayporn Apirakaramwong, Praneet Opanasopit

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Cationic niosomes formulated with Span20, cholesterol and novel synthesized spermine-cationic lipids (2-hydrocarbon tail and 4- hydrocarbon tail) in a molar ratio of 2.5:2.5:1 can mediate high gene transfection in vitro. However, the uptake mechanisms of these systems are not well clarified. In the present study, effect of endocytic inhibitors on the transfection efficiency of niosomes/DNA complexes was determined on a human cervical carcinoma cell line (HeLa cells) using the inhibitors of macropinocytosis (wortmannin), clathrin- and caveolae-mediated endocytosis (methyl-β-cyclodextrin), clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein and filipin), cytosolic transfer (ammonium chloride) and microtubules polymerization (nocodazole). The transfection of niosomes with 2-hydrocarbon tail lipid was blocked by nocodazole, genistein, ammonium chloride and filipin, respectively, whereas, the transfection of niosomes with 4-hydrocarbon tail lipid was blocked by nocodazole, genistein, ammonium chloride, methyl-β-cyclodextrin and filipin, respectively. It can be concluded that these niosomes/DNA complexes were internalized predominantly by endocytosis via clathrin and caveolae-independent pathway.

Keywords: cellular internalization, cationic niosomes, gene carriers, spermine-cationic lipids

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