Search results for: amino%20acids

Authors: Damien Rinsant, Eugen Andreiadis, Michael Carboni, Daniel Meyer

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Different types of materials have been developed for the solid/liquid uranium extraction processes, such as functionalized organic polymers, hybrid silica or inorganic adsorbents. In general, these materials exhibit a moderate affinity for uranyl ions and poor selectivity against impurities like iron, vanadium or molybdenum. Moreover, the structural organization deficiency of these materials generates ion diffusion issues inside the material. Therefore, the aim of our study is to developed efficient and organized materials, stable in the acid media encountered in uranium extraction processes. Metal organic frameworks (MOFs) are hybrid crystalline materials consisting of an inorganic part (cluster or metal ions) and tailored organic linkers connected via coordination bonds. These hierarchical materials have exceptional surface area, thermal stability and a large variety of tunable structures. However, due to the reversibility of constitutive coordination bonds, MOFs have moderate stability in strongly complexing or acidic media. Only few of them are known to be stable in aqueous media and only one example is described in strong acidic media. However, these conditions are very often encountered in the environmental pollution remediation of mine wastewaters. To tackle the challenge of developing MOFs adapted for uranium extraction from acid mine waters, we have investigated the stability of several materials. To ensure a good stability we have synthetized and characterized different materials based on highly coordinated metal clusters, such as LnOFs and Zirconium based materials. Among the latter, the UiO family shows a great stability in sulfuric acid media even in the presence of 1.4 M sodium sulfate at pH 2. However, the stability in phosphoric media is reduced due to the high affinity between zirconium and phosphate ligand. Based on these results, we have developed a tertiary amine functionalized MOF denoted UiO-68-NMe2 particularly adapted for the extraction of anionic uranyl (VI) sulfate complexes mainly present in the acid mine solutions. The adsorption capacity of the material has been determined upon varying total sulfate concentration, contact time and uranium concentration. The extraction tests put in evidence different phenomena due to the complexity of the extraction media and the interaction between the MOF and sulfate anion. Finally, the extraction mechanisms and the interaction between uranyl and the MOF structure have been investigated. The functionalized material UiO-68-NMe2 has been characterized in the presence and absence of uranium by FT-IR, UV and Raman techniques. Moreover, the stability of the protonated amino functionalized MOF has been evaluated. The synthesis, characterization and evaluation of this type of hybrid material, particularly adapted for uranium extraction in sulfuric acid media by an anionic exchange mechanism, paved the way for the development of metal organic frameworks functionalized by different other chelating motifs, such as bifunctional ligands showing an enhanced affinity and selectivity for uranium in acid and complexing media. Work in this direction is currently in progress.

Keywords: extraction, MOF, ligand, uranium

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Authors: Anna Kolanowska, Anna Kuziel, Sławomir Boncel

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Carbon nanotubes (CNTs) represent a combination of lightness and nanoscopic size with high tensile strength, excellent thermal and electrical conductivity. By now, CNTs have been used as a support in heterogeneous catalysis (CuCl anchored to pre-functionalized CNTs) in the Ullmann-type coupling with aryl halides toward formation of C-N and C-O bonds. The results indicated that the stability of the catalyst was much improved and the elaborated catalytic system was efficient and recyclable. However, CNTs have not been considered as the substrate itself in the Ullmann-type reactions. But if successful, this functionalization would open new areas of CNT chemistry leading to enhanced in-solvent/matrix nanotube individualization. The copper-catalyzed Ullmann-type reaction is an attractive method for the formation of carbon-heteroatom and carbon-carbon bonds in organic synthesis. This condensation reaction is usually conducted at temperature as high as 200 oC, often in the presence of stoichiometric amounts of copper reagent and with activated aryl halides. However, a small amount of organic additive (e.g. diamines, amino acids, diols, 1,10-phenanthroline) can be applied in order to increase the solubility and stability of copper catalyst, and at the same time to allow performing the reaction under mild conditions. The copper (pre-)catalyst is prepared by in situ mixing of copper salt and the appropriate chelator. Our research is focused on the application of Ullmann-type reaction for the covalent functionalization of CNTs. Firstly, CNTs were chlorinated by using iodine trichloride (ICl3) in carbon tetrachloride (CCl4). This method involves formation of several chemical species (ICl, Cl2 and I2Cl6), but the most reactive is the dimer. The fact (that the dimer is the main individual in CCl4) is the reason for high reactivity and possibly high functionalization levels of CNTs. This method, indeed, yielded a notable amount of chlorine onto the MWCNT surface. The next step was the reaction of CNT-Cl with three substrates: aniline, iodobenzene and phenol for the formation C-N, C-C and C-O bonds, respectively, in the presence of 1,10-phenanthroline and cesium carbonate (Cs2CO3) as a base. As the CNT substrates, two multi-wall CNT (MWCNT) types were used: commercially available Nanocyl NC7000™ (9.6 nm diameter, 1.5 µm length, 90% purity) and thicker MWCNTs (in-house) synthesized in our laboratory using catalytic chemical vapour deposition (c-CVD). In-house CNTs had diameter ranging between 60-70 nm and length up to 300 µm. Since classical Ullmann reaction was found as suffering from poor yields, we have investigated the effect of various solvents (toluene, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide) on the coupling of substrates. Owing to the fact that the aryl halides show the reactivity order of I>Br>Cl>F, we have also investigated the effect of iodine presence on CNT surface on reaction yield. In this case, in first step we have used iodine monochloride instead of iodine trichloride. Finally, we have used the optimized reaction conditions with p-bromophenol and 1,2,4-trihydroxybenzene for the control of CNT dispersion.

Keywords: carbon nanotubes, coupling reaction, functionalization, Ullmann reaction

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Authors: Azza A. Ali, Abeer I. Abd El-Fattah, Shaimaa S. Hussein, Hanan A. Abd El-Samea, Karema Abu-Elfotuh

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Background: Aluminium (Al) represents an environmental risk factor. Exposure to high levels of Al causes neurotoxic effects and different diseases. Vinpocetine is widely used to improve cognitive functions, it possesses memory-protective and memory-enhancing properties and has the ability to increase cerebral blood flow and glucose uptake. Cocoa bean represents a rich source of iron as well as a potent antioxidant. It can protect from the impact of free radicals, reduces stress as well as depression and promotes better memory and concentration. Wheatgrass is primarily used as a concentrated source of nutrients. It contains vitamins, minerals, carbohydrates, amino acids and possesses antioxidant and anti-inflammatory activities. Coenzyme Q10 (CoQ10) is an intracellular antioxidant and mitochondrial membrane stabilizer. It is effective in improving cognitive disorders and has been used as anti-aging. Zinc is a structural element of many proteins and signaling messenger that is released by neural activity at many central excitatory synapses. Objective: To study the role of some nutrients and drugs as Vinpocetine, Cocoa, Wheatgrass, CoQ10 and Zinc against neurotoxicity induced by Al in rats as well as to compare between their potency in providing protection. Methods: Seven groups of rats were used and received daily for three weeks AlCl3 (70 mg/kg, IP) for Al-toxicity model groups except for the control group which received saline. All groups of Al-toxicity model except one group (non-treated) were co-administered orally together with AlCl3 the following treatments; Vinpocetine (20mg/kg), Cocoa powder (24mg/kg), Wheat grass (100mg/kg), CoQ10 (200mg/kg) or Zinc (32mg/kg). Biochemical changes in the rat brain as acetyl cholinesterase (ACHE), Aβ, brain derived neurotrophic factor (BDNF), inflammatory mediators (TNF-α, IL-1β), oxidative parameters (MDA, SOD, TAC) were estimated for all groups besides histopathological examinations in different brain regions. Results: Neurotoxicity and neurodegenerations in the rat brain after three weeks of Al exposure were indicated by the significant increase in Aβ, ACHE, MDA, TNF-α, IL-1β, DNA fragmentation together with the significant decrease in SOD, TAC, BDNF and confirmed by the histopathological changes in the brain. On the other hand, co-administration of each of Vinpocetine, Cocoa, Wheatgrass, CoQ10 or Zinc together with AlCl3 provided protection against hazards of neurotoxicity and neurodegenerations induced by Al, their protection were indicated by the decrease in Aβ, ACHE, MDA, TNF-α, IL-1β, DNA fragmentation together with the increase in SOD, TAC, BDNF and confirmed by the histopathological examinations of different brain regions. Vinpocetine and Cocoa showed the most pronounced protection while Zinc provided the least protective effects than the other used nutrients and drugs. Conclusion: Different degrees of protection from neurotoxicity and neuronal degenerations induced by Al could be achieved through the co-administration of some nutrients and drugs during its exposure. Vinpocetine and Cocoa provided the most protection than Wheat grass, CoQ10 or Zinc which showed the least protective effects.

Keywords: aluminum, neurotoxicity, vinpocetine, cocoa, wheat grass, coenzyme Q10, Zinc, rats

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Authors: Bakytzhan Bimbetov, Abay Zhangabilov, Saule Aitbaeva, Galymzhan Meirambekov

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Mare’s milk (saumal) contains in total about 40 biological components necessary for the human body. The most significant among them are amino acids, fats, carbohydrates, enzymes (lysozyme, amylase), more minerals and vitamins which are well balanced with each other. In Kazakhstan, Company "Eurasia Invest Ltd.” produces a freeze-dried saumal in form of powder by the use of modern German innovative technology by means of evaporating at low temperature (-35°C) with an appropriate pasteurization. Research of freeze-dried biomilk for the qualitative content showed that main ingredients of freshly drown milk are being preserved. We are currently studying medical and dietary properties of freeze-dried mare's milk for diseases of the digestive system, including for nonalcoholic steatohepatitis (NASH) and liver cirrhosis (LC) viral etiology. The studied group consisted of 14 patients with NASH, and 7 patients with LC viral etiology of Class A severity degree as per Child-Pugh. Patients took freeze-dried saumal, preliminary dissolved in boiled warm water (24 g. powder per 200 ml water) 3-4 times a day for a month in conjunction with basic therapy. The results were compared to a control group (11 patients with NASH and LC) who received only basic therapy without mare’s milk. Results of preliminary research showed an improvement of subjective and objective conditions of all patients, but more significant improvement of clinical symptoms and syndromes were observed in the treatment group compared to the control one. Patients with NASH significantly over time compared to the beginning of therapy decreased asthenic and dyspeptic syndromes (p<0,01). Hepatomegaly, identified on the basis of ultrasound prior to treatment was observed in 92,8±2,4% of patients, and after combination therapy hepatomegaly the rate decreased by 14,3%, amounting to 78,5±2,8%. Patients with LC also noted the improvement of asthenic (p<0,01) and dyspeptic (p<0,05) syndromes and hemorrhagic syndrome (nosebleeds and bleeding gums when brushing your teeth, p<0,05), and jaundice. Laboratory study also showed improvement in the research group, but more significant changes were observed in the experimental group. Group of patients with NASH showed a significant improvement of index in cytolysis in conjunction with a combination therapy (p<0,05). In the control group, these indicators were also improved, but they were not statistically reliable (p>0,05). Markers of liver failure were additionally studied during the study of laboratory parameters in patients with liver cirrhosis, in particular, bilirubin, albumin and prothrombin index (PTI). Combined therapy with the use of basic treatment and mare's milk showed a significant improvement in cytolysis and bilirubin (p<0,05). In our opinion, a very important and interesting fact is that, in conjunction with basic therapy, the use of mare's milk revealed an improvement of liver function in the form of normalized PTI and albumin in patients with liver cirrhosis viral etiology. Results of this work have shown therapeutic efficiency of the use of mare's milk in complex treatment of patients with liver disease and require further in-depth study.

Keywords: liver cirrhosis, non-alcohol steatohepatitis, saumal, mare’s milk

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Authors: Maryam Nazari

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COVID-19 is a respiratory disease triggered by the novel coronavirus, SARS-CoV-2, that has reached pandemic status today. Acute inflammation and immune cells infiltration into lung injuries result in multi-organ failure. The presence of other non-communicable diseases (NCDs) with systemic inflammation derived from COVID-19 may exacerbate the patient's situation and increase the risk for adverse effects and mortality. This pandemic is a novel situation and the scientific community at this time is looking for vaccines or drugs to treat the pathology. One of the biggest challenges is focused on reducing inflammation without compromising the correct immune response of the patient. In this regard, addressing the nutritional factors should not be overlooked not only as a matter of avoiding the presence of NCDs with severe infections but also as an adjunctive way to modulate the inflammatory status of the patients. Despite the pivotal role of nutrition in modifying immune response, due to the novelty of the COVID-19 disease, information about the effects of specific dietary agents is limited in this area. From the macronutrients point of view, protein deficiency (quantity or quality) has negative effects on the number of functional immunoglobulins and gut-associated lymphoid tissue (GALT). High biological value proteins or some amino acids like arginine and glutamine are well known for their ability to augment the immune system. Among lipids, fish oil has the ability to inactivate enveloped viruses, suppress pro-inflammatory prostaglandin production and block platelet-activating factors and their receptors. In addition, protectin D1, which is an Omega-3 PUFAs derivation, is a novel antiviral drug. So it seems that these fatty acids can reduce the severity and/or improve recovery of patients with COVID-19. Carbohydrates with lower glycemic index and fibers are associated with lower levels of inflammatory cytokines (CRP, TNF-α, and IL-6). Short-Chain Fatty acids not only exert a direct anti-inflammatory effect but also provide appropriate gut microbial, which is important in gastrointestinal issues related to COVID-19. From the micronutrients point of view, Vitamins A, C, D, E, iron, magnesium, zinc, selenium and copper play a vital role in the maintenance of immune function. Inadequate status in these nutrients may result in decreased resistance against COVID-19 infection. There are specific bioactive compounds in the diet that interact with the ACE2 receptor, which is the gateway for SARS and SARS-CoV-2, and thus controls the viral infection. Regarding this, the potential benefits of probiotics, resveratrol (a polyphenol found in grape), oleoylethanolamide (derived from oleic acid), and natural peroxisome proliferator-activated receptor γ agonists in foodstuffs (like curcumin, pomegranate, hot pepper) are suggested. Yet, it should be pointed out that most of these results have been reported in animal models and further human studies are needed to be verified.

Keywords: Covid-19, inflammation, nutrition, dietary agents

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Authors: Shagufta Jabeen, Faez Jesse Firdaus Abdullah, Zunita Zakaria, Nurulfiza Mat Isa, Yung Chie Tan, Wai Yan Yee, Abdul Rahman Omar

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Pasteurella multocida is associated with acute, as well as, chronic infections in avian and bovine such as pasteurellosis and hemorrhagic septicemia (HS) in cattle and buffaloes. Iron is one of the most important nutrients for pathogenic bacteria including Pasteurella and acts as a cofactor or prosthetic group in several essential enzymes and is needed for amino acid, pyrimidine, and DNA biosynthesis. In our recent study, we showed that 2% of Pasteurella multocida serotype A strain PMTB2.1 encode for iron regulating genes (Accession number CP007205.1). Genome sequencing of other Pasteurella multocida serotypes namely PM70 and HB01 also indicated up to 2.5% of the respective genome encode for iron regulating genes, suggesting that Pasteurella multocida genome comprises of multiple systems for iron uptake. Since P. multocida PMTB2.1 has more than 40 CDs out of 2097 CDs (approximately 2%), encode for iron-regulated. The gene expression profiling of four iron-regulating genes namely fbpb, yfea, fece and fur were characterized under iron-restricted environment. The P. multocida strain PMTB2.1 was grown in broth with and without iron chelating agent and samples were collected at different time points. Relative mRNA expression profile of these genes was determined using Taqman probe based real-time PCR assay. The data analysis, normalization with two house-keeping genes and the quantification of fold changes were carried out using Bio-Rad CFX manager software version 3.1. Results of this study reflect that iron reduced environment has significant effect on expression profile of iron regulating genes (p < 0.05) when compared to control (normal broth) and all evaluated genes act differently with response to iron reduction in media. The highest relative fold change of fece gene was observed at early stage of treatment indicating that PMTB2.1 may utilize its periplasmic protein at early stage to acquire iron. Furthermore, down-regulation expression of fece with the elevated expression of other genes at later time points suggests that PMTB2.1 control their iron requirements in response to iron availability by down-regulating the expression of iron proteins. Moreover, significantly high relative fold change (p ≤ 0.05) of fbpb gene is probably associated with the ability of P. multocida to directly use host iron complex such as hem, hemoglobin. In addition, the significant increase (p ≤ 0.05) in fbpb and yfea expressions also reflects the utilization of multiple iron systems in P. multocida strain PMTB2.1. The findings of this study are very much important as relative scarcity of free iron within hosts creates a major barrier to microbial growth inside host and utilization of outer-membrane proteins system in iron acquisition probably occurred at early stage of infection with P. multocida. In conclusion, the presence and utilization of multiple iron system in P. multocida strain PMTB2.1 revealed the importance of iron in the survival of P. multocida.

Keywords: iron-related genes, real-time PCR, gene expression profiling, fold changes

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Authors: Gabriela Wyszogrodzka, Przemyslaw Dorozynski, Barbara Gil, Maciej Strzempek, Bartosz Marszalek, Piotr Kulinowski, Wladyslaw Piotr Weglarz, Elzbieta Menaszek

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MOFs (Metal-Organic Frameworks) belong to a new group of porous materials with a hybrid organic-inorganic construction. Their structure is a network consisting of metal cations or clusters (acting as metallic centers, nodes) and the organic linkers between nodes. The interest in MOFs is primarily associated with the use of their well-developed surface and large porous. Possibility to build MOFs of biocompatible components let to use them as potential drug carriers. Furthermore, forming MOFs structure from cations possessing paramagnetic properties (e.g. iron cations) allows to use them as MRI (Magnetic Resonance Imaging) contrast agents. The concept of formation of particles that combine the ability to transfer active substance with imaging properties has been called theranostic (from words combination therapy and diagnostics). By building MOF structure from iron cations it is possible to use them as theranostic agents and monitoring the distribution of the active substance after administration in real time. In the study iron-MOF: Fe-MIL-101-NH2 was chosen, consisting of iron cluster in nodes of the structure and amino-terephthalic acid as a linker. The aim of the study was to investigate the possibility of applying Fe-MIL-101-NH2 as inhalable theranostic particulate system for the first-line anti-tuberculosis antibiotic – isoniazid. The drug content incorporated into Fe-MIL-101-NH2 was evaluated by dissolution study using spectrophotometric method. Results showed isoniazid encapsulation efficiency – ca. 12.5% wt. Possibility of Fe-MIL-101-NH2 application as the MRI contrast agent was demonstrated by magnetic resonance tomography. FeMIL-101-NH2 effectively shortening T1 and T2 relaxation times (increasing R1 and R2 relaxation rates) linearly with the concentrations of suspended material. Images obtained using multi-echo magnetic resonance imaging sequence revealed possibility to use FeMIL-101-NH2 as positive and negative contrasts depending on applied repetition time. MOFs micronization via ultrasound was evaluated by XRD, nitrogen adsorption, FTIR, SEM imaging and did not influence their crystal shape and size. Ultrasonication let to break the aggregates and achieve very homogeneously looking SEM images. MOFs cytotoxicity was evaluated in in vitro test with a highly sensitive resazurin based reagent PrestoBlue™ on L929 fibroblast cell line. After 24h no inhibition of cell proliferation was observed. All results proved potential possibility of application of ironMOFs as an isoniazid carrier and as MRI contrast agent in inhalatory treatment of tuberculosis. Acknowledgments: Authors gratefully acknowledge the National Science Center Poland for providing financial support, grant no 2014/15/B/ST5/04498.

Keywords: imaging agents, metal-organic frameworks, theranostics, tuberculosis

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Authors: Hanan M. Hassan, Laila Abouzeid, Lamya H. Al-Wahaibi, George S. G. Shehatou, Ali A. El-Emam

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Cancer is a major public health problem and the second leading cause of death worldwide. In 2020, cancer diagnosis and treatment have been negatively affected by the coronavirus 2019 (COVID-19) pandemic. During the quarantine, because of the limited access to healthcare and avoiding exposure to COVID-19 as a contagious disease; patients of cancer suffered deferments in follow-up and treatment regimens leading to substantial worsening of disease, death, and increased healthcare costs. Thus, this study is designed to investigate the molecular mechanisms by which adamantne derivatives attenuate hepatocllular carcinoma experimentally and theoretically. There is a close association between increased resistance to anticancer drugs and defective apoptosis that considered a causative factor for oncogenesis. Cancer cells use different molecular pathways to inhibit apoptosis, BAX and Bcl-2 proteins have essential roles in the progression or inhibition of intrinsic apoptotic pathways triggered by mitochondrial dysfunction. Therefore, their balance ratio can promote the cellular apoptotic fate. In this study, the in vitro cytotoxic effects of seven synthetic adamantyl isothiorea derivatives were evaluated against five human tumor cell lines by MTT assay. Compounds 5 and 6 showed the best results, mostly against hepatocellular carcinoma (HCC). Hence, in vivo studies were performed in male Sprague-Dawley (SD) rats in which experimental hepatocellular carcinoma was induced with thioacetamide (TAA) (200 mg/kg, i.p., twice weekly) for 16 weeks. The most promising compounds, 5 and 6, were administered to treat liver cancer rats at a dose of 10 mg/kg/day for an additional two weeks, and the effects were compared with doxorubicin (DR), the anticancer drug. Hepatocellular carcinoma was evidenced by a dramatic increase in liver indices, oxidative stress markers, and immunohistochemical studies that were accompanied by a plethora of inflammatory mediators and alterations in the apoptotic cascade. Our results showed that treatment with adamantane derivatives 5 and 6 significantly suppressed fibrosis, inflammation, and other histopathological insults resulting in the diminished formation of hepatocyte tumorigenesis. Moreover, administration of the tested compounds resulted in amelioration of EGFR protein expression, upregulation of BAX, and lessening down of Bcl-2 levels that prove their role as apoptosis inducers. Also, the docking simulations performed for adamantane showed good fit and binding to the EGFR protein through hydrogen bond formation with conservative amino acids, which gives a shred of strong evidence for its hepatoprotective effect. In most analyses, the effects of compound 6 were more comparable to DR than compound 5. Our findings suggest that adamantane derivatives 5 and 6 are shown to have cytotoxic activity against HCC in vitro and in vivo, by more than one mechanism, possibly by inhibiting the TLR4-MyD88-NF-κB pathway and targeting EGFR signaling.

Keywords: adamantane, EGFR, HCC, apoptosis

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Authors: Weixiang Liu, Yukun Qin, Song Liu, Pengcheng Li

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Plant diseases can cause the death of crops with great economic losses. Particularly, those diseases are usually caused by pathogenic fungi. Metal fungicides are a type of pesticide that has advantages of a low-cost, broad antimicrobial spectrum and strong sterilization effect. However, the frequent and wide application of traditional metal fungicides has caused serious problems such as environmental pollution, the outbreak of mites and phytotoxicity. Therefore, it is critically necessary to discover new organic metal fungicides alternatives that have a low metal content, low toxicity, and little influence on mites. Chitosan, the second most abundant natural polysaccharide next to cellulose, was proved to have broad-spectrum antifungal activity against a variety of fungi. However, the use of chitosan was limited due to its poor solubility and weaker antifungal activity compared with commercial fungicide. Therefore, in order to improve the water solubility and antifungal activity, many researchers grafted the active groups onto chitosan. The present work was to combine free metal ions with chitosan, to prepare more potent antifungal chitosan derivatives, thus, based on condensation reaction, chitosan derivative bearing amino pyridine group was prepared and subsequently followed by coordination with cupric ions, zinc ions and nickel ions to synthesize chitosan metal complexes. The calculations by density functional theory (DFT) show that the copper ions and nickel ions underwent dsp2 hybridization, the zinc ions underwent sp3 hybridization, and all of them are coordinated by the carbon atom in the p-π conjugate group and the oxygen atoms in the acetate ion. The antifungal properties of chitosan metal complexes against Phytophthora capsici (P. capsici), Gibberella zeae (G. zeae), Fusarium oxysporum (F. oxysporum) and Botrytis cinerea (B. cinerea) were also assayed. In addition, a plant toxicity experiment was carried out. The experiments indicated that the derivatives have significantly enhanced antifungal activity after metal ions complexation compared with the original chitosan. It was shown that 0.20 mg/mL of O-CSPX-Cu can 100% inhibit the growth of P. capsici and 0.20 mg/mL of O-CSPX-Ni can 87.5% inhibit the growth of B. cinerea. In general, their activities are better than the positive control oligosaccharides. The combination of the pyridine formyl groups seems to favor biological activity. Additionally, the ligand fashion was precisely analyzed, and the results revealed that the copper ions and nickel ions underwent dsp2 hybridization, the zinc ions underwent sp3 hybridization, and the carbon atoms of the p-π conjugate group and the oxygen atoms of acetate ion are involved in the coordination of metal ions. The phytotoxicity assay of O-CSPX-M was also conducted, unlike the traditional metal fungicides, the metal complexes were not significantly toxic to the leaves of wheat. O-CSPX-Zn can even increase chlorophyll content in wheat leaves at 0.40 mg/mL. This is mainly because chitosan itself promotes plant growth and counteracts the phytotoxicity of metal ions. The chitosan derivative described here may lend themselves to future applicative studies in crop protection.

Keywords: coordination, chitosan, metal complex, antifungal properties

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Authors: Monika Patel, Kazuaki Matsumura

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Synthetic hydrogels, with their unique properties such as porosity, strength, and swelling in aqueous environment, are being used in many fields from food additives to regenerative medicines, from diagnostic and pharmaceuticals to drug delivery systems (DDS). But, hydrogels also have some limitations in terms of homogeneity of drug distribution and quantity of loaded drugs. As an alternate, polymeric micelles are extensively used as DDS. With the ease of self-assembly, and distinct stability they remarkably improve the solubility of hydrophobic drugs. However, presently, combinational therapy is the need of time and so are systems which are capable of releasing more than one drug. And it is one of the major challenges towards DDS to control the release of each drug independently, which simple DDS cannot meet. In this work, we present an amphiphilic polypeptide based micelle hydrogel composite to study the dual drug release for wound healing purposes using Amphotericin B (AmpB) and Curcumin as model drugs. Firstly, two differently charged amphiphilic polypeptide chains were prepared namely, poly L-Lysine-b-poly phenyl alanine (PLL-PPA) and poly Glutamic acid-b-poly phenyl alanine (PGA-PPA) through ring opening polymerization of amino acid N-carboxyanhydride. These polymers readily self-assemble to form micelles with hydrophobic PPA block as core and hydrophilic PLL/PGA as shell with an average diameter of about 280nm. The thus formed micelles were loaded with the model drugs. The PLL-PPA micelle was loaded with curcumin and PGA-PPA was loaded with AmpB by dialysis method. Drug loaded micelles showed a slight increase in the mean diameter and were fairly stable in solution and lyophilized forms. For forming the micelles hydrogel composite, the drug loaded micelles were dissolved and were cross linked using genipin. Genipin uses the free –NH2 groups in the PLL-PPA micelles to form a hydrogel network with free PGA-PPA micelles trapped in between the 3D scaffold formed. Different composites were tested by changing the weight ratios of the both micelles and were seen to alter its resulting surface charge from positive to negative with increase in PGA-PPA ratio. The composites with high surface charge showed a burst release of drug in initial phase, were as the composites with relatively low net charge showed a sustained release. Thus the resultant surface charge of the composite can be tuned to tune its drug release profile. Also, while studying the degree of cross linking among the PLL-PPA particles for effect on dual drug release, it was seen that as the degree of crosslinking increases, an increase in the tendency to burst release the drug (AmpB) is seen in PGA-PPA particle, were as on the contrary the PLL-PPA particles showed a slower release of Curcumin with increasing the cross linking density. Thus, two different pharmacokinetic profile of drugs were seen by changing the cross linking degree. In conclusion, a unique charged amphiphilic polypeptide based micelle hydrogel composite for dual drug delivery. This composite can be finely tuned on the basis of need of drug release profiles by changing simple parameters such as composition, cross linking and pH.

Keywords: amphiphilic polypeptide, dual drug release, micelle hydrogel composite, tunable DDS

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Authors: M. Sarraf, J. E. Moros, M. C. Sánchez

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Oleogels are self-standing systems that are able to trap edible liquid oil into a tridimensional network and also help to use less fat by forming crystallization oleogelators. There are different ways to generate oleogelation and oil structuring, including direct dispersion, structured biphasic systems, oil sorption, and indirect method (emulsion-template). The selection of processing conditions as well as the composition of the oleogels is essential to obtain a stable oleogel with characteristics suitable for its purpose. In this sense, one of the ingredients widely used in food products to produce oleogels and emulsions is polysaccharides. Basil seed gum (BSG), with the scientific name Ocimum basilicum, is a new native polysaccharide with high viscosity and pseudoplastic behavior because of its high molecular weight in the food industry. Also, proteins can stabilize oil in water due to the presence of amino and carboxyl moieties that result in surface activity. Whey proteins are widely used in the food industry due to available, cheap ingredients, nutritional and functional characteristics such as emulsifier and a gelling agent, thickening, and water-binding capacity. In general, the interaction of protein and polysaccharides has a significant effect on the food structures and their stability, like the texture of dairy products, by controlling the interactions in macromolecular systems. Using edible oleogels as oil structuring helps for targeted delivery of a component trapped in a structural network. Therefore, the development of efficient oleogel is essential in the food industry. A complete understanding of the important points, such as the ratio oil phase, processing conditions, and concentrations of biopolymers that affect the formation and stability of the emulsion, can result in crucial information in the production of a suitable oleogel. In this research, the effects of oil concentration and pressure used in the manufacture of the emulsion prior to obtaining the oleogel have been evaluated through the analysis of droplet size and rheological properties of obtained emulsions and oleogels. The results show that the emulsion prepared in the high-pressure homogenizer (HPH) at higher pressure values has smaller droplet sizes and a higher uniformity in the size distribution curve. On the other hand, in relation to the rheological characteristics of the emulsions and oleogels obtained, the predominantly elastic character of the systems must be noted, as they present values of the storage modulus higher than those of losses, also showing an important plateau zone, typical of structured systems. In the same way, if steady-state viscous flow tests have been analyzed on both emulsions and oleogels, the result is that, once again, the pressure used in the homogenizer is an important factor for obtaining emulsions with adequate droplet size and the subsequent oleogel. Thus, various routes for trapping oil inside a biopolymer matrix with adjustable mechanical properties could be applied for the creation of the three-dimensional network in order to the oil absorption and creating oleogel.

Keywords: basil seed gum, particle size, viscoelastic properties, whey protein

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Authors: M. Koksal, T. Ozyazici, E. Gurdal, M. Yarım, E. Demirpolat, M. B. Y. Aycan

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The discovery of new drugs in cancer chemotherapy is still a major topic because of severe side effects, selectivity problems and resistance development potential of existing drugs. In recent years, combined anticancer therapies or multi-acting drugs are clinically preferred over traditional cytotoxic treatment, with the aim of avoiding resistance and toxic side effects. Arrangement of multi-acting targets can be carried out either by combination of several drugs with different mechanisms or by usage of a single chemical compound capable of regulating several targets of a disease with multiple factors. In literature, several pyrimidine and piperazine derivatives have been involved in the structure of many compounds which have been used as chemotherapeutic agents along with wide clinical applications. The aim of this study is to combine pyrimidine and piperazine core structures to research and develop novel piperazinylpyrimidine derivatives with selective cytotoxicity over cancer cells. In this study, a group of novel 6-fluorophenyl-3-[2-(substitutedpiperazinyl)ethyl] hexahydropyrimidine-2,4-dione derivatives designed to observe the desired anticancer activity due to pyrimidine and piperazine based scaffolds. Target compounds were obtained by the reaction of appropriate piperazine derivatives and 6-(2/4-fluorophenyl)-3-(2-chloroethyl)hexahydropyrimidine-2,4-dione. The synthetic pathway of 6-(2/4-fluorophenyl)-3-(2-chloroethyl)hexahydropyrimidine-2,4-dione was started with Rodionov reaction using aldehyde, malonic acid and ammonium acetate in ethanol. Isolated β-fluorophenyl-β-amino acids were treated with 2-chloroethylisocyanate in the presence of an aqueous sodium hydroxide solution at room temperature to yield the sodium salts of the corresponding ureido acids. By addition of a mineral acid, ureido acids were precipitated. Later, these ureido acids were refluxed in thionyl chloride to give the 6-(2/4-fluorophenyl)-3-(2-chloroethyl)hexahydropyrimidine-2,4-di-one which were furthermore treated with secondary amines. Structures of purified compounds were characterized with IR, 1H-NMR, 13C-NMR, mass spectroscopies and elemental analysis. All of the compounds gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structures. In IR spectra of the compounds, N-H group was seen at 3230-3213 cm⁻¹. C-H was seen at 3100-2820 cm⁻¹ and C=O vibrational peaks were observed approximately at 1725 and 1665 cm⁻¹ in accordance with literature. In the NMR spectra of target compounds, the methylene protons of piperazine give two separate multiplet peaks around 3.5 and 4.5 ppm representing the successful N-alkylation of the structure. The cytotoxic activity of the synthesized compounds was investigated on human bronchial epithelial (BEAS 2B), lung (A549), colon adenocarcinoma (COLO205) and breast (MCF7) cell lines, by means of sulphorhodamine B (SRB) assays in triplicate. IC₅₀ values of the screened derivatives were found in range of 11.8-78 µM. This project was supported by The Scientific and Technological Research Council of Turkey (TUBITAK, Project no: 215S157).

Keywords: cytotoxicity, hexahydropyrimidine, piperazine, sulphorhodamine B assay

Procedia PDF Downloads 133

Authors: Jessie Rapoza, Petr Moroshkin, Jimmy Xu

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Chirality is omnipresent, in nature, in life, and in the field of physics. One intriguing example is the homochirality that has remained a great secret of life. Another is the pairs of mirror-image molecules – enantiomers. They are identical in atomic composition and therefore indistinguishable in the scalar physical properties. Yet, they can be either therapeutic or toxic, depending on their chirality. Recent studies suggest a potential link between abnormal levels of certain D-amino acids and some serious health impairments, including schizophrenia, amyotrophic lateral sclerosis, and potentially cancer. Although indistinguishable in their scalar properties, the chirality of a molecule reveals itself in interaction with the surrounding of a certain chirality, or more generally, a broken mirror-symmetry. In this work, we report on a system for chiral molecule detection, in which the mirror-symmetry is doubly broken, first by asymmetric structuring a nanopatterned plasmonic surface than by the incidence of circularly polarized light (CPL). In this system, the incident circularly-polarized light induces a surface plasmon polariton (SPP) wave, propagating along the asymmetric plasmonic surface. This SPP field itself is chiral, evanescently bound to a near-field zone on the surface (~10nm thick), but with an amplitude greatly intensified (by up to 104) over that of the incident light. It hence probes just the molecules on the surface instead of those in the volume. In coupling to molecules along its path on the surface, the chiral SPP wave favors one chirality over the other, allowing for chirality detection via the change in an optical rectification current measured at the edges of the sample. The asymmetrically structured surface converts the high-frequency electron plasmonic-oscillations in the SPP wave into a net DC drift current that can be measured at the edge of the sample via the mechanism of optical rectification. The measured results validate these design concepts and principles. The observed optical rectification current exhibits a clear differentiation between a pair of enantiomers. Experiments were performed by focusing a 1064nm CW laser light at the sample - a gold grating microchip submerged in an approximately 1.82M solution of either L-arabinose or D-arabinose and water. A measurement of the current output was then recorded under both rights and left circularly polarized lights. Measurements were recorded at various angles of incidence to optimize the coupling between the spin-momentums of the incident light and that of the SPP, that is, spin-momentum locking. In order to suppress the background, the values of the photocurrent for the right CPL are subtracted from those for the left CPL. Comparison between the two arabinose enantiomers reveals a preferential signal response of one enantiomer to left CPL and the other enantiomer to right CPL. In sum, this work reports on the first experimental evidence of the feasibility of chiral molecule detection via optical rectification in a metal meta-grating. This nanoscale interfaced electrical detection technology is advantageous over other detection methods due to its size, cost, ease of use, and integration ability with read-out electronic circuits for data processing and interpretation.

Keywords: Chirality, detection, molecule, spin

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Authors: Kyoung Lee

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Numerous aerobic bacteria have the ability to form multicellular communities on the surface layer of the air-liquid (A-L) interface as a biofilm called a pellicle. Pellicles occupied at the A-L interface will benefit from the utilization of oxygen from air and nutrient from liquid. Buoyancy of cells can be obtained by high surface tension at the A-L interface. Thus, formation of pellicles is an adaptive advantage in utilization of excess nutrients in the standing culture where oxygen depletion is easily set up due to rapid cell growth. In natural environments, pellicles are commonly observed on the surface of lake or pond contaminated with pollutants. Previously, we have shown that when cultured in standing LB media an alkylphenol-degrading bacteria Pseudomonas alkylphenolia KL28 forms pellicles in a diameter of 0.3-0.5 mm with a thickness of ca 40 µm. The pellicles have unique features for possessing flatness and unusual rigidity. In this study, the biogenesis of the circular pellicles has been investigated by observing the cell organization at early stages of pellicle formation and cell arrangements in pellicle, providing a clue for highly organized cellular arrangement to be adapted to the air-liquid niche. Here, we first monitored developmental patterns of pellicle from monolayer to multicellular organization. Pellicles were shaped by controlled growth of constituent cells which accumulate extracellular polymeric substance. The initial two-dimensional growth was transited to multilayers by a constraint force of accumulated self-produced extracellular polymeric substance. Experiments showed that pellicles are formed by clonal growth and even with knock-out of genes for flagella and pilus formation. In contrast, the mutants in the epm gene cluster for alginate-like polymer biosynthesis were incompetent in cell alignment for initial two-dimensional growth of pellicles. Electron microscopic and confocal laser scanning microscopic studies showed that the fully matured structures are highly packed by matrix-encased cells which have special arrangements. The cells on the surface of the pellicle lie relatively flat and inside longitudinally cross packed. HPLC analysis of the extrapolysaccharide (EPS) hydrolysate from the colonies from LB agar showed a composition with L-fucose, L-rhamnose, D-galactosamine, D-glucosamine, D-galactose, D-glucose, D-mannose. However, that from pellicles showed similar neutral and amino sugar profile but missing galactose. Furthermore, uronic acid analysis of EPS hydrolysates by HPLC showed that mannuronic acid was detected from pellicles not from colonies, indicating the epm-derived polymer is critical for pellicle formation as proved by the epm mutants. This study verified that for the circular pellicle architecture P. alkylphenolica KL28 cells utilized EPS building blocks different from that used for colony construction. These results indicate that P. alkylphenolica KL28 is a clever architect that dictates unique cell arrangements with selected EPS matrix material to construct sophisticated building, circular biofilm pellicles.

Keywords: biofilm, matrix, pellicle, pseudomonas

Procedia PDF Downloads 136

Authors: Parisa Shirzadeh

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Drug delivery systems in which drugs are traditionally used, multi-stage and at specified intervals by patients, do not meet the needs of the world's up-to-date drug delivery. In today's world, we are dealing with a huge number of recombinant peptide and protean drugs and analogues of hormones in the body, most of which are made with genetic engineering techniques. Most of these drugs are used to treat critical diseases such as cancer. Due to the limitations of the traditional method, researchers sought to find ways to solve the problems of the traditional method to a large extent. Following these efforts, controlled drug release systems were introduced, which have many advantages. Using controlled release of the drug in the body, the concentration of the drug is kept at a certain level, and in a short time, it is done at a higher rate. Graphene is a natural material that is biodegradable, non-toxic, and natural compared to carbon nanotubes; its price is lower than carbon nanotubes and is cost-effective for industrialization. On the other hand, the presence of highly effective surfaces and wide surfaces of graphene plates makes it more effective to modify graphene than carbon nanotubes. Graphene oxide is often synthesized using concentrated oxidizers such as sulfuric acid, nitric acid, and potassium permanganate based on Hummer 1 method. In comparison with the initial graphene, the resulting graphene oxide is heavier and has carboxyl, hydroxyl, and epoxy groups. Therefore, graphene oxide is very hydrophilic and easily dissolves in water and creates a stable solution. On the other hand, because the hydroxyl, carboxyl, and epoxy groups created on the surface are highly reactive, they have the ability to work with other functional groups such as amines, esters, polymers, etc. Connect and bring new features to the surface of graphene. In fact, it can be concluded that the creation of hydroxyl groups, Carboxyl, and epoxy and in fact graphene oxidation is the first step and step in creating other functional groups on the surface of graphene. Chitosan is a natural polymer and does not cause toxicity in the body. Due to its chemical structure and having OH and NH groups, it is suitable for binding to graphene oxide and increasing its solubility in aqueous solutions. Graphene oxide (GO) has been modified by chitosan (CS) covalently, developed for control release of doxorubicin (DOX). In this study, GO is produced by the hummer method under acidic conditions. Then, it is chlorinated by oxalyl chloride to increase its reactivity against amine. After that, in the presence of chitosan, the amino reaction was performed to form amide transplantation, and the doxorubicin was connected to the carrier surface by π-π interaction in buffer phosphate. GO, GO-CS, and GO-CS-DOX characterized by FT-IR, RAMAN, TGA, and SEM. The ability to load and release is determined by UV-Visible spectroscopy. The loading result showed a high capacity of DOX absorption (99%) and pH dependence identified as a result of DOX release from GO-CS nanosheet at pH 5.3 and 7.4, which show a fast release rate in acidic conditions.

Keywords: graphene oxide, chitosan, nanosheet, controlled drug release, doxorubicin

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Authors: Lijuan Li

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Nitric oxide (NO) has become a fascinating entity in biological chemistry over the past few years. It is a gaseous lipophilic radical molecule that plays important roles in several physiological and pathophysiological processes in mammals, including activating the immune response, serving as a neurotransmitter, regulating the cardiovascular system, and acting as an endothelium-derived relaxing factor. NO functions in eukaryotes both as a signal molecule at nanomolar concentrations and as a cytotoxic agent at micromolar concentrations. The latter arises from the ability of NO to react readily with a variety of cellular targets leading to thiol S-nitrosation, amino acid N-nitrosation, and nitrosative DNA damage. Nitric oxide can readily bind to metals to give metal-nitrosyl (M-NO) complexes. Some of these species are known to play roles in biological NO storage and transport. These complexes have different biological, photochemical, or spectroscopic properties due to distinctive structural features. These recent discoveries have spawned a great interest in the development of transition metal complexes containing NO, particularly its iron complexes that are central to the role of nitric oxide in the body. Spectroscopic evidence would appear to implicate species of “Fe(NO)2+” type in a variety of processes ranging from polymerization, carcinogenesis, to nitric oxide stores. Our research focuses on isolation and structural studies of non-heme iron nitrosyls that mimic biologically active compounds and can potentially be used for anticancer drug therapy. We have shown that reactions between Fe(NO)2(CO)2 and a series of imidazoles generated new non-heme iron nitrosyls of the form Fe(NO)2(L)2 [L = imidazole, 1-methylimidazole, 4-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, and L-histidine] and a tetrameric cluster of [Fe(NO)2(L)]4 (L=Im, 4-MeIm, BzIm, and Me2BzIm), resulted from the interactions of Fe(NO)2 with a series of substituted imidazoles was prepared. Recently, a series of sulfur bridged iron di nitrosyl complexes with the general formula of [Fe(µ-RS)(NO)2]2 (R = n-Pr, t-Bu, 6-methyl-2-pyridyl, and 4,6-dimethyl-2-pyrimidyl), were synthesized by the reaction of Fe(NO)2(CO)2 with thiols or thiolates. Their structures and properties were studied by IR, UV-vis, 1H-NMR, EPR, electrochemistry, X-ray diffraction analysis and DFT calculations. IR spectra of these complexes display one weak and two strong NO stretching frequencies (νNO) in solution, but only two strong νNO in solid. DFT calculations suggest that two spatial isomers of these complexes bear 3 Kcal energy difference in solution. The paramagnetic complexes [Fe2(µ-RS)2(NO)4]-, have also been investigated by EPR spectroscopy. Interestingly, the EPR spectra of complexes exhibit an isotropic signal of g = 1.998 - 2.004 without hyperfine splitting. The observations are consistent with the results of calculations, which reveal that the unpaired electron dominantly delocalize over the two sulfur and two iron atoms. The difference of the g values between the reduced form of iron-sulfur clusters and the typical monomeric di nitrosyl iron complexes is explained, for the first time, by of the difference in unpaired electron distributions between the two types of complexes, which provides the theoretical basis for the use of g value as a spectroscopic tool to differentiate these biologically active complexes.

Keywords: di nitrosyl iron complex, metal nitrosyl, non-heme iron, nitric oxide

Procedia PDF Downloads 282

Authors: Akanksha Agrawal, Richa Rathor, Geetha Suryakumar

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Muscle represents about ¾ of the body mass, and a healthy muscular system is required for human performance. A healthy muscular system is dynamically balanced via the catabolic and anabolic process. High altitude associated hypoxia altered this redox balance via producing reactive oxygen and nitrogen species that ultimately modulates protein structure and function, hence, disrupts proteostasis or protein homeostasis. The mechanism by which proteostasis is clinched includes regulated protein translation, protein folding, and protein degradation machinery. Perturbation in any of these mechanisms could increase proteome imbalance in the cellular processes. Altered proteostasis in skeletal muscle is likely to be responsible for contributing muscular atrophy in response to hypoxia. Therefore, we planned to elucidate the mechanism involving altered proteostasis leading to skeletal muscle atrophy under chronic hypobaric hypoxia. Material and Methods-Male Sprague Dawley rats weighing about 200-220 were divided into five groups - Control (Normoxic animals), 1d, 3d, 7d and 14d hypobaric hypoxia exposed animals. The animals were exposed to simulated hypoxia equivalent to 282 torr pressure (equivalent to an altitude of 7620m, 8% oxygen) at 25°C. On completion of chronic hypobaric hypoxia (CHH) exposure, rats were sacrificed, muscle was excised and biochemical, histopathological and protein synthesis signaling were studied. Results-A number of changes were observed with the CHH exposure time period. ROS was increased significantly on 07 and 14 days which were attributed to protein oxidation via damaging muscle protein structure by oxidation of amino acids moiety. The oxidative damage to the protein further enhanced the various protein degradation pathways. Calcium activated cysteine proteases and other intracellular proteases participate in protein turnover in muscles. Therefore, we analysed calpain and 20S proteosome activity which were noticeably increased at CHH exposure as compared to control group representing enhanced muscle protein catabolism. Since inflammatory markers (myokines) affect protein synthesis and triggers degradation machinery. So, we determined inflammatory pathway regulated under hypoxic environment. Other striking finding of the study was upregulation of Akt/PKB translational machinery that was increased on CHH exposure. Akt, p-Akt, p70 S6kinase, and GSK- 3β expression were upregulated till 7d of CHH exposure. Apoptosis related markers, caspase-3, caspase-9 and annexin V was also increased on CHH exposure. Conclusion: The present study provides evidence of disrupted proteostasis under chronic hypobaric hypoxia. A profound loss of muscle mass is accompanied by the muscle damage leading to apoptosis and cell death under CHH. These cellular stress response pathways may play a pivotal role in hypobaric hypoxia induced skeletal muscle atrophy. Further research in these signaling pathways will lead to development of therapeutic interventions for amelioration of hypoxia induced muscle atrophy.

Keywords: Akt/PKB translational machinery, chronic hypobaric hypoxia, muscle atrophy, protein degradation

Procedia PDF Downloads 238

Authors: Andre Pastori D'Aurea, Lauriston Bertelli Feranades, Luis Eduardo Ferreira, Leandro Dias Pinto, Fabiana Ayumi Shiozaki

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The aim of this study was to improve the production of beef cattle on tropical pasture without harming this environment. On tropical pastures, cattle's live weight gain is lower than feedlot, and forage production is seasonable, changing from season to season. Thus, concerned with sustainable livestock production, the Premix Company has developed strategies to improve the production of beef cattle on tropical pasture to ensure sustainability of welfare and production. There are two important principles in this productivity system: 1) increase individual gains with use of better supplementation and 2) increase the productivity units with better forage quality like corn silage or other forms of forage conservations, actually used only in winter, and adding natural additives in the diet. This production system was applied from June 2017 to May 2018 in the Research Center of Premix Company, Patrocínio Paulista, São Paulo State, Brazil. The area used had 9 hectares of pasture of Brachiaria brizantha. 36 steers Nellore were evaluated for one year. The initial weight was 253 kg. The parameters used were daily average gain and gain per area. This indicated the corrections to be made and helped design future fertilization. In this case, we fertilized the pasture with 30 kg of nitrogen per animal divided into two parts. The diet was pasture and protein-energy supplements (0.4% of live weight). The supplement used was added with natural additive Fator P® – Premix Company). Fator P® is an additive composed by amino acids (lysine, methionine and tyrosine, 16400, 2980 and 3000 mg.kg-1 respectively), minerals, probiotics (Saccharomyces cerevisiae, 7 x 10E8 CFU.kg-1) and essential fatty acids (linoleic and oleic acids, 108.9 and 99g.kg-1 respectively). Due to seasonal changes, in the winter we supplemented the diet by increasing the offer of forage, supplementing with maize silage. It was offered 1% of live weight in silage corn and 0.4% of the live weight in protein-energetic supplements with additive Fator P ®. At the end of the period, the productivity was calculated by summing the individual gains for the area used. The average daily gain of the animals were 693 grams per day and was produced 1.005 kg /hectare/year. This production is about 8 times higher than the average of Brazilian meat national production. To succeed in this project, it is necessary to increase the gains per area, so it is necessary to increase the capacity per area. Pasture management is very important to the project's success because the dietary decisions were taken from the quantity and quality of the forage. We, therefore, recommend the use of animals in the growth phase because the response to supplementation is greater in that phase and we can allocate more animals per area. This system's carbon footprint reduces emissions by 61.2 percent compared to the Brazilian average. This beef cattle production system can be efficient and environmentally friendly to the natural. Another point is that bovines will benefit from their natural environment without competing or having an impact on human food production.

Keywords: cattle production, environment, pasture, sustainability

Procedia PDF Downloads 117

Authors: Beihai Xiong, Dengke Hua, Wouter Hendriks, Wilbert Pellikaan

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To improve the energy status of dairy cows in the early lactation, lots of jobs have been done on adjusting the starch to fiber ratio in the diet. As a complex ecosystem, the rumen contains a large population of microorganisms which plays a crucial role in feed degradation. Further study on the microbiota alterations and metabolic changes under different dietary energy sources is essential and valuable to better understand the function of the ruminal microorganisms and thereby to optimize the rumen function and enlarge feed efficiency. The present study will focus on the effects of two glucogenic diets (G: ground corn and corn silage; S: steam-flaked corn and corn silage) and a lipogenic diet (L: sugar beet pulp and alfalfa silage) on rumen fermentation, gas production, the ruminal microbiota and metabolome, and also their correlations in vitro. The gas production was recorded consistently, and the gas volume and producing rate at times 6, 12, 24, 48 h were calculated separately. The fermentation end-products were measured after fermenting for 48 h. The ruminal bacteria and archaea communities were determined by 16S RNA sequencing technique, the metabolome profile was tested through LC-MS methods. Compared to the diet G and S, the L diet had a lower dry matter digestibility, propionate production, and ammonia-nitrogen concentration. The two glucogenic diets performed worse in controlling methane and lactic acid production compared to the L diet. The S diet produced the greatest cumulative gas volume at any time points during incubation compared to the G and L diet. The metabolic analysis revealed that the lipid digestion was up-regulated by the diet L than other diets. On the subclass level, most metabolites belonging to the fatty acids and conjugates were higher, but most metabolites belonging to the amino acid, peptides, and analogs were lower in diet L than others. Differences in rumen fermentation characteristics were associated with (or resulting from) changes in the relative abundance of bacterial and archaeal genera. Most highly abundant bacteria were stable or slightly influenced by diets, while several amylolytic and cellulolytic bacteria were sensitive to the dietary changes. The L diet had a significantly higher number of cellulolytic bacteria, including the genera of Ruminococcus, Butyrivibrio, Eubacterium, Lachnospira, unclassified Lachnospiraceae, and unclassified Ruminococcaceae. The relative abundances of amylolytic bacteria genera including Selenomonas_1, Ruminobacter, and Succinivibrionaceae_UCG-002 were higher in diet G and S. These affected bacteria was also proved to have high associations with certain metabolites. The Selenomonas_1 and Succinivibrionaceae_UCG-002 may contribute to the higher propionate production in the diet G and S through enhancing the succinate pathway. The results indicated that the two glucogenic diets had a greater extent of gas production, a higher dry matter digestibility, and produced more propionate than diet L. The steam-flaked corn did not show a better performance on fermentation end-products than ground corn. This study has offered a deeper understanding of ruminal microbial functions which could assistant the improvement in rumen functions and thereby in the ruminant production.

Keywords: gas production, metabolome, microbiota, rumen fermentation

Procedia PDF Downloads 126

Authors: Siddhant Sethi, Yasuharu Takashima, Shigetaka Nakamura, Kenzo Fujimoto

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Site-directed mutagenesis is a renowned technique to introduce specific mutations in the genome. To achieve site-directed mutagenesis, many chemical and enzymatic approaches have been reported in the past like disulphite induced genome editing, CRISPR-Cas9, TALEN etc. The chemical methods are invasive whereas the enzymatic approaches are time-consuming and expensive. Most of these techniques are unusable in the cellular application due to their toxicity and other limitations. Photo-chemical cytosine deamination, introduced in 2010, is one of the major technique for enzyme-free single-point mutation of cytosine to uracil in DNA and RNA, wherein, 3-cyanovinylcarbazole nucleoside (CNVK) containing oligodeoxyribonucleotide (ODN) having CNVK at -1 position to that of target cytosine is reversibly crosslinked to target DNA strand using 366 nm and then incubated at 90ºC to accommodate deamination. This technique is superior to enzymatic methods of site-directed mutagenesis but has a disadvantage that it requires the use of high temperature for the deamination step which restricts its applicability in the in vivo applications. This study has been focused on improving the technique by reducing the temperature required for deamination. Firstly, the photo-cross-linker, CNVK has been modified by replacing cyano group attached to vinyl group with methyl ester (OMeVK), amide (NH2VK), and carboxylic acid (OHVK) to observe the acceleration in the deamination of target cytosine cross-linked to vinylcarbazole derivative. Among the derivatives, OHVK has shown 2 times acceleration in deamination reaction as compared to CNVK, while the other two derivatives have shown deceleration towards deamination reaction. The trend of rate of deamination reaction follows the same order as that of hydrophilicity of the vinylcarbazole derivatives. OHVK being most hydrophilic has shown highest acceleration while OMeVK is least hydrophilic has proven to be least active for deamination. Secondly, in the related study, the counter-base of the target cytosine, guanine has been replaced by inosine, 2-aminopurine, nebularine, and 5-nitroindole having distinct hydrogen bonding patterns with target cytosine. Among the ODNs with these counter bases, ODN with inosine has shown 12 fold acceleration towards deamination of cytosine cross-linked to CNVK at physiological conditions as compared to guanosine. Whereas, when 2-aminopurine, nebularine, and 5-nitroindole were used, no deamination reaction took place. It can be concluded that inosine has potential to be used as the counter base of target cytosine for the CNVK mediated photo-cross-linking induced deamination of cytosine. The increase in rate of deamination reaction has been attributed to pattern and number of hydrogen bonding between the cytosine and counter base. One of the important factor is presence of hydrogen bond between exo-cyclic amino group of cytosine and the counter base. These results will be useful for development of more efficient technique for site-directed mutagenesis for C → U transformations in the DNA/RNA which might be used in the living system for treatment of various genetic disorders and genome engineering for making designer and non-native proteins.

Keywords: C to U transformation, DNA editing, genome engineering, ultra-fast photo-cross-linking

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Authors: Srijata Mitra, Mobina Parveen, Pranab Roy, Narayan Chandra Chattopadhyay

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Chlorinated hydrocarbon can be a major pollution problem in groundwater as well as soil. Many people interact with these chemicals on daily accidentally or by professionally in the laboratory. One of the most common sources for Chlorinated hydrocarbon contamination of soil and groundwater are industrial effluents. The wide use and discharge of Trichloroethylene (TCE), a volatile chlorohydrocarbon from chemical industry, led to major water pollution in rural areas. TCE is an mainly used as an industrial metal degreaser in industries. Biotransformation of TCE to the potent carcinogen vinyl chloride (VC) by consortia of anaerobic bacteria might have role for the above purpose. For these reasons, the aim of current study was to isolate and characterized the genes involved in TCE metabolism and also to investigate the in silico study of those genes. To our knowledge, only one aromatic dioxygenase system, the toluene dioxygenase in Pseudomonas putida F1 has been shown to be involved in TCE degradation. This is first instance where Bacillus cereus group being used in biodegradation of trichloroethylene. A novel bacterial strain 2479 was isolated from oil depot site at Rajbandh, Durgapur (West Bengal, India) by enrichment culture technique. It was identified based on polyphasic approach and ribotyping. The bacterium was gram positive, rod shaped, endospore forming and capable of degrading trichloroethylene as the sole carbon source. On the basis of phylogenetic data and Fatty Acid Methyl Ester Analysis, strain 2479 should be placed within the genus Bacillus and species cereus. However, the present isolate (strain 2479) is unique and sharply different from the usual Bacillus strains in its biodegrading nature. Fujiwara test was done to estimate that the strain 2479 could degrade TCE efficiently. The gene for TCE biodegradation was PCR amplified from genomic DNA of Bacillus cereus 2479 by using todC1 gene specific primers. The 600bp amplicon was cloned into expression vector pUC I8 in the E. coli host XL1-Blue and expressed under the control of lac promoter and nucleotide sequence was determined. The gene sequence was deposited at NCBI under the Accession no. GU183105. In Silico approach involved predicting the physico-chemical properties of deduced Tce1 protein by using ProtParam tool. The tce1 gene contained 342 bp long ORF encoding 114 amino acids with a predicted molecular weight 12.6 kDa and the theoretical pI value of the polypeptide was 5.17, molecular formula: C559H886N152O165S8, total number of atoms: 1770, aliphatic index: 101.93, instability index: 28.60, Grand Average of Hydropathicity (GRAVY): 0.152. Three differentially expressed proteins (97.1, 40 and 30 kDa) were directly involved in TCE biodegradation, found to react immunologically to the antibodies raised against TCE inducible proteins in Western blot analysis. The present study suggested that cloned gene product (TCE1) was capable of degrading TCE as verified chemically.

Keywords: cloning, Bacillus cereus, in silico analysis, TCE

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Authors: Mohammed Sabbah, Prospero Di Pierro, Raffaele Porta

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Protein-based edible films and coatings have attracted an increasing interest in recent years since they might be used to protect pharmaceuticals or improve the shelf life of different food products. Among them, several plant proteins represent an abundant, inexpensive and renewable raw source. These natural biopolymers are used as film forming agents, being able to form intermolecular linkages by various interactions. However, without the addition of a plasticizing agent, many biomaterials are brittle and, consequently, very difficult to be manipulated. Plasticizers are generally small and non-volatile organic additives used to increase film extensibility and reduce its crystallinity, brittleness and water vapor permeability. Plasticizers normally act by decreasing the intermolecular forces along the polymer chains, thus reducing the relative number of polymer-polymer contacts, producing a decrease in cohesion and tensile strength and thereby increasing film flexibility allowing its deformation without rupture. The most commonly studied plasticizers are polyols, like glycerol (GLY) and some mono or oligosaccharides. In particular, GLY not only increases film extensibility but also migrates inside the film network often causing the loss of desirable mechanical properties of the material. Therefore, replacing GLY with a different plasticizer might help to improve film characteristics allowing potential industrial applications. To improve film properties, it seemed of interest to test as plasticizers some cationic small molecules like polyamines (PAs). Putrescine, spermidine (SPD), and spermine are PAs widely distributed in nature and of particular interest for their biological activities that may have some beneficial health effects. Since PAs contains amino instead of hydroxyl functional groups, they are able to trigger ionic interactions with negatively charged proteins. Bitter vetch (Vicia ervilia; BV) is an ancient grain legume crop, originated in the Mediterranean region, which can be found today in many countries around the world. This annual Vicia genus shows several favorable features, being their seeds a cheap and abundant protein source. The main objectives of this study were to investigate the effect of different concentrations of SPD on the mechanical and permeability properties of films prepared with native or heat denatured BV proteins in the presence of different concentrations of SPD and/or GLY. Therefore, a BV seed protein concentrate (BVPC), containing about 77% proteins, was used to prepare film forming solutions (FFSs), whereas GLY and SPD were added as film plasticizers, either singly or in combination, at various concentrations. Since a primary plasticizer is generally defined as a molecule that when added to a material makes it softer, more flexible and easier to be processed, our findings lead to consider SPD as a possible primary plasticizer of protein-based films. In fact, the addition of millimolar concentrations of SPD to BVPC FFS allowed obtaining handleable biomaterials with improved properties. Moreover, SPD can be also considered as a secondary plasticizer, namely an 'extender', because of its ability even to enhance the plasticizing performance of GLY. In conclusion, our studies indicate that innovative edible protein-based films and coatings can be obtained by using PAs as new plasticizers.

Keywords: edible films, glycerol, plasticizers, polyamines, spermidine

Procedia PDF Downloads 174

Authors: Anahita Mortazavi Manesh, Mojtaba Bagherzadeh

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Metalloporphyrins are well known to mimic the activity of monooxygenase enzymes. In this regard, metalloporphyrin complexes have been largely employed as valuable biomimetic catalysts, owing to the critical roles they play in oxygen transfer processes in catalytic oxidation reactions. Investigating in this area is based on different strategies to design selective, stable and high turnover catalytic systems. Immobilization of expensive metalloporphyrin catalysts onto supports appears to be a good way to improve their stability, selectivity and the catalytic performance because of the support environment and other advantages with respect to recovery, reuse. In other words, supporting metalloporphyrins provides a physical separation of active sites, thus minimizing catalyst self-destruction and dimerization of unhindered metalloporphyrins. Furthermore, heterogeneous catalytic oxidations have become an important target since their process are used in industry, helping to minimize the problems of industrial waste treatment. Hence, the immobilization of these biomimetic catalysts is much desired. An attractive approach is the preparation of the heterogeneous catalyst involves immobilization of complexes on silica coated magnetic nano-particles. Fe3O4@SiO2 magnetic nanoparticles have been studied extensively due to their superparamagnetism property, large surface area to volume ratio and easy functionalization. Using heterogenized homogeneous catalysts is an attractive option to facile separation of catalyst, simplified product work-up and continuity of catalytic system. Homogeneous catalysts immobilized on magnetic nanoparticles (MNPs) surface occupy a unique position due to combining the advantages of both homogeneous and heterogeneous catalysts. In addition, superparamagnetic nature of MNPs enable very simple separation of the immobilized catalysts from the reaction mixture using an external magnet. In the present work, an efficient heterogeneous catalyst was prepared by immobilizing manganese porphyrin on functionalized magnetic nanoparticles through the amino propyl linkage. The prepared catalyst was characterized by elemental analysis, FT-IR spectroscopy, X-ray powder diffraction, atomic absorption spectroscopy, UV-Vis spectroscopy, and scanning electron microscopy. Application of immobilized metalloporphyrin in the oxidation of various organic substrates was explored using Gas chromatographic (GC) analyses. The results showed that the supported Mn-porphyrin catalyst (Fe3O4@SiO2-NH2@MnPor) is an efficient and reusable catalyst in oxidation reactions. Our catalytic system exhibits high catalytic activity in terms of turnover number (TON) and reaction conditions. Leaching and recycling experiments revealed that nanocatalyst can be recovered several times without loss of activity and magnetic properties. The most important advantage of this heterogenized catalytic system is the simplicity of the catalyst separation in which the catalyst can be separated from the reaction mixture by applying a magnet. Furthermore, the separation and reuse of the magnetic Fe3O4 nanoparticles were very effective and economical.

Keywords: Fe3O4 nanoparticle, immobilized metalloporphyrin, magnetically separable nanocatalyst, oxidation reactions

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Authors: Su Mon Saw, Joe Rothnagel

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Short open reading frames (sORFs) located in 5’UTR of mRNAs are known as uORFs. Characterization of uORF-encoded peptides (uPEPs) i.e., a subset of short open reading frame encoded peptides (sPEPs) and their translation regulation lead to understanding of causes of genetic disease, proteome complexity and development of treatments. Existence of uORFs within cellular proteome could be detected by LC-MS/MS. The ability of uORF to be translated into uPEP and achievement of uPEP identification will allow uPEP’s characterization, structures, functions, subcellular localization, evolutionary maintenance (conservation in human and other species) and abundance in cells. It is hypothesized that a subset of sORFs are translatable and that their encoded sPEPs are functional and are endogenously expressed contributing to the eukaryotic cellular proteome complexity. This project aimed to investigate whether sORFs encode functional peptides. Liquid chromatography-mass spectrometry (LC-MS) and bioinformatics were thus employed. Due to probable low abundance of sPEPs and small in sizes, the need for efficient peptide enrichment strategies for enriching small proteins and depleting the sub-proteome of large and abundant proteins is crucial for identifying sPEPs. Low molecular weight proteins were extracted using SDS-PAGE from Human Embryonic Kidney (HEK293) cells and Strong Cation Exchange Chromatography (SCX) from secreted HEK293 cells. Extracted proteins were digested by trypsin to peptides, which were detected by LC-MS/MS. The MS/MS data obtained was searched against Swiss-Prot using MASCOT version 2.4 to filter out known proteins, and all unmatched spectra were re-searched against human RefSeq database. ProteinPilot v5.0.1 was used to identify sPEPs by searching against human RefSeq, Vanderperre and Human Alternative Open Reading Frame (HaltORF) databases. Potential sPEPs were analyzed by bioinformatics. Since SDS PAGE electrophoresis could not separate proteins <20kDa, this could not identify sPEPs. All MASCOT-identified peptide fragments were parts of main open reading frame (mORF) by ORF Finder search and blastp search. No sPEP was detected and existence of sPEPs could not be identified in this study. 13 translated sORFs in HEK293 cells by mass spectrometry in previous studies were characterized by bioinformatics. Identified sPEPs from previous studies were <100 amino acids and <15 kDa. Bioinformatics results showed that sORFs are translated to sPEPs and contribute to proteome complexity. uPEP translated from uORF of SLC35A4 was strongly conserved in human and mouse while uPEP translated from uORF of MKKS was strongly conserved in human and Rhesus monkey. Cross-species conserved uORFs in association with protein translation strongly suggest evolutionary maintenance of coding sequence and indicate probable functional expression of peptides encoded within these uORFs. Translation of sORFs was confirmed by mass spectrometry and sPEPs were characterized with bioinformatics.

Keywords: bioinformatics, HEK293 cells, liquid chromatography-mass spectrometry, ProteinPilot, Strong Cation Exchange Chromatography, SDS-PAGE, sPEPs

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Authors: Ernest Moles, Patricia Urbán, María Belén Jiménez-Díaz, Sara Viera-Morilla, Iñigo Angulo-Barturen, Maria Antònia Busquets, Xavier Fernàndez-Busquets

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Bearing in mind the absence of an effective vaccine against malaria and its severe clinical manifestations causing nearly half a million deaths every year, this disease represents nowadays a major threat to life. Besides, the basic rationale followed by currently marketed antimalarial approaches is based on the administration of drugs on their own, promoting the emergence of drug-resistant parasites owing to the limitation in delivering drug payloads into the parasitized erythrocyte high enough to kill the intracellular pathogen while minimizing the risk of causing toxic side effects to the patient. Such dichotomy has been successfully addressed through the specific delivery of immunoliposome (iLP)-encapsulated antimalarials to Plasmodium falciparum-infected red blood cells (pRBCs). Unfortunately, this strategy has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here, we show that encapsulation efficiencies reaching >96% can be achieved for the weakly basic drugs chloroquine (CQ) and primaquine using the pH gradient active loading method in liposomes composed of neutrally charged, saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the intracellular delivery of drugs not affecting the erythrocytic metabolism. Using this strategy, we have obtained unprecedented nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Polyethylene glycol-coated liposomes conjugated with monoclonal antibodies specific for the erythrocyte surface protein glycophorin A (anti-GPA iLP) were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5 μM total lipid in the culture, with >95% of added iLPs retained into the cells. When exposed for only 15 min to P. falciparum in vitro cultures synchronized at early stages, free CQ had no significant effect over parasite viability up to 200 nM drug, whereas iLP-encapsulated 50 nM CQ completely arrested its growth. Furthermore, when assayed in vivo in P. falciparum-infected humanized mice, anti-GPA iLPs cleared the pathogen below detectable levels at a CQ dose of 0.5 mg/kg. In comparison, free CQ administered at 1.75 mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement in drug antimalarial efficacy is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.

Keywords: immunoliposomal nanoparticles, malaria, prophylactic-therapeutic polyvalent activity, targeted drug delivery

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Authors: Fatima Arrutia, Francisco Amador Riera

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The meat industry generates large volumes of blood as a result of meat processing. Several industrial procedures have been implemented in order to treat this by-product, but are focused on the production of low-value products, and in many cases, blood is simply discarded as waste. Besides, in addition to economic interests, there is an environmental concern due to bloodborne pathogens and other chemical contaminants found in blood. Consequently, there is a dire need to find extensive uses for blood that can be both applicable to industrial scale and able to yield high value-added products. Blood has been recognized as an important source of protein. The main blood serum protein in mammals is serum albumin. One of the top trends in food market is functional foods. Among them, bioactive peptides can be obtained from protein sources by microbiological fermentation or enzymatic and chemical hydrolysis. Bioactive peptides are short amino acid sequences that can have a positive impact on health when administered. The main drawback for bioactive peptide production is the high cost of the isolation, purification and characterization techniques (such as chromatography and mass spectrometry) that make unaffordable the scale-up. On the other hand, membrane technologies are very suitable to apply to the industry because they offer a very easy scale-up and are low-cost technologies, compared to other traditional separation methods. In this work, the possibility of obtaining bioactive peptide fractions from serum albumin by means of a simple procedure of only 2 steps (hydrolysis and membrane filtration) was evaluated, as an exploratory study for possible industrial application. The methodology used in this work was, firstly, a tryptic hydrolysis of serum albumin in order to release the peptides from the protein. The protein was previously subjected to a thermal treatment in order to enhance the enzyme cleavage and thus the peptide yield. Then, the obtained hydrolysate was filtered through a nanofiltration/ultrafiltration flat rig at three different pH values with two different membrane materials, so as to compare membrane performance. The corresponding permeates were analyzed by liquid chromatography-tandem mass spectrometry technology in order to obtain the peptide sequences present in each permeate. Finally, different concentrations of every permeate were evaluated for their in vitro antihypertensive and antioxidant activities though ACE-inhibition and DPPH radical scavenging tests. The hydrolysis process with the previous thermal treatment allowed achieving a degree of hydrolysis of the 49.66% of the maximum possible. It was found that peptides were best transmitted to the permeate stream at pH values that corresponded to their isoelectric points. Best selectivity between peptide groups was achieved at basic pH values. Differences in peptide content were found between membranes and also between pH values for the same membrane. The antioxidant activity of all permeates was high compared with the control only for the highest dose. However, antihypertensive activity was best for intermediate concentrations, rather than higher or lower doses. Therefore, although differences between them, all permeates were promising regarding antihypertensive and antioxidant properties.

Keywords: bioactive peptides, bovine serum albumin, hydrolysis, membrane filtration

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Authors: Rauan Sakenov, Peter Bukovics, Peter Gaszler, Veronika Tokacs-Kollar, Beata Bugyi

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FH2 (formin homology-2) domains of several proteins, collectively known as formins, including DAAM, DAAM1 and mDia1, promote G-actin nucleation and elongation. FH2 domains of these formins exist as oligomers. Chain dimerization by ring structure formation serves as a structural basis for actin polymerization function of FH2 domain. Proper single chain configuration and specific interactions between its various regions are necessary for individual chains to form a dimer functional in G-actin nucleation and elongation. FH1 and WH2 domain-containing formins were shown to behave as intrinsically disordered proteins. Thus, the aim of this research was to study structural dynamics of FH2 domain of DAAM. To investigate structural features of FH2 domain of DAAM, molecular dynamics simulation of chain A of FH2 domain of DAAM solvated in water box in 50 mM NaCl was conducted at temperatures from 293.15 to 353.15K, with VMD 1.9.2, NAMD 2.14 and Amber Tools 21 using 2z6e and 1v9d PDB structures of DAAM was obtained on I-TASSER webserver. Calcium and ATP bound G-actin 3hbt PDB structure was used as a reference protein with well-described structural dynamics of denaturation. Topology and parameter information of CHARMM 2012 additive all-atom force fields for proteins, carbohydrate derivatives, water and ions were used in NAMD 2.14 and ff19SB force field for proteins in Amber Tools 21. The systems were energy minimized for the first 1000 steps, equilibrated and produced in NPT ensemble for 1ns using stochastic Langevin dynamics and the particle mesh Ewald method. Our root-mean square deviation (RMSD) analysis of molecular dynamics of chain A of FH2 domains of DAAM revealed similar insignificant changes of total molecular average RMSD values of FH2 domain of these formins at temperatures from 293.15 to 353.15K. In contrast, total molecular average RMSD values of G-actin showed considerable increase at 328K, which corresponds to the denaturation of G-actin molecule at this temperature and its transition from native, ordered, to denatured, disordered, state which is well-described in the literature. RMSD values of lasso and tail regions of chain A of FH2 domain of DAAM exhibited higher than total molecular average RMSD at temperatures from 293.15 to 353.15K. These regions are functional in intra- and interchain interactions and contain highly conserved tryptophan residues of lasso region, highly conserved GNYMN sequence of post region and amino acids of the shell of hydrophobic pocket of the salt bridge between Arg171 and Asp321, which are important for structural stability and ordered state of FH2 domain of DAAM and its functions in FH2 domain dimerization. In conclusion, higher than total molecular average RMSD values of lasso and post regions of chain A of FH2 domain of DAAM may explain disordered state of FH2 domain of DAAM at temperatures from 293.15 to 353.15K. Finally, absence of marked transition, in terms of significant changes in average molecular RMSD values between native and denatured states of FH2 domain of DAAM at temperatures from 293.15 to 353.15K, can make it possible to attribute these formins to the group of intrinsically disordered proteins rather than to the group of intrinsically ordered proteins such as G-actin.

Keywords: FH2 domain, DAAM, formins, molecular modelling, computational biophysics

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Authors: Asma Ben Hmidene, Mizuho Hanaki, Kazuma Murakami, Kazuhiro Irie, Hiroko Isoda, Hideyuki Shigemori

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Type 2 diabetes mellitus (T2DM) and Alzheimer’s disease (AD) are characterized as a peripheral metabolic disorder and a degenerative disease of the central nervous system, respectively. It is now widely recognized that T2DM and AD share many pathophysiological features including glucose metabolism, increased oxidative stress and amyloid aggregation. Amyloid beta (Aβ) is the components of the amyloid deposits in the AD brain and while the component of the amyloidogenic peptide deposit in the pancreatic islets of Langerhans is identified as human islet amyloid polypeptide (hIAPP). These two proteins are originated from the amyloid precursor protein and have a high sequence similarity. Although the amino acid sequences of amyloidogenic proteins are diverse, they all adopt a similar structure in aggregates called cross-beta-spine. Add at that, extensive studies in the past years have found that like Aβ1-42, IAPP forms early intermediate assemblies as spherical oligomers, implicating that these oligomers possess a common folding pattern or conformation. These similarities can be used in the search for effective pharmacotherapy for DM, since potent therapeutic agents such as antioxidants with a catechol moiety, proved to inhibit Aβ aggregation, may play a key role in the inhibit the aggregation of hIAPP treatment of patients with DM. Tamarix gallica is one of the halophyte species having a powerful antioxidant system. Although it was traditionally used for the treatment of various liver metabolic disorders, there is no report about the use of this plant for the treatment or prevention of T2DM and AD. Therefore, the aim of this work is to investigate their protective effect towards T2DM and AD by isolation and identification of α-glucosidase inhibitors, with antioxidant potential, that play an important role in the glucose metabolism in diabetic patient, as well as, the polymerization of hIAPP and Aβ aggregation inhibitors. Structure-activity relationship study was conducted for both assays. And as for α-glucosidase inhibitors, their mechanism of action and their synergistic potential when applied with a very low concentration of acarbose were also suggesting that they can be used not only as α-glucosidase inhibitors but also be combined with established α-glucosidase inhibitors to reduce their adverse effect. The antioxidant potential of the purified substances was evaluated by DPPH and SOD assays. Th-T assay using 42-mer amyloid β-protein (Aβ42) for AD and hIAPP which is a 37-residue peptide secreted by the pancreatic β –cells for T2DM and Transmission electronic microscopy (TEM) were conducted to evaluate the amyloid aggragation of the actives substances. For α-glucosidase, p-NPG and glucose oxidase assays were performed for determining the inhibition potential and structure-activity relationship study. The Enzyme kinetic protocol was used to study the mechanism of action. From this research, it was concluded that polyphenols playing a role in the glucose metabolism and oxidative stress can also inhibit the amyloid aggregation, and that substances with a catechol and glucuronide moieties inhibiting amyloid-β aggregation, might be used to inhibit the aggregation of hIAPP.

Keywords: α-glucosidase inhibitors, amyloid aggregation inhibition, mechanism of action, polyphenols, structure activity relationship, synergistic potential, tamarix gallica

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Authors: Ram Sharma, Esha Chatterjee, Santosh Kumar Guru, Kunal Nepali

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A tractable anti-lung cancer agent was identified via the installation of a Ring C expanded synthetic analogue of the alkaloid vasicinone [7,8,9,10-tetrahydroazepino[2,1-b] quinazolin-12(6H)-one (TAZQ)] as a surface recognition part in the HDAC inhibitory three-component model. Noteworthy to mention that the candidature of TAZQ was deemed suitable for accommodation in HDAC inhibitory pharmacophore as per the results of the fragment recruitment process conducted by our laboratory. TAZQ was pinpointed through the fragment screening program as a synthetically flexible fragment endowed with some moderate cell growth inhibitory activity against the lung cancer cell lines, and it was anticipated that the use of the aforementioned fragment to generate hydroxamic acid functionality (zinc-binding motif) bearing HDAC inhibitors would boost the antitumor efficacy of TAZQ. Consistent with our aim of applying epigenetic targets to the treatment of lung cancer, a strikingly potent anti-lung cancer scaffold (compound 6) was pinpointed through a series of in-vitro experiments. Notably, the compounds manifested a magnificent activity profile against KRAS and EGFR mutant lung cancer cell lines (IC50 = 0.80 - 0.96 µM), and the effects were found to be mediated through preferential HDAC6 inhibition (IC50 = 12.9 nM). In addition to HDAC6 inhibition, the compounds also elicited HDAC1 and HDAC3 inhibitory activity with an IC50 value of 49.9 nM and 68.5 nM, respectively. The HDAC inhibitory ability of compound 6 was also confirmed from the results of the western blot experiment that revealed its potential to decrease the expression levels of HDAC isoforms (HDAC1, HDAC3, and HDAC6). Noteworthy to mention that complete downregulation of the HDAC6 isoform was exerted by compound 6 at 0.5 and 1 µM. Moreover, in another western blot experiment, treatment with hydroxamic acid 6 led to upregulation of H3 acK9 and α-Tubulin acK40 levels, ascertaining its inhibitory activity toward both the class I HDACs and Class II B HDACs. The results of other assays were also encouraging as treatment with compound 6 led to the suppression of the colony formation ability of A549 cells, induction of apoptosis, and increase in autophagic flux. In silico studies led us to rationalize the results of the experimental assay, and some key interactions of compound 6 with the amino acid residues of HDAC isoforms were identified. In light of the impressive activity spectrum of compound 6, a pH-responsive nanocarrier (hyaluronic acid-compound 6 nanoparticles) was prepared. The dialysis bag approach was used for the assessment of the nanoparticles under both normal and acidic circumstances, and the pH-sensitive nature of hyaluronic acid-compound 6 nanoparticles was confirmed. Delightfully, the nanoformulation was devoid of cytotoxicity against the L929 mouse fibroblast cells (normal settings) and exhibited selective cytotoxicity towards the A549 lung cancer cell lines. In a nutshell, compound 6 appears to be a promising adduct, and a detailed investigation of this compound might yield a therapeutic for the treatment of lung cancer.

Keywords: HDAC inhibitors, lung cancer, scaffold, hyaluronic acid, nanoparticles

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Authors: Milica B. Veljković, Milica B. Simović, Marija M. Ćorović, Ana D. Milivojević, Anja I. Petrov, Katarina M. Banjanac, Dejan I. Bezbradica

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In recent decades, the scientific community has recognized the growing importance of prebiotics, and therefore, numerous studies are focused on their economic production due to their low presence in natural resources. It has been confirmed that prebiotics is a source of energy for probiotics in the gastrointestinal tract (GIT) and enable their proliferation, consequently leading to the normal functioning of the intestinal microbiota. Also, products of their fermentation are short-chain fatty acids (SCFA), which play a key role in maintaining and improving the health not only of the GIT but also of the whole organism. Among several confirmed prebiotics, fructooligosaccharides (FOS) are considered interesting candidates for use in a wide range of products in the food industry. They are characterized as low-calorie and non-cariogenic substances that represent an adequate sugar substitute and can be considered suitable for use in products intended for diabetics. The subject of this research will be the production of FOS by transforming sucrose using a fructosyltransferase (FTase) present in commercial preparation Pectinex® Ultra SP-L, with special emphasis on the development of adequate FTase immobilization method that would enable selective isolation of the enzyme responsible for the synthesis of FOS from the complex enzymatic mixture. This would lead to considerable enzyme purification and allow its direct incorporation into different sucrose-based products without the fear that the action of the other hydrolytic enzymes may adversely affect the products' functional characteristics. Accordingly, the possibility of selective immobilization of the enzyme using support with primary amino groups, Purolite® A109, which was previously activated and modified using glutaraldehyde (GA), was investigated. In the initial phase of the research, the effects of individual immobilization parameters such as pH, enzyme concentration, and immobilization time were investigated to optimize the process using support chemically activated with 15% and 0.5% GA to form dimers and monomers, respectively. It was determined that highly active immobilized preparations (371.8 IU/g of support - dimer and 213.8 IU/g of support – monomer) were achieved under acidic conditions (pH 4) provided that an enzyme concentration was 50 mg/g of support after 7 h and 3 h, respectively. Bearing in mind the obtained results of the expressed activity, it is noticeable that the formation of dimers showed higher reactivity compared to the form of monomers. Also, in the case of support modification using 15% GA, the value of the ratio of FTase and pectinase (as dominant enzyme mixture component) activity immobilization yields was 16.45, indicating the high feasibility of selective immobilization of FTase on modified polystyrene resin. After obtaining immobilized preparations of satisfactory features, they were tested in a reaction of FOS synthesis under determined optimal conditions. The maximum FOS yields of approximately 50% of total carbohydrates in the reaction mixture were recorded after 21 h. Finally, it can be concluded that the examined immobilization method yielded highly active, stable and, more importantly, refined enzyme preparation that can be further utilized on a larger scale for the development of continual processes for FOS synthesis, as well as for modification of different sucrose-based mediums.

Keywords: chemical modification, fructooligosaccharides, glutaraldehyde, immobilization of fructosyltransferase

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