Search results for: melanin biosynthesis

Authors: Palaniyandi Karuppaiya, Hsin Sheng Tsay, Fang Chen

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Medicinal herbs do represent a huge and noteworthy reservoir for novel anticancer drugs discovery. Dysosma pleiantha (Hance) Woodson (Berberidaceae), one of the oldest traditional Chinese medicinal herb, highly prized by the mountain tribes of Taiwan and China for its medicinal properties contained pharmaceutically important antitumor compounds podophyllotoxin, quercetin and kaempferol. Among lignans, podophyllotoxin is an active antitumor compound and has now been modified to produce clinically useful drugs etoposide and teniposide. In recent years, natural populations of D. peliantha have declined considerably due to anthropogenic activities such as habitat destruction and commercial exploitation for medicinal applications. As to its overall conservation status, D. pleiantha has been ranked as threatened on the China Species Red List. In the present study, an efficient in vitro callus culture system of D. pleiantha was established on Gamborg’s medium with various combinations and concentrations of different auxins and cytokinins under dark condition. Best callus induction was recorded in 2 mg/L 2, 4 - Dichlorophenoxyacetic acid (2,4-D) along with 0.2 mg/L kinetin and the maximum callus proliferation was achieved at 1 mg/L 2,4-D. Among the explants tested, maximum callus induction (86 %) was achieved from tender leaves. Hence, in subsequent experiments, leaf callus was further investigated for suitable callus biomass and production level of anticancer compounds under the influence of different additives. A maximum fresh callus biomass (8.765 g) was recorded in callus proliferation medium contained 500 mg/L casein hydrolysate. High performance liquid chromatography results revealed that the addition of different concentrations of peptone (1, 2 and 4 g/L) in callus proliferation medium enhanced podophyllotoxin (16 fold), quercetin (12 fold) and kaempferol (5 fold) accumulation than control. Thus, the established in vitro callus culture under the influence of different additives may offer an alternative source of enhanced production of podophyllotoxin, kaempferol and quecertin without harming natural plant population.

Keywords: dysosma pleiantha, kaempferol, podophyllotoxin, quercetin

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Authors: Anil Kumar, Diwakar Aggarwal, M. Sudhakara Reddy

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The wood of Eucalyptus, Populus and bamboos are some important species used as raw material for the manufacture of pulp. However, higher levels of lignin pose a problem during Kraft pulping and yield of pulp is also lower. In order to increase the yield of pulp per unit wood and reduce the use of chemicals during kraft pulping it is important to reduce the lignin content and/or increase cellulose content in wood. Cellulose biosynthesis in wood takes place by the coordinated action of many enzymes. The two important enzymes are KORRIGAN and SUCROSE SYNTHASE. KORRIGAN (Endo-1,4--glucanase) is implicated in the process of editing growing cellulose chains and improvement of the crystallinity of produced cellulose, whereas SUCROSE SYNTHASE is involved in providing substrate (UDP-glucose) for growing cellulose chains. The present study was aimed at the cloning, characterization and overexpression of these genes in Eucalyptus and Populus. An efficient shoot organogenesis protocol from leaf explants taken from micro shoots of the species has been developed. Agrobacterium mediated genetic transformation using Agrobacterium tumefaciens strain EHA105 and LBA4404 harboring binary vector pBI121 was achieved. Both the genes were cloned from cDNA library of Populus deltoides. These were subsequently characterized using various bioinformatics tools. The cloned genes were then inserted into pBI121 under the CaMV35S promotors replacing GUS gene. The constructs were then mobilized into above strains of Agrobacterium and used for the transformation work. Subsequently, genetic transformation of these clones with target genes following already developed protocol is in progress. Four transgenic lines of Eucalyptus tereticornis overexpressing Korrigan gene under the strong constitutive promoters CaMV35S have been developed, which are being further evaluated. Work on development of more transgenic lines overexpressing these genes in Populus and Eucalyptus is also in progress. This presentation will focus on important developments in this direction.

Keywords: Eucalyptus tereticornis, genetic transformation, Kraft pulping Populus deltoides

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Authors: Mwafaq Ibdah, Mossab, Yahyaa, Dor Rachmany, Yoram Gerchman, Doron Holland, Liora Shaltiel-Harpaz

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Pear Psylla is the most important pest of pear in all pear-growing regions, in Asian, European, and the USA. Pear psylla damages pears in several ways: high-density populations of these insects can cause premature leaf and fruit drop, diminish plant growth, and reduce fruit size. In addition, their honeydew promotes sooty mold on leaves and russeting on fruit. Pear psyllas are also considered vectors of pear pathogens such as Candidatus Phytoplasma pyri causing pear decline that can lead to loss of crop and tree vigor, and sometimes loss of trees. Psylla control is a major obstacle to efficient integrated pest management. Recently we have identified two naturally resistance pear accessions (Py.760-261 and Py.701-202) in the Newe Ya’ar live collection. GC-MS volatile metabolic profiling identified several volatile compounds common in these accessions but lacking, or much less common, in a sensitive accession, the commercial Spadona variety. Among these volatiles were styrene and its derivatives. When the resistant accessions were used as inter-stock, the volatile compounds appear in commercial Spadona scion leaves, and it showed reduced susceptibility to pear psylla. Laboratory experiments and applications of some of these volatile compounds were very effective against psylla eggs, nymphs, and adults. The genes and enzymes involved in the specific reactions that lead to the biosynthesis of styrene in plant are unknown. We have identified a phenolic acid decarboxylase that catalyzes the formation of p-hydroxystyrene, which occurs as a styrene analog in resistant pear genotypes. The His-tagged and affinity chromatography purified E. coli-expressed pear PyPAD1 protein could decarboxylate p-coumaric acid and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene. In addition, PyPAD1 had the highest activity toward p-coumaric acid. Expression analysis of the PyPAD gene revealed that its expressed as expected, i.e., high when styrene levels and psylla resistance were high.

Keywords: pear Psylla, volatile, GC-MS, resistance

Procedia PDF Downloads 147

Authors: Emma R. Arakelova, Stepan G. Grigoryan, Flora G. Arsenyan, Nelli S. Babayan, Ruzanna M. Grigoryan, Natalia K. Sarkisyan

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Novel nanosize zinc oxide composites of doxorubicin obtained by deposition of 180 nm thick zinc oxide film on the drug surface using DC-magnetron sputtering of a zinc target in the form of gels (PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO) were studied for drug delivery applications. The cancer specificity was revealed both in in vitro and in vivo models. The cytotoxicity of the test compounds was analyzed against human cancer (HeLa) and normal (MRC5) cell lines using MTT colorimetric cell viability assay. IC50 values were determined and compared to reveal the cancer specificity of the test samples. The mechanistic study of the most active compound was investigated using Flow cytometry analyzing of the DNA content after PI (propidium iodide) staining. Data were analyzed with Tree Star FlowJo software using cell cycle analysis Dean-Jett-Fox module. The in vivo anticancer activity estimation experiments were carried out on mice with inoculated ascitic Ehrlich’s carcinoma at intraperitoneal introduction of doxorubicin and its zinc oxide compositions. It was shown that the nanosize zinc oxide film deposition on the drug surface leads to the selective anticancer activity of composites at the cellular level with the range of selectivity index (SI) from 4 (Starch+NaCMC+Dox+ZnO) to 200 (PEO(gel)+Dox+ZnO) which is higher than that of free Dox (SI = 56). The significant increase in vivo antitumor activity (by a factor of 2-2.5) and decrease of general toxicity of zinc oxide compositions of doxorubicin in the form of the above mentioned gels compared to free doxorubicin were shown on the model of inoculated Ehrlich's ascitic carcinoma. Mechanistic studies of anticancer activity revealed the cytostatic effect based on the high level of DNA biosynthesis inhibition at considerable low concentrations of zinc oxide compositions of doxorubicin. The results of studies in vitro and in vivo behavior of PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO composites confirm the high potential of the nanosize zinc oxide composites as a vector delivery system for future application in cancer chemotherapy.

Keywords: anticancer activity, cancer specificity, doxorubicin, zinc oxide

Procedia PDF Downloads 411

Authors: Mathew O. Ayoola, Foluke Aderemi, Tunde E. Lawal, Opeyemi Oladejo, Micheal A. Abiola

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Food animals reared for meat require transportation during their life cycle. The transportation procedures could initiate stressors capable of disrupting the physiological homeostasis. Such stressors associated with transportation may include; loading and unloading, crowding, environmental temperature, fear, vehicle motion/vibration, feed / water deprivation, and length of travel. This may cause oxidative stress and damage to excess free radicals or reactive oxygen species (ROS). In recent years, the application of natural products as a substitute for synthetic electrolytes and tranquilizers as anti-stress agents during the transportation is yet under investigation. Sweet orange, a predominant fruit in humid tropics, has been reported to have a good content of vitamin C (Ascorbic acid). Vitamin C, which is an active ingredient in orange juice, plays a major role in the biosynthesis of Corticosterone, a hormone that enhances energy supply during transportation and heat stress. Ninety-six, 15weeks, Isa brown pullets were allotted to four (4) oral treatments; sterile water (T1), synthetic vit C (T2), 30ml orange/liter of water (T3), 50ml orange/1 liter (T4). Physiological parameters; body temperature (BTC), rectal temperature (RTC), respiratory rate (RR), and panting rate (PR) were measured pre and post-transportation. The birds were transported with a specialized vehicle for a distance of 50km at a speed of 60 km/hr. The average environmental THI and within the vehicle was 81.8 and 74.6, respectively, and the average wind speed was 11km/hr. Treatments and periods had a significant (p>0.05) effect on all the physiological parameters investigated. Birds on T1 are significantly (p<0.05) different as compared to T2, T3, and T4. Values recorded post-transportation are significantly (p<0.05) higher as compared to pre-transportation for all parameters. In conclusion, this study showed that transportation as a stressor can affect the physiological homeostasis of pullets. Oral supplementation of electrolytes or tranquilizers is essential as an anti-stress during transportation. The application of the organic product in form of sweet orange could serve as a suitable alternative for the synthetic vitamin C.

Keywords: physiological, pullets, sweet orange, transportation stress, and vitamin C

Procedia PDF Downloads 120

Authors: Tai Chen, Caihuan Tian, Xiuxia Ren, Jingqi Xue, Xiuxin Zhang

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The herbaceous peony has become increasingly popular worldwide in recent years, especially as a cut flower with great economic value. However, peony has a very short vase life, only 3-5 d usually, which seriously affects its commodity value. In this study, we used the cut peony (Paeonia lactiflora cv. Sarah) as a material and found that melatonin treatment significantly improved its postharvest performance. In the control group, its vase life was 4.8 d, accompanied by petal dropping at last; melatonin treatment (40 μM) increased this time to 6.9 d without petal dropping at the end. Further study showed that melatonin treatment significantly increased the activity of antioxidant enzymes as well as reduced sugar content in petals, whereas the starch content in petals decreased. These results indicated that melatonin treatment may delay the oxidation reaction caused by aging, which also provides extra energy for maintaining flowering. Through full-length transcriptome sequencing, a total of 2819 differentially expressed genes (DEGs) between control and melatonin treatment groups were identified. KEGG enrichment analysis showed that these DEGs were mainly involved in three pathways, including melatonin synthesis, starch and sucrose conversion, and plant disease resistance. After the RT-qPCR verification, we identified three DEGs, named PlBAM3, PlWRKY22 and PlTIP1, and they should play major roles in melatonin-improved postharvest performance. One possible reason is that PlBAM3 caused maltose production (by starch degradation), maintained the proline biosynthesis, and then alleviated oxidative stress. Another reason is that both PlBAM3 and PlWRKY22 are key drought resistance regulators, which have the ability to alleviate osmotic stress and improve water absorption, which may also help to improve the postharvest quality of cut peony. In addition, PlTIP1 is involved in the sugar signal pathway, indicating sugar may also as a signal substance during this process. Our work may give new ideas for developing new ways to prolong the vase life of cut peony and improve its commodity value eventually.

Keywords: cut peony, melatonin, vase life, oxidation reaction, energy supply, differentially expressed genes

Procedia PDF Downloads 52

Authors: Shailendra Kumar, Kamal Kumar Gupta

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Dysdercus koenigii is an economically important pest of cotton worldwide. The pest damages the crop by sucking sap, staining lint, reducing the oil content of the seeds and deteriorating the quality of cotton. Plant possesses a plethora of secondary metabolites which are used as defense mechanism against herbivores. One of the important categories of such chemicals is insect growth regulators and the intermediates in their biosynthesis. Farnesol belongs to sesquiterpenoid. It is an intermediate in Juvenile hormone biosynthetic pathway in insects has been widely reported in the variety of plants. This chemical can disrupt the normal metabolic function and therefore, affects various life processes of the insects. Present study tested the efficacy of farnesol against Dysdercus koenigii. 2μl of 5% (100µg) and 10% (200µg) of the farnesol was applied topically on the dorsum of thoracic region of the newly emerged fifth instar nymphs of Dysdercus. The treated insects were observed daily for their survival, weight gain, and developmental anomalies for a period of ten days. The results indicated that treatment with 200µg farnesol decreased survival of the insects to 70% after 24h of exposure. At lower doses, no significant decrease in the survival was observed. However, the surviving nymphs showed alteration in growth, development, and metamorphosis. The weight gain in the treated nymphs showed deviation from control. The treated nymphs showed an increase in mortality during subsequent days and increase in the nymphal duration. The number of nymphs undergoing metamorphosis decreased to 46% and 88% in the treatments with the dose of 200µg and 100µg respectively. Severe developmental anomalies were also observed in the treated nymphs. The treated nymphs moulted into supernumerary nymphs, adultoids, adults with exuviae attached and adults with wing deformities. On treatment with 200µg; 26% adultoid, 4% adults with exuviae attached and 12% adults with wing deformed were produced. Treatment with 100µg resulted in production of 34% adultoid, 26% adults with deformed wing and 4% adults with exuviae attached. Many of the treated nymphs did not metamorphose into adults, remained in nymphal stage and died. Our results indicated potential application plant-derived secondary metabolites like farnesol in the management of Dysdercus population.

Keywords: development, Dysdercus koenigii, farnesol, survival

Procedia PDF Downloads 355

Authors: H. Jeries, N. Volkova, C. G. Iglesias, M. Najjar, M. Rosenblat, M. Aviram, T. Hayek

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Background: Synthetic forms of glucocorticoids (e.g., prednisone, prednisolone) are anti-inflammatory drugs which are widely used in clinical practice. The role of glucocorticoids (GCs) in cardiovascular diseases including atherosclerosis is highly controversial, and their impact on macrophage foam cell formation is still unknown. Our aim was to investigate the effects of prednisone or its active metabolite, prednisolone, on macrophage oxidative stress and lipid metabolism using in-vivo, ex-vivo and in-vitro systems. Methods: The in-vivo study included C57BL/6 mice which were intraperitoneally injected with prednisone or prednisolone (5mg/kg) for 4 weeks, followed by lipid metabolism analyses in the mice aorta, and in peritoneal macrophages (MPM). In the ex-vivo study, we analyzed the effect of serum samples obtained from 9 healthy volunteers before or after treatment with oral prednisone (20mg for 5 days), on J774A.1 macrophage atherogenicity. In-vitro studies were conducted using J774A.1 macrophages, human monocyte derived macrophages (HMDM) and fibroblasts. Cells were incubated with increasing concentrations (0-200 ng/ml) of prednisone or prednisolone, followed by determination of cellular oxidative status, triglyceride and cholesterol metabolism. Results: Prednisone or prednisolone treatment resulted in a significant reduction in triglycerides and mainly in cholesterol cellular accumulation in MPM or in J774A.1 macrophages incubated with human serum. Similar resulted were noted in HMDM or in J774A.1 macrophages which were directly incubated with the GCs. These effects were associated with GCs inhibitory effect on triglycerides and cholesterol biosynthesis rates, throughout downregulation of diacylglycerol acyltransferase1 (DGAT1) expression, and of the sterol regulatory element binding protein (SREBP2) and HMGCR expression, respectively. In parallel to prednisone or prednisolone induced reduction in macrophage triglyceride content, paraoxonase 2 (PON2) expression was significantly upregulated. GCs-induced reduction of cellular triglyceride and cholesterol mass was mediated by the GCs receptors on macrophages since the GCs receptor antagonist (RU 486) abolished these effects. In fibroblasts, unlike macrophages, prednisone or prednisolone showed no anti-atherogenic effects. Conclusions: Prednisone or prednisolone are anti-atherogenic since they protected macrophages from lipid accumulation and foam cell formation.

Keywords: atherosclerosis, cholesterol, foam cell, macrophage, prednisone, prednisolone, triglycerides

Procedia PDF Downloads 145

Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

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The drug discovery and development process is generally known to be a very lengthy and labor-intensive process. Therefore, in order to be able to deliver prompt and effective responses to cure certain diseases, there is an urgent need to reduce the time and resources needed to design, develop, and optimize potential drugs. Computer-aided drug design (CADD) is able to alleviate this issue by applying computational power in order to streamline the whole drug discovery process, starting from target identification to lead optimization. This drug design approach can be predominantly applied to diseases that cause major public health concerns, such as tuberculosis. Hitherto, there has been no concrete cure for this disease, especially with the continuing emergence of drug resistant strains. In this study, CADD is employed for tuberculosis by first identifying a key enzyme in the mycobacterium’s metabolic pathway that would make a good drug target. One such potential target is the lipoate protein ligase B enzyme (LipB), which is a key enzyme in the M. tuberculosis metabolic pathway involved in the biosynthesis of the lipoic acid cofactor. Its expression is considerably up-regulated in patients with multi-drug resistant tuberculosis (MDR-TB) and it has no known back-up mechanism that can take over its function when inhibited, making it an extremely attractive target. Using cutting-edge computational methods, compounds from AnalytiCon Discovery Natural Derivatives database were screened and docked against the LipB enzyme in order to rank them based on their binding affinities. Compounds which have better binding affinities than LipB’s known inhibitor, decanoic acid, were subjected to in silico toxicity evaluation using the ADMET and TOPKAT protocols. Out of the 31,692 compounds in the database, 112 of these showed better binding energies than decanoic acid. Furthermore, 12 out of the 112 compounds showed highly promising ADMET and TOPKAT properties. Future studies involving in vitro or in vivo bioassays may be done to further confirm the therapeutic efficacy of these 12 compounds, which eventually may then lead to a novel class of anti-tuberculosis drugs.

Keywords: pharmacophore, molecular docking, lipoate protein ligase B (LipB), ADMET, TOPKAT

Procedia PDF Downloads 424

Authors: Lethukuthula Ngobese, Abin Gupthar, Patrick Govender

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The ability of yeast cell wall-derived mannoproteins (glycoproteins) to positively contribute to oenological properties has been a key factor that stimulates research initiatives into these industrially important glycoproteins. In addition, and from a fundamental research perspective, yeast cell wall glycoproteins are involved in a wide range of biological interactions. To date, and to the best of our knowledge, our understanding of the fine molecular structure of these mannoproteins is fairly limited. Generally, the amino acid sequences of their protein moieties have been established from structural and functional analysis of the genomic sequence of these yeasts whilst far less information is available on the glycosyl moieties of these mannoproteins. A novel strategy was devised in this study that entails the genetic engineering of yeast strains that over-express and release cell wall-associated glycoproteins into the liquid growth medium. To this end, the Flo1p mannoprotein was overexpressed in Saccharomyces cerevisiae laboratory strains bearing a specific deletion in KNR4 and GPI7 genes involved in cell wall biosynthesis that have been previously shown to extracellularly hyper-secrete cell wall-associated glycoproteins. A polymerase chain reaction (PCR) -based cloning strategy was employed to generate transgenic yeast strains in which the native cell wall FLO1 glycoprotein-encoding gene is brought under transcriptional control of the constitutive PGK1 promoter. The modified Helm’s flocculation assay was employed to assess flocculation intensities of a Flo1p over-expressing wild type and deletion mutant as an indirect measure of their abilities to release the desired mannoprotein. The flocculation intensities of the transformed strains were assessed and all the strains showed similar intensities (>98% flocculation). To assess if mannoproteins were released into the growth medium, the supernatant of each strain was subjected to the BCA protein assay and the transformed Δknr4 strain showed a considerable increase in protein levels. This study has the potential to produce mannoproteins in sufficient quantities that may be employed in future investigations to understand their molecular structures and mechanisms of interaction to the benefit of both fundamental and industrial applications.

Keywords: glycoproteins, genetic engineering, flocculation, over-expression

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Authors: Alexandra C. Blaga, Dan Caşcaval, Alexandra Tucaliuc, Madalina Poştaru, Anca I. Galaction

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Amino acids are valuable chemical products used in in human foods, in animal feed additives and in the pharmaceutical field. Recently, there has been a noticeable rise of amino acids utilization throughout the world to include their use as raw materials in the production of various industrial chemicals: oil gelating agents (amino acid-based surfactants) to recover effluent oil in seas and rivers and poly(amino acids), which are attracting attention for biodegradable plastics manufacture. The amino acids can be obtained by biosynthesis or from protein hydrolysis, but their separation from the obtained mixtures can be challenging. In the last decades there has been a continuous interest in developing processes that will improve the selectivity and yield of downstream processing steps. The liquid-liquid extraction of amino acids (dissociated at any pH-value of the aqueous solutions) is possible only by using the reactive extraction technique, mainly with extractants of organophosphoric acid derivatives, high molecular weight amines and crown-ethers. The purpose of this study was to analyse the separation of nine amino acids of acidic character (l-aspartic acid, l-glutamic acid), basic character (l-histidine, l-lysine, l-arginine) and neutral character (l-glycine, l-tryptophan, l-cysteine, l-alanine) by reactive extraction with di-(2-ethylhexyl)phosphoric acid (D2EHPA) dissolved in butyl acetate. The results showed that the separation yield is controlled by the pH value of the aqueous phase: the reactive extraction of amino acids with D2EHPA is possible only if the amino acids exist in aqueous solution in their cationic forms (pH of aqueous phase below the isoeletric point). The studies for individual amino acids indicated the possibility of selectively separate different groups of amino acids with similar acidic properties as a function of aqueous solution pH-value: the maximum yields are reached for a pH domain of 2–3, then strongly decreasing with the pH increase. Thus, for acidic and neutral amino acids, the extraction becomes impossible at the isolelectric point (pHi) and for basic amino acids at a pH value lower than pHi, as a result of the carboxylic group dissociation. From the results obtained for the separation from the mixture of the nine amino acids, at different pH, it can be observed that all amino acids are extracted with different yields, for a pH domain of 1.5–3. Over this interval, the extract contains only the amino acids with neutral and basic character. For pH 5–6, only the neutral amino acids are extracted and for pH > 6 the extraction becomes impossible. Using this technique, the total separation of the following amino acids groups has been performed: neutral amino acids at pH 5–5.5, basic amino acids and l-cysteine at pH 4–4.5, l-histidine at pH 3–3.5 and acidic amino acids at pH 2–2.5.

Keywords: amino acids, di-(2-ethylhexyl) phosphoric acid, reactive extraction, selective extraction

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Authors: Jian Yu

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Carbo dioxide (CO2) from fossil fuel combustion is a prime green-house gas emission. It can be mitigated by microalgae through conventional photosynthesis. The algal oil is a feedstock of biodiesel, a carbon neutral liquid fuel for transportation. The conventional CO2 fixation, however, is quite slow and affected by the intermittent solar irradiation. It is also a technical challenge to reform the bio-oil into a drop-in liquid fuel that can be directly used in the modern combustion engines with expected performance. Here, an artificial photosynthesis system is presented to produce a biopolyester and liquid fuels from CO2, water, and solar power. In this green process, solar energy is captured using photovoltaic modules and converted into hydrogen as a stable energy source via water electrolysis. The solar hydrogen is then used to fix CO2 by Cupriavidus necator, a hydrogen-oxidizing bacterium. Under the autotrophic conditions, CO2 was reduced to glyceraldehyde-3-phosphate (G3P) that is further utilized for cell growth and biosynthesis of polyhydroxybutyrate (PHB). The maximum cell growth rate reached 10.1 g L-1 day-1, about 25 times faster than that of a typical bio-oil-producing microalga (Neochloris Oleoabundans) under stable indoor conditions. With nitrogen nutrient limitation, a large portion of the reduced carbon is stored in PHB (C4H6O2)n, accounting for 50-60% of dry cell mass. PHB is a biodegradable thermoplastic that can find a variety of environmentally friendly applications. It is also a platform material from which small chemicals can be derived. At a high temperature (240 - 290 oC), the biopolyester is degraded into crotonic acid (C4H6O2). On a solid phosphoric acid catalyst, PHB is deoxygenated via decarboxylation into a hydrocarbon oil (C6-C18) at 240 oC or so. Aromatics and alkenes are the major compounds, depending on the reaction conditions. A gasoline-grade liquid fuel (77 wt% oil) and a biodiesel-grade fuel (23 wt% oil) were obtained from the hydrocarbon oil via distillation. The formation routes of hydrocarbon oil from crotonic acid, the major PHB degradation intermediate, are revealed and discussed. This work shows a novel green process from which biodegradable plastics and high-grade liquid fuels can be directly produced from carbon dioxide, water and solar power. The productivity of the green polyester (5.3 g L-1 d-1) is much higher than that of microalgal oil (0.13 g L-1 d-1). Other technical merits of the new green process may include continuous operation under intermittent solar irradiation and convenient scale up in outdoor.

Keywords: bioplastics, carbon dioxide fixation, drop-in liquid fuels, green process

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Authors: Kamal Kumar Gupta, Sunil Kayesth

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Dysdercus koenigii is serious pest of cotton and other malvaceous crop. Present research work aimed at ecofriendly approach for management of pest by plant extracts. The impact of Ocimum sanctum was studied on reproductive performance of Dysdercus koenigii. The hexane extract of Ocimum leaves was prepared by ‘cold extraction method’. The newly emerged fifth instar nymphs were exposed to the extract of concentrations ranging from 0.1% to 0.00625% by ‘thin film residual method’ for a period of 24h. Reproductive fitness of the adults emerged from the treated nymphs was evaluated by assessing their courtship behaviour, oviposition behaviour, and fertility. The studies indicated that treatment of Dysdercus with the hexane extract of Ocimum altered their courtship behaviour. Consequently, the treated males exhibited less sexual activity, performed fewer mounting attempts, increased time to mate and showed decreased percent successful mating. The females often rejected courting treated male by shaking the abdomen. Similarly, the treated females in many cases remained non-receptive to the courting male. Premature termination of mating in the mating pairs prior to insemination further decreased the mating success of the treated adults. Maximum abbreviation of courtship behaviour was observed in the experimental set up where both the males and the females were treated. Only females which mate successfully were observed for study of oviposition behaviour. The treated females laid lesser number of egg batches and eggs in their life span. The eggs laid by these females were fertile indicating insemination of the female. However, percent hatchability was lesser than control. The effects of hexane extract were dose dependent. Treatment with 0.1% and 0.05% extract altered courtship behaviour. Doses of concentrations less than 0.05% did not affect courtship behaviour but altered the oviposition behaviour and fertility. Significant reduction in the fecundity and fertility was observed in the treatments at concentration as low as 0.00625%. The GCMS analysis of the extract revealed a plethora of phytochemicals including juvenile hormone mimics, and the intermediates of juvenile hormone biosynthesis. Therefore, some of these compounds individually or synergistically impair reproductive behaviour of Dysdercus. Alteration of courtship behaviour and suppression of fecundity and fertility with the help of plant extracts has wide potentials in suppression of pest population and ‘integrated pest management’.

Keywords: courtship behaviour, Dysdercus koenigii, Ocimum sanctum, oviposition behaviour

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Authors: Laura Helena Caicedo Lopez, Luis Miguel Contreras Medina, Ramon Gerardo Guevara Gonzales, Juan E. Andrade

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In this study, we evaluated the effect of three different hydric stress conditions: Low (LHS), medium (MHS), and high (HHS) on capsaicinoid content and enzyme regulation of C. annuum plants. Five main peaks were detected using a 2 Hz resolution vibrometer laser (Polytec-B&K). These peaks or “characteristic frequencies” were used as acoustic emissions (AEs) treatment, transforming these signals into audible sound with the frequency (Hz) content of each hydric stress. Capsaicinoids (CAPs) are the main, secondary metabolites of chili pepper plants and are known to increase during hydric stress conditions or short drought-periods. The AEs treatments were applied in two plant stages: the first one was in the pre-anthesis stage to evaluate the genes that encode the transcription of enzymes responsible for diverse metabolic activities of C. annuum plants. For example, the antioxidant responses such as peroxidase (POD), superoxide dismutase (Mn-SOD). Also, phenyl-alanine ammonia-lyase (PAL) involved in the biosynthesis of the phenylpropanoid compounds. The chalcone synthase (CHS) related to the natural defense mechanisms and species-specific aquaporin (CAPIP-1) that regulate the flow of water into and out of cells. The second stage was at 40 days after flowering (DAF) to evaluate the biochemical effect of AEs related to hydric stress on capsaicinoids production. These two experiments were conducted to identify the molecular responses of C. annuum plants to AE. Moreover, to define AEs could elicit any increase in the capsaicinoids content after a one-week exposition to AEs treatments. The results show that all AEs treatment signals (LHS, MHS, and HHS) were significantly different compared to the non-acoustic emission control (NAE). Also, the AEs induced the up-regulation of POD (~2.8, 2.9, and 3.6, respectively). The gene expression of another antioxidant response was particularly treatment-dependent. The HHS induced and overexpression of Mn-SOD (~0.23) and PAL (~0.33). As well, the MHS only induced an up-regulation of the CHs gene (~0.63). On the other hand, CAPIP-1 gene gas down-regulated by all AEs treatments LHS, MHS, and HHS ~ (-2.4, -0.43 and -6.4, respectively). Likewise, the down-regulation showed particularities depending on the treatment. LHS and MHS induced downregulation of the SOD gene ~ (-1.26 and -1.20 respectively) and PAL (-4.36 and 2.05, respectively). Correspondingly, the LHS and HHS showed the same tendency in the CHs gene, respectively ~ (-1.12 and -1.02, respectively). Regarding the elicitation effect of AE on the capsaicinoids content, additional treatment controls were included. A white noise treatment (WN) to prove the frequency-selectiveness of signals and a hydric stressed group (HS) to compare the CAPs content. Our findings suggest that WN and NAE did not present differences statically. Conversely, HS and all AEs treatments induced a significant increase of capsaicin (Cap) and dihydrocapsaicin (Dcap) after one-week of a treatment. Specifically, the HS plants showed an increase of 8.33 times compared to the NAE and WN treatments and 1.4 times higher than the MHS, which was the AEs treatment with a larger induction of Capsaicinoids among treatments (5.88) and compared to the controls.

Keywords: acoustic emission, capsaicinoids, elicitors, hydric stress, plant signaling

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Authors: Lenuta Kloetzer, Alexandra C. Blaga, Dan Cascaval, Alexandra Tucaliuc, Anca I. Galaction

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Rosmarinic acid, an ester of caffeic acid and 3-(3,4-dihydroxyphenyl) lactic acid, is considered a valuable compound for the pharmaceutical and cosmetic industries due to its antimicrobial, antioxidant, antiviral, anti-allergic, and anti-inflammatory effects. It can be obtained by extraction from vegetable or animal materials, by chemical synthesis and biosynthesis. Indifferent of the method used for rosmarinic acid production, the separation and purification process implies high amount of raw materials and laborious stages leading to high cost for and limitations of the separation technology. This study focused on separation of rosmarinic acid by synergic reactive extraction with a mixture of two extractants, one acidic (acid di-(2ethylhexyl) phosphoric acid, D2EHPA) and one with basic character (Amberlite LA-2). The studies were performed in experimental equipment consisting of an extraction column where the phases’ mixing was made by mean of a perforated disk with 45 mm diameter and 20% free section, maintained at the initial contact interface between the aqueous and organic phases. The vibrations had a frequency of 50 s⁻¹ and 5 mm amplitude. The extraction was carried out in two solvents with different dielectric constants (n-heptane and dichloromethane) in which the extractants mixture of varying concentration was dissolved. The pH-value of initial aqueous solution was varied between 1 and 7. The efficiency of the studied extraction systems was quantified by distribution and synergic coefficients. For calculating these parameters, the rosmarinic acid concentration in the initial aqueous solution and in the raffinate have been measured by HPLC. The influences of extractants concentrations and solvent polarity on the efficiency of rosmarinic acid separation by synergic extraction with a mixture of Amberlite LA-2 and D2EHPA have been analyzed. In the reactive extraction system with a constant concentration of Amberlite LA-2 in the organic phase, the increase of D2EHPA concentration leads to decrease of the synergic coefficient. This is because the increase of D2EHPA concentration prevents the formation of amine adducts and, consequently, affects the hydrophobicity of the interfacial complex with rosmarinic acid. For these reasons, the diminution of synergic coefficient is more important for dichloromethane. By maintaining a constant value of D2EHPA concentration and increasing the concentration of Amberlite LA-2, the synergic coefficient could become higher than 1, its highest values being reached for n-heptane. Depending on the solvent polarity and D2EHPA amount in the solvent phase, the synergic effect is observed for Amberlite LA-2 concentrations over 20 g/l dissolved in n-heptane. Thus, by increasing the concentration of D2EHPA from 5 to 40 g/l, the minimum concentration value of Amberlite LA-2 corresponding to synergism increases from 20 to 40 g/l for the solvent with lower polarity, namely, n-heptane, while there is no synergic effect recorded for dichloromethane. By analysing the influences of the main factors (organic phase polarity, extractant concentration in the mixture) on the efficiency of synergic extraction of rosmarinic acid, the most important synergic effect was found to correspond to the extractants mixture containing 5 g/l D2EHPA and 40 g/l Amberlite LA-2 dissolved in n-heptane.

Keywords: Amberlite LA-2, di(2-ethylhexyl) phosphoric acid, rosmarinic acid, synergic effect

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Authors: Ankan Roy, Niharika, Samir Kumar Patra

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Anomalous nexus of complex topological assemblies and spatiotemporal epigenetic choreography at chromosomal territory may forms the most sophisticated regulatory layer of gene expression in cancer. Colon cancer is one of the leading malignant neoplasms of the lower gastrointestinal tract worldwide. There is still a paucity of information about the complex molecular mechanisms of colonic cancerogenesis. Bioinformatics prediction and analysis helps to identify essential genes and significant pathways for monitoring and conquering this deadly disease. The present study investigates and explores potential hub genes as biomarkers and effective therapeutic targets for colon cancer treatment. Colon cancer patient sample containing gene expression profile datasets, such as GSE44076, GSE20916, and GSE37364 were downloaded from Gene Expression Omnibus (GEO) database and thoroughly screened using the GEO2R tool and Funrich software to find out common 2 differentially expressed genes (DEGs). Other approaches, including Gene Ontology (GO) and KEGG pathway analysis, Protein-Protein Interaction (PPI) network construction and hub gene investigation, Overall Survival (OS) analysis, gene correlation analysis, methylation pattern analysis, and hub gene-Transcription factors regulatory network construction, were performed and validated using various bioinformatics tool. Initially, we identified 166 DEGs, including 68 up-regulated and 98 down-regulated genes. Up-regulated genes are mainly associated with the Cytokine-cytokine receptor interaction, IL17 signaling pathway, ECM-receptor interaction, Focal adhesion and PI3K-Akt pathway. Downregulated genes are enriched in metabolic pathways, retinol metabolism, Steroid hormone biosynthesis, and bile secretion. From the protein-protein interaction network, thirty hub genes with high connectivity are selected using the MCODE and cytoHubba plugin. Survival analysis, expression validation, correlation analysis, and methylation pattern analysis were further verified using TCGA data. Finally, we predicted COL1A1, COL1A2, COL4A1, SPP1, SPARC, and THBS2 as potential master regulators in colonic cancerogenesis. Moreover, our experimental data highlights that disruption of lipid raft and RAS/MAPK signaling cascade affects this gene hub at mRNA level. We identified COL1A1, COL1A2, COL4A1, SPP1, SPARC, and THBS2 as determinant hub genes in colon cancer progression. They can be considered as biomarkers for diagnosis and promising therapeutic targets in colon cancer treatment. Additionally, our experimental data advertise that signaling pathway act as connecting link between membrane hub and gene hub.

Keywords: hub genes, colon cancer, DNA methylation, epigenetic engineering, bioinformatic predictions

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Authors: Sonia Kudlacik-Kramarczyk, Anna Drabczyk, Dagmara Malina, Bozena Tyliszczak, Agnieszka Sobczak-Kupiec

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Introduction: In the preparation of polymer materials, compounds of natural origin are currently gaining more and more interest. This is particularly noticeable in the case of synthesis of materials considered for biomedical use. Then, selected material has to meet many requirements. It should be characterized by non-toxicity, biodegradability and biocompatibility. Therefore special attention is directed to substances such as polysaccharides, proteins or substances that are the basic building components of proteins, i.e. amino acids. These compounds may be crosslinked with other reagents that leads to the preparation of polymer matrices. Such amino acids as e.g. cysteine or histidine. On the other hand, previously mentioned requirements may be met by polymers obtained as a result of biosynthesis, e.g. polyhydroxybutyrate. This polymer belongs to the group of aliphatic polyesters that is synthesized by microorganisms (selected strain of bacteria) under specific conditions. It is possible to modify matrices based on given polymer with substances of various origin. Such a modification may result in the change of their properties or/and in providing the material with new features desirable in viewpoint of specific application. Described materials are synthesized using UV radiation. Process of photopolymerization is fast, waste-free and enables to obtain final products with favorable properties. Methodology: Polymer matrices have been prepared by means of photopolymerization. First step involved the preparation of solutions of particular reagents and mixing them in the appropriate ratio. Next, crosslinking agent and photoinitiator have been added to the reaction mixture and the whole was poured into the Petri dish and treated with UV radiation. After the synthesis, polymer samples were dried at room temperature and subjected to the numerous analyses aimed at the determining their physicochemical properties. Firstly, sorption properties of obtained polymer matrices have been determined. Next, mechanical properties have been characterized, i.e. tensile strength. The ability to deformation under applied stress of all prepared polymer matrices has been checked. Such a property is important in viewpoint of the application of analyzed materials e.g. as wound dressings. Wound dressings have to be elastic because depending on the location of the wound and its mobility, such a dressing has to adhere properly to the wound. Furthermore, considering the use of the materials for biomedical purposes it is essential to determine its behavior in environments simulating these ones occurring in human body. Therefore incubation studies using selected liquids have also been conducted. Conclusions: As a result of photopolymerization process, polymer matrices based on natural compounds have been prepared. These exhibited favorable mechanical properties and swelling ability. Moreover, biocompatibility in relation to simulated body fluids has been stated. Therefore it can be concluded that analyzed polymer matrices constitute an interesting materials that may be considered for biomedical use and may be subjected to the further more advanced analyses using specific cell lines.

Keywords: photopolymerization, polymer matrices, simulated body fluids, swelling properties

Procedia PDF Downloads 128

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: Andrés Quiroz, Emilio Hormazábal, Ana Mutis, Fernando Ortega, Loreto Méndez, Leonardo Parra

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Red clover root borer, Hylastinus obscurus Marsham (Coleoptera: Curculionidae), is the main insect pest associated to red clover, Trifolium pratense L. An average of 1.5 H. obscurus per plant can cause 5.5% reduction in forage yield in pastures of two to three years old. Moreover, insect attack can reach 70% to 100% of the plants. To our knowledge, there is no a chemical strategy for controlling this pest. Therefore alternative strategies for controlling H. obscurus are a high priority for red clover producers. One of this alternative is related to the study of secondary metabolites involved in intrinsic chemical defenses developed by plants, such as isoflavonoids. The isoflavonoids formononetin and daidzein have elicited an antifeedant and phagostimult effect on H. obscurus respectively. However, we do not know how is the dynamic variation of these isoflavonoids under field conditions. The main objective of this work was to evaluate the variation of the antifeedant isoflavonoids formononetin, the phagostimulant isoflavonoids daidzein, and their respective glycosides over time in different ecotypes of red clover. Fourteen red clover ecotypes (8 cultivars and 6 experimental lines), were collected at INIA-Carillanca (La Araucanía, Chile). These plants were established in October 2015 under irrigated conditions. The cultivars were distributed in a randomized complete block with three replicates. The whole plants were sampled in four times: 15th October 2016, 12th December 2016, 27th January 2017 and 16th March 2017 with sufficient amount of soil to avoid root damage. A polar fraction of isoflavonoid was obtained from 20 mg of lyophilized root tissue extracted with 2 mL of 80% MeOH for 16 h using an orbital shaker in the dark at room temperature. After, an aliquot of 1.4 mL of the supernatant was evaporated, and the residue was resuspended in 300 µL of 45% MeOH. The identification and quantification of isoflavonoid root extracts were performed by the injection of 20 µL into a Shimadzu HPLC equipped with a C-18 column. The sample was eluted with a mobile phase composed of AcOH: H₂O (1:9 v/v) as solvent A and CH₃CN as solvent B. The detection was performed at 260 nm. The results showed that the amount of aglycones was higher than the respective glycosides. This result is according to the biosynthetic pathway of flavonoids, where the formation of glycoside is further to the glycosides biosynthesis. The amount of formononetin was higher than daidzein. In roots, where H. obscurus spent the most part of its live cycle, the highest content of formononetin was found in G 27, Pawera, Sabtoron High, Redqueli-INIA and Superqueli-INIA cvs. (2.1, 1.8, 1.8, 1.6 and 1.0 mg g⁻¹ respectively); and the lowest amount of daidzein were found Superqueli-INIA (0.32 mg g⁻¹) and in the experimental line Sel Syn Int4 (0.24 mg g⁻¹). This ecotype showed a high content of formononetin (0.9 mg g⁻¹). This information, associated with cultural practices, could help farmers and breeders to reduce H. obscurus in grassland, selecting ecotypes with high content of formononetin and low amount of daidzein in the roots of red clover plants. Acknowledgements: FONDECYT 1141245 and 11130715.

Keywords: daidzein, formononetin, isoflavonoid glycosides, trifolium pratense

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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 153

Authors: Blaga Alexandra Cristina, Kloetzer Lenuta, Bompa Amalia Stela, Galaction Anca Irina, Cascaval Dan

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Vitamin B9 is an important member of vitamins B group, being a growth factor, important for making genetic material as DNA and RNA, red blood cells, for building muscle tissues, especially during periods of infancy, adolescence and pregnancy. Its production by biosynthesis is based on the high metabolic potential of mutant Bacillus subtilis, due to a superior biodisponibility compared to that obtained by chemical pathways. Pertraction, defined as the extraction and transport through liquid membranes consists in the transfer of a solute between two aqueous phases of different pH-values, phases that are separated by a solvent layer of various sizes. The pertraction efficiency and selectivity could be significantly enhanced by adding a carrier in the liquid membrane, such as organophosphoric compounds, long chain amines or crown-ethers etc., the separation process being called facilitated pertraction. The aim of the work is to determine the impact of the presence of two extractants/carriers in the bulk liquid membrane, i.e. di(2-ethylhexyl) phosphoric acid (D2EHPA) and lauryltrialkylmetilamine (Amberlite LA2) on the transport kinetics of vitamin B9. The experiments have been carried out using two pertraction equipments for a free liquid membrane or bulk liquid membrane. One pertraction cell consists on a U-shaped glass pipe (used for the dichloromethane membrane) and the second one is an H-shaped glass pipe (used for h-heptane), having 45 mm inner diameter of the total volume of 450 mL, the volume of each compartment being of 150 mL. The aqueous solutions are independently mixed by means of double blade stirrers with 6 mm diameter and 3 mm height, having the rotation speed of 500 rpm. In order to reach high diffusional rates through the solvent layer, the organic phase has been mixed with a similar stirrer, at a similar rotation speed (500 rpm). The area of mass transfer surface, both for extraction and for reextraction, was of 1.59x10-³ m2. The study on facilitated pertraction with the mixture of two carriers, namely D2EHPA and Amberlite LA-2, dissolved in two solvents with different polarities: n-heptane and dichloromethane, indicated the possibility to obtain the synergic effect. The synergism has been analyzed by considering the vitamin initial and final mass flows, as well as the permeability factors through liquid membrane. The synergic effect has been observed at low D2EHPA concentrations and high Amberlite LA-2 concentrations, being more important for the low-polar solvent (n-heptane). The results suggest that the mechanism of synergic pertraction consists on the reaction between the organophosphoric carrier and vitamin B9 at the interface between the feed and membrane phases, while the aminic carrier enhances the hydrophobicity of this compound by solvation. However, the formation of this complex reduced the reextraction rate and, consequently, affects the synergism related to the final mass flows and permeability factor. For describing the influences of carriers concentrations on the synergistic coefficients, some equations have been proposed by taking into account the vitamin mass flows or permeability factors, with an average deviations between 4.85% and 10.73%.

Keywords: pertraction, synergism, vitamin B9, Amberlite LA-2, di(2-ethylhexyl) phosphoric acid

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Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

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Authors: Elvin P. Chizenga, Heidi Abrahamse

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Cancer cell resistance to therapy is the main cause of treatment failures and the poor prognosis of cancer convalescence. The progression of cervical cancer to other parts of the genitourinary system and the reported recurrence rates are overwhelming. Current treatments, including surgery, chemo and radiation have been inefficient in eradicating the tumor cells. These treatments are also associated with poor prognosis and reduced quality of life, including fertility loss. This has inspired the need for the development of new treatment modalities to eradicate cervical cancer successfully. Photodynamic Therapy (PDT) is a modern treatment modality that induces cell death by photochemical interactions of light and a photosensitizer, which in the presence of molecular oxygen, yields a set of chemical reactions that generate Reactive Oxygen Species (ROS) and other free radical species causing cell damage. Enhancing PDT using modified drug delivery can increase the concentration of the photosensitizer in the tumor cells, and this has the potential to maximize its therapeutic efficacy. In cervical cancer, all infected cells constitutively express genes of the E6 and E7 HPV viral oncoproteins, resulting in high concentrations of E6 and E7 in the cytoplasm. This provides an opportunity for active targeting of cervical cancer cells using immune-mediated drug delivery to maximize therapeutic efficacy. The use of nanoparticles in PDT has also proven effective in enhancing therapeutic efficacy. Gold nanoparticles (AuNps) in particular, are explored for their use in biomedicine due to their biocompatibility, low toxicity, and enhancement of drug uptake by tumor cells. In this present study, a biomolecule comprising of AuNPs, anti-E6 monoclonal antibodies, and Aluminium Phthalocyanine photosensitizer was synthesized for use in targeted PDT of cervical cancer. The AuNp-Anti-E6-Sulfonated Aluminium Phthalocyanine mix (AlPcSmix) photosensitizing biomolecule was synthesized by coupling AuNps and anti-E6 monoclonal antibodies to the AlPcSmix via Polyethylene Glycol (PEG) chemical links. The final product was characterized using Transmission Electron Microscope (TEM), Zeta Potential, Uv-Vis Spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), and X-ray diffraction (XRD), to confirm its chemical structure and functionality. To observe its therapeutic role in treating cervical cancer, cervical cancer cells, HeLa cells were seeded in 3.4 cm² diameter culture dishes at a concentration of 5x10⁵ cells/ml, in vitro. The cells were treated with varying concentrations of the photosensitizing biomolecule and irradiated using a 673.2 nm wavelength of laser light. Post irradiation cellular responses were performed to observe changes in morphology, viability, proliferation, cytotoxicity, and cell death pathways induced. Dose-Dependent response of the cells to treatment was demonstrated as significant morphologic changes, increased cytotoxicity, and decreased cell viability and proliferation This study presented a synthetic biomolecule for targeted PDT of cervical cancer. The study suggested that PDT using this AuNp- Anti-E6- AlPcSmix photosensitizing biomolecule is a very effective treatment method for the eradication of cervical cancer cells, in vitro. Further studies in vivo need to be conducted to support the use of this biomolecule in treating cervical cancer in clinical settings.

Keywords: anti-E6 monoclonal antibody, cervical cancer, gold nanoparticles, photodynamic therapy

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Authors: Cynthia K. Burzell

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Aequor's Founder, a Marine and Medical Microbiologist, discovered novel, non-toxic chemicals in the ocean that uniquely remove biofilm in minutes and prevent its formation for days. These chemicals and over 70 synthesized analogs that Aequor developed can replace thousands of toxic biocides used in consumer and industrial products and, as new drug candidates, kill biofilm-forming bacteria and fungi Superbugs -the antimicrobial-resistant (AMR) pathogens for which there is no cure. Cynthia Burzell, PhD., is a Marine and Medical Microbiologist studying natural mechanisms that inhibit biofilm formation on surfaces in contact with water. In 2002, she discovered a new genus and several new species of marine microbes that produce small molecules that remove biofilm in minutes and prevent its formation for days. The molecules include new antimicrobials that can replace thousands of toxic biocides used in consumer and industrial products and can be developed into new drug candidates to kill the biofilm-forming bacteria and fungi -- including the antimicrobial-resistant (AMR) Superbugs for which there is no cure. Today, Aequor has over 70 chemicals that are divided into categories: (1) Novel natural chemicals. Lonza validated that the primary natural chemical removed biofilm in minutes and stated: "Nothing else known can do this at non-toxic doses." (2) Specialty chemicals. 25 of these structural analogs are already approved under the U.S. Environmental Protection Agency (EPA)'s Toxic Substances Control Act, certified as "green" and available for immediate sale. These have been validated for the following agro-industrial verticals: (a) Surface cleaners: The U.S. Department of Agriculture validated that low concentrations of Aequor's formulations provide deep cleaning of inert, nano and organic surfaces and materials; (b) Water treatments: NASA validated that one dose of Aequor's treatment in the International Space Station's water reuse/recycling system lasted 15 months without replenishment. DOE validated that our treatments lower energy consumption by over 10% in buildings and industrial processes. Future validations include pilot projects with the EPA to test efficacy in hospital plumbing systems. (c) Algae cultivation and yeast fermentation: The U.S. Department of Energy (DOE) validated that Aequor's treatment boosted biomass of renewable feedstocks by 40% in half the time -- increasing the profitability of biofuels and biobased co-products. DOE also validated increased yields and crop protection of algae under cultivation in open ponds. A private oil and gas company validated decontamination of oilfield water. (3) New structural analogs. These kill Gram-negative and Gram-positive bacteria and fungi alone, in combinations with each other, and in combination with low doses of existing, ineffective antibiotics (including Penicillin), "potentiating" them to kill AMR pathogens at doses too low to trigger resistance. Both the U.S. National Institutes for Health (NIH) and Department of Defense (DOD) has executed contracts with Aequor to provide the pre-clinical trials needed for these new drug candidates to enter the regulatory approval pipelines. Aequor seeks partners/licensees to commercialize its specialty chemicals and support to evaluate the optimal methods to scale-up of several new structural analogs via activity-guided fractionation and/or biosynthesis in order to initiate the NIH and DOD pre-clinical trials.

Keywords: biofilm, potentiation, prevention, removal

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Authors: Roshni Raha, Kavya P., Gayathri M.

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India, with a humongous population, remains the world's poorest developing nation in terms of nutritional status, with iron deficiency anaemia (IDA) affecting the population. Despite efforts over the past decades, India's anaemia prevalence has not been reduced. Researchers are interested in developing therapies that will minimize the typical side effects of oral iron and optimize iron salts-based treatment through delivery methods based on the physiology of hepcidin regulation. However, they need to come up with iron therapies that will prevent making the infection worse. This article explores using bio-nanotechnology as the alternative, promising substitution of providing iron supplements for the treatment of diarrhoea and gut inflammation in kids and pregnant women. This article is an exploratory study using a literature survey and secondary research from review papers. In the realm of biotechnology, nanoparticles have become extremely famous due to unexpected variations in surface characteristics caused by particle size. Particle size distribution and shape exhibit unusual, enhanced characteristics when reduced to nanoscale. The article attempts to develop a model for a nanotechnology based solution in iron fortification to combat the problems of diarrhoea and gut inflammation. Certain dimensions that have been considered in the model include the size, shape, source, and biosynthesis of the iron nanoparticles. Another area of investigation addressed in the article is the cost-effective biocompatible production of these iron nanoparticles. Studies have demonstrated that a substantial reduction of metal ions to form nanoparticles from the bulk metal occurs in plants because of the presence of a wide diversity of biomolecules. Using this concept, the paper investigates the effectiveness and impact of how similar sources can be used for the biological synthesis of iron nanoparticles. Results showed that iron particles, when prepared in nano-metre size, offer potential advantages. When the particle size of the iron compound decreases and attains nano configuration, its surface area increases, which further improves its solubility in the gastric acid, leading to higher absorption, higher bioavailability, and producing the least organoleptic changes in food. It has no negative effects and possesses a safe, effective profile to reduce IDA. Considering all the parameters, it has been concluded that iron particles in nano configuration serve as alternative iron supplements for the complete treatment of IDA. Nanoparticles of ferric phosphate, ferric pyrophosphate, and iron oxide are the choices of iron supplements. From a sourcing perspective, the paper concludes green sources are the primary sources for the biological synthesis of iron nanoparticles. It will also be a cost-effective strategy since our goal is to treat the target population in rural India. Bio-nanotechnology serves as an alternative and promising substitution for iron supplements due to its low cost, excellent bioavailability, and strong organoleptic properties. One area of future research can be to explore the type of size and shape of iron nanoparticles that would be suitable for the different age groups of pregnant women and children and whether it would be influenced based on the topography in certain areas.

Keywords: anemia, bio-nanotechnology, iron-fortification, nanoparticle

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Authors: Marina Passero, Tianhua Zhai, Zuyi (Jacky) Huang

Abstract:

Alzheimer’s disease has become a major public health issue, as indicated by the increasing populations of Americans living with Alzheimer’s disease. After decades of extensive research in Alzheimer’s disease, only seven drugs have been approved by Food and Drug Administration (FDA) to treat Alzheimer’s disease. Five of these drugs were designed to treat the dementia symptoms, and only two drugs (i.e., Aducanumab and Lecanemab) target the progression of Alzheimer’s disease, especially the accumulation of amyloid-b plaques. However, controversial comments were raised for the accelerated approvals of either Aducanumab or Lecanemab, especially with concerns on safety and side effects of these two drugs. There is still an urgent need for further drug discovery to target the biological processes involved in the progression of Alzheimer’s disease. Excessive cholesterol has been found to accumulate in the brain of those with Alzheimer’s disease. Cholesterol can be synthesized in both the blood and the brain, but the majority of biosynthesis in the adult brain takes place in astrocytes and is then transported to the neurons via ApoE. The blood brain barrier separates cholesterol metabolism in the brain from the rest of the body. Various proteins contribute to the metabolism of cholesterol in the brain, which offer potential targets for Alzheimer’s treatment. In the astrocytes, SREBP cleavage-activating protein (SCAP) binds to Sterol Regulatory Element-binding Protein 2 (SREBP2) in order to transport the complex from the endoplasmic reticulum to the Golgi apparatus. Cholesterol is secreted out of the astrocytes by ATP-Binding Cassette A1 (ABCA1) transporter. Lipoprotein receptors such as triggering receptor expressed on myeloid cells 2 (TREM2) internalize cholesterol into the microglia, while lipoprotein receptors such as Low-density lipoprotein receptor-related protein 1 (LRP1) internalize cholesterol into the neuron. Cytochrome P450 Family 46 Subfamily A Member 1 (CYP46A1) converts excess cholesterol to 24S-hydroxycholesterol (24S-OHC). Cholesterol has been approved for its direct effect on the production of amyloid-beta and tau proteins. The addition of cholesterol to the brain promotes the activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), secretase, and amyloid precursor protein (APP), which all aid in amyloid-beta production. The reduction of cholesterol esters in the brain have been found to reduce phosphorylated tau levels in mice. In this work, a computational pipeline was developed to identify the protein targets involved in cholesterol regulation in brain and further to identify chemical compounds as the inhibitors of a selected protein target. Since extensive evidence shows the strong correlation between brain cholesterol regulation and Alzheimer’s disease, a detailed literature review on genes or pathways related to the brain cholesterol synthesis and regulation was first conducted in this work. An interaction network was then built for those genes so that the top gene targets were identified. The involvement of these genes in Alzheimer’s disease progression was discussed, which was followed by the investigation of existing clinical trials for those targets. A ligand-protein docking program was finally developed to screen 1.5 million chemical compounds for the selected protein target. A machine learning program was developed to evaluate and predict the binding interaction between chemical compounds and the protein target. The results from this work pave the way for further drug discovery to regulate brain cholesterol to combat Alzheimer’s disease.

Keywords: Alzheimer’s disease, drug discovery, ligand-protein docking, gene-network analysis, cholesterol regulation

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Authors: Junkal Gutierrez, Hernane S. Barud, Sidney J. L. Ribeiro, Agnieszka Tercjak

Abstract:

Nowadays the interest on green nanocomposites and on the development of more environmental friendly products has been increased. Bacterial cellulose has been recently investigated as an attractive environmentally friendly material for the preparation of low-cost nanocomposites. The formation of cellulose by laboratory bacterial cultures is an interesting and attractive biomimetic access to obtain pure cellulose with excellent properties. Additionally, properties as molar mass, molar mass distribution, and the supramolecular structure could be control using different bacterial strain, culture mediums and conditions, including the incorporation of different additives. This kind of cellulose is a natural nanomaterial, and therefore, it has a high surface-to-volume ratio which is highly advantageous in composites production. Such property combined with good biocompatibility, high tensile strength, and high crystallinity makes bacterial cellulose a potential material for applications in different fields. The aim of this investigation work was the fabrication of novel hybrid inorganic-organic composites based on bacterial cellulose, cultivated in our laboratory, as a template. This kind of biohybrid nanocomposites gathers together excellent properties of bacterial cellulose with the ones displayed by typical inorganic nanoparticles like optical, magnetic and electrical properties, luminescence, ionic conductivity and selectivity, as well as chemical or biochemical activity. In addition, the functionalization of cellulose with inorganic materials opens new pathways for the fabrication of novel multifunctional hybrid materials with promising properties for a wide range of applications namely electronic paper, flexible displays, solar cells, sensors, among others. In this work, different pathways for fabrication of multifunctional biohybrid nanopapers with tunable properties based on BC modified with amphiphilic poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) (EPE) block copolymer, sol-gel synthesized nanoparticles (titanium, vanadium and a mixture of both oxides) and functionalized iron oxide nanoparticles will be presented. In situ (biosynthesized) and ex situ (at post-production level) approaches were successfully used to modify BC membranes. Bacterial cellulose based biocomposites modified with different EPE block copolymer contents were developed by in situ technique. Thus, BC growth conditions were manipulated to fabricate EPE/BC nanocomposite during the biosynthesis. Additionally, hybrid inorganic/organic nanocomposites based on BC membranes and inorganic nanoparticles were designed via ex-situ method, by immersion of never-dried BC membranes into different nanoparticle solutions. On the one hand, sol-gel synthesized nanoparticles (titanium, vanadium and a mixture of both oxides) and on the other hand superparamagnetic iron oxide nanoparticles (SPION), Fe2O3-PEO solution. The morphology of designed novel bionanocomposites hybrid materials was investigated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). In order to characterized obtained materials from the point of view of future applications different techniques were employed. On the one hand, optical properties were analyzed by UV-vis spectroscopy and spectrofluorimetry and on the other hand electrical properties were studied at nano and macroscale using electric force microscopy (EFM), tunneling atomic force microscopy (TUNA) and Keithley semiconductor analyzer, respectively. Magnetic properties were measured by means of magnetic force microscopy (MFM). Additionally, mechanical properties were also analyzed.

Keywords: bacterial cellulose, block copolymer, advanced characterization techniques, nanoparticles

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Authors: F. Madani, M. Doudi, L. Rahimzadeh Torabi

Abstract:

The irregular consumption of current antibiotics contributes to an escalation in antibiotic resistance among urinary pathogens on a global scale. The objective of this research was to investigate the process of biologically synthesized silver nanoparticles through the utilization of Zataria multiflora extract. Additionally, the study aimed to evaluate the efficacy of these synthesized nanoparticles in inhibiting the growth of multi-drug resistant negative bacillus bacteria, which commonly contribute to urinary tract infections. The botanical specimen utilized in the current research investigation was Z. multiflora, and its extract was produced employing the Soxhlet extraction technique. The study examined the green synthesis conditions of silver nanoparticles by considering three key parameters: the quantity of extract used, the concentration of silver nitrate salt, and the temperature. The particle dimensions were ascertained using the Zetasizer technique. In order to identify synthesized Silver nanoparticles TEM, XRD, and FTIR methods were used. For evaluating the antibacterial effects of nanoparticles synthesized through a biological method, different concentrations of silver nanoparticles were studied on 140 cases of Multiple drug resistance (MDR) bacteria strains Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris,Citrobacter freundii, Acinetobacter bumanii and Pseudomonas aeruginosa, (each genus of bacteria, 20 samples), which all were MDR and cause urinary tract infections, for identification of bacteria were used of PCR test and laboratory methods (Agar well diffusion and Microdilution methods) to assess their sensitivity to Nanoparticles. The data were subjected to analysis using the statistical software SPSS, specifically employing nonparametric Kruskal-Wallis and Mann-Whitney tests. This study yielded noteworthy findings regarding the impacts of varying concentrations of silver nitrate, different quantities of Z. multiflora extract, and levels of temperature on nanoparticles. Specifically, it was observed that an increase in the concentration of silver nitrate, extract amount, and temperature resulted in a reduction in the size of the nanoparticles synthesized. However, the impact of the aforementioned factors on the index of particle diffusion was found to be statistically non-significant. According to the transmission electron microscopy (TEM) findings, the particles exhibited predominantly spherical morphology, with a diameter spanning from 25 to 50 nanometers. Nanoparticles in the examined sample. Nanocrystals of silver. FTIR method illustrated that the spectrums of Z. multiflora and synthesized nanoparticles had clear peaks in the ranges of 1500-2000, and 3500 - 4000. The obtained results of antibacterial effects of different concentrations of silver nanoparticles on according to agar well diffusion and microdilution method, biologically synthesized nanoparticles showed 1000 mg /ml highest and lowest mean inhibition zone diameter in E. coli, A. bumanii 23 and 15mm, respectively. MIC was observed for all of bacteria 125 mg/ml and for A. bumanii 250 mg/ml. Comparing the growth inhibitory effect of chemically synthesized the results obtained from the experiment indicated that both nanoparticles and biologically synthesized nanoparticles exhibit a notable growth inhibition effect. Specifically, the chemical method of synthesizing nanoparticles demonstrated the highest level of growth inhibition at a concentration of 62.5 mg/mL The present study demonstrated an inhibitory effect on bacterial growth, facilitating the causative factors of urine infection and multidrug resistance (MDR).

Keywords: multiple drug resistance, negative bacillus bacteria, urine infection, Zataria multiflora

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