Search results for: enzyme purification

Authors: Ibrahim Aly, Manal Ahmed, Mahmoud M. El-Shall

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Filariasis is a parasitic disease caused by small roundworms. The filarial worms are transmitted and spread by blood-feeding black flies and mosquitoes. Lymphatic filariasis (Elephantiasis) is caused by Wuchereriabancrofti, Brugiamalayi, and Brugiatimori. Elimination of Lymphatic filariasis necessitates an increasing demand for valid, reliable, and rapid diagnostic kits. Nanodiagnostics involve the use of nanotechnology in clinical diagnosis to meet the demands for increased sensitivity, specificity, and early detection in less time. The aim of this study was to evaluate the nano-based enzymelinked immunosorbent assay (ELISA) and novel nano-chip card as a rapid test for detection of filarial antigen in serum samples of human filariasis in comparison with traditional -ELISA. Serum samples were collected from an infected human with filarial gathered across Egypt's governorates. After receiving informed consenta total of 45 blood samples of infected individuals residing in different villages in Gharbea governorate, which isa nonendemic region for bancroftianfilariasis, healthy persons living in nonendemic locations (20 persons), as well as sera from 20 other parasites, affected patients were collected. The microfilaria was checked in thick smears of 20 µl night blood samples collected during 20-22 hrs. All of these individuals underwent the following procedures: history taking, clinical examination, and laboratory investigations, which included examination of blood samples for microfilaria using thick blood film and serological tests for detection of the circulating filarial antigen using polyclonal antibody- ELISA, nano-based ELISA, and nano-chip card. In the present study, a recently reported polyoclonal antibody specific to tegumental filarial antigen was used in developing nano-chip card and nano-ELISA compared to traditional ELISA for the detection of circulating filarial antigen in sera of patients with bancroftianfilariasis. The performance of the ELISA was evaluated using 45 serum samples. The ELISA was positive with sera from microfilaremicbancroftianfilariasis patients (n = 36) with a sensitivity of 80 %. Circulating filarial antigen was detected in 39/45 patients who were positive for circulating filarial antigen using nano-ELISA with a sensitivity of 86.6 %. On the other hand, 42 out of 45 patients were positive for circulating filarial antigen using nano-chip card with a sensitivity of 93.3%.In conclusion, using a novel nano-chip assay could potentially be a promising alternative antigen detection test for bancroftianfilariasis.

Keywords: lymphatic filariasis, nanotechnology, rapid diagnosis, elisa technique

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Authors: Engy Shokry, Giancarlo La Marca, Maria Luisa Della Bona

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Newborn screening for Congenital Adrenal Hyperplasia (CAH) relies on quantification of 17α-hydroxyprogesterone using enzyme immunoassays. These assays, in spite of being rapid, readily available and easy to perform, its reliability was found questionable due to lack of selectivity and specificity resulting in large number of false-positives, consequently family anxiety and associated hospitalization costs. To improve specificity of conventional 17α-hydroxyprogesterone screening which may experience false transient elevation in preterm, low birth weight or acutely ill neonates, steroid profiling by LC-MS/MS as a second-tier test was implemented. Unlike the previously applied LC-MS/MS methods, with the disadvantage of requiring a relatively high number of blood drops. Since newborn screening tests are increasing, it is necessary to minimize the sample volume requirement to make the maximum use of blood samples collected on filter paper. The proposed new method requires just one 3.2 mm dried blood spot (DBS) punch. Extraction was done using methanol: water: formic acid (90:10:0.1, v/v/v) containing deuterium labelled internal standards. Extracts were evaporated and reconstituted in 10 % acetone in water. Column switching strategy for on-line sample clean-up was applied to improve the chromatographic run. The first separative step retained the investigated steroids and passed through the majority of high molecular weight impurities. After the valve switching, the investigated steroids are back flushed from the POROS® column onto the analytical column and separated using gradient elution. Found quantitation limits were 5, 10 and 50 nmol/L for 17α-hydroxyprogesterone, androstenedione and cortisol respectively with mean recoveries of between 98.31-103.24 % and intra-/ inter-assay CV% < 10 % except at LLOQ. The method was validated using standard addition calibration and isotope dilution strategies. Reference ranges were determined by analysing samples from 896 infants of various ages at the time of sample collection. The method was also applied on patients with confirmed CAH. Our method represents an attractive combination of low sample volume requirement, minimal sample preparation time without derivatization and quick chromatography (5 min). The three steroid profile and the concentration ratios (17OHP + androstenedione/cortisol) allowed better screening outcomes of CAH reducing false positives, associated costs and anxiety.

Keywords: congenital adrenal hyperplasia (CAH), 17α-hydroxyprogesterone, androstenedione, cortisol, LC-MS/MS

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Authors: Claudio Lamilla, Andrés Santos, Vicente Llanquinao, Jocelyn Hermosilla, Leticia Barrientos

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The industry in general is very interested in improving and optimizing industrial processes in order to reduce the costs involved in obtaining raw materials and production. Thus, an interesting and cost-effective alternative is the incorporation of bioactive metabolites in such processes, being an example of this enzymes which catalyze efficiently a large number of enzymatic reactions of industrial and biotechnological interest. In the search for new sources of these active metabolites, Antarctica is one of the least explored places on our planet where the most drastic cold conditions, salinity, UVA-UVB and liquid water available are present, features that have shaped all life in this very harsh environment, especially bacteria that live in different Antarctic ecosystems, which have had to develop different strategies to adapt to these conditions, producing unique biochemical strategies. In this work the production of cellulolytic enzymes of seven bacterial strains isolated from marine sediments at different sites in the Antarctic was evaluated. Isolation of the strains was performed using serial dilutions in the culture medium at M115°C. The identification of the strains was performed using universal primers (27F and 1492R). The enzyme activity assays were performed on R2A medium, carboxy methyl cellulose (CMC)was added as substrate. Degradation of the substrate was revealed by adding Lugol. The results show that four of the tested strains produce enzymes which degrade CMC substrate. The molecular identifications, showed that these bacteria belong to the genus Streptomyces and Pseudoalteromonas, being Streptomyces strain who showed the highest activity. Only some bacteria in marine sediments have the ability to produce these enzymes, perhaps due to their greater adaptability to degrade at temperatures bordering zero degrees Celsius, some algae that are abundant in this environment and have cellulose as the main structure. The discovery of new enzymes adapted to cold is of great industrial interest, especially for paper, textiles, detergents, biofuels, food and agriculture. These enzymes represent 8% of industrial demand worldwide and is expected to increase their demand in the coming years. Mainly in the paper and food industry are required in extraction processes starch, protein and juices, as well as the animal feed industry where treating vegetables and grains helps improve the nutritional value of the food, all this clearly puts Antarctic microorganisms and their enzymes specifically as a potential contribution to industry and the novel biotechnological applications.

Keywords: antarctic, bacteria, biotechnological, cellulolytic enzymes

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Authors: Vrushali Guhe, Shailza Singh

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Cutaneous leishmaniasis is neglected tropical disease present worldwide caused by the protozoan parasite Leishmania major, the therapeutic armamentarium for leishmaniasis are showing several limitations as drugs are showing toxic effects with increasing resistance by a parasite. Thus identification of novel therapeutic targets is of paramount importance. Previous studies have shown that autophagy, a cellular process, can either facilitate infection or aid in the elimination of the parasite, depending on the specific parasite species and host background in leishmaniasis. In the present study, our objective was to target the essential autophagy protein ATG8, which plays a crucial role in the survival, infection dynamics, and differentiation of the Leishmania parasite. ATG8 in Leishmania major and its homologue, LC3, in Homo sapiens, act as autophagic markers. Present study manifested the crucial role of ATG8 protein as a potential target for combating Leishmania major infection. Through bioinformatics analysis, we identified non-conserved motifs within the ATG8 protein of Leishmania major, which are not present in LC3 of Homo sapiens. Against these two non-conserved motifs, we generated a peptide library of 60 peptides on the basis of physicochemical properties. These peptides underwent a filtering process based on various parameters, including feasibility of synthesis and purification, compatibility with Selective Reaction Monitoring (SRM)/Multiple reaction monitoring (MRM), hydrophobicity, hydropathy index, average molecular weight (Mw average), monoisotopic molecular weight (Mw monoisotopic), theoretical isoelectric point (pI), and half-life. Further filtering criterion shortlisted three peptides by using molecular docking and molecular dynamics simulations. The direct interaction between ATG8 and the shortlisted peptides was confirmed through Surface Plasmon Resonance (SPR) experiments. Notably, these peptides exhibited the remarkable ability to penetrate the parasite membrane and exert profound effects on Leishmania major. The treatment with these peptides significantly impacted parasite survival, leading to alterations in the cell cycle and morphology. Furthermore, the peptides were found to modulate autophagosome formation, particularly under starved conditions, suggesting their involvement in disrupting the regulation of autophagy within Leishmania major. In vitro, studies demonstrated that the selected peptides effectively reduced the parasite load within infected host cells. Encouragingly, these findings were corroborated by in vivo experiments, which showed a reduction in parasite burden upon peptide administration. Additionally, the peptides were observed to affect the levels of LC3II within host cells. In conclusion, our findings highlight the efficacy of these novel peptides in targeting Leishmania major’s ATG8 and disrupting parasite survival. These results provide valuable insights into the development of innovative therapeutic strategies against leishmaniasis via targeting autophagy protein ATG8 of Leishmania major.

Keywords: ATG8, leishmaniasis, surface plasmon resonance, MD simulation, molecular docking, peptide designing, therapeutics

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Authors: S. A. Krutovertsev, A. E. Tarasova, L. S. Krutovertseva, O. M. Ivanova

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The aim of the work was to get stable sensitive films with good sensitivity to ethanolamine (C2H7NO) in air. Ethanolamine is used as adsorbent in different processes of gas purification and separation. Besides it has wide industrial application. Chemical sensors of sorption type are widely used for gas analysis. Their behavior is determined by sensor characteristics of sensitive sorption layer. Forming conditions and characteristics of chemical gas sensors based on nanostructured modified silica films activated by different admixtures have been studied. As additives molybdenum containing polyoxometalates of the eighteen series were incorporated in silica films. The method of hydrolythic polycondensation from tetraethyl orthosilicate solutions was used for forming such films in this work. The method’s advantage is a possibility to introduce active additives directly into an initial solution. This method enables to obtain sensitive thin films with high specific surface at room temperature. Particular properties make polyoxometalates attractive as active additives for forming of gas-sensitive films. As catalyst of different redox processes, they can either accelerate the reaction of the matrix with analyzed gas or interact with it, and it results in changes of matrix’s electrical properties Polyoxometalates based films were deposited on the test structures manufactured by microelectronic planar technology with interdigitated electrodes. Modified silica films were deposited by a casting method from solutions based on tetraethyl orthosilicate and polyoxometalates. Polyoxometalates were directly incorporated into initial solutions. Composite nanostructured films were deposited by drop casting method on test structures with a pair of interdigital metal electrodes formed at their surface. The sensor’s active area was 4.0 x 4.0 mm, and electrode gap was egual 0.08 mm. Morphology of the layers surface were studied with Solver-P47 scanning probe microscope (NT-MDT, Russia), the infrared spectra were investigated by a Bruker EQUINOX 55 (Germany). The conditions of film formation varied during the tests. Electrical parameters of the sensors were measured electronically in real-time mode. Films had highly developed surface with value of 450 m2/g and nanoscale pores. Thickness of them was 0,2-0,3 µm. The study shows that the conditions of the environment affect markedly the sensors characteristics, which can be improved by choosing of the right procedure of forming and processing. Addition of polyoxometalate into silica film resulted in stabilization of film mass and changed markedly of electrophysical characteristics. Availability of Mn3P2Mo18O62 into silica film resulted in good sensitivity and selectivity to ethanolamine. Sensitivity maximum was observed at weight content of doping additive in range of 30–50% in matrix. With ethanolamine concentration changing from 0 to 100 ppm films’ conductivity increased by 10-12 times. The increase of sensor’s sensitivity was received owing to complexing reaction of tested substance with cationic part of polyoxometalate. This fact results in intramolecular redox reaction which sharply change electrophysical properties of polyoxometalate. This process is reversible and takes place at room temperature.

Keywords: ethanolamine, gas analysis, polyoxometalate, silica film

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Authors: V. W. Kamgang, A. Van Der Goot, N. C. Bennett, A. Ganswindt

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The roan antelope (Hippotragus equinus) is the second largest antelope species in Africa. These past decades, populations of roan antelope are declining drastically throughout Africa. This situation resulted in the development of intensive breeding programmes for this species in Southern African, where they are popular game ranching herbivores in with increasing numbers in captivity. Nowadays, avoidance of stress is important when managing wildlife to ensure animal welfare. In this regard, a non-invasive approach to monitor the adrenocortical function as a measure of stress would be preferable, since animals are not disturbed during sample collection. However, to date, a non-invasive method has not been established for the roan antelope. In this study, we validated a non-invasive technique to monitor the adrenocortical function in this species. Herein, we performed an adrenocorticotropic hormone (ACTH) stimulation test at Lapalala reserve Wilderness, South Africa, using adult captive roan antelopes to determine the stress-related physiological responses. Two individually housed roan antelope (a male and a female) received an intramuscular injection with Synacthen depot (Norvatis) loaded into a 3ml syringe (Pneu-Dart) at an estimated dose of 1 IU/kg. A total number of 86 faecal samples (male: 46, female: 40) were collected 5 days before and 3 days post-injection. All samples were then lyophilised, pulverized and extracted with 80% ethanol (0,1g/3ml) and the resulting faecal extracts were analysed for immunoreactive faecal glucocorticoid metabolite (fGCM) concentrations using five enzyme immunoassays (EIAs); (i) 11-oxoaetiocholanolone I (detecting 11,17 dioxoandrostanes), (ii) 11-oxoaetiocholanolone II (detecting fGCM with a 5α-pregnane-3α-ol-11one structure), (iii) a 5α-pregnane-3β-11β,21-triol-20-one (measuring 3β,11β-diol CM), (iv) a cortisol and (v) a corticosterone. In both animals, all EIAs detected an increase in fGCM concentration 100% post-ACTH administration. However, the 11-oxoaetiocholanolone I EIA performed best, with a 20-fold increase in the male (baseline: 0.384 µg/g, DW; peak: 8,585 µg/g DW) and a 17-fold in the female (baseline: 0.323 µg/g DW, peak: 7,276 µg/g DW), measured 17 hours and 12 hours post-administration respectively. These results are important as the ability to assess adrenocortical function non-invasively in roan can now be used as an essential prerequisite to evaluate the effects of stressful circumstances; such as variation of environmental conditions or reproduction in other to improve management strategies for the conservation of this iconic antelope species.

Keywords: adrenocorticotropic hormone challenge, adrenocortical function, captive breeding, non-invasive method, roan antelope

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Authors: Carmiña L. Vargas-Zapata, María A. Conde-Sarmiento, Maria Consuelo Maestre-Vargas

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Calcium is an essential element for good growth and development of the organism, and its requirement is increased at school age. Low socio-economic populations of developing countries such as Colombia may have food deficiency of this mineral in schoolchildren that could be reflected in calcium biochemical indicators, bone alterations and anthropometric indicators. The objective of this investigation was to evaluate some calcium biochemical indicators in a group of schoolchildren of low socioeconomic level from Barranquilla city and to correlate with body mass index. 60 schoolchildren aged 7 to 15 years were selected from Jesus’s Heart Educational Institution in Barranquilla-Atlántico, apparently healthy, without suffering from infectious or gastrointestinal diseases, without habits of drinking alcohol or smoking another hallucinogenic substance and without taking supplementation with calcium in the last six months or another substance that compromises bone metabolism. The research was approved by the ethics committee at Universidad del Atlántico. The selected children were invited to donate a blood and urine sample in a fasting time of 12 hours, the serum was separated by centrifugation and frozen at ˗20 ℃ until analyzed and the same was done with the urine sample. On the day of the biological collections, the weight and height of the students were measured to determine the nutritional status by BMI using the WHO tables. Calcium concentrations in serum and urine (SCa, UCa), alkaline phosphatase activity total and of bone origin (SAPT, SBAP) and urinary creatinine (UCr) were determined by spectrophotometric methods using commercial kits. Osteocalcin and Cross-linked N-telopeptides of type I collagen (NTx-1) in serum were measured with an enzyme-linked inmunosorbent assay. For statistical analysis the Statgraphics software Centurium XVII was used. 63% (n = 38) and 37% (n = 22) of the participants were male and female, respectively. 78% (n = 47), 5% (n = 3) and 17% (n = 10) had a normal, malnutrition and high nutritional status, respectively. The averages of evaluated indicators levels were (mean ± SD): 9.50 ± 1.06 mg/dL for SCa; 181.3 ± 64.3 U/L for SAPT, 143.8 ± 73.9 U/L for SBAP; 9.0 ± 3.48 ng/mL for osteocalcin and 101.3 ± 12.8 ng/mL for NTx-1. UCa level was 12.8 ± 7.7 mg/dL that adjusted with creatinine ranged from 0.005 to 0.395 mg/mg. Considering serum calcium values, approximately 7% of school children were hypocalcemic, 16% hypercalcemic and 77% normocalcemic. The indicators evaluated did not correlate with the BMI. Low values ​​were observed in calcium urinary excretion and high in NTx-1, suggesting that mechanisms such as increase in renal retention of calcium and in bone remodeling may be contributing to calcium homeostasis.

Keywords: calcium, calcium biochemical, indicators, school children, low socioeconomic status

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Authors: Peter. M. Kusen, Georg Wandrey, Christopher Probst, Dietrich Kohlheyer, Jochen Buchs, Jorg Pietruszkau

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Light as a stimulus provides the capability to develop regulation techniques for customizable gene expression. A great advantage is the extremely flexible and accurate dosing that can be performed in a non invasive and sterile manner even for high throughput technologies. Therefore, light regulation in a multiwell microbioreactor system was realized providing the opportunity to control gene expression with outstanding complexity. A light-regulated gene expression system in Saccharomyces cerevisiae was designed applying the strategy of caged compounds. These compounds are photo-labile protected and therefore biologically inactive regulator molecules which can be reactivated by irradiation with certain light conditions. The “caging” of a repressor molecule which is consumed after deprotection was essential to create a flexible expression system. Thereby, gene expression could be temporally repressed by irradiation and subsequent release of the active repressor molecule. Afterwards, the repressor molecule is consumed by the yeast cells leading to reactivation of gene expression. A yeast strain harboring a construct with the corresponding repressible promoter in combination with a fluorescent marker protein was applied in a Photo-BioLector platform which allows individual irradiation as well as online fluorescence and growth detection. This device was used to precisely control the repression duration by adjusting the amount of released repressor via different irradiation times. With the presented screening platform the regulation of complex expression procedures was achieved by combination of several repression/derepression intervals. In particular, a stepwise increase of temporally-constant expression levels was demonstrated which could be used to study concentration dependent effects on cell functions. Also linear expression rates with variable slopes could be shown representing a possible solution for challenging protein productions, whereby excessive production rates lead to misfolding or intoxication. Finally, the very flexible regulation enabled accurate control over the expression induction, although we used a repressible promoter. Summing up, the continuous online regulation of gene expression has the potential to synchronize gene expression levels to optimize metabolic flux, artificial enzyme cascades, growth rates for co cultivations and many other applications addicted to complex expression regulation. The developed light-regulated expression platform represents an innovative screening approach to find optimization potential for production processes.

Keywords: caged-compounds, gene expression regulation, optogenetics, photo-labile protecting group

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Authors: Ram Chandra Sah

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Few years back, naturally a very beautiful country Nepal was facing a lot of problems related to the practice of open defecation (having no toilet) by almost 98% people of the country. Now, the scenario is changed. Government of Nepal set the target of achieving the situation of basic level sanitation (toilets) facilities by 2017 AD for which the Sanitation and Hygiene Master Plan (SHMP) was brought in 2011 AD with the major beauty as institutional set up formation, local formal authority leadership, locally formulated strategic plan; partnership, harmonized and coordinated approach to working; no subsidy or support at a blanket level, community and local institutions or organizations mobilization approaches. Now, the Open Defecation Free (ODF) movement in the country is at a full swing. The Sanitation and Hygiene Master Plan (SHMP) has clearly defined Total Sanitation which is accepted to be achieved if all the households of the related boundary have achieved the 6 indicators such as the access and regular use of toilet(s), regular use of soap and water at the critical moments, regular practice of use of food hygiene behavior, regular practice of use of water hygiene behavior including household level purification of locally available drinking water, maintenance of regular personal hygiene with household level waste management and the availability of the state of overall clean environment at the concerned level of boundary. Nepal has 3158 Village Development Committees (VDC's) in the rural areas. Amarapuri VDC was selected for the purpose of achieving Total Sanitation. Based on the SHMP; different methodologies such as updating of Village Water Sanitation and Hygiene Coordination Committee (V-WASH-CC), Total Sanitation team formation including one volunteer for each indicator, campaigning through settlement meetings, midterm evaluation which revealed the need of ward level 45 (5 for all 9 wards) additional volunteers, ward wise awareness creation with the help of the volunteers, informative notice boards and hoarding boards with related messages at important locations, management of separate waste disposal rings for decomposable and non-decomposable wastes, related messages dissemination through different types of local cultural programs, public toilets construction and management by community level; mobilization of local schools, offices and health posts; reward and recognition to contributors etc. were adopted for achieving 100 % coverage of each indicator. The VDC was in a very worse situation in 2010 with just 50, 30, 60, 60, 40, 30 percent coverage of the respective indicators and became the first VDC of the country declared with Total Sanitation. The expected result of 100 percent coverage of all the indicators was achieved in 2 years 10 months and 19 days. Experiences of Amarapuri were replicated successfully in different parts of the country and many VDC's have been declared with the achievement of Total Sanitation. Thus, Community Mobilized Total Sanitation Movement in Nepal has supported a lot for achieving a Total Sanitation situation of the country with a minimal cost and it is believed that the approach can be very useful for other developing or under developed countries of the world.

Keywords: community mobilized, open defecation free, sanitation and hygiene master plan, total sanitation

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Authors: Rajeev Ravindran, Amit K. Jaiswal

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Lignocellulose is the largest reservoir of inexpensive, renewable source of carbon. It is composed of lignin, cellulose and hemicellulose. Cellulose and hemicellulose is composed of reducing sugars glucose, xylose and several other monosaccharides which can be metabolised by microorganisms to produce several value added products such as biofuels, enzymes, aminoacids etc. Enzymatic treatment of lignocellulose leads to the release of monosaccharides such as glucose and xylose. However, factors such as the presence of lignin, crystalline cellulose, acetyl groups, pectin etc. contributes to recalcitrance restricting the effective enzymatic hydrolysis of cellulose and hemicellulose. In order to overcome these problems, pre-treatment of lignocellulose is generally carried out which essentially facilitate better degradation of lignocellulose. A range of pre-treatment strategy is commonly employed based on its mode of action viz. physical, chemical, biological and physico-chemical. However, existing pretreatment strategies result in lower sugar yield and formation of inhibitory compounds. In order to overcome these problems, we proposes a novel pre-treatment, which utilises the superior oxidising capacity of alkaline potassium permanganate assisted by ultra-sonication to break the covalent bonds in spent coffee waste to remove recalcitrant compounds such as lignin. The pre-treatment was conducted for 30 minutes using 2% (w/v) potassium permanganate at room temperature with solid to liquid ratio of 1:10. The pre-treated spent coffee waste (SCW) was subjected to enzymatic hydrolysis using enzymes cellulase and hemicellulase. Shake flask experiments were conducted with a working volume of 50mL buffer containing 1% substrate. The results showed that the novel pre-treatment strategy yielded 7 g/L of reducing sugar as compared to 3.71 g/L obtained from biomass that had undergone dilute acid hydrolysis after 24 hours. From the results obtained it is fairly certain that ultrasonication assists the oxidation of recalcitrant components in lignocellulose by potassium permanganate. Enzyme hydrolysis studies suggest that ultrasound assisted alkaline potassium permanganate pre-treatment is far superior over treatment by dilute acid. Furthermore, SEM, XRD and FTIR were carried out to analyse the effect of the new pre-treatment strategy on structure and crystallinity of pre-treated spent coffee wastes. This novel one-step pre-treatment strategy was implemented under mild conditions and exhibited high efficiency in the enzymatic hydrolysis of spent coffee waste. Further study and scale up is in progress in order to realise future industrial applications.

Keywords: spent coffee waste, alkaline potassium permanganate, ultra-sonication, physical characterisation

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Authors: Priyanka Sharma, Ranjita Das, Mohan C. Kalita, Debajit Thakur

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Background: Actinomycetes are the richest source of novel bioactive secondary metabolites such as antibiotics, enzymes and other therapeutically useful metabolites with diverse biological activities. The present study aims at the antimicrobial potential and genetic diversity of culturable Actinomycetes isolated from protected forest ecosystems of Assam which includes Kaziranga National Park (26°30˝-26°45˝N and 93°08˝-93°36˝E), Pobitora Wildlife Sanctuary (26º12˝-26º16˝N and 91º58˝-92º05˝E) and Gibbon Wildlife Sanctuary (26˚40˝-26˚45˝N and 94˚20˝-94˚25˝E) which are located in the North-eastern part of India. Northeast India is a part of the Indo-Burma mega biodiversity hotspot and most of the protected forests of this region are still unexplored for the isolation of effective antibiotic-producing Actinomycetes. Thus, there is tremendous possibility that these virgin forests could be a potential storehouse of novel microorganisms, particularly Actinomycetes, exhibiting diverse biological properties. Methodology: Soil samples were collected from different ecological niches of the protected forest ecosystems of Assam and Actinomycetes were isolated by serial dilution spread plate technique using five selective isolation media. Preliminary screening of Actinomycetes for an antimicrobial activity was done by spot inoculation method and the secondary screening by disc diffusion method against several test pathogens, including multidrug resistant Staphylococcus aureus (MRSA). The strains were further screened for the presence of antibiotic synthetic genes such as type I polyketide synthases (PKS-I), type II polyketide synthases (PKS-II) and non-ribosomal peptide synthetases (NRPS) genes. Genetic diversity of the Actinomycetes producing antimicrobial metabolites was analyzed through 16S rDNA-RFLP using Hinf1 restriction endonuclease. Results: Based on the phenotypic characterization, a total of 172 morphologically distinct Actinomycetes were isolated and screened for antimicrobial activity by spot inoculation method on agar medium. Among the strains tested, 102 (59.3%) strains showed activity against Gram-positive bacteria, 98 (56.97%) against Gram-negative bacteria, 92 (53.48%) against Candida albicans MTCC 227 and 130 (75.58%) strains showed activity against at least one of the test pathogens. Twelve Actinomycetes exhibited broad spectrum antimicrobial activity in the secondary screening. The taxonomic identification of these twelve strains by 16S rDNA sequencing revealed that Streptomyces was found to be the predominant genus. The PKS-I, PKS-II and NRPS genes detection indicated diverse bioactive products of these twelve Actinomycetes. Genetic diversity by 16S rDNA-RFLP indicated that Streptomyces was the dominant genus amongst the antimicrobial metabolite producing Actinomycetes. Conclusion: These findings imply that Actinomycetes from the protected forest ecosystems of Assam, India, are a potential source of bioactive secondary metabolites. These areas are as yet poorly studied and represent diverse and largely unscreened ecosystem for the isolation of potent Actinomycetes producing antimicrobial secondary metabolites. Detailed characterization of the bioactive Actinomycetes as well as purification and structure elucidation of the bioactive compounds from the potent Actinomycetes is the subject of ongoing investigation. Thus, to exploit Actinomycetes from such unexplored forest ecosystems is a way to develop bioactive products.

Keywords: Actinomycetes, antimicrobial activity, forest ecosystems, RFLP

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Authors: Hana Al-Baghaoi, Kumar Shiva Gubbiyappa, Mayuren Candasamy, Kiruthiga Perumal Vijayaraman

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Acne is a chronic inflammatory disease of the sebaceous glands characterized by areas of skin with seborrhea, comedones, papules, pustules, nodules, and possibly scarring. Propionibacterium acnes (P. acnes), plays a key role in the pathogenesis of acne. Their colonization and proliferation trigger the host’s inflammatory response leading to the production of pro-inflammatory cytokines such as interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α). The usage of honey and natural compounds to treat skin ailments has strong support in the current trend of drug discovery. The present study was carried out evaluate antimicrobial and anti-inflammatory potential of Doani Sidr honey and its fractions against P. acnes and to screen madecassoside alone and in combination with fractions of honey. The broth dilution method was used to assess the antibacterial activity. Also, ultra structural changes in cell morphology were studied before and after exposure to Sidr honey using transmission electron microscopy (TEM). The three non-toxic concentrations of the samples were investigated for suppression of cytokines IL 8 and TNF α by testing the cell supernatants in the co-culture of the human peripheral blood mononuclear cells (hPBMCs) heat killed P. acnes using enzyme immunoassay kits (ELISA). Results obtained was evaluated by statistical analysis using Graph Pad Prism 5 software. The Doani Sidr honey and polysaccharide fractions were able to inhibit the growth of P. acnes with a noteworthy minimum inhibitory concentration (MIC) value of 18% (w/v) and 29% (w/v), respectively. The proximity of MIC and MBC values indicates that Doani Sidr honey had bactericidal effect against P. acnes which is confirmed by TEM analysis. TEM images of P. acnes after treatment with Doani Sidr honey showed completely physical membrane damage and lysis of cells; whereas non honey treated cells (control) did not show any damage. In addition, Doani Sidr honey and its fractions significantly inhibited (> 90%) of secretion of pro-inflammatory cytokines like TNF α and IL 8 by hPBMCs pretreated with heat-killed P. acnes. However, no significant inhibition was detected for madecassoside at its highest concentration tested. Our results suggested that Doani Sidr honey possesses both antimicrobial and anti-inflammatory effects against P. acnes and can possibly be used as therapeutic agents for acne. Furthermore, polysaccharide fraction derived from Doani Sidr honey showed potent inhibitory effect toward P. acnes. Hence, we hypothesize that fraction prepared from Sidr honey might be contributing to the antimicrobial and anti-inflammatory activity. Therefore, this polysaccharide fraction of Doani Sidr honey needs to be further explored and characterized for various phytochemicals which are contributing to antimicrobial and anti-inflammatory properties.

Keywords: Doani sidr honey, Propionibacterium acnes, IL-8, TNF alpha

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Authors: Hung-Chih Hsu, Pey-Jium Chang, Ching-Hsein Chen, Jer-Liang Andrew Yeh

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Osteoarthritis (OA) is a common disorder in rehabilitation clinic. The main characteristics include joint pain, localized tenderness and enlargement, joint effusion, cartilage destruction, loss of adhesion of perichondrium, synovium hyperplasia. Synoviocytes inflammation might be a cause of local tenderness and effusion. Inflammation cytokines might also play an important role in joint pain, cartilage destruction, decrease adhesion of perichondrium to the bone. Treatments of osteoarthritis include non-steroid anti-inflammation drugs (NSAID), glucosamine supplementation, hyaluronic acid, arthroscopic debridement, and total joint replacement. Total joint replacement is commonly used in patients with severe OA who failed respond to pharmacological treatment. However, some patients received surgery had serious adverse events, including instability of the implants due to insufficient adhesion to the adjacent bony tissue or synovial inflammation. We tried to develop ideal nano-topographic oxidized silicon nanosponges by using with various chemicals to produce thickness difference in nanometers in order to study more about the cell-environment interactions in vitro like the alterations of cell adhesion, morphology, extracellular matrix secretions in the pathogenesis of osteoarthritis. Cytokines studies like growth factor, reactive oxygen species, reactive inflammatory materials (Like nitrous oxide and prostaglandin E2), extracellular matrix (ECM) degradation enzymes, and synthesis of collagen will also be observed and discussed. Extracellular and intracellular expression transforming growth factor beta (TGF-β) will be studied by reverse transcription-polymerase chain reaction (RT-PCR). The degradation of ECM will be observed by the bioactivity ratio of matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase by ELISA (Enzyme-linked immunosorbent assay). When rabbit synoviocytes were cultured on these nano-topographic structures, they demonstrate better cell adhesion rate, decreased expression of MMP-2,9 and PGE2, and increased expression of TGF-β when cultured in nano-topographic oxidized silicon nanosponges than in the planar oxidized silicon ones. These results show cell behavior, cytokine production can be influenced by physical characteristics from different nano-topographic structures. Our study demonstrates the possibility of manipulating cell behavior in these nano-topographic biomaterials.

Keywords: osteoarthritis, synoviocyte, oxidized silicon surfaces, reactive oxygen species

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Authors: Neha Batra Bali, Monika Tomar, Vinay Gupta

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A reagentless biosensor for detection of urea based on ZnO-CuO composite thin film is presented in following work. Biosensors have immense potential for varied applications ranging from environmental to clinical testing, health care, and cell analysis. Immense growth in the field of biosensors is due to the huge requirement in today’s world to develop techniques which are both cost effective and accurate for prevention of disease manifestation. The human body comprises of numerous biomolecules which in their optimum levels are essential for functioning. However mismanaged levels of these biomolecules result in major health issues. Urea is one of the key biomolecules of interest. Its estimation is of paramount significance not only for healthcare sector but also from environmental perspectives. If level of urea in human blood/serum is abnormal, i.e., above or below physiological range (15-40mg/dl)), it may lead to diseases like renal failure, hepatic failure, nephritic syndrome, cachexia, urinary tract obstruction, dehydration, shock, burns and gastrointestinal, etc. Various metal nanoparticles, conducting polymer, metal oxide thin films, etc. have been exploited to act as matrix to immobilize urease to fabricate urea biosensor. Amongst them, Zinc Oxide (ZnO), a semiconductor metal oxide with a wide band gap is of immense interest as an efficient matrix in biosensors by virtue of its natural abundance, biocompatibility, good electron communication feature and high isoelectric point (9.5). In spite of being such an attractive candidate, ZnO does not possess a redox couple of its own which necessitates the use of electroactive mediators for electron transfer between the enzyme and the electrode, thereby causing hindrance in realization of integrated and implantable biosensor. In the present work, an effort has been made to fabricate a matrix based on ZnO-CuO composite prepared by pulsed laser deposition (PLD) technique in order to incorporate redox properties in ZnO matrix and to utilize the same for reagentless biosensing applications. The prepared bioelectrode Urs/(ZnO-CuO)/ITO/glass exhibits high sensitivity (70µAmM⁻¹cm⁻²) for detection of urea (5-200 mg/dl) with high stability (shelf life ˃ 10 weeks) and good selectivity (interference ˂ 4%). The enhanced sensing response obtained for composite matrix is attributed to the efficient electron exchange between ZnO-CuO matrix and immobilized enzymes, and subsequently fast transfer of generated electrons to the electrode via matrix. The response is encouraging for fabricating reagentless urea biosensor based on ZnO-CuO matrix.

Keywords: biosensor, reagentless, urea, ZnO-CuO composite

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Authors: Mustafa M. Donma, Orkide Donma

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The impact of metabolic syndrome (MetS) on cognition and functions of the brain is being investigated. Iron deficiency and deficiencies of B9 (folate) as well as B12 (cobalamin) vitamins are best-known nutritional anemias. They are associated with cognitive disorders and learning difficulties. The antidepressant effects of vitamin D are known and the deficiency state affects mental functions negatively. The aim of this study is to investigate possible correlations of MetS with serum brain-derived neurotrophic factor (BDNF), iron, folate, cobalamin and vitamin D in pediatric patients. 30 children, whose age- and sex-dependent body mass index (BMI) percentiles vary between 85 and 15, 60 morbid obese children with above 99^th percentiles constituted the study population. Anthropometric measurements were taken. BMI values were calculated. Age- and sex-dependent BMI percentile values were obtained using the appropriate tables prepared by the World Health Organization (WHO). Obesity classification was performed according to WHO criteria. Those with MetS were evaluated according to MetS criteria. Serum BDNF was determined by enzyme-linked immunosorbent assay. Serum folate was analyzed by an immunoassay analyzer. Serum cobalamin concentrations were measured using electrochemiluminescence immunoassay. Vitamin D status was determined by the measurement of 25-hydroxycholecalciferol [25-hydroxy vitamin D3, 25(OH)D] using high performance liquid chromatography. Statistical evaluations were performed using SPSS for Windows, version 16. The p values less than 0.05 were accepted as statistically significant. Although statistically insignificant, lower folate and cobalamin values were found in MO children compared to those observed for children with normal BMI. For iron and BDNF values, no alterations were detected among the groups. Significantly decreased vitamin D concentrations were noted in MO children with MetS in comparison with those in children with normal BMI (p ≤ 0.05). The positive correlation observed between iron and BDNF in normal-BMI group was not found in two MO groups. In THE MetS group, the partial correlation among iron, BDNF, folate, cobalamin, vitamin D controlling for waist circumference and BMI was r = -0.501; p ≤ 0.05. None was calculated in MO and normal BMI groups. In conclusion, vitamin D should also be considered during the assessment of pediatric MetS. Waist circumference and BMI should collectively be evaluated during the evaluation of MetS in children. Within this context, BDNF appears to be a key biochemical parameter during the examination of obesity degree in terms of mental functions, cognition and learning capacity. The association observed between iron and BDNF in children with normal BMI was not detected in MO groups possibly due to development of inflammation and other obesity-related pathologies. It was suggested that this finding may contribute to mental function impairments commonly observed among obese children.

Keywords: brain-derived neurotrophic factor, iron, vitamin B9, vitamin B12, vitamin D

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Authors: Haiying Zong, Yi Yuan, Pengcheng Li

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Cadmium (Cd), one of the toxic heavy metals, has high solubility and mobility in agricultural soils and is readily taken up by roots and transported to the vegetative and reproductive organs which can cause deleterious effects on crop yield and quality. Excess Cd in plants can interfere with many metabolic processes, such as photosynthesis, transpiration, respiration or nutrients homeostasis. Generally, the main methods to reduce Cd accumulation in plants are to decrease the concentration of Cd in the soil solution through reduction of Cd influx into the soil system, site selection, and management practices. However, these approaches can be very costly and consume a lot of energy Therefore, it is critical to develop effective approaches to reduce the Cd concentration in plants. It is proved that chitooligosaccharide (COS) can enhance the plant's tolerance to abiotic stress including drought stress, salinity stress, and toxic metal stress. However, so far little information is known about whether foliar application with COS modulates Cd-induced toxicity in plants. The metal detoxification processes of plants treated with COS also remain unclear. In this study, edible rape (Brassica rapa L.), one of the most widely consumed leafy vegetables, was selected as an experimental mode plant. The effect of foliar application with COS on reducing Cd accumulation in edible rape was investigated. Moreover, Cd subcellular distribution pattern in response to Cd stress in the rape plant sprayed with COS was further tested in order to explore the potential detoxification mechanisms in plants. The results demonstrated that spraying COS at different concentrations (25, 50,100 and 200 mg L-1) possess diverse functions including growth-promoting,chlorophyll contents-enhancing, malondialdehyde (MDA) level-decreasing in leaves, Cd2+ concentration-decreasingin shoots and roots of edible rape under Cd stress. In addition, it was found that COS can also dramatically improve superoxide dismutase (SOD) activity, catalase (CAT) activity and peroxidase (POX) activity of edible rape leaves. The relievingeffect of COS was related to theconcentration and COS with 50-100 mg L-1 displayed the best activity. Furtherly, theexperiments results exhibitedthat COS could decrease the proportion of Cd in the organelle fraction of leaves by 40.1% while enhance the proportion of Cd in the soluble fraction by 13.2% at the concentration of 50 mg L-1. The above results showed that COS may have thepotential to improve plant resistance to Cd via promoting antioxidant enzyme activities and altering Cd subcellular distribution. All the results described here open up a new way to study the protection role of COS in alleviating Cd tolerance and lay the foundation for future research about the detoxification mechanism at subcellular level.

Keywords: chitooligosaccharide, cadmium, edible rape (Brassica rapa L.), subcellular distribution

Procedia PDF Downloads 273

Authors: José Maria, Vânia Laranjo, Luís Abrunhosa, António Inês

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The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Keywords: carboxypeptidase, lactic acid bacteria, mycotoxins, ochratoxin a.

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Authors: Martyna Chotomska, Aleksandra Studzinska, Marta Lisowska, Justyna Szubert, Aleksandra Tabis, Jacek Bania, Arkadiusz Miazek

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Objectives: Assessing antigen-specific T cell responses is of utmost importance for the pre-clinical testing of prototype vaccines against intracellular pathogens and tumor antigens. Mainly two types of in vitro assays are used for this purpose 1) enzyme-linked immunospot (ELISpot) and 2) intracellular cytokine staining (ICS). Both are time-consuming, relatively expensive, and require manual dexterity. Here, we assess if a straightforward detection of luciferase activity in blood samples of transgenic reporter mice expressing a secreted Lucia luciferase under the transcriptional control of IFN-γ promoter parallels the sensitivity of IFNγ ELISpot assay. Methods: IFN-γ-LUCIA mouse strain carrying multiple copies of Lucia luciferase transgene under the transcriptional control of IFNγ minimal promoter were generated by pronuclear injection of linear DNA. The specificity of transgene expression and mobilization was assessed in vitro using transgenic splenocytes exposed to various mitogens. The IFN-γ-LUCIA mice were immunized with 50mg of ovalbumin (OVA) emulsified in incomplete Freund’s adjuvant three times every two weeks by subcutaneous injections. Blood samples were collected before and five days after each immunization. Luciferase activity was assessed in blood serum. Peripheral blood mononuclear cells were separated and assessed for frequencies of OVA-specific IFNγ-secreting T cells. Results: We show that in vitro cultured splenocytes of IFN-γ-LUCIA mice respond by 2 and 3 fold increase in secreted luciferase activity to T cell mitogens concanavalin A and phorbol myristate acetate, respectively but fail to respond to B cell-stimulating E.coli lipopolysaccharide. Immunization of IFN-γ-LUCIA mice with OVA leads to over 4 fold increase in luciferase activity in blood serum five days post-immunization with a barely detectable increase in OVA-specific, IFNγ-secreting T cells by ELISpot. Second and third immunizations, further increase the luciferase activity and coincidently also increase the frequencies of OVA-specific T cells by ELISpot. Conclusions: We conclude that minimally invasive monitoring of luciferase secretions in blood serum of IFN-γ-LUCIA mice constitutes a sensitive method for evaluating primary and memory Th1 responses to protein antigens. As such, this method may complement existing methods for rapid immunogenicity assessment of prototype vaccines.

Keywords: ELISpot, immunogenicity, interferon-gamma, reporter mice, vaccines

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Authors: Auteleyeva Laura, Maikanov Balgabai, Smagulova Ayana

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In order to find and select detoxification methods, patent and information research was conducted, as a result of which 68 patents for inventions were found, among them from the near abroad - 14 (Russia), from far abroad: China – 27, USA - 6, South Korea–1, Germany - 2, Mexico – 4, Yugoslavia – 7, Austria, Taiwan, Belarus, Denmark, Italy, Japan, Canada for 1 security document. Aflatoxin B₁ in various nuts was determined by two methods: enzyme immunoassay "RIDASCREEN ® FAST Aflatoxin" with determination of optical density on a microplate spectrophotometer RIDA®ABSORPTION 96 with RIDASOFT® software Win.NET (Germany) and the method of high-performance liquid chromatography (HPLC Corporation Water, USA) according to GOST 307112001. For experimental contamination of nuts, the cultivation of strain A was carried out. flavus KWIK-STIK on the medium of Chapek (France) with subsequent infection of various nuts (peanuts, peanuts with shells, badam, walnuts with and without shells, pistachios).Based on our research, we have selected 2 detoxification methods: method 1 – combined (5% citric acid solution + microwave for 640 W for 3 min + UV for 20 min) and a chemical method with various leaves of plants: Artemisia terra-albae, Thymus vulgaris, Callogonum affilium, collected in the territory of Akmola region (Artemisia terra-albae, Thymus vulgaris) and Western Kazakhstan (Callogonum affilium). The first stage was the production of ethanol extracts of Artemisia terraea-albae, Thymus vulgaris, Callogonum affilium. To obtain them, 100 g of vegetable raw materials were taken, which was dissolved in 70% ethyl alcohol. Extraction was carried out for 2 hours at the boiling point of the solvent with a reverse refrigerator using an ultrasonic bath "Sapphire". The obtained extracts were evaporated on a rotary evaporator IKA RV 10. At the second stage, the three samples obtained were tested for antimicrobial and antifungal activity. Extracts of Thymus vulgaris and Callogonum affilium showed high antimicrobial and antifungal activity. Artemisia terraea-albae extract showed high antimicrobial activity and low antifungal activity. When testing method 1, it was found that in the first and third experimental groups there was a decrease in the concentration of aflatoxin B1 in walnut samples by 63 and 65%, respectively, but these values also exceeded the maximum permissible concentrations, while the nuts in the second and third experimental groups had a tart lemon flavor; When testing method 2, a decrease in the concentration of aflatoxin B1 to a safe level was observed by 91% (0.0038 mg/kg) in nuts of the 1st and 2nd experimental groups (Artemisia terra-albae, Thymus vulgaris), while in samples of the 2nd and 3rd experimental groups, a decrease in the amount of aflatoxin in 1 to a safe level was observed.

Keywords: nuts, aflatoxin B1, my, mycotoxins

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Authors: Clara Tramuta, Floris Irene, Daniela Manila Bianchi, Monica Pitti, Giulia Federica Cazzaniga, Lucia Decastelli

Abstract:

This work presents the results obtained by the Regional Reference Centre for Salmonella Typing (CeRTiS) in a retrospective study aimed to investigate, through Pulsed-field Gel Electrophoresis (PFGE) analysis, the genetic relatedness of emerging Salmonella serotypes of human origin circulating in North-West of Italy. Furthermore, the goal of this work was to create a Regional database to facilitate foodborne outbreak investigation and to monitor them at an earlier stage. A total of 112 strains, isolated from 2016 to 2018 in hospital laboratories, were included in this study. The isolates were previously identified as Salmonella according to standard microbiological techniques and serotyping was performed according to ISO 6579-3 and the Kaufmann-White scheme using O and H antisera (Statens Serum Institut®). All strains were characterized by PFGE: analysis was conducted according to a standardized PulseNet protocol. The restriction enzyme XbaI was used to generate several distinguishable genomic fragments on the agarose gel. PFGE was performed on a CHEF Mapper system, separating large fragments and generating comparable genetic patterns. The agarose gel was then stained with GelRed® and photographed under ultraviolet transillumination. The PFGE patterns obtained from the 112 strains were compared using Bionumerics version 7.6 software with the Dice coefficient with 2% band tolerance and 2% optimization. For each serotype, the data obtained with the PFGE were compared according to the geographical origin and the year in which they were isolated. Salmonella strains were identified as follow: S. Derby n. 34; S. Infantis n. 38; S. Napoli n. 40. All the isolates had appreciable restricted digestion patterns ranging from approximately 40 to 1100 kb. In general, a fairly heterogeneous distribution of pulsotypes has emerged in the different provinces. Cluster analysis indicated high genetic similarity (≥ 83%) among strains of S. Derby (n. 30; 88%), S. Infantis (n. 36; 95%) and S. Napoli (n. 38; 95%) circulating in north-western Italy. The study underlines the genomic similarities shared by the emerging Salmonella strains in Northwest Italy and allowed to create a database to detect outbreaks in an early stage. Therefore, the results confirmed that PFGE is a powerful and discriminatory tool to investigate the genetic relationships among strains in order to monitoring and control Salmonellosis outbreak spread. Pulsed-field gel electrophoresis (PFGE) still represents one of the most suitable approaches to characterize strains, in particular for the laboratories for which NGS techniques are not available.

Keywords: emerging Salmonella serotypes, genetic characterization, human strains, PFGE

Procedia PDF Downloads 67

Authors: Elif Erdem, Nalan Kaya, Gonca Ozan, Durrin Ozlem Dabak, Enver Ozan

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This study was carried out to determine the histological and biochemical changes in the lungs of the rat pups exposed to tobacco smoke during pregnancy period and to investigate the protective effects of alpha lipoic acid, which is administered during pregnancy, on these changes. In our study, 24 six-week old Spraque-Dawley female rats weighing 160 ± 10 g were used (n:7). Rats were randomly divided into four equal groups: group I (control), group II (tobacco smoke), group III (tobacco smoke + alpha lipoic acid) and group IV (alpha lipoic acid). Rats in the group II, group III were exposed to tobacco smoke twice a day for one hour starting from eight weeks before mating and during pregnancy. In addition to tobacco smoke, 20 mg/kg of alpha lipoic acid was administered via oral gavage to the rats in the group III. Only alpha lipoic acid was administered to the rats in the group IV. Once after the delivery, all administrations were stopped. On the 7 and 21th days, the seven pups of all groups were decapitated. A portion of the lung was taken and stained with HE, PAS and Masson. In addition to immunohistochemical staining of surfactant protein A, vascular endothelial growth factor, caspase-3, TUNEL method was also used to determine apoptosis. Biochemical analyzes were performed with some part of the lung tissue specimens. In the histological evaluations performed under light microscopy, inflammatory cell increase, hemorrhagic areas, edema, interalveolar septal thickening, alveolar numbers decrease, degeneration of some bronchi and bronchial epithelium, epithelial cells that were fallen into the lumen and hyaline membrane formation were observed in tobacco smoke group. These findings were ameliorated in tobacco smoke + ALA group. Hyaline membrane formation was not detected in this group. The TUNEL positive cell numbers a significant increase was detected in the tobacco smoke group, whereas a significant decrease was detected in the tobacco smoke + ALA group. In terms of the immunoreactivity of both SP-A and VEGF, a significant decrease was observed in the tobacco smoke group, and a significant increase was observed in the tobacco smoke + ALA group. Regarding the immunoreactivity of caspase-3, there was a significant increase in the group of tobacco smoke and a significant decrease in the group of tobacco smoke + ALA. The malondialdehyde levels were determined to be significantly increased in the tobacco smoke group, and a significant decreased in the tobacco smoke + ALA. Glutathione and superoxide dismutase enzyme activities showed a significant decrease in the group of tobacco smoke and a significant increase in the tobacco smoke + ALA group. In conclusion, we suggest that the exposure to tobacco smoke during pregnancy leads to morphological, histopathological and functional changes on lung development by causing oxidative damage in lung tissues of neonatal rats and the maternal use of alpha lipoic acid can provide a protective effect on the neonatal lung development against this oxidative stress originating from tobacco smoke.

Keywords: alpha lipoic acid, lung, neonate, tobacco smoke, pregnancy

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Authors: Gustavo Axel Elizalde-Velázquez

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Metformin is the first line of oral therapy to treat type II diabetes and is also employed as a treatment for other indications, such as polycystic ovary syndrome, cancer, and COVID-19. Recent data suggest it is the aspirin of the 21st century due to its antioxidant and anti-aging effects. However, increasingly current articles indicate its long-term consumption generates mitochondrial impairment. Up to date, it is known metformin increases the biogenesis of Alzheimer's amyloid peptides via up-regulating BACE1 transcription, but further information related to brain damage after its consumption is missing. Bearing in mind the above, this work aimed to establish whether or not chronic exposure to metformin may alter swimming behavior and induce neurotoxicity in Danio rerio adults. For this purpose, 250 Danio rerio grown-ups were assigned to six tanks of 50 L of capacity. Four of the six systems contained 50 fish, while the remaining two had 25 fish (≈1 male:1 female ratio). Every system with 50 fish was allocated one of the three metformin treatment concentrations (1, 20, and 40 μg/L), with one system as the control treatment. Systems with 25 fish, on the other hand, were used as positive controls for acetylcholinesterase (10 μg/L of Atrazine) and oxidative stress (3 μg/L of Atrazine). After four months of exposure, a mean of 32 fish (S.D. ± 2) per group of MET treatment survived, which were used for the evaluation of behavior with the Novel Tank test. Moreover, after the behavioral assessment, we aimed to collect the blood and brains of all fish from all treatment groups. For blood collection, fish were anesthetized with an MS-222 solution (150 mg/L), while for brain gathering, fish were euthanized using the hypothermic shock method (2–4 °C). Blood was employed to determine CASP3 activity and the percentage of apoptotic cells with the TUNEL assay, and brains were used to evaluate acetylcholinesterase activity, oxidative damage, and gene expression. After chronic exposure, MET-exposed fish exhibited less swimming activity when compared to control fish. Moreover, compared with the control group, MET significantly inhibited the activity of AChE and induced oxidative damage in the brain of fish. Concerning gene expression, MET significantly upregulated the expression of Nrf1, Nrf2, BAX, p53, BACE1, APP, PSEN1, and downregulated CASP3 and CASP9. Although MET did not overexpress the CASP3 gene, we saw a meaningful rise in the activity of this enzyme in the blood of fish exposed to MET compared to the control group, which we then confirmed by a high number of apoptotic cells in the TUNEL assay. To the best of our understanding, this is the first study that delivers evidence of oxidative impairment, apoptosis, AChE alteration, and overexpression of B- amyloid-related genes in the brain of fish exposed to metformin.

Keywords: AChE inhibition, CASP3 activity, NovelTank test, oxidative damage, TUNEL assay

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Authors: Suman Som, Abhijit Maity, Sunil B. Daschakraborty, Sujit Chaudhuri, Manik Pradhan

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Background: Helicobacter pylori is a common and important human pathogen and the primary cause of peptic ulcer disease and gastric cancer. Currently H. pylori infection is detected by both invasive and non-invasive way but the diagnostic accuracy is not up to the mark. Aim: To set up an optimal diagnostic cut-off value of 13C-Urea Breath Test to detect H. pylori infection and evaluate a novel c-PDR methodology to overcome of inconclusive grey zone. Materials and Methods: All 83 subjects first underwent upper-gastrointestinal endoscopy followed by rapid urease test and histopathology and depending on these results; we classified 49 subjects as H. pylori positive and 34 negative. After an overnight, fast patients are taken 4 gm of citric acid in 200 ml water solution and 10 minute after ingestion of the test meal, a baseline exhaled breath sample was collected. Thereafter an oral dose of 75 mg 13C-Urea dissolved in 50 ml water was given and breath samples were collected upto 90 minute for 15 minute intervals and analysed by laser based high precisional cavity enhanced spectroscopy. Results: We studied the excretion kinetics of 13C isotope enrichment (expressed as δDOB13C ‰) of exhaled breath samples and found maximum enrichment around 30 minute of H. pylori positive patients, it is due to the acid mediated stimulated urease enzyme activity and maximum acidification happened within 30 minute but no such significant isotopic enrichment observed for H. pylori negative individuals. Using Receiver Operating Characteristic (ROC) curve an optimal diagnostic cut-off value, δDOB13C ‰ = 3.14 was determined at 30 minute exhibiting 89.16% accuracy. Now to overcome grey zone problem we explore percentage dose of 13C recovered per hour, i.e. 13C-PDR (%/hr) and cumulative percentage dose of 13C recovered, i.e. c-PDR (%) in exhaled breath samples for the present 13C-UBT. We further explored the diagnostic accuracy of 13C-UBT by constructing ROC curve using c-PDR (%) values and an optimal cut-off value was estimated to be c-PDR = 1.47 (%) at 60 minute, exhibiting 100 % diagnostic sensitivity , 100 % specificity and 100 % accuracy of 13C-UBT for detection of H. pylori infection. We also elucidate the gastric emptying process of present 13C-UBT for H. pylori positive patients. The maximal emptying rate found at 36 minute and half empting time of present 13C-UBT was found at 45 minute. Conclusions: The present study exhibiting the importance of c-PDR methodology to overcome of grey zone problem in 13C-UBT for accurate determination of infection without any risk of diagnostic errors and making it sufficiently robust and novel method for an accurate and fast non-invasive diagnosis of H. pylori infection for large scale screening purposes.

Keywords: 13C-Urea breath test, c-PDR methodology, grey zone, Helicobacter pylori

Procedia PDF Downloads 279

Authors: I. Nava-Arenas, N. Ruiz-Ordaz, C. J. Galindez-Mayer, M. L. Luna-Guido, S. L. Ruiz-López, A. Cabrera-Orozco, D. Nava-Arenas

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Among the most important herbicides, the organochlorine compounds are of considerable interest due to their recalcitrance to the chemical, biological, and photolytic degradation, their persistence in the environment, their mobility, and their bioacummulation. The most widely used herbicides in North America are primarily 2,4-dichlorophenoxyacetic acid (2,4-D), the triazines (atrazine and simazine), and to a lesser extent diuron. The contamination of soils and water bodies frequently occurs by mixtures of these xenobiotics. For this reason, in this work, the operational stability to changes in the composition of the medium supplied to an aerobic biofilm reactor was studied. The reactor was packed with fragments of volcanic rock that retained a complex microbial film, able to degrade a mixture of organochlorine herbicides atrazine, simazine, diuron and 2,4-D, and whose members have microbial genes encoding the main catabolic enzymes atzABCD, tfdACD and puhB. To acclimate the attached microbial community, the biofilm reactor was fed continuously with a mineral minimal medium containing the herbicides (in mg•L-1): diuron, 20.4; atrazine, 14.2, simazine, 11.4, and 2,4-D, 59.7, as carbon and nitrogen sources. Throughout the bioprocess, removal efficiencies of 92-100% for herbicides, 78-90% for COD, 92-96% for TOC and 61-83% for dehalogenation were reached. In the microbial community, the genes encoding catabolic enzymes of different herbicides tfdACD, puhB and, occasionally, the genes atzA and atzC were detected. After the acclimatization, the triazine herbicides were eliminated from the mixture formulation. Volumetric loading rates of the mixture 2,4-D and diuron were continuously supplied to the reactor (1.9-21.5 mg herbicides •L-1 •h-1). Along the bioprocess, the removal efficiencies obtained were 86-100% for the mixture of herbicides, 63-94% for for COD, 90-100% for COT, and dehalogenation values of 63-100%. It was also observed that the genes encoding the enzymes in the catabolism of both herbicides, tfdACD and puhB, were consistently detected; and, occasionally, the atzA and atzC. Subsequently, the triazine herbicide atrazine and simazine were restored to the medium supply. Different volumetric charges of this mixture were continuously fed to the reactor (2.9 to 12.6 mg herbicides •L-1 •h-1). During this new treatment process, removal efficiencies of 65-95% for the mixture of herbicides, 63-92% for COD, 66-89% for TOC and 73-94% of dehalogenation were observed. In this last case, the genes tfdACD, puhB and atzABC encoding for the enzymes involved in the catabolism of the distinct herbicides were consistently detected. The atzD gene, encoding the cyanuric hydrolase enzyme, could not be detected, though it was determined that there was partial degradation of cyanuric acid. In general, the community in the biofilm reactor showed some catabolic stability, adapting to changes in loading rates and composition of the mixture of herbicides, and preserving their ability to degrade the four herbicides tested; although, there was a significant delay in the response time to recover to degradation of the herbicides.

Keywords: biodegradation, biofilm reactor, microbial community, organochlorine herbicides

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Authors: Avijit Dey, Antony Gomes, Subir Chandra Dasgupta

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Tea (Camellia sinensis) is the most popular beverages in the world and is ranked second after the water. Tea has been considered as a health promoting beverage since ancient times due to its health-promoting activity. Recently, immunomodulatory, anti-arthritic, antioxidant, anticancer and cardioprotective activity of tea has been established. Very few studies have demonstrated the effect of high dose of black tea on health. The aim of the present study was to evaluate the role of low & high dose of Black Tea Extract (BTE) on the different physiological parameters of mother and pups during prenatal and postnatal developmental period in the experimental rodent. BTE was orally administered in LD (50mg BTE/kg/day) and HD (100mg BTE/kg/day) except control groups of rats (n=6/group) throughout the prenatal (day 0-21) and postnatal (day 21-42) periods. During prenatal period (0, 7th, 14th, 20th days) body weight, urinary calcium, magnesium, urea and creatinine was measured. In postnatal period physical (0, 10th, 21th days) parameters of pups like body weight, cranial length, cranial diameter, neck width, tail length, craniosacral length of pups were analyzed. Liver and lungs from pups and kidney spleen, etc. from mothers were collected on day 42 for histopathological studies. The comparative urine strip and morphology of RBC was also analyzed by SEM from mothers of different groups on day 42. The level of cytokines like IL-1alpha, IL-1beta, IL-6, IL-10, TNF-alpha were analysed by enzyme-linked immunosorbent assay (ELISA) on day 0, day 20 and day 42. The body weight of LD and HD mothers were also significantly (P<0.05) less than control mothers at 20th day of pregnancy and there was also significant changes in urinary calcium, urea, creatinine. The bio morphometric analysis of pups showed significant alteration (P<0.05) in HD groups relative to control. Some histological alterations were also observed in pups and mothers. Comparative urine strip analysis and morphology of RBC showed significant changes in treated groups. LD and HD treated mothers showed an increase in proinflammatory cytokines like IL-1beta, TNF-alpha and decrease in anti-inflammatory cytokine-like IL-10 on day 20 compared to PC mothers. This study clearly indicated that high dose of BTE possesses detrimental effect on pregnant mother and the pup. Further studies are in progress to elucidate the molecular mechanism of actions. This project work has been sponsored by National Tea Research Foundation vide Project Sanction No.: 17 (305)/2013/4423 dated 11th March, 2014. All experimental protocols described in the study were approved by animal ethics committee.

Keywords: black tea extract, pregnancy, prenatal and postnatal development, inflammation

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Authors: Sudarat Jiamyangyuen, Tipawan Thongsook, Riantong Singanusong, Chanida Saengtubtim

Abstract:

Rice is a staple food as well as exported commodity of Thailand. Rice bran, a 10.5% constituent of rice grain, is a by-product of rice milling process. Rice bran is normally used as a raw material for rice bran oil production or sold as feed with a low price. Therefore, this study aimed to increase value of defatted rice bran as obtained after extracting of rice bran oil. Conventionally, the protein in defatted rice bran was extracted using alkaline extraction and acid precipitation, which results in reduction of nutritious components in rice bran. Rice bran protein concentrate is suitable for those who are allergenic of protein from other sources eg. milk, wheat. In addition to its hypoallergenic property, rice bran protein also contains good quantity of lysine. Thus it may act as a suitable ingredient for infant food formulations while adding variety to the restricted diets of children with food allergies. The objectives of this study were to compare properties of rice bran protein concentrate (RBPC) extracted from defatted rice bran using enzymes together with precipitation step using polysaccharides (alginate and carrageenan) to those of a control sample extracted using a conventional method. The results showed that extraction of protein from rice bran using enzymes exhibited the higher protein recovery compared to that extraction with alkaline. The extraction conditions using alcalase 2% (v/w) at 50 C, pH 9.5 gave the highest protein (2.44%) and yield (32.09%) in extracted solution compared to other enzymes. Rice bran protein concentrate powder prepared by a precipitation step using alginate (protein in solution: alginate 1:0.006) exhibited the highest protein (27.55%) and yield (6.62%). Precipitation using alginate was better than that of acid. RBPC extracted with alkaline (ALK) or enzyme alcalase (ALC), then precipitated with alginate (AL) (samples RBP-ALK-AL and RBP-ALC-AL) yielded the precipitation rate of 75% and 91.30%, respectively. Therefore, protein precipitation using alginate was then selected. Amino acid profile of control sample, and sample precipitated with alginate, as compared to casein and soy protein isolated, showed that control sample showed the highest content among all sample. Functional property study of RBP showed that the highest nitrogen solubility occurred in pH 8-10. There was no statically significant between emulsion capacity and emulsion stability of control and sample precipitated by alginate. However, control sample showed a higher of foaming and lower foam stability compared to those of sample precipitated with alginate. The finding was successful in terms of minimizing chemicals used in extraction and precipitation steps in preparation of rice bran protein concentrate. This research involves in a production of value-added product in which the double amount of protein (28%) compared to original amount (14%) contained in rice bran could be beneficial in terms of adding to food products eg. healthy drink with high protein and fiber. In addition, the basic knowledge of functional property of rice bran protein concentrate was obtained, which can be used to appropriately select the application of this value-added product from rice bran.

Keywords: alginate, carrageenan, rice bran, rice bran protein

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Authors: Osman Turkoglu, Alvin Estilo, Ritu Gupta, Liliam Pineda-Salgado, Rajesh Pandey

Abstract:

Background: Hepatotoxicity can be linked to a variety of clinical symptoms and histopathological signs, posing a great challenge in the surveillance of suspected drug-induced liver injury (DILI) cases in the safety database. Additionally, the majority of such cases are rare, idiosyncratic, highly unpredictable, and tend to demonstrate unique individual susceptibility; these qualities, in turn, lend to a pharmacovigilance monitoring process that is often tedious and time-consuming. Objective: Develop a multifactorial algorithm to assist pharmacovigilance physicians in identifying high-risk hepatotoxicity cases associated with DILI from the sponsor’s safety database (Argus). Methods: Multifactorial selection criteria were established using Structured Query Language (SQL) and the TIBCO Spotfire® visualization tool, via a combination of word fragments, wildcard strings, and mathematical constructs, based on Hy’s law criteria and pattern of injury (R-value). These criteria excluded non-eligible cases from monthly line listings mined from the Argus safety database. The capabilities and limitations of these criteria were verified by comparing a manual review of all monthly cases with system-generated monthly listings over six months. Results: On an average, over a period of six months, the algorithm accurately identified 92% of DILI cases meeting established criteria. The automated process easily compared liver enzyme elevations with baseline values, reducing the screening time to under 15 minutes as opposed to multiple hours exhausted using a cognitively laborious, manual process. Limitations of the algorithm include its inability to identify cases associated with non-standard laboratory tests, naming conventions, and/or incomplete/incorrectly entered laboratory values. Conclusions: The newly developed multifactorial algorithm proved to be extremely useful in detecting potential DILI cases, while heightening the vigilance of the drug safety department. Additionally, the application of this algorithm may be useful in identifying a potential signal for DILI in drugs not yet known to cause liver injury (e.g., drugs in the initial phases of development). This algorithm also carries the potential for universal application, due to its product-agnostic data and keyword mining features. Plans for the tool include improving it into a fully automated application, thereby completely eliminating a manual screening process.

Keywords: automation, drug-induced liver injury, pharmacovigilance, post-marketing

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Authors: Jyoti Singh, Ranju Bansal

Abstract:

Fighting pain and inflammation is a common problem faced by physicians while dealing with a wide variety of diseases. Since ancient time nonsteroidal anti-inflammatory agents (NSAIDs) and opioids have been the cornerstone of treatment therapy, however, the usefulness of both these classes is limited due to severe side effects. NSAIDs, which are mainly used to treat mild to moderate inflammatory pain, induce gastric irritation and nephrotoxicity whereas opioids show an array of adverse reactions such as respiratory depression, sedation, and constipation. Moreover, repeated administration of these drugs induces tolerance to the analgesic effects and physical dependence. Further discovery of selective COX-2 inhibitors (coxibs) suggested safety without any ulcerogenic side effects; however, long-term use of these drugs resulted in kidney and hepatic toxicity along with an increased risk of secondary cardiovascular effects. The basic approaches towards inflammation and pain treatment are constantly changing, and researchers are continuously trying to develop safer and effective anti-inflammatory drug candidates for the treatment of different inflammatory conditions such as osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, psoriasis and multiple sclerosis. Synthetic 3(2H)-pyridazinones constitute an important scaffold for drug discovery. Structure-activity relationship studies on pyridazinones have shown that attachment of a lactam at N-2 of the pyridazinone ring through a methylene spacer results in significantly increased anti-inflammatory and analgesic properties of the derivatives. Further introduction of the heterocyclic ring at lactam nitrogen results in improvement of biological activities. Keeping in mind these SAR studies, a new series of compounds were synthesized as shown in scheme 1 and investigated for anti-inflammatory, analgesic, anti-platelet activities and docking studies. The structures of newly synthesized compounds have been established by various spectroscopic techniques. All the synthesized pyridazinone derivatives exhibited potent anti-inflammatory and analgesic activity. Homoveratryl substituted derivative was found to possess highest anti-inflammatory and analgesic activity displaying 73.60 % inhibition of edema at 40 mg/kg with no ulcerogenic activity when compared to standard drugs indomethacin. Moreover, 2-substituted-4-benzo[d][1,3]dioxole-6-phenylpyridazin-3(2H)-ones derivatives did not produce significant changes in bleeding time and emerged as safe agents. Molecular docking studies also illustrated good binding interactions at the active site of the cyclooxygenase-2 (hCox-2) enzyme.

Keywords: anti-inflammatory, analgesic, pyridazin-3(2H)-one, selective COX-2 inhibitors

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Authors: M. Iqbal Hossain, Azharul Islam Khan, S. M. Rafiqul Islam, Tahmeed Ahmed

Abstract:

Objective: Moderate acute malnutrition (MAM) is a major cause of morbidity and mortality in under-5 children of low-income countries. Approximately 14.6% of all under-5 mortality worldwide is attributed to MAM with >3 times increased risk of death compared to well-nourished peers. Prevalence of MAM among under-5 children in Bangladesh is ~12% (~1.7 million). Providing a diet containing adequate nutrients is the mainstay of treatment of children with MAM. It is now possible to process fish into fish peptides with longer shelf-life without refrigerator, known as ‘Fish Surimi peptide’ and this could be an attractive alternative to supply fish protein in the diet of children in low-income countries like Bangladesh. We conducted this study to assess the acceptability of Fish Surimi peptide given with various foods/meals in 2-5 years old children with MAM. Design/methods: Fish Surimi peptide is broken down from white fish meat using plant-derived enzyme and the ingredient is just fish meat consisted of 20 different kinds of amino acids including nine essential amino acids. In a convenience sample of 34 children we completed the study ward of Dhaka Hospital of icddr,b in Bangladesh during November 2014 through February 2015. For each child the study was for two consecutive days: i.e. direct observation of food intake of two lunches and two suppers. In a randomly and blinded manner and cross over design an individual child received Fish Surimi peptide (5g at lunch and 5g at supper) mixed meal [e.g. 30g rice and 30g dahl (thick lentil soup) or 60g of a vegetables-lentil-rice mixed local dish known as khichuri in one day and the same meal on other day without any Fish Surimi peptide. We observed the completeness and eagerness of eating and any possible side effect (e.g. allergy, vomiting, diarrhea etc.) over these two days. Results: The mean±SD age of the enrolled children was 38.4±9.4 months, weight 11.22±1.41 kg, height 91.0±6.3 cm, and WHZ was -2.13±0.76. Their mean±SD total feeding time (minutes) for lunch was 25.4±13.6 vs. 20.6±11.1 (p=0.130) and supper was 22.3±9.7 vs. 19.7±11.2 (p=0.297), and total amount (g) of food eaten in lunch and supper was found similar 116.1±7.0 vs. 117.7±8.0 (p=3.01) in A (Fish Surimi) and B group respectively. Score in Hedonic scale by mother on test of food given to children at lunch or supper was 3.9±0.2 vs. 4.0±0.2 (p=0.317) and on overall acceptance (including the texture, smell, and appearance) of food at lunch or supper was 3.9±0.2 vs. 4.0±0.2 (p=0.317) for A and B group respectively. No adverse event was observed in any food group during the study period. Conclusions: Fish Surimi peptide may be a cost effective supplementary food, which should be tested by appropriately designed randomized community level intervention trial both in wasted children and stunted children.

Keywords: protein-energy malnutrition, moderate acute malnutrition, weight-for-height z-score, mid upper arm circumference, acceptability, fish surimi peptide, under-5 children

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

Abstract:

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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