Search results for: streptomyces rochei subsp. chromatogenes neae-k

Authors: Endang Lukitaningsih, Fathul Jannah, Arief R. Hakim, Ratna D. Puspita, Zullies Ikawati

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Rifampicin is a semisynthetic antibiotic derivative of rifamycin B produced by Streptomyces mediterranei. RIF has been used worldwide as first line drug-prescribed throughout tuberculosis therapy. This study aims to develop and to validate an HPLC method couple with a UV detection for determination of rifampicin in spiked human plasma and its application for bioequivalence study. The chromatographic separation was achieved on an RP-C18 column (LachromHitachi, 250 x 4.6 mm., 5μm), utilizing a mobile phase of phosphate buffer/acetonitrile (55:45, v/v, pH 6.8 ± 0.1) at a flow of 1.5 mL/min. Detection was carried out at 337 nm by using spectrophotometer. The developed method was statistically validated for the linearity, accuracy, limit of detection, limit of quantitation, precise and specifity. The specifity of the method was ascertained by comparing chromatograms of blank plasma and plasma containing rifampicin; the matrix and rifampicin were well separated. The limit of detection and limit of quantification were 0.7 µg/mL and 2.3 µg/mL, respectively. The regression curve of standard was linear (r > 0.999) over a range concentration of 20.0 – 100.0 µg/mL. The mean recovery of the method was 96.68 ± 8.06 %. Both intraday and interday precision data showed reproducibility (R.S.D. 2.98% and 1.13 %, respectively). Therefore, the method can be used for routine analysis of rifampicin in human plasma and in bioequivalence study. The validated method was successfully applied in pharmacokinetic and bioequivalence study of rifampicin tablet in a limited number of subjects (under an Ethical Clearance No. KE/FK/6201/EC/2015). The mean values of Cmax, Tmax, AUC(0-24) and AUC(o-∞) for the test formulation of rifampicin were 5.81 ± 0.88 µg/mL, 1.25 hour, 29.16 ± 4.05 µg/mL. h. and 29.41 ± 4.07 µg/mL. h., respectively. Meanwhile for the reference formulation, the values were 5.04 ± 0.54 µg/mL, 1.31 hour, 27.20 ± 3.98 µg/mL.h. and 27.49 ± 4.01 µg/mL.h. From bioequivalence study, the 90% CIs for the test formulation/reference formulation ratio for the logarithmic transformations of Cmax and AUC(0-24) were 97.96-129.48% and 99.13-120.02%, respectively. According to the bioequivamence test guidelines of the European Commission-European Medicines Agency, it can be concluded that the test formulation of rifampicin is bioequivalence with the reference formulation.

Keywords: validation, HPLC, plasma, bioequivalence

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Authors: Alok Kumar, Hari Ram, Lebin Thomas, Ved Pal Singh

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Organic solvent tolerant amylases and proteases of microbial origin are in great demand for their application in transglycosylation of water-insoluble flavanoids and in peptide synthesizing reaction in organic media. Most of the amylases and proteases are unstable in presence of organic solvent. In the present work two different bacterial strains M-11 and VP-07 were isolated from the soil sample of a dairy farm in Delhi, India, for the efficient production of extracellular amylase and protease through their screening on starch agar (SA) and skimmed milk agar (SMA) plates, respectively. Both the strains (M-11 and VP-07) were identified based on morphological, biochemical and 16S rRNA gene sequencing methods. After analysis through Ez-Taxon software, the strains M-11 and VP-07 were found to have maximum pairwise similarity of 98.63% and 100% with Bacillus subtilis subsp. inaquosorum BGSC 3A28 and Bacillus anthracis ATCC 14578 and were therefore identified as Bacillus sp. UKS1 and Bacillus sp. UKS2, respectively. Time course study of enzyme activity and bacterial growth has shown that both strains exhibited typical sigmoid growth behavior and maximum production of amylase (180 U/ml) and protease (78 U/ml) by these strains (UKS1 and UKS2) was commenced during stationary phase of growth at 24 and 20 h, respectively. Thereafter, both amylase and protease were tested for their tolerance towards organic solvents and were found to be active as well stable in p-xylene (130% and 115%), chloroform (110% and 112%), isooctane (119% and 107%), benzene (121% and 104%), n-hexane (116% and 103%) and toluene (112% and 101%, respectively). Owing to such properties, these enzymes can be exploited for their potential application in industries for organic synthesis.

Keywords: amylase, enzyme activity, industrial applications, organic solvent tolerant, protease

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Authors: Nurul Aqilah Mohd Zaini, Afroditi Chatzifragkou, Dimitris Charalampopoulos

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D-Lactic acid production is gaining increasing attention due to the thermostable properties of its polymer, Polylactic Acid (PLA). In this study, D-lactic acid was produced in microbial cultures using Lactobacillus coryniformis subsp. torquens as D-lactic acid producer and hydrolysates of Dried Distillers Grains with Solubles (DDGS) as fermentation substrate. Prior to fermentation, DDGS was first alkaline pretreated with 5% (w/v) NaOH, for 15 minutes (121oC/ ~16 psi). This led to the generation of DDGS solid residues, rich in carbohydrates and especially cellulose (~52%). The carbohydrate-rich solids were then subjected to enzymatic hydrolysis with Accellerase® 1500. For Separate Hydrolysis and Fermentation (SHF), enzymatic hydrolysis was carried out at 50oC for 24 hours, followed by fermentation of D-lactic acid at 37oC in controlled pH 6. The obtained hydrolysate contained 24 g/l glucose, 5.4 g/l xylose and 0.6 g/l arabinose. In the case of Simultaneous Saccharification and Fermentation (SSF), hydrolysis and fermentation were conducted in a single step process at 37oC in pH 5. The enzymatic hydrolysis of DGGS pretreated solids took place mostly during lag phase of L. coryniformis fermentation, with only a small amount of glucose consumed during the first 6 h. When exponential phase was started, glucose generation reduced as the microorganism started to consume glucose for D-lactic acid production. Higher concentrations of D-lactic acid were produced when SSF approach was applied, with 28 g/l D-lactic acid after 24 h of fermentation (84.5% yield). In contrast, 21.2 g/l D-lactic acid were produced when SHF was used. The optical pu rity of D-lactic acid produced from both experiments was 99.9%. Besides, approximately 2 g/l acetic acid was also generated due to lactic acid degradation after glucose depletion in SHF. SSF was proved an efficient towards DDGS ulilisation and D-lactic acid production, by reducing the overall processing time, yielding sufficient D-lactic acid concentrations without the generation of fermentation by-products.

Keywords: DDGS, alkaline pretreatment, SSF, D-lactic acid

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Authors: Molin A., De Ramos J. M., Cadion L. G., Pico R. L.

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Malnutrition and food insecurity represent significant global challenges affecting millions of individuals, particularly in low-income and developing regions. The researchers created a solar-powered enzymatic composting bioreactor with an Arduino-based monitoring system for pH, humidity, and temperature. It manages mixed municipal solid wastes incorporating industrial enzymes and whey additives for accelerated composting and minimized carbon footprint. Within 15 days, the bioreactor yielded 54.54% compost compared to 44.85% from traditional methods, increasing yield by nearly 10%. Tests showed that the bioreactor compost had 4.84% NPK, passing metal analysis standards, while the traditional pit compost had 3.86% NPK; both are suitable for agriculture. Statistical analyses, including ANOVA and Tukey's HSD test, revealed significant differences in agricultural yield across different compost types based on leaf length, width, and number of leaves. The study compared the effects of different composts on Brassica rapa subsp. Chinesis (Petchay) and Brassica juncea (Mustasa) plant growth. For Pechay, significant effects of compost type on plant leaf length (F(5,84) = 62.33, η² = 0.79) and leaf width (F(5,84) = 12.35, η² = 0.42) were found. For Mustasa, significant effects of compost type on leaf length (F(4,70) = 20.61, η² = 0.54), leaf width (F(4,70) = 19.24, η² = 0.52), and number of leaves (F(4,70) = 13.17, η² = 0.43) were observed. This study explores the effectiveness of the enzymatic composting bioreactor and its viability in promoting carbon farming as a solution to food insecurity and malnutrition.

Keywords: malnutrition, food insecurity, enzymatic composting bioreactor, arduino-based monitoring system, enzymes, carbon farming, whey additive, NPK level

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Authors: Lee A. Browne, K. Rumbold

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The first fluorinating enzyme, 5'-fluoro-5'-deoxyadenosine synthase (fluorinase) was isolated from the soil bacterium Streptomyces cattleya. Such an enzyme, with the ability to catalyze a C-F bond, presents great potential as a biocatalyst. Naturally fluorinated compounds are extremely rare in nature. As a result, the number of fluorinases identified remains relatively few. The field of fluorination is almost completely synthetic. However, with the increasing demand for fluorinated organic compounds of commercial value in the agrochemical, pharmaceutical and materials industries, it has become necessary to utilize biologically based methods such as biocatalysts. A key step in this crucial process is the large-scale production of the fluorinase enzyme in considerable quantities for industrial applications. Thus, this study aimed to optimize expression of the fluorinase enzyme in both prokaryotic and eukaryotic expression systems in order to obtain high protein yields. The fluorinase gene was cloned into the pET 41b(+) and pPinkα-HC vectors and used to transform the expression hosts, E.coli BL21(DE3) and Pichia pastoris (PichiaPink™ strains) respectively. Expression trials were conducted to select optimal conditions for expression in both expression systems. Fluorinase catalyses a reaction between S-adenosyl-L-Methionine (SAM) and fluoride ion to produce 5'-fluorodeoxyadenosine (5'FDA) and L-Methionine. The activity of the enzyme was determined using HPLC by measuring the product of the reaction 5'FDA. A gradient mobile phase of 95:5 v/v 50mM potassium phosphate buffer to a final mobile phase containing 80:20 v/v 50mM potassium phosphate buffer and acetonitrile were used. This resulted in the complete separation of SAM and 5’-FDA which eluted at 1.3 minutes and 3.4 minutes respectively. This proved that the fluorinase enzyme was active. Optimising expression of the fluorinase enzyme was successful in both E.coli and PichiaPink™ where high expression levels in both expression systems were achieved. Protein production will be scaled up in PichiaPink™ using fermentation to achieve large-scale protein production. High level expression of protein is essential in biocatalysis for the availability of enzymes for industrial applications.

Keywords: biocatalyst, expression, fluorinase, PichiaPink™

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Authors: Mohammadreza Hajialigol, Nargues Falahi Charkhabi, Fatemeh Shahryari, Saadat Sarikhani

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BACKGROUND AND OBJECTIVES Iran is the third producer of Persian walnut worldwide. However, its walnut trees have been under threat from decline during last decade. Walnut canker caused by B. nigrifluens and B. rubrifaciens was recorded in multiple regions of Iran. Furthermore, Brenneria rosae subsp. rosae and Gibbsiella quercinecans were recently recognized as responsible for walnut decline in northwestern Iran. This study aimed to identify the causal agent of walnut decline in Kermanshah and Isfahan. MATERIAL AND METHODS Symptomatic samples were collected from affected walnut trees of Kermanshah and Isfahan provinces. The pathogenicity of strains was proved on immature walnut fruits cv. ‘Hartley’ and young green twigs of two-year-old walnut seedling cv. ‘Chandler’. Pathogenic strains were subjected to conventional phenotypic tests. 16S rRNA, gyrB, and infB genes were partially amplified and sequenced. RESULTS Irregular longitudinal cankers and dark lesions were observed in the outer and inner bark, respectively. Twenty-four strains were isolated on EMB-agar media. Fourteen strains were able to cause necrosis and a dark-colored region in the mesocarp and on young green twigs around the inoculation site 14 and 30 days post-inoculation, respectively. Strains were able to hydrolyze Tween 20, Tween 80, gelatin and esculin, however, did not produce indole or urease. Pairwise comparison, the 16S rRNA gene nucleotide sequences of strain I2 were 100% identical with those of Rahnella victoriana FRB 225T. Moreover, a phylogenetic tree reconstructed based on the concatenated sequences of two housekeeping gene fragments, gyrB (601 bp) and infB (615 bp), revealed that the strains I2, I5, and KE6 were clustered with R. victoriana FRB 225T. CONCLUSION To the best of our knowledge, this is the first report of R. victoriana in association with walnut decline. This result is necessary to find resistant genotypes.

Keywords: emerging pathogens, Iran, juglans regia, MLSA

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Authors: Mervat Kassem, Nourhan Fanaki, F. Dabbous, Hamida Abou-Shleib, Y. R. Abdel-Fattah

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With increasing environmental awareness and emphasis on a sustainable society in harmony with the global environment, biosurfactants are gaining prominence and have already taken over for a number of important industrial uses. They are produced by living organisms, for examples Pseudomonas aeruginosa which produces rhamnolipids, Candida (formerly Torulopsis) bombicola, which produces high yields of sophorolipids from vegetable oils and sugars and Bacillus subtilis which produces a lipopeptide called surfactin. The main goal of this work was to optimize biosurfactants production by an environmental Gram positive isolate for large scale production with maximum yield and low cost. After molecular characterization, phylogenetic tree was constructed where it was found to be B. subtilis, which close matches to B. subtilis subsp. subtilis strain CICC 10260. For optimizing its biosurfactants production, sequential statistical design using Plackett-Burman and response surface methodology, was applied where 11 variables were screened. When analyzing the regression coefficients for the 11 variables, pH, glucose, glycerol, yeast extract, ammonium chloride and ammonium nitrate were found to have a positive effect on the biosurfactants production. Ammonium nitrate, pH and glucose were further studied as significant independent variables for Box-Behnken design and their optimal levels were estimated and were found to be 7.328 pH value, 3 g% glucose and 0.21g % ammonium nitrate yielding high biosurfactants concentration that reduced the surface tension of the culture medium from 72 to 18.16 mN/m. Next, kinetics of cell growth and biosurfactants production by the tested B. subtilis isolate, in bioreactor was compared with that of shake flask where the maximum growth and specific growth (µ) in the bioreactor was higher by about 25 and 53%, respectively, than in shake flask experiment, while the biosurfactants production kinetics was almost the same in both shake flask and bioreactor experiments.

Keywords: biosurfactants, B. subtilis, molecular identification, phylogenetic trees, Plackett-Burman design, Box-Behnken design, 16S rRNA

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Authors: H. Alijani, M. Jabari, S. Matroodi, H. Zolqarnein, A. Sharafi, I. Zamani

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Actinomycetes, Gram-positive bacteria, are interesting as a main producer of secondary metabolites and are important industrially and pharmaceutically. The marine environment is a potential source for new actinomycetes, which can provide novel bioactive compounds and industrially important enzymes. The aims of this study were to isolate and identify novel actinomycetes from Persian Gulf sediments and screen these isolates for the production of secondary metabolites, especially antibiotics, Using phylogenetic (16S rRNA gene sequence), morphological and biochemical analyses. 15 different actinomycete strains from Persian Gulf sediments at a depth of 5-10 m were identified. DNA extraction was done using Cinnapure DNA Kit. PCR amplification of 16S rDNA gene was performed using F27 and R1492 primers. Phylogenetic tree analysis was performed using the MEGA 6 software. Most of the isolated strains belong to the genus namely Streptomyces (14), followed by Nocardiopsis (1). Antibacterial assay of the isolates supernatant was performed using a standard disc diffusion assay with replication (n=3). The results of disk diffusion assay showed that most active strain against Proteus volgaris and Bacillus cereus was AMJ1 (16.46±0.2mm and 13.78±0.2mm, respectively), against Salmonella sp. AMJ7 was the most effective strain (10.13±0.2mm), and AMJ1 and AHA5 showed more inhibitory activity against Escherichia coli (8.04±0.02 mm and 8.2±0.03 ). The AMJ6 strain showed best antibacterial activity against Klebsiella sp. (8.03±0.02mm). Antifungal activity of AMJ2 showed that it was most active strain against complex (16.05±0.02mm) and against Aspergillus flavus strain AMJ1 was most active strain (16.4±0.2mm) and highest antifungal activity against Trichophyton mentagrophytes, Microsporum gyp serum and Candida albicans, were shown by AHA1 (21.03±0.02mm), AHA3 and AHA7 (18±0.03mm), AMJ6 (21.03±0.2mm) respectively. Our results revealed that the marine actinomycetes of Persian Gulf sediments were potent source of novel antibiotics and bioactive compounds and indicated that the antimicrobial metabolites were extracellular. Most of the secondary metabolites and antibiotics are extracellular in nature and extracellular products of actinomycetes show potent antimicrobial activities.

Keywords: antibacterial activity, antifungal activity, marine actinomycetes, Persian Gulf

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Authors: Suleyman Kizil, Tahsin Sogut, Khalid M. Khawar

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Genus Allium includes 600 to 750 species and most of these including Allium tuncelianum (Kollman) N. Ozhatay, B. Mathew & Siraneci; Syn; A. macrochaetum Boiss. and Hausskn. subsp. tuncelianum Kollman] or Tunceli garlic is endemic to Eastern Turkish Province of Tunceli and Munzur mountains. They are edible, bear attractive white-to-purple flowers and fertile black seeds with deep seed dormancy. This study aimed to break seed dormancy of Tunceli garlic and determine the conditions for induction of bulblets on these seeds and increase their diameter by culturing them on MS medium supplemented different strengths of KNO3. Tunceli garlic seeds were collected from field grown plants. They were germinated on MS medium with or without 20 g/l sucrose followed by their culture on 1 × 1900 mg/l, 2 × 1900 mg/l, 4 ×1900 mg/l and 6 × 1900 mg/l mg/l KNO3 supplemented with 20 g/l sucrose to increase bulb diameter. Improved seeds germination was noted on MS medium with and without sucrose but with variation compared to previous reports. The bulb development percentage on each of the sprouted seeds was not parallel to the percentage of seed germination. The results showed 34% and 28.5% bulb induction was noted on germinated seeds after 150 and 158 days on MS medium containing 20 g l-1 sucrose and no sucrose respectively showing a delay of 8 days on the latter compared to the former. The results emphatically noted role of cold stratification on agar solidified MS medium supplemented with sucrose to improve seed germination. The best increase in bulb diameter was noted on MS medium containing 1 × 1900 mg/l KNO3 after 178 days with bulblet diameter and bulblet weight of 0.54 cm and 0.048 g, respectively. Consequently, the bulbs induced on sucrose containing MS medium could be transferred to pots earlier. Increased (>1 × 1900 mg/l KNO3) strengths of KNO3 induced negative effect on growth and development of Tunceli garlic bulbs. The strategy of seed germination and bulblet induction reported in this study could be positively used for conservation of this endemic plant species.

Keywords: Tunceli garlic, seed, dormancy, bulblets, bulb growth

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Authors: Ahilya Singh, Brad Carte, Ramesh Subramani, William Aalbersberg

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A total of 37 actinomycete strains were purified from 25 Solomon Islands marine sediments using four different types of isolation media. Among them, 54% of the strains had obligate requirement of seawater for growth. The ethyl acetate extract of 100 ml fermentation product of each strain was screened for antimicrobial activity against multidrug resistant human pathogens and cytotoxic activity against brine shrimps. A total of 67% of the ethyl acetate extracts showed antimicrobial and/or cytotoxic activities. A strain F-1915 was selected for isolation and evaluation of bioactive compound(s) based on its bioactive properties and chemical profile analysis using the LC-MS. The strain F-1915 was identified to have 96% sequence similarity to Streptomyces violaceusniger on the basis of 16S rDNA sequences using BLAST analysis. The 16S rDNA revealed that the strain F-1915 is a new member of MAR4 clade of actinomycetes. The MAR4 clade is an interesting clade of actinomycetes known for the production of pharmaceutically important hybrid isoprenoid compounds. The ethyl acetate extract of the fermentation product of this strain was purified by silica gel column chromatography and afforded the isolation of one bioactive pure compound. Based on the 1D and 2D NMR spectral data of compound 1 it was identified as a new mono-brominated phenazinone, Marinophenazimycin A, a structure which has already been studied by external collaborators at Scripps Institution of Oceanography but is yet to be published. Compound 1 displayed significant antimicrobial activity against drug resistant human pathogens. The minimum inhibitory concentration (MIC) of compound 1 was against Methicillin Resistant Staphylococcus aureus (MRSA) was about 1.9 μg/ml and MIC recorded against Amphotericin Resistant Candida albicans (ARCA) was about 0.24 μg/ml. The bioactivity of compound 1 against ARCA was found to be better than the standard antifungal agent amphotericin B. Compound 1 however did not show any cytotoxic activity against brine shrimps.

Keywords: actinomycetes, antimicrobial activity, brominated phenazine, MAR4 clade, marine natural products, multidrug resistent, 1D and 2D NMR

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Authors: Ofonime U. M. John, Justina I. R. Udotong, Victor O. Nwaugo, Ime R. Udotong

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Oily wastes from oil and gas exploration and production (O&G E&P) activities were remediated for twelve weeks using Soil-Poultry dropping amendment. Culture-dependent microbiological, chemical and enzymatic techniques were employed to assess the efficacy of remediation process. Microbiological activities of the remediated wastes showed increased hydrocarbonoclastic microbial populations with increased remediation time; 2.7±0.1 x 105cfu/g to 8.3 ± 0.04 x106cfu/g for hydrocarbon utilizing bacteria, 1.7 ± 0.2 x103cfu/g to 6.0 ± 0.01 x 104cfu/g for hydrocarbon utilizing fungi and 2.2 ± 0.1 x 102cfu/g to 6.7 ± 0.1 x 103cfu/g for hydrocarbon utilizing actinomycetes. Bacteria associated with the remediated wastes after the remediation period included the genera Bacillus, Psuedomonas, Beijerinckia, Acinetobacter, Alcaligenes and Serratia. Fungal isolates included species of Penicillium, Aspergillus and Cladosporium, while the Actinomycetes included species of Rhodococcus, Nocardia and Streptomyces. Slight fluctuations in pH values between 6.5± 0.2 and 7.1 ± 0.08 were recorded throughout the process, while total petroleum hydrocarbon (TPH) content decreased from 89, 900 ± 0.03mg/kg to 425 ± 0.1 mg/kg after twelve weeks of remediation. The polycyclic aromatic hydrocarbon (PAH) levels decreased with increased remediation time; naphthalene, flourene, pheneanthrene, anthracene, pyrene, chrysene and benzo(b)flouranthene showed decreased values < 0.01 after twelve weeks of remediation. Enzyme activities revealed increased dehydrogenase and urease activities with increased remediation time and decreased phenol oxidase activity with increased remediation period. There was a positive linear correlation between densities of hydrocarbonoclastic microbes and dehydrogenase activity. On the contrary, phenol oxidase and urease activities showed negative correlation with microbial population. Results of this study confirmed that remediation of oily wastes using soil-poultry dropping amendment can result in eco-friendly O&G E&P wastes. It also indicates that urease and phenol oxidase activities can be reliable indices/tools to monitor PAH levels and rates of petroleum hydrocarbon degradation.

Keywords: dehydrogenase activity, oily wastes, remediation, soil-poultry dropping amendment

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Authors: Mindaye Teshome, Nesibu Yahya, Carlos Moreira Miquelino Eleto Torres, Pedro Manuel Villaa, Mehari Alebachew

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Arbagugu forest is one of the remnant dry Afromontane forests under severe anthropogenic disturbances in central Ethiopia. Despite this fact, up-to-date information is lacking about the status of the forest and its role in climate change mitigation. In this study, we evaluated the woody species composition, structure, biomass, and carbon stock in this forest. We employed a systematic random sampling design and established fifty-three sample plots (20 × 100 m) to collect the vegetation data. A total of 37 woody species belonging to 25 families were recorded. The density of seedlings, saplings, and matured trees were 1174, 101, and 84 stems ha-1, respectively. The total basal area of trees with DBH (diameter at breast height) ≥ 2 cm was 21.3 m2 ha-1. The characteristic trees of dry Afromontane Forest such as Podocarpus falcatus, Juniperus procera, and Olea europaea subsp. cuspidata exhibited a fair regeneration status. On the contrary, the least abundant species Lepidotrichilia volkensii, Canthium oligocarpum, Dovyalis verrucosa, Calpurnia aurea, and Maesa lanceolata exhibited good regeneration status. Some tree species such as Polyscias fulva, Schefflera abyssinica, Erythrina brucei, and Apodytes dimidiata lack regeneration. The total carbon stored in the forest ranged between 6.3 Mg C ha-1 and 835.6 Mg C ha-1. This value is equivalent to 639.6 Mg C ha-1. The forest had a very low number of woody species composition and diversity. The regeneration study also revealed that a significant number of tree species had unsatisfactory regeneration status. Besides, the forest had a lower carbon stock density compared with other dry Afromontane forests. This implies the urgent need for forest conservation and restoration activities by the local government, conservation practitioners, and other concerned bodies to maintain the forest and sustain the various ecosystem goods and services provided by the Arbagugu forest.

Keywords: aboveground biomass, forest regeneration, climate change, biodiversity conservation, restoration

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Authors: F. Z. Aliyat, M. El Guilli, L. Nassiri, J. Ibijbijen

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Climate change is becoming a crucial factor that can significantly impact all ecosystems. It has a negative impact on the environment in many parts of the planet. Agriculture is the main sector affected by climate change. Particularly, the salinity of agricultural soils is among the problems caused by climate change. The use of phosphate solubilizing bacteria (PSB) as a biofertilizer requires previous research on their tolerance to abiotic stress, specifically saline stress tolerance, before the formation of biofertilizers. In this context, the main goal of this research was to assess the salinity tolerance of four strains: Serratia rubidaea strain JCM1240, Enterobacter bugandensis strain 247BMC, Pantoea agglomerans strain ATCC 27155, Pseudomonas brassicacearum subsp. Neoaurantiaca strain CIP109457, which was isolated from solid phosphate sludge. Additionally, their capacity to solubilize potassium and zinc, as well as their effect on Wheat (Triticum Durum 'Karim') germination. The four PSB strains were tested for their ability to solubilize phosphate in NBRIP medium with tricalcium phosphate (TCP) as the sole source of phosphorus under salt stress. Five concentrations of NaCl were used (0%, 0.5%, 1%, 2.5%, 5%). Their phosphate solubilizing activity was estimated by the vanadate-molybdate method. The potassium and zinc solubilization has been tested qualitatively and separately on solid media with mica and zinc oxide as the only sources of potassium and zinc, respectively. The result showed that the solubilization decreases with the increase in the concentration of NaCl; all the strains solubilize the TCP even with 5% NaCl, with a significant difference among the four strains. The Serratia rubidaea strain was the most tolerant strain. In addition, the four strains solubilize the potassium and the zinc. The Serratia rubidaea strain was the most efficient. Therefore, biofertilization with PSB salt-tolerant strains could be a climate-change-preparedness strategy for agriculture in salt soil.

Keywords: bioavailability of mineral nutrients, phosphate solid sludge; phosphate solubilization, potassium solubilization, salt stress, zinc solubilization.

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Authors: Raviv Harris, Maggie Levy

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Natural product-based pesticides may serve as an alternative to the traditional synthetic pesticides, which have a potentially damaging effect, both to human health and for the environment. Along with plants, microorganisms are a prospective source of such biological pesticides. A unique and active strain of P. aphidis (designated isolate L12, Israel 2004), an epiphytic and non-pathogenic basidiomycete yeast, was isolated in our lab from strawberry leaves. P. aphidis L12 secretions were found to inhibit broad range of plant pathogens. This work demonstrates that metabolites isolated from the biocontrol agent P. aphidis (isolate L12) can inhibit varied fungal and bacterial phytopathogens. Biologically active metabolites were extracted from P. aphidis biomass, using the organic solvent ethyl acetate. The antimicrobial activity of the extract was demonstrated, both in vitro and in planta. Using disk diffusion assays, the following inhibition zones were obtained: 43cm² for Pseudomonas syringae pv. tomato, 28.5cm² for Xanthomonas campestris pv. vesicatoria, 59cm² for Clavibacter michiganensis subsp. michiganensis, 34cm² for Erwinia amylovora and 34cm² for Agrobacterium tumefaciens. Additionally, strong inhibitory activity of the extract against fungi mycelial growth was established, with IC₅₀ values of 606µg ml⁻¹ for Botrytis cinerea, 221µg ml⁻¹ for Pythium spp., 519µg ml⁻¹ for Rhizoctonia solani, 455µg ml⁻¹ for Sclerotinia sclerotiorum, 2270µg ml⁻¹ for Fusarium oxysporum f. sp. lycopersici, and 2038µg ml⁻¹ for Alternaria alternata. The results of the in planta experiments demonstrated a dose-dependent reduction in disease infection. Significant inhibition of B. cinerea lesions on tomato plants was obtained when a spore suspension of this pathogen was treated with extract concentrations higher than 4.2mg ml⁻¹. Concentration of 7mg ml⁻¹ caused a reduction of over 95% in the lesion size of B. cinerea on tomato plants. The strong antimicrobial activity demonstrated both in vitro and in planta against varied phytopathogens, may indicate that the extracted antimicrobial metabolites have potential to serve as natural pesticides in the field.

Keywords: antimicrobial, B. cinerea, metabolites, natural pesticides, P. aphidis

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Authors: Miaomiao Zhang, Sabrina Beckmann, Haluk Ertan, Rocky Chau, Mike Manefield

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Phenazines are a large class of nitrogen-containing aromatic heterocyclic compounds, which are almost exclusively produced by bacteria from diverse genera including Pseudomonas and Streptomyces. Phenazine-1-carboxylic acid (PCA) as one of 'core' phenazines are converted from chorismic acid before modified to other phenazine derivatives in different cells. Phenazines have attracted enormous interests because of their multiple roles on biocontrol, bacterial interaction, biofilm formation and fitness of their producers. However, in spite of ecological importance, degradation as a part of phenazines’ fate only have extremely limited attention now. Here, to isolate PCA-degrading bacteria, 200 mg L-1 PCA was supplied as sole carbon, nitrogen and energy source in minimal mineral medium. Quantitative PCR and Reverse-transcript PCR were employed to study abundance and activity of functional gene MFORT 16269 in PCA degradation, respectively. Intermediates and products of PCA degradation were identified with LC-MS/MS. After enrichment and isolation, a PCA-degrading strain was selected from soil and was designated as Rhodanobacter sp. PCA2 based on full 16S rRNA sequencing. As determined by HPLC, strain PCA2 consumed 200 mg L-1 (836 µM) PCA at a rate of 17.4 µM h-1, accompanying with significant cells yield from 1.92 × 105 to 3.11 × 106 cells per mL. Strain PCA2 was capable of degrading other phenazines as well, including phenazine (4.27 µM h-1), pyocyanin (2.72 µM h-1), neutral red (1.30 µM h-1) and 1-hydroxyphenazine (0.55 µM h-1). Moreover, during the incubation, transcript copies of MFORT 16269 gene increased significantly from 2.13 × 106 to 8.82 × 107 copies mL-1, which was 2.77 times faster than that of the corresponding gene copy number (2.20 × 106 to 3.32 × 107 copies mL-1), indicating that MFORT 16269 gene was activated and played roles on PCA degradation. As analyzed by LC-MS/MS, decarboxylation from the ring structure was determined as the first step of PCA degradation, followed by cleavage of nitrogen-containing ring by dioxygenase which catalyzed phenazine to nitrosobenzene. Subsequently, phenylhydroxylamine was detected after incubation for two days and was then transferred to aniline and catechol. Additionally, genomic and proteomic analyses were also carried out for strain PCA2. Overall, the findings presented here showed that a newly isolated strain Rhodanobacter sp. PCA2 was capable of degrading phenazines through decarboxylation and cleavage of nitrogen-containing ring, during which MFORT 16269 gene was activated and played important roles.

Keywords: decarboxylation, MFORT16269 gene, phenazine-1-carboxylic acid degradation, Rhodanobacter sp. PCA2

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Authors: Natan C. G. Silva, Carlos A. C. Girao Neto, Marcele M. S. Vasconcelos, Luciana R. B. Goncalves, Maria Valderez P. Rocha

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Galactooligosaccharides (GOS) are sugars with prebiotic function that can be synthesized chemically or enzymatically, and this last one can be promoted by the action of β-galactosidases. In addition to favoring the transgalactosylation reaction to form GOS, these enzymes can also catalyze the hydrolysis of lactose. A highly studied type of GOS is lactulose because it presents therapeutic properties and is a health promoter. Among the different raw materials that can be used to produce lactulose, whey stands out as the main by-product of cheese manufacturing, and its discarded is harmful to the environment due to the residual lactose present. Therefore, its use is a promising alternative to solve this environmental problem. Thus, lactose from whey is hydrolyzed into glucose and galactose by β-galactosidases. However, in order to favor the transgalactosylation reaction, the medium must contain fructose, due this sugar reacts with galactose to produce lactulose. Then, the glucose-isomerase enzyme can be used for this purpose, since it promotes the isomerization of glucose into fructose. In this scenario, the aim of the present work was first to develop β-galactosidase biocatalysts of Kluyveromyces lactis and to apply it in the integrated reactions of hydrolysis, isomerization (with the glucose-isomerase from Streptomyces murinus) and transgalactosylation reaction, using whey as a substrate. The immobilization of β-galactosidase in chitosan previously functionalized with 0.8% glutaraldehyde was evaluated using different enzymatic loads (2, 5, 7, 10, and 12 mg/g). Subsequently, the hydrolysis and transgalactosylation reactions were studied and conducted at 50°C, 120 RPM for 20 minutes. In parallel, the isomerization of glucose into fructose was evaluated under conditions of 70°C, 750 RPM for 90 min. After, the integration of the three processes for the production of lactulose was investigated. Among the evaluated loads, 7 mg/g was chosen because the best activity of the derivative (44.3 U/g) was obtained, being this parameter determinant for the reaction stages. The other parameters of immobilization yield (87.58%) and recovered activity (46.47%) were also satisfactory compared to the other conditions. Regarding the integrated process, 94.96% of lactose was converted, achieving 37.56 g/L and 37.97 g/L of glucose and galactose, respectively. In the isomerization step, conversion of 38.40% of glucose was observed, obtaining a concentration of 12.47 g/L fructose. In the transgalactosylation reaction was produced 13.15 g/L lactulose after 5 min. However, in the integrated process, there was no formation of lactulose, but it was produced other GOS at the same time. The high galactose concentration in the medium probably favored the reaction of synthesis of these other GOS. Therefore, the integrated process proved feasible for possible production of prebiotics. In addition, this process can be economically viable due to the use of an industrial residue as a substrate, but it is necessary a more detailed investigation of the transgalactosilation reaction.

Keywords: beta-galactosidase, glucose-isomerase, galactooligosaccharides, lactulose, whey

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Authors: Iva Blazkova, Amitava Moulick, Vedran Milosavljevic, Pavel Kopel, Marketa Vaculovicova, Vojtech Adam, Rene Kizek

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Doxorubicin is one of the most commonly used and the most effective chemotherapeutic drugs. This antracycline drug isolated from the bacteria Streptomyces peuceticus var. caesius is sold under the trade name Adriamycin (hydroxydaunomycin, hydroxydaunorubicin). Doxorubicin is used in single therapy to treat hematological malignancies (blood cancers, leukaemia, lymphoma), many types of carcinoma (solid tumors) and soft tissue sarcomas. It has many serious side effects like nausea and vomiting, hair lost, myelosupression, oral mucositis, skin reactions and redness, but the most serious one is the cardiotoxicity. Because of the risk of heart attack and congestive heart failure, the total dose administered to patients has to be accurately monitored. With the aim to lower the side effects and to targeted delivery of doxorubicin into the tumor tissue, the different nanoparticles are studied. The drug can be bound on a surface of nanoparticle, encapsulated in the inner cavity, or incorporated into the structure of nanoparticle. Among others, carbon nanoparticles (graphene, carbon nanotubes, fullerenes) are highly studied. Besides the number of inorganic nanoparticles, a great potential exhibit also organic ones mainly lipid-based and polymeric nanoparticle. The aim of this work was to perform a toxicity study of free doxorubicin compared to doxorubicin conjugated with various nanotransporters. The effect of liposomes, fullerenes, graphene, and carbon nanotubes on the toxicity was analyzed. As a first step, the binding efficacy of between doxorubicin and the nanotransporter was determined. The highest efficacy was detected in case of liposomes (85% of applied drug was encapsulated) followed by graphene, carbon nanotubes and fullerenes. For the toxicological studies, the chicken embryos incubated under controlled conditions (37.5 °C, 45% rH, rotation every 2 hours) were used. In 7th developmental day of chicken embryos doxorubicin or doxorubicin-nanotransporter complex was applied on the chorioallantoic membrane of the eggs and the viability was analyzed every day till the 17th developmental day. Then the embryos were extracted from the shell and the distribution of doxorubicin in the body was analyzed by measurement of organs extracts using laser induce fluorescence detection. The chicken embryo mortality caused by free doxorubicin (30%) was significantly lowered by using the conjugation with nanomaterials. The highest accumulation of doxorubicin and doxorubicin nanotransporter complexes was observed in the liver tissue

Keywords: doxorubicin, chicken embryos, nanotransporters, toxicity

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Authors: Nehir Nebioglu

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Chemotherapy is widely used for the treatment of cancer. Doxorubicin is an anti-cancer chemotherapy drug that is classified as an anthracycline antibiotic. Antitumor antibiotics consist of natural products produced by species of the soil fungus Streptomyces. These drugs act in multiple phases of the cell cycle and are known cell-cycle specific. Although DOX is a precious clinical antineoplastic agent, resistance is also a problem that limits its utility besides cardiotoxicity problem. The drug resistance of cancer cells results from multiple factors including individual variation, genetic heterogeneity within a tumor, and cellular evolution. The mechanism of resistance is thought to involve, in particular, ABCB1 (MDR1, Pgp) and ABCC1 (MRP1) as well as other transporters. Several studies on DOX-resistant cell lines have shown that resistance can be overcome by an inhibition of ABCB1, ABCC1, and ABCC2. This study attempts to understand the effects of different concentration levels of glucose treatment and starvation on the proliferation of Doxorubicin resistant cancer cells lines. To understand the effect of starvation, K562/Dox and K562 cell lines were treated with 0, 5 nM, 50 nM, 500 nM, 5 uM and 50 uM Dox concentrations in both starvation and normal medium conditions. In addition to this, to interpret the effect of glucose treatment, different concentrations (0, 1 mM, 5 mM, 25 mM) of glucose were applied to Dox-treated (with 0, 5 nM, 50 nM, 500 nM, 5 uM and 50 uM) K562/Dox and K652 cell lines. All results show significant decreasing in the cell count of K562/Dox, when cells were starved. However, while proliferation of K562/Dox lines decrease is associated with the increasingly applied Dox concentration, K562/Dox starved ones remain at the same proliferation level. Thus, the results imply that an amount of K562/Dox lines gain starvation resistance and remain resistant. Furthermore, for K562/Dox, there is no clear effect of glucose treatment in terms of cell proliferation. In the presence of a moderate level of glucose (5 mM), proliferation increases compared to other concentration of glucose for each different Dox application. On the other hand, a significant increase in cell proliferation in moderate level of glucose is only observed in 5 uM Dox concentration. The moderate concentration level of Dox can be examined in further studies. For the high amount of glucose (25 mM), cell proliferation levels are lower than moderate glucose application. The reason could be high amount of glucose may not be absorbable by cells. Also, in the presence of low amount of glucose, proliferation is decreasing in an orderly manner of increase in Dox concentration. This situation can be explained by the glucose depletion -Warburg effect- in the literature.

Keywords: drug resistance, cancer cells, chemotherapy, doxorubicin

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Authors: Carlos Y. Soto, Camila A. Lota, G. Astrid Garzón

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Bacterial biofilms cause an ongoing problem for food safety. They are formed when microorganisms aggregate to form a community that attaches to solid surfaces. Biofilms increase the resistance of pathogens to cleaning, disinfection and antibacterial products. This resistance gives rise to problems for human health, industry, and agriculture. At present, plant extracts rich in polyphenolics are being investigated as natural alternatives to degrade bacterial biofilms. The pomace of the tropical Berry Vaccinium meridionale S. contains high amounts of phenolic compounds. Therefore, in the current study, the antimicrobial and antibiofilm effects of extracts from the pomace of Vaccinium meridionale S. were tested on three foodborne pathogens: Enterohaemorrhagic Escherichia coli O157:H7 (ATCC®700728TM), Staphylococcus aureus subsp. aureus (ATCC® 6538TM), and Salmonella enterica serovar Enteritidis (ATCC® 13076TM). Microwave-assisted extraction was used to extract polyphenols with aqueous methanol (80% v/v) at a solid to solvent ratio of 1:10 (w/v) for 20 min. The magnetic stirring was set at 400 rpm, and the microwave power was adjusted to 400 W. The antimicrobial effect of the extract was assessed by determining the half maximal inhibitory concentration (IC50) against the three food poisoning pathogens at concentrations ranging from 50 to 2,850 μg gallic acid equivalents (GAE)/mL of the extract. Biofilm inhibition was assessed using a crystal violet assay applying the same range of concentration. Three replications of the experiments were carried out, and all analyses were run in triplicate. IC50 values were determined using the GraphPad Prism8® program. Significant differences (P<0.05) among means were identified using one-factor analysis of variance (ANOVA) and the post-hoc least significant difference (LSD) test using the Statgraphics plus program, version 2.1.There was significant difference among the mean IC50 values for the tested bacteria. The IC50 for S. aureus was 48 ± 9 μg GAE/mL, followed by 123 ± 49 μg GAE/mL for Salmonella and 376 ± 32 μg GAE/mL for E. coli. The percent inhibition of the extract on biofilm formation was significantly higher for S. aureus (85.8  0.3), followed by E. coli (74.5  1.0) and Salmonella (53.6  9.7). These findings suggest that polyphenolic extracts obtained from the pomace of V. meridionale S. might be used as natural antimicrobial and anti-biofilm natural agents, effective against S. aureus, E. coli and Salmonella enterica.

Keywords: antibiofilm, antimicrobial, E. coli, S. aureus, salmonella, IC50, pomace, V. meridionale

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Authors: Siti Hajar Muhamad Rosli, Xin Yi Lim, Terence Yew Chin Tan, Muhammad nor Farhan Sa’At, Syazwani Sirdar Ali, Ami Fazlin Syed Mohamed

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A growing interest on therapeutic benefits of hemp (Cannabis sativa subsp. sativa) is evident in the pharmaceutical market, attributed to its lower levels of psychoactive constituent delta-9-tetrahydronannabidiol (THC). Deemed as a legal and safer alternative to its counterpart marijuana, the use of medicinal hemp is highly debatable as current scientific evidence on the efficacy for clinical use is yet to be established This study was aimed to provide an overview of the current landscape of hemp research, through recent clinical findings specific to the pharmacological properties of the hemp plant and its derived compounds. A systematic search was conducted following the Preferred Reporting Items for Systematic Review and Meta-Analysis-ScR (PRISMA) checklist on electronic databases (MEDLINE, OVID, Cochrane Library Central, and Clinicaltrials.gov) for articles published from 2009 to 2019. With predetermined inclusion criteria, all human trials with hemp intervention were included. A total of 18 human trials were identified, investigating therapeutic effects on the neuronal, gastrointestinal, musculoskeletal and immune system, with sample sizes ranging from one to 194 subjects. Three randomised controlled trials showed hempseed pills (in Traditional Chinese Medicine formulation MaZiRenWan) consumption significantly improved spontaneous bowel movement in functional constipation. The use of commercial cannabidiol (CBD) sourced from hemp suggested benefits in cannabis dependence, epilepsy, and anxiety disorders. However, there was insufficient evidence to suggest analgesic or anxiolytics effects of hemp being equivalent to marijuana. All clinical trials reviewed varied in terms of test item formulation and standardisation, which made it challenging to confirm overall efficacy for a specific disease or condition. Published efficacy data on hemp are still at a preliminary level, with limited high quality clinical evidence for any specific therapeutic indication. With multiple variants of this plant having different phytochemical and bioactive compounds, future empirical research should focus on uniformity in experimental designs to further strengthen the notion of using medicinal hemp.

Keywords: cannabis, complementary medicine, hemp, herbal medicine.

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Authors: Ajuna B. Henry

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In recent decades, climate change has demanded biological pesticides; more Bt strains are being discovered worldwide, some containing novel insecticidal genes while others have been modified through molecular approaches for increased yield, toxicity, and wider host target. In this study, B. thuringiensis strain AH-2 (Bt-2) was isolated from the soil and tested for insecticidal activity against Aphis gossypii (Hemiptera: Aphididae) and Lepidoptera insect pests: fall webworm (Hyphantria cunea) and diamondback moth (Plutella xylostella). A commercial strain B. thuringiensis subsp. kurstaki (Btk), and a chemical pesticide, imidacloprid (for Hemiptera) and chlorantraniliprole (for Lepidoptera), were used as positive control and the same media (without bacterial inoculum) as a negative control. For aphidicidal activity, Bt-2 caused a mortality rate of 70.2%, 78.1% or 88.4% in third instar nymphs of A. gossypii (3N) at 10%, 25% or 50% culture concentrations, respectively. Moreover, Bt-2 was effectively produced in cost-effective (PB) supplemented with either glucose (PBG) or sucrose (PBS) and maintained high aphicidal efficacy with 3N mortality rates of 85.9%, 82.9% or 82.2% in TSB, PBG or PBS media, respectively at 50% culture concentration. Bt-2 also suppressed adult fecundity by 98.3% compared to only 65.8% suppression by Btk at similar concentrations but was slightly lower than chemical treatment, which caused 100% suppression. Partial purification of 60 – 80% (NH4)2SO4 fraction of Bt-2 aphicidal proteins purified on anion exchange (DEAE-FF) column revealed a 105 kDa aphicidal protein with LC50 = 55.0 ng/µℓ. For Lepidoptera pests, chemical pesticide, Bt-2, and Btk cultures, mortality of 86.7%, 60%, and 60% in 3rd instar larvae of P. xylostella, and 96.7%, 80.0%, and 93.3% in 6th instar larvae of H. cunea, after 72h of exposure. When the entomopathogenic strains were cultured in a cost-effective PBG or PBS, the insecticidal activity in all strains was not significantly different compared to the use of a commercial medium (TSB). Bt-2 caused a mortality rate of 60.0%, 63.3%, and 50.0% against P. xylostella larvae and 76.7%, 83.3%, and 73.3% against H. cunea when grown in TSB, PBG, and PBS media, respectively. Bt-2 (grown in cost-effective PBG medium) caused a dose-dependent toxicity of 26.7%, 40.0%, and 63.3% against P. xylostella and 46.7%, 53.3%, and 76.7% against H. cunea at 10%, 25% and 50% culture concentration, respectively. The partially purified Bt-2 insecticidal proteins fractions F1, F2, F3, and F4 (extracted at different ratios of organic solvent) caused low toxicity (50.0%, 40.0%, 36.7%, and 30.0%) against P. xylostella and relatively high toxicity (56.7%, 76.7%, 66.7%, and 63.3%) against H. cunea at 100 µg/g of artificial diets. SDS-PAGE analysis revealed that a128kDa protein is associated with toxicity of Bt-2. Our result demonstrates a medium and strong larvicidal activity of Bt-2 against P. xylostella and H. cunea, respectively. Moreover, Bt-2 could be potentially produced using a cost-effective PBG medium which makes it an effective alternative biocontrol strategy to reduce chemical pesticide application.

Keywords: biocontrol, insect pests, larvae/nymph mortality, cost-effective media, aphis gossypii, plutella xylostella, hyphantria cunea, bacillus thuringiensi

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Authors: Javier Enciso-Benavides, Fredy Fabian, Carlos Castaneda, Luis Alfaro, Alex Choque, Aparicio Aguilar, Javier Enciso

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Background: The use of biomarkers in breast cancer diagnosis, therapy, and prognosis has gained increasing interest. Cancer stem cells (CSCs) are a subpopulation of tumor cells that can drive tumor initiation and may cause relapse. Therefore, due to the importance of diagnosis, therapy, and prognosis, several biomarkers that characterize CSCs have been identified; however, in treatment-naïve triple-negative breast tumors, there is an urgent need to identify new biomarkers and therapeutic targets. According to this, the aim of this study was to identify serum proteins associated with cancer stem cells and pluripotency in women with triple-negative breast tumors in order to subsequently identify a biomarker for this type of breast tumor. Material and Methods: Whole blood samples from 12 women with histopathologically diagnosed triple-negative breast tumors were used after obtaining informed consent from the patient. Blood serum was obtained by conventional procedure and frozen at -80ºC. Identification of cancer stem cell-associated proteins was performed by matrix-assisted laser desorption/ionisation-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS), protein analysis was obtained using the AB Sciex TOF/TOF™ 5800 system (AB Sciex, USA). Sequences not aligned by ProteinPilot™ software were analyzed by Protein BLAST. Results: The following proteins related to pluripotency and cancer stem cells were identified by MALDI TOF/TOF mass spectrometry: A-chain, Serpin A12 [Homo sapiens], AIEBP [Homo sapiens], Alpha-one antitrypsin, AT {internal fragment} [human, partial peptide, 20 aa] [Homo sapiens], collagen alpha 1 chain precursor variant [Homo sapiens], retinoblastoma-associated protein variant [Homo sapiens], insulin receptor, CRA_c isoform [Homo sapiens], Hydroxyisourate hydrolase [Streptomyces scopuliridis], MUCIN-6 [Macaca mulatta], Alpha-actinin-3 [Chrysochloris asiatica], Polyprotein M, CRA_d isoform, partial [Homo sapiens], Transcription factor SOX-12 [Homo sapiens]. Recommendations: The serum proteins identified in this study should be investigated in the exosome of triple-negative breast cancer stem cells and in the blood serum of women without breast cancer. Subsequently, proteins found only in the blood serum of women with triple-negative breast cancer should be identified in situ in triple-negative breast cancer tissue in order to identify a biomarker to study the evolution of this type of cancer, or that could be a therapeutic target. Conclusions: Eleven cancer stem cell-related serum proteins were identified in 12 women with triple-negative breast cancer, of which MUCIN-6, retinoblastoma-associated protein variant, transcription factor SOX-12, and collagen alpha 1 chain are the most representative and have not been studied so far in this type of breast tumor. Acknowledgement: This work was supported by Proyecto CONCYTEC–Banco Mundial “Mejoramiento y Ampliacion de los Servicios del Sistema Nacional de Ciencia Tecnología e Innovacion Tecnologica” 8682-PE (104-2018-FONDECYT-BM-IADT-AV).

Keywords: triple-negative breast cancer, MALDI TOF/TOF MS, serum proteins, cancer stem cells

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Authors: Michelle C. S. Azevedo, Priscila M. Colavite, Carolina F. Francisconi, Ana P. Trombone, Gustavo P. Garlet

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VIP (vasoactive intestinal peptide) know as a potential protective factor in the view of its marked immunosuppressive properties. In this work, we investigated a possible association of VIP with the clinical status of experimental periapical granulomas and the association with expression markers in the lesions potentially associated with periapical lesions pathogenesis. C57BL/6WT mice were treated or not with recombinant VIP. Animals with active/progressive (N=40), inactive/stable (N=70) periapical granulomas and controls (N=50) were anesthetized and the right mandibular first molar was surgically opened, allowing exposure of dental pulp. Endodontic pathogenic bacterial strains were inoculated: Porphyromonas gingivalis, Prevotella nigrescens, Actinomyces viscosus, and Fusobacterium nucleatum subsp. polymorphum. The cavity was not sealed after bacterial inoculation. During lesion development, animals were treated or not with recombinant VIP 3 days post infection. Animals were killed after 3, 7, 14, and 21 days of infection and the jaws were dissected. The extraction of total RNA from periodontal tissues was performed and the integrity of samples was checked. qPCR reaction using TaqMan chemistry with inventoried primers were performed in ViiA7 equipment. The results, depicted as the relative levels of gene expression, were calculated in reference to GAPDH and β-actin expression. Periodontal tissues from upper molars were vested and incubated supplemented RPMI, followed by processing with 0.05% DNase. Cell viability and couting were determined by Neubauer chamber analysis. For flow cytometry analysis, after cell counting the cells were stained with the optimal dilution of each antibody; (PE)-conjugated and (FITC)-conjugated antibodies against CD4, CD25, FOXP3, IL-4, IL-17 and IFN-γ antibodies, as well their respective isotype controls. Cells were analyzed by FACScan and CellQuest software. Results are presented as the number of cells in the periodontal tissues or the number of positive cells for each marker in the CD4+FOXp3+, CD4+IL-4+, CD4+IFNg+ and CD4+IL-17+ subpopulations. The levels mRNA were measured by qPCR. The VIP expression was predominated in inactive lesions, as well part of the clusters of cytokine/Th markers identified as protective factors and a negative correlation between VIP expression and lesion evolution was observed. A quantitative analysis of IL1β, IL17, TNF, IFN, MMP2, RANKL, OPG, IL10, TGFβ, CTLA4, COL5A1, CTGF, CXCL11, FGF7, ITGA4, ITGA5, SERP1 and VTN expression was measured in experimental periapical lesions treated with VIP 7 and 14 days after lesion induction and healthy animals. After 7 days, all targets presented a significate increase in comparison to untreated animals. About migration kinetics, profile of chemokine receptors expression of TCD4+ subsets and phenotypic analysis of Tregs, Th1, Th2 and Th17 cells during the course of experimental periodontal disease evaluated by flow cytometry and depicted as the number of positive cells for each marker. CD4+IFNg+ and CD4+FOXp3+ cells migration were significate increased 7 days post VIP treatment. CD4+IL17+ cells migration were significate increased 7 and 14 days post VIP treatment, CD4+IL4+ cells migration were significate increased 14 and 21 days post VIP treatment compared to the control group. In conclusion, our experimental data support VIP involvement in determining the inactivity of periapical lesions. Financial support: FAPESP #2015/25618-2.

Keywords: chronic inflammation, cytokines, osteolytic lesions, VIP (Vasoactive Intestinal Peptide)

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