Search results for: microbial pathogenesis

Authors: A. K. Tursunova, O. V. Chebonenko, A. Zh. Amirkulova, A. O. Abaildayev, O. A. Sapko, Y. M. Dyo, A. Sh. Utarbaeva

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Fusarium solani and its variants cause root and stem rot of plants. Dry rot is the most common disease of potato tubers during storage. The causative agents of fusariosis in contact with plants behave as antagonists, growth stimulants or parasites. The diversity of host-parasite relationships is explained by the parasite’s ability to produce a wide spectrum of biologically active compounds including toxins, enzymes, oligosaccharides, antibiotic substances, enniatins and gibberellins. Many of these metabolites contribute to the creation of compatible relations; others behave as elicitors, inducing various protective responses in plants. An important part of the strategy for developing plant resistance against pathogens is the activation of protein synthesis to produce protective ‘pathogenesis-related’ proteins. The family of PR-proteins known to confer the most protective response is chitinases (EC 3.2.1.14, Cht) and β-1,3-glucanases (EC 3.2.1.39, Glu). PR-proteins also include a large multigene family of peroxidases (EC 1.11.1.7, Pod), and increased activity of Pod and expression of the Pod genes leads to the development of resistance to a broad class of pathogens. Despite intensive research on the role of PR-proteins, the question of their participation in the mechanisms of formation of the F.solani–S.tuberosum pathosуstem is not sufficiently studied. Our aim was to investigate the effect of different classes of F. solani metabolites on the activity of chitinase, β-1,3-glucanases and peroxidases in tubers of Solanum tuberosum. Metabolite culture filtrate (CF) and cytoplasmic components were fractionated by extraction of the mycelium with organic solvents, salting out techniques, dialysis, column chromatography and ultrafiltration. Protein, lipid, carbohydrate and polyphenolic fractions of fungal metabolites were derived. Using enzymatic hydrolysis we obtained oligo glycans from fungal cell walls with different molecular weights. The activity of the metabolites was tested using potato tuber discs (d = 16mm, h = 5mm). The activity of PR-proteins of tubers was analyzed in a time course of 2–24 hours. The involvement of the analysed metabolites in the modulation of both early non-specific and late related to pathogenesis reactions was demonstrated. The most effective inducer was isolated from the CF (fraction of total phenolic compounds including naphtazarins). Induction of PR-activity by this fraction was: chitinase - 340-360%, glucanase - 435-450%, soluble forms of peroxidase - 400-560%, related forms of peroxidase - 215-237%. High-inducing activity was observed by the chloroform and acetonitrile extracts of the mycelium (induction of chitinase and glucanase activity was 176-240%, of soluble and bound forms of peroxidase - 190-400%). The fraction of oligo glycans mycelium cell walls of 1.2 kDa induced chitinase and β-1,3-glucanase to 239-320%; soluble forms and related peroxidase to 198-426%. Oligo glycans cell walls of 5-10 kDa had a weak suppressor effect - chitinase (21-25%) and glucanase (25-28%) activity; had no effect on soluble forms of peroxidase, but induced to 250-270% activity related forms. The CF polysaccharides of 8.5 kDa and 3.1 kDa inhibited synchronously the glucanase and chitinase specific response in step (after 24 hours at 42-50%) and the step response induced nonspecific peroxidase activity: soluble forms 4.8 -5.2 times, associated forms 1.4-1.6 times.

Keywords: fusarium solani, PR-proteins, peroxidase, solanum tuberosum

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Authors: K. Premakumar, R. G. Lakmali, S. M. A. C. U. Senarathna

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In the present world, production and consumption of fruit and vegetable beverages have increased owing to the healthy life style of the people. Therefore, a study was conducted to develop composite vegetable squash by incorporating nutritional, medicinal and organoleptic properties of tomato, pumpkin and ginger. Considering the finding of several preliminary studies, five formulations in different combinations tomato pumpkin were taken and their physico-chemical parameters such as pH, TSS, titrable acidity, ascorbic acid content and total sugar and organoleptic parameters such as colour, aroma, taste, nature, overall acceptability were analyzed. Then the best sample was improved by using 1 % ginger (50% tomato+ 50% pumpkin+ 1% ginger). Best three formulations were selected for storage studied. The formulations were stored at 30 °C room temperature and 70-75% of RH for 12 weeks. Physicochemical parameters , organoleptic and microbial activity (total plate count, yeast and mold, E-coil) were analyzed during storage periods and protein content, fat content, ash were also analysed%.The study on the comparison of physico-chemical and sensory qualities of stored Squashes was done up to 12 weeks storage periods. The nutritional analysis of freshly prepared tomato pumpkin vegetable squash formulations showed increasing trend in titratable acidity, pH, total sugar, non -reducing sugar, total soluble solids and decreasing trend in ascorbic acid and reducing sugar with storage periods. The results of chemical analysis showed that, there were the significant different difference (p < 0.05) between tested formulations. Also, sensory analysis also showed that there were significant differences (p < 0.05) for organoleptic character characters between squash formulations. The highest overall acceptability was observed in formulation with 50% tomato+ 50% pumpkin+1% ginger and all the all the formulations were microbiologically safe for consumption. Based on the result of physico-chemical characteristics, sensory attributes and microbial test, the Composite Vegetable squash with 50% tomato+50% pumpkin+1% ginger was selected as best formulation and could be stored for 12 weeks without any significant changes in quality characteristics.

Keywords: nutritional analysis, formulations, sensory attributes, squash

Procedia PDF Downloads 177

Authors: Apichaya Aneksampant, Xuefei Tu, Masami Fukushima, Mitsuo Yamamoto

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The restoration of seaweed beds recovery has been developed using a fertilization technique for supplying dissolved iron to barren coastal areas. The fertilizer is composed of iron oxides as a source of iron and compost as humic substance (HS) source, which can serve as chelator of iron to stabilize the dissolved species under oxic seawater condition. However, elution mechanisms of iron from iron oxide surfaces have not sufficiently elucidated. In particular, roles of microbial activities in the elution of iron from the fertilizer are not sufficiently understood. In the present study, a fertilizer (iron oxide/compost = 1/1, v/v) was incubated in a water tank at Mashike coast, Hokkaido Japan. Microorganisms in the 6-month fertilizer were isolated and identified as Exiguobacterium oxidotolerans sp. (T-2-2). The identified bacteria were inoculated to perform iron elution test in a postgate B medium, prepared in artificial seawater. Hematite was used as a model iron oxide and anthraquinone-2,7-disolfonate (AQDS) as a model for HSs. The elution test performed in presence and absence of bacteria inoculation. ICP-AES was used to analyze total iron and a colorimetric technique using ferrozine employed for the determination of ferrous ion. During the incubation period, sample contained hematite and T-2-2 in both presence and absence of AQDS continuously showed the iron elution and reached at the highest concentration after 9 days of incubation and then slightly decrease to stabilize within 20 days. Comparison to the sample without T-2-2, trace amount of iron was observed, suggesting that iron elution to seawater can be attributed to bacterial activities. The levels of total organic carbon (TOC) in the culture solution with hematite decreased. This may be to the adsorption of organic compound, AQDS, to hematite surfaces. The decrease in UV-vis absorption of AQDS in the culture solution also support the results of TOC that AQDS was adsorbed to hematite surfaces. AQDS can enhance the iron elution, while the adsorption of organic matter suppresses the iron elution from hematite.

Keywords: anthraquinone-2, 7-disolfonate, barren ground, E.oxidotolerans sp., hematite, humic substances, iron elution

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Authors: Malory Beaujolais

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Chlordecone is an organochlorine pesticide with a bishomocubane structure which led to high stability in organic matter. Microbial fuel cell is a type of electrochemical system that can convert organic matters into electricity thanks to electroactive bacteria. This technique has been used with mangrove swamp from Martinique to try to reduce chlordecone rates. Those experiments led to characterize the behavior of the electroactive biofilm formed at the cathode, without added redox mediator. The designed bioelectrochemical system seems to provide the necessary conditions for chlordecone degradation.

Keywords: bioelectrochemistry, bioremediation, chlordecone, mangrove swamp

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Authors: Christopher Oyediran, Nicola Ubayasiri, Christopher Gough

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Splenic artery aneurysms are often clinically occult, occasionally identified incidentally with imaging. The pathogenesis of aneurysms is complex, but certain factors are thought to contribute to their development. Given the potential fatal complications of rupture, a high index of suspicion is required to make an early diagnosis. We present a case of a 36-year-old female with a history of endometriosis and multiple sclerosis who presented to the Emergency Department with sudden onset epigastric pain and collapse. On arrival, she was pale and clammy with profound tachycardia and hypotension. An ultrasound done in the resuscitation department revealed abdominal free fluid. She was resuscitated with blood and transferred for emergent laparotomy. Laparotomy revealed massive haemoperitoneum from the spleen. She underwent emergency splenectomy and inspection of the spleen revealed a splenic artery aneurysm. She received our massive transfusion protocol followed by a short stay on ITU, making a good post-operative recovery and was discharged home a week later.

Keywords: aneurysm, human chorionic gonadotrophin (hCG), resuscitation, laparotomy

Procedia PDF Downloads 407

Authors: Sachin Mulmi Shrestha, Xin Fang, Hui Ye, Lihua Ren, Qinghua Ji, Ruihua Shi

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Background: Esophageal squamous cell carcinoma (ESCC) is a tumor arising from esophageal epithelial cells and is one of the major disease subtype in Asian countries, including China. Esophageal cancer is the 7th highest incidence based on the 2020 data of GLOBOCAN. The pathogenesis of cancer is still not well understood as many molecular and genetic basis of esophageal carcinogenesis has yet to be clearly elucidated. Circular RNAs are RNA molecules that are formed by back-splicing covalently joined 3′- and 5′-endsrather than canonical splicing, and recent data suggest circular RNAs could sponge miRNAs and are enriched with functional miRNA binding sites. Hence, we studied the mechanism of circular RNA, its biological function, and the relationship between microRNA in the carcinogenesis of ESCC. Methods: 4 pairs of normal and esophageal cancer tissues were collected in Zhongda hospital, affiliated to Southeast University, and high-throughput RNA sequencing was done. The result revealed that circ_0032746 was upregulated, and thus we selected circ_0032746 for further study. The backsplice junction of circRNA was validated by sanger sequence, and stability was determined by RNASE R assay. The binding site of circRNA and microRNA was predicted by circinteractome,mirandaand RNAhybrid database. Furthermore, circRNA was silenced by siRNA and then by lentivirus. The regulatory axis of circ0032746/miR4270 was validated by shRNA, mimic, and inhibitor transfection. Then, in vitro experiments were performed to assess the role of circ0032746 on proliferation (CCK-8 assay and colon formation assay), migration and invasion (Transewell assay), and apoptosis of ESCC. Results: The upregulated circ0032746 was validated in 9 pairs of tissues and 5 types of cell lines by qPCR, which showed high expression and was statistically significant (P<0.005) ). Upregulated circ0032746 was silenced by shRNA, which showed significant knockdown in KYSE 30 and TE-1 cell lines expression compared to control. Nuclear and cytoplasmic mRNA fraction experiment displayed the cytoplasmic location of circ0032746. The sponging of miR4270 was validated by co-transfection of sh-circ0032746 and mimic or inhibitor. Transfection with mimic showed the decreased expression of circ_0032746, whereas inhibitor inhibited the result. In vitro experiments showed that silencing of circ_0032746 inhibited the proliferation, migration, and invasion compared to the negative control group. The apoptosis was seen higher in a knockdown group than in the control group. Furthermore, 11 common mircoRNA target mRNAs were predicted by Targetscan, MirTarbase, and miRanda database, which may further play role in the pathogenesis. Conclusion: Our results showed that novel circ_0032746 is upregulated in ESCC and plays role in itsoncogenicity. Silencing of circ_0032746 inhibits the proliferation and migration of ESCC whereas increases the apoptosis of cancer cells. Hence, circ0032746 acts as an oncogene in ESCC by sponging with miR4270 and could be a potential biomarker in the diagnosis of ESCC in the future.

Keywords: circRNA, esophageal squamous cell carcinoma, microRNA, upregulated

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Authors: Ali Bayram, Burak Uz, Remzi Yiğiter

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The impairment of lipid metabolism in the central nervous system has been suggested as a critical factor of Alzheimer’s disease (AD) pathogenesis. Homo sapiens acyl-coenyme A synthetase short-chain family member 2 (ACSS2) gene encodes the enzyme acetyl-Coenzyme A synthetase (AMP forming; AceCS) providing acetyl-coenzyme A (Ac-CoA) for various physiological processes, such as cholesterol and fatty acid synthesis, as well as the citric acid cycle. We investigated ACSS2, transcript variant 1 (ACSS2*1), mRNA levels in the peripheral blood mononuclear cells (PBMC) of patients with AD and compared them with the controls. The study group comprised 50 patients with the diagnosis of AD who have applied to Gaziantep University Faculty of Medicine, and Department of Neurology. 49 healthy individuals without any neurodegenerative disease are included as controls. ACSS2 mRNA expression in PBMC of AD/control patients was 0.495 (95% confidence interval: 0.410-0.598), p= .000000001902). Further studies are needed to better clarify this association.

Keywords: Alzheimer’s disease, ACSS2 Genes, mRNA expression, RT-PCR

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Authors: Peli̇n Yilmaz, Gi̇zemnur Yildiz Uysal, Emi̇ne Bi̇rci̇, Berk Özcan, Burak Koca, Ehsan Tuzcuoğlu, Fati̇h Kasap

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Development of Energy Efficient Programs (EEP) is one of the most significant trends in the wet appliance industry of the recent years. Thanks to the EEP, the energy consumption of a washing machine as one of the most energy-consuming home appliances can shrink considerably, while its washing performance and the textile hygiene should remain almost unchanged. Here in, the goal of the present study is to achieve an optimum EEP algorithm providing excellent textile hygiene results as well as cleaning performance in a domestic washing machine. In this regard, steam-pretreated cold wash approach with a combination of innovative algorithm solution in a relatively short washing cycle duration was implemented. For the parametric study, steam exposure time, washing load, total water consumption, main-washing time, and spinning rpm as the significant parameters affecting the textile hygiene and cleaning performance were investigated within a Design of Experiment study using Minitab 2021 statistical program. For the textile hygiene studies, specific loads containing the contaminated cotton carriers with Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa bacteria were washed. Then, the microbial removal performance of the designed programs was expressed as log reduction calculated as a difference of microbial count per ml of the liquids in which the cotton carriers before and after washing. For the cleaning performance studies, tests were carried out with various types of detergents and EMPA Standard Stain Strip. According to the results, the optimum EEP program provided an excellent hygiene performance of more than 2 log reduction of microorganism and a perfect Washing Efficiency Index (Iw) of 1.035, which is greater than the value specified by EU ecodesign regulation 2019/2023.

Keywords: washing machine, energy efficient programs, hygiene, washing efficiency index, microorganism, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, laundry

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Authors: Danyi Wang, Andrew Schriefer, Dennis O'Rourke, Brajendra Kumar, Yang Liu, Fei Zhong, Juergen Scheuenpflug, Zheng Feng

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Purpose: It has been recognized that the microbiome plays critical roles in disease pathogenesis, including cancer, autoimmune disease, and multiple sclerosis. To develop a clinical-grade assay for exploring microbiome-derived clinical biomarkers across disease areas, a two-phase approach is implemented. 1) Identification of the optimal sample preparation reagents using pre-mixed bacteria and healthy donor stool samples coupled with proprietary Sigma-Aldrich® bioinformatics solution. 2) Exploratory analysis of patient samples for enabling precision medicine. Study Procedure: In phase 1 study, we first compared the 16S sequencing results of two ATCC® microbiome standards (MSA 2002 and MSA 2003) across five different extraction kits (Kit A, B, C, D & E). Both microbiome standards samples were extracted in triplicate across all extraction kits. Following isolation, DNA quantity was determined by Qubit assay. DNA quality was assessed to determine purity and to confirm extracted DNA is of high molecular weight. Bacterial 16S ribosomal ribonucleic acid (rRNA) amplicons were generated via amplification of the V3/V4 hypervariable region of the 16S rRNA. Sequencing was performed using a 2x300 bp paired-end configuration on the Illumina MiSeq. Fastq files were analyzed using the Sigma-Aldrich® Microbiome Platform. The Microbiome Platform is a cloud-based service that offers best-in-class 16S-seq and WGS analysis pipelines and databases. The Platform and its methods have been extensively benchmarked using microbiome standards generated internally by MilliporeSigma and other external providers. Data Summary: The DNA yield using the extraction kit D and E is below the limit of detection (100 pg/µl) of Qubit assay as both extraction kits are intended for samples with low bacterial counts. The pre-mixed bacterial pellets at high concentrations with an input of 2 x106 cells for MSA-2002 and 1 x106 cells from MSA-2003 were not compatible with the kits. Among the remaining 3 extraction kits, kit A produced the greatest yield whereas kit B provided the least yield (Kit-A/MSA-2002: 174.25 ± 34.98; Kit-A/MSA-2003: 179.89 ± 30.18; Kit-B/MSA-2002: 27.86 ± 9.35; Kit-B/MSA-2003: 23.14 ± 6.39; Kit-C/MSA-2002: 55.19 ± 10.18; Kit-C/MSA-2003: 35.80 ± 11.41 (Mean ± SD)). Also, kit A produced the greatest yield, whereas kit B provided the least yield. The PCoA 3D visualization of the Weighted Unifrac beta diversity shows that kits A and C cluster closely together while kit B appears as an outlier. The kit A sequencing samples cluster more closely together than both the other kits. The taxonomic profiles of kit B have lower recall when compared to the known mixture profiles indicating that kit B was inefficient at detecting some of the bacteria. Conclusion: Our data demonstrated that the DNA extraction method impacts DNA concentration, purity, and microbial communities detected by next-generation sequencing analysis. Further microbiome analysis performance comparison of using healthy stool samples is underway; also, colorectal cancer patients' samples will be acquired for further explore the clinical utilities. Collectively, our comprehensive qualification approach, including the evaluation of optimal DNA extraction conditions, the inclusion of positive controls, and the implementation of a robust qualified bioinformatics pipeline, assures accurate characterization of the microbiota in a complex matrix for deciphering the deep biology and enabling precision medicine.

Keywords: 16S rRNA sequencing, analytical validation, bioinformatics pipeline, metagenomics

Procedia PDF Downloads 134

Authors: Moatlhodi Wise Letshwenyo, Lesedi Lebogang

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Botswana is an arid country that needs to start reusing wastewater as part of its water security plan. Pilot scale slow sand filtration in combination with roughing filter was investigated for the treatment of effluent from Botswana International University of Science and Technology to meet Botswana irrigation standards. The system was operated at hydraulic loading rates of 0.04 m/hr and 0.12 m/hr. The results show that the system was able to reduce turbidity from 262 Nephelometric Turbidity Units to a range between 18 and 0 Nephelometric Turbidity Units which was below 30 Nephelometric Turbidity Units threshold limit. The overall efficacy ranged between 61% and 100%. Suspended solids, Biochemical Oxygen Demand, and Chemical Oxygen Demand removal efficiency averaged 42.6%, 45.5%, and 77% respectively and all within irrigation standards. Other physio-chemical parameters were within irrigation standards except for bicarbonate ion which averaged 297.7±44 mg L^-1 in the influent and 196.22±50 mg L^-1 in the effluent which was above the limit of 92 mg L^-1, therefore averaging a reduction of 34.1% by the system. Total coliforms, fecal coliforms, and Escherichia coli in the effluent were initially averaging 1.1 log counts, 0.5 log counts, and 1.3 log counts respectively compared to corresponding influent log counts of 3.4, 2.7 and 4.1, respectively. As time passed, it was observed that only roughing filter was able to reach reductions of 97.5%, 86% and 100% respectively for faecal coliforms, Escherichia coli, and total coliforms. These organism numbers were observed to have increased in slow sand filter effluent suggesting multiplication in the tank. Water quality index value of 22.79 for the physio-chemical parameters suggests that the effluent is of excellent quality and can be used for irrigation purposes. However, the water quality index value for the microbial parameters (1820) renders the quality unsuitable for irrigation. It is concluded that slow sand filtration in combination with roughing filter is a viable option for the treatment of secondary effluent for reuse purposes. However, further studies should be conducted especially for the removal of microbial parameters using the system.

Keywords: irrigation, slow sand filter, turbidity, wastewater reuse

Procedia PDF Downloads 129

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: Yi H. Wong, Li L. Chan, Chee O. Leong, Stephen Ambu, Joon W. Mak, Priyadashi S. Sahu

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Background: Acanthamoeba is a free-living opportunistic protist which is ubiquitously distributed in the environment. Virulent Acanthamoeba can cause fatal encephalitis in immunocompromised patients and potential blinding keratitis in immunocompetent contact lens wearers. Approximately 24 species have been identified but only the A. castellanii, A. polyphaga and A. culbertsoni are commonly associated with human infections. Until to date, the precise molecular basis for Acanthamoeba pathogenesis remains unclear. Previous studies reported that Acanthamoeba virulence can be diminished through prolonged axenic culture but revived through serial mouse passages. As no clear explanation on this reversible pathogenesis is established, hereby, we postulate that the epigenetic regulators, DNA-methyltransferases (DNMT) and histone-deacetylases (HDAC), could possibly be involved in granting the virulence plasticity of Acanthamoeba spp. Methods: Four rounds of mouse passages were conducted to revive the virulence potential of the virulence-attenuated Acanthamoeba castellanii strain (ATCC 50492). Briefly, each mouse (n=6/group) was inoculated intraperitoneally with Acanthamoebae cells (2x 105 trophozoites/mouse) and incubated for 2 months. Acanthamoebae cells were isolated from infected mouse organs by culture method and subjected to subsequent mouse passage. In vitro cytopathic, encystment and gelatinolytic assays were conducted to evaluate the virulence characteristics of Acanthamoebae isolates for each passage. PCR primers which targeted on the 2 members (DNMT1 and DNMT2) and 5 members (HDAC1 to 5) of the DNMT and HDAC gene families respectively were custom designed. Quantitative real-time PCR (qPCR) was performed to detect and quantify the relative expression of the two gene families in each Acanthamoeba isolates. Beta-tubulin of A. castellanii (Genbank accession no: XP_004353728) was included as housekeeping gene for data normalisation. PCR mixtures were also analyzed by electrophoresis for amplicons detection. All statistical analyses were performed using the paired one-tailed Student’s t test. Results: Our pathogenicity tests showed that the virulence-reactivated Acanthamoeba had a higher degree of cytopathic effect on vero cells, a better resistance to encystment challenge and a higher gelatinolytic activity which was catalysed by serine protease. qPCR assay showed that DNMT1 expression was significantly higher in the virulence-reactivated compared to the virulence-attenuated Acanthamoeba strain (p ≤ 0.01). The specificity of primers which targeted on DNMT1 was confirmed by sequence analysis of PCR amplicons, which showed a 97% similarity to the published DNA-methyltransferase gene of A. castellanii (GenBank accession no: XM_004332804.1). Out of the five primer pairs which targeted on the HDAC family genes, only HDAC4 expression was significantly difference between the two variant strains. In contrast to DNMT1, HDAC4 expression was much higher in the virulence-attenuated Acanthamoeba strain. Conclusion: Our mouse passages had successfully restored the virulence of the attenuated strain. Our findings suggested that DNA-methyltransferase (DNMT1) and histone deacetylase (HDAC4) expressions are associated with virulence potential of Acanthamoeba spp.

Keywords: acanthamoeba, DNA-methyltransferase, histone deacetylase, virulence-associated proteins

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Authors: Patricia O. Carvalho, Marcia C. F. Messias, Laura Credidio, Carlos A. R. Martinez

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Lipidomic strategy can provide important information regarding cancer pathogenesis mechanisms and could reveal new biomarkers to enable early diagnosis of rectal adenocarcinoma (RAC). This study set out to evaluate lipoperoxidation biomarkers, and lipidomic signature by gas chromatography (GC) and electrospray ionization-qToF-mass spectrometry (ESI-qToF-MS) combined with multivariate data analysis in plasma from 23 RAC patients (early- or advanced-stages cancer) and 18 healthy controls. The most abundant ions identified in the RAC patients were those of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) while those of lisophosphatidylcholine (LPC), identified as LPC (16:1), LPC (18:1) and LPC (18:2), were down-regulated. LPC plasmalogen containing palmitoleic acid (LPC (P-16:1)), with highest VIP score, showed a low tendency in the cancer patients. Malondialdehyde plasma levels were higher in patients with advanced cancer (III/IV stages) than in the early stages groups and the healthy group (p<0.05). No differences in F2-isoprostane levels were observed between these groups. This study shows that the reduction in plasma levels of LPC plasmalogens associated to an increase in MDA levels may indicate increased oxidative stress in these patients and identify the metabolite LPC (P-16:1) as new biomarkers for RAC.

Keywords: biomarkers, lipidomic, plasmalogen, rectal adenocarcinoma

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Authors: Ali Bayram, Burak Uz, Remzi Yiğiter

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The aim of the present study was to investigate the expression levels of homo sapiens nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, transcript variant 1 (NFKB1*1) mRNA in the peripheral blood of patients with Parkinson to elucidate the role in the pathogenesis of Parkinson disease (PD). The study group comprised 50 patients with the diagnosis of PD who have applied to Gaziantep University Faculty of Medicine, and Department of Neurology. 50 healthy individuals without any neuro degenerative disease are included as controls. Ribonucleic acid (RNA) was obtained from blood samples of patient and control groups. Complementary deoxyribonucleic acid (cDNA) was obtained from RNA samples using reverse transcription polymerase chain reaction (RT-PCR) technique. The gene expression of NFKB1*1 in patient/control groups were observed to decrease significantly, and the differences between groups with the Mann-Whitney method within 95% confidence interval (p<0.05) were analyzed. This salient finding provide a clue for our hypothesis that reduced activity of NFKB1*1 gene might play a role, at least partly, in the pathophysiology of PD.

Keywords: Parkinson’s Disease, NFKB1, mRNA expression, RT-PCR

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Authors: Rijnbout-St.James Willem, Lindner Volkhard, Scholand Mary Beth, Ashton M. Tillett, Di Gennaro Michael Jude, Smith Silvia Enrica

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Introduction: Fibrosing lung diseases are characterized by changes in the lung interstitium and are classified based on etiology: 1) environmental/exposure-related, 2) autoimmune-related, 3) sarcoidosis, 4) interstitial pneumonia, and 4) idiopathic. Among interstitial lung diseases (ILD) idiopathic forms, idiopathic pulmonary fibrosis (IPF) is the most severe. Pathogenesis of IPF is characterized by an increased presence of proinflammatory mediators, resulting in alveolar injury, where injury to alveolar epithelium precipitates an increase in collagen deposition, subsequently thickening the alveolar septum and decreasing gas exchange. Identifying biomarkers implicated in the pathogenesis of lung fibrosis is key to developing new therapies and improving the efficacy of existing therapies. The transforming growth factor-beta (TGF-B1), a mediator of tissue repair associated with WNT5A signaling, is partially responsible for fibroblast proliferation in ILD and is the target of Pirfenidone, one of the antifibrotic therapies used for patients with IPF. Canonical TGF-B signaling is mediated by the proteins SMAD 2/3, which are, in turn, indirectly regulated by Collagen Triple Helix Repeat Containing 1 (CTHRC1). In this study, we tested the following hypotheses: 1) CTHRC1 is more elevated in the ILD cohort compared to unaffected controls, and 2) CTHRC1 is differently expressed among ILD types. Material and Methods: CTHRC1 levels were measured by ELISA in 171 plasma samples from the deidentified University of Utah ILD cohort. Data represent a cohort of 131 ILD-affected participants and 40 unaffected controls. CTHRC1 samples were categorized by a pulmonologist based on affectation status and disease subtypes: IPF (n = 45), sarcoidosis (4), nonspecific interstitial pneumonia (16), hypersensitivity pneumonitis (n = 7), interstitial pneumonia (n=13), autoimmune (n = 15), other ILD - a category that includes undifferentiated ILD diagnoses (n = 31), and unaffected controls (n = 40). We conducted a single-factor ANOVA of plasma CTHRC1 levels to test whether CTHRC1 variance among affected and non-affected participants is statistically significantly different. In-silico analysis was performed with Ingenuity Pathway Analysis® to characterize the role of CTHRC1 in the pathway of lung fibrosis. Results: Statistical analyses of CTHRC1 in plasma samples indicate that the average CTHRC1 level is significantly higher in ILD-affected participants than controls, with the autoimmune ILD being higher than other ILD types, thus supporting our hypotheses. In-silico analyses show that CTHRC1 indirectly activates and phosphorylates SMAD3, which in turn cross-regulates TGF-B1. CTHRC1 also may regulate the expression and transcription of TGFB-1 via WNT5A and its regulatory relationship with CTNNB1. Conclusion: In-silico pathway analyses demonstrate that CTHRC1 may be an important biomarker in ILD. Analysis of plasma samples indicates that CTHRC1 expression is positively associated with ILD affectation, with autoimmune ILD having the highest average CTHRC1 values. While characterizing CTHRC1 levels in plasma can help to differentiate among ILD types and predict response to Pirfenidone, the extent to which plasma CTHRC1 level is a function of ILD severity or chronicity is unknown.

Keywords: interstitial lung disease, CTHRC1, idiopathic pulmonary fibrosis, pathway analyses

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Authors: Ariel Vilchez, Francisca Acevedo, Rodrigo Navia

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The wound healing process can be accelerated and improved by the action of antioxidants such as curcumin (Cur) over the tissues; however, the efficacy of curcumin used through the digestive system is not enough to exploit its benefits. Electrospinning presents an alternative to carry curcumin directly to the wounds, and polyhydroxybutyrate (PHB) is proposed as the matrix to load curcumin owing to its biodegradable and biocompatible properties. PHB is among 150 types of Polyhydroxyalkanoates (PHAs) identified, it is a natural thermoplastic polyester produced by microbial fermentation obtained from microorganisms. The proposed objective is to develop electrospun bacterial PHB-based microfibers containing curcumin for possible biomedical applications. Commercial PHB was solved in Chloroform: Dimethylformamide (4:1) to a final concentration of 7% m/V. Curcumin was added to the polymeric solution at 1%, and 7% m/m regarding PHB. The electrospinning equipment (NEU-BM, China) with a rotary collector was used to obtain Cur-PHB fibers at different voltages and flow rate of the polymeric solution considering a distance of 20 cm from the needle to the collector. Scanning electron microscopy (SEM) was used to determine the diameter and morphology of the obtained fibers. Thermal stability was obtained from Thermogravimetric (TGA) analysis, and Fourier Transform Infrared Spectroscopy (FT-IR) was carried out in order to study the chemical bonds and interactions. A preliminary curcumin release to Phosphate Buffer Saline (PBS) pH = 7.4 was obtained in vitro and measured by spectrophotometry. PHB fibers presented an intact chemical composition regarding the original condition (dust) according to FTIR spectra, the diameter fluctuates between 0.761 ± 0.123 and 2.157 ± 0.882 μm, with different qualities according to their morphology. The best fibers in terms of quality and diameter resulted in sample 2 and sample 6, obtained at 0-10kV and 0.5 mL/hr, and 0-10kV and 1.5 mL/hr, respectively. The melting temperature resulted near 178 °C, according to the bibliography. The crystallinity of fibers decreases while curcumin concentration increases for the studied interval. The curcumin release reaches near 14% at 37 °C at 54h in PBS adjusted to a quasi-Fickian Diffusion. We conclude that it is possible to load curcumin in PHB to obtain continuous, homogeneous, and solvent-free microfibers by electrospinning. Between 0% and 7% of curcumin, the crystallinity of fibers decreases as the concentration of curcumin increases. Thus, curcumin enhances the flexibility of the obtained material. HPLC should be used in further analysis of curcumin release.

Keywords: antioxidant, curcumin, polyhydroxybutyrate, wound healing

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Authors: Hosein Maghsoudi

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Rheumatois arthritis (RA) is progressive inflammatory autoimmune diseases that primarily affect the joints, characterized by synovial hyperplasia and inflammatory cell infiltration, deformed and painful joints, which can lead tissue destruction, functional disability systemic complications, and early dead and socioeconomic costs. The cause of rheumatoid arthritis is unknown, but genetic and environmental factors are contributory and the prognosis is guarded. However, advances in understanding the pathogenesis of the disease have fostered the development of new therapeutics, with improved outcomes. The current treatment strategy, which reflects this progress, is to initiate aggressive therapy soon after diagnosis and to escalate the therapy, guided by an assessment of disease activity, in pursuit of clinical remission. The pathobiology of RA is multifaceted and involves T cells, B cells, fibroblast-like synoviocyte (FLSc) and the complex interaction of many pro-inflammatory cytokine. Novel biologic agents that target tumor necrosis or interlukin (IL)-1 and Il-6, in addition T- and B-cells inhibitors, have resulted in favorable clinical outcomes in patients with RA. Despite this, at least 30% of RA patients are résistance to available therapies, suggesting novel mediators should be identified that can target other disease-specific pathway or cell lineage. Among the inflammatory cell population that might participated in RA pathogenesis, FLSc are crucial in initiaing and driving RA in concert of cartilage and bone by secreting metalloproteinase (MMPs) into the synovial fluid and by direct invasion into extracellular matrix (ECM), further exacerbating joint damage. Invasion of fibroblast-like synoviocytes (FLSc) is critical in the pathogenesis of rheumatoid-arthritis. The metalloproteinase (MMPs) and activator of Toll-like receptor 4 (TLR4)/nuclear factor- κB pthway play a critical role in RA-FLS invasion induced by lipopolysaccharide (LPS). The present study aimed to explore the anti-invasion activity of Glycyrrhizic Acid as a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1beta and TNF-alpha signalling pathways in Bovine fibroblast-like synoviocyte ex- vitro, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Results showed that Glycyrrhizic Acid suppressed LPS-stimulated bovine FLS migration and invasion by inhibition MMP-9 expression and activity. In addition our results revealed that Glycyrrhizic Acid inhibited the transcriptional activity of MMP-9 by suppression the nbinding activity of NF- κB in the MMP-9 promoter pathway. The extract of licorice (Glycyrrhiza glabra L.) has been widely used for many centuries in the traditional Chinese medicine as native anti-allergic agent. Glycyrrhizin (GL), a triterpenoidsaponin, extracted from the roots of licorice is the most effective compound for inflammation and allergic diseases in human body. The biological and pharmacological studies revealed that GL possesses many pharmacological effects, such as anti-inflammatory, anti-viral and liver protective effects, and the biological effects, such as induction of cytokines (interferon-γ and IL-12), chemokines as well as extrathymic T and anti-type 2 T cells. GL is known in the traditional Chinese medicine for its anti-inflammatory effect, which is originally described by Finney in 1959. The mechanism of the GL-induced anti-inflammatory effect is based on different pathways of the GL-induced selective inhibition of the prostaglandin E2 production, the CK-II- mediated activation of both GL-binding lipoxygenas (gbLOX; 17) and PLA2, an anti-thrombin action of GL and production of the reactive oxygen species (ROS; GL exerts liver protection properties by inhibiting PLA2 or by the hydroxyl radical trapping action, leading to the lowering of serum alanine and aspartate transaminase levels. The present study was undertaken to examine the possible mechanism of anti-inflammatory properties GL on IL-1beta and TNF-alpha signalling pathways in bovine fibroblast-like synoviocyte ex-vivo, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Our results clearly showed that treatment of bovine fibroblast-like synoviocyte with GL suppressed LPS-induced cell migration and invasion. Furthermore, it revealed that GL inhibited the transcription activity of MMP-9 by suppressing the binding activity of NF-κB in the MM-9 promoter. MMP-9 is an important ECM-degrading enzyme and overexpression of MMPs in important of RA-FLSs. LPS can stimulate bovine FLS to secret MMPs, and this induction is regulated at the transcription and translational levels. In this study, LPS treatment of bovine FLS caused an increase in MMP-2 and MMP-9 levels. The increase in MMP-9 expression and secretion was inhibited by ex- vitro. Furthermore, these effects were mimicked by MMP-9 siRNA. These result therefore indicate the the inhibition of LPS-induced bovine FLS invasion by GL occurs primarily by inhibiting MMP-9 expression and activity. Next we analyzed the functional significance of NF-κB transcription of MMP-9 activation in Bovine FLSs. Results from EMSA showed that GL suppressed LPS-induced NF-κB binding to the MMP-9 promotor, as NF-κB regulates transcriptional activation of multiple inflammatory cytokines, we predicted that GL might target NF-κB to suppress MMP-9 transcription by LPS. Myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NF-κB and apoptotic signaling pathways, GL inhibited the expression of TLR4 and MYD88. These results demonstrated that GL suppress LPS-induced MMP-9 expression through the inhibition of the induced TLR4/NFκB signaling pathway. Taken together, our results provide evidence that GL exerts anti-inflammatory effects by inhibition LPS-induced bovine FLSs migration and invasion, and the mechanisms may involve the suppression of TLR4/NFκB –mediated MMP-9 expression. Although further work is needed to clarify the complicated mechanism of GL-induced anti-invasion of bovine FLSs, GL might be used as a further anti-invasion drug with therapeutic efficacy in the treatment of immune-mediated inflammatory disease such as RA.

Keywords: glycyrrhizic acid, bovine fibroblast-like synoviocyte, tlr4/nf-κb, metalloproteinase-9

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Authors: Ahmad Ali, Muhammad Imran Ghani, Haiyan Ding, Zhihui Cheng, Muhammad Iqbal

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Background and Aims: In recent years, protected cultivation trend, especially in the northern parts of China, spread dynamically where production area, structure, and crops diversity have expanded gradually under plastic greenhouse vegetable cropping (PGVC) system. Under this growing system, continuous monoculture with excessive synthetic fertilizers inputs are common cultivation practices frequently adopted by commercial producers. Such long-term cumulative wild exercise year after year sponsor the continuous cropping obstacles in PGVC soil, which have greatly threatened the regional soil eco-sustainability and further impose the continuous assault on soil ecological diversity leading to the exhaustion of agriculture productivity. The aim of this study was to develop new allelopathic insights by exploiting available biological resources in the favor of sustainable PGVC to illuminate the continuous obstacle factors in plastic greenhouse. Method: A greenhouse study was executed under plastic tunnel located at the Horticulture Experimental Station of the College of Horticulture, Northwest A&F University, Yangling, Shaanxi Province, one of the prominent regions for intensive commercial PGVC in China. Post-harvest garlic residues (stalk, leaves) mechanically smashed, homogenized into powder size and incorporated at the ratio of 1:100; 3:100; 5:100 as a soil amendment in a replanted soil that have been used for continuous cucumber monoculture for 7 years (annually double cropping system in a greenhouse). Results: Incorporated C-rich garlic stalk significantly influenced the soil condition through various ways; organic matter decomposition and mineralization, moderately adjusted the soil pH, enhanced the soil nutrient availability, increased enzymatic activities, and promoted 20% more cucumber yield in short-time. Using Illumina MiSeq sequencing analysis of bacterial 16S rRNA and fungal 18S rDNA genes, the current study revealed that addition of garlic stalk/residue could also improve the microbial abundance and community composition in extensively exploited soil, and contributed in soil functionality, caused prosper changes in soil characteristics, reinforced to good crop yield. Conclusion: Our study provided evidence that addition of garlic stalk as soil fertility amendment is a feasible, cost-effective and efficient resource utilization way for renovation of degraded soil health, ameliorate soil quality components and improve ecological environment in short duration. Our study may provide a better scientific understanding for efficient crop residue management typically from allelopathic source.

Keywords: garlic stalk, microbial community dynamics, plant growth, soil amendment, soil-plant system

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Authors: Shima Mahmoudi, Setareh Mamishi, Babak Pourakbari, Majid Marjani

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In the last years, the potential role of distinct T-cell subsets as biomarkers of active tuberculosis TB and/or latent tuberculosis infection (LTBI) has been studied. The aim of this study was to investigate the potential role of interleukin-2 (IL-2) in whole blood stimulated with M. tuberculosis-specific antigens in the QuantiFERON-TB Gold In Tube (QFT-G-IT) for the discrimination of active and latent tuberculosis. After 72-h of stimulation by antigens from the QFT-G-IT assay, IL-2 secretion was quantitated in supernatants by using ELISA (Mabtech AB, Sweden). Observing the level of IL-2 released after 72-h of incubation, we found that the level of IL-2 were significantly higher in LTBI group than in patients with active TB infection or control group (P value=0.019, Kruskal–Wallis test). The discrimination performance (assessed by the area under ROC curve) between LTBI and patients with active TB was 0.816 (95%CI: 0.72-0.97). Maximum discrimination was reached at a cut-off of 13.9 pg/mL for IL-2 following stimulation with 82% sensitivity and 86% specificity. In conclusion, although cytokine analysis has greatly contributed to the understanding of TB pathogenesis, data on cytokine profiles that might distinguish progression from latency of TB infection are scarce and even controversial. Our data indicate that the concomitant evaluation of IFN- γ and IL-2 could be instrumental in discriminating of active and latent TB infection.

Keywords: interleukin-2, discrimination, active TB, latent TB

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Authors: Ivana Cabarkapa, Zorica Tomicic, Olivera Duragic

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Antimicrobial resistance represents one of the major challenges facing humanity in the last decades. Increasing antibiotic-resistant pathogens indicates the need for the development of alternative antibacterial drugs and new treatment strategies. One of the innovative emerging treatments in overcoming multidrug-resistant pathogens certainly represents the inhibition anti-quorum sensing system. For most of the food-borne pathogens, the expression of the virulence depends on their capability communication with other members of the population by means of quorum sensing (QS). QS represents a specific way of bacterial intercellular communication, which enabled owing to their ability to detect and to respond to cell population density by gene regulation. QS mechanisms are responsible for controls the pathogenesis, virulence luminescence, motility, sporulation and biofilm formation of many organisms by regulating gene expression. Therefore, research in this field is being an attractive target for the development of new natural antibacterial agents. Anti-QS compounds are known to have the ability to prohibit bacterial pathogenicity. Considering the importance of quorum sensing during bacterial pathogenesis, this research has been focused on evaluation anti - QS properties of four essential oils (EOs) Origanum heracleoticum, Origanum vulgare, Thymus vulgare, and Thymus serpyllum, using biomonitor strain of Chromobacterium violaceum CV026. Tests conducted on Luria Bertani agar supplemented with N hexanol DL homoserine lacton (HHL) 10µl/50ml of agar. The anti-QS potential of the EOs was assayed in a range of concentrations of 200 – 0.39 µl/ml using the disc diffusion method. EOs of Th. vulgaris and T. serpyllum were exhibited anti-QS activity indicated by a non- pigmented ring with a dilution-dependent manner. The lowest dilution of EOs T. vulgaris and T. serpyllum in which they exhibited visually detectable inhibition of violacein synthesis was 6.25 µl/ml for both tested EOs. EOs of O. heracleoticum and O. vulgare were displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by the outer non-pigmented ring, in a concentration-dependent manner. The lowest dilution of EOs of O. heracleoticum and O. vulgare in which exhibited visually detectable inhibition of violacein synthesis was 1.56 and 3.25 µl/ml, respectively. Considering that, the main constituents of the tested EOs represented by monoterpenes (carvacrol, thymol, γ-terpinene, and p-cymene), anti - QS properties of tested EOs can be mainly attributed to their activity. In particular, from the scientific literature, carvacrol and thymol show a sub-inhibitory effect against foodborne pathogens. Previous studies indicated that sub-lethal concentrations of carvacrol reduced the mobility of bacteria due to the ability of interference using QS mechanism between the bacterial cells, and thereby reducing the ability of biofilm formation The precise mechanism by which carvacrol inhibits biofilm formation is still not fully understood. Our results indicated that EOs displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by an outer non- pigmented ring with visually detectable inhibition of violacein. Preliminary results suggest that EOs represent a promising alternative for effective control of the emergence and spread of resistant pathogens.

Keywords: anti-quorum sensing activity, Chromobacterium violaceum, essential oils, violacein

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Authors: Austin Belfiori, Kevin Rinek, Zach Barcroft, Jennifer Berglind

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Diet influences the composition of the gut microbiota and host's health. Disruption of the balance among the microbiota, epithelial cells, and resident immune cells in the intestine is involved in the pathogenesis of inflammatory bowel disease (IBD). To study the role of gut microbiota in intestinal inflammation, the microbiome of control mice (C57BL6) given a gluten-containing standard diet versus C57BL6 mice given the gluten-free (GF) feed (n=10 in each group) was examined. All mice received the 3% DSS for 5 days. Throughout the study, feces were collected and processed for DNA extraction and MiSeq Illumina sequencing of V4 region of bacterial 16S rRNA gene. Alpha and beta diversities and compositional differences at phylum and genus levels were determined in intestinal microbiota. The mice receiving the GF diet showed a significantly increased abundance of Firmicutes and a decrease of Bacteroides and Lactobacillus at phylum level. Therefore, the gluten free diet led to reductions in beneficial gut bacteria populations. These findings indicate a role of wheat gluten in dysbiosis of the intestinal microbiota.

Keywords: gluten, colitis, microbiota, DSS, dextran sulphate sodium

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Authors: Awei Bainivalu, Joachim Larsen, Logesh Panneerselvan, Toby Mills, Brett Neilan, Megharaj Mallavarapu

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World War II (WWII) shipwrecks harbour up to 20 million tonnes of oil. More than 3000 wrecks are in the Pacific Ocean; 300 are oil tankers. Compared to other oil removal methods, bioremediation is environmentally friendly and cost-effective. Oil's microbial community and hydrocarbon properties from the Pacific WWII wrecks were identified. Dominant phyla are Proteobacteria, Actinobacteria, and Firmicutes. Native marine bacteria oil-degraders were isolated for bioremediation. Petroleum degradation data from the bacterial consortium will be analyzed over the next three months.

Keywords: oil bioremediation, marine bacteria, WWII shipwrecks, pacific

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Authors: O. P. Yadav

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Ag-N co-doped ZnS and ZnS/Fe2O3 composite nanoparticles have been synthesized by chemical and sol-gel methods. As-synthesized nanomaterial have been characterized by XRD and TEM techniques and their antimicrobial effects were studied using paper disc diffusion technique against gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria. As-synthesized nanomaterial showed potent antimicrobial activity against studied bacterial strains. Antimicrobial activity of synthesized nanomaterial has also been compared with some commonly used antibiotics.

Keywords: antibiotic, Escherichia coli, nanomaterial, TEM, Staphylococcus aureus

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Authors: Iñaki Iturria, Pasquale Russo, Montserrat Nacher-Vázquez, Giuseppe Spano, Paloma López, Miguel Angel Pardo

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Introduction: It is known that some Lactic Acid Bacteria (LAB) present an interesting probiotic effect. Probiotic bacteria stimulate host resistance to microbial pathogens and thereby aid in immune response, and modulate the host's immune responses to antigens with a potential to down-regulate hypersensitivity reactions. Therefore, probiotic therapy is valuable against intestinal infections and may be beneficial in the treatment of Inflammatory Bowel Disease (IBD). Several in vitro tests are available to evaluate the probiotic potential of a LAB strain. However, an in vivo model is required to understand the interaction between the host immune system and the bacteria. During the last few years, zebrafish (Danio rerio) has gained interest as a promising vertebrate model in this field. This organism has been extensively used to study the interaction between the host and the microbiota, as well as the host immune response under several microbial infections. In this work, we report on the use of the zebrafish model to investigate in vivo the colonizing ability and the immunomodulatory effect of probiotic LAB. Methods: Lactobacillus strains belonging to different LAB species were fluorescently tagged and used to colonize germ-free zebrafish larvae gastrointestinal tract (GIT). Some of the strains had a well-documented probiotic effect (L. acidophilus LA5); while others presented an exopolysaccharide (EPS) producing phenotype, thus allowing evaluating the influence of EPS in the colonization and immunomodulatory effect. Bacteria colonization was monitored for 72 h by direct observation in real time using fluorescent microscopy. CFU count per larva was also evaluated at different times. The immunomodulatory effect was assessed analysing the differential expression of several innate immune system genes (MyD88, NF-κB, Tlr4, Il1β and Il10) by qRT- PCR. The anti-inflammatory effect was evaluated using a chemical enterocolitis zebrafish model. The protective effect against a pathogen was also studied. To that end, a challenge test was developed using a fluorescently tagged pathogen (Vibrio anguillarum-GFP+). The progression of the infection was monitored up to 3 days using a fluorescent stereomicroscope. Mortality rates and CFU counts were also registered. Results and conclusions: Larvae exposed to EPS-producing bacteria showed a higher fluorescence and CFU count than those colonized with no-EPS phenotype LAB. In the same way, qRT-PCR results revealed an immunomodulatory effect on the host after the administration of the strains with probiotic activity. A downregulation of proinflammatory cytoquines as well as other cellular mediators of inflammation was observed. The anti-inflammatory effect was found to be particularly marked following exposure to LA% strain, as well as EPS producing strains. Furthermore, the challenge test revealed a protective effect of probiotic administration. As a matter of fact, larvae fed with probiotics showed a decrease in the mortality rate ranging from 20 to 35%. Discussion: In this work, we developed a promising model, based on the use of gnotobiotic zebrafish coupled with a bacterial fluorescent tagging in order to evaluate the probiotic potential of different LAB strains. We have successfully used this system to monitor in real time the colonization and persistence of exogenous LAB within the gut of zebrafish larvae, to evaluate their immunomodulatory effect and for in vivo competition assays. This approach could bring further insights into the complex microbial-host interactions at intestinal level.

Keywords: gnotobiotic, immune system, lactic acid bacteria, probiotics, zebrafish

Procedia PDF Downloads 299

Authors: Feleke Terefe Fanta

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The existence of heavy metals and microbial contaminants in aquatic system of Akaki river basin, a sub city of Addis Ababa, has become a public concern as human population increases and land development continues. This is because effluents from chemical and pharmaceutical industries are directly discharged onto surrounding land, irrigation fields and surface water bodies. In the present study, we synthesised zeolites and copper- zeolite composite based adsorbent through cost effective and simple approach to mitigate the problem. The study presents determination of heavy metal content and microbial contamination level of waste water sample collected from Akaki river using zeolites and copper- doped zeolites as adsorbents. The synthesis of copper- zeolite X composite was carried out by ion exchange method of copper ions into zeolites frameworks. The optimum amount of copper ions loaded into the zeolites frameworks were studied using the pore size determination concept via iodine test. The copper- loaded zeolites were characterized by X-ray diffraction (XRD). The XRD analysis showed clear difference in phase purity of zeolite before and after copper ion exchange. The concentration of Cd, Cr, and Pb were determined in waste water sample using atomic absorption spectrophotometry. The mean concentrations of Cd, Cr, and Pb in untreated sample were 0.795, 0.654 and 0.7025 mg/L respectively. The concentration of Cd, Cr, and Pb decreased to 0.005, 0.052 and BDL mg/L for sample treated with bare zeolite X while a further decrease in concentration of Cd, Cr, and Pb (0.005, BDL and BDL) mg/L respectively was observed for the sample treated with copper- zeolite composite. The antimicrobial activity was investigated by exposing the total coliform to the Zeolite X and Copper-modified Zeolite X. Zeolite X and Copper-modified Zeolite X showed complete elimination of microbilas after 90 and 50 minutes contact time respectively. This demonstrates effectiveness of copper- zeolite composite as efficient disinfectant. To understand the mode of heavy metals removal and antimicrobial activity of the copper-loaded zeolites; the adsorbent dose, contact time, temperature was studied. Overall, the results obtained in this study showed high antimicrobial disinfection and heavy metal removal efficiencies of the synthesized adsorbent.

Keywords: waste water, copper doped zeolite x, adsorption heavy metal, disinfection

Procedia PDF Downloads 51

Authors: Hyvee Gean Cabuso, Arvin Taruc, Danielle Villanueva, Channela Anais Hipolito, Jia Bianca Alfonso

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Introduction: Denture adhesive aids to provide additional retention, support and comfort for patients with loose dentures, as well as for patients who seek to achieve optimal denture adhesion. But due to its growing popularity, arising oral health issues should be considered, including its possible impact that may alter the microbiological condition of the denture. Changes as such may further resolve to denture-related oral diseases that can affect the day-to-day lives of patients. Purpose: The study aims to assess and compare the microbiological status of dentures without adhesives versus dentures when adhesives were applied. The study also intends to identify the presence of specific microorganisms, their colony concentration and their possible effects on the oral microflora. This study also aims to educate subjects by introducing an alternative denture cleaning method as well as denture and oral health care. Methodology: Edentulous subjects age 50-80 years old, both physically and medically fit, were selected to participate. Before obtaining samples for the study, the alternative cleaning method was introduced by demonstrating a step-by-step cleaning process. Samples were obtained by swabbing the intaglio surface of their upper and lower prosthesis. These swabs were placed in a thioglycollate broth, which served as a transport and enrichment medium. The swabs were then processed through bacterial culture. The colony-forming units (CFUs) were calculated on MacConkey Agar Plate (MAP) and Blood Agar Plate (BAP) in order to identify and assess the microbiological status, including species identification and microbial counting. Result: Upon evaluation and analysis of collected data, the microbiological assessment of the upper dentures with adhesives showed little to no difference compared to dentures without adhesives, but for the lower dentures, (P=0.005), which is less than α = 0.05; therefore, the researchers reject (Ho) and that there is a significant difference between the mean ranks of the lower denture without adhesive to those with, implying that there is a significant decrease in the bacterial count. Conclusion: These results findings may implicate the possibility that the addition of denture adhesives may contribute to the significant decrease of microbial colonization on the dentures.

Keywords: denture, denture adhesive, denture-related, microbiological assessment

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Authors: Tommaso Adamo, Gianpaolo Ghiani, Antonio Domenico Grieco, Emanuela Guerriero

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Batch production plants provide a wide range of scheduling problems. In pharmaceutical industries a batch process is usually described by a recipe, consisting of an ordering of tasks to produce the desired product. In this research work we focused on pharmaceutical production processes requiring the culture of a microorganism population (i.e. bacteria, yeasts or antibiotics). Several sources of uncertainty may influence the yield of the culture processes, including (i) low performance and quality of the cultured microorganism population or (ii) microbial contamination. For these reasons, robustness is a valuable property for the considered application context. In particular, a robust schedule will not collapse immediately when a cell of microorganisms has to be thrown away due to a microbial contamination. Indeed, a robust schedule should change locally in small proportions and the overall performance measure (i.e. makespan, lateness) should change a little if at all. In this research work we formulated a constraint programming optimization (COP) model for the robust planning of antibiotics production. We developed a discrete-time model with a multi-criteria objective, ordering the different criteria and performing a lexicographic optimization. A feasible solution of the proposed COP model is a schedule of a given set of tasks onto available resources. The schedule has to satisfy tasks precedence constraints, resource capacity constraints and time constraints. In particular time constraints model tasks duedates and resource availability time windows constraints. To improve the schedule robustness, we modeled the concept of (a, b) super-solutions, where (a, b) are input parameters of the COP model. An (a, b) super-solution is one in which if a variables (i.e. the completion times of a culture tasks) lose their values (i.e. cultures are contaminated), the solution can be repaired by assigning these variables values with a new values (i.e. the completion times of a backup culture tasks) and at most b other variables (i.e. delaying the completion of at most b other tasks). The efficiency and applicability of the proposed model is demonstrated by solving instances taken from Sanofi Aventis, a French pharmaceutical company. Computational results showed that the determined super-solutions are near-optimal.

Keywords: constraint programming, super-solutions, robust scheduling, batch process, pharmaceutical industries

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Authors: M. A. Hashem, I. Jahan

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The experiment was conducted to find out the effect of different levels of Pomegranate (Punica granatum) extract and synthetic antioxidant BHA (Beta Hydroxyl Anisole) on fresh and preserved beef meatballs in order to make functional food. For this purpose, ground beef samples were divided into five treatment groups. They were treated as control group, 0.1% synthetic antioxidant group, 0.1%, 0.2% and 0.3% pomegranate extract group as T1, T2, T3, T4 and T5 respectively. Proximate analysis, sensory tests (color, flavor, tenderness, juiciness, overall acceptability), cooking loss, pH value, free fatty acids (FFA), thiobarbituric acid values (TBARS), peroxide value (POV) and microbiological examination were determined in order to evaluate the effect of pomegranate extract as natural antioxidant and antimicrobial activities compared to BHA (Beta Hydroxyl Anisole) at first day before freezing and for maintaining meatballs qualities on the shelf life of beef meat balls stored for 60 days under frozen condition. Freezing temperature was -20˚C. Days of intervals of experiment were on 0, 15th, 30th and 60th days. Dry matter content of all the treatment groups differ significantly (p<0.05). On the contrary, DM content increased significantly (p<0.05) with the advancement of different days of intervals. CP content of all the treatments were increased significantly (p<0.05) among the different treatment groups. EE and Ash content were decreased significantly (p<0.05) at different treatment levels. FFA values, TBARS, POV were decreased significantly (p<0.05) at different treatment levels. Color, odor, tenderness, juiciness, overall acceptability decreased significantly (p<0.05) at different days of intervals. Raw PH, cooked pH were increased at different treatment levels significantly (p<0.05). The cooking loss (%) at different treatment levels were differ significantly (p<0.05). TVC (logCFU/g), TCC (logCFU/g) and TYMC (logCFU/g) was decreased significantly (p<0.05) at different treatment levels and at different days of intervals comparison to control. Considering CP, tenderness, juiciness, overall acceptability, cooking loss, FFA, POV, TBARS value and microbial analysis it can be concluded that pomegranate extract at 0.1%, 0.2% and 0.3% can be used instead of synthetic antioxidant BHA in beef meatballs. On the basis of sensory evaluation, nutrient quality, physicochemical properties, biochemical analysis and microbial analysis 0.3% Pomegranate extract can be recommended for formulation of value added beef meatball enriched with natural antioxidant.

Keywords: antioxidant, pomegranate, BHA, value added meat products

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Authors: Dana Gabriková, Martin Mistrík, Jarmila Bernasovská, Iveta Tóthová, Jana Kisková

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The Roma (Gypsies) is a transnational minority with a high degree of consanguineous marriages. Similar to other genetically isolated founder populations, the Roma harbor a number of unique or rare genetic disorders. This paper discusses about a rare form of Charcot-Marie-Tooth disease – type 4G (CMT4G), also called Hereditary Motor and Sensory Neuropathy type Russe, an autosomal recessive disease caused by mutation private to Roma characterized by abnormally increased density of non-myelinated axons. CMT4G was originally found in Bulgarian Roma and in 2009 two putative causative mutations in the HK1 gene were identified. Since then, several cases were reported in Roma families mainly from Bulgaria and Spain. Here we present a Slovak Roma family in which CMT4G was diagnosed on the basis of clinical examination and genetic testing. This case is a further proof of the role of the HK1 gene in pathogenesis of the disease. It confirms that mutation in the HK1 gene is a common cause of autosomal recessive CMT disease in Roma and should be considered as a common part of a diagnostic procedure.

Keywords: gypsies, HK1, HSMN-Russe, rare disease

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Authors: Xingxin Wu, Fenli Shao, Tao Tan, Yang Tan, Yang Sun, Qiang Xu

Abstract:

Psoriasis is a chronic inflammatory skin disease that affects about 2% of the world's population. IL-23 signaling plays a key role in the pathogenesis of psoriasis. Control of IL-23 release by small molecule compounds during developing psoriasis has not been well established. Here, we show that compound 1, a small molecule nature product, protected mice from imiquimod-induced psoriasis with improved skin lesions, reduced skin thickness, and reduced IL-23 mRNA expression in the skin tissue. FACS results showed compound 1 reduced the number of dendritic cells in the skin. Interestingly, compound 1 was not able to ameliorate IL-23-induced psoriasis-like skin inflammation in mice. Further, compound 1 inhibited MyD88-dependent IL-23 mRNA expression induced by LPS, CpG and imiquimod in BMDC cells, but not MyD88-independent CD80 and CD86 expression induced by LPS. The methods included real-time PCR, western blot, H & E staining, FACS and ELISA et al. In conclusion, compound 1 regulates MyD88-dependent signaling to control IL-23 release in dendritic cells, which improves imiquimod-induced psoriasis.

Keywords: dendritic cells, IL-23, toll-like receptor signaling, psoriasis

Procedia PDF Downloads 617