Search results for: mammalian pathogenicity

Authors: Khaled S. Al Salhen

Abstract:

Aldehyde oxidase (AO) and xanthine oxidoreductase (XOR) catalyze the oxidation of many different N-heterocyclic compounds as well as aliphatic and aromatic aldehydes to their corresponding lactam and carboxylic acids respectively. The present study examines the oxidation of dimethylamino-cinnamaldehyde (DMAC), vanillin and phenanthridine by AO and xanthine by XOR from Drosophila cytosol. Therefore, the results obtained in the present study showed the DMAC, vanillin and phenanthridine substrates used were found to be good substrates of Drosophila AO and xanthine is the preferred substrate for Drosophila XOR. Km values of AO substrates were observed with DMAC (50±5.4 µM), phenanthridine (80±9.1 µM) and vanillin (303±11.7 µM) respectively for Drosophila cytosol. The Km values for DMAC and phenanthridine were ~6 and ~4 fold lower than that for vanillin as a substrate. The Km for XOR with xanthine using NAD+ as an electron acceptor was 27±4.1 µM. Relatively low Vmax values were obtained with phenanthridine (1.78±0.38 nmol/min/mg protein) and DMAC (1.80±0.35 nmol/min/mg protein). The highest Vmax was obtained from Drosophila cytosol with vanillin (7.58±2.11 nmol/min/mg protein). It is concluded these results that AO and XOR in Drosophila were able to catalyse the biotransformation of numerous substrates of the well-characterised mammalian AO and XOR. The kinetic parameters have indicated that the activity of AO of Drosophila may be a significant factor the oxidation of aromatic aldehyde compounds.

Keywords: aldehyde oxidase, xanthine oxidoreductase, dimethylamino-cinnamaldehyde, vanillin, phenanthridine, Drosophila melanogaster

Procedia PDF Downloads 425

Authors: Akash Bera, Suraj Singh, Jacinta Dsouza, Ramakrishna V. Hosur, Pushpa Mishra

Abstract:

Ultraviolet C (UVC) radiation induces apoptosis in mammalian cells and it is suggested that the mechanism by which this occurs is the mitochondrial pathway of apoptosis through the release of cytochrome c from the mitochondria into the cytosol. The Bcl-2 family of proteins pro-and anti-apoptotic is the regulators of the mitochondrial pathway of apoptosis. Upon UVC irradiation, the proliferation of apoptosis is enhanced through the downregulation of the anti-apoptotic protein Bcl-xl and up-regulation of Bax. Although the participation of the Bcl-2 family of proteins in apoptosis appears responsive to UVC radiation, to the author's best knowledge, it is unknown how the structure and, effectively, the function of these proteins are directly impacted by UVC exposure. In this background, we present here a structural rationale for the effect of UVC irradiation in restoring apoptosis using two of the relevant proteins, namely, Bid-FL and Bcl-xl ΔC, whose solution structures have been reported previously. Using a variety of biophysical tools such as circular dichroism, fluorescence and NMR spectroscopy, we show that following UVC irradiation, the structures of Bcl-xlΔC and Bid-FL are irreversibly altered. Bcl-xLΔC is found to be more sensitive to UV exposure than Bid-FL. From the NMR data, dramatic structural perturbations (α-helix to β-sheet) are seen to occur in the BH3 binding region, a crucial segment of Bcl-xlΔC which impacts the efficacy of its interactions with pro-apoptotic tBid. These results explain the regulation of apoptosis by UVC irradiation. Our results on irradiation dosage dependence of the structural changes have therapeutic potential for the treatment of cancer.

Keywords: Bid, Bcl-xl, UVC, apoptosis

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Authors: Arunas Stirke, Neringa Bakute, Gatis Mozolevskis

Abstract:

A technology of microfluidics is an emerging tool in the field of biology, medicine and chemistry. Microfluidic device is also known as ‘lab-on-a-chip’ technology [1]. In moving from macro- to microscale, there is unprecedented control over spatial and temporal gradients and patterns that cannot be captured in conventional Petri dishes and well plates [2]. However, there is not a single standard microfluidic chip designated for all purposes – every different field of studies needs a specific microchip with certain geometries, inlet/outlet, channel depth and other parameters to precisely regulate the required function. Since our group is studying an effect of pulsed electric field (PEF) to the cells, we have manufactured a microfluidic chip designated for high-throughput electroporation of cells. In our microchip, a cell culture chamber is divided into two parallel channels by a membrane, meanwhile electrodes for electroporation are attached to the wall of the channels. Both microchannels have their own inlet and outlet, enabling injection of transfection material separately. Our perspective is to perform electroporation of mammalian cells in two different ways: (1) plasmid and cells are injected in the same microchannel and (2) injected into separate microchannels. Moreover, oxygen and pH sensors are integrated on order to analyse cell viability parameters after PEF treatment.

Keywords: microfluidics, chip, fabrication, electroporation

Procedia PDF Downloads 63

Authors: Hamid Hossein Khezri, Afsaneh Javdani-Mallak

Abstract:

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Cannabidiol (CBD) is one of the members of cannabinoid compounds found in cannabis. CBD and Cannabinol (CBN), as the other extract of plant Cannabis were able to reduce myofascial pain in rats with immunosuppressive and anti-inflammatory activities. In this study, we accomplished protein-protein BLAST, and the sequence was found to be for Gamma-aminobutyric acid receptor subunit alpha-1 (GBRA1) chain A and its 3D structure was subsequently downloaded from Protein Data Bank. The structures of the ligands, cannabinol, and cannabidiol, were obtained from PubChem. After the necessary process of the obtained files, AutoDock Vina was used to perform molecular docking. Docking between the ligands and GBRA1 chain A revealed that cannabinol has a higher affinity to GBRA1 (binding energy = -7.5 kcal/mol) compared to cannabidiol (binding energy = -6.5 kcal/mol). Furthermore, cannabinol seems to be able to interact with 10 residues of the protein, out of which 3 are in the neurotransmitter-gated ion-channel transmembrane domain of GBRA1, whereas cannabidiol interacts with two other residues. Although the results of this project do not indicate the activating /or inhibitory capability of the studied compounds, it suggests that cannabinol can act as a relatively strong ligand for GBRA1.

Keywords: protein-ligand docking, cannabinol, cannabidiol, GBRA1

Procedia PDF Downloads 95

Authors: Hamid Hossein Khezri, Afsaneh Javdani-Mallak

Abstract:

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Cannabidiol (CBD) is one of the members of cannabinoid compounds found in cannabis. CBD and Cannabinol (CBN), as the other extract of plant Cannabis, were able to reduce myofascial pain in rats with immunosuppressive and anti-inflammatory activities. In this study, we accomplished protein-protein BLAST and the sequence was found to be for Gamma-aminobutyric acid receptor subunit alpha-1 (GBRA1) chain A and its 3D structure was subsequently downloaded from Protein Data Bank. The structures of the ligands cannabinol and cannabidiol were obtained from PubChem. After a necessary process of the obtained files, AutoDock Vina was used to performing molecular docking. Docking between the ligands and GBRA1 chain A revealed that cannabinol has a higher affinity to GBRA1 (binding energy = -7.5 kcal/mol) compared to cannabidiol (binding energy = -6.5 kcal/mol). Furthermore, cannabinol seems to be able to interact with 10 residues of the protein, out of which 3 are in the neurotransmitter-gated ion-channel transmembrane domain of GBRA1, whereas cannabidiol interacts with two other residues. Although the results of this project do not indicate the activating /or inhibitory capability of the studied compounds, it suggests that cannabinol can act as a relatively strong ligand for GBRA1.

Keywords: protein-ligand docking, cannabinol, cannabidiol, GBRA1

Procedia PDF Downloads 99

Authors: Bingjie Wang, Su-Yeon Kwon, Ik-Joong Kang

Abstract:

A Surface-enhanced Raman Scattering (SERS) as the principle for enhancing Raman scattering by molecules adsorbed on rough metal surfaces or by nanostructures is used to detect the concentration change of γ-Aminobutyric Acid (GABA). As for the gold-chitosan nanoshell, it is made by using chitosan nanoparticles crosslinking with sodium tripolyphosphate(TPP) for the first step to form the chitosan nanoparticles, which would be covered with the gold sequentially. The size of the fabricated product was around 100nm. Based on the method that the sulfur end of the MPA linked to gold can form the very strong S–Au bond, and the carboxyl group, the other end of the MPA, can easily absorb the GABA. GABA is the mainly inhibitory neurotransmitter in the mammalian central nervous system in the human body. It plays such significant role in reducing neuronal excitability throughout the nervous system. When the system formed, it generated SERS, which made a clear difference in the intensity of Raman scattering within the range of GABA concentration. So it is obtained from the experiment that the calibration curve according to the GABA concentration relevant with the SERS scattering. In this study, DLS, SEM, FT-IR, UV, SERS were used to analyze the products to obtain the conclusion.

Keywords: chitosan-gold nanoshell, mercaptopropionic acid, γ-aminobutyric acid, surface-enhanced Raman scattering

Procedia PDF Downloads 246

Authors: Khaled S. Al Salhen

Abstract:

Aldehyde oxidase is molybdo-flavoenzyme involved in the oxidation of hundreds of endogenous and exogenous and N-heterocyclic compounds and environmental pollutants. Uncharged N-heterocyclic aromatic compounds such phenanthridine are commonly distributed pollutants in soil, air, sediments, surface water and groundwater, and in animal and plant tissues. Phenanthridine as uncharged N-heterocyclic aromatic compound was incubated with partially purified aldehyde oxidase from rainbow trout fish liver. Reversed-phase HLPC method was used to separate the oxidation products from phenanthridine and the metabolite was identified. The 6(5H)-phenanthridinone was identified the major metabolite by partially purified aldehyde oxidase from fish liver. Kinetic constant for the oxidation reactions were determined spectrophotometrically and showed that this substrate has a good affinity (Km = 78 ± 7.6 µM) for hepatic aldehyde oxidase, coupled with a relatively high oxidation rate (0.77± 0.03 nmol/min/mg protein). In addition, the kinetic parameters of hepatic fish aldehyde oxidase towards the phenanthridine substrate indicate that in vitro biotransformation by hepatic fish aldehyde oxidase will be a significant pathway. This study confirms that partially purified aldehyde oxidase from fish liver is indeed the enzyme responsible for the in vitro production 6(5H)-phenanthridinone metabolite as it is a major metabolite by mammalian aldehyde oxidase.

Keywords: aldehyde oxidase, fish, phenanthridine, specificity

Procedia PDF Downloads 347

Authors: Salim Cerig, Fatime Geyikoglu, Murat Bakir, Suat Colak, Merve Sonmez, Kubra Koc

Abstract:

Cisplatin (CIS) is one of the most effective an anticancer drug and also toxic to cells by activating oxidative stress. Oleuropein (OLE) has key role against oxidative stress in mammalian cells, but the role of this antioxidant in the toxicity of CIS remains unknown. The aim of the present study was to investigate the efficacy of OLE on CIS-induced liver damages in male rats. With this aim, male Sprague Dawley rats were randomly assigned to one of eight groups: Control group; the group treated with 7 mg/kg/day CIS; the groups treated with 50, 100 and 200 mg/kg/day OLE (i.p.); and the groups treated with OLE for three days starting at 24 h following CIS injection. After 4 days of injections, serum was provided to assess the blood AST, ALT and LDH values. The liver tissues were removed for histological, biochemical (TAC, TOS and MDA) and genotoxic evaluations. In the CIS treated group, the whole liver tissue showed significant histological changes. Also, CIS significantly increased both the incidence of oxidative stress and the induction of 8-hydroxy-deoxyguanosine (8-OH-dG). Moreover, the rats taking CIS have abnormal results on liver function tests. However, these parameters reached to the normal range after administration of OLE for 3 days. Finally, OLE demonstrated an acceptable high potential and was effective in attenuating CIS-induced liver injury. In this trial, the 200 mg/kg dose of OLE firstly appeared to induce the most optimal protective response.

Keywords: antioxidant response, cisplatin, histology, liver, oleuropein, 8-OhdG

Procedia PDF Downloads 329

Authors: Bingjie Wang, Su-Yeon Kwon, Ik-Joong Kang

Abstract:

The goal of this experiment is to develop a sensor that can quickly check the concentration by using the nanoparticles made by chitosan and gold. Using chitosan nanoparticles crosslinking with sodium tripolyphosphate(TPP) is the first step to form the chitosan nanoparticles, which would be covered with the gold sequentially. The size of the fabricated product was around 100nm. Based on the method that the sulfur end of the MPA linked to gold can form the very strong S–Au bond, and the carboxyl group, the other end of the MPA, can easily absorb the GABA. As for the GABA, what is the primary inhibitory neurotransmitter in the mammalian central nervous system in the human body. It plays such significant role in reducing neuronal excitability pass through the nervous system. A Surface-enhanced Raman Scattering (SERS) as the principle for enhancing Raman scattering by molecules adsorbed on rough metal surfaces or by nanostructures is used to detect the concentration change of γ-Aminobutyric Acid (GABA). When the system is formed, it generated SERS, which made a clear difference in the intensity of Raman scattering within the range of GABA concentration. So it is obtained from the experiment that the calibration curve according to the GABA concentration relevant with the SERS scattering. In this study, DLS, SEM, FT-IR, UV, SERS were used to analyze the products to obtain the conclusion.

Keywords: mercaptopropionic acid, chitosan-gold nanoshell, γ-aminobutyric acid, surface-enhanced raman scattering

Procedia PDF Downloads 260

Authors: Dike H. Ogbuagu, Etsede J. Oritsematosan

Abstract:

This work investigated possible inductions of CaC₂, often misused by fruit vendors to stimulate artificial ripening, on mammalian sperm morphology and viability. Thirty isogenic strains of male albino mice, Mus musculus (age≈ 8weeks; weight= 32.5±2.0g) were acclimatized (ambient temperature 28.0±1.0°C) for 2 weeks and fed standard growers mash and water ad libutum. They were later exposed to graded toxicant concentrations (w/w) of 2.5000, 1.2500, 0.6250, and 0.3125% in 4 cages. A control cage was also established. After 5 weeks, 3 animals from each cage were sacrificed by cervical dislocation and the cauda epididymis excised. Sperm morphology and viability were determined by microscopic procedures. The ANOVA, means plots, Student’s t-test and variation plots were used to analyze data. The common abnormalities observed included Double Head, Pin Head, Knobbed Head, No Tail and With Hook. The higher toxicant concentrations induced significantly lower body weights [F(829.899) ˃ Fcrit(4.19)] and more abnormalities [F(26.52) ˃ Fcrit(4.00)] at P˂0.05. Sperm cells in the control setup were significantly more viable than those in the 0.625% (t=0.005) and 2.500% toxicant doses (t=0.018) at the 95% confidence limit. CaC₂ appeared to induced morphological abnormalities and reduced viability in sperm cells of M. musculus.

Keywords: artificial ripening, calcium carbide, fruit vendors, sperm morphology, sperm viability

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Authors: Anjali Verma, Mahesh Pal, Veena Pande, Dalip Kumar Upreti

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The present study is carried out to explore the anti-hyperglycemic activity of Leucas cephalotes plant parts. A fruit, leaves, stems, and roots part of the Leucas cephalotes has been extracted in ethanol and have been evaluated for anti-hyperglycemic activity. The present study indicated that, ethanolic extract of fruit and leaves have shown significant α- amylase inhibitory activity with IC50 value of 92.86 ± 0.89 μg/mL and 98.09 ± 0.69 μg/mL respectively. Two known compounds β-sitosterol and lupeol were isolated from ethanolic extract of L. cephalotes leaves and were subjected to anti-hyperglycemic activity. Lupeol shows the best activity with IC50 55.73 ± 0.47 μg/mL and the results were verified by docking study of these compounds with mammalian α-amylase was carried out on its active site. It was concluded from the study that β-sitosterol and lupeol form one H-bond interactions with the active site residues either Asp212 or Thr21. The estimated free energy binding of β-sitosterol was found to be -9.47 kcal mol-1 with an estimated inhibition constant (Ki) of 558.94 nmol whereas the estimated free energy binding of lupeol was -11.73 kcal mol-1 with an estimated inhibition constant (Ki) of 476.71pmmol. The present study clearly showed that lupeol is more potent in comparison to β-sitosterol. The study indicates that L. cephalotes have significant potential to inhibit α-amylase enzyme.

Keywords: alpha-amylase, beta-sitosterol, hyperglycemia, lupeol

Procedia PDF Downloads 197

Authors: Mohammed N. Al Kindi, Mazin Al Khabouri, Khalsa Al Lamki, Tommasso Pappuci, Giovani Romeo, Nadia Al Wardy

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Hereditary hearing loss is a heterogeneous group of complex disorders with an overall incidence of one in every five hundred newborns presented as syndromic and non-syndromic forms. Cadherin-related 23 (CDH23) is one of the listed deafness causative genes. CDH23 is found to be expressed in the stereocilia of hair cells and the retina photoreceptor cells. Defective CDH23 has been associated mostly with prelingual severe-to-profound sensorineural hearing loss (SNHL) in either syndromic (USH1D) or non-syndromic SNHL (DFNB12). An Omani family diagnosed clinically with severe-profound sensorineural hearing loss was genetically analysed by whole exome sequencing technique. A novel homozygous missense variant, c.A7451C (p.D2484A), in exon 53 of CDH23 was detected. One hundred and thirty control samples were analysed where all were negative for the detected variant. The variant was analysed in silico for pathogenicity verification using several mutation prediction software. The variant proved to be a pathogenic mutation and is reported for the first time in Oman and worldwide. It is concluded that in silico mutation prediction analysis might be used as a useful molecular diagnostics tool benefiting both genetic counseling and mutation verification. The aspartic acid 2484 alanine missense substitution might be the main disease-causing mutation that damages CDH23 function and could be used as a genetic hearing loss marker for this particular Omani family.

Keywords: Cdh23, d2484a, in silico, Oman

Procedia PDF Downloads 197

Authors: V. V. Lakshmi, Y. V. S. Annapurna

Abstract:

Urinary tract infection (UTI) is one of the most common infectious diseases seen in the community. Susceptible individuals experience multiple episodes, and progress to acute pyelonephritis or uro-sepsis or develop asymptomatic bacteriuria (ABU). Ability to cause extraintestinal infections depends on several virulence factors required for survival at extraintestinal sites. Presence of virulence phenotypes enhances the pathogenicity of these otherwise commensal organisms and thus augments its ability to cause extraintestinal infections, the most frequent in urinary tract infections(UTI). The present study focuses on detection of the virulence characters exhibited by the uropathogenic organism and most common factors exhibited in the local pathogens. A total of 700 isolates of E.coli and Klebsiella spp were included in the study. These were isolated from patients from local hospitals reported to be suffering with UTI over a period of three years. Isolation and identification was done based on Gram character and IMVIC reactions. Antibiotic sensitivity profile was carried out by disc diffusion method and multi drug resistant strains with MAR index of 0.7 were further selected.. Virulence features examined included their ability to produce exopolysaccharides, protease- gelatinase production, hemolysin production, haemagglutination and hydrophobicity test. Exopolysaccharide production was most predominant virulence feature among the isolates when checked by congo red method. The biofilms production examined by microtitre plates using ELISA reader confirmed that this is the major factor contributing to virulencity of the pathogens followed by hemolysin production

Keywords: Escherichia coli, Klebsiella sp, Uropathogens, Virulence features.

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Authors: Liu Xiaohan

Abstract:

Epstein–Barr virus (EBV) is a human herpesvirus and is closely related to many malignancies of lymphocyte and epithelial origins, such as gastric cancer, Burkitt’s lymphoma, and nasopharyngeal carcinoma (NPC). NPC is a malignant epithelial tumor which is 100% associated with EBV latent infection. Most of the NPC cases are densely populated in southern China, especially in Guangdong and Hong Kong. To our knowledge, overexpression of pro-inflammatory cytokines may result in a loss of balance of the immune system and cause damage to human bodies. Interleukin-6 (IL6) is a pro-inflammatory cytokine which plays an important role in tumor progression. In addition, gene expression is regulated by both transcriptional and post-transcriptional pathways, while post-transcriptional regulation is an important mechanism to modulate the mature mRNA level in mammalian cells. AU-rich element binding factor 1 (AUF1)/heterogeneous nuclear RNP D (hnRNP D) is known for its function in destabilizing mRNAs, including cytokines and cell cycle regulators. Previous studies have found that overexpression of hnRNP D would lead to tumorigenesis. In this project, our aim is to determine the role played by hnRNP D in EBV-infected cells and how our anti-EBV agents can affect the function of hnRNP D. The results of this study will provide a new insight into how the pro-inflammatory cytokine expression can be regulated by EBV.

Keywords: interleukin 6 (IL6), epstein-barr virus (EBV), nasopharyngeal carcinoma (NPC, epstein-barr nuclear antigen-1 (EBNA1)

Procedia PDF Downloads 43

Authors: Victor Iorungwa Gwa, Ebenezer Jonathan Ekefan

Abstract:

Potential of Trichoderma harzianum for biological control of postharvest fungal rot of white yam (Dioscorea rotundata Poir) tubers in storage was studied. Pathogenicity test revealed the susceptibility of healthy looking yam tubers to Aspergillus niger, Botryodiplodia theobromae, and Fusarium oxysporum f. sp. melonganae after fourteen days of inoculation. Treatments comprising A. niger, B. theobromae, and F. oxysporum each paired with T. harzianum and were arranged in completely randomized design and stored for five months. Experiments were conducted between December 2015 and April 2016 and December 2016 and April 2017. Results showed that tubers treated with the pathogenic fungi alone caused mean percentage rot of between 6.67 % (F. oxysporum) and 22.22 % (A. niger) while the paired treatments produced only between 2.22 % (T. harzianum by F. oxysporum) and 6.67 % (T. harzianum by A. niger). In the second year of storage, mean percentage rot was found to be between 13.33 % (F. oxysporum) and 28.89 % (A. niger) while in the paired treatment rot was only between 6.67 % (F. oxysporum) and 8.89% (A. niger). Tubers treated with antagonist alone produced 0.00 % and 2.22 % in the first and second year, respectively. Result revealed that there was a significant difference (P ≤ 0.05) in mean percentage rot between the first year and the second year except where B. theobromae was inoculated alone, A. niger and T. harzianum paired and B. theobromae and T. harzianum paired. The most antagonised fungus in paired treatment for both years was F. oxysporum f. sp. melonganae, while the least antagonised, was A. niger and B. theobromae. It is, therefore, concluded that T. harzianum has potentials to control rot causing pathogens of yam tubers in storage. This can compliment or provide better alternative ways of reducing rot in yam tubers than by the use of chemical fungicides which are not environmentally friendly.

Keywords: biological control, fungal rot, postharvest, Trichoderma harzianum, white yam

Procedia PDF Downloads 137

Authors: V. V. Lakshmi, Y. V. S. Annapurna

Abstract:

Urinary tract infection (UTI) is one of the most common infectious diseases seen in the community. Susceptible individuals experience multiple episodes, and progress to acute pyelonephritis or uro-sepsis or develop asymptomatic bacteriuria (ABU). Ability to cause extraintestinal infections depends on several virulence factors required for survival at extraintestinal sites. Presence of virulence phenotypes enhances the pathogenicity of these otherwise commensal organisms and thus augments its ability to cause extraintestinal infections, the most frequent in urinary tract infections(UTI). The present study focuses on detection of the virulence characters exhibited by the uropathogenic organism and most common factors exhibited in the local pathogens. A total of 700 isolates of E.coli and Klebsiella spp were included in the study.These were isolated from patients from local hospitals reported to be suffering with UTI over a period of three years. Isolation and identification was done based on Gram character and IMVIC reactions. Antibiotic sensitivity profile was carried out by disc diffusion method and multi drug resistant strains with MAR index of 0.7 were further selected. Virulence features examined included their ability to produce exopolysaccharides, protease- gelatinase production, hemolysin production, haemagglutination and hydrophobicity test. Exopolysaccharide production was most predominant virulence feature among the isolates when checked by congo red method. The biofilms production examined by microtitre plates using ELISA reader confirmed that this is the major factor contributing to virulencity of the pathogens followed by hemolysin production.

Keywords: Escherichia coli, Klebsiella spp, Uropathogens, virulence features

Procedia PDF Downloads 302

Authors: Anderson Chidi Amadioha, Promise Chidi Kenkwo, A. A. Markson

Abstract:

Cassava (Manihot esculenta Crantz) is an important food and cash crop that provide cheap source of carbohydrate for food, feed and raw material for industries hence a commodity for feature economic development of developing countries. Despite the importance, its production potentials is undermined by disease agents that greatly reduce yield and render it unfit for human consumption and industrial use. Pathogenicity tests on fungal isolates from infected cassava revealed Aspergillus flavus, Rhizopus stolonifer, Aspergillus niger, and Trichodderma viride as rot-causing organisms. Water and ethanol extracts of Piper guineense, Ocimum graticimum, Cassia alata, and Tagetes erecta at 50% concentration significantly inhibited the radial growth of the pathogens in vitro and their development and spread in vivo. Low cassava rot incidence and severity was recorded when the extracts were applied before than after spray inoculating with spore suspension (1x105 spores/ml of distilled water) of the pathogenic organisms. The plant materials are readily available, and their extracts are biodegradable and cost effective. The fungitoxic potentials of extracts of these plant materials could be exploited as potent biopesticides in the management of postharvest fungal deterioration of cassava especially in developing countries where synthetic fungicides are not only scarce but also expensive for resource poor farmers who produce over 95% of the food consumed.

Keywords: cassava, biopesticides, in vitro, in vivo, pathogens, plant extracts

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Authors: Wendu Admasu, Assefa Sintayehu, Alemu Gezahgne, Zewdu Terefework

Abstract:

Eucalyptus is the most widely planted forest tree species in the world. In Ethiopia, pathogenic fungi pose an increasing threat to Eucalyptus species. Due to limited research, there is insufficient information on the associated diseases and pathogens. This study investigated Eucalyptus diseases, the extent of their damage, and the causal fungal pathogens. A Eucalyptus disease survey was conducted in the Eucalyptus forestry areas of Ethiopia during the growth years 2019/20 and 2020/21. Disease assessment and sampling were carried out in eighteen plantations at nine locations. E. camaldulensis was the most dominant species planted in the surveyed areas. The field study shows a high incidence and severity of canker diseases. Diseased stem and branch samples were collected, cultured on malt extract agar media and studied. The results of morphological and ITS sequence analysis confirmed that the fungal species Neofusicoccum parvum, Lasiodiplodia theobromae, and Aplosporella hesperidica caused the observed canker symptoms. This is the first report of Lasiodiplodia theobromae and Aplosporella hesperidica causing diseases in Eucalyptus plants in Ethiopia. Changes in global climate and environmental factors, such as altitude, are believed to have a strong impact on the susceptibility of Eucalyptus plants to diseases. Strict quarantine practices and continuous monitoring of pathogenic and endophytic fungal species associated with Eucalyptus trees are issued to be prioritized to effectively control and manage the disease.

Keywords: Neofusicoccum, Lasiodiplodia, Aplosporella, pathogenicity, phylogeny, severity

Procedia PDF Downloads 39

Authors: Ranjith Kumar Kankala, Wei-Zhi Lin, Chia-Hung Lee

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Antibiotic resistance in bacteria has become an emergency situation clinically. To improve the efficacy of antibiotics in resistant strains, advancement of nanoparticles is inevitable than ever. Herewith, we demonstrate a design by immobilizing tetracycline (TET) in copper substituted mesoporous silica nanoparticles (Cu-MSNs) through a pH-sensitive coordination link, enabling its release in the acidic environment. Subsequently, MSNs are coated with silver nanoparticles stabilized polyethyleneimine (PEI-SNP) to act against drug-resistant (MDR) bacterial strains. Silver ions released from SNP are capable of sensitizing the resistant strains and facilitate the generation of free radicals capable of damaging the cell components. In addition, copper ions in the framework are also capable of generating free radicals through Fenton-like reaction. Furthermore, the nanoparticles are well-characterized physically, and various antibacterial efficacious tests against isolated multidrug resistant bacterial strain were highly commendable. However, this formulation has no significant toxic effect on normal mammalian fibroblast cells accounting its high biocompatibility. These MSN trio-hybrids, i.e., SNP, tetracycline, and copper ions result in synergistic effects, and their advancement could bypass resistance and allow synergism for effective treatment of antibiotic clinically.

Keywords: antibiotic resistance, copper, mesoporous silica nanoparticles, Ph-sensitive release, polyethyleneimine, silver, tetracycline

Procedia PDF Downloads 182

Authors: Sumeeta Khurana, Kirti Megha, Amit Gupta, Rakesh Sehgal

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Background: Amoebic keratitis is a painful vision threatening infection caused by a free living pathogenic amoeba Acanthamoeba. It can be misdiagnosed and very difficult to treat if not suspected early. The epidemiology of Acanthamoeba genotypes causing infection in our geographical area is not yet known to the best of our knowledge. Objective: To characterize Acanthamoeba isolates from amoebic keratitis patients. Methods: A total of 19 isolates obtained from patients with amoebic keratitis presenting to the Advanced Eye Centre at Postgraduate Institute of Medical Education and Research, a tertiary care centre of North India over a period of last 10 years were included. Their corneal scrapings, lens solution and lens case (in case of lens wearer) were collected for microscopic examination, culture and molecular diagnosis. All the isolates were maintained in the Non Nutrient agar culture medium overlaid with E.coli and 13 strains were axenised and maintained in modified Peptone Yeast Dextrose Agar. Identification of Acanthamoeba genotypes was based on amplification of diagnostic fragment 3 (DF3) region of the 18srRNA gene followed by sequencing. Nucleotide similarity search was performed by BLAST search of sequenced amplicons in GenBank database (http//www.ncbi.nlm.nih.gov/blast). Multiple Sequence alignments were determined by using CLUSTAL X. Results: Nine out of 19 Acanthamoeba isolates were found to belong to Genotype T4 followed by 6 isolates of genotype T11, 3 T5 and 1 T3 genotype. Conclusion: T4 is the predominant Acanthamoeba genotype in our geographical area. Further studies should focus on differences in pathogenicity of these genotypes and their clinical significance.

Keywords: Acanthamoeba, free living amoeba, keratitis, genotype, ocular

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Authors: Kkochorong Park, Keun Cheon Kim, Hyoban Lee, Eun Ju Lee, Bongsoo Kim

Abstract:

Introduction of biomaterials such as DNA, RNA, proteins is important for many research areas. There are many methods to introduce biomaterials into living organisms like tissue and cells. To introduce biomaterials, several indirect methods including virus‐mediated delivery, chemical reagent (i.e., lipofectamine), electrophoresis have been used. Such methods are passive delivery using an endocytosis process of cell, reducing an efficiency of delivery. Unlike the indirect delivery method, it has been reported that a direct delivery of exogenous biomolecules into nucleus have been more efficient to expression or integration of biomolecules. Nano-sized material is beneficial for detect signal from cell or deliver stimuli/materials into the cell at cellular and molecular levels, due to its similar physical scale. Especially, because 1 dimensional (1D) nanomaterials such as nanotube, nanorod and nanowire with high‐aspect ratio have nanoscale geometry and excellent mechanical, electrical, and chemical properties, they could play an important role in molecular and cellular biology. In this study, by using single crystalline 1D noble metal nanowire, we fabricated nano-sized 1D injector which can successfully interface with living cells and directly deliver biomolecules into several types of cell line (i.e., stem cell, mammalian embryo) without inducing detrimental damages on living cell. This nano-bio technology could be a promising and robust tool for introducing exogenous biomaterials into living organism.

Keywords: DNA, gene delivery, nanoinjector, nanowire

Procedia PDF Downloads 258

Authors: Anyaphat Srithanasuwan, Noppason Pangprasit, Montira Intanon, Phongsakorn Chuammitri, Witaya Suriyasathaporn, Ynte H. Schukken

Abstract:

Streptococcus uberis is one of the most common mastitis-causing pathogens, with a wide range of intramammary infection (IMI) durations and pathogenicity. This study aimed to compare shared or unique virulence factor gene clusters distinguishing persistent and transient strains of S. uberis. A total of 139 S. uberis strains were isolated from three small-holder dairy herds with a high prevalence of S. uberis mastitis. The duration of IMI was used to categorize bacteria into two groups: transient and persistent strains with an IMI duration of less than 1 month and longer than 2 months, respectively. Six representative S. uberis strains, three from each group (transience and persistence) were selected for analysis. All transient strains exhibited multi-locus sequence types (MLST), indicating a highly diverse population of transient S. uberis. In contrast, MLST of persistent strains was available in an online database (pubMLST). Identification of virulence genes was performed using whole-genome sequencing (WGS) data. Differences in genomic size and number of virulent genes were found. For example, the BCA gene or alpha-c protein and the gene associated with capsule formation (hasAB), found in persistent strains, are important for attachment and invasion, as well as the evasion of the antimicrobial mechanisms and survival persistence, respectively. These findings suggest a genetic-level difference between the two strain types. Consequently, a comprehensive study of 139 S. uberis isolates will be conducted to perform an in-depth genetic assessment through WGS analysis on an Illumina platform.

Keywords: Streptococcus Uberis, mastitis, whole genome sequence, intramammary infection, persistent S. Uberis, transient s. Uberis

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Authors: Hajar Hosseini Khorami

Abstract:

Porphyrins are organic, aromatic compounds found in heme, cytochrome, cobalamin, chlorophyll , and many other natural products with essential roles in biological processes that their cationic forms have been used as groups of favorable non-viral vectors recently. Cationic porphyrins are self-chromogenic reagents with a high capacity for modifications, great interaction with DNA and protection of DNA from nuclease during delivery of it into a cell with low toxicity. In order to have high efficient gene transfection into the cell while causing low toxicity, genetically manipulations of the non-viral vector, cationic porphyrin, would be useful. In this study newly modified cationic porphyrin derivative, 5, 10-dihexyl-15, 20bis (N-methyl-4-pyridyl) porphyrin was applied. Cytotoxicity of synthesized cationic porphyrin on Chinese Hamster Ovarian (CHO) cells was evaluated by using MTT assay. This cationic derivative is dose-dependent, with low cytotoxicity at the ranges from 100 μM to 0.01μM. It was uptake by cells at high concentration. Using direct non-viral gene transfection method and different concentration of cationic porphyrin were tested on transfection of CHO cells by applying derived transfection reagent with X-tremeGENE HP DNA as a positive control. However, no transfection observed by porphyrin derivative and the parameters tested except for positive control. Results of this study suggested that applying different protocol, and also trying other concentration of cationic porphyrins and DNA for forming a strong complex would increase the possibility of efficient gene transfection by using cationic porphyrins.

Keywords: cationic porphyrins, gene delivery, non-viral vectors, transfection reagents

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Authors: Saba Atefyekta

Abstract:

Aim: Control of bacterial bioburden in wounds is an important step for minimizing the risk of wound infection. An antimicrobial hydrogel wound dressing is developed out of soft polymeric hydrogels that contain antimicrobial peptides (AMPs). Such wound dressings can bind and kill all types of bacteria, even the resistance types at the wound site. Methods: AMPs are permanently bonded onto a soft nanostructured polymer via covalent attachment and physical entanglement. This improves stability, rapid antibacterial activity, and, most importantly, prevents the leaching of AMPs. Major Findings: Antimicrobial analysis of antimicrobial hydrogels using in-vitro wound models confirmed >99% killing efficiency against multiple bacterial trains, including MRSA, MDR, E. Coli. Furthermore, the hydrogel retained its antibacterial activity for up to 4 days when exposed to human serum. Tests confirmed no release of AMPs, and it was proven non-toxic to mammalian cells. An in-vivo study on human intact skin showed a significant reduction of bacteria for part of the subject’s skin treated with antibacterial hydrogels. A similar result was detected through a qualitative study in veterinary trials on different types of surgery wounds in cats, dogs, and horses. Conclusions: Antimicrobial hydrogels wound dressings developed by permanent attachment of AMPs can effectively and rapidly kill bacteria in contact. Such antibacterial hydrogel wound dressings are non-toxic and do not release any substances into the wound.

Keywords: antibacterial wound dressing, antimicrobial peptides, post-surgical wounds, infection

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Authors: Sangyeop Shin, D. C. M. Kulatunga, S. H. S. Dananjaya, Chamilani Nikapitiya, Jehee Lee, Mahanama De Zoysa

Abstract:

Saprolegniasis is one of the most devastating fungal diseases in freshwater fish which is caused by species in the genus Saprolegnia including Saprolegnia parasitica. In this study, we isolated the strain of S. parasitica from diseased rainbow trout in Korea. Morphological and molecular based identification confirmed that isolated fungi belong to the member of S. parasitica, supported by its typical fungal features including cotton-like whitish mycelium, zoospores (primary and secondary) and phylogenetic analysis with internal transcribed spacer (ITS) region. Pathogenicity of isolated S. parasitica was developed in embryo, larvae, juvenile and adult zebrafish as a disease model. Up regulation of host genes encoding ZfTnf-α, Zfc-Rel, ZfIl-12, ZfLyz-c, Zfβ-def, and ZfHsp-70 was identified in zebrafish larvae after experimental challenge of S. parasitica showing the host immune responses against the S. parasitica. Survival of the juveniles upon fungal infection might be due to the increased immune protection in the host. Investigation of antifungal susceptibility of S. parasitica with natural lawsone (2-hydroxy-1,4-naphthoquinone) revealed the minimum inhibitory concentration (MIC) and percentage inhibition of radial growth (PIRG %) as 200 µg/mL and 31.8%, respectively. Lawsone was able to change the membrane permeability, and cause irreversible damage and disintegration to the cellular membranes of S. parasitica which might have effect on fungi growth inhibition. Moreover, the mycelium exposed to lawsone (MIC level) changed the transcriptional responses of S. parasitica genes. Overall results indicate that lawsone could be a potential and novel anti-S. parasitica agent for controlling S. parasitica infection.

Keywords: host-pathogen interactions, lawsone, rainbow trout, Saprolegnia parasitica, Saprolegniasis, zebrafish

Procedia PDF Downloads 229

Authors: Constantine Uwaremwe, Wenjie Bao, Bachir Goudia Daoura, Sandhya Mishra, Xianxian Zhang, Lingjie Shen, Shangwen Xia, Xiaodong Yang

Abstract:

Background: Plukenetia volubilis Linneo is an oleaginous plant belonging to the family Euphorbiaceae. Due to its seeds containing a high content of edible oil and rich in vitamins, P. volubilis is cultivated as an economical plant worldwide. However, the cultivation and growth of P. volubilis is challenged by phytopathogen invasion leading to production loss. Methods: In the current study, we tested the pathogenicity of fungal pathogens isolated from root rot infected P. volubilis plant tissues by inoculating them into healthy P. volubilis seedlings. Metagenomic sequencing was used to assess the shift in the fungal community of P. volubilis rhizosphere soil after root rot infection. Results: Four Fusarium isolates and two Rhizopus isolates were found to be root rot causative agents of P. volubilis as they induced typical root rot symptoms in healthy seedlings. The metagenomic sequencing data showed that root rot infection altered the rhizosphere fungal community. In root rot infected soil, the richness and diversity indices increased or decreased depending on pathogens. The four most abundant phyla across all samples were Ascomycota, Glomeromycota, Basidiomycota, and Mortierellomycota. In infected soil, the relative abundance of each phylum increased or decreased depending on the pathogen and functional taxonomic classification. Conclusions: Based on our results, we concluded that Fusarium and Rhizopus species cause root rot infection of P. volubilis. In root rot infected P. volubilis, the shift in the rhizosphere fungal community was pathogen-dependent. These findings may serve as a key point for a future study on the biocontrol of root rot of P. volubilis.

Keywords: fusarium spp., plukenetia volubilis l., rhizopus spp., rhizosphere fungal community, root rot

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Authors: Abdelkarim Mahdhi, Fdhila Kais, Faouzi Lamari, Zeineb Hmila, Fathi Kamoun, Maria Ángeles Esteban, Amina Bakhrouf

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Aquaculture is the world’s fastest growing food-production sector. However, one of the most serious problems regarding the culture of marine fishes is the mortality associated with pathogenic bacteria that occurs in the critical phases of larval development. Conventional approaches, such as the use of antimicrobial drugs to control diseases, have had limited success in the prevention or cure of aquatic diseases. Promising alternatives to antibiotics are probiotics, which are food supplements consisting of live microorganisms that benefit the host organism. In the search for more effective and environmentally friendly treatments with probionts against pathogenic species in shrimp larval culture, the probiotic properties of Bacillus strains isolated from Artemia culture such as antibacterial activity, adhesion, pathogenicity, toxicity and the effect of marine stress on viability and survival were investigated, as well as the changes occurring in their properties. Analyses showed that these bacteria corresponded to the genus Bacillus sp. Antagonism and adherence assays revealed that these strains have an inhibitory effect against pathogenic bacteria in vitro and in vivo conditions and are fairly adherent. Challenge tests performed with Artemia larvae provided evidence that the tested Bacillus strains were neither pathogenic nor toxic to the host. The tested strains maintained their viability and their probiotic properties during the period of study. The results suggest that the tested strains have suffered changes allowing them to survive in seawater in the absence of nutrients and outside their natural host, identifying them as potential probiotic candidates for Artemia culture.

Keywords: bacillus, probiotic, cell viability, stress response

Procedia PDF Downloads 370

Authors: Milena Trevisan Pelegrino, Bruna De Araujo Lima, Mônica H. M. Do Nascimento, Christiane B. Lombello, Marcelo Brocchi, Amedea B. Seabra

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Nitric oxide (NO) is a small molecule involved in a wide range of physiological and pathophysiological processes, including vasodilatation, control of inflammatory pain, wound healing, and antibacterial activities. As NO is a free radical, the design of drugs that generates therapeutic amounts of NO in controlled spatial and time manners is still a challenge. In this study, the NO donor S-nitrosoglutathione (GSNO) was incorporated into the thermoresponsive Pluronic F-127 (PL) - chitosan (CS) hydrogel, in an easy and economically feasible methodology. CS is a polysaccharide with known antimicrobial and biocompatibility properties. Scanning electron microscopy, rheology and differential scanning calorimetry techniques were used for hydrogel characterization. The results demonstrated that the hydrogel has a smooth surface, thermoresponsive behavior, and good mechanical stability. The kinetics of NO release and GSNO diffusion from GSNO-containing PL/CS hydrogel demonstrated a sustained NO/GSNO release, in concentrations suitable for biomedical applications, at physiological and skin temperatures. The GSNO-PL/CS hydrogel demonstrated a concentration-dependent toxicity to Vero cells, and antimicrobial activity to Pseudomonas aeruginosa (minimum inhibitory concentration and minimum bactericidal concentration values of 0.5 µg·mL-1 of hydrogel, which correspondents to 1 mmol·L-1 of GSNO). Interesting, the concentration range in which the NO-releasing hydrogel demonstrated antibacterial effect was not found toxic to Vero mammalian cell. Thus, GSNO-PL/CS hydrogel is suitable biomaterial for topical NO delivery applications.

Keywords: antimicrobial, chitosan, biocompatibility, S-nitrosothiols

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Authors: V. Viswanathan, Md. Irshad Ahmad, Prashant K. Singh, Nayeem Ahmad, Pradeep Sharma, Sujata Sharma, Tej P Singh

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Lactoperoxidase (LPO) is a heme containing mammalian enzyme which uses hydrogen peroxide (H2O2) to catalyze the conversion of substrates into oxidized products. LPO is found in body fluids and tissues such as milk, saliva, tears, mucosa and other body secretions. The previous structural studies have shown that LPO converts substrates, thiocyanate (SCN‾) and iodide (I‾) ions into oxidized products, hypothiocyanite (OSCN‾) and hypoiodite (IO‾) ions, respectively. We report here a new structure of the complex of LPO with an oxidized product, nitrite (NO2‾). This product was generated from NO using the two step reaction of LPO by adding hydrogen peroxide (H2O2) in the solution of LPO in 0.1M phosphate buffer at pH 6.8 as the first step. In the second step, NO gas was added to the above mixture. This was crystallized using 20% (w/v) PEG-3350 and 0.2M ammonium iodide at pH 6.8. The structure determination showed the presence of NO2‾ ion in the distal heme cavity of the substrate binding site of LPO. The structure also showed that the propionate group, which is linked to pyrrole ring D of the heme moiety, was disordered. Similarly, the side chain of Asp108, which is covalently linked to heme moiety, was also split into two components. As a result of these changes, the conformation of the side chain of Arg255 was altered, allowing it to form new interactions with the disordered carboxylic group of propionate moiety. These structural changes are indicative of an intermediate state in the catalytic reaction pathway of LPO.

Keywords: lactoperoxidase, structure, nitric oxide, nitrite ion, intermediate, complex

Procedia PDF Downloads 79

Authors: Affiah D. U., Katuri I. P., Emefiene M. E., Amienyo C. A.

Abstract:

Broccoli (Brassica oleraceae L., var. italica) is one of the most important vegetables that is high in nutrients and bioactive compounds. It easily grown on a wide range of soil types and is adaptable to many different climatic conditions. This study was carried out within Jos North and environs in vitro to evaluate Neem (Azadirachta indica) leaves extract against Albugo candida, the causative agent of white blisters disease of broccoli. Through the survey, prevalence and incidence were accessed and a fluffy white growth symptom on the underside of leaves was also observed on the field. Infected leaves samples were collected from three different farms namely: Farin Gada, Naraguta, and Juth and the organism associated with the disease was isolated. Pathogenicity test carried out revealed the fungal isolate Albugo candida to be responsible for the disease. Antimicrobial susceptibility test was performed using agar well diffusion method to determine the minimum inhibitory concentrations of two extract of Azadirachta indica leaves against the organism. Ethanolic extract had the highest antifungal activities of 3.30±0.21 - 17.61± 0.11 while aqueous extract had the least antifungal activities of 0.00±0.00 - 13.23±0.12. The minimum inhibitory concentration of aqueous was 100 mg/ml while its minimum fungicidal concentration was at 200 mg/ml. For ethanol, the minimum inhibitory concentration was 50 mg/ml while its minimum fungicidal concentration was 100 mg/ml. Plants being less toxic in usage over synthetic or inorganic chemicals makes them easy to handle, easily accessible and renewable. Due to the biosafety of plant extracts and its availability since the plant-based extracts of the two different solvents were found to be effective against the test organism hence, it is recommended for in-depth research to make it readily available for control of other pathogens and pests.

Keywords: antifungal, biocontrol, broccoli, fungi

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