Search results for: cytochrome c oxidase subunit I

Authors: Yasser M. Saad, Heba El-Sebaie Abd El-Sadek

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Some Metapenaeus monoceros cox1 gene fragments were isolated, purified, sequenced, and comparatively analyzed with some other Crustacean Cox1 gene sequences (obtained from National Center for Biotechnology Information). This work was designed for testing the efficiency of this system in reconstruction of phylogenetic relations among some Crustacean species belonging to four genera (Metapenaeus, Artemia, Daphnia and Calanus). The single nucleotide polymorphism and haplotype diversity were calculated for all estimated mt-DNA fragments. The genetic distance values were 0.292, 0.015, 0.151, and 0.09 within Metapenaeus species, Calanus species, Artemia species, and Daphnia species, respectively. The reconstructed phylogenetic tree is clustered into some unique clades. Cytochrome oxidase subunit 1 gene (cox1) was a powerful system in reconstruction of phylogenetic relations among evaluated crustacean species.

Keywords: crustaceans, genetics, Cox1, phylogeny

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Authors: Yijun Lai, Saber Khederzadeh, Lingshaung Han

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Species in Thunnus are organized due to the similarity between them. The closeness between T. maccoyii, T. thynnus, T. Tonggol, T. atlanticus, T. albacares, T. obsesus, T. alalunga, and T. orientails are in different degrees. However, the genetic pattern of differentiation has not been presented based on individuals yet, to the author’s best knowledge. Hence, we aimed to analyze the difference in individuals level of tuna species to identify the factors that contribute to the maternal lineage variety using Cytochrome c oxidase subunit I (COXI) gene sequences. Our analyses provided evidence of sharing lineages in the Thunnus. A phylogenetic analysis revealed that these lineages are basal to the other sequences. We also showed a close connection between the T. tonggol, T. thynnus, and T. albacares populations. Also, the majority of the T. orientalis samples were clustered with the T. alalunga and, then, T. atlanticus populations. Phylogenetic trees and migration modeling revealed high proximity of T. thynnus sequences to a few T. orientalis and suggested possible gene flow with T. tonggol and T. albacares lineages, while all T. obsesus samples indicated unique clustering with each other. Our results support the presence of old maternal lineages in Thunnus, as a legacy of an ancient wave of colonization or migration.

Keywords: Thunnus Tuna, phylogeny, maternal lineage, COXI gene

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Authors: R. M. Srivastava, Subbanna A.R.N.S, Md Abbas Ahmad, S. P.More, Shivashankar, B. Kalyanbabu

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The similar morphology associated with high genetic variability poses problems in phylogenetic studies of Helicoverpa armigera (Hubner). To identify genetic variation of North Western Himalayan population’s, partial (Mid to terminal region) cytochrome c oxidase subunit I (COX-1) gene was amplified and sequenced for three populations collected from Pantnagar, Almora, and Chinyalisaur. The alignment of sequences with other two populations, Nagpur representing central India population and Anhui, China representing complete COX-1 sequence revealed unanimity in middle region with eleven single nucleotide polymorphisms (SNPs) in Nagpur populations. However, the consensus is missing when approaching towards terminal region, which is associated with 15 each SNPs and pair base substitutions in Chinyalisaur populations. In minimum evolution tree, all the five populations were majorly separated into two clades, one comprising of only Nagpur population and the other with rest. Amongst, North Western populations, Chinyalisaur one is promising by farming a separate clade. The pairwise genetic distance ranges from 0.025 to 0.192 with the maximum between H. armigera populations of Nagpur and Chinyalisaur. This genetic isolation of populations can be attributed to a key role of topological barriers of weather and mountain ranges and temporal barriers due to cropping patterns.

Keywords: cytochrome c oxidase subunit I, northwestern Himalayan population, Helicoverpa armigera (Noctuidae: Lepidoptera), phylogenetic relationship, genetic variation

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Authors: Zain Ul Abdin, Mirza Aizaz Asim, Rao Sohail Ahmad Khan, Luqman Amrao, Fiaz Hussain, Hasooba Hira, Saqi Kosar Abbas

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The success of Integrated vector management programmes mainly depends on the correct identification of insect vector species involved in vector borne diseases. Based on molecular data the most important insect species involved as vectors for Zoonotic diseases in Pakistan were identified. The precise and accurate identification of such type of organism is only possible through molecular based techniques like “DNA barcoding”. Morphological species identification in insects at any life stage, is very challenging, therefore, DNA barcoding was used as a tool for rapid and accurate species identification in a wide variety of taxa across the globe and parallel studies revealed that DNA barcoding data can be effectively used in resolving taxonomic ambiguities, detection of cryptic diversity, invasion biology, description of new species etc. A comprehensive survey was carried out for the collection of insects (both adult and immature stages) in district Faisalabad, Pakistan and their DNA was extracted and mitochondrial cytochrome oxidase subunit I (COI-59) barcode sequences was used for molecular identification of immature and adult life stage.This preliminary research work opens new frontiers for developing sustainable insect vectors management programmes for saving lives of mankind from fatal diseases.

Keywords: zoonotic diseases, cytochrome oxidase, and insect vectors, CO1

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Authors: Makhosi Buthelezi

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Mitochondrial DNA cytochrome c oxidase I (COI) gene analyses linked the South African groundnut leaf miner (GLM) to the Australian soya bean moth Aproaerema simplexella (Walker) and Indian Aproaerema modicella (Deventer). Thus, the genetic relatedness of GLM, A. simplexela, and A. modicella was examined by performing mitochondrial and nuclear (COI, cytochrome oxidase subunit II (COII), mitochondrial cytochrome b (CYTB), nuclear ribosomal 28S (28S) and intergenic spacer elongation factor-1 alpha ( EF-1 ALPHA) on 44 specimens collected from South Africa, four from Mozambique, and three each from single locations in India and Australia. Phylogenetic analyses were conducted using the Maximum Parsimony (MP) and Neighbour-Joining (NJ) methods. All of the datasets of the five DNA gene regions that were sequenced were also analyzed using the Basic Local Alignment Search Tool (BLAST) to find the closest matches for inclusion in the phylogenetic trees as outgroups and for purposes of information. In the phylogenetic trees for COI, COII, cytb and EF-1 ALPHA, a similar pattern was observed in the way that the sequences assembled into different groups; i.e., some sequences of A. simplexella from Australia were grouped separately from the others, but some Australian sequences grouped with those of the GLM from South Africa, India, and Mozambique. In the phylogenetic tree for 28S, all sequences from South Africa, Australia, India, and Mozambique grouped together and formed one group. For COI, genetic pairwise distance ranged from 0.97 to 3.60 %, for COII it ranged from 0.19% to 2.32%, for cytb it ranged from 0.25 to 9.77% and for EF-1 ALPHA it ranged 0.48 to 6.99%. Results of this study indicate that these populations are genetically related and presumably constitute a single species. Thus, further molecular and morphological studies need to be undertaken in order to resolve this apparent conundrum on the taxonomy of these populations.

Keywords: aproaerema modicella, aproaerema simplexella, mitochondrial DNA, nuclear DNA

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Authors: M. Y. Abubakar, Ipinjolu J. K., Yuzine B. Esa, Magawata I., Hassan W. A., Turaki A. A.

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Catfish (Heterobranchus spp.) is a major freshwater fish that are widely distributed in Nigeria waters and are gaining rapid aquaculture expansion. However, indiscriminate artificial crossbreeding of the species with others poses a threat to their biodiversity. There is a paucity of information about the genetic variability, hence this insight on the genetic variability is badly needed, not only for the species conservation but for aquaculture expansion. In this study, we tested the level of Genetic diversity, population differentiation and phylogenetic relationship analysis on 35 individuals of two populations of Heterobranchus bidorsalis and 29 individuals of three populations of Heterobranchus longifilis using the mitochondrial cytochrome c oxidase subunit I (mtDNA COI) gene sequence. Nucleotide sequences of 650 bp fragment of the COI gene of the two species were compared. In the whole 4 and 5 haplotypes were distinguished in the populations of H. bidorsalis & H. longifilis with accession numbers (MG334168 - MG334171 & MG334172 to MG334176) respectively. Haplotypes diversity indices revealed a range of 0.59 ± 0.08 to 0.57 ± 0.09 in H. bidorsalis and 0.000 to 0.001051 ± 0.000945 in H. longifilis population, respectively. Analysis of molecular variance (AMOVA) revealed no significant variation among H. bidorsalis population of the Niger & Benue Rivers, detected significant genetic variation was between the Rivers of Niger, Kaduna and Benue population of H. longifilis. Two main clades were recovered, showing a clear separation between H. bidorsalis and H. longifilis in the phylogenetic tree. The mtDNA COI genes studied revealed high gene flow between populations with no distinct genetic differentiation between the populations as measured by the fixation index (FST) statistic. However, a proportion of population-specific haplotypes was observed in the two species studied, suggesting a substantial degree of genetic distinctiveness for each of the population investigated. These findings present the description of the species character and accessions of the fish’s genetic resources, through gene sequence submitted in Genetic database. The data will help to protect their valuable wild resource and contribute to their recovery and selective breeding in Nigeria.

Keywords: AMOVA, genetic diversity, Heterobranchus spp., mtDNA COI, phylogenetic tree

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Authors: Ayesha Imtiaz, Darlina Md. Naim

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Family Nemipteridae is among the most abundantly distributed family in Malaysian fish markets due to its high contribution to landing sites of Malaysia. Using an advanced molecular approach that used two mitochondrial (Cytochrome oxidase c I and Cytochrome oxidase b) and one nuclear gene (Recombination activating gene, RAGI) to expose cryptic diversity and phylogenetic relationships among commercially important species of family Nemipteridae. Our research covered all genera (including 31 species out total 45 species) of family Nemipteridae, distributed in Malaysia. We also found certain type of geographical barriers in the South China sea that reduces dispersal and stops a few species to intermix. Northside of the South China Sea (near Vietnam) does not allow genetic diversity to mix with the Southern side of the South China sea (Sarawak) and reduces dispersal. Straits of Malacca reduce the intermixing genetic diversity of South China Sea and the Indian Ocean.

Keywords: Nemipteridae, RAG I, south east Asia, Malaysia

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Authors: M. Omer Faruk, A. M. A. M. Zonaed Siddiki, M. Fazal Karim, Md. Masuduzzaman, S. Chowdhury, Md. Shafiqul Islam, M. Alamgir Hossain

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The dog tapeworms Echinococcus granulosus develop hydatid cysts in various organs in human and domestic animals worldwide including Bangladesh. The aim of this study was to identify and characterize the genotype of E. granulosus isolated from cattle using 12S rRNA and Cytochrome oxidase 1 (COX 1) genes. A total of 43 hydatid cyst samples were collected from 390 examined cattle samples derived from slaughterhouses. Among them, three cysts were fertile. Genomic DNA was extracted from germinal membrane and/or protoscoleces followed by PCR amplification of mitochondrial 12S rRNA and Cytochrome oxidase 1 gene fragments. The sequence data revealed existence of G1 (64.28%) and possible G3 (21.43%) genotypes for the first time in Bangladesh. The study indicates that common sheep strain G1 is the dominant subtype of E. granulosus in Chittagong region of Bangladesh. This will increase our understanding of the epidemiology of hydatidosis in the southern part of the country and will be useful to plan suitable control measures in the long run.

Keywords: Echinococcus granulosus, Cox1, 12S rRNA, molecular characterization, Bangladesh

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Authors: B. M. Md-Zain, N. A. Rahman, M. A. B. Abdul-Latiff, W. M. R. Idris

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We conducted molecular research to portray phylogenetic relationships of Malaysian primates particularly in the genus of Macaca. We have sequenced cytochrome C oxidase subunit I (COI) of mitochondrial DNA of several individuals from M. fascicularis and M. arctoides. PCR amplifications were performed and COI DNA sequences were aligned using ClustalW. Phylogenetic trees were constructed using distance analyses by employing neighbor-joining algorithm (NJ). We managed to sequence 700 bp of COI DNA sequences. The tree topology showed that M. fascicularis did not clump based on phyleogeography division in Peninsular Malaysia. Individuals from Negeri Sembilan merged together with samples from Perak and Penang into one clade. In addition, phylogenetic analyses indicated that M. arctoides was classified into sinica group instead of fascicularis group supported by genetic distance data. COI gene is an effective locus to clarify phylogenetic position of M. arctoides but not in discriminating M. fascicularis population in Peninsular Malaysia.

Keywords: cercopithecine, long-tailed macaque, Macaca fascicularis, Macaca arctoides

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Authors: C. I. Boshoff, S. Steenkamp-Jonker

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Cystic echinococcosis (CE), caused by Echinococcus granulosus is an important parasitic infection in livestock worldwide, with severe zoonotic potential. It is important to understand the variability of Echinococcus granulosus, as genotype variations may influence lifecycle patterns, development rate, and transmission. Cystic Echinococcus samples were collected from domestic animals in Eastern Cape Province, South Africa. A molecular study was performed on 14 hydatid cysts obtained from caprine, ovine and bovine livers in order to determine the Echinococcus granulosus strain present in these hosts. The sequencing of the mitochondrial cytochrome C oxidase subunit I (coxI) gene of the hydatid cysts produced sequences of 400 bp for each sample analysed. These sequences were aligned with those present in GenBank and a phylogenetic tree was constructed. Based on coxI genotype the isolates could be grouped into E. granulosus sensu stricto. The findings of the study represent a pilot molecular study on Echinococcus from domestic animals undertaken in South Africa.

Keywords: Echinococcus granulosus, genotypes, livestock, South Africa

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Authors: Khaled S. Al Salhen

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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

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Authors: Sara Arafeh, Kovriguine Evguine

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Cytochromes P450 are enzymes that require a supply of electrons to catalyze the synthesis of steroid hormones, fatty acids, and prostaglandin hormone. Cytochrome P450 Oxidoreductase (CYPOR), a membrane bound enzyme, provides these electrons in its open conformation. CYPOR has two cytosolic domains (FAD domain and FMN domain) and an N-terminal in the membrane. In its open conformation, electrons flow from NADPH, FAD, and finally to FMN where cytochrome P450 picks up these electrons. In the closed conformation, cytochrome P450 does not bind to the FMN domain to take the electrons. It was found that when the cytosolic domains are isolated, CYPOR could not bind to cytochrome P450. This suggested that the membrane environment is important for CYPOR function. This project takes the initiative to better understand the dynamics of CYPOR in its full length. Here, we determine the distance between specific sites in the FAD and FMN binding domains in CYPOR by Forster Resonance Energy Transfer (FRET) and Ultrafast TA spectroscopy with and without NADPH. The approach to determine these distances will rely on labeling these sites with red and infrared fluorophores. Mimic membrane attachment is done by inserting CYPOR in lipid nanodiscs. By determining the distances between the donor-acceptor sites in these domains, we can observe the open/closed conformations upon reducing CYPOR in the presence and absence of cytochrome P450. Such study is important to better understand CYPOR mechanism of action in various endosomal membranes including hepatic CYPOR which is vital in plasma cholesterol homeostasis. By investigating the conformational cycles of CYPOR, we can synthesize drugs that would be more efficient in affecting the steroid hormonal levels and metabolism of toxins catalyzed by Cytochrome P450.

Keywords: conformational cycle of CYPOR, cytochrome P450, cytochrome P450 oxidoreductase, FAD domain, FMN domain, FRET, Ultrafast TA Spectroscopy

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Authors: Rahile Ozturk, Esra Maltas

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This study aimed to determine the effect of vinclozolin on some biochemical characteristics of Galleria mellonella (Lepidoptera: Pyralidae) which is an economically harmful species damaging the honeycomb in beekeeping. For experimental groups, the eggs obtained from stock were dropped into the mixed feed of vinclozolin at different doses (20, 40 and 60 ppm) and had the larvae fed with this feed. As result of the addition of vinclozolin at concentrations of 20, 40 and 60 ppm, glycogen contents of G. mellonella were determined and a significant reduction in the amount of glycogen was observed with increasing concentration of vinclozolin. In this study, activity of catalase enzyme, particularly effective in defense mechanism, activity of xanthine oxidase involved in nucleotide metabolism and activity of glucose oxidase in the metabolism of carbohydrates were measured. When compared with the results from control groups, the enzyme activities of the larvaes fed with the feed including 20, 40 and 60 ppm of vinclozolin were observed to vary or remain constant. Accordingly, glucose oxidase and catalase activities increased with the increase in amount of vinclozolin in the feed and the activity of xanthine oxidase remained stable.

Keywords: Catalase, Galleria mellonella, glucose oxidase, vinclozolin, xanthine oxidase.

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Authors: Asma Karim

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Fish taxonomy plays a fundamental role in the study of biodiversity. However, traditional methods of fish taxonomy rely on morphological features, which can lead to confusion due to great similarities between closely related species. To overcome this limitation, modern taxonomy employs DNA barcoding as a species identification method. This involves using a short standardized mitochondrial DNA region as a barcode, specifically a 658 base pair fragment near the 5′ ends of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, to exploit the diversity in this region for identification of species. To test the effectiveness and reliability of DNA barcoding, 25 fish specimens from nine different fish species found in various rivers of Pakistan were identified morphologically using a dichotomous key at the start of the study. Comprising nine freshwater fish species, including Mystus cavasius, Mystus bleekeri, Osteobrama cotio, Labeo rohita, Labeo culbasu, Labeo gonius, Cyprinus carpio, Catla catla and Cirrhinus mrigala from different rivers of Pakistan were used in the present study. DNA was extracted from one of the pectoral fins and a partial sequence of CO1 gene was amplified using the conventional PCR method. Analysis of the barcodes confirmed that genetically identified fishes were the same as those identified morphologically at the beginning of the study. The sequences were also analyzed for biodiversity and phylogenetic studies. Based on the results of the study, it can be concluded that DNA barcoding is an effective and reliable method for studying biodiversity and conducting phylogenetic analysis of different fish species in Pakistan.

Keywords: DNA barcoding, fresh water fishes, taxonomy, biodiversity, Pakistan

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Authors: Foluke E. Sola-Ojo, Ibraheem A. Abubakar, Semiu F. Bello, Isiaka H. Fatima, Sule Bisola, Adesina M. Olusegun, Adeniyi C. Adeola

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The domesticated pigeon, Columba livia domestica, has many valuable characteristics, including high nutritional value and fast growth rate. There is a lack of information on its genetic diversity in Nigeria; thus, the genetic variability in mitochondrial cytochrome oxidase subunit I (COI) sequences of 150 domestic pigeons from four different locations was examined. Three haplotypes (HT) were identified in Nigerian populations; the most common haplotype, HT1, was shared with wild and domestic pigeons from Europe, America, and Asia, while HT2 and HT3 were unique to Nigeria. The overall haplotype diversity was 0.052± 0.025, and nucleotide diversity was 0.026± 0.068 across the four investigated populations. The phylogenetic tree showed significant clustering and genetic relationship of Nigerian domestic pigeons with other global pigeons. The median-joining network showed a star-like pattern suggesting population expansion. AMOVA results indicated that genetic variations in Nigerian pigeons mainly occurred within populations (99.93%), while the Neutrality tests results suggested that the Nigerian domestic pigeons’ population experienced recent expansion. This study showed a low genetic diversity and population differentiation among Nigerian domestic pigeons consistent with a relatively conservative COI sequence with few polymorphic sites. Furthermore, the COI gene could serve as a candidate molecular marker to investigate the genetic diversity and origin of pigeon species. The current data is insufficient for further conclusions; therefore, more research evidence from multiple molecular markers is required.

Keywords: Nigeria pigeon, COI, genetic diversity, genetic variation, conservation

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Authors: M. S. Kilic, S. Korkut, B. Hazer

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Newly synthesized Polypropylene-g-Polyethylene glycol polymer was first time used for a compartment-less enzymatic fuel cell. Working electrodes based on Polypropylene-g-Polyethylene glycol were operated as unmediated and mediated system (with ferrocene and gold/cobalt oxide nanoparticles). Glucose oxidase and bilirubin oxidase was selected as anodic and cathodic enzyme, respectively. Glucose was used as fuel in a single-compartment and membrane-less cell. Maximum power density was obtained as 0.65 nW cm-2, 65 nW cm-2, and 23500 nW cm-2 from the unmediated, ferrocene and gold/cobalt oxide modified polymeric film, respectively. Power density was calculated to be ~16000 nW cm-2 for undiluted wastewater sample with gold/cobalt oxide nanoparticles including system.

Keywords: bilirubin oxidase, enzymatic fuel cell, glucose oxidase, nanoparticles

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Authors: Mosaab A. Omara, Layla O. Elmajdoubb, Mohammad Saleh Al-Aboodyc, Ahmed ElSifyd, Ahmed O. Elkhtamd

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The aim of this study is to present the molecular characterization of cysticercus tenuicolis of Taenia hydatigena from livestock isolates in Egypt, using the amplification of sequencing of the mt-CO1 gene. We introduce a detailed image of the Cysticercus tenuicolis infection in ruminant animals in Upper Egypt. Cysticercus tenuicolis inhabits such organs in ruminants as the omentum, viscera, and liver. In the present study, the infection rate of Cysticercus tenuicolis was found to be 16% and 19% in sheep and goat sample respectively. Firstly we report one larval stage of Taenia hydatigena detected in the camel liver in Egypt. Cysticercus tenuicolis infection manifested a higher prevalence in females than in males. Those above 2 years of age manifested a higher infection rate than younger animals. The preferred site for the infection was the omentum: a 70% preference in sheep and a 68% preference in goat samples. The molecular characterization using the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene of isolates from sheep, goats and camels corresponded to T. hydatigena. For this study, molecular characterizations of T. hydatigena were done for the first time in Egypt. Molecular tools are of great assistance in characterizing the Cysticercus tenuicolis parasite especially when the morphological character cannot be detected because the metacestodes are frequently confused with infection by the Hydatid cyst, especially when these occur in the visceral organs. In the present study, Cysticercus tenuicolis manifested high identity in the goat and sheep samples, while differences were found more frequently in the camel samples (10 pairbase). Clearly molecular diagnosis for Cysticercus tenuicolis infection significantly helps to differentiate it from such other metacestodes.

Keywords: cysticercus tenuicolis, its2, genetic, qena, molecular and taenia hydatigena

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Authors: Hanouf A. S. Al Nuwaysir, Nadine Moubayed, Abir Ben Bacha, Islem Abid

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Consumption of waste water continues to cause significant problems for human health in both developed and developing countries. Many regulations have been implied by different world authorities controlling water quality for the presence of coliforms used as standard indicators of water quality deterioration and historically leading health protection concept. In this study, the European directive for the detection of Escherichia coli, ISO 9308-1, was applied to examine and monitor coliforms in water samples collected from Wadi Hanifa and neighboring wells, Riyadh governorate, kingdom of Saudi Arabia, which is used for irrigation and industrial purposes. Samples were taken from different locations for 8 months consecutively, chlorine concentration ranging from 0.1- 0.4 mg/l, was determined using the DPD FREE CHLORINE HACH kit. Water samples were then analyzed following the ISO protocol which relies on the membrane filtration technique (0.45µm, pore size membrane filter) and a chromogenic medium TTC, a lactose based medium used for the detection and enumeration of total coliforms and E.coli. Data showed that the number of bacterial isolates ranged from 60 to 300 colonies/100ml for well and surface water samples respectively; where higher numbers were attributed to the surface samples. Organisms which apparently ferment lactose on TTC agar plates, appearing as orange colonies, were selected and additionally cultured on EMB and MacConkey agar for a further differentiation among E.coli and coliform bacteria. Two additional biochemical tests (Cytochrome oxidase and indole from tryptophan) were also investigated to detect and differentiate the presence of E.coli from other coliforms, E. coli was identified in an average of 5 to 7colonies among 25 selected colonies.On the other hand, a more rapid, specific and sensitive analytical molecular detection namely single colony PCR was also performed targeting hha gene to sensitively detect E.coli, giving more accurate and time consuming identification of colonies considered presumptively as E.coli. Comparative methodologies, such as ultrafiltration and direct DNA extraction from membrane filters (MoBio, Grermany) were also applied; however, results were not as accurate as the membrane filtration, making it a technique of choice for the detection and enumeration of water coliforms, followed by sufficiently specific enzymatic confirmatory stage.

Keywords: coliform, cytochrome oxidase, hha primer, membrane filtration, single colony PCR

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Authors: Yu-Chieh Chen, Li-Yun Wang, Chi-Chih Chen, Huy Hùng Đào, Ya-Mei Chen, Ming-Chu Cheng, Wen-Bin Chung, Hso-Chi Chaung, Guan-Ming Ke

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Many recent researches have demonstrated that CD154, a protein primarily expressed on activated T cell molecules, has potentially acted as a molecular adjuvant to improve the immunogenicity of subunit vaccines against viral infections. Classical swine fever (CSF) affects the swine industry worldwide that is one of the most devastating and highly contagious pig diseases. It is listed by the World Organization for Animal Health (OIE) as an infectious animal disease that must be reported. Although pigs vaccinated with subunit vaccines can be differentially diagnosed from those infected animals, subunit vaccines usually need adjuvants to enhance and elicit immune responses. In this study, CD154 was linked with CSFV E2 sequences and then expressed in CHO cells to produce the fusion protein as E2-CD154. The porcine specific CpG adjuvant was also used in one of the formulations. The specific pathogen-free pigs (SPF) at the age of 4-week-old were randomly separated into four groups, vaccinated with E2-CpG, E2-CD154, E2-CD154-CpG or the commercial Bayovac® CSF-E2 vaccine and boosted two weeks after primary vaccination. The results showed that the percentages of CD4+ and CD4+IL2+ in peripheral blood mononuclear cells (PBMC) in E2-CD154 vaccinated piglets seven days after primary vaccination were gained by 1-5% relative to the control group. In addition, the percentages of CD4+IFNγ+ T cells had slightly edged up 0.1-0.3% compared with the control group. Also, increased E2-specific IFNγ levels had edged up CD4+CD8+ T cells found in E2-CD154 and E2-CD154-CpG groups, particularly in the E2-CD154-CpG group. These results implicate that CD154 may enhance cellular immunity and synergistically act with species-specific CpG adjuvant as a dual-phase adjuvant. Therefore, the CD154 may be beneficial as a promising adjuvant in subunit vaccines.

Keywords: CD154, CpG adjuvant, cellular immunity, subunit vaccine, pig

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Authors: Sundus Mona, Atif Adnan, Babar Ali, Fareeha Arshad, Allah Rakha

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Molecular diversity is the variation in the abundance of species. Forensic entomology is a neglected field in Pakistan. Insects collected from the crime scene should be handled by forensic entomologists who are currently virtually non-existent in Pakistan. Correct identification of insect specimen along with knowledge of their biodiversity can aid in solving many problems related to complicated forensic cases. Inadequate morphological identification and insufficient thermal biological studies limit the entomological utility in Forensic Medicine. Recently molecular identification of entomological evidence has gained attention globally. DNA barcoding is the latest and established method for species identification. Only proper identification can provide a precise estimation of postmortem intervals. Arthropods are known to be the first tourists scavenging on decomposing dead matter. The objective of the proposed study was to identify species by molecular techniques and analyze their phylogenetic importance with barcoded necrophagous insect species of early succession on human cadavers. Based upon this identification, the study outcomes will be the utilization of established DNA bar codes to identify carrion feeding insect species for concordant estimation of post mortem interval. A molecular identification method involving sequencing of a 658bp ‘barcode’ fragment of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene from collected specimens of unknown dipteral species from cadavers of Lahore was evaluated. Nucleotide sequence divergences were calculated using MEGA 7 and Arlequin, and a neighbor-joining phylogenetic tree was generated. Three species were identified, Chrysomya megacephala, Chrysomya saffranea, and Chrysomya rufifacies with low genetic diversity. The fixation index was 0.83992 that suggests a need for further studies to identify and classify forensically relevant insects in Pakistan. There is an exigency demand for further research especially when immature forms of arthropods are recovered from the crime scene.

Keywords: molecular diversity, DNA barcoding, species identification, forensically relevant

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Authors: Kyung-Soon Choi, Ji-Young Hwang, Young-Hee Pyo

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Vinegars have been used as an alternative remedy for treating gout, but the scientific basis remains to be elucidated. In this study, acetic acid fermentation was applied for the first time to Monascus-fermented soybeans to examine its effect on the bioactive components together with the xanthine oxidase inhibitory (XOI) activity of the soy vinegar. The content of total phenols (0.47~0.97 mg gallic acid equivalents/mL) and flavonoids (0.18~0.39 mg quercetin equivallents/mL) were spectrophotometrically determined, and the content of organic acid (10.22~59.76 mg/mL) and isoflavones (6.79~7.46 mg/mL) were determined using HPLC-UV. The analytical method for ubiquinones (0.079~0.276 μg/mL) employed saponification before solvent extraction and quantification using LC-MS. Soy vinegar also showed significant XOI (95.3%) after 20 days of acetic acid fermentation at 30 °C. The results suggest that soy vinegar has potential as a novel medicinal food.

Keywords: acetic acid fermentation, bioactive component, soy vinegar, xanthine oxidase inhibitory activity

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Authors: Seyhan Sahan-Firat, Meryem Temiz-Resitoglu, Demet Sinem Guden, Sefika Pinar Kucukkavruk, Bahar Tunctan, Ayse Nihal Sari, Zumrut Kocak

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We hypothesized that mTOR inhibition may prevent the multiple organ failures following severe multiple tissue injury associated with increased NADPH oxidase system activity occur in zymosan-induced systemic inflammation. Therefore, we investigated the role of mTOR in oxidative/nitrosative stress associated with increase in NADPH oxidase activity in zymosan-induced systemic inflammation model in rats. Male Wistar rats received saline (4 ml/kg, i.p.) and zymosan (500 mg/kg, i.p.) at time 0. Saline, or zymosan-treated rats were given rapamycin (1 mg/kg, i.p.) 1 h after saline or zymosan injections. Rats were sacrified 4 h after zymosan challenge and kidney, heart, thoracic aorta, and superior mesenteric artery were collected. NADPH oxidase activity, p22phox, gp91phox, and p47phox protein expression and nitrotyrosine levels were measured in tissue samples. Zymosan administration caused an increase in NADPH oxidase activity, p22phox, gp91phox, and p47phox protein expression and nitrotyrosine levels in kidney, heart, thoracic aorta, and superior mesenteric artery. These changes caused by zymosan reversed by rapamycin, a selective mTOR inhibitor. Rapamycin alone had no effect on the parameters measured. Our results demonstrated that zymosan-induced oxidative/nitrosative stress presumably due to enhanced activity of NADPH oxidase, expression of p22phox, gp91phox, and p47phox and production of peroxynitrite were mediated by mTOR. [This work was financially supported by Research Foundation of Mersin University (2016-2-AP3-1900)].

Keywords: oxidative stress, mTOR, nitrosative stress, zymosan

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Authors: Jung-Feng Hsieh, Chu Ze Chen

Abstract:

Curcumin is the main active compound of turmeric that can inhibit the activities of α-glucosidase and xanthine oxidase (XO). α-Glucosidase and XO inhibitors are widely used to treat patients with diabetes mellitus and gout, respectively; therefore, the objective of this research was to evaluate the inhibitory activities of curcumin and its analogs against α-glucosidase and XO. Our results demonstrated that CM-F had the strongest antioxidant activity with a half-maximal effective concentration (EC50) of 9.39 ± 0.16 μM, which was superior to vitamin E (EC50=17.03 ± 0.09 μM). CM-F also exhibited potent inhibitory activity against XO with an IC50 value of 6.14 ± 0.38 μM and enzyme kinetic results revealed competitive inhibition of XO. We also found that CM-1 and CM-2 inhibited α-glucosidase with IC50 values of 21.06 ± 0.92 μM and 5.95 ± 0.09 μM, respectively, and kinetic studies indicated that both CM-1 and CM-2 are mixed competitive inhibitors of α-glucosidase. Furthermore, docking simulation identified five hydrogen bonds between XO and CM-F; however, only one and two hydrogen bonds are involved in CM-1 and CM-2 binding to α-glucosidase, respectively. Accordingly, curcumin and its analogs have the potential to be used in the treatment of patients with diabetes mellitus and gout.

Keywords: curcumin, α-glucosidase, inhibitor, xanthine oxidase

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Authors: Othman Elmahdy Othman, Agnés Germot, Daniel Petit, Muhammad Khodary, Abderrahman Maftah

Abstract:

Recently, the control (D-loop) and cytochrome b (Cyt b) regions of mtDNA have received more attention due to their role in the genetic diversity and phylogenetic studies in different livestock which give important knowledge towards the genetic resource conservation. Studies based on sequencing of sheep mitochondrial DNA showed that there are five maternal lineages in the world for domestic sheep breeds; A, B, C, D and E. By using cytochrome B sequencing, we aimed to clarify the genetic affinities and phylogeny of six Egyptian sheep breeds. Blood samples were collected from 111 animals belonging to six Egyptian sheep breeds; Barki, Rahmani, Ossimi, Saidi, Sohagi and Fallahi. The total DNA was extracted and the specific primers were used for conventional PCR amplification of the cytochrome B region of mtDNA. PCR amplified products were purified and sequenced. The alignment of sequences was done using BioEdit software and DnaSP 5.00 software was used to identify the sequence variation and polymorphic sites in the aligned sequences. The result showed that the presence of 39 polymorphic sites leading to the formation of 29 haplotypes. The haplotype diversity in six tested breeds ranged from 0.643 in Rahmani breed to 0.871 in Barki breed. The lowest genetic distance was observed between Rahmani and Saidi (D: 1.436 and Dxy: 0.00127) while the highest distance was observed between Ossimi and Sohagi (D: 6.050 and Dxy: 0.00534). Neighbour-joining (Phylogeny) tree was constructed using Mega 5.0 software. The sequences of 111 analyzed samples were aligned with references sequences of different haplogroups; A, B, C, D and E. The phylogeny result showed the presence of four haplogroups; HapA, HapB, HapC and HapE in the examined samples whereas the haplogroup D was not found. The result showed that 88 out of 111 tested animals cluster with haplogroup B (79.28%), whereas 12 tested animals cluster with haplogroup A (10.81%), 10 animals cluster with haplogroup C (9.01%) and one animal belongs to haplogroup E (0.90%).

Keywords: phylogeny, genetic biodiversity, MtDNA, cytochrome B, Egyptian sheep

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Authors: Maria Ostroukhova, Zhanneta Zalutskaya, Elena Ermilova

Abstract:

The alternative oxidase (AOX) mediates cyanide-resistant respiration, which bypasses proton-pumping complexes III and IV of the cytochrome pathway to directly transfer electrons from reduced ubiquinone to molecular oxygen. In Chlamydomonas reinhardtii, AOX is a monomeric protein that is encoded by two genes of discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In C. reinhardtii, AOX2 was initially identified as one of constitutively low expressed genes. Like other photosynthetic organisms C. reinhardtii cells frequently experience periods of hypoxia. To examine AOX2 transcriptional regulation and role of AOX2 in hypoxia adaptation, real-time PCR analysis and artificial microRNA method were employed. Two experimental approaches have been used to induce the anoxic conditions: dark-anaerobic and light-anaerobic conditions. C. reinhardtii cells exposed to the oxygen deprivation have shown increased AOX2 mRNA levels. By contrast, AOX1 was not an anoxia-responsive gene. In C. reinhardtii, a subset of genes is regulated by transcription factor CRR1 in anaerobic conditions. Notable, the AOX2 promoter region contains the potential motif for CRR1 binding. Therefore, the role of CRR1 in the control of AOX2 transcription was tested. The CRR1-underexpressing strains, that were generated and characterized in this work, exhibited low levels of AOX2 transcripts under anoxic conditions. However, the transformants still slightly induced AOX2 gene expression in the darkness. These confirmed our suggestions that darkness is a regulatory stimulus for AOX genes in C. reinhardtii. Thus, other factors must contribute to AOX2 promoter activity under dark-anoxic conditions. Moreover, knock-down of CRR1 caused a complete reduction of AOX2 expression under light-anoxic conditions. These results indicate that (1) CRR1 is required for AOX2 expression during hypoxia, and (2) AOX2 gene is regulated by CRR1 together with yet-unknown regulatory factor(s). In addition, the AOX2-underexpressing strains were generated. The analysis of amiRNA-AOX2 strains suggested a role of this alternative oxidase in hypoxia adaptation of the alga. In conclusion, the results reported here show that C. reinhardtii AOX2 gene is stress inducible. CRR1 transcriptional factor is involved in the regulation of the AOX2 gene expression in the absence of oxygen. Moreover, AOX2 but not AOX1 functions under oxygen deprivation. This work was supported by Russian Science Foundation (research grant № 16-14-10004).

Keywords: alternative oxidase 2, artificial microRNA approach, chlamydomonas reinhardtii, hypoxia

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Authors: R. D. Esposito, V. M. Barberá, P. García Morales, P. Dorado Rodríguez, J. Sanz, M. Fuentes, D. Planes Meseguer, M. Saceda, L. Fernández Fornos, M. P. Ventero

Abstract:

Results obtained by our group on glioblastoma multiforme (GBM) primary cultures , show a dramatic potentiation of radiation effects when 2 units/ml of D-amino acid oxidase (DAO) enzyme are added, free or immobilized in magnetic nanoparticles, to irradiated samples just after the irradiation. Cell cultures were exposed to radiation doses of 7Gy and 15Gy of 6 MV photons from a clinical linear accelerator. At both doses, we observed a clear enhancing effect of radiation-induced damages due to the addition of DAO.

Keywords: D-amino Acid Oxidase (DAO) enzyme, magnetic particles, nanotechnology, radiation therapy enhancement

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Authors: Abdelbary Prince, Said Moussa, Hyungkyu Kim, Eman Gouda, Jin Han

Abstract:

We compared the dynamic alterations of mitochondrial proteome of control, ischemia-reperfusion (IR) and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 29 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For two of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included succinate dehydrogenase complex, Acyl-CoA dehydrogenase, carnitine acetyltransferase, dihydrolipoamide dehydrogenase, Atpase, ATP synthase, dihydrolipoamide succinyltransferase, ubiquinol-cytochrome c reductase, translation elongation factor, acyl-CoA dehydrogenase, actin alpha, succinyl-CoA Ligase, dihydrolipoamide S-succinyltransferase, citrate synthase, acetyl-Coenzyme A dehydrogenase, creatine kinase, isocitrate dehydrogenase, pyruvate dehydrogenase, prohibitin, NADH dehydrogenase (ubiquinone) Fe-S protein, enoyl Coenzyme A hydratase, superoxide dismutase [Mn], and 24-kDa subunit of complex I. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain, antioxidant enzyme system, and energy metabolism. The results provide clues as to the cardioprotective mechanism of ischemic preconditioning at the protein level and may serve as potential biomarkers for detection of ischemia-induced cardiac injury.

Keywords: ischemic preconditioning, mitochondria, proteome, cardioprotection

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Authors: Thi Thao Ninh, Yuri Trusov, José Ramón Botella

Abstract:

Heterotrimeric G proteins, comprised of three subunits, α, β and γ, are involved in signal transduction pathways that mediate a vast number of processes across the eukaryotic kingdom. 23 Gα subunits are present in humans whereas most plant genomes encode for only one canonical Gα. The disparity observed between Arabidopsis, rice, and maize Gα-deficient mutant phenotypes suggest that Gα functions have diversified between eudicots and monocots during evolution. Alternatively, since the only Gα mutations available in dicots have been produced in Arabidopsis, the possibility exists that this species might be an exception to the rule. In order to test this hypothesis, we studied the G protein α subunit (TGA1) in tomato. Four tga1 knockout lines were generated in tomato cultivar Moneymaker using CRISPR/Cas9. The tga1 mutants exhibit a number of auxin-related phenotypes including changes in leaf shape, reduced plant height, fruit size and number of seeds per fruit. In addition, tga1 mutants have increased sensitivity to abscisic acid during seed germination, reduced sensitivity to exogenous auxin during adventitious root formation from cotyledons and excised hypocotyl explants. Our results suggest that Gα mutant phenotypes in tomato are very similar to those observed in monocots, i.e. rice and maize, and cast doubts about the validity of using Arabidopsis as a model system for plant G protein studies.

Keywords: auxin-related phenotypes, CRISPR/Cas9, G protein α subunit, heterotrimeric G proteins, tomato

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Authors: Pawasuth Saengdee, Chamras Promptmas, Ting Zeng, Silke Leimkühler, Ulla Wollenberger

Abstract:

Sulfite has been used as a versatile preservative to limit the microbial growth and to control the taste in some food and beverage. However, it has been reported to cause a wide spectrum of severe adverse reactions. Therefore, it is important to determine the amount of sulfite in food and beverage to ensure consumer safety. An efficient electrocatalytic biosensor for sulfite detection was developed by immobilizing of human sulfite oxidase (hSO) on 3-aminoproplytriethoxysilane (APTES) modified indium tin oxide (ITO) electrode. Cyclic voltammetry was employed to investigate the electrochemical characteristics of the hSO modified ITO electrode for various pretreatment and binding conditions. Amperometry was also utilized to demonstrate the current responses of the sulfite sensor toward sodium sulfite in an aqueous solution at a potential of 0 V (vs. Ag/AgCl 1 M KCl). The proposed sulfite sensor has a linear range between 0.5 to 2 mM with a correlation coefficient 0.972. Then, the additional polymer layer of PVA was introduced to extend the linear range of sulfite sensor and protect the enzyme. The linear range of sulfite sensor with 5% coverage increases from 2.8 to 20 mM at a correlation coefficient of 0.983. In addition, the stability of sulfite sensor with 5% PVA coverage increases until 14 days when kept in 0.5 mM Tris-buffer, pH 7.0 at 4 8C. Therefore, this sensor could be applied for the detection of sulfite in the real sample, especially in food and beverage.

Keywords: sulfite oxidase, bioelectrocatalytsis, indium tin oxide, direct electrochemistry, sulfite sensor

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Authors: Bhavini Patel, Aslihan Ugun-Klusek, Ellen Billet

Abstract:

The two main contributing factors to familial and idiopathic form of Parkinson’s disease (PD) are oxidative stress and altered proteolysis. Monoamine oxidase-A (MAO-A) plays a significant role in redox homeostasis by producing reactive oxygen species (ROS) via deamination of for example, dopamine. The ROS generated induces chemical modification of proteins resulting in altered biological function. The ubiquitin-proteasome system, which consists of three different types or proteolytic activity, namely “chymotrypsin-like” activity (CLA), “trypsin-like” activity (TLA) and “post acidic-like” activity (PLA), is responsible for the degradation of ubiquitinated proteins. Defects in UPS are known to be strongly correlated to PD. Herein, the effect of ROS generated by MAO-A on proteasome activity and the effects of proteasome inhibition on MAO-A protein levels in WT, mock and MAO-A overexpressed (MAO-A+) SHSY5Y neuroblastoma cell lines were investigated. The data in this study report increased proteolytic activity when MAO-A protein levels are significantly increased, in particular CLA and PLA. Additionally, 20S proteasome inhibition induced a decrease in MAO-A levels in WT and mock cells in comparison to MAO-A+ cells in which 20S proteasome inhibition induced increased MAO-A levels to be further increased at 48 hours of inhibition. This study supports the fact that MAO-A could be a potential pharmaceutical target for neuronal protection as data suggests that endogenous MAO-A levels may be essential for modulating cell death and survival.

Keywords: monoamine oxidase, neurodegeneration, Parkinson's disease, proteasome

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