Search results for: low molecular methyl siloxanes

Authors: Olena Mayboroda, Angel Gonzalez Benito, Jonathan Sabate Del Rio, Marketa Svobodova, Sandra Julich, Herbert Tomaso, Ciara K. O'Sullivan, Ioanis Katakis

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DNA amplification is required for most molecular diagnostic applications but conventional PCR has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the Recombinase Polymerase Amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 hour at a constant temperature of 37oC. Single-stranded and double-stranded DNA sequences were detected achieving detection limits of 4.04*10-13 M and 3.14*10-16 M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.

Keywords: recombinase polymerase amplification, Yersinia pestis, solid-phase detection, ELONA

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Authors: Raja Chinnappan, Huda Alanazi, Shanmugam Easwaramoorthi, Tanveer Mir, Balamurugan Kanagasabai, Ahmed Yaqinuddin, Sandhanasamy Devanesan, Mohamad S. AlSalhi

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Serum albumin (SA) is a highly rich water-soluble protein in plasma. It is known to maintain the living organisms' health and help to maintain the proper liver function, kidney function, and plasma osmolality in the body. Low levels of serum albumin are an indication of liver failure and chronic hepatitis. Therefore, it is important to have a low-cost, accurate and rapid method. In this study, we designed a fluorescent probe, triphenylamine rhodanine-3-acetic acid (mRA), which triggers the fluorescence signal upon binding with serum albumin (SA). mRA is a bifunctional molecule with twisted intramolecular charge transfer (TICT)-induced emission characteristics. An aqueous solution of mRA has an insignificant fluorescence signal; however, when mRA binds to SA, it undergoes TICT and turns on the fluorescence emission. A SA dose-dependent fluorescence signal was performed, and the limit of detection was found to be less than ng/mL. The specific binding of SA was tested from the cross-reactivity study using similar structural or functional proteins.

Keywords: serum albumin, fluorescent sensing probe, liver diseases, twisted intramolecular charge transfer

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Authors: Marcela Parra-Vargas, Ana Sandoval-Rodriguez, Roberto Rodriguez-Echevarria, Jose Dominguez-Rosales, Juan Armendariz-Borunda

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Non-alcoholic fatty liver disease (NAFLD) is characterized by an excess of hepatic lipids, and it is to author’s best knowledge, the most prevalent chronic liver disorder. Anthocyanin-rich food consumption is linked to health benefits in metabolic disorders associated with obesity and NAFLD, although the precise functional role of anthocyanidin delphinidin (Dp) has yet to be established. The aim of this study was to investigate the effect of the Dp in NAFLD metabolic alterations by evaluating prevention or amelioration of hepatic lipid accumulation, as well as molecular mechanisms in two experimental obesity-related models of NALFD. In vitro: HepG2 cells were incubated with sodium palmitate (PA, 1 mM) to induce lipotoxic damage, and concomitantly treated with Dp (180 uM) for 24 h. Subsequently, total lipid accumulation was measured by colorimetric staining with Oil Red O, and total intrahepatic triglycerides were determined by an enzymatic assay. To assess molecular mechanisms, cells were pre-treated with PA for 24 h and then exposed to Dp for 1 h. In vivo: four-week-old male C57BL/6Nhsd mice were allocated in two main groups. Mice were fed with standard diet (control) or high-fat and high-carbohydrate diet (45% fat, HFD) for 16 wk to induce NAFLD. Then HFD was divided into subgroups: one treated orally with Dp (15 mg/kg bw, HFD-Dp) every day for 4 wk, while HFD group treated with vehicle (DMSO). Weight and fasting glucose were recorded weekly, while dietary ingestion was measured daily. Insulin tolerance test was performed at the end of treatment. Liver histology was evaluated with H&E and Masson’s trichrome stain. RT-PCR was used to evaluate gene expression and Western Blot to determine levels of protein in both experimental models. Parametric data were analyzed with one-way ANOVA and Tukey’s post-hoc test. Kruskal-Wallis and Mann-Whitney U test for non-parametric data, and P < 0.5 were considered significant. Dp prevented hepatic lipid accumulation by PA in HepG2 hepatocytes. Furthermore, Dp down-regulated gene expression of SREBP1c, FAS, and CPT1a without modifying AMPK phosphorylation levels. In vivo, Dp oral administration did not ameliorate lipid metabolic alterations raised by HFD. Adiposity, dietary ingestion, fasting glucose, and insulin sensitivity after Dp treatment remained similar to HFD group. Histological analysis showed hepatic damage in HFD groups and no differences between HFD and HFD-Dp groups were found. Hepatic gene expression of ACC and FAS were not altered by HFD. SREBP1c was similar in both HFD and HFD-Dp groups. No significant changes were observed in SREBP1c, ACC, and FAS adipose tissue gene expression by HFD or Dp treatment. Additionally, immunoblotting analysis revealed no changes in pathway SIRT1-LKB-AMPK and PPAR alpha by both HFD groups compared to control. In conclusion, the antioxidant Dp may provoke beneficial effects in the prevention of hepatic lipid accumulation. Nevertheless, the oral dose administrated in mice that simulated the total intake of anthocyanins consumed daily by humans has no effect as a treatment on hepatic lipid metabolic alterations and histological abnormalities associated with exposure to chronic HFD. A healthy lifestyle with regular intake of antioxidants such as anthocyanins may prevent metabolic alterations in NAFLD.

Keywords: anthocyanins, antioxidants, delphinidin, non-alcoholic fatty liver disease, obesity

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Authors: Mir Hussain Nawaz, Lyudmila Nedyalkova, Haizhong Zhu, Wael M. Rabeh

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In this study, we aim to biochemically and structurally characterize the interactions of human HK2 with the mitochondria in addition to the role of its N-terminal domain in catalysis and stability of the full-length enzyme. Here, we solved the crystal structure of human HK2 in complex with glucose and glucose-6-phosphate (PDB code: 2NZT), where it is a homodimer with catalytically active N- and C-terminal domains linked by a seven-turn α-helix. Different from the inactive N-terminal domains of isozymes 1 and 3, the N- domain of HK2 not only capable to catalyze a reaction but it is responsible for the thermodynamic stabilizes of the full-length enzyme. Deletion of first α-helix of the N-domain that binds to the mitochondria altered the stability and catalytic activity of the full-length HK2. In addition, we found the linker helix between the N- and C-terminal domains to play an important role in controlling the catalytic activity of the N-terminal domain. HK2 is a major step in the regulation of glucose metabolism in cancer making it an ideal target for the development of new anticancer therapeutics. Characterizing the structural and molecular mechanisms of human HK2 and its role in cancer metabolism will accelerate the design and development of new cancer therapeutics that are safe and cancer specific.

Keywords: cancer metabolism, enzymology, drug discovery, protein stability

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Authors: L. Bouzina, A. Bensafi, M. Duval, C. Mathis, M. Rawiso

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We study the stability and structural properties of mixtures of model nanoparticles and non-adsorbing polymers in the 'protein limit', where the size of polymers exceeds the particle size substantially. We have synthesized in institute (Charles Sadron Strasbourg) model nanoparticles by coating fullerene C60 molecules with low molecular weight polystyrene (PS) chains (6 PS chains with a degree of polymerization close to 25 and 50 are grafted on each fullerene C60 molecule. We will present a Small Angle Neutron scattering (SANS) study of Tetrahydrofuran (THF) solutions involving long polystyrene (PS) chains and fullerene (C60) nanoparticles. Long PS chains and C60 nanoparticles with different arm lengths were synthesized either hydrogenated or deuteriated. They were characterized through Size Exclusion Chromatography (SEC) and Quasielastic Light Scattering (QLS). In this way, the solubility of the C60 nanoparticles in the usual good solvents of PS was controlled. SANS experiments were performed by use of the contrast variation method in order to measure the partial scattering functions related to both components. They allow us to obtain information about the dispersion state of the C60 nanoparticles as well as the average conformation of the long PS chains. Specifically, they show that the addition of long polymer chains leads to the existence of an additional attractive interaction in between soft nanoparticles.

Keywords: fulleren nanoparticles, polymer, small angle neutron scattering, solubility

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Authors: M. Doudi, M. H. Pazandeh, L. Rahimzadeh Torabi

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Bedsores are pressure ulcers that occur on the skin or tissue due to being immobile and lying in bed for extended periods. Bedsores have the potential to progress into open ulcers, increasing the possibility of variety of bacterial infection. Acinetobacter baumannii, a pathogen of considerable clinical importance, exhibited a significant correlation with Bedsores (pressure ulcers) infections, thereby manifesting a wide spectrum of antibiotic resistance. The emergence of drug resistance has led researchers to focus on alternative methods, particularly phage therapy, for tackling bacterial infections. Phage therapy has emerged as a novel therapeutic approach to regulate the activity of these agents. The management of bacterial infections greatly benefits from the clinical utilization of bacteriophages as a valuable antimicrobial intervention. The primary objective of this investigation consisted of isolating and discerning potent bacteriophage capable of targeting multi drug-resistant (MDR) and extensively drug-resistant (XDR) bacteria obtained from pressure ulcers. In present study, analyzed and isolated A. baumannii strains obtained from a cohort of patients suffering from pressure ulcers at Taleghani Hospital in Ahvaz, Iran. An approach that included biochemical and molecular identification techniques was used to determine the taxonomic classification of bacterial isolates at the genus and species levels. The molecular identification process was facilitated by using the 16S rRNA gene in combination with universal primers 27 F, and 1492 R. Bacteriophage was obtained through the isolation process conducted on treatment plant sewage located in Isfahan, Iran. The main goal of this study was to evaluate different characteristics of phage, such as their appearance, range of hosts they can infect, how quickly they can enter a host, their stability at varying temperatures and pH levels, their effectiveness in killing bacteria, the growth pattern of a single phage stage, mapping of enzymatic digestion, and identification of proteomics patterns. The findings demonstrated that an examination was conducted on a sample of 50 specimens, wherein 15 instances of A. baumannii were identified. These microorganisms are the predominant Gram-negative agents known to cause wound infections in individuals suffering from bedsores. The study's findings indicated a high prevalence of antibiotic resistance in the strains isolated from pressure ulcers, excluding the clinical strains that exhibited responsiveness to colistin.According to the findings obtained from assessments of host range and morphological characteristics of bacteriophage VbɸAB-1, it can be concluded that this phage possesses specificity towards A. Baumannii BAH_Glau1001 was classified as a member of the Plasmaviridae family. The bacteriophage mentioned earlier showed the strongest antibacterial effect at a temperature of 18 °C and a pH of 6.5. Through the utilization of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on protein fragments, it was established that the bacteriophage VbɸAB-1 exhibited a size range between 50 and 75 kilodaltons (KDa). The numerous research findings on the effectiveness of phages and the safety studies conducted suggest that the phages studied in this research can be considered as a practical solution and recommended approach for controlling and treating stubborn pathogens in burn wounds among hospitalized patients.

Keywords: acinetobacter baumannii, extremely drug- resistant, phage therapy, surgery wound

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Authors: Prabhat Kashyap, Krishan Kumar

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Delhi, the capital of India, is one of the most populated and polluted city among the world. In terms of air quality, Delhi’s air is degrading day by day & becomes worst of any major city in the world. The role of biogenic volatile organic compounds (BVOCs) is not much studied in cities like Delhi as a culprit for degraded air quality. They not only play a critical role in rural areas but also determine the atmospheric chemistry of urban areas as well. Particularly, Isoprene (2-methyl 1,3-butadiene, C5H8) is the single largest emitted compound among other BVOCs globally, that influence the tropospheric ozone chemistry in urban environment as the ozone forming potential of isoprene is very high. It is mainly emitted by vegetation & a small but significant portion is also released by vehicular exhaust of petrol operated vehicles. This study investigates the spatial and temporal variations of quantitative measurements of isoprene emissions along with different traffic tracers in 2 different seasons (post-monsoon & winter) at four different locations of Delhi. For the quantification of anthropogenic and biogenic isoprene, two sites from traffic intersections (Punjabi Bagh & CRRI) and two sites from vegetative locations (JNU & Yamuna Biodiversity Park) were selected in the vicinity of isoprene emitting tree species like Ficus religiosa, Dalbergia sissoo, Eucalyptus species etc. The concentrations of traffic tracers like benzene, toluene were also determined & their robust ratios with isoprene were used to differentiate anthropogenic isoprene with biogenic portion at each site. The ozone forming potential (OFP) of all selected species along with isoprene was also estimated. For collection of intra-day samples (3 times a day) in each season, a pre-conditioned fenceline monitoring (FLM) carbopack X thermal desorption tubes were used and further analysis was done with Gas chromatography attached with mass spectrometry (GC-MS). The results of the study proposed that the ambient air isoprene is always higher in post-monsoon season as compared to winter season at all the sites because of high temperature & intense sunlight. The maximum isoprene emission flux was always observed during afternoon hours in both seasons at all sites. The maximum isoprene concentration was found to be 13.95 ppbv at Biodiversity Park during afternoon time in post monsoon season while the lower concentration was observed as low as 0.07 ppbv at the same location during morning hours in winter season. OFP of isoprene at vegetation sites is very high during post-monsoon because of high concentrations. However, OFP for other traffic tracers were high during winter seasons & at traffic locations. Furthermore, high correlation between isoprene emissions with traffic volume at traffic sites revealed that a noteworthy share of its emission also originates from road traffic.

Keywords: biogenic VOCs, isoprene emission, anthropogenic isoprene, urban vegetation

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Authors: Dawit Gebreegzabher Hagos, Yazezew Kebede Kiro, Mahmud Abdulkader, Henk H. D. F. Schallig, Dawit Wolday

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Rapid and accurate visceral leishmaniasis (VL) diagnosis is needed to initiate prompt treatment to reduce morbidity and mortality. Here, we evaluated the performance of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of VL from blood in an endemic area in Ethiopia. LAMP was positive in 117/122 confirmed VL cases and negative in 149/152 controls, resulting in a sensitivity of 95.9% (95% CI: 90.69–98.66) and a specificity of 98.0% (95% CI: 94.34–99.59), respectively. The sensitivity of the LAMP assay was 95.0% (95% CI: 88.61–98.34) in HIV-negatives and 100% (95% CI: 85.18–100.0) in HIV-positives. Compared with microscopy, LAMP detected 82/87 (94.3%, 95% CI: 87.10–98.11) of the microscopy1 cases and was negative in 11/27 (40.7%, 95% CI: 22.39–61.20) of the microscopy2 cases. Compared with the rK39 serology, LAMP detected 113/120 (94.2%, 95% CI: 88.35–97.62) of the rK391 cases and was negative in 149/154 (96.8%, 95% CI: 92.59–98.94) of the rK392 cases. However, when compared with microscopy only, rK39 detected 83/87 (95.4%, 95% CI: 88.64–98.73) of the microscopy1 cases and negative in only 12/27 (44.4%, 95% CI: 25.48–64.67) of the microscopy– cases. There was an excellent agreement between rK39 and LAMP (Kappa 5 0.91, 95% CI: 0.86–0.96). Furthermore, an algorithm using rK39 followed by LAMP would yield a sensitivity of 99.2% (95%CI: 95.52–99.89) and a specificity of 98.0% (95% CI: 94.34–99.59). The findings demonstrate that the LAMP assay is an accurate and rapid molecular assay for VL diagnosis, including in HIV-1 co-infected patients, in an endemic setting.

Keywords: visceral leishmaniasis, HIV, diagnosis, LAMP, Ethiopia

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Authors: Rajan Vohra, Ravinder Singh Sawhney, Kunwar Partap Singh

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Electrical charge transport through two basic strands thymine and adenine of DNA have been investigated and analyzed using the jellium model approach. The FFT-2D computations have been performed for semi-empirical Extended Huckel Theory using atomistic tool kit to contemplate the charge transport metrics like current and conductance. The envisaged data is further evaluated in terms of transmission spectrum, HOMO-LUMO Gap and number of electrons. We have scrutinized the behavior of the devices in the range of -2V to 2V for a step size of 0.2V. We observe that both thymine and adenine can act as molecular devices when sandwiched between two gold probes. A prominent observation is a drop in HLGs of adenine and thymine when working as a device as compared to their intrinsic values and this is comparative more visible in case of adenine. The current in the thymine based device exhibit linear increase with voltage in spite of having low conductance. Further, the broader transmission peaks represent the strong coupling of electrodes to the scattering molecule (thymine). Moreover, the observed current in case of thymine is almost 3-4 times than that of observed for adenine. The NDR effect has been perceived in case of adenine based device for higher bias voltages and can be utilized in various future electronics applications.

Keywords: adenine, DNA, extended Huckel, thymine, transmission spectra

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Authors: Jyothi Nagraj, Sudeshna Mukherjee, Rajdeep Chowdhury

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Autophagy has recently been linked with cancer cell survival post drug insult contributing to acquisition of resistance. However, the molecular signaling governing autophagic survival response is poorly explored. In our study, in osteosarcoma (OS) cells cisplatin shock was found to activate both MAPK and autophagy signaling. An activation of JNK and autophagy acted as pro-survival strategy, while ERK1/2 triggered apoptotic signals upon cisplatin stress. An increased sensitivity of the cells to cisplatin was obtained with simultaneous inhibition of both autophagy and JNK pathway. Furthermore, we observed that the autophagic stimulation upon drug stress regulates other developmentally active signaling pathways like the Hippo pathway in OS cells. Cisplatin resistant cells were thereafter developed by repetitive drug exposure followed by clonal selection. Basal levels of autophagy were found to be high in resistant cells to. However, the signaling mechanism leading to autophagic up-regulation and its regulatory effect differed in OS cells upon attaining drug resistance. Our results provide valuable clues to regulatory dynamics of autophagy that can be considered for development of improved therapeutic strategy against resistant type cancers.

Keywords: JNK, autophagy, drug resistance, cancer

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Authors: Ching Shya Lee, Mohamed Kheireddine Aroua, Wan Mohd Ashri Wan Daud, Patrick Cognet, Yolande Peres, Mohammed Ajeel

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In recent years, with the decreasing supply of fossil fuel, renewable energy has received a significant demand. Biodiesel which is well known as vegetable oil based fatty acid methyl ester is an alternative fuel for diesel. It can be produced from transesterification of vegetable oils, such as palm oil, sunflower oil, rapeseed oil, etc., with methanol. During the transesterification process, crude glycerol is formed as a by-product, resulting in 10% wt of the total biodiesel production. To date, due to the fast growing of biodiesel production in worldwide, the crude glycerol supply has also increased rapidly and resulted in a significant price drop for glycerol. Therefore, extensive research has been developed to use glycerol as feedstock to produce various added-value chemicals, such as tartronic acid, mesoxalic acid, glycolic acid, glyceric acid, propanediol, acrolein etc. The industrial processes that usually involved are selective oxidation, biofermentation, esterification, and hydrolysis. However, the conversion of glycerol into added-value compounds by electrochemical approach is rarely discussed. Currently, the approach is mainly focused on the electro-oxidation study of glycerol under potentiostatic mode for cogenerating energy with other chemicals. The electro-organic synthesis study from glycerol under galvanostatic mode is seldom reviewed. In this study, the glycerol was converted into various added-value compounds by electrochemical method under galvanostatic mode. This work aimed to study the possible compounds produced from glycerol by electrochemical technique in a one-pot electrolysis cell. The electro-organic synthesis study from glycerol was carried out in a single compartment reactor for 8 hours, over the platinum cathode and anode electrodes under acidic condition. Various parameters such as electric current (1.0 A to 3.0 A) and reaction temperature (27 °C to 80 °C) were evaluated. The products obtained were characterized by using gas chromatography-mass spectroscopy equipped with an aqueous-stable polyethylene glycol stationary phase column. Under the optimized reaction condition, the glycerol conversion achieved as high as 95%. The glycerol was successfully converted into various added-value chemicals such as ethylene glycol, glycolic acid, glyceric acid, acetaldehyde, formic acid, and glyceraldehyde; given the yield of 1%, 45%, 27%, 4%, 0.7% and 5%, respectively. Based on the products obtained from this study, the reaction mechanism of this process is proposed. In conclusion, this study has successfully converted glycerol into a wide variety of added-value compounds. These chemicals are found to have high market value; they can be used in the pharmaceutical, food and cosmetic industries. This study effectively opens a new approach for the electrochemical conversion of glycerol. For further enhancement on the product selectivity, electrode material is an important parameter to be considered.

Keywords: biodiesel, glycerol, electrochemical conversion, galvanostatic mode

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Authors: Maryam Vaezjalali, Koroush Rahimian, Maryam Asli, Tahmineh Kandelouei, Foad Davoodbeglou, Amir H. Kashi

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Objectives: The present study was conducted to assess the HBV molecular profiles including genotypes, subgenotypes, subtypes & mutations in hepatitis B genes. Materials/Patients and Methods: This study was conducted on 229 intravenous drug users who referred to three Drop- in-Centers and a hospital in Tehran. HBV DNA was extracted from HBsAg positive serum samples and amplified by Nested PCR. HBV genotype, subgenotypes, subtype and genes mutation were determined by direct sequencing. Phylogenetic tree was constructed using neighbor- joining (NJ) method. Statistical analyses were carried out by SPSS 20. Results: HBV DNA was found in 3 HBsAg positive cases. Phylogenetic tree of derived HBV DNAs showed the existence of genotype D (subgenotype D1, subtype ayw2). Also immune escape mutations were determined in S gene. Conclusion: There were a few variations and genotypes and subtypes among infected intravenous drug users. This study showed the predominance of genotype D among intravenous drug users. Our study concurs with other reports from Iran, that all showing currently only genotype D is the only detectable genotype in Iran.

Keywords: drug users, genotype, HBV, phylogenetic tree

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Authors: Selena Gutiérrez, Araceli Martínez

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One of the most important challenges worldwide, scientific and technological, is to have a sustainable energy source; friendly to the environment and widely available. Currently, the 85% of the energy used comes from the fossil sources. Another important environmental problem is that several rubber products (tires, gloves, hoses, among others) are discarded practically without any treatment. In nature, the degradation of such products will take at least 500 years. In 2009, the worldwide rubber production was about 23.6 million tons. In order to solve this problems, our research focus in an alternative synthesis of biofuels in a two-step approach: The metathesis degradation of industrial rubber (models of rubber waste), and the oligomers transesterification. Thus, cis-1,4-polybutadiene (Mn= 9.1x105, Mw/Mn= 2.2) and styrene-butadiene block copolymers with 30% (Mn= 1.61x105; Mw/Mn= 1.3) and 21% wt styrene (Mn= 1.92x105; Mw/Mn= 1.4) were degraded via metathesis with soybean oil as chain transfer agent (CTA) and green solvent; using [(PCy3)2Cl2Ru=CHPh] and [(1,3-diphenyl-4,5-dihydroimidazol-2-ylidene)(PCy3)Ru=CHPh] catalysts. Afterwards, the products were transesterified by basic homogeneous catalysis. Before transesterification, the polystyrene microblocks (Mn= 16,761; Mw/Mn= 1.2) were isolated. Finally, the biofuels obtained (BO) were purified, characterized and showed similar properties to standards biodiesel (SB) (Norms: EN 14214-03 and ASTM D6751-02), i.e. (SB / BO): molecular weight [Daltons] (570 / 543-596), density [g/cm3] (0.86-0.90 / 0.88), kinematic viscosity [mm2/s] (1.90-6.0 / 3.5-4.5), iodine (97 / 97-98) and cetane number (Min.47 / 56-58).

Keywords: biofuels, industrial rubber, metathesis, vegetable oils

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Authors: Patsarawadee Paojinda, Waranya Imprasittichai, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

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Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, can be stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to 3 orders of magnitude, while the Km was not much affected and remained at low µM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivaties and metabolic functions on molecular evolution.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

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Authors: Renu Yadav, Sanjeev Kumar

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In northern India, chickpea (Cicer arietinum L.) come across with terminal high-temperature stress during reproductive stage which leads to reduced yield. Hence, stable production of chickpea will depend on the development of new methods like ‘priming’ which allow improved adaptation to the drought and heat stress. In the present experiment, 11-day chickpea seedling was primed with mild drought stress and put on recovery stage by irrigating and finally 30-day seedlings were exposed to heat stress 38°C (4 hours), 35°C (8 hours) and 32°C (12 hours). To study the effect of combinatorial stress, heat and drought stress was applied simultaneously. Analyses of various physiological parameters like membrane damage assay, photosynthetic pigments, antioxidative enzyme, total sugars were estimated at all stages. To study the effect of heat stress on the metabolites of the plants, GC-MS and HPLC were performed, while at transcriptional level Real-Time PCR of predicted heat stress-related genes was done. It was concluded that the heat stress significantly affected the chickpea plant at physiological and molecular level in all the five varieties. Results also show less damaging effect in primed plants by increasing the activity of antioxidative enzymes and increased expression of heat shock proteins and heat shock factors.

Keywords: chickpea, combinatorial stress, heat stress, oxidative stress, priming, RT-PCR

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Authors: Nermin Isik, Ozlem Derinbay Ekici

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Bovine coccidiosis is a protozoan disease caused by various species of Eimeria and most signs of disease are chronic or subclinical. The aim of this study was to determine the prevalence of Eimeria spp. in calves in Konya, in Turkey. The study, conducted from January- February 2015, involved 240 faecal samples of calves in the age groups of <1 month, 1-3 months and >3 months in Konya city centre, in Turkey. In a retrospective study from these faecal samples of calves submitted to the University of Selcuk, Faculty of Veterinary Medicine, Laboratory of Parasitology were evaluated regarding the prevalence of Eimeria spp. Faecal samples were examined by Fulleborn saturated salt floatation technique. Eimeria oocysts were found in 8.33% of all samples. The positivity rates in each of the age groups were different. According to the age groups (<1 month, 1-3 months and >3 months), the Eimeria spp. were determined as 0.83, 22.73 and 7.41%, respectively. After examination of stool, detected oocysts were sporulated in 2.5% potassium dichromate at 22º C and species were identified as E. cylindrica, E. zuernii, E. ellipsoidalis, E. subspherica, E. bovis, E. auburnensis, E. canadensis, E. illinoisensis and E. brasiliensis in infected calves. In conclusion, the highest prevalence was observed in the age group of 1-3 months. The presence of Eimeria species in calves demonstrated for the first time in the Konya region in Turkey. Other etiologic agents should also be investigated in calves more seriously. Further molecular epidemiological studies should be performed in this community.

Keywords: Eimeria spp., calves, diarrhea, bovine coccidiosis

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Authors: Taehoon Kwon, Hyeongrae Cho, Dirk Henkensmeier, Youngjong Kang, Chong Min Koo

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Ionic polymer actuators have been of interest in the bio-inspired artificial muscle devices. However, the relatively slow response and low power density were the obstacles for practical applications. In this study, ionic polymer actuators are fabricated with ionic polymer composite membranes based on sulfonated poly(arylene ether sulfone) (SPAES) and copper(II) phthalocyanine tetrasulfonic acid (CuPCSA). CuPCSA is an organic filler with very high ion exchange capacity (IEC, 4.5 mmol H+/g) that can be homogeneously dispersed on the molecular scale into the SPAES membrane. SPAES/CuPCSA actuators show larger ionic conductivity, mechanical properties, bending deformation, exceptional faster response to electrical stimuli, and larger mechanical power density (3028 W m–3) than Nafion actuators. This outstanding actuation performance of SPAES/CuPCSA composite membrane actuators makes them attractive for next generation transducers with high power density, which are currently developed biomimetic devices such as endoscopic surgery.

Keywords: actuation performance, composite membranes, ionic polymer actuators, organic filler

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Authors: Mondal Indranil, Bhakat Debjyoti, Mukhopadayay Asish K., Chatterjee Nabendu S.

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CS6 is the prevalent CF in our region and deciphering its molecular regulators would play a pivotal role in reducing the burden of ETEC pathogenesis. In prokaryotes, most of the genes are under the control of one operon and the promoter present upstream of the gene regulates the transcription of that gene. Here the promoter of CS6 was characterized by computational method and further analyzed by β-galactosidase assay and sequencing. Promoter constructs and deletions were prepared as required to analyze promoter activity. The effect of different additives on the CS6 promoter was analysed by the β-galactosidase assay. Bioinformatics analysis done by Softberry/BPROM predicted fur, lrp, and crp boxes, -10 and -35 region upstream of the CS6 gene. The promoter construction in no promoter plasmid pTL61T showed that region -573 to +1 is actually the promoter region as predicted. Sequential deletion of the region upstream of CS6 revealed that promoter activity remains the same when -573bp to -350bp is deleted. But after the deletion of the upstream region -350 bp to -255bp, promoter expression decreases drastically to 26%. Further deletion also decreases promoter activity up to a little range. So the region -355bp to -255bp holds the promoter sequence for the CS6 gene. Additives like iron, NaCl, etc., modulate promoter activity in a dose-dependent manner. From the promoter analysis, it can be said that the minimum region lies between -254 and +1. Important region(s) lies between -350 bp to -255 bp upstream in the promoter, which might have important elements needed to control CS6 gene expression.

Keywords: microbiology, promoter, colonization factor, ETEC

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Authors: Hyun Joon Chang, Jae In Kim, Sungsoo Na

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Kinesin-1 (hereafter called kinesin) is a molecular motor protein that moves cargos toward the end of microtubules using the energy of adenosine triphosphate (ATP) hydrolysis. When kinesin is inactive, its tail autoinhibits the motor chain in order to prevent from reacting with the ATP by cross-linking of the tail domain to the motor domains at two positions. However, the morphological study of kinesin during autoinhibition is yet remained obscured. In this study, we report the effect of the binding site of the tail domain using the normal mode analysis of the elastic network model on kinesin in the tail-free form and tail-bind form. Considering the relationship between the connectivity of conventional network model with respect to the cutoff length and the functionality of the binding site of the tail, we revaluated the network model to observe the key role of the tail domain in its structural aspect. Contingent on the existence of the tail domain, the results suggest the morphological stability of the motor domain. Furthermore, employing the results from normal mode analysis, we have determined the strain energy of the neck linker, an essential portion of the motor domain for ATP hydrolysis. The results of the neck linker also converge to the same indication, i.e. the morphological analysis of the motor domain.

Keywords: elastic network model, Kinesin-1, autoinhibition

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Authors: Yomna Ibrahim El-Gazzar, Hussien Ibrahim El-Subbagh, Hanan Hanaa Georgey, Ghada S. Hassan Hassan

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Dihydrofolate reductase (DHFR) is an enzyme that has pivotal importance in biochemistry and medicinal chemistry. It catalyzes the reduction of dihydrofolate to tetrahydrofolate and intimately couples with thymidylate synthase. Thymidylate synthase is a crucial enzyme that catalyzes the reductive methylation of (dUMP) to (dTMP) utilizing N5, N10-methylenetetrahydrofolate as a cofactor. A new series of 2-substituted thio-quinazolin-4-one analogs was designed that possessed electron withdrawing or donating functional groups (Cl or OCH3) at position 6- or 7-, 4-methoxyphenyl function at position 3-.The thiol function is used to connect to either 1,2,4-triazole, or 1,3,4-thiadiazole via a methylene bridge. Most of the functional groups designed to be accommodated on the quinazoline ring such as thioether, alkyl to increase lipid solubility of polar compounds, a character very much needed in the nonclassical DHFR inhibitors. The target compounds were verified with spectral data and elemental analysis. DHFR inhibitions, as well as antitumor activity, were applied on three cell lines (MCF-7, CACO-2, HEPG-2).

Keywords: nonclassical antifolates, DHFR Inhibitors, antitumor activity, quinazoline ring

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Authors: Yahia I. Mohamed, Ahmed I. Marzouk, Mohamed A. Yacout

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Aim of this work was to study the genetic basis for oil accumulation in olive fruit via tracking DGAT2 (Diacylglycerol acyltransferase type-2) gene in three Egyptian Origen Olive cultivars namely Toffahi, Hamed and Maraki using molecular marker techniques and bioinformatics tools. Results illustrate that, firstly: specific genomic band of Maraki cultivars was identified as DGAT2 (Diacylglycerol acyltransferase type-2) and identical for this gene in Olea europaea with 100 % of similarity. Secondly, differential genomic band of Maraki cultivars which produced from RAPD fingerprinting technique reflected predicted distinguished sequence which identified as DGAT2 (Diacylglycerol acyltransferase type-2) in Fragaria vesca subsp. Vesca with 76% of sequential similarity. Third and finally, specific genomic specific band of Hamed cultivars was indentified as two fragments, 1-Olea europaea cultivar Koroneiki diacylglycerol acyltransferase type 2 mRNA, complete cds with two matches regions with 99% or 2-PREDICTED: Fragaria vesca subsp. vesca diacylglycerol O-acyltransferase 2-like (LOC101313050), mRNA with 86% of similarity.

Keywords: Olea europaea, fingerprinting, diacylglycerol acyltransferase type-2 (DGAT2), Egypt

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Authors: Hiroyuki Aoki

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Molecular imaging has attracted much attention recently, which visualizes biological molecules, cells, tissue, and so on. Among various in vivo imaging techniques, the fluorescence imaging method has been widely employed as a useful modality for small animals in pre-clinical researches. However, the higher signal intensity is needed for highly sensitive in vivo imaging. The objective of the current study is the development of a fluorescent imaging agent with high brightness for the tumor imaging of a mouse. The strategy to enhance the fluorescence signal of a bio-imaging agent is the increase of the absorption of the excitation light and the fluorescence conversion efficiency. We developed a nano-particle fluorescence imaging agent consisting of a π-conjugated polymer emitting a fluorescence signal in a near infrared region. A large absorption coefficient and high emission intensity at a near infrared optical window for biological tissue enabled highly sensitive in vivo imaging with a tumor-targeting ability by an EPR (enhanced permeation and retention) effect. The signal intensity from the π-conjugated fluorescence imaging agent is larger by two orders of magnitude compared to a quantum dot, which has been known as the brightest imaging agent. The π-conjugated polymer nano-particle would be a promising candidate in the in vivo imaging of small animals.

Keywords: fluorescence, conjugated polymer, in vivo imaging, nano-particle, near-infrared

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Authors: R. Pérez Castells, J. M. Carbajo

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Sepiolite is used as a processing aid in the manufacture of fibre cement from the start of the replacement of asbestos in the 80s. Sepiolite increases the inter-laminar bond between cement layers and improves homogeneity of the slurries. A new type of sepiolite processed product, Wollatrop TF/C, has been checked as a retention agent for fine particles in the production of fibre cement in a Hatschek machine. The effect of Wollatrop T/FC on filtering and fine particle losses was studied as well as the interaction with anionic polyacrylamide and microsilica. The design of the experiments were factorial and the VDT equipment used for measuring retention and drainage was modified Rapid Köethen laboratory sheet former. Wollatrop TF/C increased the fine particle retention improving the economy of the process and reducing the accumulation of solids in recycled process water. At the same time, drainage time increased sharply at high concentration, however drainage time can be improved by adjusting APAM concentration. Wollatrop TF/C and microsilica are having very small interactions among them. Microsilica does not control fine particle losses while Wollatrop TF/C does efficiently. Further research on APAM type (molecular weight and anionic character) is advisable to improve drainage.

Keywords: drainage, fibre-reinforced cement, fine particle losses, flocculation, microsilica, sepiolite

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Authors: Sangram Shamrao Patil, Hara Mohan Jena

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Phthalates are among the most common organic pollutant since they have become widespread in the environment and found in sediments, natural waters, soils, plants, landfill leachates, biota including human tissue and aquatic organisms. Diethyl phthalate (DEP) is a low molecular weight phthalate which has wide applications as plasticizer and become a major cause of environmental pollution. Environmental protection agency (EPA) listed DEP as priority pollutant because of its toxicity and they recommended human health ambient water quality criterion for diethyl phthalate (DEP) as 4 mg/l. Therefore, wastes containing phthalates require proper treatment before being discharged into the environment. Biodegradation is attractive and efficient treatment method as it is cost effective and produces non-toxic end products. In the present study, a DEP degrading aerobic bacterium was isolated from soil contaminated with plastic industry wastewater. Morphological and biochemical characteristics of isolate were performed. 16S rRNA sequencing and phylogenetic analysis of isolate was carried out and it was identified as Empedobacter brevis. Isolate has been found to tolerate up to 1650 mg/l of DEP. This study will be significant for exploring an application of microbes for remediation of phthalates and development of a suitable bioreactor.

Keywords: diethyl phthalate, plasticizer, pollutant, biodegradation

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Authors: Banu Aytül Ekmekçi

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Salinity stress has negative effects on agricultural yield throughout the world, affecting production whether it is for subsistence or economic gain. This study investigates the inductive role of vitamin C and its application mode in mitigating the detrimental effects of irrigation with diluted (10, 20 and 30 %) NaCl + water on carthamus tinctorius plants. The results show that 10% of salt water exhibited insignificant changes, while the higher levels impaired growth by reducing seed germination, dry weights of shoot and root, water status and chlorophyll contents. However, irrigation with salt water enhanced carotenoids and antioxidant enzyme activities. The detrimental effects of salt water were ameliorated by application of 100 ppm ascorbic acid (vitamin C). The inductive role of vitamin was associated with the improvement of seed germination, growth, plant water status, carotenoids, endogenous ascorbic acid and antioxidant enzyme activities. Moreover, vitamin C alone or in combination with 30% NaCl water increased the intensity of protein bands as well as synthesized additional new proteins with molecular weights of 205, 87, 84, 65 and 45 kDa. This could increase tolerance mechanisms of treated plants towards water salinity.

Keywords: salinity, stress, vitamin c, antioxidant, NaCl, enzyme

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Authors: Jingyuan Hu, Zhandong Liu

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CRISPR genome editing technology has transformed molecular biology by accurately targeting and altering an organism’s DNA. Despite the state-of-art precision of CRISPR genome editing, the imprecise mutation outcome and off-target effects present considerable risk, potentially leading to unintended genetic changes. Targeted deep sequencing, combined with bioinformatics sequence alignment, can detect such unwanted mutations. Nevertheless, the classical method, Needleman-Wunsch (NW) algorithm may produce false alignment outcomes, resulting in inaccurate mutation identification. The key to precisely identifying CRISPR-induced mutations lies in determining optimal parameters for the sequence alignment algorithm. Hidden Markov models (HMM) are ideally suited for this task, offering flexibility across CRISPR systems by leveraging forward-backward algorithms for parameter estimation. In this study, we introduce CRISPR-HMM, a statistical software to precisely call CRISPR-induced mutations. We demonstrate that the software significantly improves precision in identifying CRISPR-induced mutations compared to NW-based alignment, thereby enhancing the overall understanding of the CRISPR gene-editing process.

Keywords: CRISPR, HMM, sequence alignment, gene editing

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Authors: Fayyaz Rasool, Shakeela Parveen

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The studies on genetic diversity of Labeo rohita by using molecular markers were carried out to investigate the genetic structure by RAPAD marker and the levels of polymorphism and similarity amongst the different groups of five populations of wild and farmed types. The samples were collected from different five locations as representatives of wild and hatchery raised populations. RAPAD data for Jaccard’s coefficient by following the un-weighted Pair Group Method with Arithmetic Mean (UPGMA) for Hierarchical Clustering of the similar groups on the basis of similarity amongst the genotypes and the dendrogram generated divided the randomly selected individuals of the five populations into three classes/clusters. The variance decomposition for the optimal classification values remained as 52.11% for within class variation, while 47.89% for the between class differences. The Principal Component Analysis (PCA) for grouping of the different genotypes from the different environmental conditions was done by Spearman Varimax rotation method for bi-plot generation of the co-occurrence of the same genotypes with similar genetic properties and specificity of different primers indicated clearly that the increase in the number of factors or components was correlated with the decrease in eigenvalues. The Kaiser Criterion based upon the eigenvalues greater than one, first two main factors accounted for 58.177% of cumulative variability.

Keywords: variation, clustering, PCA, wild, hatchery, RAPAD, Labeo rohita

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Authors: A. Baqqal, O. Abduhamid, H. Abdul-Hameed, T. Messager, G. Ayoub

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In this contribution, the effect of crystal content on the cyclic response of semi-crystalline polyethylene is studied over a large strain range. Experimental observations on a high-density polyethylene with 72% crystal content and an ultralow density polyethylene with 15% crystal content are reported. The cyclic stretching does appear a thermoplastic-like response for high crystallinity and an elastomeric-like response for low crystallinity, both characterized by a stress-softening, a hysteresis and a residual strain, whose amount depends on the crystallinity and the applied strain. Based on the experimental observations, a unified viscoelastic-viscoplastic constitutive model capturing the polyethylene cyclic response features is proposed. A two-phase representation of the polyethylene microstructure allows taking into consideration the effective contribution of the crystalline and amorphous phases to the intermolecular resistance to deformation which is coupled, to capture the strain hardening, to a resistance to molecular orientation. The polyethylene cyclic response features are captured by introducing evolution laws for the model parameters affected by the microstructure alteration due to the cyclic stretching.

Keywords: cyclic loading unloading, polyethylene, semi-crystalline polymer, viscoelastic-viscoplastic constitutive model

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Authors: Abdallah Abusam, Andrzej Mydlarczyk, Fadila Al-Salameen, Moh Elmuntasir Ahmed

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Treated municipal wastewater produced in Kuwait is used mainly in agricultural and greenery landscape irrigations. However, there are strong doubts that severe sludge bulking and foaming problems, particularly during winter seasons, may render the treated wastewater to be unsuitable for irrigation purposes. To assess the impact of sludge bulking and foaming problems on the quality of treated effluents, samples were collected weekly for nine months (January to September 2014) from the secondary effluents, tertiary effluents and sludge-mixed liquor streams of the two plants that severely suffer from sludge bulking and foaming problems. Dominant filamentous bacteria were identified and quantified using a molecular method called VIT (Vermicon Identification Technology). Quality of the treated effluents was determined according to water and wastewater standard methods. Obtained results were then statistically analyzed and compared to irrigation water standards. Statistical results indicated that secondary effluents were greatly impacted by sludge bulking and foaming problems, while tertiary effluents were slightly affected. This finding highlights the importance of having tertiary treatment units in plants that encountering sludge bulking and foaming problems.

Keywords: agriculture, filamentous bacteria, reclamation, reuse, wastewater

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Authors: Naouelle Azzag, Nadia Haddad, Benoit Durand, Elisabeth Petit, Ali Ammouche, Bruno Chomel, Henri J. Boulouis

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Bartonella henselae is a small, gram negative, arthropod-borne bacterium that has been shown to cause multiple clinical manifestations in humans including cat scratch disease, bacillary angiomatosis, endocarditis, and bacteremia. In this research, we report the results of a cross sectional study of Bartonella henselae bacteremia in stray cats from Algiers. Whole blood of 227 stray cats from Algiers was tested for the presence of Bartonella species by culture and for the evaluation of the genetic diversity of B. henselae strains by multi-locus variable number of tandem repeats assay (MLVA). Bacteremia prevalence was 17% and only B. henselae was identified. Type I was the predominant type (64%). MLVA typing of 259 strains from 30 bacteremic cats revealed 52 different profiles. 51 of these profiles were specific to Algerian cats/identified for the first time. 20/30 cats (67%) harbored 2 to 7 MLVA profiles simultaneously. The similarity of MLVA profiles obtained from the same cat, neighbor-joining clustering and structure-neighbor clustering showed that such a diversity likely results from two different mechanisms occurring either independently or simultaneously independent infections and genetic drift from a primary strain.

Keywords: Bartonella, cat, MLVA, genetic

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