Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry

The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct

A Control Design Technique for Grinding Systems with Feedforward Undercompensation and Feedback Control

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Experimental evaluation of infection, dissemination, and transmission rates for two West Nile virus strains in European Aedes japonicus under a fluctuating temperature regime

West Nile virus (WNV) is continuously spreading in Eastern and Southern Europe. However, the extent of vector competence of Aedes japonicus (Theobald, 1901) is controversial. In this work, we elucidated the dynamics of virus growth in this invasive mosquito species. Females of Ae. japonicus were reared from eggs collected in the field in Switzerland and fed on bovine blood spiked with two WNV lineage 1 strains (FIN, Italy; NY99, USA). Fully engorged females were incubated for 14 days under a fluctuating temperature regime of 24 ± 7 °C (average 24 °C), 45–90% relative humidity, which is realistic for a Central European mid-summer day. Infection, dissemination, and transmission rates were assessed from individual mosquitoes by analyzing the abdomen, legs and wings, and saliva for the presence of viral RNA. Saliva was also investigated for the presence of infectious virus particles. Overall, 302 females were exposed to WNV strain FIN and 293 to strain NY99. A higher infection rate was observed for NY99 (57.4%) compared to FIN (30.4%) (p = 0.003). There was no statistical evidence that the dissemination rate (viral RNA in legs and wings) was different between females infected with FIN (57.1%) compared to NY99 (35.5%) (p = 0.16). Viral RNA load of FIN compared to NY99 was significantly higher in the hemocoel (p = 0.031) of exposed females but not at other sites (legs and wings, saliva). This is the first study describing the vector competence parameters for two WNV strains in a European population of Ae. japonicus. The high dissemination and transmission rates for WNV under a realistic temperature regime in Ae. japonicus together with recent findings on its opportunistic feeding behavior (mammals and birds) indicate its potential role in WNV transmission in Central Europe where it is highly abundant

Genetic variants of Anaplasma phagocytophilum from 14 equine granulocytic anaplasmosis cases

<p>Abstract</p> <p>Background</p> <p>Equine Granulocytic Anaplasmosis (EGA) is caused by <it>Anaplasma phagocytophilum</it>, a tick-transmitted, obligate intracellular bacterium. In Europe, it is transmitted by <it>Ixodes ricinus</it>. A large number of genetic variants of <it>A. phagocytophilum </it>circulate in nature and have been found in ticks and different animals. Attempts have been made to assign certain genetic variants to certain host species or pathologies, but have not been successful so far. The purpose of this study was to investigate the causing agent <it>A. phagocytophilum </it>of 14 cases of EGA in naturally infected horses with molecular methods on the basis of 4 partial genes (<it>16S rRNA</it>, <it>groEL</it>, <it>msp2</it>, and <it>msp4</it>).</p> <p>Results</p> <p>All DNA extracts of EDTA-blood samples of the horses gave bands of the correct nucleotide size in all four genotyping PCRs. Sequence analysis revealed 4 different variants in the partial <it>16S rRNA</it>, <it>groEL </it>gene and <it>msp2 </it>genes, and 3 in the <it>msp4 </it>gene. One <it>16S rRNA </it>gene variant involved in 11 of the 14 cases was identical to the "prototype" variant causing disease in humans in the amplified part [GenBank: <ext-link ext-link-id="U02521" ext-link-type="gen">U02521</ext-link>]. Phylogenetic analysis revealed as expected for the <it>groEL </it>gene that sequences from horses clustered separately from roe deer. Sequences of the partial <it>msp2 </it>gene from this study formed a separate cluster from ruminant variants in Europe and from all US variants.</p> <p>Conclusions</p> <p>The results show that more than one variant of <it>A. phagocytophilum </it>seems to be involved in EGA in Germany. The comparative genetic analysis of the variants involved points towards different natural cycles in the epidemiology of <it>A. phagocytophilum</it>, possibly involving different reservoir hosts or host adaptation, rather than a strict species separation.</p

Diverse tick-borne microorganisms identified in free-living ungulates in Slovakia

Background: Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. Results: Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. Conclusions: The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.Fil: Kazimírová, Mária. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Hamšíková, Zuzana. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Spitalská, Eva. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Minichová, Lenka. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Mahríková, Lenka. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Caban, Radoslav. Široká ; EslovaquiaFil: Sprong, Hein. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Fonville, Manoj. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kocianová, Elena. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaqui

Engineering tyrosine-based electron flow pathways in proteins: The case of aplysia myoglobin

Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed. © 2012 American Chemical Society

Babesia spp. in ticks and wildlife in different habitat types of Slovakia

Background: Babesiosis is an emerging and potentially zoonotic disease caused by tick-borne piroplasmids of the Babesia genus. New genetic variants of piroplasmids with unknown associations to vectors and hosts are recognized. Data on the occurrence of Babesia spp. in ticks and wildlife widen the knowledge on the geographical distribution and circulation of piroplasmids in natural foci. Questing and rodent-attached ticks, rodents, and birds were screened for the presence of Babesia-specific DNA using molecular methods. Spatial and temporal differences of Babesia spp. prevalence in ticks and rodents from two contrasting habitats of Slovakia with sympatric occurrence of Ixodes ricinus and Haemaphysalis concinna ticks and co-infections of Candidatus N. mikurensis and Anaplasma phagocytophilum were investigated. Results: Babesia spp. were detected in 1.5 % and 6.6 % of questing I. ricinus and H. concinna, respectively. Prevalence of Babesia-infected I. ricinus was higher in a natural than an urban/suburban habitat. Phylogenetic analysis showed that Babesia spp. from I. ricinus clustered with Babesia microti, Babesia venatorum, Babesia canis, Babesia capreoli/Babesia divergens, and Babesia odocoilei. Babesia spp. amplified from H. concinna segregated into two monophyletic clades, designated Babesia sp. 1 (Eurasia) and Babesia sp. 2 (Eurasia), each of which represents a yet undescribed novel species. The prevalence of infection in rodents (with Apodemus flavicollis and Myodes glareolus prevailing) with B. microti was 1.3 % in an urban/suburban and 4.2 % in a natural habitat. The majority of infected rodents (81.3 %) were positive for spleen and blood and the remaining for lungs and/or skin. Rodent-attached I. ricinus (accounting for 96.3 %) and H. concinna were infected with B. microti, B. venatorum, B. capreoli/B. divergens, Babesia sp. 1 (Eurasia), and Babesia sp. 2 (Eurasia). All B. microti and B. venatorum isolates were identical to known zoonotic strains from Europe. Less than 1.0 % of Babesia-positive ticks and rodents carried Candidatus N. mikurensis or A. phagocytophilum.Inst. de PatobiologíaFil: Hamsikova, Zuzana. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Kazimirová, Mária. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Harustiakova, Danka. Masaryk University. Faculty of Medicine and Faculty of Science, Institute of Biostatistics and Analyses; República ChecaFil: Mahrikova, Lenka. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Slovak, Mirko. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Berthova, Lenka. Slovak Academy of Sciences. Biomedical Research Center. Institute of Virology; EslovaquiaFil: Kocianova, Elena. Slovak Academy of Sciences. Biomedical Research Center. Institute of Virology; EslovaquiaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Evidence of West Nile virus (WNV) circulation in wild birds and WNV RNA negativity in mosquitoes of the Danube Delta Biosphere Reserve, Romania, 2016

West Nile virus (WNV) is a zoonotic flavivirus whose transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. Bridge vectors can transmit WNV to mammal species potentially causing West Nile Fever. Wild bird migration is a mode of WNV introduction into new areas. The Danube Delta Biosphere Reserve (DDBR) is a major stopover of wild birds migrating between Europe and Africa. The aim of this study was to investigate the presence of WNV in the DDBR during the 2016 transmission season in wild birds and mosquitoes. Blood from 68 wild birds (nine different species) trapped at four different locations was analyzed by competitive ELISA and Virus Neutralization Test (VNT), revealing positive results in 8/68 (11.8%) of the wild birds by ELISA of which six samples (three from juvenile birds) were confirmed seropositive by VNT. Mosquitoes (n = 6523, 5 genera) were trapped with CDC Mini Light traps at two locations and in one location resting mosquitoes were caught. The presence of WNV RNA was tested in 134 pools by reverse transcription quantitative PCR (RT-qPCR). None of the pools was positive for WNV-specific RNA. Based on the obtained results, WNV was circulating in the DDBR during 2016