Long-term monitoring and population dynamics of ixodid ticks in Tomsk city (Western Siberia).

Monitoring of two tick species (Ixodes persulcatus Schulze 1930 and Ixodes pavlovskyi Pomerantsev) was performed in four city parks of Tomsk, Russia (Camp Garden, University Park, Southern Cemetery, and Polytechnic Stadium) and in a control wild biotope in city environs (Kolarovo) in 2002-2013. Ticks were collected by flagging repeatedly after each 10 ± 1 days, starting from the disappearance of the snow cover till the end of tick activity (April-August). Gradual penetration of both tick species into city parks was demonstrated. In the wild biotope, I. persulcatus dominated during the whole period of monitoring, forming about 95% of tick population, whereas in urban biotopes I. pavlovskyi became the dominant species. Two peaks of population density during each season of spring-summer activity were observed in both species. Possible explanations of the phenomena are discussed.

Pulsed Radiofrequency Increases Nestin and Matrix Metalloproteinase-2 Expression in Porcine Lumbar Dorsal Root Ganglion.

Background: Pulsed radiofrequency (PRF) has been used for the treatment of chronic lumbar radicular pain and other chronic pain states. The dorsal root ganglion (DRG) consists of primary afferent somatic and visceral nerve cell bodies that transduce sensory signals from the periphery to the central part of the nervous system. It is a very important part of acute nociception, as well as the development and maintenance of chronic pain. Methods: A total of seven domestic pigs were investigated. All pigs underwent a PRF procedure while under general anesthesia and with X-ray imaging. Four lumbar DRGs were randomly treated. We used the opposite side of the DRGs as controls. The lumbar region of the spine was placed in 10% formaldehyde for one month. After this fixation, DRG samples were prepared for slide analysis. Results: Nestin (Nes, code-Nr. AB 5968, dilution 1:250, rabbit, Abcam, United Kingdom) and matrix metallopeptidase 2 (MMP-2, code-Nr. DUB 03, dilution 1:100, goat) expressions were detected by immunohistochemical staining. The cell numbers with Nes (28.4 ± 3.3 vs. 16.1 ± 3.4; P < 0.05) and MMP-2 (26.2 ± 3.2 vs. 14.1 ± 2.3; P < 0.05) expressions were larger on the PRF side compared to the control side. The glial cells in the spinal ganglia on both sides showed immunoreactivity. Conclusions: The increase of MMP-2-containing gangliocytes one month after PRF procedures highlights active neural cell proliferation. Increased Nes factor expression in spinal gangliocytes of the lumbar region indicates neural remodeling and regeneration.

Detection of tick-borne pathogens in wild birds and their ticks in Western Siberia and high level of their mismatch.

The Tomsk region located in the south of Western Siberia is one of the most high-risk areas for tick-borne diseases due to elevated incidence of tick-borne encephalitis and Lyme disease in humans. Wild birds may be considered as one of the reservoirs for tick-borne pathogens and hosts for infected ticks. A high mobility of wild birds leads to unpredictable possibilities for the dissemination of tick-borne pathogens into new geographical regions. The primary goal of this study was to evaluate the prevalence of tick-borne pathogens in wild birds and ticks that feed on them as well as to determine the role of different species of birds in maintaining the tick-borne infectious foci. We analysed the samples of 443 wild birds (60 species) and 378 ticks belonging to the genus Ixodes Latraille, 1795 collected from the wild birds, for detecting occurrence of eight tick-borne pathogens, the namely tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and species of Borrelia, Rickettsia, Ehrlichia, Anaplasma, Bartonella and Babesia Starcovici, 1893, using RT-PCR/or PCR and enzyme immunoassay. One or more tick-borne infection markers were detected in 43 species of birds. All markers were detected in samples collected from fieldfare Turdus pilaris Linnaeus, Blyth's reed warbler Acrocephalus dumetorum Blyth, common redstart Phoenicurus phoenicurus (Linnaeus), and common chaffinch Fringilla coelebs Linnaeus. Although all pathogens have been identified in birds and ticks, we found that in the majority of cases (75.5 %), there were mismatches of pathogens in birds and ticks collected from them. Wild birds and their ticks may play an extremely important role in the dissemination of tick-borne pathogens into different geographical regions.

Diagnosis and management of migraine in ten steps.

Migraine is a disabling primary headache disorder that directly affects more than one billion people worldwide. Despite its widespread prevalence, migraine remains under-diagnosed and under-treated. To support clinical decision-making, we convened a European panel of experts to develop a ten-step approach to the diagnosis and management of migraine. Each step was established by expert consensus and supported by a review of current literature, and the Consensus Statement is endorsed by the European Headache Federation and the European Academy of Neurology. In this Consensus Statement, we introduce typical clinical features, diagnostic criteria and differential diagnoses of migraine. We then emphasize the value of patient centricity and patient education to ensure treatment adherence and satisfaction with care provision. Further, we outline best practices for acute and preventive treatment of migraine in various patient populations, including adults, children and adolescents, pregnant and breastfeeding women, and older people. In addition, we provide recommendations for evaluating treatment response and managing treatment failure. Lastly, we discuss the management of complications and comorbidities as well as the importance of planning long-term follow-up.

Complete mitogenome of the ixodid tick Dermacentor reticulatus (Acari: Ixodida).

Here, we present the complete mitochondrial DNA sequence of Dermacentor reticulatus. The mitogenome is 14,806 bp and contains 13 protein-coding, 2 rRNA, and 22 tRNA genes, along with 2 control regions. Dermacentor reticulatus mitogenome has the common mitochondrial gene order of Metastriata ticks. It is phylogenetically close to the mitogenomes of Dermacentor ticks, of which D. everestanus mitogenome is the closest with 85.7% similarity. These data provide insights into the phylogenetic relations among Dermacentor ticks.

Development and pharmaceutical evaluation of the anticancer Anthrafuran/Cavitron complex, a prototypic parenteral drug formulation.

To improve the water solubility of the anticancer drug candidate LCTA-2034 (A1), we investigated the formation of complexes of this anthrax[2,3-b]furan congener with the solubilizing 2-hydroxypropyl derivative of ß-cyclodextrin HP-ßCD (Cavitron®). The interaction of A1 with HP-ßCD resulted in the inclusion complex A1/HP-ßCD in 1:1 stoichiometry. The A1/HP-ßCD complex was used to develop a prototype of a lyophilised drug formulation with enhanced (>10-fold) aqueous solubility than A1 and a long-term stability. The use of HP-ßCD decreased the acute toxicity of A1 by >30%. The A1/HP-ßCD drug formulation as well as A1 in equal doses (5×30mg/kg) to increase the lifespan by up to 140% for mice with i.p. transplanted P388 leukaemia. Furthermore, the A1/HP-ßCD formulation demonstrated a significant and reliable antitumor efficacy in a Р388/ADR drug resistant leukaemia and B16/F10 melanoma, proving a perspective of investigations of toxicology, biodistribution and pharmacokinetics.

Complete mitogenome of the ixodid tick Ixodes pavlovskyi (Acari: Ixodida).

Here, we present complete mitochondrial DNA sequence of Ixodes pavlovskyi Pom., 1946 for the first time. The mitogenome is 14,575 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The overall base composition is 40.1% T, 13.8% C, 37.9% A and 8.1% G. Four protein-coding genes are initiated by ATT codon, three genes--by ATA codon and ATG start codon is found for six genes. Only tRNA-Lys, tRNA-Ile, tRNA-Arg are folded into the cloverleaf secondary structure, other tRNA have atypical structure with reduced T- or D-arms.

Discovery of antitumor anthra[2,3-b]furan-3-carboxamides: Optimization of synthesis and evaluation of antitumor properties.

Anthraquinones and their analogues, in particular heteroarene-fused anthracendiones, are prospective scaffolds for new compounds with improved antitumor characteristics. We herein report the use of a 'scaffold hopping' approach for the replacement of the core structure in the previously discovered hit compound naphtho[2,3-f]indole-5,10-dione 2 with an alternative anthra[2,3-b]furan-5,10-dione scaffold. Among 13 newly synthesized derivatives the majority of 4,11-dihydroxy-2-methyl-5,10-dioxoanthra[2,3-b]furan-3-carboxamides demonstrated a high antiproliferative potency against a panel of wild type and drug resistant tumor cell lines, a property superior over the reference drug doxorubicin or lead naphtho[2,3-f]indole-5,10-dione 2. At low micromolar concentrations the selected derivative of (R)-3-aminopyrrolidine 3c and its stereoisomer (S)-3-aminopyrrolidine 3d caused an apoptotic cell death preceded by an arrest in the G2/M phase. Studies of intracellular targets showed that 3c and 3d formed stable intercalative complexes with the duplex DNA as determined by spectral analysis and molecular docking. Both 3c and 3d attenuated topoisomerase 1 and 2 mediated unwinding of the supercoiled DNA via a mechanism different from conventional DNA-enzyme tertiary complex formation. Furthermore, 3d decreased the activity of selected human protein kinases in vitro, indicating multiple targeting by the new chemotype. Finally, 3d demonstrated an antitumor activity in a model of murine intraperitoneally transplanted P388 leukemia, achieving the increase of animal life span up to 262% at tolerable doses. Altogether, the 'scaffold hopping' demonstrated its productivity for obtaining new perspective antitumor drug candidates.

Synthesis and evaluation of new antitumor 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones.

A series of new 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-5,10-diones 6-13 bearing the cyclic diamine in the position 3 of the indole ring was synthesized. The majority of new compounds demonstrated a superior cytotoxicity than doxorubicin against a panel of mammalian tumor cells with determinants of altered drug response, that is, Pgp expression or p53 inactivation. For naphtho[2,3-f]indole-5,10-diones 6-9 bearing 3-aminopyrrolidine in the side chains, the ability to bind double-stranded DNA and inhibit topoisomerases 1 and 2 mediated relaxation of supercoiled DNA were demonstrated. Only one isomer, (R)-4,11-dihydroxy-3-((pyrrolidin-3-ylamino)methyl)-1H-naphtho[2,3-f]indole-5,10-dione (7) induced the formation of specific DNA cleavage products similar to the known topoisomerase 1 inhibitors camptothecin and indenoisoquinoline MJ-III-65, suggesting a role of the structure of the side chain of 3-aminomethylnaphtho[2,3-f]indole-5,10-diones in interaction with the target. Compound 7 demonstrated an antitumor activity in mice with P388 leukemia transplants whereas its enantiomer 6 was inactive. Thus, 3-aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione emerge as a new prospective chemotype for the search of antitumor agents.

Surveillance of tick-borne encephalitis virus in wild birds and ticks in Tomsk city and its suburbs (Western Siberia).

To study the role of wild birds in the transmission of tick borne encephalitis virus (TBEV), we investigated randomly captured wild birds bearing ixodid ticks in a very highly endemic TBE region located in Tomsk city and its suburbs in the south of Western Siberia, Russia. The 779 wild birds representing 60 species were captured carrying a total of 841 ticks, Ixodes pavlovskyi Pom., 1946 (n=531), Ixodes persulcatus P. Sch., 1930 (n=244), and Ixodes plumbeus Leach. 1815 (n=66). The highest average number of ticks per bird in a particular species was found for the fieldfare (Turdus pilaris Linnaeus, 1758) (5.60 ticks/bird) and the tree pipit (Anthus trivialis Linnaeus, 1758) (13.25 ticks/bird). Samples from wild birds and ticks collected in highly endemic periods from 2006 to 2011 were tested for the TBEV markers using monoclonal modified enzyme immunoassay (EIA) and RT-PCR. TBEV RNA and antigen were found in 9.7% and 22.8% samples collected from wild birds, respectively. TBEV markers were also detected in 14.1% I. persulcatus ticks, 5.2% I. pavlovskyi, and 4.2% I. plumbeus ticks collected from wild birds. Two TBEV strains were also isolated on PKE (pig kidney embryo) cells from fieldfare and Blyth's reed warbler (Acrocephalus dumetorum Blyth, 1849). Sequencing of 5'-NCR of TBEV revealed that all TBEV isolates belong to Far Eastern (dominate) and Siberian genotypes. Several phylogenetic subgroups included TBEV sequences novel for the Tomsk region. Our data suggest that wild birds are potential disseminators of TBEV, TBEV-infected ixodid ticks, and possibly other tick-borne infections.

The tick Ixodes pavlovskyi as a host of spirochetes pathogenic for humans and its possible role in the epizootiology and epidemiology of borrelioses.

The tick Ixodes pavlovskyi is taxonomically, morphologically, and ecologically close to the taiga tick Ixodes persulcatus, a major host and vector of Borrelia spirochetes. The recent range of I. pavlovskyi is disjoined into the Western Siberian and Far Eastern parts, with this tick being almost always sympatric with I. persulcatus. A total of 56 unfed adult I. pavlovskyi ticks from the biotope where this species was absolutely dominant (within the city limits of Tomsk, Western Siberia) and 50 I. persulcatus ticks from the vicinity of this city, where I. pavlovskyi was almost absent, were collected by flagging in May-June 2006, at the seasonal peak of their abundance. The guts and internal organs of individual ticks were inoculated into the Barbour-Stoenner-Kelly (BSK) medium. Thus, 35 Borrelia isolates were obtained and identified by means of polymerase chain reaction-restriction fragment length polymorphism analysis and sequencing of the rrfA-rrlB spacer and, selectively, an rrs gene fragment. The Borrelia infection rate in I. pavlovskyi (35.7 +/- 12.8%) was almost the same as in I. persulcatus (30.0 +/- 13.0%). Such a high infection rate in I. pavlovskyi shows that this vector can itself maintain natural foci of borreliosis, regardless of very low abundance or even absence of I. persulcatus. In both foci compared, Borrelia garinii prevailed in ixodid ticks (31 isolates). Three B. afzelii VS461 isolates were obtained from I. pavlovskyi. Therefore, independently of whether I. pavlovskyi or I. persulcatus is the main vector, B. garinii of two genomic groups, 20047 and NT29, can circulate in a natural focus, but B. garinii NT29 is more closely associated with I. persulcatus. Moreover, two isolates (one from I. pavlovskyi and one from I. persulcatus) proved to be completely identical to B. garinii ChY13p from I. persulcatus collected in China. The hypothesis is that these and other similar isolates described previously comprise an individual genomic group of B. garinii.

Variability of the tick-borne encephalitis virus genome in the 5' noncoding region derived from ticks Ixodes persulcatus and Ixodes pavlovskyi in Western Siberia.

We report the prevalence of Siberian and Far Eastern subtypes of tick-borne encephalitis virus (TBEV) in Ixodes persulcatus and Ix. pavlovskyi ticks collected in Tomsk and its suburbs during 2006-2008. The TBEV was detected in 5.7% ticks collected in the city, where Ix. pavlovskyi ticks were dominated and 7.5% ticks from suburban foci with prevalence Ix. persulcatus ticks. Genotyping of the virus showed that Siberian subtype (89.5%) is predominant in individual ticks of Tomsk suburbs; however, the proportion of Far Eastern subtype in two urban sites reached 47%. Phylogenetic analysis demonstrated that Siberian subtype variants from individual ticks were quite divergent and original. Only one subclade was found to be similar to Zausaev strain of TBEV, which is the etiological agent of lethal chronic form of tick-borne encephalitis infection. The average level of homology of 5' noncoding region (5'-NCR) of TBEV in the individual ticks was 95% for Far Eastern subtype and 89% for Siberian subtype of TBEV. Multiple substitutions in 5'-NCR were found in viral RNA derived from individual ticks. The A2 and C1 elements of Y-shaped structure and putative site for viral RNA polymerase were most variable regions for TBEV 5'-NCR. The B1 and B2 elements and the start codon were practically conserved. The viral RNA from three TBEV-infected pig kidney embryo cells after three passages (out of 21 polymerase chain reaction-positive ticks) were found to multiple substitutions in 5'-NCR in comparison with viral RNA from individual parent tick. However, these three variants did not replicate efficiently in pig kidney embryo cells that may be connected with a considerable modification of Y-shaped structure of 5'-NCR. The efficiently replicating isolate Kolarovo had only seven substitutions in the 5'-NCR and typical Y-shaped structure for Siberian subtype of TBEV. Our data support the idea that hypervariability of the 5'-NCR reflects viral strategy to select the fittest RNA molecule for productive viral infection in mammalian and tick cells.