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The prevalence and outcomes of the unicuspid aortic valve (UAV) in patients undergoing the Ross procedure have been strongly underreported in the current literature. We sought to evaluate this in comparison with bicuspid (BAV) and tricuspid valve (TAV) in our Ross cohort.This was a retrospective observational study of patients undergoing the Ross procedure at 2 dedicated centers between 2009 and 2020. Primary end points were the risks of midterm autograft reoperation and the onset of at least moderate aortic regurgitation during follow-up. The secondary end point was to compare the perioperative outcomes between the groups.Included in the analysis were 286 patients, of those 39% with UAV, 52% with BAV, and 9% with TAV. UAV patients were operated on at the youngest age (P.001) and more often for a combined hemodynamic aortic valve pathology (P = .02). They had the largest aortic root dimensions: annulus (P = .01), Valsalva sinuses (P = .11), sinotubular junction (P = .001), and ascending aorta (P.0001). The risks of reoperation (P = .86) and the onset of aortic regurgitation (P = .75) were comparable among the groups over the follow-up of 4.1 years. There was no difference in perioperative outcomes.UAV is a separate unit characterized by a distinct hemodynamic pathology and generated aortopathy. It is not associated with a higher risk of reoperation or new onset of aortic regurgitation after the Ross procedure in the midterm postoperatively. At the current state, UAV remains acceptable for the Ross procedure.

Typically, laterally patterned films are fabricated by lithographic techniques, external fields, or di-block copolymer self-assembly. We investigate the self-patterning of polyelectrolyte multilayers, poly(diallyldimethylammonium) (PDADMA)/poly(styrenesulfonate) (PSS)short. The low PSS molecular weight (Mw(PSSshort) = 10.7 kDa) is necessary because PSSshort is somewhat mobile within a PDADMA/PSSshort film, as demonstrated by the exponential growth regime at the beginning of the PDADMA/PSSshort multilayer build-up. No self-patterning was observed when the PDADMA/PSS film consisted of only immobile polyelectrolytes. Atomic force microscopy images show that self-patterning begins when the film consists of seven deposited PDADMA/PSSshort bilayers. When more bilayers are added, the surface ribbing evolved into bands, and circular domains were finally observed. The mean distance between the surface structures increased monotonously with the film thickness, from 70 to 250 nm. Scanning electron microscopy images showed that exposure to vacuum resulted in thinning of the film and an increase in the mean distance between domains. The effect is weaker for PSSshort-terminated films than for PDADMA-terminated films. The mechanism leading to domain formation during film build-up and the effect of post-preparation treatment are discussed.

Decorin binding proteins (Dbps) mediate attachment of spirochetes in host organisms during the early stages of Lyme disease infection. Previously, different binding mechanisms of Dbps to glycosaminoglycans have been elucidated for the pathogenic species Borrelia burgdorferi sensu stricto and B. afzelii. We are investigating various European Borrelia spirochetes and their interactions at the atomic level using NMR. We report preparative scale recombinant expression of uniformly stable isotope enriched B. afzelii DbpA in Escherichia coli, its chromatographic purification, and solution NMR assignments of its backbone and sidechain 1H, 13C, and 15N atoms. This data was used to predict secondary structure propensity, which we compared to the North American B. burgdorferi sensu stricto and European B. garinii DbpA for which solution NMR structures had been determined previously. Backbone dynamics of DbpA from B. afzelii were elucidated from spin relaxation and heteronuclear NOE experiments. NMR-based secondary structure analysis together with the backbone dynamics characterization provided a first look into structural differences of B. afzelii DbpA compared to the North American species and will serve as the basis for further investigation of how these changes affect interactions with host components.

Made available in DSpace on 2020-12-10T20:09:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-10 Ministry of Education, Youth and Sports of the Czech Republic INTER-ACTION project Grant Agency of the Czech Republic Czech research infrastructure for systems biology C4SYS United Kingdom Biotechnology and Biological Sciences Research Council Ministry of Education, Youth and Sports of the Czech Republic under the Projects CESNET CERIT-Scientific Cloud Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and other aspects of tick cellular processes. Tick cell cultures are often continuously cultivated, as freezing can affect their viability. However, the long-term cultivation of tick cells can influence their genome stability. In the present study, we investigated karyotype and genome size of tick cell lines. Though 16S rDNA sequencing showed the similarity between Ixodes spp. cell lines at different passages, their karyotypes differed from 2n=28 chromosomes for parental Ixodes spp. ticks, and both increase and decrease in chromosome numbers were observed. For example, the highly passaged Ixodes scapularis cell line ISE18 and Ixodes ricinus cell lines IRE/CTVM19 and IRE/CTVM20 had modal chromosome numbers 48, 23 and 48, respectively. Also, the Ornithodoros moubata cell line OME/CTVM22 had the modal chromosome number 33 instead of 2n=20 chromosomes for Ornithodoros spp. ticks. All studied tick cell lines had a larger genome size in comparison to the genomes of the parental ticks. Thus, highly passaged tick cell lines can be used for research purposes, but possible differences in encoded genetic information and downstream cellular processes, between different cell populations, should be taken into account. Czech Acad Sci, Inst Parasitol, Biol Ctr, Branisovska 31, Ceske Budejovice 37005, Czech Republic Univ South Bohemia, Fac Sci, Branisovska 1760, Ceske Budejovice 37005, Czech Republic Masaryk Univ, Cent European Inst Technol, Kamenice 5, Brno 62500, Czech Republic Sao Paulo State Univ, Dept Gen & Appl Biol, Rio Claro, SP, Brazil Univ Castilla La Mancha, Reg Ctr Biomed Res CRIB, Ciudad Real Med Sch, Neuroplastic & Neurodegenerat Grp, Ciudad Real 13005, Spain Univ Liverpool, Inst Infect Vet & Ecol Sci, Dept Infect Biol & Microbiomes, Liverpool L3 5RF, Merseyside, England Sao Paulo State Univ, Dept Gen & Appl Biol, Rio Claro, SP, Brazil Ministry of Education, Youth and Sports of the Czech Republic INTER-ACTION project: LTARF 18021 Grant Agency of the Czech Republic: 15-03044S Grant Agency of the Czech Republic: 18-27204S Czech research infrastructure for systems biology C4SYS: LM2015055 United Kingdom Biotechnology and Biological Sciences Research Council: BB/P024270/1 Ministry of Education, Youth and Sports of the Czech Republic under the Projects CESNET: LM2015042 CERIT-Scientific Cloud: LM2015085

Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by ‘non-culturability’–a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, ‘non-culturable’ M. tuberculosis cells in an in vitro model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions—the raw data are available via ProteomeXchange with identifier PXD028849. This approach revealed the preservation of 1379 proteins in cells after 5 months of storage in dormancy; among them, 468 proteins were statistically different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system PhoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages: especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current in vitro model of M. tuberculosis self-acidification reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins displaying antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

The article deals with the creation of a program for stopping an autonomous robotic vehicle Robotino® 4. generation at a defined distance from an obstacle. One of the nine infrared distance sensors located on the frame of the robotic vehicle in the front part of the frame is used for this application task. The infrared distance sensor characteristic is created from the measured experimental data, which is then linearized in the given section. The main aim of the experiment is to find such an equation of a line that corresponds to the stopping of a robotic vehicle with a given accuracy from an obstacle. The determined equation of the line is applied to the resulting program for autonomous control of the robotic vehicle. This issue is one of the many tasks performed by AGV in the industry. The introduction of AGVs into the industry is one of the many possibilities within Industry 4.0.

Dormant cells ofMycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by ‘non-culturability’ – a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, ‘non-culturable’M. tuberculosiscells in anin vitromodel of self-acidification of mycobacteria in the post-stationary phase, simulating thein vivopersistence conditions. This approach revealed the accumulation of a significant number of proteins (1379) in cells after 4 months of storage in dormancy; among them, 468 proteins were significantly different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system phoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages, especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the currentin vitromodel ofM. tuberculosisself-acidification reflects the biochemical adaptation of these bacteria to persistencein vivo. Comparative analysis with published proteins with antigenic properties makes it possible to distinguish immunoreactive proteins (40) among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

Background: Complex cardiovascular procedures may initiate a systemic inflammatory response syndrome (SIRS) with a massive cytokine release, which is involved in postoperative myocardial injury. Intraoperative cytokine hemoadsorption (HA) mitigates the inflammatory response. Micro ribonucleic acids (miRNAs) are emerging as a marker of myocardial injury. Methods: This study evaluated if intraoperative cytokine reduction by HA modulates SIRS and affects myocardial injury as measured by miRNA-126, 223 and miRNA-1, 133a, respectively. Twenty-eight patients were assigned into HA (n = 15) and control (C) (n = 13) groups. HA was performed by integrating CytoSorb™ into the extracorporeal circuit. Results: MiRNA-133a plasma levels were increased postoperatively in both groups but were much higher in the HA group than in the C group at 3 h (P = 0.037) and 18 h (P = 0.017) after reperfusion. MiRNA-1 and miRNA-223 plasma levels were significantly increased postoperatively, but did not differ between groups. The vascular miRNA-126 was not affected. Conclusion: Intraoperative cytokine HA in cardiovascular operations increased the plasma levels of miRNA-133a, suggesting higher myocardial injury. J Clin Med Res. 2019;11(12):789-797 doi: https://doi.org/10.14740/jocmr3989

The article deals with the measurement of conveyor belt noise and vibration. The conveyor belt was supported on six rollers fixed on three plates of stand, return and drive drum. These six rollers were produced in two sets (convectional tube and accurate tube). Rollers were driven by a rubber belt. The noise was measured and evaluated on both sides of the conveyor. Vibration of the conveyor was measured at four locations of the conveyor construction. All measurements were performed in an unloaded condition. In conclusion of the article is a summary and evaluation of all measurements. The vibration of the conveyor is comparable in both cases, however, the rollers from accuracy tube excite the construction less, and this results in a lower total noise. By comparing the acceleration to the dominant frequency, it is obvious that the noise and the vibration of the belt conveyor construction were reduced by using rollers from accuracy tube

Made available in DSpace on 2019-10-04T12:37:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-01 Czech Science Foundation Ministry of Education, Youth and Sports of the Czech Republic -project CENAKVA project Biodiversity (Reproductive and genetic procedures for preserving fish biodiversity and aquaculture) All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 +/- 1% saturated FAs, 28 +/- 1% monounsaturated FAs (MUFAs), and 41 +/- 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 +/- 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity. (C) 2019 Elsevier Inc. All rights reserved. Univ South Bohemia Ceske Budejovice, Fac Fisheries & Protect Waters, Bohemian Res Ctr Aquaculture & Biodivers Hydrocen, Zatisi 728-2, Vodnany 38925, Czech Republic Sao Paulo State Univ, Fac Engn, Dept Biol & Zootechny, Neotrop Ichthyol Lab LINEO, Moncao St 226, BR-15385000 Ilha Solteira, SP, Brazil Swedish Univ Agr Sci, Dept Mol Sci, POB 7015, S-75007 Uppsala, Sweden Univ South Bohemia Ceske Budejovice, Inst Chem, Fac Sci, Branisovska 1760, Ceske Budejovice 37005, Czech Republic Biol Ctr Czech Acad Sci, Inst Parasitol, Branisovska 31, Ceske Budejovice 37005, Czech Republic Sao Paulo State Univ, Fac Engn, Dept Biol & Zootechny, Neotrop Ichthyol Lab LINEO, Moncao St 226, BR-15385000 Ilha Solteira, SP, Brazil Czech Science Foundation: 16-03754S Ministry of Education, Youth and Sports of the Czech Republic -project CENAKVA: LM2018099 project Biodiversity (Reproductive and genetic procedures for preserving fish biodiversity and aquaculture): CZ.02.1.01./0.0/0.0/16_025/0007370

OBJECTIVES The choice of optimal surgical treatment for young and middle-aged adults with aortic valve disease remains a challenge. Mechanical aortic valve replacement (mAVR) is generally preferred despite promising recent outcomes of the Ross procedure. Our goal was to compare the strategies at a nationwide level. METHODS This study was a retrospective analysis of prospectively recorded data from the National Registry of Cardiac Surgery of the Czech Republic. Using propensity score matching, we compared the outcomes of patients undergoing the Ross procedure in 2 dedicated centres with all mAVRs performed in country between 2009 and 2020. RESULTS Throughout the study period, 296 adults underwent the Ross procedure and 5120 had an mAVR. We found and compared 291 matched pairs. There were no in-hospital deaths, and the risk of perioperative complications was similar in both groups. Over the average follow-up period of 4.1 vs 6.1 years, the Ross group had a lower all-cause mortality (0.7 vs 6.5%; P = 0.015). This result remained significant even when accounting for cardiac- and valve-related deaths only (P = 0.048). Unlike the Ross group, the mAVR group had a significantly lower relative survival compared with the age- and sex-matched general population. There was no difference in the risk of reoperation (4.5 vs 5.5%; P = 0.66). CONCLUSIONS The Ross procedure offers a significant midterm survival benefit over mAVR. The procedures have a comparable risk of perioperative complications. Patients after mAVR have reduced survival. Thus, the Ross procedure should be the preferred treatment option for young and middle-aged adults with aortic valve disease in dedicated centres.

Background The castor bean tick Ixodes ricinus is an important vector of several clinically important diseases, whose prevalence increases with accelerating global climate changes. Characterization of a tick life-cycle is thus of great importance. However, researchers mainly focus on specific organs of fed life stages, while early development of this tick species is largely neglected. Methods In an attempt to better understand the life-cycle of this widespread arthropod parasite, we sequenced the transcriptomes of four life stages (egg, larva, nymph and adult female), including unfed and partially blood-fed individuals. To enable a more reliable identification of transcripts and their comparison in all five transcriptome libraries, we validated an improved-fit set of five I. ricinus-specific reference genes for internal standard normalization of our transcriptomes. Then, we mapped biological functions to transcripts identified in different life stages (clusters) to elucidate life stage-specific processes. Finally, we drew conclusions from the functional enrichment of these clusters specifically assigned to each transcriptome, also in the context of recently published transcriptomic studies in ticks. Results We found that reproduction-related transcripts are present in both fed nymphs and fed females, underlining the poorly documented importance of ovaries as moulting regulators in ticks. Additionally, we identified transposase transcripts in tick eggs suggesting elevated transposition during embryogenesis, co-activated with factors driving developmental regulation of gene expression. Our findings also highlight the importance of the regulation of energetic metabolism in tick eggs during embryonic development and glutamate metabolism in nymphs. Conclusions Our study presents novel insights into stage-specific transcriptomes of I. ricinus and extends the current knowledge of this medically important pathogen, especially in the early phases of its development.

Abstract Vector-borne diseases constitute 17% of all infectious diseases in the world; among the blood-feeding arthropods, ticks transmit the highest number of pathogens. Understanding the interactions between the tick vector, the mammalian host and the pathogens circulating between them is the basis for the successful development of vaccines against ticks or the tick-transmitted pathogens as well as for the development of specific treatments against tick-borne infections. A lot of effort has been put into transcriptomic and proteomic analyses; however, the protein-carbohydrate interactions and the overall glycobiology of ticks and tick-borne pathogens has not been given the importance or priority deserved. Novel (bio)analytical techniques and their availability have immensely increased the possibilities in glycobiology research and thus novel information in the glycobiology of ticks and tick-borne pathogens is being generated at a faster pace each year. This review brings a comprehensive summary of the knowledge on both the glycosylated proteins and the glycan-binding proteins of the ticks as well as the tick-transmitted pathogens, with emphasis on the interactions allowing the infection of both the ticks and the hosts by various bacteria and tick-borne encephalitis virus.

Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 5%) was less at 15 s post-activation. No difference (ANOVA; P > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation.

We report the proteomic datasets on the mouse macrophage cell line PMJ2R infected with tick-borne encephalitis virus (TBEV) for two and six days. Data were acquired using shotgun ultra-high resolution mass spectrometry. Peptide identifications were done using the Mascot version 2.4 (Matrix Science), and quantification was performed by a label-free approach. Protein profiles of early (two days) and late (six days) stages of infection were compared between each other and the respective control samples. Protein profiles of infected and control samples differed in the number of identified proteins and their relative abundances. Proteins detected in the TBEV-infected cells were involved in various processes related to the infection, including defense response against the virus, regulation of viral process, negative regulation of viral genome replication, RNA binding, or innate immune response. Also, proteins specific for the early and late stages of infection were identified.

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

Background The availability of tick in vitro cell culture systems has facilitated many aspects of tick research, including proteomics. However, certain cell lines have shown a tissue-specific response to infection. Thus, a more thorough characterization of tick cell lines is necessary. Proteomic comparative studies of various tick cell lines will contribute to more efficient application of tick cell lines as model systems for investigation of host-vector-pathogen interactions. Results Three cell lines obtained from a hard tick, Ixodes ricinus, and two from I. scapularis were investigated. A cell mass spectrometry approach (MALDI-TOF MS) was applied, as well as classical proteomic workflows. Using PCA, tick cell line MS profiles were grouped into three clusters comprising IRE/CTVM19 and ISE18, IRE11 and IRE/CTVM20, and ISE6 cell lines. Two other approaches confirmed the results of PCA: in-solution digestion followed by nanoLC-ESI-Q-TOF MS/MS and 2D electrophoresis. The comparison of MS spectra of the cell lines and I. ricinus tick organs revealed 29 shared peaks. Of these, five were specific for ovaries, three each for gut and salivary glands, and one for Malpighian tubules. For the first time, characteristic peaks in MS profiles of tick cell lines were assigned to proteins identified in acidic extracts of corresponding cell lines. Conclusions Several organ-specific MS signals were revealed in the profiles of tick cell lines. Electronic supplementary material The online version of this article (10.1186/s13071-019-3460-5) contains supplementary material, which is available to authorized users.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Ticks, being vectors for a variety of pathogens such as tick-borne encephalitis virus (TBEV), have developed defense mechanisms and pathways against infections, allowing them to control the virus at a level that does not hinder their fitness and development. At the present moment, only a few studies focused on interactions between ticks and TBEV on a molecular level have been published. Here, a possible application of MALDI-TOF MS as a research tool for the investigation of tick-virus interactions was shown. Mass spectrometry (MS) profiles of TBEV-infected and non-infected IRE/CTVM19 tick cell line were compared using principal component analysis. MS spectra were clustered based on the cultivation time of cells, but not their infection status. Nevertheless, the analysis of loading plots revealed different factors (peaks) being involved in the clustering of infected and non-infected cells. Out of them, nine were assigned with proteins: five and four for non-infected and infected cells, respectively. Peak with m/z 8565 was found to be of interest because it was suppressed upon TBEV infection and assigned to proteasome subunit alpha type (B7QE67).

DNA methylation at the fifth position of cytosine (5mC) and at the sixth position of adenine (6 mA) plays an important role in the regulation of the gene expression and, in eukaryotes, is essential for normal development. For Ixodes ricinus, the most common European arthropod vector of human and animal pathogens, the DNA methylation profile and the role of DNA methylation in tick development are still under discussion. Our goal was to analyze the status of I. ricinus DNA methylation at different life stages and identify enzymes that produce this type of DNA modification. We found that 5mC and 6mA are present in I. ricinus genomic DNA at all life stages. In the transcriptome of I. ricinus, we identified the sequences of the putative IrDNMT1, IrDNMT3, and IrDAMT enzymes, and bioinformatic analysis and three-dimensional modeling predicted their DNA methylation activity. This confirms that I. ricinus possesses a complete DNA methylation toolkit. Our results suggest that DNA methylation is important for the physiology and transstadial development of ticks.

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85 ± 4% motility and 160 ± 2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44 ± 9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58 ± 14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5 ± 6% live cells, while the cryopreserved sperm only contained 26.6 ± 14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.

Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L−1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L−1 contained 42% PHB.

Spatial distribution of Ixodes ricinus tick host-seeking activity, as well as prevalence of Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV) were studied in the TBE endemic area of South Bohemia (Czech Republic). High variability in tick abundance detected in a network of 30 study sites was most closely associated with characteristics of vegetation cover. Of 11,182 tested tick samples, 12% carried DNA of spirochete from B. burgdorferi s.l. complex. B. afzelii and B. garinii prevailed among spirochete species. The presence of B. spielmanii in the region was confirmed. The median number of borrelial genome copies in positive samples reached 6.6 × 10(3) by real-time PCR. The total prevalence of TBEV in pooled samples reached 0.32% (20,057 samples tested), at least one TBEV positive tick was present in 21 out of 30 sampling sites.

We describe the detection of sialylated N-linked glycans in partially fed Ixodes ricinus tick females using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Sialylated glycans were detected in salivary glands as well as in tick guts and we propose the host origin of these structures. In addition, we mapped the transport of sialylated structures from the blood meal through the gut to the salivary glands using electron microscopy. Specific localization of sialylated glycans to basement membranes of salivary glands was observed. Finally, the influence of the sample preparation methods for electron microscopy on ultrastructure and immunogold labeling was evaluated.

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al., Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rick-ettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma prote-ome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission., This research was supported by MEC, Spain, grant BFU2011-23896 (JF), European Union FP7 ANTIGONE project number 278976 (JF) and Postdok BIOGLOBE, Czech Republic CZ.1.07/2.3.00/30.0032 co-financed by the European Social Fund and the state budget of the Czech Republic (JS). N. Ayllón was funded by Ministerio de Educacion, Cultura y Deporte, Spain. S. Weisheit was supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7-PEOPLE-ITN programme (EU Grant No. 238511).

Background Tick carrier proteins are able to bind, transport, and store host-blood heme, and thus they function also as antioxidants. Nevertheless, the role of carrier proteins in ticks is not fully understood. Some of them are found also in tick males which do not feed on hosts to such an extent such as females (there are differences in male feeding in different tick species) and thus they are not dealing with such an excess of heme; some of the carrier proteins were found in salivary glands where the processing of blood and thus release of heme does not occur. Besides, the carrier proteins bind relatively low amounts of heme (in one case only two molecules of heme per protein) compared to their sizes (above 200 kDa). The main aim of this study is the biochemical characterization of a carrier protein from the ornate sheep tick Dermacentor marginatus, hemelipoglycoprotein, with emphasis on its size in native conditions, its glycosylation and identification of its modifying glycans, and examining its carbohydrate-binding specificity. Results Hemelipoglycoprotein from D. marginatus plasma was purified in native state by immunoprecipitation and denatured using electroelution from SDS-PAGE separated plasma. The protein (290 kDa) contains two subunits with molecular weights 100 and 95 kDa. It is glycosylated by high-mannose and complex N-glycans HexNAc2Hex9, HexNAc2Hex10, HexNAc4Hex7, and HexNAc4Hex8. The purified protein is able to agglutinate red blood cells and has galactose- and mannose-binding specificity. The protein is recognized by antibodies directed against plasma proteins with hemagglutination activity and against fibrinogen-related lectin Dorin M from the tick Ornithodoros moubata. It forms high-molecular weight complexes with putative fibrinogen-related proteins and other unknown proteins under native conditions in tick plasma. Feeding does not increase its amounts in male plasma. The hemelipoglycoprotein was detected also in hemocytes, salivary glands, and gut. In salivary glands, the protein was present in both glycosylated and nonglycosylated forms. Conclusion A 290 kDa hemelipoglycoprotein from the tick Dermacentor marginatus, was characterized. The protein has two subunits with 95 and 100 kDa, and bears high-mannose and complex N-linked glycans. In hemolymph, it is present in complexes with putative fibrinogen-related proteins. This, together with its carbohydrate-binding activity, suggests its possible involvement in tick innate immunity. In fed female salivary glands, it was found also in a form corresponding to the deglycosylated protein.