Additional file 2: of Genome scaffolding and annotation for the pathogen vector Ixodes ricinus by ultra-long single molecule sequencing

Figure S1. Sequence distribution as percent identity between I. ricinus and I. scapularis.I. ricinus scaffolds were blasted against I. scapularis scaffolds. Only sequences passing the threshold of a maximum e-value of 1.0 e-5 and minimum 80% identity are included. (PDF 229 kb

Additional file 1: Figure S1. of Identification of a novel β-adrenergic octopamine receptor-like gene (βAOR-like) and increased ATP-binding cassette B10 (ABCB10) expression in a Rhipicephalus microplus cell line derived from acaricide-resistant ticks

βAOR gene in earlier passages of Rhipicephalus cell lines BME/CTVM5 and BME/CTVM6. Detection of βAOR gene in Rhipicephalus microplus cell lines BME/CTVM5 passage 8 (5p8) and BME/CTVM6 passages 32, 221 and 243 (6p32, 6p221 and 6p243, respectively). Amplicons of 183 bp, 220 bp and 245 bp were detected in the gDNA of BME/CTVM5 and amplicon of only 220 bp was detected in BME/CTVM6 passages. M = Marker. (TIF 2846 kb

Additional file 1: Table S1. of Tick-borne pathogens induce differential expression of genes promoting cell survival and host resistance in Ixodes ricinus cells

Primers used in this study for detection of viral RNA (LIV/TBEV) from infected I. ricinus IRE/CTVM20 cells, along with host gene transcripts in RNA extracted from I. ricinus IRE/CTVM20 cells infected with A. phagocytophilum, LIV or TBEV. (DOC 38 kb

Additional file 3: of Ixodes scapularis and Ixodes ricinus tick cell lines respond to infection with tick-borne encephalitis virus: transcriptomic and proteomic analysis

TBEV infection levels in mock-infected and TBEV-infected tick cells. Numbers of copies of TBEV NS5 were determined by qRT-PCR using NS5 primers and the linearised plasmid pJET-NS5 to create a standard curve. Copy numbers were normalised to 1 μg of total RNA. The limit of detection was derived from the number of NS5 copies in the highest dilution which was still detectable with a variance less than one Ct and was normalised to 1 μg of total RNA. (A) IDE8 infected and mock-infected (control) at days 2 (2d) and 6 (6d) p.i. (B) IRE/CTVM19 infected and mock-infected (control) at days 2 and 6 p.i.. Error bars are standard deviations. Samples marked with + passed both RNA and protein quality checks and were used in transcriptomic and proteomic analyses

Pressing the Nuts to Sheet Metal Blanks

The bachelor thesis consists of the theoretical introduction into pressing the nuts, familiarization with used machines followed by testing and evaluating of outcomes. Pressing the nuts is tested for torque and pulling force. In the practical part of the thesis chosen press nuts will be tested. Objective of this thesis is to find out suitable pressing parameters, which can substitute present unsuitable practices

Toscana virus (TOSV) shows remarkable adaptability to the endosomal environment to penetrate cells.

(A) The binding of R18-TOSV (MOI ∼10) to A549 cells was synchronized on ice. Cells were then rapidly warmed to 37°C, and virus fusion was triggered by adding acidic buffers within a fluorometer while recording the fluorescence signal. The virus fusion-specific R18 release is shown. Data were normalized to those at the time point 0. RU, relative unit. (B) The half-maximal fluorescence intensity (t1/2) was measured in the series of data obtained in A. n > 3. (C) R18-TOSV particles were pretreated at pH 6.0 or 7.4 and then neutralized as described in Fig 8A before being assessed and analyzed as in panel A. The t1/2 of fusion was calculated from n > 7. T-test with Welch’s correction was applied. *, pD) After the synchronization of TOSV binding at MOI 1 on ice, A549 cells were rapidly warmed to 37°C in the presence of NH4Cl (50 mM). NH4Cl was then washed out at the indicated times to allow endosomal acidification and the acid-activated penetration of infectious TOSV particles. Samples were harvested 6 h later, and infection was analyzed by flow cytometry. Values were normalized to those from samples for which NH4Cl was removed at t0.</p

Structure of the <i>I</i>. <i>scapularis</i> Tudor-SN.

<p>Tudor-SN amino acid sequence alignment between <i>I</i>. <i>scapularis</i> (B7QIP4_IXOSC), <i>I</i>. <i>ricinus</i> (V5GZ29_IXORI) and <i>Danio rerio</i> (Q5RGK8_DANRE). Conserved domains in <i>I</i>. <i>scapularis</i> Tudor-SN contain conserved staphylococcal nuclease homologues (SN, cd00175; red) and Tudor domain (cd04508; blue).</p

Entry of Toscana virus (TOSV) into cells.

The time and pH scale on the right side illustrates the typical acidity and timing of cellular cargoes to reach the respective endosomal compartments. +/++ indicate the amount of Rab7a and LAMP-1.</p

This Excel workbook includes all the numerical values used to produce the graphs shown in this study.

This Excel workbook includes all the numerical values used to produce the graphs shown in this study.</p

Toscana virus (TOSV) penetrates host cells by acid-activated membrane fusion.

(A) A549 cells were exposed to TOSV (MOI ∼10) on ice, subjected to various pH values at 37°C for 90 sec to trigger the virus fusion at the plasma membrane, and then incubated for 7 h at 37°C in the presence of 50 mM NH4Cl to prevent viral penetration from endosomes. Infection was quantified by flow cytometry, and the data were normalized to those from samples where the infection was triggered with a buffer at pH 5.0. (B and C) TOSV particles were bound to A549 cells (B) and iPSC-derived neurons (C) at MOIs 1 and 15, respectively, on ice and then rapidly shifted to 37°C to allow virus internalization. 50 mM NH4Cl was added at the indicated times to block further viral penetration. Infected cells were quantified by flow cytometry, and data were normalized to the samples where NH4Cl was added 80 min (B) and 90 min (C) post-warming. (D) R18-TOSV was bound at MOI 10 to A549 cells on ice and rapidly warmed to 37°C. The increase in fluorescence resulted from the dequenching of the lipid dye R18 after virus fusion with cell membranes, as measured by fluorometry (black line). NH4Cl was used to block virus fusion by neutralizing endosomal pH and, thus, to define the fluorescence background due to spontaneous translocation of the R18 dyes between the viral envelope and the neighboring cell membrane (grey line). The red line shows the virus fusion-specific R18 release, i.e., the black line (fusion + free diffusion) minus the grey line (free diffusion). RU, relative unit. (E) TOSV and Semliki Forest virus (SFV) were bound to A549 cells on ice, and samples were shifted to indicated temperatures for 50 min. Infected cells were then incubated at 37°C for 6 h in the presence of NH4Cl and quantified by flow cytometry. Infection was normalized to that in samples incubated throughout at 37°C. (F) TOSV fusion was assessed in A549 cells for efficiency at various temperatures using the assay in A. Data were normalized to those of samples incubated throughout at 37°C. (G) The binding of R18-TOSV (MOI ∼10) to A549 cells was synchronized on ice. Samples were then warmed to 37°C in a fluorometer, and the fluorescence signal was monitored over 10 min. “+pH” indicates when buffers at pH 5.0 or 7.4 were added to trigger virus fusion. Data were normalized to those at the time point 0. The red line shows the virus fusion-specific R18 release at pH ∼5.0. RU, relative unit. (H) R18-TOSV penetration into A549 cells at MOI 10 was recorded in real time for 90 min using the protocol in D, and triton X-100 was added at the end to induce the dequenching of all R18 molecules associated with bound and internalized virions. The data show the ratio between the fluorescence resulting from viral fusion and that associated with all virions in the cells.</p