The Role of a Forensic Expert in the Field of Self-Defense Under Current Legislation and Beyond

A forensic expert in the field of self-defense is a specialist with extensive knowledge in defence methods, close combat, defensive means, or coercive means. The core of the forensic expert's work in this field is the assessment of the facts of conflict situations in which physical violence between two or more persons has been used. The role of a forensic expert is to answer questions related to the parties' roles in the conflict, factors that influenced the conduct of the fight, the course of the fight and the possibility of resolving the conflict situation. Self-defense expertise is sometimes associated or confused with martial arts or combat sports expertise. This judgment is based on the assumption that most forensic experts in the field of self-defense also have experience or expertise in martial arts and combat sports. Such knowledge is an advantage for the profession of forensic expert in the field of self-defense, similar to knowledge in the field of weapons and ammunition, but it is not the same area as expertise in the field of self-defense or a condition for the exercise of this profession. The new Act No. 254/2019 Coll., on forensic experts, expert offices and expert institutes, faces professional criticism in several areas (expert duties, sanctions, division of specializations, etc.) and is likely to lead to pressure for further amendments. The article deals with these changes and the perspective in the coming years with the expert opinion of current forensic experts on the field of self-defense on the appropriate inclusion of this specialization in the future categorization of specializations

Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis

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 rickettsial 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 proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission. (Résumé d'auteur

Determination of surface properties of treated cement pastes by acoustic methods and scratch test - pilot experiments

The concrete has been and at the same time will be the most used construction material worldwide. It is exposed to various physical and chemical degradation processes that deteriorate its properties and shorten its service life in most applications. As most of the detrimental effects on concrete come from the ambient environment, the quality of concrete surface plays a key role in the overall concrete performance. It should be resistant to abrasion, free of microcracks and open pores to prevent ingress of water, aggressive solutions, and gases. To enhance the properties of the concrete surface, various approaches can be used. The treatment via silicate-based sealers is becoming increasingly popular in concrete technology, especially in preventing deterioration when exposed to highly aggressive environments. This contribution focuses on applying unconventional test methods (e.g. combined scratch/acoustic emission method) to detect the fundamental properties of the treated cementitious surfaces will also provide a new perspective approach of material testing, which may be in the future advantageously used in technical practice. The present combined scratch/acoustic emission test evaluation will provide an excellent insight into lithium silicate sealers’ physical behaviour on fine-grained cement-based materials during this test

Three-dimensional reconstruction of the feeding apparatus of the tick Ixodes ricinus (Acari: Ixodidae): a new insight into the mechanism of blood-feeding

International audienceThe different components of the mouthparts of hard ticks (Ixodidae) enable these parasites to penetrate host skin, secrete saliva, embed, and suck blood. Moreover, the tick's mouthparts represent a key route for saliva-assisted pathogen transmission as well as pathogen acquisition from blood meal during the tick feeding process. Much has been learned about the basic anatomy of the tick's mouthparts and in the broad outlines of how they function in previous studies. However, the precise mechanics of these functions are little understood. Here, we propose for the first time an animated model of the orchestration of the tick mouthparts and associated structures during blood meal acquisition and salivation. These two actions are known to alternate during tick engorgement. Specifically, our attention has been paid to the mechanism underlining the blood meal uptake into the pharynx through the mouth and how ticks prevent mixing the uptaken blood with secreted saliva. We animated function of muscles attached to the salivarium and their possible opening /closing of the salivarium, with a plausible explanation of the movement of saliva within the salivarium and massive outpouring of saliva

Antibodies against recombinant proteins recognize <i>A</i>. <i>phagocytophilum</i> in infected tick cells and ticks by immunofluorescence.

<p>(A) Uninfected and <i>A</i>. <i>phagocytophilum</i> (NY18)-infected ISE6 tick cells were characterized by immunofluorescence in (a, c, e, g) uninfected and (b, d, f, h) infected cells. Representative immunofluorescence images are shown for tick cells stained with rabbit preimmune (control) or anti-<i>A</i>. <i>phagocytophilum</i> protein antibodies (green, FITC; blue, DAPI). Arrows show the localization of <i>A</i>. <i>phagocytophilum</i> proteins in infected cells. Bars, 5 μm. (B) Sections were made from <i>I</i>. <i>scapularis</i> female ticks after feeding on an uninfected (a, c, e) or <i>A</i>. <i>phagocytophilum</i> (NY18)-infected (b, d, f) sheep. Representative immunofluorescence images are shown for salivary gland sections stained with rabbit preimmune (control) or anti-<i>A</i>. <i>phagocytophilum</i> protein antibodies (green, FITC). Arrows show the localization of <i>A</i>. <i>phagocytophilum</i> proteins in infected cells. Bars, 10 μm. (C) IDE8 tick cells were collected in low and high percentage <i>A</i>. <i>phagocytophilum</i> (L610)-infected cells and representative immunofluorescence images are shown. (a, b) Bright-field images of Giemsa-stained (a) low percentage and (b) high percentage infected tick cells. Bacteria stain purple (arrows) and host nuclei stain pink. (c) Low percentage and (d) high percentage infected tick cells were stained with rabbit anti-<i>A</i>. <i>phagocytophilum</i> HSP70 protein antibodies (green, FITC). Arrows show the localization of <i>A</i>. <i>phagocytophilum</i> proteins in infected cells. Bar, 5 μm. (D) Flow cytometry profile showing MFI values determined using a FITC-conjugated secondary antibody. <i>A</i>. <i>phagocytophilum</i> (NY18)-infected and uninfected control ISE6 tick cells were washed, fixed, permeabilized and incubated with primary unlabeled antibody (preimmune IgG isotype control, MSP4, SOD, HSP70 and GroEL), washed in PBS and incubated with FITC-goat anti-rabbit IgG. MFI was calculated as the MFI of the test-labeled sample minus the MFI of the isotype control, shown as Ave+SD and compared between infected and uninfected tick cells by Student's t-test (*P<0.05) (N = 3).</p

Proposed mechanisms of how stress response and surface proteins facilitate <i>A</i>. <i>phagocytophilum</i> infection in high percentage infected tick cells and tick salivary glands.

<p>The levels of certain bacterial stress response and surface proteins are higher in high percentage <i>A</i>. <i>phagocytophilum</i>-infected tick cells and tick salivary glands. MSP4, GroEL and HSP70 interact and bind to tick cells, thus facilitating rickettsia-tick interactions and infection. In high percentage infected tick cells and tick salivary glands, bacteria reduce multiplication once they infect the cells but infection is required to complete the life cycle and get ready for transmission. The activation of stress response proteins in <i>A</i>. <i>phagocytophilum</i> may represent a mechanism by which rickettsiae increase infection by facilitating interaction with tick cells and protecting bacteria against stress. The T4SS may be associated with the secretion of HSP70 and other stress response proteins. Abbreviations: T4SS, Type IV Secretion System; Question mark indicates that secretion of HSP70 in a T4SS-dependent manner remains to be proved.</p

Characterization of the mRNA levels for selected genes encoding for <i>A</i>. <i>phagocytophilum</i> over-represented proteins.

<p>The mRNA levels for <i>groEL</i>, <i>msp4</i> and <i>hsp70</i> were determined by real-time RT-PCR in low and high percentage infected ISE6 tick cells. Amplification efficiencies were normalized against tick <i>16S rRNA</i> and mRNA levels expressed in arbitrary units. The ratio of normalized mRNA levels in high to low percentage-infected cells was represented as Ave+SD. Normalized Ct values were compared between low and high percentage infected tick cells by Student's t-test (*P<0.05) (N = 5).</p

Inhibition of <i>A</i>. <i>phagocytophilum</i> infection by antibodies against over-represented proteins.

<p>The antibodies against surface-exposed proteins, GroEL, HSP70 and MSP4, were used to characterize the inhibition of pathogen infection in ISE6 tick cells. Tick cells were treated with different rabbit antibodies and then infected with <i>A</i>. <i>phagocytophilum</i> (NY18). Treatments included rabbit pre-immune serum, anti-<i>A</i>. <i>phagocytophilum</i> GroEL, HSP70 and MSP4 protein antibodies and anti-tick Porin antibodies. Untreated cells were left uninfected or infected with <i>A</i>. <i>phagocytophilum</i> (NY18). <i>A</i>. <i>phagocytophilum</i> infection levels were determined by <i>16S rDNA</i> and <i>msp4</i> PCR and normalized against tick <i>16S</i> mitochondrial <i>rDNA</i> with similar results. Normalized <i>msp4</i> levels are shown in arbitrary units as Ave+S.D and were compared between infected and uninfected untreated cells and between infected cells treated with the pre-immune serum and antigen-specific antibodies by Student’s t-test with unequal variance (P<0.05; N = 4 replicates per treatment).</p

Electron micrographs of ISE6 tick cells reacted with recombinant <i>E</i>. <i>coli</i> producing <i>A</i>. <i>phagocytophilum</i> recombinant proteins.

<p>Representative images of recombinant <i>E</i>. <i>coli</i> shown by arrowheads adhered to tick cells. (a, b) <i>A</i>. <i>marginale</i> MSP1a positive control; (c-f) MSP4; (g-k) HSP70; (l) GroEL; (m) Thioredoxin negative control. Microtubules are shown by arrows in (d). Scale bars, 1 μm (a, c, e, g, i, k-m), 500 nm (b, d, f, h, j).</p

Characterization of <i>A</i>. <i>phagocytophilum</i> protein-protein interactions.

<p>(A) Protein-protein interactions were characterized <i>in silico</i> using STRING 8.3 (<a href="http://string-db.org" target="_blank">http://string-db.org</a>). The STRING score value is shown, defined as threshold of significance to include the interaction (maximum value = 1) computed by combining the probabilities from the different evidence channels, correcting for the probability of randomly observing an interaction. (B) Protein-protein interactions were characterized <i>in vitro</i> using <i>A</i>. <i>phagocytophilum</i> HSP70 (red arrow) and GroEL (blue arrow) recombinant proteins and tick Porin as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137237#pone.0137237.ref015" target="_blank">15</a>]. The proteins were mixed in equimolar amounts and immunoprecipitated using anti-GroEL or anti-HSP70 antibodies and Protein G Dynabeads. The purified proteins were eluted using Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70, anti-GroEL or anti-Porin antibodies. (C) Protein-protein interactions were characterized <i>in vitro</i> using <i>A</i>. <i>phagocytophilum</i> protein extracts, recombinant HSP70 (red arrow) and GroEL (blue arrow) proteins and tick Porin as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137237#pone.0137237.ref015" target="_blank">15</a>]. Protein G Dynabeads were incubated with purified anti-HSP70, anti-GroEL or anti-Porin antibodies and then 130 μg of <i>A</i>. <i>phagocytophilum</i> proteins were added. Unbound proteins were removed and the beads were washed three times with PBS with addition of 0.1% Triton X-100, resuspended in Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70 or anti-GroEL antibodies. (D) Protein-protein interactions were characterized <i>in vitro</i> using <i>A</i>. <i>phagocytophilum</i> HSP70 (red arrow), GroEL (blue arrow) and MSP4 (green arrows) recombinant proteins and tick Porin as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137237#pone.0137237.ref015" target="_blank">15</a>]. Nickel beads were covered with histidine-tagged MSP4, washed and incubated with GroEL or HSP70, MSP4 or Porin as control. After incubation, beads were washed and proteins eluted in Laemmli sample buffer and loaded onto a 15% SDS-PAGE gel.</p