A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assay

Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection

Secretory glands in cercaria of the neuropathogenic schistosome Trichobilharzia regenti - ultrastructural characterization, 3-D modelling, volume and pH estimations

<p>Abstract</p> <p>Background</p> <p>Cercariae of schistosomes employ bioactive molecules for penetration into their hosts. These are released from specialized unicellular glands upon stimuli from host skin. The glands were previously well-described in the human pathogen <it>Schistosoma mansoni</it>. As bird schistosomes can also penetrate human skin and cause cercarial dermatitis, our aim was to characterize the architecture and ultrastructure of glands in the neurotropic bird schistosome <it>Trichobilharzia regenti </it>and compare it with <it>S. mansoni</it>. In the context of different histolytic enzymes used by these two species, we focused also on the estimations of gland volumes and pH in <it>T. regenti</it>.</p> <p>Results</p> <p>The architecture and 3-D models of two types of acetabular penetration glands, their ducts and of the head gland are shown here. We characterized secretory vesicles in all three gland types by means of TEM and confirmed accuracy of the models obtained by confocal microscopy. The results of two independent approaches showed that the glands occupy ca. one third of cercarial body volume (postacetabular glands ca. 15%, circumacetabular 12% and head gland 6%). The inner environment within the two types of acetabular glands differed significantly as evidenced by dissimilar ability to bind fluorescent markers and by pH value which was higher in circumacetabular (7.44) than in postacetabular (7.08) glands.</p> <p>Conclusions</p> <p>As far as we know, this is the first presentation of a 3-D model of cercarial glands and the first exact estimation of the volumes of the three gland types in schistosomes. Our comparisons between <it>T. regenti </it>and <it>S. mansoni </it>implied that the architecture and ultrastructure of the glands is most likely conserved within the family. Only minor variations were found between the two species. It seems that the differences in molecular composition have no effect on general appearance of the secretory cells in TEM. Fluorescent markers employed in this study, distinguishing between secretory vesicles and gland types, can be useful in further studies of mechanisms used by cercariae for host invasion. Results of the first attempts to estimate pH within schistosome glands may help further understanding of regulation of enzymatic activities present within the glands.</p

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Increasing importance of anthelmintic resistance in European livestock: creation and meta-analysis of an open database

peer reviewedHelminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke (Fasciola hepatica) was collated by members of the European COST Action “COMBAR” (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980–2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0–100% (BZ and ML), 0–17% (LEV) and 0–73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread

Influence of 'Trichobilharzia regenti' (Digenea: Schistosomatidae) on the defence activity of 'Radix lagotis' (Lymnaeidae) haemocytes

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2–36 h post exposure (p.e.) to the parasite. At later time points, 44–92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae

Individual Professional Practice in the Company

Import 03/08/2012Tato práce je zaměřena na odbornou praxi ve společnosti Tieto Czech s.r.o. v oddělení MOSS Testing na pozici tester (Test Engineer). Jedná se o testování MOSS webových aplikací pro zákazníky. Hlavní náplní praxe byla spolupráce na testování aspx webových aplikací, které mají společnou platformu SharePoint Server založenou na ASP.NET. Součástí mé bakalářské práce bylo seznámení s prostředím MOSS, testovacími postupy ve firmě Tieto a prostudování nástrojů a prostředků, které se k testování používají.This thesis is focused to the Professional Practice in the Company Tieto Czech s.r.o. in Department MOSS Testing to possition Test Engineer. It is testing MOSS web applications for Customers. The~ main scope of practical experience was cooperation on testing aspx web applications which they have together platform SharePoint Server based on ASP.NET. Part of my thesis was introduction with a working environment MOSS, Test Procedures in the Company Tieto and I studied tools and resources which used for testing.460 - Katedra informatikyvýborn

Motivational Aspects of the Organization Human Potential Development

Import 04/07/2011Bakalářská práce s názvem „Motivační aspekty rozvoje lidského potenciálu organizace“ se zabývá motivací, rozvojem lidského potenciálu a jak spolu tyto pojmy souvisí. Cílem této práce bylo zjistit, které motivační prvky působí na rozvoj lidí, a jakým způsobem. Analyzován byl především systém vzdělání, motivace, odměňování a firemní kultury ve společnosti CS CABOT, spol. s r.o.. Tato analýza probíhala metodou studia dokumentů společnosti a polostandardizovanými rozhovory se členy managementu a administrativy. První část práce se zabývá teoretickými východisky motivace a rozvoje lidského potenciálu, druhá část popisuje analyzovanou společnost, třetí kapitola obsahuje analytickou část práce a ve čtvrté části se nachází shrnutí poznatků analýzy a doporučení pro management společnosti.Bachelor thesis named "Motivational aspects of human development potential of the organization" is concerned with motivation, development of human potential and howthese concepts are related. The aim of this study was to find out which incentives work to develop people and how. Was analyzed in particular the system of education, motivation, compensation and corporate culture in CS CABOT, spol s r.o.. The analysis is performed by the method of study of documents and semi interviews with members of management and administration. The first part contains the theoretical bases of motivation and development of human potential, the second part describes the analyzed company, in the third part is the analytical part and the fourth is a summary of the analysis, findings and recommendations for management of this company.115 - Katedra managementuvelmi dobř

Design of System for Monitoring of Vehicles Shutdowning in the Depot

Import 03/08/2012Diplomová práce se zabývá problematikou sledování a prezentace odstavených vozidel v depu kolejových vozidel. Cílem je navrhnout počítačový systém pro sledování odstavených vozidel, který by nahradil dosavadní systém sledování pomocí magnetické tabule. Výstupem práce je sešit Microsoft Excel obsahující systém pro prezentaci umístění lokomotiv na jednotlivých stáních v SOKV Ostrava a seznam lokomotiv, které má SOKV Ostrava v inventárním stavu. V úvodu je provedena analýza jednotlivých činností a postupů řízení provozních procesů při organizování činnosti hnacího vozidla v depu. Dále je z hlediska ovladatelnosti a funkčnosti popsán navrhovaný systém. V závěru této práce je provedeno celkové zhodnocení vytvořeného počítačového systému.The present thesis deals with the issue of the monitoring and the presentation of the vehicles stabled in the depot of the track vehicles. The thesis target is to project the computer system for monitoring of the stabled vehicles that would replace the actual monitoring system based on the usage of the magnetic board. The output of the thesis is the Microsoft Excel file containing the diagrammatic scheme of the yard in SOKV Ostrava in the inventory stage. In the introduction there is done the analysis of the particular activities and of the control procedures of the operating processes during the organization of the driving vehicle activity in the depot. Afterwards the manipulability and functionality aspects of the designed system are described. In the thesis conclusion there is presented the total evaluation of the created computer system.342 - Institut dopravyvelmi dobř

Peptidases of Trematodes

90 3. SUMMARY The text above refers about the majority of characterized trematode peptidases; the fundamental enzymes for trematode existence, which are integrated in many physiological processes like pathogenesis, tissue invasion/migration, nutrition, immune evasion and host-parasite interactions. In the history (until 1996), the peptidase catalytic activities in trematode extracts have been monitored. During 1980s and 1990s, the information of first cloned trematode peptidase genes were published and during last three decades cca 90 trematode peptidase sequences belonging to 19 peptidase families of 5 clans have been identified. The most studied trematode peptidases have been of Schistosoma mansoni origin: the serine peptidase - cercarial elastase (of cercariae), cysteine peptidases - cathepsins B, L, F, C plus the asparaginyl endopeptidase SmAE and the aspartic peptidase - cathepsin D (of adult worms and some other life stages). The recent computational cluster analysis revealed that the sequence S. mansoni elastase (the main cercarial penetration enzyme) is quite divergent from other serine peptidases of the S1 family. Cercarial elastase gene was proved in S. mansoni, S. haematobium and Schistosomatium douthitti, but not in the related S. japonicum. Mass spectrometry analysis confirmed cercarial..