The rapid detection of procalcitonin in septic serum using immunoaffinity MALDI chips.

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

Protecting Occupational Health and Safety in Construction Companies in the Czech Republic.

Currently it is the third year when the global COVID-19 pandemic is having a negative impact on the lives of individuals, on the activities of economic entities of all sizes and on the economies of countries around the world. Following the partial calming in this area, a crisis linked to the war in Ukraine hit Europe in early 2022. This has a negative impact on economic production and the associated decline in the standard of living. The prices of materials, products and transport are rising, causing a sharp increase in construction prices in the construction sector. Protecting the occupational health of workers and providing a safe environment for their work form an essential part of all construction projects. This article deals with the research into occupational health and safety on construction sites in the Czech Republic. The research described in this article was conducted in several successive steps. In the first step, a research design was drawn up, in the second step data collection was carried out, and in the third step data analysis and compilation of results were carried out. Qualitative approaches to data collection and analysis, namely in-depth interviews and the coding method, were used in the companies under research. Open-ended questions relating to respondents' opinions, experience and overall perceptions of the issues were developed in the preparatory phase of the research. The aim was to obtain credible answers to the questions raised. The research lasted for six months and involved 19 medium and large companies from the Czech Republic. The purpose of the research described in this article was to find out the situation focusing on providing the health and safety of workers during the implementation of construction. The costs of implementing the necessary measures in this field were also investigated.

Hymenal atresia - a rare congenital anomaly with the risk of late diagnosis.

OBJECTIVE: Comprehensive analysis of causes, clinical signs, dia-gnostic process, differential dia-gnosis and therapy of hymenal atresia. METHODS: Literature search using the Web of Science, Google Scholar and PubMed databases with keywords and analysis of articles published in high impact and reviewed journals. RESULTS: Hymenal atresia is a congenital malformation of a womans genitals, which is manifested by complete obstruction of the vaginal introitus by a closed hymen. It should be dia-gnosed in the neonatal period, but clinically it usually manifests itself only during puberty as a result of menstrual blood retention (cryptomenorrhea) with the cyclic abdominal pain at monthly intervals. The therapy is based on optimally timed surgical creation of communication in the hymen (hymenotomy, hymenectomy) enabling free evacuation of menstrual contents. The aim of this simple treatment method is immediate subjective relief from pain and a permanent solution to this congenital anomaly. CONCLUSION: Knowledge of all types of congenital malformations of the female genitalia is a basic condition for an early and effective dia-gnostic process in adolescent girls with abdominal pain. The girl who has not yet menstruated and has cyclic lower abdominal pain and a tumor behind the pubic symphysis should be examined by a specialist in pediatric and adolescent gynecology who will confirm hymenal atresia according to a bluish and closed hymen, and suggest prompt and effective therapy.

Impact of the COVID-19 Pandemic on Construction Companies in the Czech Republic.

The global COVID-19 pandemic has been a part of our lives for the second year in a row and it has affected the activities of all economic entities across individual fields and industries. This article deals with research into the impact of the COVID-19 pandemic on construction companies in the Czech Republic. The research presented in this paper consists of several consecutive steps: designing a research plan, performing data collection and analysis and compiling research results. Qualitative approaches to data collection and evaluation, especially in-depth interviews and the coding method, were used for contextual understanding and comprehension of the situation in the companies under research. Open questions (topics) related to the respondents' experience, perceptions and opinions were created as a part of the preparation process. The aim was to obtain reliable and relevant answers to the questions asked. The research, which lasted for 5 months, involved 16 medium-scale and large companies from the Czech Republic. The aim of the research described in this article was to find out about the development of construction companies in the first half of 2021 and to specify their results in selected areas of activity.

Safety assessment of a novel thermostable phytase.

A novel 6-phytase (Phytase TSP, trade name OptiPhos® PLUS) with improved thermostability has been developed for use in animal feed. The safety of the new phytase was evaluated by testing for genotoxicity and subchronic toxicity. In in vitro and in vivo genotoxicity assays Phytase TSP concentrate was not mutagenic and did not induce biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes. In a subchronic toxicity study, male and female rats administered 100, 500 or 1000 mg/kg body weight/day of Phytase TSP concentrate via oral gavage for 90 days had no mortalities, and no treatment-related effects on body weight, food consumption, clinical observations or ophthalmology. Furthermore, there were no changes in haematology, clinical chemistry, urinalysis, gross pathology, organ weights or histopathology that could be attributed to the test article. Several endpoints exhibited statistically significant effects, but none was dose-related or considered to be of toxicological relevance. Based on these results, Phytase TSP concentrate (OptiPhos® PLUS) was not genotoxic and the No Observed Adverse Effect Level (NOAEL) for male and female rats was 1000 mg/kg body weight/day.

Effect of Alkali-Free Synthesis and Post-Synthetic Treatment on Acid Sites in Beta Zeolites.

Beta zeolites with Si/Al around 14 were prepared using three new alkali-free synthesis methods based on the application of amorphous aluminosilicate precursor and calcined in ammonia or air. All samples exhibit structural and textural properties of standard beta zeolite. Comprehensive study by 27Al and 29Si MAS NMR, together with FTIR adsorption of d3-acetonitrile and pyridine were used to characterize the influence of both the synthesis and calcination procedure on the framework Al atoms and related Brønsted and Lewis acid sites. While calcination in ammonia preserves all framework Al atoms, calcination in air results in 15% release of framework Al, but without restrictions of the accessibility of the beta zeolite channel system for bulky pyridine molecules. Terminal (SiO)3AlOH groups present in the hydrated zeolites were suggested as a precursor of framework Al-Lewis sites. Surprisingly, the mild dealumination of the air-calcined zeolites result in an increase of the concentration of Brønsted acid sites and a decrease of the total concentration of Lewis sites with the formation of the extra-framework ones.

The MEK-ERK-MST1 Axis Potentiates the Activation of the Extrinsic Apoptotic Pathway during GDC-0941 Treatment in Jurkat T Cells.

The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4⁺ T cells to the effector stage. MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies.

Quambalarine B, a Secondary Metabolite from Quambalaria cyanescens with Potential Anticancer Properties.

Quambalarine B (QB) is a secondary metabolite produced by the basidiomycete Quambalaria cyanescens with potential anticancer activity. Here we report that QB at low micromolar concentration inhibits proliferation of several model leukemic cell lines (Jurkat, NALM6, and REH), whereas higher concentrations induce cell death. By contrast, the effect of QB on primary leukocytes (peripheral blood mononuclear cells) is significantly milder with lower toxicity and cytostatic activity. Moreover, QB inhibited expression of the C-MYC oncoprotein and mRNA expression of its target genes, LDHA, PKM2, and GLS. Finally, QB blocked the phosphorylation of P70S6K, a downstream effector kinase in mTOR signaling that regulates translation of C-MYC. This observation could explain the molecular mechanism behind the antiproliferative and cytotoxic effects of QB on leukemic cells. Altogether, our results establish QB as a promising molecule in anticancer treatment.

Planar Functionalized Surfaces for Direct Immunoaffinity Desorption/Ionization Mass Spectrometry.

BACKGROUND: Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay. METHODS: MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script. RESULTS: The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found. CONCLUSIONS: MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.

PAR-2, IL-4R, TGF-ß and TNF-α in bronchoalveolar lavage distinguishes extrinsic allergic alveolitis from sarcoidosis.

Sarcoidosis (SARC) and extrinsic allergic alveolitis (EAA) share certain markers, making a differential diagnosis difficult even with histopathological investigation. In lung tissue, proteinase-activated receptor-2 (PAR-2) is primarily investigated with regard to epithelial and inflammatory perspectives. Varying levels of certain chemokines can be a useful tool for distinguishing EAA and SARC. Thus, in the present study, differences in the levels of transforming growth factor (TGF)-ß1, tumor necrosis factor (TNF)-α, interleukin-4 receptor (IL-4R) and PAR-2 in bronchoalveolar lavage fluid (BALF) were compared, using an ELISA method, between 14 patients with EAA and six patients with SARC. Statistically significant higher levels of IL-4R, PAR-2 and the PAR-2/TGF-ß1 and PAR-2/TNF-α ratios were observed in EAA patients as compared with SARC patients. Furthermore, the ratios of TNF-α/total protein, TGF-ß1/PAR-2 and TNF-α/PAR-2 were significantly lower in EAA patients than in SARC patients. The results indicated a higher detection of PAR-2 in EAA samples in association with TNF-α and TGF-ß levels. As EAA and PAR-2 in parallel belong to the Th2-mediated pathway, the results significantly indicated an association between this receptor and etiology. In addition, the results indicated that SARC is predominantly a granulomatous inflammatory disease, thus, higher levels of TNF-α are observed. Therefore, the detection of PAR-2 and investigated chemokines in BALF may serve as a useful tool in the differential diagnosis between EAA and SARC.

Biodegradation of spilled diesel fuel in agricultural soil: effect of humates, zeolite, and bioaugmentation.

Possible enhancement of biodegradation of petroleum hydrocarbons in agricultural soil after tank truck accident (~5000 mg/kg dry soil initial concentration) by bioaugmentation of diesel degrading Pseudomonas fluorescens strain and addition of abiotic additives (humates, zeolite) was studied in a 9-month pot experiment. The biodegradation process was followed by means of analytical parameters (hydrocarbon index expressed as content of C10-C40 aliphatic hydrocarbons, ratio pristane/C17, and total organic carbon content) and characterization of soil microbial community (content of phospholipid fatty acids (PLFA) as an indicator of living microbial biomass, respiration, and dehydrogenase activity). The concentration of petroleum hydrocarbons (C10-C40) was successfully reduced by ~60% in all 15 experiment variants. The bioaugmentation resulted in faster hydrocarbon elimination. On the contrary, the addition of humates and zeolite caused only a negligible increase in the degradation rate. These factors, however, affected significantly the amount of PLFA. The humates caused significantly faster increase of the total PLFA suggesting improvement of the soil microenvironment. Zeolite caused significantly slower increase of the total PLFA; nevertheless it aided in homogenization of the soil. Comparison of microbial activities and total PLFA revealed that only a small fraction of autochthonous microbes took part in the biodegradation which confirms that bioaugmentation was the most important treatment.

Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids.

High transformation competency of Escherichia coli is one of the critical factors in the bacterial artificial chromosome (BAC)-based DNA library construction. Many electroporation protocols have been published until now, but the majority of them was optimized for transformation of small plasmids. Large plasmids with a size above 50 kbp display reduced transformation efficiency and thereby require specific conditions in the preparation and electroporation of electrocompetent cells. In the present work, we have optimized the parameters critical to the application of BAC DNA electrotransformation into E. coli. Systematic evaluation of electroporation variables has revealed several key factors like temperature of growth, media supplements, washing buffer, and cell concentration. Improvements made in the transformation protocol have led to electrocompetent cells with transformation efficiency up to 7 × 10(8) transformants per microgram of 120 kbp BAC plasmid DNA. We have successfully used in-house prepared competent cells, the quality of which is comparable with those produced by different companies, in the construction of metagenomic libraries from the soil. Our protocol can also be beneficial for other application with limited DNA source.

Detection of the GPI-anchorless prion protein fragment PrP226* in human brain.

BACKGROUND: The accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2. METHODS: We developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot. RESULTS: Our results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD). CONCLUSIONS: In the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.

Mechanism of framework oxygen exchange in Fe-zeolites: a combined DFT and mass spectrometry study.

The role of framework oxygen atoms in N(2)O decomposition [N(2)O(g)→N(2)(g) and 1/2O(2)(g)] over Fe-ferrierite is investigated employing a combined experimental (N(2)(18)O decomposition in batch experiments followed by mass spectroscopy measurements) and theoretical (density functional theory calculations) approach. The occurrence of the isotope exchange indicates that framework oxygen atoms are involved in the N(2)O decomposition catalyzed by Fe-ferrierite. Our study, using an Fe-ferrierite sample with iron exclusively present as Fe(II) cations accommodated in the cationic sites, shows that the mobility of framework oxygen atoms in the temperature range: 553 to 593 K is limited to the four framework oxygen atoms of the two AlO(4)(-) tetrahedra forming cationic sites that accomodate Fe(II). They exchange with the Fe extra-framework (18)O atom originating from the decomposed N(2)(18)O. We found, using DFT calculations, that O(2) molecules facilitate the oxygen exchange. However, the corresponding calculated energy barrier of 87 kcal mol(-1) is still very high and it is higher than the assumed experimental value based on the occurrence of the sluggish oxygen exchange at 553 K.

Disorders of balance and gait in essential tremor are associated with midline tremor and age.

Disorders of balance and gait have been observed in patients with essential tremor (ET), but their association with tremor severity remains unclear. This study aimed to evaluate postural instability and gait changes in ET patients and to investigate their relationship to tremor characteristics with regard to cerebellar dysfunction as a possible common pathogenetic mechanism in ET. Thirty ET patients (8F, mean (SD) age 55.8 (17.8), range 19-81 years) and 25 normal controls (7F, 53.0 (17.7), 19-81) were tested with the scales of Activities-specific Balance Confidence (ABC), Fullerton Advanced Balance (FAB), and International Cooperative Ataxia Rating Scale (ICARS). Posturography and gait were assessed using a Footscan® system. Tremor was evaluated by the Fahn-Tolosa-Marin Tremor Rating Scale (TRS) and accelerometry in five upper limb positions. A mean (SD) TRS sum score of 27.0 (13.2) corresponded to mild to moderate tremor severity in most patients. In comparison with controls, ET subjects exhibited lower tandem gait velocity (0.21 vs. 0.26 m/s, P = 0.028), more missteps (0.57 vs. 0.12, P = 0.039), and increased postural sway in tandem stance (sway area 301.1 vs. 202.9 mm(2), P = 0.045). In normal gait, step width increased with the midline tremor subscore of TRS (Pearson r = 0.60, P = 0.046). Moreover, significant correlations were found between age and quantitative measures of normal and tandem gait in ET patients but not in controls. ABC, FAB, and ICARS scores did not significantly differ between patients and controls. In conclusion, gait and balance alterations in ET patients occur even without subjective complaints. Their relationship with midline tremor and dependence on age suggest a connection with cerebellar dysfunction.

Gerstmann-Sträussler-Scheinker syndrome with the P102L pathogenic mutation presenting as familial Creutzfeldt-Jakob disease: a case report and review of the literature.

Gerstmann-Sträussler-Scheinker syndrome is a rare autosomal dominant disease caused by a mutation in the prion gene, usually manifesting as progressive ataxia with late cognitive decline. A 44-year-old woman with a positive family history developed early personality and behavior changes, followed by paresthesias and ataxia, later associated with memory problems, pyramidal signs, anosognosia and very late myoclonus, spasticity, and severe dysexecutive impairment. Magnetic resonance showed caudate, mesio-frontal, and insular hyper-intensities, electroencephalography revealed generalized triphasic periodic complexes. A pathogenic P102L mutation in the prion gene was detected. Our case differed from classical Gerstmann-Sträussler-Scheinker syndrome by rapid progression, severe dementia, abnormal electroencephalography and magnetic resonance findings, which were highly suggestive of familial Creutzfeldt-Jakob disease.

The assessment of pathogenic prions in the brains of eye tissue donors: 2-years experience in the Czech Republic.

PURPOSE: The aim of this study was to assess the presence of pathogenic prions in the brain tissue of eye donors and to evaluate the benefits of 2-year obligatory testing in the Czech Republic. METHODS: Brain tissue was retrieved during autopsies of eye donors of 3 tissue banks in the Czech Republic. The frozen specimens obtained from the frontal lobe were transported to the Czech National Reference Laboratory for the diagnosis of human prion disorders. The presence of pathogenic prions was tested using the Prionics-Check WESTERN kit. Confirmative Western blotting using 1 of 2 different clones of monoclonal anti-PrP antibody was performed as well. RESULTS: No pathogenic prions were found in any of the 1142 tested specimens. One specimen revealed weak positivity at initial screening; however, repeated examination of the specimen and other specimens from different locations in the brain of the same donor did not confirm the presence of pathogenic prions. The negative result was confirmed by the National CJD Surveillance Unit, University of Edinburgh, United Kingdom. CONCLUSION: The absence of pathogenic prions from all of the 1142 tested specimens corresponds to the presumed very low risk of transmission of Creutzfeldt-Jakob disease through corneal graft transplantation. As a result of this disorder's rarity, a larger series of tested samples should be evaluated to obtain statistically significant findings. Although such testing increases the safety of donor eye tissue, it also increases the expense, causes organizational difficulties, and may extend the time needed to release the tissue for grafting.

Leptospira interrogans binds to human cell surface receptors including proteoglycans.

Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.

MicroRNA involvement in glioblastoma pathogenesis.

MicroRNAs are endogenously expressed regulatory noncoding RNAs. Altered expression levels of several microRNAs have been observed in glioblastomas. Functions and direct mRNA targets for these microRNAs have been relatively well studied over the last years. According to these data, it is now evident, that impairment of microRNA regulatory network is one of the key mechanisms in glioblastoma pathogenesis. MicroRNA deregulation is involved in processes such as cell proliferation, apoptosis, cell cycle regulation, invasion, glioma stem cell behavior, and angiogenesis. In this review, we summarize the current knowledge of miRNA functions in glioblastoma with an emphasis on its significance in glioblastoma oncogenic signaling and its potential to serve as a disease biomarker and a novel therapeutic target in oncology.