UNLABELLED: The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 μL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used (P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE: Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.

• MeSH
• Molecular Diagnostic Techniques standards   methods   MeSH
• DNA, Fungal * blood   genetics   MeSH
• Real-Time Polymerase Chain Reaction * standards   methods   MeSH
• Humans MeSH
• Mucorales * genetics   isolation & purification   MeSH
• Mucormycosis * diagnosis   microbiology   blood   MeSH
• Sensitivity and Specificity * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH

Atlas a přehled, který se zaměřuje na biochemii a fyziologii člověka. Určeno odborné veřejnosti.; Biochemie člověka přehledně na 223 barevných schématech. Grafické znázornění je základem knihy – texty slouží především k rozšíření a doplnění legendy k vyobrazením. Autoři krátce uvádějí čtenáře do hlavní problematiky chemie a biochemie, zmiňují provázanost mezi chemickou strukturou a biologickou funkcí nebo patologickými procesy. V knize čtenář najde aktuální informace, vývoj a poznatky z oboru, strukturu důležitých molekul a mnohé další informace. Nejvíce místa je v tomto atlasu samozřejmě věnováno biochemii člověka, zahrnuje však i biochemii dalších živočichů, rostlin a mikroorganismů. Didaktické přednosti, které je nutné ocenit: * Efektivní poměr mezi barevnou grafikou a vysvětlujícím textem. * Unifikované barevné zobrazení atomů, koenzymů, chemických tříd a buněčných organel, umožňující rychlou orientaci a pochopení ve všech zobrazených systémech. * Moderní zobrazení mnoha důležitých molekul. * Rychlé orientaci pomáhá barevné kódování a užití různých symbolů, vysvětlivky jsou umístěny na vnitřních stranách obálky. O úspěšnosti knihy vypovídá i seznam předchozích vydání: české 2012; německé 1994, 1997, 2003, 2009; francouzské 1994, 1999, 2004, 2011; anglické 1996, 2004, 2012; japonské 1997, 2007, 2015; portugalské 2005, 2013; ruské 2000, 2017; řecké 1999, 2007; španělské 2004, 2012; turecké 2002, 2016; čínské 2008; indonéské 2002; italské 1997; korejské 2008; nizozemské 2004; polské 2005.

• MeSH
• Biochemistry MeSH
• Physiology MeSH
• Publication type
• Atlas MeSH
• Review MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• biochemie

The aim of this study is to evaluate opportunistic pathogenic bacteria of the genus Pseudomonas in anthropogenically impacted bathing waters, primarily focusing on bathing ponds. The findings include the detection of these bacteria, their susceptibility to selected antibiotics, and the determination of the Exotoxin A (exoA) gene using PCR method. P. aeruginosa was present in most samples, albeit in low concentrations (1-14 CFU/100 mL). The presence of P. otitidis, which is associated with ear infection, in this type of bathing water, was not rare (up to 90 CFU/100 mL). This species would not be detected by the standard methods, including tests on acetamid medium, used for P. aeruginosa in water. The isolated strains of P. otitidis lack the exoA gene and exhibited higher resistance to meropenem compared to P. aeruginosa.

• MeSH
• ADP Ribose Transferases genetics   MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Drug Resistance, Bacterial MeSH
• Bacterial Proteins genetics   MeSH
• Bacterial Toxins genetics   MeSH
• Pseudomonas aeruginosa Exotoxin A MeSH
• Exotoxins genetics   MeSH
• Virulence Factors genetics   MeSH
• Microbial Sensitivity Tests * MeSH
• Water Microbiology * MeSH
• Polymerase Chain Reaction MeSH
• Pseudomonas * genetics   isolation & purification   classification   drug effects   MeSH
• Ponds * microbiology   MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Cell cycle progression and leukemia development are tightly regulated processes in which even a small imbalance in the expression of cell cycle regulatory molecules and microRNAs (miRNAs) can lead to an increased risk of cancer/leukemia development. Here, we focus on the study of a ubiquitous, multifunctional, and oncogenic miRNA-hsa-miR-155-5p (miR-155, MIR155HG), which is overexpressed in malignancies including chronic lymphocytic leukemia (CLL). Nonetheless, the precise mechanism of how miR-155 regulates the cell cycle in leukemic cells remains the subject of extensive research. METHODS: We edited the CLL cell line MEC-1 by CRISPR/Cas9 to introduce a short deletion within the MIR155HG gene. To describe changes at the transcriptome and miRNome level in miR-155-deficient cells, we performed mRNA-seq/miRNA-seq and validated changes by qRT-PCR. Flow cytometry was used to measure cell cycle kinetics. A WST-1 assay, hemocytometer, and Annexin V/PI staining assessed cell viability and proliferation. RESULTS: The limited but phenotypically robust miR-155 modification impaired cell proliferation, cell cycle, and cell ploidy. This was accompanied by overexpression of the negative cell cycle regulator p21/CDKN1A and Cyclin D1 (CCND1). We confirmed the overexpression of canonical miR-155 targets such as PU.1, FOS, SHIP-1, TP53INP1 and revealed new potential targets (FCRL5, ISG15, and MX1). CONCLUSIONS: We demonstrate that miR-155 deficiency impairs cell proliferation, cell cycle, transcriptome, and miRNome via deregulation of the MIR155HG/TP53INP1/CDKN1A/CCND1 axis. Our CLL model is valuable for further studies to manipulate miRNA levels to revert highly aggressive leukemic cells to nearly benign or non-leukemic types.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * genetics   pathology   MeSH
• Cyclin D1 genetics   metabolism   MeSH
• Cyclin-Dependent Kinase Inhibitor p21 * genetics   metabolism   MeSH
• Cell Cycle Checkpoints * genetics   MeSH
• Humans MeSH
• MicroRNAs * genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Cell Proliferation genetics   MeSH
• Heat-Shock Proteins MeSH
• Gene Expression Regulation, Leukemic MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The spread of multidrug-resistant Escherichia coli in healthcare facilities is a global challenge. Hospital-acquired infections produced by Escherichia coli include gastrointestinal, blood-borne, urinary tract, surgical sites, and neonatal infections. Therefore, novel approaches are needed to deal with this pathogen and its rising resistance. The concept of attenuating virulence factors is an alternative strategy that might lead to low levels of resistance and combat this pathogen. A sub-inhibitory concentration (1⁄4 MIC) of sitagliptin and nitazoxanide was used for phenotypic assessments of Escherichia coli virulence factors such as biofilm production, swimming motility, serum resistance, and protease production. Moreover, qRT-PCR was used to determine the impact of sub-MIC of the tested drugs on the relative expression levels of papC, fimH, fliC, kpsMTII, ompT_m, and stcE genes encoding virulence factors in Escherichia coli. Also, an in vivo model was conducted as a confirmatory test. Phenotypically, our findings demonstrated that the tested strains showed a significant decrease in all the tested virulence factors. Moreover, the genotypic results showed a significant downregulation in the relative expression levels of all the tested genes. Besides, the examined drugs were found to be effective in protecting mice against Escherichia coli pathogenesis. Sitagliptin and nitazoxanide exhibited strong anti-virulence activities against Escherichia coli. In addition, it is recommended that they might function as adjuvant in the management of Escherichia coli infections with either conventional antimicrobial agents or alone as alternative treatment measures.

• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Biofilms drug effects   MeSH
• Nitro Compounds MeSH
• Escherichia coli * drug effects   pathogenicity   genetics   MeSH
• Virulence Factors genetics   metabolism   MeSH
• Escherichia coli Infections * drug therapy   microbiology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial MeSH
• Mice MeSH
• Escherichia coli Proteins genetics   MeSH
• Sitagliptin Phosphate * pharmacology   MeSH
• Thiazoles * pharmacology   MeSH
• Virulence drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

In 2019, Pantoea piersonii was initially isolated from the interior surfaces of the International Space Station. This microorganism is a species within the genus Pantoea in the family Erwiniaceae, belonging to the order Enterobacterales. Recent literature has documented four cases of its isolation. Despite initial predictions suggesting the non-pathogenicity of P. piersonii strains, evidence from observed cases indicates potential pathogenicity. According to documented evidence in the literature, this microorganism is capable of causing severe and life-threatening conditions, including sepsis. Traditional tests, as well as automated systems, may fail to provide complete differentiation due to these similarities. While MALDI-TOF MS is a valuable tool for identification in clinical diagnostic microbiology, sequencing may be necessary for precise identification. To determine the antibiotic susceptibility profile, various methods can be utilized, including minimum inhibitory concentration determination, disk diffusion testing (Kirby-Bauer test), genotypic resistance assays (PCR and sequencing), and automated systems. The literature reports a limited number of cases associating P. piersonii with human infection. This study contributes to this body of knowledge by reporting a novel case in which P. piersonii was isolated from a tissue sample for the first time. In this case report, the patient achieved recovery following the administration of appropriate antibiotic treatment based on the diagnosis. It underscores the need for precise identification and understanding of its pathogenicity.

• MeSH
• Anti-Bacterial Agents * pharmacology   therapeutic use   MeSH
• Enterobacteriaceae Infections * microbiology   diagnosis   drug therapy   MeSH
• Humans MeSH
• Microbial Sensitivity Tests * MeSH
• Pantoea * isolation & purification   genetics   pathogenicity   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521 bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500 pg/μL. DNA concentration as low as 10 pg/μL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100 pg/μL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

• MeSH
• Alcohol Oxidoreductases * genetics   MeSH
• Bacillus subtilis * genetics   isolation & purification   enzymology   MeSH
• Bacterial Proteins genetics   MeSH
• Molecular Diagnostic Techniques * methods   MeSH
• Colorimetry * methods   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Soil Microbiology MeSH
• Sensitivity and Specificity MeSH
• Nucleic Acid Amplification Techniques * methods   MeSH
• Publication type
• Journal Article MeSH

In this study, lactic acid bacteria (LAB) isolation from fermented foods and molecular identification using magnetic bead technology were performed. And then exopolysaccharide (EPS) production possibility was tested in agar medium, and the positive ones were selected for the next step. The bacteria that could produce higher carbohydrate level were grown in MRS medium fortified with whey and pumpkin waste. In our study, 19 different LAB species were identified from fermented products collected from different places in Hatay (Türkiye) province. In molecular identification, universal primer pairs, p806R/p8FPL, and PEU7/DG74 were used for PCR amplification. After that, PCR products purified using paramagnetic bead technology were sequenced by the Sanger sequencing method. The dominant species, 23.8% of the isolates, were identified as Lactiplantibacillus plantarum. As a technological property of LAB, exopolysaccharide production capability of forty-two LAB isolate was tested in agar medium, and after eleven isolates were selected as positive. Two LAB (Latilactobacillus curvatus SHA2-3B and Loigolactobacillus coryniformis SHA6-3B) had higher EPS production capability when they were grown in MRS broth fortified with pumpkin waste and whey. The highest EPS content (1750 mg/L glucose equivalent) was determined in Loigolactobacillus coryniformis SHA6-3B grown in MRS broth fortified with 10% pumpkin waste. Besides the produced EPS samples were validated with FTIR and SEM methods.

• MeSH
• Polysaccharides, Bacterial * biosynthesis   metabolism   MeSH
• Cucurbita microbiology   MeSH
• Fermentation MeSH
• Fermented Foods * microbiology   MeSH
• Phylogeny MeSH
• Culture Media chemistry   MeSH
• Lactobacillales * isolation & purification   classification   genetics   metabolism   MeSH
• Waste Products * analysis   MeSH
• Food Microbiology * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Whey MeSH
• Publication type
• Journal Article MeSH

Laboratorní diagnostika infekčního agens je založena na metodách přímého průkazu (kultivace, mikroskopie, PCR, antigenní test) a na metodách nepřímého průkazu (detekce protilátek). Sérologie zůstává důležitou součástí laboratorní diagnostiky infekčních onemocnění, přestože význam některých sérologických testů klesá s rozvojem molekulárně biologických metod. Pro pochopení, jakou roli hrají protilátky v procesu imunitní odpovědi na infekční agens, je nutné znát obecné mechanismy, které jsou s tvorbou protilátek spojené. Interpretace sérologických výsledků je složitá a závisí na fázi imunitní odpovědi, antigenních vlastnostech patogenu, použité metodě, senzitivitě a specificitě testů a klinickém kontextu konkrétního pacienta. Správná interpretace sérologických testů vyžaduje hlubokou znalost patogeneze infekce (specifická reakce na bakterie, viry nebo prvoky), metodických limitací a klinických souvislostí, což je zásadní pro efektivní diagnostiku a léčbu pacientů v klinické praxi.

Martinek J, Lochmanová A, Maďar R. Interpretation of serological results in the diagnosis of infectious diseases Laboratory diagnosis of infectious agents is based on direct detection methods (culture, microscopy, PCR, antigen test) and indirect detection methods (antibody detection). Serology remains an important part of the laboratory diagnosis of infectious diseases, although the importance of some serological tests is declining with the development of molecular biological methods. To understand the role of antibodies in the immune response to infectious agents, it is necessary to know the general mechanisms involved in antibody production. Interpretation of serological results is complex and depends on the phase of the immune response, the antigenic properties of the pathogen, the method used, the sensitivity and specificity of the tests, and the clinical context of the individual patient. Correct interpretation of serological tests requires an in-depth knowledge of the pathogenesis of infection (specific response to bacteria, viruses or protozoa), methodological limitations and clinical context, which is essential for effective diagnosis and treatment of patients in clinical practice.

BackgroundDuring the COVID-19 pandemic, non-pharmaceutical interventions (NPIs) such as social distancing, lockdowns and enhanced hygiene led to a decrease in respiratory pathogens. However, as NPIs were relaxed, a resurgence in several respiratory pathogens was observed including one local Chlamydia pneumoniae outbreak in Switzerland, prompting the need for a better understanding of C. pneumoniae epidemiology.AimTo assess temporal and geographical variations in C. pneumoniae detection before, during and after the COVID-19 pandemic.MethodsData on C. pneumoniae PCR detection ratios (number of positive tests/ total number of tests) across pre-pandemic (2018-2019), pandemic (2020-2022) and post-pandemic (2023) periods were collected via a global survey disseminated through various professional networks.ResultsC. pneumoniae detection ratios were analysed across 28 sites (27 in Europe, one in Taiwan) in 2023 (Dataset A, n = 172,223 tests) and 20 sites from 2018 to 2023 (Dataset B, n = 693,106 tests). Twenty-seven sites were laboratories (hospital or clinical) and one a surveillance system (Denmark). A significant decrease in detection ratios was observed during the pandemic period (from 1.05% to 0.23%, p < 0.001). In 2023, detection ratios increased to 0.28% (p < 0.002). Notable regional variations were found, with statistically significant increases in detection ratios at six sites located in Switzerland and Slovenia, where ratios ranged from 0.52% to 3.25%.DiscussionThe study highlights how NPIs influenced C. pneumoniae epidemiology, with reduced detection during the pandemic and partial resurgence afterwards. Regional variations suggest differing NPI impacts and underscore the need for continued surveillance.

• MeSH
• Chlamydophila pneumoniae * isolation & purification   genetics   MeSH
• COVID-19 * epidemiology   MeSH
• Chlamydophila Infections * epidemiology   diagnosis   MeSH
• Humans MeSH
• Pandemics MeSH
• Polymerase Chain Reaction MeSH
• SARS-CoV-2 MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH
• Taiwan MeSH