Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 μl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.

• MeSH
• analýza nákladů a výnosů MeSH
• buněčné kultury metody   ekonomika   MeSH
• komplementární DNA * genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• leukocyty mononukleární cytologie   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• stanovení celkové genové exprese metody   ekonomika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.

• MeSH
• komplementární DNA MeSH
• lidé MeSH
• nanopórové sekvenování * MeSH
• opičí neštovice * MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• dataset MeSH

CONTEXT: Testing for BCR-ABL1 kinase domain (KD) mutations should always be performed before tyrosine kinase inhibitor (TKI) changes. Next-generation sequencing (NGS) is the best approach to highlight emerging mutations in patients not responding adequately to TKI therapy. However, NGS requires sample centralization and batch analysis and has a non-negligible time to results. In this study, we set up and validated a novel droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harboring mutations impacting TKI selection. METHODS: In collaboration with Bio-Rad, a 3-tube ddPCR strategy was designed that enables identification and quantitation of 16 nucleotide substitutions encoding the 13 mutations associated with resistance to one or more second-generation TKI (2GTKI). Primers and FAM-or FAM/HEX-labelled probes were grouped on a TKI-specific basis and generated clusters of droplets mapping to spatially distinct areas of the 2D plot based on resistance profiles. Each tube also incorporated primers and HEX-labelled probes for e13a2, e14a2, and e1a2 BCR::ABL1 fusion transcripts to express results as the percentage of mutation-positive over total BCR::ABL1 transcripts. For validation, a total of 101 RNA samples from healthy donors, TKI-sensitive and -resistant patients, and BCR::ABL1-positive and -negative cell lines were used. cDNA (125 ng) obtained with ABL1-specific primers was analyzed in duplicate on a QX200 ddPCR system (Bio-Rad). RESULTS: The limit of blank was determined using 60 blank samples. Accuracy and specificity were confirmed using 48 samples positive for one or more 2GTKI-resistant mutations or mutations at nearby codons (to exclude cross-reactivity). Analysis of serial dilutions of cell line mixtures made using BCR::ABL1-positive mutation-positive cells, BCR::ABL1-positive unmutated cells, and BCR::ABL1-negative cells to mimic different mutation frequencies (70%, 5%, and 0.5%) and different transcript levels (MR0 to MR3) showed that a 0.5% lower detection limit could be consistently achieved irrespective of BCR::ABL1 levels. CONCLUSIONS: ddPCR proved highly sensitive and accurate. Total hands-on time was approximately 2 hrs, and time from sample to results was 2 days. Therefore, ddPCR may be integrated into diagnostic algorithms of CML (and Ph+ ALL) patients as a convenient first-level screening tool for mutations impacting TKI selection.

• MeSH
• bcr-abl fúzní proteiny * genetika   metabolismus   MeSH
• chemorezistence genetika   MeSH
• chronická myeloidní leukemie * diagnóza   farmakoterapie   genetika   MeSH
• inhibitory proteinkinas farmakologie   terapeutické užití   MeSH
• komplementární DNA farmakologie   MeSH
• lidé MeSH
• mutace MeSH
• nukleotidy MeSH
• polymerázová řetězová reakce MeSH
• RNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Wilt (Fusarium oxysporum f. sp. lentis; Fol) is one of the major diseases of lentil worldwide. Two hundred and thirty-five isolates of the pathogen collected from 8 states of India showed substantial variations in morphological characters such as colony texture and pattern, pigmentation and growth rate. The isolates were grouped as slow (47 isolates), medium (118 isolates) and fast (70 isolates) growing. The macroconidia and microconidia (3.0-77.5 × 1.3-8.8 μm for macroconidia and 1.8-22.5 × 0.8-8.0 μm for microconidia for length × width) were variable in size and considering the morphological features, the populations were grouped into 12 categories. Seventy representative isolates based on their morphological variability and place of origin were selected for further study. A set of 10 differential genotypes was identified for virulence analysis and based on virulence patterns on these 10 genotypes, 70 Fol isolates were grouped into 7 races. Random amplified polymorphic DNA (RAPD), universal rice primers (URPs), inter simple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) were used for genetic diversity analysis. URPs, ISSR and SRAP markers gave 100% polymorphism while RAPD gave 98.9% polymorphism. The isolates were grouped into seven clusters at genetic similarities ranging from 21 to 80% using unweighted paired group method with arithmetic average analysis. The major clusters include the populations from northern and central regions of India in distinct groups. All these three markers proved suitable for diversity analysis, but their combined use was better to resolve the area specific grouping of the isolates. The sequences of rDNA ITS and TEF-1α genes of the representative isolates were analysed. Phylogenetic analysis of ITS region grouped the isolates into two major clades representing various races. In TEF-1α analysis, the isolates were grouped into two major clades with 28 isolates into one clade and 4 remaining isolates in another clade. The molecular groups partially correspond to the lentil growing regions of the isolates and races of the pathogen.

• MeSH
• biologická variabilita populace MeSH
• Cicer * MeSH
• čočka * genetika   MeSH
• DNA primery MeSH
• Fusarium * genetika   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• nemoci rostlin MeSH
• ribozomální DNA MeSH
• technika náhodné amplifikace polymorfní DNA MeSH
• Publikační typ
• časopisecké články MeSH

During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.

• MeSH
• DNA primery metabolismus   MeSH
• DNA-primasa * metabolismus   MeSH
• DNA genetika   MeSH
• katalytická doména MeSH
• replikace DNA * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

• MeSH
• bakteriální RNA MeSH
• dospělí MeSH
• esenciální geny * MeSH
• exprese genu * MeSH
• komplementární DNA MeSH
• kontaminace DNA MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• RNA izolace a purifikace   MeSH
• sliny metabolismus   MeSH
• stanovení celkové genové exprese metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores showed solvatochromic fluorescence with strong fluorescence at 433-457 nm in non-polar solvents and very weak fluorescence at 567 nm in alcohols. The nucleosides and nucleotides also showed only negligible fluorescence in alcohols or water. The triphosphates were substrates for DNA polymerase in the enzymatic synthesis of modified DNA probes that showed only very weak fluorescence in aqueous buffer but a significant light-up and blue shift were observed when they interacted with proteins (histone H3.1 or p53 for double-stranded DNA probes or single-strand binding protein for single-stranded oligonucleotide probes). Hence, nucleotides have good potential in the construction of DNA sensors for studying protein-DNA interactions. The modified dNTPs were also transported into cells using a cyclodextrin-based transporter but they were not incorporated into the genomic DNA.

• MeSH
• chromony chemie   MeSH
• DNA sondy chemie   MeSH
• DNA chemie   MeSH
• enzymy chemie   MeSH
• fluorescence MeSH
• fluorescenční barviva chemie   MeSH
• nukleotidy chemie   MeSH
• proteiny chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Routinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726-0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726-0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones'.

• MeSH
• bakteriální geny * MeSH
• denaturace nukleových kyselin MeSH
• DNA bakterií chemie   genetika   MeSH
• DNA primery MeSH
• epidemický výskyt choroby MeSH
• Escherichia coli klasifikace   genetika   izolace a purifikace   MeSH
• genom bakteriální MeSH
• genotypizační techniky * MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• jednonukleotidový polymorfismus * MeSH
• multilokusová sekvenční typizace metody   MeSH
• počítačová simulace MeSH
• polymerázová řetězová reakce metody   MeSH
• repetitivní sekvence nukleových kyselin MeSH
• sekvenování celého genomu MeSH
• surveillance populace MeSH
• techniky typizace bakterií * MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

• MeSH
• Bacteria enzymologie   genetika   MeSH
• Bacteroidetes enzymologie   genetika   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Cas proteiny metabolismus   MeSH
• CRISPR-Cas systémy * MeSH
• dimerizace MeSH
• DNA primery biosyntéza   MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• DNA-primasa genetika   metabolismus   MeSH
• Escherichia coli metabolismus   MeSH
• exprese genu MeSH
• fylogeneze MeSH
• mutace MeSH
• prokaryotické buňky metabolismus   MeSH
• rekombinantní proteiny MeSH
• ribonukleotidy metabolismus   MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.

• MeSH
• Bacteria enzymologie   genetika   izolace a purifikace   MeSH
• bakteriologické techniky * MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• beta-laktamová rezistence genetika   MeSH
• DNA bakterií genetika   MeSH
• DNA primery MeSH
• lidé MeSH
• polymerázová řetězová reakce * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH