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.
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.
Although triple labeling of molecular beacons has been documented to improve quenching efficiencies and studies generally assume similar benefits at long TaqMan probes, a limited number of works have studied this issue in TaqMan probes. We therefore prepared a series of long triple-labeled oligodeoxynucleotide probes with 6-carboxyfluorescein as a fluorophore at the 5'-end and BlackBerry (BBQ-650) or azaphthalocyanine quenchers at the 3'-end and in the intrastrand position and systematically compared their quenching efficiencies with those of the corresponding double-labeled probes including important control probes. A model polymerase chain reaction (PCR) assay enabled the determination of the quenching efficiencies of static and Förster resonance energy transfer (FRET) quenching in the target probes. The type of probe had no effect on the static quenching ability. Importantly, FRET quenching of double-labeled probes with a quencher at the 3'-end showed a statistically insignificant difference from the control probe without any quencher, indicating the need to shift the quencher closer to the fluorophore in long probes. Shortening the distance between the fluorophore and the quencher played a key role in FRET quenching, whereas the introduction of an additional quencher only slightly improved the quenching efficiency. BBQ-labeled probes had lower quenching efficiencies than azaphthalocyanine probes. The methodologies and relationships described above seem, however, to be universal and applicable to any quencher.
Microbial strains isolated from extreme and understudied environments, such as caves, are still poorly investigated for the production of bioactive secondary metabolites. Investigation of the ethyl acetate extract from the growth medium produced by the soil-derived fungus Aspergillus sp. SDC28, isolated from a Brazilian cave, yielded two anthraquinones: versicolorin C (1) and versiconol (2). The complete assignment of nuclear magnetic resonance and mass spectroscopic data of 1 and 2 was performed for the first time. Moreover, the yet unreported absolute configuration of both compounds was unambiguously established by analysis of experimental and theoretical electronic circular dichroism data. Vibrational circular dichroism was also applied to confirm the absolute stereochemistry of 2. Compounds 1 and 2 showed cytotoxic activity against human ovarian cancer cells (OVCAR3).
- MeSH
- Anthraquinones pharmacology MeSH
- Apoptosis MeSH
- Aspergillus chemistry metabolism MeSH
- Circular Dichroism MeSH
- Caves * MeSH
- Humans MeSH
- Molecular Structure MeSH
- Cell Line, Tumor MeSH
- Ovarian Neoplasms * MeSH
- Oligodeoxyribonucleotides MeSH
- Soil MeSH
- Thionucleotides MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Brazil MeSH
The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (Tm) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.
The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.
- MeSH
- Acetonitriles chemistry MeSH
- Aza Compounds * analysis chemistry MeSH
- Fluorescent Dyes * analysis chemistry MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Molecular Probes * analysis chemistry MeSH
- Oligodeoxyribonucleotides * analysis chemistry MeSH
- Solvents MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article 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
- Genes, Bacterial * MeSH
- Nucleic Acid Denaturation MeSH
- DNA, Bacterial chemistry genetics MeSH
- DNA Primers MeSH
- Disease Outbreaks MeSH
- Escherichia coli classification genetics isolation & purification MeSH
- Genome, Bacterial MeSH
- Genotyping Techniques * MeSH
- Escherichia coli Infections microbiology MeSH
- Polymorphism, Single Nucleotide * MeSH
- Multilocus Sequence Typing methods MeSH
- Computer Simulation MeSH
- Polymerase Chain Reaction methods MeSH
- Repetitive Sequences, Nucleic Acid MeSH
- Whole Genome Sequencing MeSH
- Population Surveillance MeSH
- Bacterial Typing Techniques * MeSH
- Base Composition MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg2+. The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management.
- MeSH
- Antiviral Agents * chemistry pharmacokinetics pharmacology MeSH
- Aptamers, Nucleotide * chemistry pharmacokinetics pharmacology MeSH
- HeLa Cells MeSH
- Drug Delivery Systems * MeSH
- Humans MeSH
- Uterine Cervical Neoplasms drug therapy metabolism MeSH
- Oligodeoxyribonucleotides * chemistry pharmacokinetics pharmacology MeSH
- Cell Proliferation drug effects MeSH
- Antineoplastic Agents * chemistry pharmacokinetics pharmacology MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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 enzymology genetics MeSH
- Bacteroidetes enzymology genetics MeSH
- Bacterial Proteins genetics metabolism MeSH
- CRISPR-Associated Proteins metabolism MeSH
- CRISPR-Cas Systems * MeSH
- Dimerization MeSH
- DNA Primers biosynthesis MeSH
- DNA-Directed DNA Polymerase genetics metabolism MeSH
- DNA Primase genetics metabolism MeSH
- Escherichia coli metabolism MeSH
- Gene Expression MeSH
- Phylogeny MeSH
- Mutation MeSH
- Prokaryotic Cells metabolism MeSH
- Recombinant Proteins MeSH
- Ribonucleotides metabolism MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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 enzymology genetics isolation & purification MeSH
- Bacteriological Techniques * MeSH
- beta-Lactamases genetics metabolism MeSH
- beta-Lactam Resistance genetics MeSH
- DNA, Bacterial genetics MeSH
- DNA Primers MeSH
- Humans MeSH
- Polymerase Chain Reaction * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
