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Introduction: Intersectional hybrids in lilies possess significant breeding value, but the lack of complete lily genomes and complex genotypes pose challenges for early identification of lily hybrids. This study aimed to use intersectional hybrid cultivars as female parents and wild lilies as male parents to facilitate early identification of hybrid offsprings and enhance the efficiency and convenience of the process. Methods: We investigated the nature of cross combinations using Non-denaturing Fluorescence In Situ Hybridization (ND-FISH) and Genomic In Situ Hybridization (GISH) techniques. Three novel oligonucleotide probes—Oligo-pTa794, Oligo-pITS and Oligo-telo—were developed for lily chromosome research. Results: Our results demonstrated successful hybridization between wild lilies and intersectional hybrid cultivars, producing a total of 130 hybrid progenies. The combination of ND-FISH and GISH analyses effectively revealed the genomic composition of the hybrid progeny and determined the parental origin of specific chromosomes. Discussion: This research provides significant guidance for lily breeding practices and offers a valuable reference for the application of ND-FISH and GISH techniques in interspecific hybridization breeding and molecular cytogenetic research across various plant species. The methods developed enable more precise, efficient, and convenient identification of hybrid offsprings. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Single‐nucleotide variants (SNVs) are crucial in disease development, but their accurate detection is challenging due to their low abundance and interference from wild‐type targets. Although nucleic acid analogs like peptide nucleic acids (PNAs) have been used for SNV detection, they often lack programmable sensitivity and specificity due to poorly calculated thermodynamics and kinetics. Here, we present a computational method for calculating the stacking energy of PNA and DNA hybrids, leveraging nearest neighbor parameters. Validation against experimental data from 16 sequences under varied hybridization conditions yielded good agreement using Bland‐Altman analysis, with all data points falling within the confidence interval. Our findings indicate that PNA‐DNA hybridization is thermodynamically more stable and exhibits kinetics 140‐fold faster than DNA‐DNA hybridization for identical sequences. Utilizing this computational framework, we designed PNA toehold probes, which were screened via simulations and experiments. This combined approach facilitated the identification of highly sensitive and specific PNA toehold probes for single point mutation detection via strand displacement reaction. Our results demonstrate the successful application of PNA toehold probes for detecting point mutations with high sensitivity and specificity, achieving a selective amplification of approximately 200‐fold for variants with a variant allele frequency (VAF) of 0.5% using quantitative polymerase chain reaction. [ABSTRACT FROM AUTHOR]

Nucleic acid‐based molecular recognition plays crucial roles in various fields like biosensing and disease diagnostics. To achieve optimal detection and analysis, it is essential to regulate the response performance of nucleic acid probes or switches to match specific application requirements by regulating thermodynamics and kinetics properties. However, the impacts of thermodynamics and kinetics theories on recognition performance are sometimes obscure and the relative conclusions are not intuitive. To promote the thorough understanding and rational utilization of thermodynamics and kinetics theories, this review focuses on the landmarks and recent advances of nucleic acid thermodynamics and kinetics and summarizes the nucleic acid thermodynamics and kinetics‐based strategies for regulation of nucleic acid‐based molecular recognition. This work hopes such a review can provide reference and guidance for the development and optimization of nucleic acid probes and switches in the future, as well as for advancements in other nucleic acid‐related fields. [ABSTRACT FROM AUTHOR]

While it is recognized that early diagnosis of cancer-related biomarkers can become an effective avenue for timely treatment and successfully improve patient survival, it remains challenging to get accurate inspection results. Currently, most reported cancer biomarker sensing methods are focused on the quantitative detection of a single type of biomarker, which makes accurate medical diagnostics difficult. In this work, we constructed a DNA walker nanomachine aptasensor based on gold nanoparticles for the simultaneous sensing of dual cancer biomarkers. The aptamers, labelled with a fluorophore, hybridized with complementary strands on the gold nanoparticle surface, serve as a walking track. Target analytes bind to their specific aptamers, leading to the dissociation of the unstable double-strand spherical nucleic acid. Exonuclease I (Exo I) selectively digested the aptamers bound with the target analytes, then the released targets go back to the next apamers on the gold nanopareticles surface for walking. The use of spherical nucleic acid probes improved the sensitivity of analyte detection. Exo I provided a driving power for target recycling and considerably improved the sensitivity of the aptasensor as well. The DNA walker nanomachine aptasensor was successfully applied for the detection of carcinoembryonic antigen (CEA) in the range of 0.167 to 3.34 ng mL−1, and mucin-1 (MUC-1) in the same range. Moreover, we used the two aptamers to construct the DNA walker nanomachine and achieved the simultaneous detection of CEA and MUC-1, thus having great potential for biomolecular logic gate construction and early disease diagnosis. [ABSTRACT FROM AUTHOR]

The 18-exon human TKFC gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as tkfc type by their intron-17 splice), databases contain evidence for alternative TKFC transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no. DQ344550). It results from an alternative 3′ splice-site in intron 17. The cloned cDNA contains an ORF also spanning from exon 2 to exon 18 of the TKFC gene but with a 56-nt insertion between exons 17 and 18 (classified as tkfc_ins56 type). This insertion introduces an in-frame stop, and the resulting ORF codes for a shorter TKFC variant, which, after expression, is enzymatically inactive. TKFC intron-17 splicing was found to be differentially expressed in human tissues. In a multiple-tissue northern blot using oligonucleotide probes, the liver showed a strong expression of the tkfc-like splice of intron 17, and the heart preferentially expressed the tkfc_ins56-like splice. Through a comparison to global expression data from massive-expression studies of human tissues, it was inferred that the intestine preferentially expresses TKFC transcripts that contain neither of those splices. An analysis of transcript levels quantified by RNA-Seq in the GTEX database revealed an exception to this picture due to the occurrence of a non-coding short transcript with a tkfc-like splice. Altogether, the results support the occurrence of potentially relevant transcript variants of the TKFC gene, differentially expressed in human tissues. (This work is dedicated in memoriam to Professor Antonio Sillero, 1938–2024, for his lifelong mentoring and his pioneering work on triokinase). [ABSTRACT FROM AUTHOR]

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents. [ABSTRACT FROM AUTHOR]

Objective: Identification of driver mutations in tumors is an extremely important task in oncology for the choice of treatment strategy and assessment of therapy efficacy. In many cases, especially in disease monitoring, there is a need to detect a small number of copies of the mutant allele against the background of excessive content of wild-type DNA. Methods: Genomic DNA was isolated from tumor tissue in paraffine blocks and amplified using polymerase-chain reaction (PCR) with blocking of wild-type DNA amplification via addition of locked-nucleic acid (LNA) oligonucleotides. Fluorescently labelled PCR-product enriched by IDH-mutant alleles was hybridized on a biochip with immobilized oligonucleotide probes which was able to determine 5 mutations in the IDH1 gene and 2 mutations in the IDH2 gene. Results and Discussion: The method was developed and tested on a collection of 26 samples of paraffinized tumor tissue (glioma, glioblastoma, chondrosarcoma). In three cases, R132C, R132L, and R132H mutations in the IDH1 gene were detected in tumor samples with low representation of the mutant allele. The limit of detection of mutant DNA was determined to be 0.1% in the wild-type DNA background. The advantages of the method are simultaneous analysis of multiple targets, simplicity, reliability, and cost-effectiveness. Conclusions: A highly sensitive method for detecting somatic mutations in the IDH1/2 genes by LNA-mediated blocking of amplification of wild-type alleles and hybridization in a biological microchip was developed. We believe that the method may be useful for detecting low-abundance mutations in tumor samples, as well as in circular tumor DNA. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for assessing the therapeutic efficacy of milk powder. In this study, we introduced a novel propidium monoazide (PMA)-antibiotic fluorescence in situ hybridization (AFISH)-flow cytometry (FC) method to rapidly and accurately quantify viable Bifidobacterium cells in milk powder. Briefly, Bifidobacterium cells were treated with chloramphenicol (CM) to increase their rRNA content, followed by staining with RNA-binding oligonucleotide probes, based on the AFISH technique. Then, the DNA-binding dye PMA was used to differentiate between viable and nonviable cells. The PMA-AFISH-FC method, including sample pretreatment, CM treatment, dual staining, and FC analysis, required approximately 2 h and was found to be better than the current methods. This is the first study to implement FC combined with PMA and an oligonucleotide probe for detecting Bifidobacterium. [Display omitted] [ABSTRACT FROM AUTHOR]

The demand for highly sensitive methods of pathogen detection drives the development of new diagnostic assays. While nucleic acid amplification methods such as PCR are very sensitive and remain widely used, they may be limited in complex sample matrices due to the presence of polymerase inhibitors. On the other hand, the direct detection of nucleic acids by DNA hybridization assays is simple but typically less sensitive. This work combines a branched DNA (bDNA) hybridization assay with upconversion nanoparticle (UCNP) labels to enhance the sensitivity of DNA detection. The anti‐Stokes emission of UCNP labels enables measurements without optical background interference. The bDNA assay relies on a series of oligonucleotide probes creating a branched structure with several binding sites for biotinylated amplification probes and streptavidin‐conjugated UCNPs. Several configurations of the bDNA assay are investigated to achieve the highest signal amplification and the lowest background signal. The optimal configuration of bDNA assay yields a limit of detection (LOD) of 5.9 × 104 cfu mL−1 for the target DNA of the bacteriophage M13. The upconversion‐linked bDNA assay is easily adaptable to other target DNAs by adjusting the oligonucleotide probes. [ABSTRACT FROM AUTHOR]

Background: microRNAs (miRNAs) were recognized as a promising source of diagnostic biomarker. Herein, we aim to evaluate the performance of an ultrasensitive method for detecting serum miRNAs using single molecule arrays (Simoa). Methods: In this study, candidate miRNAs were trained and tested by RT-qPCR in a cohort of PTB patients. Besides that, ultrasensitive serum miRNA detection were developed using the Single Molecule Array (Simoa) platform. In this ultrasensitive sandwich assay, two target-specific LNA-modified oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA respectively. We characterized its analytical performance and measured miRNAs in the serum of patients with pulmonary tuberculosis and healthy individuals. Results: We identified a five signature including three upregulated (miR-101, miR-196b, miR-29a) and two downregulated (miR-320b, miR-99b) miRNAs for distinguishing PTB patients from HCs, and validated in our 104 PTB patients. On the basis of Simoa technology, we developed a novel, fully automated digital analyser, which can be used to directly detect miRNAs in serum samples without pre-amplification. We successfully detected miRNAs at femtomolar concentrations (with limits of detection [LODs] ranging from 0.449 to 1.889 fM). Simoa-determined serum miR-29a and miR-99b concentrations in patients with PTB ((median 6.06 fM [range 0.00-75.22]), (median 2.53 fM [range 0.00-24.95]), respectively) were significantly higher than those in HCs ((median 2.42 fM [range 0.00-28.64]) (P < 0.05), (median 0.54 fM [range 0.00-9.12] (P < 0.0001), respectively). Serum levels of miR-320b were significantly reduced in patients with PTB (median 2.11 fM [range 0.00-39.30]) compared with those in the HCs (median 4.76 fM [range 0.00-25.10]) (P < 0.001). A combination of three miRNAs (miR-29a, miR-99b, and miR-320b) exhibited a good capacity to distinguish PTB from HCs, with an area under the curve (AUC) of 0.818 (sensitivity: 83.9%; specificity: 79.7%). Conclusions: This study benchmarks the role of Simoa as a promising tool for monitoring miRNAs in serum and offers considerable potential as a non-invasive platform for the early diagnosis of PTB. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Time to receive effective therapy is a primary determinant of mortality in patients with sepsis. Blood culture is the reference standard for the microbiological diagnosis of bloodstream infections, despite its low sensitivity and prolonged time to receive a pathogen detection. In recent years, rapid tests for pathogen identification, antimicrobial susceptibility, and sepsis identification have emerged, both culture-based and culture-independent methods. This rapid narrative review presents currently commercially available approved diagnostic molecular technologies in bloodstream infections, including their clinical performance and impact on patient outcome, when available. Peer-reviewed publications relevant to the topic were searched through PubMed, and manufacturer websites of commercially available assays identified were also consulted as further sources of information. We have reviewed data about the following technologies for pathogen identification: fluorescence in situ hybridization with peptide nucleic acid probes (Accelerate PhenoTM), microarray-based assay (Verigene®), multiplex polymerase chain reaction (cobas® eplex, BioFire® FilmArray®, Molecular Mouse, Unyvero BCU SystemTM), matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Rapid MBT Sepsityper®), T2 magnetic resonance (T2Bacteria Panel), and metagenomics-based assays (Karius©, DISQVER®, Day Zero Diagnostics). Technologies for antimicrobial susceptibility testing included the following: Alfed 60 ASTTM, VITEK® REVEALTM, dRASTTM, ASTar®, Fastinov®, QuickMIC®, ResistellTM, and LifeScale. Characteristics, microbiological performance, and issues of each method are described, as well as their clinical performance, when available. [ABSTRACT FROM AUTHOR]

Carbon dots (CDs), including carbon quantum dots, graphene quantum dots, carbon nanodots, and polymer dots, have gained significant attention due to their unique structural and fluorescence characteristics. This review provides a comprehensive overview of the classification, structural characteristics, and fluorescence properties of CDs, followed by an exploration of various fluorescence sensing mechanisms and their applications in gene detection, nucleolus imaging, and gene delivery. Furthermore, the functionalization of CDs with diverse surface ligand molecules, including dye molecules, nucleic acid probes, and metal derivatives, for sensitive nucleic acid detection is systematically examined. Fluorescence imaging of the cell nucleolus plays a vital role in examining intracellular processes and the dynamics of subcellular structures. By analyzing the mechanism of fluorescence and structure–function relationships inherent in CDs, the nucleolus targeting abilities of CDs in various cell lines have been discussed. Additionally, challenges such as the insufficient organelle specificity of CDs and the inconsistent mechanisms underlying nucleolus targeting have also been highlighted. The unique physical and chemical properties of CDs, particularly their strong affinity toward deoxyribonucleic acid (DNA), have spurred interest in gene delivery applications. The use of nuclear-targeting peptides, polymers, and ligands in conjunction with CDs for improved gene delivery applications have been systematically reviewed. Through a comprehensive analysis, the review aims to contribute to a deeper understanding of the potential and challenges associated with CDs in biomedical applications. [ABSTRACT FROM AUTHOR]

Fluorescence-based oligonucleotide probes have a great importance in research of molecular interactions. Molecular beacons (MBs) are special case of fluorescent probes that form a stem-loop shape, bringing together a fluorophore and quencher, thus emitting fluorescence only when hybridized to a complementary target. Here we describe a new method for the quantitation of MB hybridization based on the measurement of changes in free energy instead of the fluorescence intensity. The MB energy state can be measured by micro-fluorescence detection. The approach allowed to determine hybridization energy of the MB with target nucleotide directly from fluorescence spectra and distinguish the MB in unfolded and hybridized states. Moreover, the method enabled us to discriminate between DNA duplexes with perfect complementarity or a single-nucleotide mismatch, based on the first direct experimental prove of enthalpy-entropy compensation. [ABSTRACT FROM AUTHOR]

Purpose: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the 'smart-activatable' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus. Methods: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria. Results: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min. Conclusion: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials. [ABSTRACT FROM AUTHOR]

Sequence‐independent detection of low concentrations of nucleic acids is important for applications in forensics and diagnostics. An emission‐based probe for detecting and quantifying DNA and RNA utilizing a water‐soluble dicationic tetraphenylethene (TPE) derivativewas developed. The recognition is based on the electrostatic and other non‐covalent interactions between the phosphate backbone of nucleic acids and the cationic probe, which cause the restriction of rotation of the aryl units of the probe, ensuing in the enhancement of the fluorescence signal. The binding was validated by different spectroscopic techniques and also by electrophoretic mobility shift assay. The probable mode of binding with the nucleic acids was studied by blind‐docking studies that correlated well with the experimental results. [ABSTRACT FROM AUTHOR]

Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, −2DL3, −2DL5, −2DS2, −2DS3, −2DS4, −2DS5, and −3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, −2DS4, and −2DS5 were independent predictors of NCWS. Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, −2DS4, and −2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings. [ABSTRACT FROM AUTHOR]

A new rapid dual detection method was established to distinguish Bupleurum scorzonerifolium Willd. (BS) and Bupleurum chinense DC. (BC) simultaneously using the proofreading enzyme-mediated probe cleavage coupled with ladder-shape melting temperature isothermal amplification (proofman–LMTIA). The internal transcribed spacer (ITS) sequences of BS and BC were selected as targets for designs of proofman–LMTIA primers and proofman fluorescence probes labeled with FAM or JOE. The reaction temperature optimization, repeatability and reliability assessment, specificity assessment and sensitivity assessment of the proofman–LMTIA performed for dual detection of BS and BC and its application. The results showed that the optimal reaction temperature of the proofman–LMTIA method was at 63℃, which had strongly specificity, repeatability and reliability, as well as sensitivity, and the detection was completed within 20 min with a detection sensitivity of 1 pg/μL. The proofman–LMTIA method realized dual rapid detection of BS and BC, which showed a strong practical value. The 4 kinds were BC, 1 kind was BS, and 2 kinds were counterfeit in the detection of 7 kinds of BS or BC samples from different habitats. Our study successfully established a new approach for dual rapid detection of BS and BC using the proofman–LMTIA, which will provide an effective technique or method for the authenticity detection of authentic Chinese medicinal materials and present a very important practical significance. [ABSTRACT FROM AUTHOR]

Both tight and specific binding of folded biological mRNA is required for gene silencing by oligonucleotide gene therapy agents. However, this is fundamentally impossible using the conventional oligonucleotide probes according to the affinity/specificity dilemma. This study addresses this problem for cleaving folded RNA by using multicomponent agents (dubbed 'DNA nanomachine' or DNM). DNMs bind RNA by four short RNA binding arms, which ensure tight and highly selective RNA binding. Along with the improved affinity, DNM maintain the high sequence selectivity of the conventional DNAzymes. DNM enabled up to 3‐fold improvement in DNAzymes catalytic efficiency (kcat/Km) by facilitating both RNA substrate binding and product release steps of the catalytic cycle. This study demonstrates that multicomponent probes organized in sophisticated structures can help to achieve the balance between affinity and selectivity in recognizing folded RNA and thus creates a foundation for applying complex DNA nanostructures derived by DNA nanotechnology in gene therapy. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a rare neurodegenerative neuromuscular disorder with a wide phenotypic spectrum of severity. SMA was previously life limiting for patients with the most severe phenotype and resulted in progressive disability for those with less severe phenotypes. This has changed dramatically in the past few years with the approvals of three disease-modifying treatments. We review the evidence supporting the use of currently approved SMA treatments (nusinersen, onasemnogene abeparvovec, and risdiplam), focusing on mechanisms of action, side effect profiles, published clinical trial data, health economics, and pending questions. Whilst there is robust data from clinical trials of efficacy and side effect profile for individual drugs in select SMA populations, there are no comparative head-to-head clinical trials. This presents a challenge for clinicians who need to make recommendations on the best treatment option for an individual patient and we hope to provide a pragmatic approach for clinicians across each SMA profile based on current evidence. [ABSTRACT FROM AUTHOR]

The WHO currently recommends dolutegravir (DTG)-based ART for persons living with HIV infection in resource-limited-settings (RLS). To expand access to testing for HIV drug resistance (DR) to DTG in RLS, we developed probes for use in the oligonucleotide ligation assay (OLA)-Simple, a near-point of care HIV DR kit. Genotypic data from clinical trials and case reports were used to determine the mutations in HIV-1 integrase critical to identifying individuals with DTG-resistance at virologic failure of DTG-based ART. Probes to detect G118R, Q148H/K/R, N155H and R263K in HIV-1 subtypes A, B, C, D and CRF01_AE were designed using sequence alignments from the Los Alamos database and validated using 61 clinical samples of HIV-1 subtypes A, B, C, D, CRF01_AE genotyped by PacBio (n = 15) or Sanger (n = 46). Initial OLA probes failed to ligate for 16/244 (6.5%) codons (9 at G118R and 7 at Q148H/K/R). Probes revised to accommodate polymorphisms interfering with ligation at codons G118R and Q148R reduced indeterminates to 3.7% (5 at G118R and 4 at Q148H/K/R) and detected DTG-mutations with a sensitivity of 96.5% and 100% specificity. These OLA DTG resistance probes appear highly sensitive and specific across HIV-1 subtypes common in RLS with high burden of HIV infection. [ABSTRACT FROM AUTHOR]

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement. [ABSTRACT FROM AUTHOR]

Background Asymptomatic SARS-CoV-2 infection in children is highly prevalent but its acute and chronic implications have been minimally described. Methods In this controlled case-ascertained household transmission study, we recruited asymptomatic children <18 years with SARS-CoV-2 nucleic acid testing performed at 12 tertiary care pediatric institutions in Canada and the United States. We attempted to recruit all test-positive children and 1 to 3 test-negative, site-matched controls. After 14 days' follow-up we assessed the clinical (ie, symptomatic) and combined (ie, test-positive, or symptomatic) secondary attack rates (SARs) among household contacts. Additionally, post–COVID-19 condition (PCC) was assessed in SARS-CoV-2–positive participating children after 90 days' follow-up. Results A total of 111 test-positive and 256 SARS-CoV-2 test-negative asymptomatic children were enrolled between January 2021 and April 2022. After 14 days, excluding households with co-primary cases, the clinical SAR among household contacts of SARS-CoV-2–positive and –negative index children was 10.6% (19/179; 95% CI: 6.5%–16.1%) and 2.0% (13/663; 95% CI: 1.0%–3.3%), respectively (relative risk = 5.4; 95% CI: 2.7–10.7). In households with a SARS-CoV-2–positive index child, age <5 years, being pre-symptomatic (ie, developed symptoms after test), and testing positive during Omicron and Delta circulation periods (vs earlier) were associated with increased clinical and combined SARs among household contacts. Among 77 asymptomatic SARS-CoV-2–infected children with 90-day follow-up, 6 (7.8%; 95% CI: 2.9%–16.2%) reported PCC. Conclusions Asymptomatic SARS-CoV-2–infected children, especially those <5 years, are important contributors to household transmission, with 1 in 10 exposed household contacts developing symptomatic illness within 14 days. Asymptomatic SARS-CoV-2–infected children may develop PCC. [ABSTRACT FROM AUTHOR]

The early diagnosis of cancer is vital for effective treatment and improved prognosis. Tumor biomarkers, which can be used for the early diagnosis, treatment, and prognostic evaluation of cancer, have emerged as a topic of intense research interest in recent years. Nucleic acid, as a type of tumor biomarker, contains vital genetic information, which is of great significance for the occurrence and development of cancer. Currently, living cell nucleic acid probes, which enable the in situ imaging and dynamic monitoring of nucleic acids, have become a rapidly developing field. This review focuses on living cell nucleic acid probes that can be used for the early diagnosis of tumors. We describe the fundamental design of the probe in terms of three units and focus on the roles of different nanomaterials in probe delivery. [ABSTRACT FROM AUTHOR]

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile. [ABSTRACT FROM AUTHOR]

A method has been developed for manufacturing biological microchips on an aluminum substrate with hydrophilic cells from brush copolymers with the formation of a matrix of cells using photolithography. The surface of aluminum substrates was previously coated with a thin, durable, moderately hydrophobic layer of cross-linked polymer to prevent contact with the aluminum surface of the components used in the analysis of nucleic acids. Aluminum biochip substrates have high thermal conductivity and low heat capacity, which is important for the development of methods for multiplex PCR analysis on a chip. Oligonucleotide probes were covalently immobilized in the cells of the biochip. The preservation of the hybridization activity of the immobilized DNA probes was demonstrated in a hybridization analysis with a synthetic DNA target representing a section of the sequence of the seventh exon of the human ABO gene. The methods developed can be used in the development of a technology for parallel multiple rapid microanalysis of nucleic acids "lab on a chip" for the detection of human somatic and infectious diseases. [ABSTRACT FROM AUTHOR]

The method for a site-specific RNA acetylation using a novel oligonucleotide acetylating probe (Ac-probe) has been developed. It was confirmed that the acetyl group was site-specifically introduced to a 2′-OH of the sugar part of the target RNA via the formation of the RNA-probe duplex. [ABSTRACT FROM AUTHOR]

This document compares the standard plaque assay (SPA) with an unnamed alternative method for measuring viral load in a laboratory setting. The alternative method is described as more reproducible, accurate, sensitive, and capable of processing a larger number of samples compared to SPA. It is also unaffected by material compatibility. However, the document does not provide further details about the alternative method or its specific benefits. Further studies are needed to validate its use in assessing the protective performance of personal protective equipment (PPE). [Extracted from the article]

With both foodborne illness and food spoilage detrimentally impacting human health and the economy, there is growing interest in the development of in situ sensors that offer real-time monitoring of food quality within enclosed food packages. While oligonucleotide-based fluorescent sensors have illustrated significant promise, the development of such on-food sensors requires consideration towards sensing-relevant fluorescence properties of target food products—information that has not yet been reported. To address this need, comprehensive fluorescence profiles for various contamination-prone food products are established in this study across several wavelengths and timepoints. The intensity of these food backgrounds is further contextualized to biomolecule-mediated sensing using overlaid fluorescent oligonucleotide arrays, which offer perspective towards the viability of distinct wavelengths and fluorophores for in situ food monitoring. Results show that biosensing in the Cyanine3 range is optimal for all tested foods, with the Cyanine5 range offering comparable performance with meat products specifically. Moreover, recognizing that mass fabrication of on-food sensors requires rapid and simple deposition of sensing agents onto packaging substrates, RNA-cleaving fluorescent nucleic acid probes are successfully deposited via microcontact printing for the first time. Direct incorporation onto food packaging yields cost-effective sensors with performance comparable to ones produced using conventional deposition strategies. [ABSTRACT FROM AUTHOR]

This article discusses recent advances in the synthesis of 5-(hetero)aryl uracil derivatives, which are important compounds in the field of life sciences. The article focuses on various synthesis strategies for these derivatives, including selective coupling of pre-functionalized uracils, transition-metal-catalyzed or photo-prompted modification of uracil moieties, substitution of unactivated uracils with (hetero)aromatic components, and oxidative coupling of un-functionalized uracils. The article also discusses specific coupling reactions, such as Negishi coupling and Suzuki-Miyaura coupling, that have been used to synthesize these compounds. The development of new derivatives and synthesis strategies for these compounds is of great interest due to their wide-ranging biological potential. [Extracted from the article]

SUMMARY: Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope‐ and biotin‐ (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope‐based probe is limited by strict environmental regulation and availability in many places in the world while biotin‐based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase‐based method for incorporation of a cluster of 33 biotin‐labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P‐labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low‐abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus‐based miRNA silencing in pepper, knocking down accumulation of Can‐miR6027a and Can‐miR6149L. Importantly, further analysis showed that knocking‐down Can‐miR6027a led to upregulation of a nucleotide binding‐leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici. Significance Statement: A highly sensitive and convenient method is developed for synthesis of biotin‐based probe and validated in small RNA northern blot. Can‐miR6027a is identified as a negative regulator of immunity against Phytophthora capsici in pepper using virus‐based miRNA silencing technology in conjunction with newly developed northern blot method. [ABSTRACT FROM AUTHOR]

Presented is an infrared-induced sensor constructed of upconversion nanoparticles and DNAzyme for ultra-sensitive, and rapid detection of uranyl ions. The detection method eliminates the use of chemically labeled nucleic acid probes, possesses high selectivity, and a uranyl detection limit of 39 nM. The technique is successfully applied to the detection of uranyl in various water samples as well as human serum, illustrating the excellent sensitivity and specificity of the quantification platform. [ABSTRACT FROM AUTHOR]

Mutations in the hydroxymethylbilane synthase (HMBS) gene can lead to a deficiency of the HMBS enzyme, allowing porphyrins to accumulate to toxic levels in the liver and other organs, leading to acute intermittent porphyria (AIP). This case report describes the medical journey of a 20‑year‑old female, previously in good health, who experienced multiple hospitalizations and clinic visits due to severe abdominal pain episodes and remained undiagnosed for over 6 years. Despite the nonspecific nature of these symptoms, a suspicion of acute porphyria confirmed by genetic analysis revealed a splice pathogenic variant (c.826‑2A>T) in the HMBS gene in a heterozygous state. As the disease progressed, the patient developed a series of complications, including hyponatremia, autonomic instability, and motor neuropathy, culminating in complete paralysis (quadriplegia) and respiratory failure. The case highlights the importance of early recognition and differential diagnoses in managing AIP, with genetic testing playing a crucial role in confirming the diagnosis. [ABSTRACT FROM AUTHOR]

This work reports on the surface functionalization of a nanomaterial supporting localized surface plasmon resonances (LSPRs) with (synthetic) thiolated oligonucleotide-based biorecognition elements, envisaging the development of selective LSPR-based DNA biosensors. The LSPR thin-film transducers are composed of noble metal nanoparticles (NPs) embedded in a TiO2 dielectric matrix, produced cost-effectively and sustainably by magnetron sputtering. The study focused on the immobilization kinetics of thiolated oligonucleotide probes as biorecognition elements, followed by the evaluation of hybridization events with the target probe. The interaction between the thiolated oligonucleotide probe and the transducer's surface was assessed by monitoring the LSPR signal with successive additions of probe solution through a microfluidic device. The device was specifically designed and fabricated for this work and adapted to a high-resolution LSPR spectroscopy system with portable characteristics. Benefiting from the synergetic characteristics of Ag and Au in the form of bimetallic nanoparticles, the Au-Ag/TiO2 thin film proved to be more sensitive to thiolated oligonucleotide binding events. Despite the successful surface functionalization with the biorecognition element, the detection of complementary oligonucleotides revealed electrostatic repulsion and steric hindrance, which hindered hybridization with the target oligonucleotide. This study points to an effect that is still poorly described in the literature and affects the design of LSPR biosensors based on nanoplasmonic thin films. [ABSTRACT FROM AUTHOR]

The New Journal of Chemistry, published by The Royal Society of Chemistry, has released its latest issue which includes a variety of articles covering different topics in the field of chemistry. The papers discuss recent progress in areas such as nitrogen-heterocycles, chemisorption of gases, photoactive derivatives, and potential applications of nanoribbons. The journal aims to connect the global community with the chemical sciences and provides scientific insights and advancements in various fields of study. [Extracted from the article]

Three novel monomethine cyanine dyes were synthesized carrying electron donating groups to obtain even brighter nucleic acids probes. Photophysical properties of these dyes were evaluated with DNA and selected dyes with RNA. A great variation in brightness and binding properties of the dyes was observed. Moreover the X-ray crystal structures are presented for two dyes. [ABSTRACT FROM AUTHOR]

Circulating tumor DNA (ctDNA) is a highly promising biomarker for the early diagnosis and treatment of gastric cancer (GC). However, there is still a lack of effective and practical ctDNA detection methods. In this work, a simple and economical capillary non-gel sieving electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform coupled with catalytic hairpin assembly (CHA) as the signal amplification strategy is proposed for quantitative detection of PIK3CA E542K and TP53 (two types of ctDNA associated with GC). We have reasonably designed two pairs of programmable oligonucleotide hairpin probes for PIK3CA E542K and TP53. Using a one-pot reaction, the presence of ctDNA triggers the cyclic amplification of CHA, forming numerous thermodynamically stable H1/H2 double-strands. The H1/H2 double-stranded DNA catalyzed by PIK3CA E542K and TP53 can be easily separated by NGCE due to their different lengths, enabling simultaneous detection of both ctDNAs. Under optimal experimental conditions, the detection limits of this strategy for detecting GC-related biomarkers PIK3CA E542K and TP53 are 20.35 pM and 19.61 pM, respectively, and can achieve 730-fold signal amplification. This strategy has a good recovery in the serum matrix. The results of this study show that this strategy has significant advantages such as high selectivity, a simple process, no special instruments and equipment, no need for fluorescence modification of hairpin probes in advance, high automation, low cost, and minimal sample consumption. This provides a powerful method for the detection of trace cancer biomarkers in the serum matrix with good application prospects. [ABSTRACT FROM AUTHOR]

Discrimination of chromosome is essential for chromosome manipulation or visual chromosome characterization. Oligonucleotide probes can be employed to simplify the procedures of chromosome identification in molecular cytogenetics due to its simplicity, fastness, cost-effectiveness, and high efficiency. So far, however, visual identification of cotton chromosomes remains unsolved. Here, we developed 16 oligonucleotide probes for rapid and accurate identification of chromosomes in Gossypium hirsutum: 9 probes, of which each is able to distinguish individually one pair of chromosomes, and seven probes, of which each distinguishes multiple pairs of chromosomes. Besides the identification of Chrs. A09 and D09, we first find Chr. D08, which carries both 45S and 5S rDNA sequences. Interestingly, we also find Chr. A07 has a small 45S rDNA size, suggesting that the size of this site on Chr. A07 may have reduced during evolution. By the combination of 45S and 5S rDNA sequences and oligonucleotide probes developed, 10 chromosomes (Chrs. 3–7, and 9–13) in A subgenome and 7 (Chrs. 1–2, 4–5, and 7–9) in D subgenome of cotton are able to be recognized. This study establishes cotton oligonucleotide fluorescence in situ hybridization technology for discrimination of chromosomes, which supports and guides for sequence assembling, particularly, for tandem repeat sequences in cotton. [ABSTRACT FROM AUTHOR]

This article reports three new two-photon absorption (TPA) materials that are quinolinium-carbazole derivates. They are 3-(N-methyl-4-ethylquinolinium iodide)-9-ethylcarbazole (M4), 3-(N-methyl-4-ethylquinolinium iodide)-9-ethylcarbazole (H2), and 3-(N-methyl-4-ethylquinolinium iodide)-9-ethylcarbazole (H4). Their TPA cross-sections are 491, 515, and 512 GM, respectively. Under the excitation of near-infrared light, their fluorescence emission is about 650 nm. The compounds can stain nucleic acid DNA with the same level of nuclear localization as Hoechst 33342. Under continuous irradiation with a near-infrared laser, the three new compounds showed less fluorescence decay than DAPI, and the average fluorescence decay rates were 0.016%/s, 0.020%/s, and 0.023%/s. They are expected to become new two-photon fluorescent probes of nucleic acid DNA because of their excellent performance. [ABSTRACT FROM AUTHOR]

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection. [ABSTRACT FROM AUTHOR]

Over the last several decades, a range of experimental techniques from x-ray crystallography and atomic force microscopy to nuclear magnetic resonance and small angle x-ray scattering have probed nucleic acid structure and conformation with high resolution both in the condensed state and in solution. We present a computational study that examines the prospect of using electrostatic free energy measurements to detect 3D conformational properties of nucleic acid molecules in solution. As an example, we consider the conformational difference between A- and B-form double helices whose structures differ in the values of two key parameters—the helical radius and rise per basepair. Mapping the double helix onto a smooth charged cylinder reveals that electrostatic free energies for molecular helices can, indeed, be described by two parameters: the axial charge spacing and the radius of a corresponding equivalent cylinder. We show that electrostatic free energies are also sensitive to the local structure of the molecular interface with the surrounding electrolyte. A free energy measurement accuracy of 1%, achievable using the escape time electrometry (ETe) technique, could be expected to offer a measurement precision on the radius of the double helix of approximately 1 Å. Electrostatic free energy measurements may, therefore, not only provide information on the structure and conformation of biomolecules but could also shed light on the interfacial hydration layer and the size and arrangement of counterions at the molecular interface in solution. [ABSTRACT FROM AUTHOR]

Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) – ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality. Fluorescence-based oligonucleotide probes, also known as molecular beacons (MBs), are popular for detecting nucleic acids with high specificity. Here, the authors demonstrate self-sequestration of MB-based biosensors and target strands within peptide-based coacervates, increasing local concentrations and significantly increasing the sensitivity and kinetics of the DNA biosensors. [ABSTRACT FROM AUTHOR]

We describe a microfluidic system for conducting thermal lysis, polymerase chain reaction (PCR) amplification, hybridization, and colorimetric detection of foodborne viral organisms in a sample-to-answer format. The on-chip protocol entails 24 steps which are conducted by a centrifugal platform that allows for actuating liquids pneumatically during rotation and so facilitates automation of the workflow. The microfluidic cartridge is fabricated from transparent thermoplastic polymers and accommodates assay components along with an embedded micropillar array for detection and read-out. A panel of oligonucleotide primers and probes has been developed to perform PCR and hybridization assays that allows for identification of five different viruses, including pathogens such as norovirus and hepatitis A virus (HAV) in a multiplexed format using digoxigenin-labelled amplicons and immunoenzymatic conversion of a chromogenic substrate. Using endpoint detection, we demonstrate that the system can accurately and repetitively (n = 3) discriminate positive and negative signals for HAV at 350 genome copies per μL. As part of the characterization and optimization process, we show that the implementation of multiple (e.g., seven) micropillar arrays in a narrow fluidic pathway can lead to variation (up to 50% or more) in the distribution of colorimetric signal deriving from the assay. Numerical modeling of flow behaviour was used to substantiate these findings. The technology—by virtue of automation—can provide a pathway toward rapid detection of viral pathogens, shortening response time in food safety surveillance, compliance, and enforcement as well as outbreak investigations. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae (S. pneumoniae) represents a significant pathogenic threat, often responsible for community-acquired pneumonia with potentially life-threatening consequences if left untreated. This underscores the pressing clinical need for rapid and accurate detection of this harmful bacteria. In this study, we report the screening and discovery of a novel biomarker for S. pneumoniae detection. We used S. pneumoniae nucleases as biomarker and we have identified a specific oligonucleotide that works as substrate. This biomarker relies on a specific nuclease activity found on the bacterial membrane, forming the basis for the development of both fluorescence and electrochemical biosensors. We observed an exceptionally high sensitivity in the performance of the electrochemical biosensor, detecting as low as 102 CFU mL−1, whereas the fluorescence sensor demonstrated comparatively lower efficiency, with a detection limit of 106 CFU mL−1. Moreover, the specificity studies have demonstrated the biosensors' remarkable capacity to identify S. pneumoniae from other pathogenic bacteria. Significantly, both biosensors have demonstrated the ability to identify S. pneumoniae cultured from clinical samples, providing compelling evidence of the potential clinical utility of this innovative detection system. [ABSTRACT FROM AUTHOR]

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides. [ABSTRACT FROM AUTHOR]

Despite significant advancements in cancer research, real-time monitoring and effective treatment of cancer through non-invasive techniques remain a challenge. Herein, a novel polydopamine (PDA) nucleic acid nanoprobe has been developed for imaging signal amplification of intracellular mRNA and precise photothermal therapy guidance in cancer cells. The PDA nucleic acid nanoprobe (PDA@DNA) is constructed by assembling an aptamer hairpin (H1) labeled with the Cy5 fluorophore and another nucleic acid recognition hairpin (H2) onto PDA nanoparticles (PDA NPs), which have exceptionally high fluorescence quenching ability and excellent photothermal conversion properties. The nanoprobe could facilitate cellular uptake of DNA molecules and their protection from nuclease degradation. Upon recognition and binding to the intracellular mRNA target, a catalytic hairpin assembly (CHA) reaction occurs. The stem of H1 unfolds upon binding, allowing the exposed H1 to hybridize with H2, forming a flat and sturdy DNA double-stranded structure that detaches from the surface of PDA NPs. At the same time, the target mRNA is displaced and engages in a new cyclic reaction, resulting in the recovery and significant amplification of Cy5 fluorescence. Using thymidine kinase1 (TK1) mRNA as a model mRNA, this nanoprobe enables the analysis of TK1 mRNA with a detection limit of 9.34 pM, which is at least two orders of magnitude lower than that of a non-amplifying imaging nucleic acid probe. Moreover, with its outstanding performance for in vitro detection, this nanoprobe excels in precisely imaging tumor cells. Through live-cell TK1 mRNA imaging, it can accurately distinguish between tumor cells and normal cells. Furthermore, when exposed to 808-nm laser irradiation, the nanoprobe fully harnesses exceptional photothermal conversion properties of PDA NPs. This results in a localized temperature increase within tumor cells, which ultimately triggers apoptosis in these tumor cells. The integration of PDA@DNA presents innovative prospects for tumor diagnosis and image-guided tumor therapy, offering the potential for high-precision diagnosis and treatment of tumors. [ABSTRACT FROM AUTHOR]

Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars. [ABSTRACT FROM AUTHOR]

Foodborne pathogens can be found in various foods, and it is important to detect foodborne pathogens to provide a safe food supply and to prevent foodborne diseases. The nucleic acid base detection method is one of the most rapid and widely used methods in the detection of foodborne pathogens; it depends on hybridizing the target nucleic acid sequence to a synthetic oligonucleotide (probes or primers) that is complementary to the target sequence. Designing primers and probes for this method is a preliminary and critical step. However, new bioinformatics tools are needed to automate, specific and improve the design sets to be used in the nucleic acid‒base method. Thus, we developed foodborne pathogen primer probe design (FBPP), an open-source, user-friendly graphical interface Python-based application supported by the SQL database for foodborne pathogen virulence factors, for (i) designing primers/probes for detection purposes, (ii) PCR and gel electrophoresis photo simulation, and (iii) checking the specificity of primers/probes. [ABSTRACT FROM AUTHOR]

A new green procedure has been applied for the synthesis and purification of asymmetric monomethine cyanine dyes. The photophysical properties of the newly synthesized compounds have been examined by combined application of spectroscopic and theoretical methods. The structural characteristics of the molecules and dimer formation were characterized by quantum chemical computation and juxtaposed to the aggregachromism in UV/Vis spectra. The applicability of the dyes as fluorogenic nucleic acid probes has been proven by fluorescence titration, and their binding constants have been calculated. The mode of ligand–dsDNA/RNA interaction was rationalized by means of CD spectroscopy, molecular docking analysis, and fluorescent intercalator displacement experiments. [ABSTRACT FROM AUTHOR]

A comparative karyotype analysis of four species of yellow-flowered Eranthis sect. Eranthis, i.e., E. bulgarica, E. cilicica, E. hyemalis, and E. longistipitata from different areas, has been carried out for the first time. All the studied specimens had somatic chromosome number 2n = 16 with basic chromosome number x = 8. Karyotypes of the investigated plants included five pairs of metacentric chromosomes and three pairs of submetacentric/subtelocentric chromosomes. The chromosome sets of the investigated species differ mainly in the ratio of submetacentric/subtelocentric chromosomes, their relative lengths, and arm ratios. A new oligonucleotide probe was developed and tested to detect 45S rDNA clusters. Using this probe and an oligonucleotide probe to 5S rDNA, 45S and 5S rDNA clusters were localized for the first time on chromosomes of E. cilicica, E. hyemalis, and E. longistipitata. Major 45S rDNA clusters were identified on satellite chromosomes in all the species; in E. cilicica, minor clusters were also identified in the terminal regions of one metacentric chromosome pair. The number and distribution of 5S rDNA clusters is more specific. In E. cilicica, two major clusters were identified in the pericentromeric region of a pair of metacentric chromosomes. Two major clusters in the pericentromeric region of a pair of submetacentric chromosomes and two major clusters in the interstitial region of a pair of metacentric chromosomes were observed in E. longistipitata. E. hyemalis has many clusters of different sizes, localized mainly in the pericentromeric regions. Summarizing new data on the karyotype structure of E. sect. Eranthis and previously obtained data on E. sect. Shibateranthis allowed conclusions to be formed about the clear interspecific karyological differences of the genus Eranthis. [ABSTRACT FROM AUTHOR]

Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides (MLs), but, presently, resistance to MLs has been a matter of close clinical concern. This assay is intended to contribute to resistance detection of M. pneumoniae in clinical practice. A novel real-time PCR assay with two non-overlapping probes on the same nucleic acid strand was designed in this study. It could effectively detect all mutation types of M. pneumoniae in 23S rRNA at loci 2063 and 2064. The results were determined by the following methods: ΔCT < 0.5 for MLs-sensitive M. pneumoniae; ΔCT > 2.0 for MLs-resistant M. pneumoniae; 10 copies as a limit of detection for all types. For detection of M. pneumoniae in 92 clinical specimens, the consistency between the results of this assay and the frequently used real-time PCR results was 95.65%. The consistency of MLs resistance results between PCR sequencing and this assay was 100% in all 43 specimens. The assay could not only cover a comprehensive range of targets and have high detection sensitivity but is also directly used for detection and MLs analysis of M. pneumoniae in specimens. [ABSTRACT FROM AUTHOR]