Search Results (117)

Capillary electrophoresis based on laser-induced fluorescence (CE-LIF) plays an important role in the analysis of nucleic acids. However, the commercial CE-LIF is not only quite expensive but also inflexible, thus hindering its widespread use in the lab. Herein, we proposed a compact, low-cost, and flexible CE-LIF system. We also investigated its stability by separating the DNA ladders. Experiments demonstrated that the relative standard error of the relative fluorescence intensity and migration time was lower than 6.2% and 1.1%, respectively. The aperture size of the light source illuminating the capillary can affect the separation performance. Smaller apertures offer higher resolution length for the adjacent DNA fragments but may reduce the number of theoretical plates. Various fluorescent dyes (e.g., SYBR Green I, Gel Green, EvaGreen) can be employed in the self-built system. The limit of detection of dsDNA was as low as 0.05 ng/μL. The working range for DNA was 0.05 ng/μL~10 ng/μL. Finally, we have successfully separated the PCR products of the target gene of Porphyromonas gingivalis and Candida albicans in the home-built CE system. Such a robust CE-LIF system is easy to assemble in the lab. The total cost of the assembled CE system did not exceed 1100 USD. We believe this work can advance the application of CE and hope it will facilitate the easy assembly of flexible CE instruments in labs. Full article

High-quality RNA is crucial in clinical diagnostics and precision medicine. Formalin-fixed and paraffin-embedded (FFPE) tissues pose a challenge due to nucleic acid fragmentation and crosslinking. In this pilot study, various commercially available techniques for extracting RNA from small FFPE samples were compared. We evaluated the KingFisher Duo automated system or the manual MagMAX FFPE DNA/RNA Ultra Kit as an RNA extraction method combined with either a xylene, d-limonene, or AutoLys M tubes deparaffinization method. Additionally, the automated Maxwell RSC RNA FFPE kit and the High Pure FFPET RNA Isolation Kit were examined using FFPE samples from inflammatory bowel disease (IBD) patients, as well as samples from ovarian, kidney, and breast cancer and the skin. The KingFisher Duo system gave a higher yield and more consistent RNA quantities, especially from small volumes of IBD samples, compared to manual extraction. The deparaffinization method also impacted results, with AutoLys M tubes proving effective in combination with the KingFisher Duo system. Conversely, the High Pure kit exhibited higher yields for larger FFPE samples. While RNA integrity is a critical factor, particularly for messenger RNA (mRNA) expression studies, its role is less prominent in microRNA (miRNA) analyses. Recognizing this, our study focused on RNA yield and purity (A260/A230) to evaluate RNA extraction methods for various sample types. These findings emphasize the importance of selecting appropriate RNA extraction methods based on sample characteristics and research goals, highlighting the performance of automated methods and the impact of deparaffinization choices. The findings contribute to refining RNA extraction for molecular biology analyses, suggesting avenues for further exploration, including cost-effectiveness under specific experimental conditions. Full article

Head lice infestation (HLI), caused by Pediculus humanus capitis De Geer, 1767, has long been a common global problem of school children. Permethrin is an old pyrethroid derivative that has been used commonly for its treatment, and it exerts its activity over the voltage-sensitive calcium channels (VSCC) of the lice. There has been a growing list of persistent HLI cases lately in the world among patients using permethrin, and knockdown resistance (kdr)-related point mutations on VSCC have been identified and reported from those resistant lice samples. The aim of this study was to investigate the gene mutations associated with permethrin resistance in head lice collected from primary school children in Istanbul (Türkiye) and Nagarkot (Nepal) for the first time. A total of 192 P. h. capitis adults were collected from school children aged 6–12 years in two cities (96 lice each). Following DNA isolation, the fragment of the VSCC a-subunit gene, which contained the possible mutation sites ((kdr-like M815I (ATG > ATT), kdr T917I (ACA > ATA), and kdr-like L920F (CTT > TTT)), was amplified in each louse by PCR, and the PCR products were sequenced and aligned, followed by frequency calculations for alleles, genotypes, and haplotypes. Using nucleic acid sequence analysis, it was revealed that M815I, T917I, or L920F mutations were present on the VSCC genes in the lice samples from both Türkiye and Nepal. In addition, genotypic analyses indicated the presence of all three mutations in the lice samples from Türkiye, while the T917I mutation was detected in none of the lice collected in Nepal. This is the first report of gene mutations associated with permethrin resistance in head lice collected from a group of primary school children in the largest city of Türkiye (Istanbul) and Nagarkot. High mutation rates were identified in the lice, especially those from Istanbul, which is concordant with our previous unpublished study, in which almost 60% of the examined lice of the school children (in the same school selected in this study) remained alive despite long-term exposure to permethrin in the laboratory. These initial results show that gene mutations associated with permethrin resistance are common in lice samples in Istanbul and Nagarkot, which may suggest the current need for the selection of new pediculicidal agents in HLI treatment. Full article

Antimetabolite antitumor drugs interfere with nucleic acid and DNA synthesis, causing cancer cell death. However, they also affect rapidly dividing normal cells and cause serious side effects. Doxifluridine (5′-deoxy-5-fluorouridine [5′-DFUR]), a 5-fluorouracil (5-FU) prodrug converted to 5-FU by thymidine phosphorylase (TP), exerts antitumor effects. Since TP is distributed in tumor and normal tissues, 5′-DFUR features side effects. Here we designed a series of novel 5′-DFUR derivatives based on high nitroreductase (NTR) levels in the hypoxic microenvironment of tumor tissues by introducing nitro-containing moieties into the 5′-DFUR structure. These derivatives exert their antitumor effects by producing 5-FU under the dual action of TP and NTR in the tumor microenvironment. The derivatives were synthesized and their stability, release, and cytotoxicity evaluated in vitro and antitumor activity evaluated in vivo. Compound 2c, featuring nitrofuran fragments, was stable in phosphate-buffered saline and plasma at different pH values and reduced rapidly in the presence of NTR. The in vitro cytotoxicity evaluation indicated that compound 2c showed excellent selectivity in the MCF-7 and HT29 cell lines. Moreover, it exhibited antitumor effects comparable to those of 5′-DFUR in vivo without significant toxic side effects. These results suggest that compound 2c is a promising antitumor prodrug. Full article

Single nucleotide variant (SNV) detection is pivotal in various fields, including disease diagnosis, viral screening, genetically modified organism (GMO) identification, and genotyping. However, detecting SNVs presents significant challenges due to the fragmentation of nucleic acids caused by cellular apoptosis, molecular shearing, and physical degradation processes such as heating. Fragmented nucleic acids often exhibit variable lengths and inconsistent breakpoints, complicating the accurate detection of SNVs. This article delves into the underlying causes of nucleic acid fragmentation and synthesizes the strengths and limitations of next-generation sequencing technology, high-resolution melting curves, molecular probes, and CRISPR-based approaches for SNV detection in fragmented nucleic acids. By providing a detailed comparative analysis, it seeks to offer valuable insights for researchers working to overcome the challenges of SNV detection in fragmented samples, ultimately advancing the accurate and efficient detection of single nucleotide variants across diverse applications. Full article

Adenosine is an endogenous molecule that plays a vital role in biological processes. Research indicates that abnormal adenosine levels are associated with a range of diseases. The development of sensors capable of detecting adenosine is pivotal for early diagnosis of disease. For example, elevated adenosine levels are closely associated with the onset and progression of cancer. In this study, we designed a novel DNA biosensor utilizing chaperone copolymer-assisted catalytic hairpin assembly for highly sensitive detection of adenosine. The functional probe comprises streptavidin magnetic beads, an aptamer, and a catalytic chain. In the presence of adenosine, it selectively binds to the aptamer, displacing the catalytic chain into the solution. The cyclic portion of H1 hybridizes with the catalytic strand, while H2 hybridizes with the exposed H1 fragment to form an H1/H2 complex containing a G-quadruplex. Thioflavin T binds specifically to the G-quadruplex, generating a fluorescent signal. As a nucleic acid chaperone, PLL-g-Dex expedites the strand exchange reaction, enhancing the efficiency of catalytic hairpin assembly, thus amplifying the signal and reducing detection time. The optimal detection conditions were determined to be a temperature of 25 °C and a reaction time of 10 min. Demonstrating remarkable sensitivity and selectivity, the sensor achieved a lowest limit of detection of 9.82 nM. Furthermore, it exhibited resilience to interference in complex environments such as serum, presenting an effective approach for rapid and sensitive adenosine detection. Full article

Accurate calculation of non-covalent interaction energies in nucleotides is crucial for understanding the driving forces governing nucleic acid structure and function, as well as developing advanced molecular mechanics forcefields or machine learning potentials tailored to nucleic acids. Here, we dissect the nucleotides’ structure into three main constituents: nucleobases (A, G, C, T, and U), sugar moieties (ribose and deoxyribose), and phosphate group. The interactions among these fragments and between fragments and water were analyzed. Different quantum mechanical methods were compared for their accuracy in capturing the interaction energy. The non-covalent interaction energy was decomposed into electrostatics, exchange-repulsion, dispersion, and induction using two ab initio methods: Symmetry-Adapted Perturbation Theory (SAPT) and Absolutely Localized Molecular Orbitals (ALMO). These calculations provide a benchmark for different QM methods, in addition to providing a valuable understanding of the roles of various intermolecular forces in hydrogen bonding and aromatic stacking. With SAPT, a higher theory level and/or larger basis set did not necessarily give more accuracy. It is hard to know which combination would be best for a given system. In contrast, ALMO EDA2 did not show dependence on theory level or basis set; additionally, it is faster. Full article

Nucleic acid tests are key tools for the detection and diagnosis of many diseases. In many cases, the amplification of the nucleic acids is required to reach a detectable level. To make nucleic acid amplification tests more accessible to a point-of-care (POC) setting, isothermal amplification can be performed with a simple heating source. Although these tests are being performed in bulk reactions, the quantification is not as accurate as it would be with digital amplification. Here, we introduce the use of the vibrating sharp-tip capillary for a simple and portable system for tunable on-demand droplet generation. Because of the large range of droplet sizes possible and the tunability of the vibrating sharp-tip capillary, a high dynamic range (~2 to 6000 copies/µL) digital droplet loop-mediated isothermal amplification (ddLAMP) system has been developed. It was also noted that by changing the type of capillary on the vibrating sharp-tip capillary, the same mechanism can be used for simple and portable DNA fragmentation. With the incorporation of these elements, the present work paves the way for achieving digital nucleic acid tests in a POC setting with limited resources. Full article

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders. Full article

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization. Full article

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids. Full article

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids’ quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6–8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6–8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond. Full article

Bovine parainfluenza-3 virus (BPI3V) is an important respiratory pathogen in cattle, contributing to syndromes in the bovine respiratory disease complex (BRDC). Despite its significance, the understanding of its prevalence remains fragmented, especially within the larger framework of BRDC. This systematic review and meta-analysis aimed to determine the global prevalence of BPI3V in cattle using varied detection methods and to highlight associated risk factors. Of 2187 initially retrieved articles, 71 were selected for analysis, covering 32 countries. Depending on the detection method employed, the meta-analysis revealed significant variations in BPI3V prevalence. In the general cattle population, the highest prevalence was observed using the antibody detection method, with a proportion of 0.64. In contrast, in cattle with BRDC, a prevalence of 0.75 was observed. For the antigen detection method, a prevalence of 0.15 was observed, exclusively in cattle with BRDC. In nucleic acid detection, a prevalence of 0.05 or 0.10 was observed in the general and BRDC cattle populations, respectively. In virus isolation methods, a prevalence of 0.05 or 0.04 was observed in the general and BRDC cattle populations, respectively. These findings highlight the differences in the detection ability of different methods in identifying BPI3V. Other factors, such as country, study year, coinfections, farm size, the presence of respiratory signs, sex, and body weight, may also affect the prevalence. Most studies were anchored within broader BRDC investigations or aimed at detecting other diseases, indicating a potential under-representation of focused BPI3V research. BPI3V plays an important role in BRDC, with its prevalence varying significantly based on the detection methodology. To further understand its unique role within BRDC and pave the way for targeted interventions, there is an evident need for independent, dedicated research on BPI3V. Full article

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leucosis (EBL), which is the most significant neoplastic disease in cattle. Although EBL has been successfully eradicated in most European countries, infections continue to rise in Argentina, Brazil, Canada, Japan, and the United States. BLV imposes a substantial economic burden on the cattle industry, particularly in dairy farming, as it leads to a decline in animal production performance and increases the risk of disease. Moreover, trade restrictions on diseased animals and products between countries and regions further exacerbate the problem. Recent studies have also identified fragments of BLV nucleic acid in human breast cancer tissues, raising concerns for public health. Due to the absence of an effective vaccine, controlling the disease is challenging. Therefore, it is crucial to accurately detect and diagnose BLV at an early stage to control its spread and minimize economic losses. This review provides a comprehensive examination of BLV, encompassing its genomic structure, epidemiology, modes of transmission, clinical symptoms, detection methods, hazards, and control strategies. The aim is to provide strategic information for future BLV research. Full article

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 10⁷ times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient–dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection. Full article