Search Results (81)

The global antimicrobial resistance crisis demands innovative strategies to combat bacterial infections, including those caused by drug-sensitive pathogens that evade treatment through biofilm formation or metabolic adaptations. Here, we demonstrate that Squama Manitis extract (SME)—a traditional Chinese medicine component—exhibits broad-spectrum bactericidal activity against clinically significant pathogens, including both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) species (MIC = 31.25 mg/mL), achieving significant reduction in bacterial viability within 24 h. Through integrated multi-omics analysis combining scanning electron microscopy and RNA sequencing, we reveal SME’s unprecedented tripartite mechanism of action: (1) direct membrane disruption causing cell envelope collapse, (2) metabolic paralysis through coordinated suppression of TCA cycle and fatty acid degradation pathways, and (3) inhibition of DNA repair systems (SOS response and recombination downregulation). Despite its potent activity, SME shows low cytotoxicity toward mammalian cells (>90% viability) and can penetrate Gram-negative outer membranes. These features highlight SME’s potential to address drug-resistant infections through synthetic lethality across stress response, energy metabolism, and DNA integrity pathways. While advocating for synthetic alternatives to endangered animal products, this study establishes SME as a polypharmacological template for resistance-resilient antimicrobial design, demonstrating how traditional knowledge and modern systems biology can converge to guide sustainable anti-infective development. Full article

DNA and RNA nucleotide sequences are ubiquitous in all biological cells, serving as both a comprehensive library of capabilities for the cells and as an impressive regulatory system to control cellular function. The multi-alignment framework (MAF) provided in this study offers a user-friendly platform for sequence alignment and quantification. It is adaptable to various research needs and can incorporate different tools and parameters for in-depth analysis, especially in low read rate scenarios. This framework can be used to compare results from different alignment programs and algorithms on the same dataset, allowing for a comprehensive analysis of subtle to significant differences. This concept is demonstrated in a small RNA case study. MAF is specifically designed for the Linux platform, commonly used in bioinformatics. Its script structure streamlines processing steps, saving time when repeating procedures with various datasets. While the focus is on microRNA analysis, the templates provided can be adapted for all transcriptomic and genomic analyses. The template structure allows for flexible integration of pre- and post-processing steps. MicroRNA analysis indicates that STAR and Bowtie2 alignment programs are more effective than BBMap. Combining STAR with the Salmon quantifier or, with some limitations, the Samtools quantification, appears to be the most reliable approach. This method is ideal for scientists who want to thoroughly analyze their alignment results to ensure quality. The detailed microRNA analysis demonstrates the quality of three alignment and two quantification methods, offering guidance on assessing result quality and reducing false positives. Full article

Clinical studies indicate the etiology of liver disease to be unknown in 5% to 30% of patients. A long-standing hypothesis is the existence of unknown viruses beyond hepatitis A through E virus. We conducted serum virome sequencing in nine patients with cryptogenic liver disease and identified eight contigs that could not be annotated. One was determined to be a contaminant, while two of seven contigs from an individual (Patient 3) were validated by reverse transcription and polymerase chain reaction (RT-PCR) and Sanger sequencing. The possibility of contamination was completely excluded through PCR, with templates extracted using different methods from samples taken at different time points. One of the contigs, Seq260, was characterized as negative-sense single-stranded DNA via enzymatic digestion and genome walking. Digital-droplet PCR revealed the copy number of Seq260 to be low: 343 copies/mL. Seq260-based nested PCR screening was negative in 200 blood donors and 225 patients with liver disease with/without known etiologies. None of the seven contigs from Patient 3 was mapped onto 118,713 viral metagenomic data. Conclusively, we discovered seven unknown contigs from a patient with cryptogenic liver cirrhosis. These sequences are likely from a novel human virus with a negative-sense, linear single-stranded DNA genome. Full article

Rolling circle amplification (RCA) at ambient temperature is prone to false positive signals during nucleic acid detection, which makes it challenging to establish an efficient RCA detection method. The false positive signals are primarily caused by binding of non-target nucleic acids to the circular single-stranded template, leading to non-specific amplification. Here, we present an RCA method for miRNA detection at 37 °C using two circular ssDNAs, each of which is formed by ligating the intramolecularly formed nick (without any splint) in a secondary structure. The specific target recognition is realized by utilizing low concentrations (0.1 nM) of circular ssDNA1 (C1). A phosphorothioate modification is present at G*AATTC on C1 to generate a nick for primer extension during the primer self-generated rolling circle amplification (PG-RCA). The fragmented amplification products are used as primers for the following RCA that serves as signal amplification using circular ssDNA2 (C2). Notably, the absence of splints and the low concentration of C1 significantly inhibits non-target binding, thus minimizing false positive signals. A high concentration (10 nM) of C2 is used to carry out linear rolling circle amplification (LRCA), which is highly specific. This strategy demonstrates a good linear response to 0.01–100 pM of miRNA with a detection limit of 7.76 fM (miR-155). Moreover, it can distinguish single-nucleotide mismatch in the target miRNA, enabling the rapid one-pot detection of miRNA at 37 °C. Accordingly, this method performs with high specificity and sensitivity. This approach is suitable for clinical serum sample analysis and offers a strategy for developing specific biosensors and diagnostic tools. Full article

Low-density DNA microarrays are crucial in molecular diagnostics due to their cost-effectiveness and high sensitivity. However, reliable spot localization remains challenging due to positional variations and image artifacts. Traditional intensity-based methods often struggle with weak fluorescence signals. To address this, we propose a rapid spot localization method that combines template matching with point pattern matching, enhanced through vectorized programming and square (box) templates. Vectorized programming accelerated the most time-consuming calculation by 82 times on a PC and was 6000 times faster on a Raspberry Pi compared to a for-loop implementation. While this improvement applies to the vectorized square calculation alone, substantial performance gains were still achieved in the overall process. Additionally, replacing circular templates with square templates resulted in a fourfold reduction in processing time without compromising detection performance. The proposed method effectively reduces computational overhead, making it suitable for high-throughput and resource-constrained applications. The method was validated using HPV genotyping images from commercial DNA microarrays, demonstrating its practical applicability and robust performance in clinical settings. Full article

Background/Objectives: DNA extraction from dried blood spot (DBS) samples is often applied in neonatal screening programs. Although various methods to extract DNA from DBSs have been described, the optimal approach remains unclear. Therefore, this study aimed to compare and optimize extraction methods to establish a reliable and efficient protocol for human DNA extraction from DBSs. Methods: We conducted a back-to-back comparison of five different DNA extraction methods on 20 DBS samples: three column-based kits (QIAamp DNA mini kit, High Pure PCR Template Preparation kit, DNeasy Blood & Tissue kit) and two in-house boiling methods (one using TE buffer, one using Chelex-100 resin). DNA recovery was measured with DeNovix DS-11 and ACTB qPCR. Further optimization of elution volumes and starting material was performed on the best-performing methods (sample size = 5). Additionally, T-cell receptor excision circle (TREC) DNA was assessed by qPCR as an application. Results: The Chelex boiling method yielded significantly (p < 0.0001) higher ACTB DNA concentrations compared to the other methods. Column-based methods showed low DNA recovery, except for Roche, which showed significantly (p < 0.0001) higher DNA concentrations than the other column-based methods, as measured by DeNovix DS-11. Decreasing elution volumes (150 vs. 100 vs. 50 µL) increased ACTB DNA concentrations significantly, while increasing starting material (two vs. one 6 mm spot) did not. Conclusions: We identified an easy and cost-effective optimized DNA extraction method using Chelex from DBSs, with an elution volume of 50 µL and 1 × 6 mm DBS punch, which is particularly advantageous for research in low-resource settings and large populations, such as neonatal screening programs. Full article

Background: Epigenetic biomarkers, particularly CpG methylation, are increasingly employed in clinical and forensic settings. However, we still lack a cost-effective, sensitive, medium-scale method for the analysis of hundreds to thousands of user-defined CpGs suitable for minute DNA input amounts (<10 ng). In this study, motivated by promising results in the genetics field, we investigated single-molecule molecular inversion probes (smMIPs) for simultaneous analysis of hundreds of CpGs by using an example set of 514 age-associated CpGs (Zhang model). Methods: First, we developed a novel smMIP design tool to suit bisulfite-converted DNA (Locksmith). Then, to optimize the capture process, we performed single-probe capture for ten selected, representative smMIPs. Based on this pilot, the full smMIP panel was tested under varying capture conditions, including hybridization and elongation temperature, smMIP and template DNA amounts, dNTP concentration and elongation time. Results: Overall, we found that the capture efficiency was highly probe-(and hence, sequence-) dependent, with a heterogeneous coverage distribution across CpGs higher than the 1000-fold range. Considering CpGs with at least 20X coverage, we yielded robust methylation detection with levels comparable to those obtained from the gold standard EPIC microarray analysis (Pearsons’s r: 0.96). Conclusions: The observed low specificity and uniformity indicate that smMIPs in their current form are not compatible with the lowered complexity of bisulfite-converted DNA. Full article

Background. The increase in antibiotic resistance in human-impacted environments, such as coastal waters with aquaculture activity, is related to the widespread use of antibiotics, even at sub-lethal concentrations. In Chile, the world’s second largest producer of salmon, aquaculture is considered the main source of antibiotics in coastal waters. In this work, we aimed to characterize the genetic and phenotypic profiles of antibiotic resistance in bacterial communities from salmon farms. Methods. Bacterial metagenomes from an intensive aquaculture zone in southern Chile were sequenced, and the composition, abundance and sequence of antibiotic resistance genes (ARGs) were analyzed using assembled and raw read data. Total DNA from bacterial communities was used as a template to recover floR gene variants, which were tested by heterologous expression and functional characterization of phenicol resistance. Results. Prediction of ARGs in salmon farm metagenomes using more permissive parameters yielded significantly more results than the default Resistance Gene Identifier (RGI) software. ARGs grouped into drug classes showed similar abundance profiles to global ocean bacteria. The floR gene was the most abundant phenicol-resistance gene with the lowest gene counts, showing a conserved sequence although with variations from the reference floR. These differences were recovered by RGI prediction and, in greater depth, by mapping reads to the floR sequence using SNP base-calling. These variants were analyzed by heterologous expression, revealing the co-existence of high- and low-resistance sequences in the environmental bacteria. Conclusions. This study highlights the importance of combining metagenomic and phenotypic approaches to study the genetic variability in and evolution of antibiotic-resistant bacteria associated with salmon farms. Full article

Background/Objectives: One of the significant challenges in forensic casework is the analysis of samples with degraded or poorly concentrated genetic material. The utilisation of the GlobalFiler™ and Yfiler Plus™ kits has unquestionably enhanced the efficacy of genetic profiling in challenging samples, facilitating the analysis of alleles that were previously undetectable with alternative kits. Therefore, the main objective of this work was to verify the efficiency of these kits in analysing forensic samples, progressively reducing the amplification volumes. To further optimise genetic profiling, it was essential not only to assess the behaviour of the alleles but also to prevent allelic loss. Methods: A series of reaction volume reduction studies were conducted, evaluating the performance of genetic profiles in both controlled samples (positive controls) and low template DNA samples (0.01 ng/µL). Results: The results demonstrate that it is effective to obtain complete genetic profiles from the amplification of optimal samples in reduced volumes of 12, 6 or 3 µL with GlobalFiler™ and Yfiler™ Plus. Conclusions: The limiting factor in obtaining complete genetic profiles is the amount of DNA available, rather than the amplification volume. Furthermore, reducing the amplification volume from DNA extracts of low template DNA samples proportionally increases the number of allelic dropouts. Full article

The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay’s working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon’s loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies. Full article

Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed. Methods: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity. Results: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker. Full article

As nanocarriers of a new generation, biomimetic nanovesicles are an emerging class of therapeutic tools whose surface is integrated or fabricated with biomaterials capable of mimicking the biological features and functions of native cells. Thanks to this, biomimetic nanovesicles, in particular, those made by plasma membrane moieties, possess greatly improved biocompatibility, high target specificity, a long retention time, and minimal undesired immune responses. For these reasons, a multitude of progenitor cells including cancer ones were employed as templates to generate biomimetic or membrane-camouflaged nanovesicles hosting different therapeutic compounds. In this contribution, different membrane-derived biomimetic vesicles (M-NVs) were generated by osmotic lysis or plasma membrane isolation approaches from normal and cancer cell lines and assayed against in vitro models of human glioblastoma. M-NVs were compared in their cellular internalization degrees of DNA and proteins, morphologically and molecularly characterized, expressing an extracellular membrane-associated marker. Then, Rose Bengal (RB), a photoactivable drug characterized by a relatively low cellular uptake, was incorporated into nascent glioblastoma-derived M-NVs and finally administered to homotypic receiving cells, showing an increased degree of internalization as well as induced cytotoxic effects, even in the absence of photodynamic direct stimulation. Similar results were also obtained assaying lyophilized M-NVs loaded with RB. In conclusion, M-NVs generated by cell membranes effectively deliver several cargoes, including therapeutic molecules, maintain functionality after lyophilization, and show significant internalization effects, making them a promising strategy for therapeutic applications against human glioblastoma cells. Full article

Mantidis Ootheca (sangpiaoxiao), the egg case of the mantis, is a type of insect-derived traditional medicine widely used in East Asia. However, species identification based on egg morphology is challenging, leading to the distribution of counterfeit and adulterated products. The use of inauthentic ingredients can pose serious health risks to consumers. This study aimed to develop PCR markers that can rapidly and accurately differentiate between authentic and counterfeit Mantidis Ootheca. The mitochondrial cytochrome c oxidase I (COI) region was sequenced in thirteen samples from four mantis species: Tenodera angustipennis, Statilia maculata, Hierodula patellifera, and T. sinensis. Four sets of SCAR primers were designed based on species-specific nucleotide polymorphisms, and a multiplex SCAR assay was developed by combining all sets of the primers. The sequence-characterized amplified region (SCAR) primers successfully produced amplicons for each target species, even with low-DNA templates or templates containing DNA from multiple samples. No amplification was observed for nontarget species. This study presents a novel approach for identifying authentic Mantidis Ootheca species using DNA-based diagnostic marker assays, which enable rapid and precise species identification. The SCAR assays developed in this study will aid in maintaining quality control and promoting the standardization of commercial Mantidis Ootheca products. Full article

The accurate diagnosis and identification of Leishmania species are crucial for the therapeutic selection and effective treatment of leishmaniasis. This study aims to develop and evaluate the use of high-resolution melting curve analysis (HRM)-PCR for Leishmania species identification causing visceral leishmaniasis (VL), post-kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL) in the Indian subcontinent. Two multi-copy targets (ITS-1 and 7SL-RNA genes) were selected, and an HRM-PCR assay was established using L. donovani, L. major, and L. tropica standard strain DNA. The assay was applied on 93 clinical samples with confirmed Leishmania infection, including VL (n = 30), PKDL (n = 50), and CL (n = 13) cases. The ITS-1 HRM-PCR assay detected as little as 0.01 pg of template DNA for L. major and up to 0.1 pg for L. donovani and L. tropica. The detection limit for the 7SL-RNA HRM-PCR was 1 pg for L. major and 10 pg for L. donovani and L. tropica. The ITS-1 HRM-PCR identified 68 out of 93 (73.11%) leishmaniasis cases, whereas 7SL-RNA HRM-PCR could only detect 18 out of 93 (19.35%) cases. A significant correlation was observed between the kDNA-based low Ct values and ITS-1 HRM-PCR positivity in the VL (p = 0.007), PKDL (p = 0.0002), and CL (p = 0.03) samples. The ITS-1 HRM-PCR assay could identify Leishmania spp. causing different clinical forms of leishmaniasis in the Indian subcontinent, providing rapid and accurate results that can guide clinical management and treatment decisions. Full article

Recombination hotspot-activating DNA sites (e.g., M26, CCAAT, Oligo-C) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe. We used a fission yeast-optimized CRISPR-Cas9 system (SpEDIT) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus. Full article