DNA Products Research Articles

On-Chip DNA Assembly via Dielectrophoresis

On-chip gene synthesis has the potential to improve the synthesis throughput and reduce the cost exponentially. While there exist several microarray-based oligo synthesis technologies, on-chip gene assembly has yet to be demonstrated. This work introduces a novel on-chip DNA assembly method via dielectrophoresis (DEP) that can potentially be integrated with microarray-based oligo synthesis on the same chip. Our DEP chip can selectively manipulate oligos and guide their movement without perturbing the surrounding fluid medium, thus aiding in DNA assembly. Helical forked electrode design has been optimized for compatibility with DEP, ensuring efficient control over target oligos. By applying an alternating current signal set at 2 MHz, we successfully achieve the desired directed movement of oligonucleotides. Additionally, chemical treatments combined with photoirradiation enabled the connection of complementary gene sequences and the subsequent release of single-stranded DNA products. Sequencing results validate the effective assembly of DNA fragments, approximately 500 base pairs in length, using our DEP device.

Influence of Seven Ion Species on Osteogenic Differentiation of Mesenchymal Stem Cells Stimulated by Macrophages in Indirect and Direct Coculture Systems.

Implanted biomaterials release inorganic ions that trigger inflammatory responses, which recruit immune cells whose biochemical signals affect bone tissue regeneration. In this study, we evaluated how mouse macrophages (RAW264, RAW) and mesenchymal stem cells (KUSA-A1, MSCs) respond to seven types of ions (silicon, calcium, magnesium, zinc, strontium, copper, and cobalt) that reportedly stimulate cells related to bone formation. The collagen synthesis, alkaline phosphatase activity, and osteocalcin production of the MSCs varied by ion dose and type after culture in the secretome of RAW cells. However, DNA production was relatively unaffected. The MSC secretome may also stimulate RAW cells in coculture and, therefore, affect osteogenic differentiation of MSCs. Overall, the ions often exerted different effects on each cell type. This study guides future work that explores the mechanisms behind ion-dependent osteogenic differentiation and cell functions.

Efficient Control of Fusarium Head Blight and Reduction of Deoxynivalenol Accumulation by a Novel Nanopartner-Based Strategy.

Chemical control of Fusarium head blight (FHB) in wheat plants is often challenged by the resistance outbreak and deoxynivalenol (DON) accumulation. Developing green partners for fungicides is crucial for reducing fungal growth, mycotoxin contamination, and agricultural fungicides input. Herein, we investigated the mechanism of MgO nanoparticles (NPs) in controlling FHB. The EC50 of MgO NPs on mycelial growth was 105.2 μg/mL. At this concentration, they inhibited the spore germination, DON production, and wheat colonization of Fusarium graminearum by 56.0%, 24.5%, and 43.8%, respectively, exhibiting superior performance compared to nine other bioactive NPs such as ZnO and TiO2. Importantly, MgO NPs showed an additive effect with carbendazim and azoxystrobin in inhibiting F. graminearum. The extracellular toxicity of MgO NPs against F. graminearum was mainly attributed to the inhibition of fungal growth and germination by oxidative damage, alkaline damage, and cell structure damage. Although MgO NPs could not be absorbed into mycelia, they (EC90) decreased the soluble protein content and DNA concentration of mycelia by 27.8% and 42.3%, respectively, and increased the pyruvate content by 67.4%, demonstrating that the intracellular toxicity was mainly based on their inhibition of protein and DNA production and promotion of carbohydrate degradation. With low risks to nontarget organisms, MgO NPs could be a promising nanopartner for fungicides to protect wheat from FHB and mitigate fungicide overuse.

Advanced Ligase Chain Reaction Strategy to Generate a Circular DNA Walker for Electrochemiluminescent Detection of Single Nucleotide Polymorphism.

Single nucleotide polymorphism (SNP) primarily refers to DNA sequence polymorphism caused by variations in a single nucleotide, which is closely associated with many diseases such as genetic disorders and tumors. However, trace DNA mutants typically exist in a large pool of wild-type DNA, making it challenging to establish accurate and sensitive approaches for SNP detection. Herein, we developed an advanced ligase chain reaction (LCR) strategy to output the circular DNA walker for signal amplification, which realized accuracy and sensitive SNP detection based on the electrochemiluminescent (ECL) platform. Unlike the general LCR system that utilizes two sets of short single-stranded DNA (ssDNA) primers to generate double-stranded DNA amplification products, we ingeniously designed a long single-stranded DNA primer to replace one set of short ssDNA primers, allowing for the generation of circular DNA products upon complementing the target. Noticeably, the circular DNA serves as a DNA walker that can be easily purified by nucleases to eliminate unreacted primers and byproducts, significantly improving accuracy and sensitivity. Then, the circular DNA walker moved along a linearly ordered DNA quenching probe track modified on the ECL sensing interface, restoring the ECL signals by cleaving the quenching probes labeled on the DNA track with the help of apurinic/apyrimidinic endonuclease 1. Employing the p53 gene as a model, we realized the sensitive detection of mutant p53 in the range from 10 aM to 10 pM, with a detection limit of 6 aM, providing a promising platform for SNP detection.

Selenium Ameliorates Aluminum Poisoning-Induced Impaired Production of Neutrophil Extracellular Traps in Chicken.

Neutrophil extracellular traps (NETs) are released by neutrophils to modulate the immune response. Aluminum (Al) poisoning is linked to immunotoxicity, and selenium (Se) can maintain immune homeostasis. In this study, we investigated the toxic effects of Al on the release of NETs, the antagonistic effect of Se on Al-induced toxicity, and the potential molecular mechanisms underlying these processes. We assessed the cytotoxicity of aluminum on neutrophils using CCK-8 assay, visualized the structure of selenium/aluminum-induced NETs through immunofluorescence and scanning electron microscope, quantified ROS release during NETs formation using fluorescence microplate analysis, and employed the selenoprotein levels to dissect the mechanisms underlying selenium and aluminum-induced NETs release. Peripheral blood neutrophils were exposed to zymosan for a duration of 3h to induce the formation of NETs. Microscopic analysis indicated that NETs formation was inhibited in the presence of aluminum. Furthermore, assessments using a multifunctional microplate reader demonstrated that aluminum suppressed both the production of extracellular DNA and the reactive oxygen species burst in neutrophils. Western blot analysis revealed that aluminum altered the levels of cellular selenoproteins. In contrast, Se reduced the Al-induced toxic reaction including restored NETs production, ROS burst, and selenoprotein levels. These results indicate that Al decreases the formation of NETs induced by Zym, while Se inhibits the Al toxicity, promoting the formation of NETs by modulating the expression of selenoprotein.

Harnessing Demethylase-Regulated Catalytic DNA Circuit for In-Situ Investigation of the Regulatory Connection with MicroRNA.

Insight into the epigenetic modulation-correlated molecule interactions has significant implications for the in-depth understanding of intracellular intricate biological networks. However, there is currently a lack of reliable biological tools for elucidating the potential correlation between epigenetic regulators and relevant genes, e.g., microRNAs (miRNAs). Herein, an alkB homologue 5 (ALKBH5, a key epigenetic regulator)-modulated catalytic DNA circuit (ACD) was constructed by grafting a N6-methyladenosine (m6A)-caged I-R3 DNAzyme into the circuitry components for achieving the on-site miRNA imaging in living cells. Specifically, the catalytic activity of I-R3 DNAzyme could be effectively suppressed by the m6A modification situated at its highly sequence-conserved core region and then be selectively restored through the ALKBH5-mediated demethylation pathway. And the ALKBH5-activated I-R3 DNAzyme allowed the highly efficient DNA cleaving reaction in the presence of DNAzyme cofactors, resulting in the liberation of catalytic hairpin assembly (CHA) reactants. Subsequently, target miRNA triggered the CHA circuit to produce a duplex DNA product while releasing the miRNA analyte. The liberated miRNA could autonomously trigger the next round of the CHA assembly cycle for generating the amplified fluorescence readout. By virtue of the stimuli-responsive activation and the CHA amplification circuit, the ACD system achieved highly specific and sensitive imaging of miRNA in tumor cells. Moreover, this efficiently and reliably ALKBH5-activated DNA circuit is demonstrated to reveal the underlying relationship between activator ALKBH5 and miRNA. Overall, the developed ACD system provides a promising tool for the robust on-site profiling of epigenetic-involved signal pathways, thus displaying great potential in bioanalytical applications.

High throughput variant libraries and machine learning yield design rules for retron gene editors.

The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply this new information to a specific application: the use of the retron to produce a precise genome editing donor in combination with a CRISPR-Cas9 RNA-guided nuclease (an editron). We use high-throughput libraries in Saccharomyces cerevisiae to additionally define design rules for editrons. We extend our new knowledge of retron DNA production and editron design rules to human genome editing to achieve the highest efficiency Retron-Eco1 editrons to date.

Early Infection for Mass Production of Artificial Single-Stranded DNA with Escherichia coli

Large quantities of artificial single-stranded DNA (ssDNA) with user-defined sequences are increasingly required to exploit the potential of DNA nanotechnology. Cross-contamination-free ssDNA production can be achieved using Escherichia coli with an optimized helper plasmid in high-cell-density cultivation via the secretion of phagemid particles containing ssDNA with user-defined sequences. In our study, we aimed to reduce the number of phagemid particles for the initiation of ssDNA production. We tested different infection densities, ranging from a multiplicity of infection (MOI) of 10−6–10−2 tfu cfu−1 at the start of the initial batch phase in a 2.5 L stirred tank bioreactor. A MOI of 10−3 tfu cfu−1 was the best compromise between process time and ssDNA concentration. Early initiation of ssDNA production with low MOI reduced the number of phagemid particles by a factor of 250,000. The early infection strategy was successfully scaled up to the 25 L scale, resulting in ssDNA concentrations of >100 mg L−1 within a process time of one day. Transferring the infection strategy to a 1000 L scale gained 65 mg L−1 ssDNA because of incomplete initial infection. The versatility of the early infection strategy was further proven with a second prolonged, user-defined ssDNA sequence.

Methionine Synthase Reductase A66G Variant in Pediatric Acute Lymphoblastic Leukemia Patients.

Methionine synthase reductase, which is encoded by the methionine synthase reductase (MTRR) gene, plays a crucial role in the methylation reactions and the production of DNA and its epigenetic processes. There was a correlation between the MTRR (A66G) polymorphism and the likelihood of developing acute lymphoblastic leukemia (ALL). This study was carried out to investigate the correlation among pediatric ALL cases. Within the participant population of this case-control study, there were 86 individuals who had been diagnosed with ALL, and there were also 150 healthy persons who acted as the control group. To determine the MTRR (A66G) polymorphism, DNA was first extracted and then observed through the use of real-time polymerase chain reaction. The results of the flow cytometry analysis showed that the prevalence of B-cell ALL (B-ALL) was much higher than that of T-cell ALL (T-ALL), which accounted for only 20 cases (23.3%). Upon comparing the hematological parameters of ALL subtypes in patients with T-ALL, it was discovered that there was a statistically significant higher mean total white blood count (P < 0.0005) and mean blast percentage (P = 0.050). Upon examination, it was discovered that both of these figures were much higher than the average. In accordance with the results of the molecular analysis, the occurrence of the MTRR homozygous GG genotype was found to be considerably lower in the patients' group (4.65%) than in the control group (20.67%). However, the MTRR homozygous AA and heterozygous AG were nearly similar in the two groups. The risk of acute lymphoblastic leukemia and MTRR genotypes, on the other hand, exhibited a correlation that was not statistically significant (P = 0.082). The study's findings showed that among pediatric ALL patients, the MTRR A66G polymorphism was not linked to an increased risk of ALL.

An Escherichia coli Strain for Plasmid DNA Production with a Low Endotoxin Level

Plasmid DNA (pDNA) is one of the possible types of vectors used in gene therapy. Escherichia coli is the most common bacterium for the production of pDNA. Lipopolysaccharides are the main component of the outer membrane of E. coli. Lipopolysaccharides are strong endotoxins, and their presence in pharmacological substances is highly undesirable. One of the approaches to eliminating endotoxin contamination of target substances is knocking out the genes of the lipopolysaccharide biosynthesis pathway. Using the CRISPR/Cas9 genome editing technology, we obtained a mutant form of the E. coli strain SCS110. We knocked out the genes kdsD, gutQ, lpxM, lpxL, lpxP, pagP, and eptA. As a result, lipopolysaccharide contamination of pDNA samples isolated by different methods from the mutant E. coli SCS 110 was significantly lower (19 to 295 times) than in pDNA samples from the original strain.

DNA sequence and lesion-dependent mitochondrial transcription factor A (TFAM)-DNA-binding modulates DNA repair activities and products.

Mitochondrial DNA (mtDNA) is indispensable for mitochondrial function and is maintained by DNA repair, turnover, mitochondrial dynamics and mitophagy, along with the inherent redundancy of mtDNA. Base excision repair (BER) is a major DNA repair mechanism in mammalian mitochondria. Mitochondrial BER enzymes are implicated in mtDNA-mediated immune response and inflammation. mtDNA is organized into mitochondrial nucleoids by mitochondrial transcription factor A (TFAM). The regulation of DNA repair activities by TFAM-DNA interactions remains understudied. Here, we demonstrate the modulation of DNA repair enzymes by TFAM concentrations, DNA sequences and DNA modifications. Unlike previously reported inhibitory effects, we observed that human uracil-DNA glycosylase 1 (UNG1) and AP endonuclease I (APE1) have optimal activities at specific TFAM/DNA molar ratios. High TFAM/DNA ratios inhibited other enzymes, OGG1 and AAG. In addition, TFAM reduces the accumulation of certain repair intermediates. Molecular dynamics simulations and DNA-binding experiments demonstrate that the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in certain sequence motifs enhances TFAM-DNA binding, partially explaining the inhibition of OGG1 activity. Bioinformatic analysis of published 8-oxodG, dU, and TFAM-footprint maps reveals a correlation between 8-oxodG and TFAM locations in mtDNA. Collectively, these results highlight the complex regulation of mtDNA repair by DNA sequence, TFAM concentrations, lesions and repair enzymes.

Anticariogenic activity of marine brown algae Padina boergesenii and its active components towards Streptococcus mutans.

Streptococcus mutans is a well-recognized bacterium that plays a predominant role in the progression of dental caries. Its pathogenicity is linked to several key characteristics, including the ability to produce organic acids (acidogenicity), thrive in low pH environments (aciduricity), synthesize exopolysaccharides (EPS) via glucosyltransferases, and form retentive biofilms. The treatment of dental caries with conventional antibiotics is often ineffective due to the bacterium's capacity to form recalcitrant biofilms. To address these challenges, strategies that specifically target the pathogen's virulence without affecting its viability have emerged as promising alternatives. In this context, we investigated the anticariogenic properties of the methanolic extract of Padina boergesenii (MEPB). MEPB demonstrated substantial, dose-dependent antibiofilm activity, with a maximum inhibition of 93% at 128 μg/mL, without compromising the viability of S. mutans. Anti-virulence assays using sub-MIC (minimum inhibitory concentration) levels of MEPB showed significant reductions in key virulence factors: 75% reduction in sucrose-dependent adherence, 65% reduction in sucrose-independent adherence, along with notable decreases in acid production, acid tolerance, and water-insoluble (85%) and water-soluble (52%) glucan synthesis. Additionally, MEPB significantly reduced cell surface hydrophobicity (55%) and extracellular DNA (eDNA) production (64%). qPCR analysis corroborated these in vitro findings, revealing that MEPB suppresses the expression of genes involved in S. mutans virulence, particularly genes related to EPS synthesis (gtfB, gtfC & gtfD) biofilm formation(gbpB & gbpC) and two-component regulatory system (vicR) were downregulated. Toxicity testing on human buccal epithelial cells confirmed the non-toxic nature of MEPB, suggesting its safety for potential therapeutic use. Furthermore, GC-MS/MS analysis identified palmitic acid, myristic acid, and stearic acid as the major active constituents of the MEPB extract. Subsequent biofilm inhibitory assays confirmed the potent antibiofilm efficacy of these compounds: palmitic acid (85%), myristic acid (72%) and stearic acid (83%). In conclusion, this study identifies P. boergesenii and its active biomolecules as potential anticariogenic agents, offering an alternative approach to combat dental caries by targeting bacterial virulence mechanisms rather than viability.

Removal, detection, and limits of endotoxin in the industry of recombinant proteins

With the advancement of synthetic biology, recombinant proteins are poised to play a significant role in medical applications. The scaled manufacturing is a pillar for the extensive application and development of recombinant proteins across various fields. In the large-scale production process of recombinant proteins, the removal and detection of endotoxins are essential to reduce their levels to safe thresholds in the final products. Currently, establishing stringent endotoxin limits for different recombinant protein products is a crucial aspect of safety assessment. This review begins by shedding light on the pathogenicity of endotoxins and discusses the methods for the removal and determination of endotoxins during the production processes of recombinant proteins. Subsequently, this review summarizes the endotoxin limits in industries such as biologics, medical devices, and human recombinant DNA products, particularly those in recombinant protein injection products. It is highlighted that regardless of whether the hosts for recombinant protein expression are bacteria or not, endotoxin testing is required for the final products of injectable recombinant proteins, and compliance with relevant industry standards is necessary. This review aims to provide a reference for the research on endotoxins in the large-scale production process of recombinant proteins.

In-situ nucleic acid amplification induced by DNA self-assembly for rapid and ultrasensitive detection of miRNA

BackgroundTo improve the sensitivity and specificity of nucleic acid detection, coupling two or more signal amplification systems is a feasible pattern, such as nucleic acid isothermal amplification coupling genome-editing technology, and cascaded DNA self-assembly circuits. And representative signal amplification strategies include loop-mediated isothermal amplification (LAMP), clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas) systems, and catalyzed hairpin assembly (CHA). However, these detection strategies often require the enrichment of intermediate products, the replacement of reaction conditions and the design of multiple probes, which may seriously affect the reliability of detection results. ResultsHerein, we propose a novel nucleic acid detection system which is named as catalyzed hairpin assembly (CHA) coupled with embedded primer triggered isothermal amplification (CEA for short). DNA self-assembly probes in CEA contain a specially designed primer. When target nucleic acid (e.g., miRNA) initiates CHA reaction (the first signal amplification), the self-assembly product of CHA will expose a primer (named as embedded primer). The embedded primer will trigger a special nucleic acid isothermal amplification in situ, then generate plenty of double-stranded DNA products in 30 min with varying lengths (the second signal amplification). Compared to that of a typical CHA reaction, the sensitivity of CEA has increased by three orders of magnitude and the detection limit is as low as 0.228 fM. Besides, it has excellent detection performance in cancer and stem cell samples. SignificanceBy coupling embedded primer with DNA self-assembly system, a new nucleic acid detection system (CEA) with one-step operation and dual signal amplification has been successfully established. Compared with traditional dual signal amplification systems, CEA can not only significantly improve the reaction efficiency, but also greatly reduce the difficulty of detection system design and experimental operation.

NS1-mediated DNMT1 degradation regulates human bocavirus 1 replication and RNA processing.

Methylation of the DNA genome plays an important role in viral gene inactivation. However, the role of DNA methylation in human bocavirus (HBoV) remains unclear. In this study, the HBoV1 genomic DNA was found extensively methylated at the CHG and CHH sites. Inhibiting DNA methylation with 5-aza-2'-deoxycytidine (DAC) altered the methylation status and reduced viral DNA production, while enhanced the RNA splicing at D1 and D3 sites and the polyadenylation at the proximal polyadenylation site, (pA)p. Knockdown of DNA methyltransferase 1 (DNMT1) had the same effect on viral DNA synthesis and RNA processing as the DAC treatment, indicating that DNMT1 is the major host methyltransferase involved in viral DNA methylation. In addition, the nonstructural protein NS1 promoted DNMT1 degradation through the ubiquitin-proteasome pathway to regulate viral replication and RNA processing. Collectively, the results suggest that DNA methylation and DNMT1 facilitate HBoV replication and are essential for appropriate NS1 localization in the nucleus. DNMT1 degradation through NS1 promotes the virus RNA processing, leading to viral protein expression.

Genotyping of human isolates from human toxoplasmosis outbreak: Restriction Fragment Length Polymorphism and Sanger Sequencing.

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii, capable of infecting a wide range of hosts. The parasite exhibits a broad genetic diversity, necessitating genotypic characterization for genotype identification and associations with epidemiological information. Therefore, the Restriction Fragment Length Polymorphism (RFLP) technique is used for characterization. This study aimed to perform genotypic characterization of isolates from pregnant women infected during a human toxoplasmosis outbreak, comparing the RFLP and Sanger Sequencing methodologies. For this purpose, six human isolates were subjected to conventional PCR, Multiplex PCR, Nested PCR, Enzymatic Digestion, and Sanger Sequencing. Additionally, the standard strains GTI (Type I), PTG (Type II), and CTG (Type III) were also subjected to the same techniques described above. Subsequently, the amplified DNA products were compared with the standard strains. As a result, it was observed that Sanger Sequencing provides the same information as RFLP PCR, as well as the possibility of cost reduction for genotypic characterization, and providing greater agility in issuing results. Additionally, Sanger Sequencing of T. gondii isolates allows for detailed evaluation of nucleotide sequences, including the assessment of SNPs and enzymatic restriction sites, which the RFLP technique does not.

First report of lily mottle virus naturally infecting lily (Lilium asiatica) in Texas, USA.

Lily (Lilium asiatica) is an important plant grown for its range of flower colors and heavy scent. In March 2024, potyvirus-like symptoms consisting of light-yellow mottling and mosaic were noticed on 12/20 lily plants in a private property in Weslaco, Hidalgo County, Texas. Two symptomatic plants (WTX1 and WTX2) were sampled randomly for virus diagnosis. The leaf extracts of both samples were negative for the potyvirus group using Agdia's Poty ImmunoStrip® (Agdia, Inc., Elkhart, IN, USA). However, rub-inoculation of the extracts onto healthy Nicotiana benthamiana and Vigna unguiculata plants (n=4, each) induced mild mottle symptoms on the systemic leaves of both herbaceous test plants 20 to 28 days post inoculation, indicating the presence of a mechanically transmissible agent in the samples. No virus-like symptoms were observed on the mock-inoculated plants (n=1, each) of both species. To test for suspected potyvirus infection, 2-µg of total nucleic acid extracts (Dellaporta et al. 1983) from WTX1 and WTX2 were used for complimentary DNA (cDNA) synthesis with Oligo(dT) primer and the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, USA). One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. LMoV is a quarantine pest and was previously reported in the U.S. (Brierley and Smith, 1944) and other lily-producing parts of the world, such as the Netherlands, China, Korea, and Brazil. To our knowledge, this is the first confirmed report of the virus in Texas, thus expanding its geographical range. Testing of lily foundation stocks before propagation is essential to prevent further spread of LMoV via planting material.

Association of Peripheral Markers of Oxidative Stress with Clinical Parameters and Inflammatory Factors in Alcoholic Patients

One of the fundamental problems in studying addiction is elucidation of mechanisms of alcohol dependence (AD) development. Disturbances of cellular redox balance and inflammation play an important role in AD pathogenesis. Deciphering associations between biological and clinical indicators can elucidate molecular mechanisms of disease pathogenesis. The aim of the work was to study peripheral markers of oxidative stress in patients with AD during early abstinence period and to identify their relationship with clinical parameters of the disease and inflammatory factors. In total, 84 patients with AD were studied (average age, 44.3 ± 8.2 years). The analyzed clinical parameters included patient’s age, age of alcohol withdrawal syndrome (AWS) formation, disease duration, and AWS duration. The markers of oxidative stress determined in the blood plasma were oxidation products of proteins (protein carbonyls, PCs), lipids (thiobarbituric acid-reactive products, TBA-RPs), and DNA (8-hydroxy-2′-deoxyguanosine, 8-OH-dG). The content of inflammatory mediators, such as proinflammatory cytokines (IFNγ, IL-1β, IL-6, IL-8, IL-17A, TNFα) was determined in the blood serum. Blood samples of 80 conditionally healthy men (average age, 40.9 ± 9.6 years) were used as a control. Patients with AD demonstrated an increase in the content of PCs, TBA-RPs, and all analyzed cytokines (but not 8-OH-dG) compared to the control individuals. There was a direct correlation between the TBA-RP content and disease duration and inverse correlation of the PC content with the age of AWS formation and AWS duration. The content of PCs demonstrated an inverse correlation with the IL-6 concentration in the blood plasma. We also observed a positive correlation between 8-OH-dG and IL-6, TBA-RPs and IL-8, and TBA-RPs and TNFα. Therefore, the early abstinence period in patients with AD was characterized by a pronounced oxidative stress and inflammation. The obtained results expand the knowledge on the integrative contribution of oxidative stress and inflammatory factors to the AD pathogenesis and can be used in the development of new therapies.

Optimization and Clinical Application Potential of Single Nucleotide Polymorphism Detection Method Based on CRISPR/Cas12a and Recombinase Polymerase Amplification.

Conventional methods for detecting single nucleotide polymorphisms (SNPs) in clinical practice often require substantial time, labor, and specialized equipment, limiting their widespread application. To address this limitation, we refined our previous SNP detection method, IMAS-RPA [introducing an extra mismatched base adjacent to the single-base mutant site by recombinase polymerase amplification (RPA)], resulting in an updated version termed IMAS-RPAv2. We began by introducing a suboptimal protospacer adjacent motif (PAM) sequence, GTTG, into the double-stranded DNA (dsDNA) products using either RPA or reverse transcription RPA. This modification decreased the efficiency with which CRISPR RNA (crRNA) recognizes the PAM and locally unwinds the dsDNA to form an R loop. After a delay, the R loop forms. However, due to the intentional incorporation of a mismatched base on the crRNA relative to the wild-type double-stranded DNA (WT-dsDNA), a continuous two-base mismatch is established between the crRNA and WT-dsDNA. Consequently, WT-dsDNA does not activate CRISPR/Cas12a's cleavage activity within a short time, while variant-type dsDNA continues to activate CRISPR/Cas12a and produce a robust fluorescence signal. This improvement significantly enhances the SNP discrimination sensitivity, allowing for detection at the single-copy level. Results were observed using both a conventional microplate reader and a specially designed portable device created through 3D printing. This device allows a direct fluorescence observation without the need for additional equipment. Consequently, the entire detection process becomes independent of large-scale equipment. This greatly expands its range of applications and offers promising prospects for clinical use.

Human identification of single hair shaft using a mass spectrometry mRNA typing system

Hair is one of the most common forms of forensic biological material at various crime scenes. So far, human identification cannot be effectively accomplished with a single telogen hair encountered in forensic casework due to the detection limit. Emerging studies have revealed RNA as a promising biomarker in hair shafts, while the single telogen hair could not be successfully genotyped even after being examined with the recently developed mRNA typing system. MALDI-TOF MS, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enables sensitive and accurate measurement of DNA products. To address this problem and further develop the analysis technology of hairs, we established a mass spectrometry system for human identification based on a single hair shaft using 25 polymorphic SNPs located on 18 mRNA molecules (KRT31, RFK, KRT86, KRT35, PABPC1, KMT2D, LEMD2, TBC1D4, CTC1, PPP1R15A, RBM33, LRRC15, KRT33A, KRTAP12–2, KRT81, AHNAK, KRTAP4–8, FLG2). The forensic application of the detection system was evaluated, and all hair samples used were collected from individuals in Shanxi province. Firstly, we demonstrated that the RNA typing results of a single hair shaft were in perfect concordance with DNA typing results and confirmed the consistency between hairs from different body parts. To assess the potential influence of positions along the hair shaft, 6 cm long hair shafts from the distal end were examined by the MALDI-TOF MS system, whose genotype could be successfully detected. The system was capable of detecting aged samples stored for 390 days and could also be employed on various types of hair samples, such as white hair and permed or dyed hair. Finally, 50 unrelated individuals from Shanxi province were genotyped for the population study, and the CDP of the system in the Shanxi population is 0.998928. In this study, we established a mass spectrometry system for human identification based on a single hair shaft. We used a single hair shaft, rather than multiple hair shafts reported in our previous report, to get a full typing profile. The system sensitivity was substantially enhanced, which provided a valuable strategy for forensic practice to perform human identification using hairs.