Rapid DNA Research Articles

Hypoxic Effects on Matrix Metalloproteinases’ Expression in the Tumor Microenvironment and Therapeutic Perspectives

The tumor microenvironment (TME) is characterized by an acidic pH and low oxygen concentrations. Hypoxia induces neoplastic cell evasion of the immune surveillance, rapid DNA repair, metabolic reprogramming, and metastasis, mainly as a response to the hypoxic inducible factors (HIFs). Likewise, cancer cells increase matrix metalloproteinases’ (MMPs) expression in response to TME conditions, allowing them to migrate from the primary tumor to different tissues. Since HIFs and MMPs are augmented in the hypoxic TME, it is easy to consider that HIFs participate directly in their expression regulation. However, not all MMPs have a hypoxia response element (HRE)-HIF binding site. Moreover, different transcription factors and signaling pathways activated in hypoxia conditions through HIFs or in a HIF-independent manner participate in MMPs’ transcription. The present review focuses on MMPs’ expression in normal and hypoxic conditions, considering HIFs and a HIF-independent transcription control. In addition, since the hypoxic TME causes resistance to anticancer conventional therapy, treatment approaches using MMPs as a target alone, or in combination with other therapies, are also discussed.

Detection of Esca‐associated fungi in grapevine trunks using loop‐mediated isothermal amplification (LAMP) assays

AbstractEsca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH) and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1460 [FMED]; 1950 [PMI]; 342 [PCH] genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease. The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases.

Molecular wrench activity of DNA helicases: Keys to modulation of rapid kinetics in DNA repair.

DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.

Processing biological samples from simulated radiological terrorist events using Rapid DNA instruments

Two commercially available portable Rapid DNA instruments were evaluated for their ability to process 1 µL and 10 µL saliva samples deposited on metal and plastic surfaces and contaminated with surrogates of cesium (Cs)-137, strontium (Sr)-90 and cobalt (Co)-60; radioactive materials potentially released during a nuclear weapon accident or a radiological dispersal device detonation. A comparable success rate was noted for both Rapid DNA instruments when considering the number of complete and balanced DNA profiles, the number of profiles with a minimum of 10 autosomal STR loci (out of 23 [FlexPlex™ 27] or 21 [GlobalFiler™ Express]), and the possibility to search a national DNA database in Canada and the United States. Cobalt had an adverse impact on the quality of the megaplex short tandem repeat (STR) DNA profiles derived on each instrument for two of the three contamination levels tested in this study, i.e., 0.05 M and 0.1 M as reflected by a reduced number of detected alleles and decreased profile peak heights. Strontium exhibited some adverse effect on the Rapid DNA results when used at the highest contamination level (0.1 M) whereas cesium had none. No new artifacts were observed in the Rapid DNA profiles of samples spiked with the non-radiogenic surrogates. Importantly, in the context of a radiological/nuclear (RN) event, the ANDE™ 6C offers the possibility to dispose of all radioactive materials associated with contaminated samples quickly using a chip on which all steps of the Rapid DNA process are performed whereas the RapidHIT™ ID accumulates radioactive materials for many days before disposal. An individual handling 25 samples in a week (5 per day) on the RapidHIT™ ID at a 30.5 cm distance with a 5 min exposure to the radioactive source estimated at every run would exceed the 0.042 µSv/5 min limit with gamma dose rates for Cs at 0.13 mSv and for Co at 3.8 mSv. Beta dose rates calculated for the surrogate isotopes at the three concentrations tested were also above the recommended radiation exposure limit of 1 mSv/yr (0.042 µSv/5 min). Various potential mechanisms of action behind the interference noted for Sr and Co at high concentrations are presented. These elements may play a role in the steps prior to PCR (at the DNA molecule by binding to bases or to phosphate groups), during PCR (at the DNA polymerase as cofactors for catalytic sites), or even during amplified DNA fragment detection (as fluorescence quenchers).

Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance

Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use. In this study, we developed a rapid on-site applicable DNA extraction and library preparation approach for nanopore sequencing, using portable devices. The optimized method consists of a portable mechanical lysis approach followed by magnetic bead-based DNA purification and automated sequencing library preparation, and resulted in a throughput comparable to a current optimal, laboratory-based protocol using enzymatic digestion to lyse cells. By using spike-in reference communities, we compared the on-site method with other workflows, and demonstrated reliable taxonomic profiling, despite method-specific biases. We also demonstrated the added value of long-read sequencing by recovering reads containing full-length antimicrobial resistance genes, and attributing them to a host species based on the additional genomic information they contain. Our method may provide a rapid, widely-applicable approach for microbial detection and surveillance in a variety of on-site settings.

DNAR-02. MICROGLIA IN THE GLIOBLASTOMA MICROENVIRONMENT ASSIST REPAIR OF RADIATION INDUCED DNA DAMAGE IN A CONTACT INDEPENDENT MANNER

Abstract Rapid repair of DNA damage mediates genotoxic therapy resistance and poor prognosis in glioblastoma (GBM). The GBM tumor microenvironment (TME) is heterogenous, and we hypothesized that non-malignant cells in the GBM TME support the repair of DNA damage in glioblastoma cells. Consistent with this hypothesis, we found that GBM tumors grown intracranially in mice had rapid DNA repair and were resistant to radiation when compared to tumors of identical genotype grown extracranially in mouse flanks. Further interrogation of irradiated intracranial tumors using gamma-H2AX immunofluorescence revealed spatial heterogeneity in DNA damage repair, with spatially separate regions of rapid and slow gamma-H2AX resolution. Paired spatial transcriptomic analysis and gamma-H2AX immunofluorescence revealed that tumor regions with rapid DNA repair were rich in microglia and enriched for inflammatory response and epithelial to mesenchymal transition (EMT) pathway transcripts. Spatial transcriptomic analysis of patient tissue revealed that regions enriched in DNA damage repair pathways are also enriched for microglia and not other non-malignant cell types. Having nominated microglia, we then cocultured GBM cells and microglia and found that GBM cells cocultured with microglia repair DNA damage faster in a contact-independent manner. Hence, we show that microglia in the GBM microenvironment assist GBM cells to repair radiation induced DNA damage through inflammatory response and EMT and hence, mediate treatment resistance.

A Loop-Mediated Isothermal Amplification Assay for the Identification of Botrytis fragariae in Strawberry.

Gray mold in strawberry is caused by multiple species of Botrytis, including Botrytis cinerea, B. pseudocinerea, B. fragariae, and B. mali. The species B. cinerea and B. fragariae are widespread in production regions of the eastern United States and Germany, and their distinction is important for disease management strategies. Currently, the only way to differentiate these species in field samples is by PCR, which is time consuming, labor intensive, and costly. In this study, a loop-mediated isothermal amplification (LAMP) technique was developed based on species-specific NEP2 gene nucleotide sequences. The designed primer set specifically amplified B. fragariae DNA and no other Botrytis spp. (B. cinerea, B. mali, and B. pseudocinerea) or plant pathogens. The LAMP assay was able to amplify fragments from DNA extracted from infected fruit using a rapid DNA extraction protocol, confirming its ability to detect low amounts of B. fragaria DNA from field-infected fruit. In addition, a blind test was performed to identify B. fragariae in 51 samples collected from strawberry fields in the eastern United States using the LAMP technique. The B. fragariae samples were identified with a reliability of 93.5% (29 of 32), and none of the B. cinerea, B. pseudocinerea, or B. mali samples included in the test were amplified in 10 min. Our results show that the LAMP technique is a specific and reliable method for the detection of B. fragariae from infected fruit tissue and can help to control this important disease in the field.

Temperature-induced DNA density transition in phage λ capsid revealed with contrast-matching SANS.

Structural details of a genome packaged in a viral capsid are essential for understanding how the structural arrangement of a viral genome in a capsid controls its release dynamics during infection, which critically affects viral replication. We previously found a temperature-induced, solid-like to fluid-like mechanical transition of packaged λ-genome that leads to rapid DNA ejection. However, an understanding of the structural origin of this transition was lacking. Here, we use small-angle neutron scattering (SANS) to reveal the scattering form factor of dsDNA packaged in phage λ capsid by contrast matching the scattering signal from the viral capsid with deuterated buffer. We used small-angle X-ray scattering and cryoelectron microscopy reconstructions to determine the initial structural input parameters for intracapsid DNA, which allows accurate modeling of our SANS data. As result, we show a temperature-dependent density transition of intracapsid DNA occurring between two coexisting phases-a hexagonally ordered high-density DNA phase in the capsid periphery and a low-density, less-ordered DNA phase in the core. As the temperature is increased from 20 °C to 40 °C, we found that the core-DNA phase undergoes a density and volume transition close to the physiological temperature of infection (~37 °C). The transition yields a lower energy state of DNA in the capsid core due to lower density and reduced packing defects. This increases DNA mobility, which is required to initiate rapid genome ejection from the virus capsid into a host cell, causing infection. These data reconcile our earlier findings of mechanical DNA transition in phage.

Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales.

Trade in pangolins is illegal, and yet tons of their scales and products are seized at various ports. These large seizures are challenging to process and comprehensively genotype for upstream provenance tracing and species identification for prosecution. We implemented a scalable DNA barcoding pipeline in which rapid DNA extraction and MinION sequencing were used to genotype a substantial proportion of pangolin scales subsampled from 2 record shipments seized in Singapore in 2019 (37.5t). We used reference sequences to match the scales to phylogeographical regions of origin. In total, we identified 2346 cytochrome b (cytb) barcodes of white-bellied (Phataginus tricuspis) (from 1091 scales), black-bellied (Phataginus tetradactyla) (227 scales), and giant (Smutsia gigantea) (1028 scales) pangolins. Haplotype diversity was higher for P. tricuspis scales (121 haplotypes, 66 novel) than that for P. tetradactyla (22 haplotypes, 15 novel) and S. gigantea (25 haplotypes, 21 novel) scales. Of the novel haplotypes, 74.2% were likely from western and west-central Africa, suggesting potential resurgence of poaching and newly exploited populations in these regions. Our results illustrate the utility of extensively subsampling large seizures and outline an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies.

A Simplified, Efficient and Rapid DNA Extraction Protocol from Rice Grains and Leaf

DNA extraction is very complicated with different plant species because the presence of secondary metabolites that hinder with DNA isolation processes and application like DNA restriction, Gene amplification, as well as gene cloning. A simple method for preparation of rice genomic DNA was developed. The current study expressed comparatively rapid, cost-effective, less time consuming, methods for DNA extraction from seed, young leaves and old leaves of rice genotypes without using liquid nitrogen, phenol, and β–mercaptoethanol in extraction buffer. Using this method, high quality and quantity of genomic DNA was get from 0.5 g of rice seed, young leaves and 75 days old leavesand extracted total nucleic acids (genomic DNA) were amplified by employing single sequence repeats (SSR) or microsatellite markers, which produced reproducible results.This protocol resolves the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides.

Comparative Study of Genomic DNA Extraction Methods for Common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is one of the significant grains used in the human diet, accounting for half of all grain legumes consumed globally. To enhance production, conventional breeding and molecular approaches have been used so far. An efficient and rapid genomic DNA extraction method is required for these molecular approaches. The aim of this study was to compare and optimize an efficient and rapid DNA extraction protocol for common bean. Modified cetyl trimethylammonium bromide (CTAB) and potassium chloride (KCL) extraction methods were used. The mean DNA yield per nanoliter was 209 µg from modified CTAB and 150.3 µg from the KCL method. The concentration of gDNA was significantly (P< 0.05) higher for the KCL method, which was 5.01 µg/µl and 2.09 µg/µl for the CTAB method. The obtained DNA was also pure, with an absorbance ratio at 260 nm to an absorbance of 280 nm (A260/280) of 1.75-2.23 for the KCL method and 1.86-2.09 for the modified CTAB method. Gel electrophoresis separation was used to evaluate the quality of the total DNA extracted by the present protocols. The results showed that intense bands close to the gel wells were obtained from both extraction methods. DNA isolated with the two methods was successfully used for PCR-based downstream analysis, which includes random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. In this study, it took approximately 150 minutes for KCL and 240 minutes for the CTAB for whole process. In contrast to the CTAB method, the KCL method uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. Therefore, it is more convenient and economical than the traditional technique.

Success rate of rapid DNA technology in kinship analysis for forensic purpose.

Reducing the time required for DNA analysis in forensic genetics can yield significant benefits, both in determining genealogical relationships for legal proceedings and in criminal cases. Swift identification of individuals plays a pivotal role in solving crimes and apprehending perpetrators. Additionally, in situations like mass disasters, prompt victim identification holds utmost importance. The Rapid DNA technology, introduced in 2012 to expedite DNA analysis, has evolved to streamline the process into a single step. This advancement not only minimizes the risk of human error and contamination, but also boasts a remarkable time advantage, delivering results in as little as 90min. In this study, DNA profiles of 30 families (consisting of mothers, fathers, and children) were analyzed using the RapidHITTM ID System. The system automatically calculated maternity-paternity probabilities to assess the suitability of Rapid DNA technology for kinship analysis. For validation, DNA profiles were also generated using the 3500 GA method. The study revealed that 9 out of 30 families exhibited discrepancies in DNA profiling, leading to inaccuracies in automatic kinship analysis. Consequently, while the method offers rapid and user-friendly advantages for forensic sciences, the software underlying the system requires re-evaluation. Issues such as maternal-paternal exclusion in kinship analyses, arising from challenges like un-called alleles, warrant further attention.

EP20.43: The rapid long read DNA sequencing method identified bacterial species causing silent intra‐amniotic infection

EP20.43: The rapid long read DNA sequencing method identified bacterial species causing silent intra‐amniotic infection

A Phase II Trial Evaluating Rapid Mid-Treatment Nodal Shrinkage to Select for Adaptive Deescalation in p16+ Oropharyngeal Cancer Patients Undergoing Definitive Chemoradiation

The purpose of this study is to determine if rapid mid-treatment nodal shrinkage (RMNS) can identify patients with p16+ oropharyngeal cancer (OPC) who can be safely deescalated with reduced dose chemoradiation therapy (CRT). The primary endpoint was 2-year progression free survival (PFS). Inclusion criteria were as follows: T1-3, N1, M0 (AJCC 8th edition) p16+ OPC with <10 pack-year smoking history. All patients were initially planned for standard dose CRT (70 Gy) and weekly cisplatin. Patients were evaluated with a CT scan at week 4 for RMNS, defined as >40% nodal volumetric reduction from baseline. If RMNS was achieved, they proceeded to deescalated CRT (60 Gy). If not, they received standard CRT. Biomarker correlates were collected at baseline and week 4 of CRT including plasma TTMV (tumor tissue modified viral) HPV DNA and MRI diffusion weighted imaging (DWI). Univariate logistic regression analyses (UVA) were performed to evaluate predictors of RMNS. Odds ratios with 95% CI are reported, using a p<0.05 for statistical significance with a two-sided test. Wilcoxon rank sum tests were used to evaluate differences between the two groups using p < 0.05, 2-sided) for statistical significance. All statistical procedures were performed using R () with no adjustments for multiple testing. Thirty-six patients were enrolled: median age: 60 years; 81% male; primary site: 36% base of tongue, 53% tonsil, 11% both; T-stage: 39% T1, 50% T2, 11% T3; N-stage: 100% N1; any smoking history: 58% yes, 42% no; 67% (n = 24) had RMNS and received deescalated CRT while the remaining proceeded to standard CRT. At a median follow-up of 32.4 months, 2-year PFS between the standard and deescalated groups were 91.7% vs 90.9%, respectively (p = 0.97). All patients with recurrence underwent successful salvage treatment with 2-year OS 100% for all patients. On UVA, rapid TTMV HPV DNA clearance (baseline to week 4) (OR 12.0 [1.65-250], p = 0.034), lower MRI diffusivity (ADC) at baseline (OR 0.79 [0.61-0.97], p = 0.042) and week 4 (OR 0.76 [0.60-0.91], p = 0.009), and higher MRI diffusional kurtosis at baseline (OR 1.09 [1.01-1.21], p = 0.051) and week 4 (OR 1.24 [1.09-1.52], p = 0.009) were significantly associated with RMNS. When comparing the deescalated and standard cohorts, the mean baseline and week 4 MRI ADC were significantly lower and week 4 MRI diffusional kurtosis was significantly higher in the deescalated group. In this phase II study, rapid mid-treatment nodal shrinkage appeared to select favorable risk p16+ oropharynx cancer patients for treatment de-escalation. Rapid clearance of TTMV HPV DNA at week 4 as well as MRI DWI biomarkers of low ADC and high diffusional kurtosis values were correlated with RMNS. A larger study is planned to incorporate RMNS and biomarkers for further treatment de-escalation. Additional trial information is available at ClinicalTrials.gov (Identifier: NCT03215719).

Exploring the World of Mungbean: Uncovering its Origins, Taxonomy, Genetic Resources and Research Approaches

The mungbean is one of the important leguminous crops, holds great importance in agriculture and nutrition in Asian countries. It is a vital source of protein, playing a key role in vegetarian diets and offering versatility in culinary applications. The crop wild relatives and landraces are invaluable sources of novel genes and alleles, augmenting mungbean resilience against both biotic and abiotic stresses. Researchers have meticulously examined the roles of additive and dominant gene effects in governing vital traits, including seed weight, seed yield, plant height, days to flowering, and yield components. However, despite their potential, these genetic resources remain underutilized due to several constraints, encompassing interspecific hybridization barriers, limited trait evaluation data, and the absence of advanced breeding tools. Announcement of the VC1973A draft reference genome utilizing next-generation sequencing, have facilitated rapid DNA marker development, gene mapping, and the identification of candidate genes for complex traits, predominantly within the last decade. Reports on GBS analysis of mungbean relatively limited and mostly includes investigating population structure and LD in mungbean. This review offers a comprehensive overview of the origin, taxonomy, gene pool dynamics, and the pivotal role played by mungbean CWRs in ensuring agricultural sustainability and elevating crop improvement initiatives. Additionally, it discusses about genotypic characterization of CWRs, advances in sequencing technologies, QTLs mapping for various economically important traits and validation techniques that aid in confirming the QTLs found through GWAS.

Rapid and Sensitive Detection of Toxigenic Fusarium asiaticum Integrating Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Techniques.

Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. Both Fusarium graminearum and F. asiaticum are the causal agents of FHB in China. F. asiaticum is the predominant species in the Middle-Lower Reaches of the Yangtze River (MLRYR) and southwest China. Therefore, detecting F. asiaticum in a timely manner is crucial for controlling the disease and preventing mycotoxins from entering the food chain. Here, we combined rapid genomic DNA extraction, recombinase polymerase amplification, Cas12a cleavage, and lateral flow detection techniques to develop a method for the rapid detection of F. asiaticum. The reaction conditions were optimized to provide a rapid, sensitive, and cost-effective method for F. asiaticum detection. The optimized method demonstrated exceptional specificity in detecting F. asiaticum while not detecting any of the 14 other Fusarium strains and 3 non-Fusarium species. Additionally, it could detect F. asiaticum DNA at concentrations as low as 20 ag/μL, allowing for the diagnosis of F. asiaticum infection in maize and wheat kernels even after 3 days of inoculation. The developed assay will provide an efficient and robust detection platform to accelerate plant pathogen detection.

Disposable and instrument-free nucleic acid lateral flow cassette for rapid and on-site identification of adulterated goat milk

Species identification has become a significant concern due to the growing use of food alternatives that may cause allergies and reduce nutritional value. To address the issue of fraudulent adulteration of goat milk products with cow milk, we have developed an affordable, portable, and user-friendly platform called microfluidic-integrated nucleic acid lateral flow strips (LFS). This platform enables simultaneous detection of components derived from both goats and cows in goat milk. In this study, we have introduced an innovative nucleic acid labeling method. The loop primers of loop-mediated isothermal amplification (LAMP) have been modified with amplification terminator spacer C3 and an oligonucleotide sequence, thus eliminating the requirement for costly antibodies in traditional nucleic acid LFS. This modification not only lowers costs but also enables multiple detections. Additionally, we have integrated the LAMP and LFS assay steps into a microfluidic chip, allowing convenient on-site detection while effectively preventing aerosol contamination of LAMP products. The testing process includes rapid DNA extraction, followed by a short nucleic acid addition and incubation for visualized results in about 50 min. This platform is user-friendly, requiring no specialized equipment or extensive training, making it suitable for rapid on-site detection of dairy products by personnel in diverse fields.

A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1.

The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an 'Escaping' effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for 'Escaping'. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.

A rapid, efficient and cost-effective DNA isolation protocol for various herbal products which is appropriate for different downstream genomics applications

A rapid, efficient and cost-effective DNA isolation protocol for various herbal products which is appropriate for different downstream genomics applications

A simple and rapid DNA extraction method from meat by microneedle patch for detection of adulterated components

ABSTRACT The simple and rapid identification of meat via DNA-based methods is still subject to the conditions of complex and time-consuming preprocessing. It usually takes 3–4 h to obtain DNA sample using conventionally available DNA extraction kits. The existing approaches are cumbersome, require large equipment, and are not suitable for field operation. This study developed a swelling microneedle patch, constituted by micron-sized copolymers of methyl vinyl ether and maleic acid (PMVE/MA). The microneedle patch was applied to the meat sample for 1 min before being pulled out and rinsed with Tris-EDTA (TE) buffer to obtain DNA, which could be used for polymerase chain reaction (PCR) without purification. The DNA extracted by the microneedle patch could meet the needs for amplification and identification of meat samples. The developed approach which combines the patch-based extraction method and conventional PCR amplification is quick, simple, and reliable for DNA extraction and identification of meat samples.