Showing papers on "genomic DNA published in 2022"
TL;DR: In this article , a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae was explored.
Abstract: In this study a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae was explored. For this purpose, using Chattonella marina as a test algal species, PCR targeting the D1-D2 region of large subunit ribosomal gene of this alga was performed with the tagged specific primers. The amplicons were then analyzed with the manually prepared dot lateral flow strip, and the strip could produce a test dot and a control dot that are naked-eye detectable, indicating the successful establishment of PCDS. The established PCDS assay does not require expensive instruments for the detection, and the results can be observed visually after adding 7.5 μL of PCR amplicons in combination with 92.5 μL of chromatography buffer to the sample pad of the strip for about 10 min. The PCR conditions were optimized to enhance the effectiveness of detection. The cross-reactivity test with 23 microalgae species, including Chattonella marina, showed good specificity of the PCDS. The detection limit of PCDS was 1.25 × 10-2 ng µL-1 for genomic DNA and 101 cells mL-1 for crude cell extracts, which can meet the detection needs. In summary, the PCDS proposed in this study has low cost, clear, and intuitive detection results and good specificity and sensitivity, providing a novel detection method for C. marina.The online version contains supplementary material available at 10.1007/s10811-021-02667-x.
46 citations
TL;DR: GuIDE-tag as mentioned in this paper uses tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of Nuclease-mediated double-strand break (DSB) DNA repair factors.
Abstract: Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.
17 citations
TL;DR: In this paper , the authors showed that YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNA polymerase that has the ability to replicate the genome.
Abstract: Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 Å in other phages, spacings of 33-36 Å between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome.
15 citations
TL;DR: In this article , the reverse transcriptase (RT) was fused with nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment, which simultaneously introduced a double strand break (DSB) and a single 3' extended flap in the target site.
Abstract: Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3' extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3' terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.
13 citations
TL;DR: In this paper , the authors developed a novel method of the on-site detection for Salmonella in food by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA).
Abstract: Salmonella species are common foodborne pathogenic bacteria. At present, most detection methods for Salmonella are unsuitable for on-site applications because they require large instruments or complicated procedures. This study developed a novel method of the on-site detection for Salmonella in food by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA). The optimal concentration ratio of Cas12a enzyme, CRISPR RNA (crRNA) and FQ-probe is 1 : 1: 2.5. The detection limit of the RPA–CRISPR/Cas12a method was 1 × 10−4 ng/μL for genomic DNA (gDNA), and 102 CFU/mL for bacterial liquid. The method exhibited no cross-reactivity to 4 common pathogenic bacteria. A simple boiling method was used to pretreat chicken and egg samples to meet on-site testing requirements. The detection limits of chicken and egg samples were 103 CFU/mL initially and could reach 102 CFU/mL and 101 CFU/mL, respectively, after 3 h enrichment at 37 °C. The sensitivity of the RPA–CRISPR/Cas12a method was comparable to that of the quantitative polymerase chain reaction (qPCR) technique. In addition, the operation of this method is simpler and faster than qPCR. Thus, the method is applicable to the on-site detection for Salmonella in food.
11 citations
TL;DR: In this paper , the authors investigated the effects of the DNA sequences flanking the target 5mC site on TET activity and found that flanking sequence preferences were similar for TET1 and TET2 and also for 5mc and 5hmC substrates.
Abstract: TET dioxygenases convert 5-methylcytosine (5mC) preferentially in a CpG context into 5-hydroxymethylcytosine (5hmC) and higher oxidized forms, thereby initiating DNA demethylation, but details regarding the effects of the DNA sequences flanking the target 5mC site on TET activity are unknown. We investigated oxidation of libraries of DNA substrates containing one 5mC or 5hmC residue in randomized sequence context using single molecule readout of oxidation activity and sequence and show pronounced 20 and 70-fold flanking sequence effects on the catalytic activities of TET1 and TET2, respectively. Flanking sequence preferences were similar for TET1 and TET2 and also for 5mC and 5hmC substrates. Enhanced flanking sequence preferences were observed at non-CpG sites together with profound effects of flanking sequences on the specificity of TET2. TET flanking sequence preferences are reflected in genome-wide and local patterns of 5hmC and DNA demethylation in human and mouse cells indicating that they influence genomic DNA modification patterns in combination with locus specific targeting of TET enzymes.
11 citations
TL;DR: Recombinant vaccines against COVID-19 are continuously being applied, and new clinical trials have been tested by interchangeability studies of viral vaccines developed by classical and next-generation platforms.
Abstract: Several coronaviruses (CoVs) have been identified as human pathogens, including the α-CoVs strains HCoV-229E and HCoV-NL63 and the β-CoVs strains HCoV-HKU1 and HCoV-OC43. SARS-CoV, MERS-CoV, and SARS-CoV-2 are also classified as β-coronavirus. New SARS-CoV-2 spike genomic variants are responsible for human-to-human and interspecies transmissibility, consequences of adaptations of strains from animals to humans. The receptor-binding domain (RBD) of SARS-CoV-2 binds to receptor ACE2 in humans and animal species with high affinity, suggesting there have been adaptive genomic variants. New genomic variants including the incorporation, replacement, or deletion of the amino acids at a variety of positions in the S protein have been documented and are associated with the emergence of new strains adapted to different hosts. Interactions between mutated residues and RBD have been demonstrated by structural modelling of variants including D614G, B.1.1.7, B1.351, P.1, P2; other genomic variants allow escape from antibodies generated by vaccines. Epidemiological and molecular tools are being used for real-time tracking of pathogen evolution and particularly new SARS-CoV-2 variants. COVID-19 vaccines obtained from classical and next-generation vaccine production platforms have entered clinicals trials. Biotechnology strategies of the first generation (attenuated and inactivated virus–CoronaVac, CoVaxin; BBIBP-CorV), second generation (replicating-incompetent vector vaccines–ChAdOx-1; Ad5-nCoV; Sputnik V; JNJ-78436735 vaccine-replicating-competent vector, protein subunits, virus-like particles–NVX-CoV2373 vaccine), and third generation (nucleic-acid vaccines–INO-4800 (DNA); mRNA-1273 and BNT 162b (RNA vaccines) have been used. Additionally, dendritic cells (LV-SMENP-DC) and artificial antigen-presenting (aAPC) cells modified with lentiviral vector have also been developed to inhibit viral activity. Recombinant vaccines against COVID-19 are continuously being applied, and new clinical trials have been tested by interchangeability studies of viral vaccines developed by classical and next-generation platforms.
9 citations
TL;DR: In this article , the authors investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5'-C-phosphate-G-3' (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of syphilis patients.
Abstract: Downstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5'-C-phosphate-G-3' (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93 to 98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit-propagated isolates, containing >14 T. pallidum genomic copies/μL of sample for SWGA and >129 genomic copies/μL for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole-genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which has been challenging until now. IMPORTANCE Syphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions and, if left untreated, can progress through multiple stages with various clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past 2 decades, WGS data are needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of an SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.
8 citations
TL;DR: This paper constitutes a comprehensive summary on the role of exosome gDNA on CRC with the intent of providing a theoretical basis and reference for early diagnosis and clinical treatment of cancer.
Abstract: Exosomes are extracellular vesicles that mediate cell-to-cell communication. Bioactive substances such as DNA, RNA, lipids, and proteins are present in it, and they play an essential role in the pathogenesis of colorectal cancer (CRC). The role of RNA and protein in exosomes has been extensively studied. Exosome DNA has recently attracted the attention of a great deal of scientists. According to studies, exosome DNA mainly contains genomic DNA (gDNA) and mitochondrial DNA (mtDNA), of which exosome gDNA is widely used in liquid biopsy of CRC. It includes a variety of clinically relevant tumor-specific mutation genes. In addition to liquid biopsy, researchers find that exosome gDNA regulates immune and metabolic functions in CRC, making it an important research object. However, the primary research on exosome gDNA is still limited. Here, we describe the occurrence and composition of exosomes. Summarize the essential characteristics and mode of action of exosome gDNA. Remarkably, this paper constitutes a comprehensive summary on the role of exosome gDNA on CRC with the intent of providing a theoretical basis and reference for early diagnosis and clinical treatment of cancer.
8 citations
TL;DR: It is found that extensive genomic disruptions by Cas9, involving the allelic cooccurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements led to functional aberrant DNA fragments and can alter cell proliferation.
Abstract: The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.
7 citations
TL;DR: In this article , a colorimetric based PSR technique was used for the detection of trypanosoma evansi in the blood of the host by targeting the 196bp Invariable Surface Glycoprotein (ISG) gene of T. evansis.
TL;DR: In this article , a two-tube hexaplex polymerase chain reaction (PCR) method was used to authenticate twelve meat species in actual adulteration event using genomic DNA isolated from both boiled and microwave-cooked meat as templates, PCR amplification generated expected PCR products.
Abstract: Frequent meat frauds have aroused significant social attention. The aim of this study is to construct a two-tube hexaplex polymerase chain reaction (PCR) method offering accurate molecular authentication of twelve meat species in actual adulteration event. Deoxyribonucleic acid (DNA) sequencing demonstrates that designed primers can specifically amplify target species from genomic DNA mixture of six species in each tube reaction, which showed 100% accuracy of horse (148 bp), pigeon (218 bp), camel (283 bp), rabbit (370 bp), ostrich (536 bp), and beef (610 bp) as well as turkey (124 bp), dog (149 bp), chicken (196 bp), duck (277 bp), cat (380 bp), and goose (468 bp). A species-specific primer pair produced the target band in the presence of target genomic DNA but not non-target species. Through multiplex PCR assays with serial concentration of the DNA mixture of six species in each PCR reaction, the detection limit (LOD) of the two-tube hexaplex PCR assay reached up to 0.05–0.1 ng. Using genomic DNA isolated from both boiled and microwave-cooked meat as templates, PCR amplification generated expected PCR products. These findings demonstrate that the proposed method is specific, sensitive and reproducible, and is adequate for food inspection. Most importantly, this method was successfully applied to detect meat frauds in commercial meat products. Therefore, this method is of great importance with a good application foreground.
TL;DR: A diagnostic method using recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) for the rapid on-site detection of B. xylophilus and showed potential for point-of-care testing (POCT) in resource-limited areas or in field.
Abstract: The pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most lethal nematode species, which causes pine wilt disease (PWD), a devastating forest disease. To date, no effective methods have been developed to control the disease; hence, rapid precise detection of B. xylophilus is of great significance. Traditional molecular diagnostic methods are time-consuming and require sophisticated instruments or skilled operators, which are unavailable in resource-limited settings. A specific, sensitive, and field-applicable diagnostic method is urgently needed. In this study, we developed a diagnostic method using recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) for the rapid on-site detection of B. xylophilus. The false-positive signals from primer-dependent artifacts were eliminated using a probe, and base substitutions were included in the primer and probe. The entire detection process for the RPA-LFD assay can be completed under 38°C within approximately 30 min, including 15 min for crude nematode genomic DNA (gDNA) extraction and master mix preparation, 15 min for the RPA-LFD assay. This assay displayed high specificity toward B. xylophilus and showed no cross-reactions with closely related species, including Bursaphelenchus mucronatus and Bursaphelenchus doui. The sensitivity of this assay had a detection limit as low as 1 pg of B. xylophilus purified genomic DNA. Furthermore, the application of the RPA-LFD assay in simulated spiked pinewood samples showed accurate detection results. The RPA-LFD assay in this study successfully detected B. xylophilus in less than 30 min, providing a novel alternative for the simple, sensitive, and specific detection of B. xylophilus and showed potential for B. xylophilus point-of-care testing (POCT) in resource-limited areas or in field.
TL;DR: Wang et al. as discussed by the authors developed a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.
TL;DR: The Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting as mentioned in this paper .
Abstract: Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR.DNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €).The Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.
TL;DR: It is demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.
Abstract: 5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD (R2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng μL-1. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.
TL;DR: In this article , a quick and inexpensive sonication technique was described to extract genomic DNA from filamentous fungi without buffer which can be used to perform PCR in under an hour, and the extracted fungal DNA was used for multiplex PCR to detect virulence genes from Zymoseptoria tritici.
TL;DR: In this paper , the authors demonstrate that when quantifying bacterial load in mixed samples, reaction efficiency should not be used as a calibration curve since gDNA isolation efficiency is neglected, significantly impacting quantification.
TL;DR: Based on phylogenetic analyses and whole genomic comparisons, strain SM2-42 T represented a novel species, for which the name Oceanobacter mangrovi sp.
TL;DR: In this paper , the authors exploit surface-enhanced Raman scattering (SERS) to investigate aqueous droplets of genomic DNA deposited onto silver-coated silicon nanowires, and show that it is possible to efficiently discriminate between spectra of tumoral and healthy cells.
Abstract: We exploit Surface-Enhanced Raman Scattering (SERS) to investigate aqueous droplets of genomic DNA deposited onto silver-coated silicon nanowires, and we show that it is possible to efficiently discriminate between spectra of tumoral and healthy cells. To assess the robustness of the proposed technique, we develop two different statistical approaches, one based on the Principal Components Analysis of spectral data and one based on the computation of the ℓ2 distance between spectra. Both methods prove to be highly efficient, and we test their accuracy via the Cohen’s κ statistics. We show that the synergistic combination of the SERS spectroscopy and the statistical analysis methods leads to efficient and fast cancer diagnostic applications allowing rapid and unexpansive discrimination between healthy and tumoral genomic DNA alternative to the more complex and expensive DNA sequencing.
TL;DR: This study supports the premise that m5C can serve as a marker of plant tissue viability whereas oxidized nucleobases, although indicating a cellular redox state, cannot, and demonstrates that tissue desiccation induces a similar trend in changes in the global level of hm5C and 8-oxoG.
Abstract: Modifications of DNA nucleobases are present in all forms of life. The purpose of these modifications in eukaryotic cells, however, is not always clear. Although the role of 5-methylcytosine (m5C) in epigenetic regulation and the maintenance of stability in plant genomes is becoming better understood, knowledge pertaining to the origin and function of oxidized nucleobases is still scarce. The formation of 5-hydroxymetylcytosine (hm5C) in plant genomes is especially debatable. DNA modifications, functioning as regulatory factors or serving as DNA injury markers, may have an effect on DNA structure and the interaction of genomic DNA with proteins. Thus, these modifications can influence plant development and adaptation to environmental stress. Here, for the first time, the changes in DNA global levels of m5C, hm5C, and 8-oxo-7,8-dihydroguanine (8-oxoG) measured by ELISA have been documented in recalcitrant embryonic axes subjected to desiccation and accelerated aging. We demonstrated that tissue desiccation induces a similar trend in changes in the global level of hm5C and 8-oxoG, which may suggest that they both originate from the activity of reactive oxygen species (ROS). Our study supports the premise that m5C can serve as a marker of plant tissue viability whereas oxidized nucleobases, although indicating a cellular redox state, cannot.
TL;DR: Wang et al. as discussed by the authors identified white-pigmented, Gram-staining-positive, strictly aerobic, non-spore-forming, irregular rod-shaped bacteria from the faeces of bats collected from Guangxi autonomous region (22°20'54″N, 106°49'20″E; July 28, 2011) and Chongqing city (30°02'15''N, 107°07'4″E) of South China.
TL;DR: In this article , four strains were isolated and identified as Leuconostoc mesenteroides SG315 (LA), L. citreum SG255 (LB), L lactis CCK940 (LC), and L. lactis SBC001(LD).
Abstract: Leuconostoc is mostly found in food, plants, and dairy products. Due to their innate genomic features, such as the presence of carbohydrate-active enzymes, bacteriocins, and plasmids, Leuconostoc spp. have great biotechnological potential. In this study, four strains were isolated and identified as Leuconostoc mesenteroides SG315 (LA), L. citreum SG255 (LB), L. lactis CCK940 (LC), and L. lactis SBC001(LD). Comparative analysis was performed using their draft genome sequences. Differences among the four strains were analyzed using the average nucleotide identity, dot plot, and multiple alignments of conserved genomic sequences. Functional profiling revealed 2134, 1917, 1751, and 1816 open reading frames; 2023, 1823, 1655, and 1699 protein-coding genes; 60, 57, 83, and 82 RNA-coding genes; and GC content of 37.5 %, 38.8 %, 43.3 %, and 43.2 %, in LA, LB, LC, and LD, respectively. The total number of genes encoding carbohydrate-active enzymes was 76 (LA), 73 (LB), 57 (LC), and 67 (LD). These results indicate that the four strains shared a large number of genes, but their gene content is different. Furthermore, most genes with unknown functions were observed in the prophage regions of the genome. This study also elucidated the oligosaccharide utilization and folate biosynthesis pathways in Leuconostoc spp. Taken together, our findings provide useful information on the genomic diversity of CAZymes in the four Leuconostoc strains and suggest that these species could be used for potent exploitation.
TL;DR: A novel software, Genomic Region sets Enrichment Analysis Platform (GREAP), which provides comprehensive region annotation and enrichment analysis capabilities and a customizable genome browser containing >400 000 000 customizable tracks for visualization is designed.
Abstract: The rapid development of genomic high-throughput sequencing has identified a large number of DNA regulatory elements with abundant epigenetics markers, which promotes the rapid accumulation of functional genomic region data. The comprehensively understanding and research of human functional genomic regions is still a relatively urgent work at present. However, the existing analysis tools lack extensive annotation and enrichment analytical abilities for these regions. Here, we designed a novel software, Genomic Region sets Enrichment Analysis Platform (GREAP), which provides comprehensive region annotation and enrichment analysis capabilities. Currently, GREAP supports 85 370 genomic region reference sets, which cover 634 681 107 regions across 11 different data types, including super enhancers, transcription factors, accessible chromatins, etc. GREAP provides widespread annotation and enrichment analysis of genomic regions. To reflect the significance of enrichment analysis, we used the hypergeometric test and also provided a Locus Overlap Analysis. In summary, GREAP is a powerful platform that provides many types of genomic region sets for users and supports genomic region annotations and enrichment analyses. In addition, we developed a customizable genome browser containing >400 000 000 customizable tracks for visualization. The platform is freely available at http://www.liclab.net/Greap/view/index.
TL;DR: Based on the phenotypic, phylogenetic, genomic and chemotaxonomic features, strain SCR006T represents a novel species, for which the name Proteiniclasticum aestuarii sp.
Abstract: A novel bacterium, designated SCR006T, was isolated from tidal flat sediment from Suncheon Bay, Republic of Korea. Cells of strain SCR006T were strictly anaerobic, motile cocci, Gram-reaction-negative, and catalase- and oxidase-negative. Growth was observed at 4-41 °C (optimum, 34-37 °C), at pH 6.5-10.0 (optimum, pH 7.0-7.5) and in presence of 0-8 % NaCl (optimum, 0-2 %). Fermentation products of peptone-yeast-glucose medium were acetate and ethanol. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that strain SCR006T had high sequence similarity to Proteiniclasticum ruminis D3RC-2T (97.9 %), followed by Youngiibacter multivorans DSM 6139T (95.9 %) and Youngiibacter fragilis 232.1T (95.0 %). The average nucleotide identity value between strain SCR006T and P. ruminis DSM 24773T was 72.7 %, which strongly supported that strain SCR006T reresents a novel species within the genus Proteiniclasticum. The major cellular fatty acids are iso-C15 : 0 (27.2 %) and anteiso-C15 : 0 (16.9 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unidentified phospholipids, an unidentified aminolipid and five unidentified lipids. The genomic size was 3.2 Mb with genomic DNA G+C content of 45.6 mol%. The results of 16S rRNA-based and genome-based phylogenetic tree analyses indicated that SCR006T should be assigned to the genus Proteiniclasticum. Strain SCR006T could be distinguished from P. ruminis D3RC-2T by its growth conditions, cell morphology and genomic characteristics. Based on the phenotypic, phylogenetic, genomic and chemotaxonomic features, strain SCR006T represents a novel species, for which the name Proteiniclasticum aestuarii sp. nov. is proposed, with the type strain SCR006T (=KCTC 25245T= JCM 34531T).
TL;DR: In this article, high sensitive quantitative polymerase chain reaction (qPCR) and digital droplet polymerase Chain reaction (ddPCR), were developed to detect and quantify total eumycetes with potential application in several food matrices and specifically determine the level of contamination by Saccharomycopsis fibuligera and Wickerhamomyces anomalus cells directly in bread.
TL;DR: In this paper , a suppression thermo-interlaced (STI) PCR method was proposed for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools.
TL;DR: In this article , high sensitive quantitative polymerase chain reaction (qPCR) and digital droplet polymerase Chain Reaction (ddPCR)-based assays were developed to detect and quantify total eumycetes with potential application in several food matrices and to specifically determine the level of contamination by Saccharomycopsis fibuligera and Wickerhamomyces anomalus cells directly in bread.