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The problem that this study addresses is to understand how microwave radiation is able to degrade genomic DNA of E. coli. In addition, a comparative study was made to evaluate the suitability of a high-throughput automated electrophoresis platform for quantifying the DNA degradation under microwave radiation. Overall, this study investigated the genomic DNA degradation of E. coli under microwave radiation using automated gel electrophoresis. To examine the viable organisms and degradation of genomic DNA under microwave exposure, we used three methods: (1) post-microwave exposure, where E. coli was enumerated using modified mTEC agar method using membrane filtration technique; (2) extracted genomic DNA of microwaved sample was quantified using the Qubit method; and (3) automated gel electrophoresis, the TapeStation 4200, was used to examine the bands of extracted DNA of microwaved samples. In addition, to examine the impacts of microwaves, E. coli colonies were isolated from a fecal sample (dairy cow manure), these colonies were grown overnight to prepare fresh E. coli culture, and this culture was exposed to microwave radiation for three durations: (1) 2 min; (2) 5 min; and (3) 8 min. In general, Qubit values (ng/µL) were proportional to the results of automated gel electrophoresis, TapeStation 4200, DNA integrity numbers (DINs). Samples from exposure studies (2 min, 5 min, and 8 min) showed no viable E. coli. Initial E. coli levels (at 0 min microwave exposure) were 5 × 108 CFU/mL, and the E. coli level was reduced to a non-detectable level within 2 min of microwave exposure. The relationships between Qubit and TapeStation measurements was linear, except for when the DNA level was lower than 2 ng/µL. In 8 min of microwave exposure, E. coli DNA integrity was reduced by 61.7%, and DNA concentration was reduced by 81.6%. The overall conclusion of this study is that microwave radiation had a significant impact on the genomic DNA of E. coli, and prolonged exposure of E. coli to microwaves can thus lead to a loss of genomic DNA integrity and DNA concentrations.

Implementing nanofertilizers in cultivation to enhance food security is important and gaining great significance, as they have good properties to improve plant production, phytochemicals, and nutrient efficiency and thereby meet the demands of the increasing world population for food. This work demonstrated the impact of calcium carbonate nanoparticles (Ca-NPs) and Ca bulk at three concentrations (0, 100, and 200 mg L−1) on growth, productivity, photosynthetic pigments, phytochemical content, antioxidant activity enzymes, minerals, toxicity, and genomic DNA of lettuce plants. In this regard, Ca-NPs at a concentration of 200 mg L−1 reinforced the vegetative growth characteristics of lettuce plants, increasing head length by 15.7 and 19.2%, head diameter by 20.3 and 19.9%, head fresh weight by 54.4 and 52.9%, and production per hectare by 54.7 and 52.8% as compared to the control during the two growing seasons. Furthermore, the percentages of total chlorophyll (62.6 and 59.5%), carotenoids (48.4 and 56.5%), total phenolics (63.6 and 65.7%), total indoles (39.4 and 36.4%), vitamin C (39.7 and 39.6%), antioxidant activity (57.8 and 53.7%), nitrogen (70.5 and 67.5%), phosphorus (120 and 110.5%), potassium (33.0 and 33.2%), and calcium (67.14 and 63.2%) were also increased compared with the control during two consecutive growing seasons. Additionally, Ca-NPs and Ca bulk had an impact on the plants' genomic DNA compared to the control. In addition, lettuce plants treated with Ca-NPs were proven to be nontoxic and safe for humans by using the Microtox 500 analyzer. [ABSTRACT FROM AUTHOR]

AbstractIt is very challenging to distinguish between Panax ginseng (P. ginseng) and Panax quinquefolius (P. quinquefolius) based on their physical appearances. Therefore, a molecular test that can be performed in commercial settings is needed to address this challenge. We have selected a locus containing a single nucleotide polymorphism (SNP) site in the Dammarendiol-II Synthase (DS) gene to differentiate between P. ginseng and P. quinquefolius. An isothermal nucleic acid amplification test (NAAT) developed previously was adapted and used on a spot test (ST) format consisting of a sample pad, a nitrocellulose membrane, and an adsorbent pad. An ST with a colorimetric detection method has been developed with visual detection based on 20-nm gold nanoparticles. The presence of the target DNA will generate a red color signal, which is visible to the naked eye. The assay comprises the capture probes, constructed based on the DS gene of ginseng, and the ends of the probe sequences sit on the SNP site for the P. ginseng and P. quinquefolius sequences; the detection probe employed in the assay is tagged with gold. The NAAT amplifies the extracted plant genomic samples and enhances the detection of specific SNPs. This NAAT technique was used to authenticate two ginseng root samples, showing greater specificity ratios than our previous work. [ABSTRACT FROM AUTHOR]

With advancements in DNA amplification research, isothermal amplification technology has emerged as an attractive method for detecting target DNA. Here, we describe primer generation-rolling circle amplification (PG-RCA) as an isothermal amplification method for detecting Escherichia coli O157:H7, Salmonella Typhimurium, Bacillus cereus, and Listeria monocytogenes. To improve PG-RCA sensitivity, the concentrations of the reaction components, dNTPs, phi29 DNA polymerase, and circular probes were optimized; the optimized conditions were applied to detect each target bacterium. A pair of forward and reverse circular probes that hybridized to the sense and anti-sense target genes was used in PG-RCA, exhibiting target selectivity. PG-RCA, which generated additional primers simultaneously with linear RCA and comprised multiple reaction cycles, resulted in higher accumulation of amplified DNA products than did linear RCA within the same reaction period. The threshold time (Tt) for each target gene concentration was determined based on the threshold value set in the amplification plot for PG-RCA, and a linear correlation between the Tt value and genomic DNA concentration was proven for each of the four bacteria. The PG-RCA-based assay could be applied to gene-based detection of various microorganisms and may be a useful isothermal amplification method for replacing traditional PCR methods. [ABSTRACT FROM AUTHOR]

Conformational changes in DNA and protein biomolecules were studied in ischemic stroke (IS) cases varying severity by Raman spectroscopy. The conformational structure of hematoporphyrin was found to change in IS patients, leading to a higher (I1355/I1550)/(I1375/I1580) ratio (hemoglobin affinity of for ligands) and an increase in I1375/I1172 (a change in pyrrole conformation). Changes in genomic DNA spectra were observed at frequencies caused by stretching vibrations of primary amines (3400 cm–1), secondary amines and hydroxyls involved in hydrogen bonding (3100 cm–1), and CH2 groups of sugar phosphates (2900 cm–1) and vibrations of vibrational bonds between nitrogenous bases and sugars (1400 cm–1). The significant changes observed in genomic DNA and hemoglobin spectra were assumed to indicate conformational rearrangements of the molecules in IS. Severe IS was associated with maximum changes in DNA and hemoglobin spectra. [ABSTRACT FROM AUTHOR]

Pepper anthracnose, caused by Colletotrichum spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of Colletotrichum spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.

Greipsson, S.; Stover, K.; Whitney, S., and McElroy, T., 2024. Arbuscular mycorrhizal fungi and soil fungi associated with Uniola paniculata L. (sea oats) on restored and natural sand dunes on the Georgia Coast, U.S.A. Journal of Coastal Research, 40(5), 875–886. Charlotte (North Carolina), ISSN 0749-0208. Coastal sand dunes are fragile ecosystems that serve as natural defenses to protect barrier islands and the adjacent mainland during severe storms. A high frequency of disturbances may result in dune erosion. Several strategies have been used for coastal restoration; however, previous practices often lack the connection between plants and below-ground microbial communities. Symbiotic arbuscular mycorrhizal fungi (AMF) are beneficial in supplying a range of limiting nutrients to the host plant while improving dune stability. This study assessed the diversity of the indigenous AMF and soil fungal communities associated with Uniola paniculata L. (sea oats) on natural dunes and restored dunes at Jekyll and Tybee Islands on U.S. Georgia's coast. The phosphorus (P) concentrations of dune sand were also analyzed. At each site, samples were collected from 10 dune ridges that were randomly selected along a 100-m transect where sea oats were growing. The genomic DNA was extracted from 60 sand and sea oats root samples. A total of 13 amplicon sequence variants of AMF were identified; seven were classifiable to the species level. The most abundant genera of AMF were Glomus; the most abundant species were unclassified Glomus spp., followed by Glomus custos and G. deserticola. Using the Sorensen-Dice coefficient, the study showed that AMF assemblages of the natural dunes of both sites shared a high similarity (0.73). However, AMF assemblages of the Jekyll Island natural site had no (0) similarity to Jekyll Island restored site 1. The greatest number of classes of soil fungi were found at Jekyll Island's restored site 1 (18 classes); conversely, the lowest number was found at Jekyll Island's natural site (nine classes). Natural dunes contained significantly greater levels of P in the sand compared with restored dunes. These findings demonstrate the need to manage sand material and AMF assemblages during coastal dune restoration. [ABSTRACT FROM AUTHOR]

Genomic studies make it possible to breakthrough in many fields such as biochemistry, physiology, phylogenetics, etc., though they are unworkable without sequences of genomic DNA of an organism. The terrestrial mollusks’ genomes would benefit gastropod biology investigations, that are unavailable so far due to problems in DNA integrity and quality after the isolation procedures. Here we describe a fast and handy protocol for genomic DNA extraction from the tissues of Helix lucorum, which allows to yield high-quality samples applicable for downstream analysis such as high-throughput DNA sequencing. Troubleshooting revealed the nuclease activity of snail tissue lysate, which may be avoided by heating the lysate and decreasing the incubation time. [ABSTRACT FROM AUTHOR]

Celosia argentea is an annual leafy vegetable popularly known for its dietary and medicinal values. Hence, it is important to preserve and further improve this vegetable to enhance its numerous benefits. This study therefore investigated the genetic variability among different genotypes of C. argentea using ISSR primers. A total of 15 C. argentea genotypes were sourced from National Centre for Genetic Resources and Biotechnology (NACGRAB) in Ibadan, Nigeria and 10 genotypes sourced from different markets. The open field experiment was set up in a completely randomized design. Seeds of each cultivar were grown and seedlings transplanted. Fresh young apical leaves were harvested. DNA was extracted from young frozen apical leaves. Six ISSR primers were optimized and used in PCR with a touch-down procedure in a thermocycler. Agarose gel electrophoresis was performed, and bands were visualized. Molecular data was analyzed for total gene diversity, while morphological data was analyzed using ANOVA. The genotypes of NGB recorded the highest mean performance for plant height, leaf biomass and seed weight, while the A00 genotypes were observed to have higher values of leaf length, leaf area and root biomass. The principal component analysis showed that the first component accounted for 42% of the total variation. The correlation matrix for growth, agronomic and yield characters show highly significant positive relationship among the growth characters at P<0.05. Primer UBC-866(CTC)6 was highly polymorphic. Genotype A005 performed best for growth characters while NGB00182 performed best for yield characters. Genetic assessment and improvements in C. argenta germplasm play key role in future studies and improvements of vegetable crop. [ABSTRACT FROM AUTHOR]

Implementing nanofertilizers in cultivation to enhance food security is important and gaining great significance, as they have good properties to improve plant production, phytochemicals, and nutrient efficiency and thereby meet the demands of the increasing world population for food. This work demonstrated the impact of calcium carbonate nanoparticles (Ca-NPs) and Ca bulk at three concentrations (0, 100, and 200 mg L−1) on growth, productivity, photosynthetic pigments, phytochemical content, antioxidant activity enzymes, minerals, toxicity, and genomic DNA of lettuce plants. In this regard, Ca-NPs at a concentration of 200 mg L−1 reinforced the vegetative growth characteristics of lettuce plants, increasing head length by 15.7 and 19.2%, head diameter by 20.3 and 19.9%, head fresh weight by 54.4 and 52.9%, and production per hectare by 54.7 and 52.8% as compared to the control during the two growing seasons. Furthermore, the percentages of total chlorophyll (62.6 and 59.5%), carotenoids (48.4 and 56.5%), total phenolics (63.6 and 65.7%), total indoles (39.4 and 36.4%), vitamin C (39.7 and 39.6%), antioxidant activity (57.8 and 53.7%), nitrogen (70.5 and 67.5%), phosphorus (120 and 110.5%), potassium (33.0 and 33.2%), and calcium (67.14 and 63.2%) were also increased compared with the control during two consecutive growing seasons. Additionally, Ca-NPs and Ca bulk had an impact on the plants’ genomic DNA compared to the control. In addition, lettuce plants treated with Ca-NPs were proven to be nontoxic and safe for humans by using the Microtox 500 analyzer.

The genetically modified (GM) maize DBN9936 with a biosafety certificate will soon undergo commercial application. To monitor the safety of DBN9936 maize, three genomic DNA (gDNA) reference materials (RMs) (DBN9936a, DBN9936b, and DBN9936c) were prepared with nominal copy number ratios of 100%, 3%, and 1% for the DBN9936 event, respectively. DBN9936a was prepared from the leaf tissue gDNA of DBN9936 homozygotes, while DBN9936b and DBN9936c were prepared by the quantitative mixing of gDNA from the leaf tissues of DBN9936 homozygotes and non-GM counterparts. Validated DBN9936/zSSIIb duplex droplet digital PCR was demonstrated to be an accurate reference method for conducting homogeneity study, stability study, and collaborative characterization. The minimum intake for one measurement was determined to be 2 μL, and the gDNA RMs were stable during transport at 37 °C for 14 days and storage at −20 °C for 18 months. Each gDNA RM was certified for three property values: DBN9936 event copy number concentration, zSSIIb reference gene copy number concentration, and DBN9936/zSSIIb copy number ratio. The measurement uncertainty of the certified values took the uncertainty components related to possible inhomogeneity, instability, and characterization into account. This batch of gDNA RMs can be used for calibration and quality control when quantifying DBN9936 events. [ABSTRACT FROM AUTHOR]

Objective To explore the pathogenic genes in a Chinese family affected by nonsyndromic tooth agenesis so as to study the pathogenesis of oligodontia. Methods Hospital ethical approval and informed consent of the patients and family members were obtained. Clinical data of the proband and close family members were collected, peripheral venous blood was collected, and DNA was extracted. Gene sequencing was performed through whole-exome sequencing, and then the screened pathogenic genes were verified by Sanger sequencing. The three-dimensional structure of the mutant proteins was analyzed and compared with the wild-type using bioinformatics tools. Results The two patients with congenital majority tooth loss in this family were cousins, and there were no other patients with congenital majority tooth loss in the family. Besides congenital multiple tooth loss, the two patients had no obvious hair abnormalities, finger/toe abnormalities, sweating abnormalities or other abnormal manifestations of ectodermal tissue. We found a mutant gene that in this family by carrying out gene sequencing of the patients and their close family members. A novel EDA (ectodysplasin A) missense mutation c.983C>T (p. Pro328Leu) was identified, which changed the encoded amino acid from proline (Pro) to leucine (Leu). Analysis of the mutation site showed that the site was highly conserved, and three-dimensional structure modeling also found that it changed the structure of EDA. Conclusion A novel EDA missense variant (c.983C>T, p.Pro328Leu) was first identified in a Chinese family with nonsyndromic tooth agenesis, extending the mutation spectrum of the EDA gene.

This article examines the reproducibility of a previous study on B chromosomes in three species. The authors discovered inconsistencies and errors in the original study, resulting in false positive results and unsupported claims. They stress the significance of rigorous reproducibility practices in bioinformatics analyses and propose enhancements to the review process for such studies. The article includes supplementary materials that compare the authors' analyses with those of the original study. [Extracted from the article]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The Argentine ant, Linepithema humile (Mayr, 1868), native to the Paraguay River in South America, was first discovered in Korea in 2019. With increasing reports of L. humile, its genetic variation according to domestic growth colonies and its effects on the domestic ecosystem should be studied. Here, the genomes of L. humile specimens found at three locations in Busan were analyzed for genetic changes. First, morphological observation of L. humile samples collected from the three sites showed no phenotypic differences among them. Next, single‐nucleotide polymorphism (SNP)/insertion and deletion (INDEL) analyses on the genomic DNA from the three groups showed that in the 1‐NIE sample, the most frequent mutations were G → A and C → T. The mutations A → C and T → G were confirmed in the 14‐NIE and 19‐NIE samples. Although the number of SNPs in the N section was small, sequences of 4681 bp (1‐NIE), 4217 bp (14‐NIE) and 4631 bp (19‐NIE) in length were identified. From the INDEL length distribution of the three samples, most changes were associated with insertions and deletions of 1–2 bp. However, no heterogeneity was found in the population samples analyzed based on SNP data. Comparative analysis of the SNPs investigated revealed that 760 819 (11.72%) of the total 6 492 517 SNPs were found in common, demonstrating that the three groups analyzed had different genetic backgrounds. Overall, we have developed a method for analyzing the genetic diversity of L. humile invading the Republic of Korea, precisely classified its genetic characteristics and obtained genomic data on interspecies mutations, according to the local environment. [ABSTRACT FROM AUTHOR]

The toxic side effects of chemotherapy have long limited its efficacy, prompting expensive and long-drawn efforts to develop more targeted cancer therapeutics. An alternative approach to mitigate off-target toxicity is to develop a device that can sequester chemotherapeutic agents from the veins that drain the target organ before they enter systemic circulation. This effectively localizes the chemotherapy to the target organ, minimizing any hazardous side effects. 3D printing is ideal for fabricating these devices, as the geometric control afforded allows us to precisely dictate its hemodynamic performance in vivo. However, the existing materials compatible with 3D printing do not have drug-binding capabilities. Here, we report the stable coating of genomic DNA on a 3D-printed structure for the capture of doxorubicin. Genomic DNA is an effective chemotherapeutic-agent capture material due to the intrinsic DNA-targeting mechanism of action of these drugs. Stable DNA coatings were achieved through a combination of electrostatic interactions and ultraviolet C (UVC, 254 nm) cross-linking. These UVC cross-linked DNA coatings were extremely stable-leaching on average 100 pg of genomic DNA per mm2 of 3D-printed structure over a period of 30 min. In vitro studies of these materials in phosphate buffered saline and human serum demonstrated that they were able to capture, on average, 72 and 60 ng of doxorubicin per mm2 of structure, respectively. The stability and efficacy of these genomic DNA-coated 3D-printed materials represent a significant step forward towards the translation of these devices to clinical applications for the potential improvement of chemotherapy treatment.

The expediency of using unique SSR DNA-markers of Schizophyllum commune for population genetic assays in various fungal species has been demonstrated. In Auricularia auricula-judae and Irpex lacteus, we observed formation of heterogeneous amplicons mostly up to 500 bp in length that ensured their high resolution and facilitated data analysis. The established sets of molecular markers are efficient for DNA-fingerprinting of S. commune, I. lacteus, and A. auricula-judae, as well as are prospective for species of the genus Pleurotus, but need to be further enlarged.

The problem that this study addresses is to understand how microwave radiation is able to degrade genomic DNA of E. coli. In addition, a comparative study was made to evaluate the suitability of a high-throughput automated electrophoresis platform for quantifying the DNA degradation under microwave radiation. Overall, this study investigated the genomic DNA degradation of E. coli under microwave radiation using automated gel electrophoresis. To examine the viable organisms and degradation of genomic DNA under microwave exposure, we used three methods: (1) post-microwave exposure, where E. coli was enumerated using modified mTEC agar method using membrane filtration technique; (2) extracted genomic DNA of microwaved sample was quantified using the Qubit method; and (3) automated gel electrophoresis, the TapeStation 4200, was used to examine the bands of extracted DNA of microwaved samples. In addition, to examine the impacts of microwaves, E. coli colonies were isolated from a fecal sample (dairy cow manure), these colonies were grown overnight to prepare fresh E. coli culture, and this culture was exposed to microwave radiation for three durations: (1) 2 min; (2) 5 min; and (3) 8 min. In general, Qubit values (ng/µL) were proportional to the results of automated gel electrophoresis, TapeStation 4200, DNA integrity numbers (DINs). Samples from exposure studies (2 min, 5 min, and 8 min) showed no viable E. coli. Initial E. coli levels (at 0 min microwave exposure) were 5 × 108 CFU/mL, and the E. coli level was reduced to a non-detectable level within 2 min of microwave exposure. The relationships between Qubit and TapeStation measurements was linear, except for when the DNA level was lower than 2 ng/µL. In 8 min of microwave exposure, E. coli DNA integrity was reduced by 61.7%, and DNA concentration was reduced by 81.6%. The overall conclusion of this study is that microwave radiation had a significant impact on the genomic DNA of E. coli, and prolonged exposure of E. coli to microwaves can thus lead to a loss of genomic DNA integrity and DNA concentrations.

High-quality genomic DNA extraction is fundamental for the study of gene cloning and expression in plants. Therefore, this study evaluated several methods for extracting genomic DNA from shoots of four Dendrocalamus species to determine the optimal technique. Genomic DNA was extracted using three different methods: a commercial DNA extraction kit method, a modified cetyltrimethylammonium bromide method and a sodium dodecyl sulfate method. A membership function analysis was employed to compare these methods. The results demonstrated that the commercial DNA extraction kit method was the most effective and comprehensive approach for extracting genomic DNA from shoots of four Dendrocalamus species. Furthermore, this study provided valuable insights into optimizing techniques for extracting genomic DNA in other bamboo species.

Background: Accurate identification of Chinese herbal medicines is the basis for their research and utilization. Molecular identification can effectively differentiate original plants from counterfeit plants. The quality of genomic DNA is an important factor affecting molecular identification. However, the processing can lead to DNA degradation of the herbal medicines, which can make it difficult for their molecular identification. Objectives: To establish a genomic DNA degradation model of Gentiana crassicaulis Radix to evaluate the effects of processing methods and storage times on genomic DNA integrity. Materials and Methods: A genomic DNA degradation model of G. crassicaulis —the original plant source of the Chinese herbal medicine G. crassicaulis Radix—was established using a steam heating method. Genomic DNA integrity of G. crassicaulis Radix was evaluated using capillary electrochromatography (CEC) fingerprinting and polymerase chain reaction (PCR) of DNA barcoding markers following different processing and drying methods, including slicing (sliced roots), no slicing (whole roots), stoving, air drying, and sweating. Results: CEC fingerprinting and DNA barcoding PCR effectively evaluated genomic DNA integrity. Compared to whole roots, sliced roots better helped maintain genomic DNA integrity. As the storage time increased, the integrity of the genomic DNA reduced; the integrity of the genomic DNA of sliced roots was greater than that of whole roots. Furthermore, the interactions between slicing and drying methods possibly reduced the genomic DNA integrity. Conclusion: A genomic DNA degradation model and an evaluation system for herbal medicines were established. Our findings can help optimize the method for processing G. crassicaulis Radix and establish the traceability of genuine herbal medicines. Key Message: The quality of genomic DNA is an important factor affecting molecular identification, which is essential to determine original plants. The results of our study can help develop a method for processing G. crassicaulis Radix and enable the traceability of genuine herbal medicines. [ABSTRACT FROM AUTHOR]

Copyright of Ecosistemas y Recursos Agropecuarios is the property of Universidad Juarez Autonoma de Tabasco and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Mammalian ribonuclease (RNase) H2 is a trimer consisting of catalytic A and accessory B and C subunits. RNase H2 is involved in the removal of misincorporated ribonucleotides from genomic DNA. In humans, mutations in RNase H2 gene cause a severe neuroinflammatory disorder, Aicardi–Goutières syndrome (AGS). Here, we constructed RNase H2 C subunit (RH2C)-knockout mouse fibroblast NIH3T3 cells. Compared with the wild-type NIH3T3 cells, the knockout cells exhibited a decreased single ribonucleotide-hydrolyzing activity and an increased accumulation of ribonucleotides in genomic DNA. Transient expression of wild-type RH2C in the knockout cells increased this activity and decreased this ribonucleotide accumulation. Same events were observed when RH2C variants with an AGS-causing mutation, R69W or K145I, were expressed. These results corresponded with our previous results on the RNase H2 A subunit (RH2A)-knockout NIH3T3 cells and the expression of wild-type RH2A or RH2A variants with an AGS-causing mutation, N213I and R293H, in the RH2A-knockout cells. [ABSTRACT FROM AUTHOR]

A sequence of the COI-COII intergenic region of the mitochondrial DNA of honeybees (Apis mellifera) was conducted to characterize the genetic basis of Ethiopian honeybee populations and elucidate the existing controversial concepts among morphologically identified local honeybee subspecies in Ethiopia. Samples of 204 honeybees were collected from 51 localities from eight regions (Oromia, Amhara, South Nation and Nationality people, Tigray, Afar, Somali, Beneshagul Gumaz and Gambella). Total genomic DNA was extracted from adult worker honeybee and amplified using E2 and H2 universal primers. Sequences of the COI-COII intergenic region generated from a total of 199 honeybees resulted in 15 haplotypes with a diversity value of 0.469. Within genetic variation (73.62%) was higher compared to among the population (26.38%). Lineages Y, A, and O were well separated, in which A and O are the first report for Ethiopia. The presence of four distinct groups of honey bee subspecies (A. m. jemenitica, A. m. litorea, A. m. syriaca, and A. m. iberiensis) could be now evident. A. m. jemenitica, categorized under lineage Y covers a large geographic area (83.43%) as compared to A. m. litorea (7.03%), A. m. syriaca (2.51%) and A. m. iberiensis (2.51%) that are categorized under linage A and O. Honeybees native to Saudi Arabia, carried by the reference subspecies A.m. jemenitica, categorized under lineage O were also accounted for 4.52%. A. m. jemenitica is the first report of honey bee subspecies in Ethiopia. [ABSTRACT FROM AUTHOR]

The industrial hemp plant Cannabis sativa is a source of vegetable fiber for both textiles and biocomposite applications. After harvesting, the plant stems are laid out on the ground and colonized by microorganisms (bacteria and fungi) naturally present in the soil and on the stems. By producing hydrolytic enzymes that degrade the plant wall polymers, the natural cement that binds the fiber bundles together is removed, thus facilitating their dissociation (retting process) which is required for producing high-performant fibers. To investigate temporal dynamics of retting microbial communities (density levels, diversity, and structure), a reliable protocol for extracting genomic DNA from stems is mandatory. However, very little attention has been paid to the methodological aspects of nucleic acid extraction, although they are crucial for the significance of the final result. Three protocols were selected and tested: a commercial kit (FastDNA™ Spin Kit for soil), the Gns-GII procedure, and a custom procedure from the Genosol platform. A comparative analysis was carried out on soil and two different varieties of hemp stem. The efficiency of each method was measured by evaluating both the quantity and quality of the extracted DNA and the abundance and taxonomy of bacterial and fungal populations. The Genosol protocol provides interesting yields in terms of quantity and quality of genomic DNA compared to the other two protocols. However, no major difference was observed in microbial diversity between the two extraction procedures (FastDNA™ SPIN Kit and Genosol protocol). Based on these results, the FastDNA™ SPIN kit or the Genosol procedure seems to be suitable for studying bacterial and fungal communities of the retting process. It should be noted that this work has demonstrated the importance of evaluating biases associated with DNA recovery from hemp stems. Key points: • Metagenomic DNA was successfully extracted from hemp stem samples using three different protocols. • Further evaluation was performed in terms of DNA yield and purity, abundance level, and microbial community structure. • This work exhibited the crucial importance of DNA recovery bias evaluation. [ABSTRACT FROM AUTHOR]

The isolation of intact and high-molecular mass genomic DNA is an initial and essential step for many molecular biology applications like restriction enzyme fingerprinting, polymerase chain reaction (PCR) etc. The presence of essential oils, polysaccharides, polyphenols and other secondary metabolites in the medicinal plants can hamper and reduce the yield and purity of extracted DNA. In this protocol, we describe simple modifications to the conventional cetyl trimethylammonium bromide (CTAB) method for reliable isolation of high molecular weight genomic DNA from Gloriosa superba - a potent medicinal plant. The purity of genomic DNA was confirmed by excellent absorbance (A260/A280) ratio of ~1.8 calculated from the spectrophotometric readings and the DNA concentration ranged from 75 to 308 µg/g of leaves. The PCR amplification from the isolated DNA was optimized to produce clear amplification in all the germplasm tested as mentioned in our earlier research. Besides being rapid, effective and technically easy, the protocol can also be used for the isolation of genomic DNA from other crop plants too. [ABSTRACT FROM AUTHOR]

Background: A simple and effective modified ethanol precipitation-based protocol was described for the extraction of genomic DNA from ancient human bones. The qualitative and quantitative evaluation of genomic DNA was done based on DNA purity (260/280) and the PCR method. Method and Materials: In this study, a total of 50 embalmed ancient bones, including 20 long and 30 hip bone samples, were taken for genomic DNA extraction. The efficiency of the genomic DNA extraction was demonstrated on >50-year-old ancient human HIP and long bone samples. In vitro quantitative and qualitative analysis of extracted genomic DNA was performed by 0.8% agarose gel electrophoresis and PCR amplification. To assess the quality of extracted genomic DNA, a mitochondrial-specific ATPase6 gene primer was used to obtain sequence information of 675 bp. Result: Our data show that a concentration of genomic DNA between 1.6 and 2.0 at 260/280 resulted in successful PCR amplification. Our results demonstrated that the extraction of DNA from ancient bone samples with a manual approach will increase the amplification efficiency of the polymerase chain reaction (PCR). Conclusion: In the present study, a simple, time-saving, and cost-effective protocol is described for the extraction of genomic DNA from ancient human bones. Further, we believe the extraction of genomic DNA from ancient bone samples with this approach will increase the success rate of PCR amplification.

A novel strain YR1T, Gram-stain-negative, rod-shaped, catalase- and oxidase-positive, and aerobic bacterium, was isolated from the feces of Ceratotherium simum. The strain grew at 9–42 °C (optimal temperature, 30 °C), at pH 6.0–10.0 (optimal pH, 7.0), and in the presence of 0–3% (w/v) NaCl (optimal salinity, 0%). Phylogenetic analyses based on 16S rRNA gene sequencing indicated that strain YR1T was most closely related to Rheinheimera soli BD-d46T (98.6%), R. riviphila KYPC3T (98.6%), and R. mangrovi LHK 132T (98.1%). Moreover, the average nucleotide identity, average amino acid identity, and digital DNA–DNA hybridization values between strain YR1T and R. mangrovi LHK 132 T were 88.3%, 92.1%, and 35.3%, respectively, indicating that strain YR1T is a novel species in the genus Rheinheimera. The genome size and genomic DNA G + C content of strain YR1T were 4.5 Mbp and 46.37%, respectively. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol, while the predominant respiratory quinone was Q-8. Summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16: 0, and summed feature 8 (C18:1ω7c) were the primary cellular fatty acids (> 16%). Based on these genotypic and phenotypic characteristics, strain YR1T was identified as a novel species in the genus Rheinheimera, for which the name Rheinheimera faecalis sp. nov. is proposed, with the type strain is YR1T (= KACC 22402T = JCM 34823T). [ABSTRACT FROM AUTHOR]

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Periplaneta americana is a cosmopolitan pest cockroach endemic to tropical and subtropical climates. It occurs frequently in urban sewer and wastewater system and transit in human proximities, spreading pathogens that causes serious public health concerns such as asthma, allergies, and others. By using the Next-generation Sequencing (NGS) known as Illumina NovaSeq 6000, this article documents for the draft genome data set of P. americana collected in Penang Island, Malaysia. This article displays the pair-end 150 bp genome dataset and results on the sequence quality. This genome dataset presents the information for further understanding of P. americana populations at molecular level and the opportunity to develop effective control and management strategies for the species. This dataset is available under Sequence Read Archive (SRA) databases with the SRR23867103.

The genetically modified (GM) maize DBN9936 with a biosafety certificate will soon undergo commercial application. To monitor the safety of DBN9936 maize, three genomic DNA (gDNA) reference materials (RMs) (DBN9936a, DBN9936b, and DBN9936c) were prepared with nominal copy number ratios of 100%, 3%, and 1% for the DBN9936 event, respectively. DBN9936a was prepared from the leaf tissue gDNA of DBN9936 homozygotes, while DBN9936b and DBN9936c were prepared by the quantitative mixing of gDNA from the leaf tissues of DBN9936 homozygotes and non-GM counterparts. Validated DBN9936/zSSIIb duplex droplet digital PCR was demonstrated to be an accurate reference method for conducting homogeneity study, stability study, and collaborative characterization. The minimum intake for one measurement was determined to be 2 μL, and the gDNA RMs were stable during transport at 37 °C for 14 days and storage at −20 °C for 18 months. Each gDNA RM was certified for three property values: DBN9936 event copy number concentration, zSSIIb reference gene copy number concentration, and DBN9936/zSSIIb copy number ratio. The measurement uncertainty of the certified values took the uncertainty components related to possible inhomogeneity, instability, and characterization into account. This batch of gDNA RMs can be used for calibration and quality control when quantifying DBN9936 events.

After the onset of COVID-19 pandemic, a sharp surge in the usage of the face-masks throughout the globe has been observed. Pre-experiment survey of 252 individuals indicated a higher use of cotton-make masks (41%), followed by N-95 make (31%), and surgical disposable masks (26%). It was also further revealed that a higher fraction of individuals wear a face-mask more than 3 times (37%) before its disposal. In order to assess the potential usability of different mask types as forensic DNA evidence, a study was conducted on 50 healthy individuals. DNA content of different fractions such as the portion of mask covering the mouth region and the ear-piece showed a good source of host DNA. Though no statistically significant difference (P < 0.05) was found in the DNA quantity obtained from different face mask types, an increasing trend was obtained in the order: cloth make type (7.031 ± 0.31 ng), N-95 make (4.711 ± 0.15 ng), and surgical disposable type (2.17 ± 0.13 ng). The time of wearing of a face-mask showed a positive correlation with the yield of DNA irrespective of the face-mask type used. Samples retrieved from both the portions covering the mouth area and the ear-piece showed a good source of genomic DNA yielding an average of 4.82 ± 0.11 ng and 4.44 ± 0.10 ng of DNA, respectively. Irrespective of the face-mask types, number of reuse, and the portion of the mask, 66.66–96.11% of samples showed a complete autosomal STR DNA profile. This suggests that if a face-mask is found at the crime scene, it should be collected and preserved as a potential source of DNA evidence for routine forensic DNA analysis. [ABSTRACT FROM AUTHOR]

Cryptosporidium, a protozoan parasite, in wastewater presents a major public health concern for water safety. However, bactericidal efficiencies of conventional disinfection methods towards Cryptosporidium oocysts are still hampered owing to the presence of their thick outer wall. In this study, we present a novel UV inactivation process where the efficiency has been significantly enhanced by addition of a trypsin pretreatment stage. Notably, inactivation (log-reduction) of oocysts was noted to be 73.75–294.72% higher than that obtained by UV irradiation alone, under identical conditions. Experimental observations and supporting mechanistic analyses suggest that trypsin led to cleavage of the protein layers on the oocyst wall, facilitating penetration of UV radiation into the oocysts leading to degradation of their genomic DNA (gDNA). The dissociative effect of trypsin on the oocyst wall was indicated by the fact that 64.50% of oocysts displayed early apoptosis after trypsinization. Imaging by scanning electron microscopy indicated that this combined treatment led to substantial disruption of the oocyst coat, deforming their shape. This resulted in the release of cellular proteins and gDNA, their concentrations in bulk solution increasing by 1.22–8.60 times. As UV irradiation time was prolonged, gDNA was degraded into smaller fragments with lower molecular masses. Both laddering and diffuse smear patterns in gel analysis indicated significantly detrimental effects on gDNA and viability of oocysts. Overall, this study demonstrated enhancement of UV inactivation of Cryptosporidium oocysts by trypsin and explored the underlying mechanisms for the process. [ABSTRACT FROM AUTHOR]

Since the importance of spices in the global nutritional challenges has been understudied, our aim was to assess the arbuscular mycorrhizal diversity associated with one of the most expensive spices - Saffron (Crocus sativus) in the Kashmir Himalaya. We used both morphological and molecular approaches to characterize the arbuscular mycorrhizal fungal (AMF) diversity associated with saffron targeting nuclear ribosomal DNA sequences. In order to capture the entire AMF diversity associated with saffron, we assessed the spore density and diversity in rhizospheric soils, and sampled roots from sampling sites spread across major saffron growing area of Kashmir overlying a grid map on the target fields. Genomic DNA was extracted from roots, amplified using nested PCR with two set of primers, sequenced and phylogenetically analysed. Based on morphological and molecular characterization, 15 different AMF species were found associated with saffron in Kashmir Himalaya, India. The most dominant species was Rhizophagus intraradices followed by Funneliformis mosseae, while the least dominant one was Acaulospora trappei. In view of the deteriorating quality and yield of saffron over the years in this Himalayan valley, tapping the beneficial soil microorganisms, such as AMF promises to be of help. The implications of these results for formulation of effective biofertilizers for improving yield and quality of saffron vis-à-vis regional nutritional security are discussed. [ABSTRACT FROM AUTHOR]

The available information on the abundance of restorative plants on earth is incomplete, and the data regarding botanicals from various countries differ significantly. Thesubstantial development of theworldwide natural botanical market is attributable tothe expanding revenue ofglobal drug companies trading herbal medicines. This essential type of traditional medical care isdepended onbyapprox. 72–80% of individuals. Even though numerous restorative plants are readily used, they have never been subject tothe same strict quality guidelines as conventional drugs. Nonetheless, it is vital to have specific organic, phytochemical, and molecular tools and methods for identifying restorative plant species so that traditional and novel plant products can be safely used inmodern medicine. Molecular biotechnology approaches provide areliable and accurate way toidentify botanicals and can be used toensure thesafety and efficacy ofplant-based products. This review explores various molecular biotechnology approaches and methods for identifying botanicals. [ABSTRACT FROM AUTHOR]

Background: A simple and effective modified ethanol precipitation-based protocol was described for the extraction of genomic DNA from ancient human bones. The qualitative and quantitative evaluation of genomic DNA was done based on DNA purity (260/280) and the PCR method. Method and Materials: In this study, a total of 50 embalmed ancient bones, including 20 long and 30 hip bone samples, were taken for genomic DNA extraction. The efficiency of the genomic DNA extraction was demonstrated on >50-year-old ancient human HIP and long bone samples. In vitro quantitative and qualitative analysis of extracted genomic DNA was performed by 0.8% agarose gel electrophoresis and PCR amplification. To assess the quality of extracted genomic DNA, a mitochondrial-specific ATPase6 gene primer was used to obtain sequence information of 675 bp. Result: Our data show that a concentration of genomic DNA between 1.6 and 2.0 at 260/280 resulted in successful PCR amplification. Our results demonstrated that the extraction of DNA from ancient bone samples with a manual approach will increase the amplification efficiency of the polymerase chain reaction (PCR). Conclusion: In the present study, a simple, time-saving, and cost-effective protocol is described for the extraction of genomic DNA from ancient human bones. Further, we believe the extraction of genomic DNA from ancient bone samples with this approach will increase the success rate of PCR amplification. [ABSTRACT FROM AUTHOR]

In polymerase chain reaction (PCR)-based nucleic acid quantification, the DNA template type, primer/probe sequence, and instrument platform such as real-time quantitative PCR (qPCR) and digital PCR (dPCR) affect the accuracy and reliability of quantitative results. In this study, a plasmid DNA (pDNA) pBI121-screening, genetically modified (GM) rice SDrice genomic DNA (gDNA), and GM rapeseed SDrape gDNA, all carrying the same 11 screening elements, were used to prepare samples of different levels of gDNA and pDNA in a non-GM gDNA background. The comparison of the dPCR assays targeting the 11 screening elements revealed that the primer/probe set is a key factor that affects the accuracy of dPCR quantification. The optimal PCR method for the 11 screening elements was screened out from among the validated qPCR methods. The accuracy of the qPCR quantification of the low-level pDNA and gDNA test samples was low when pDNA was used as a calibrator, whereas that of the dPCR quantification was high and not affected by variations in template type and detection target. The validated dPCR assays targeting one or two elements can be randomly selected to characterize multiple-target pDNA reference materials (RMs). Low-level pDNA RMs with certified values can be used as quality controls for dPCR assays to avoid significant bias in gDNA quantification. [ABSTRACT FROM AUTHOR]

Cepola schlegelii (Bleeker 1854) belongs to the genus Cepola in the family Cepolidae and order Priacanthiformes. The complete mitochondrial genome of C. schlegelii was sequenced and analyzed by a high-throughput sequencing approach. The full length of the genome is 17,020 bp, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a non-coding control region (D-loop). Phylogenetic analysis based on complete mitochondrial genomes revealed that C. schlegelii was most closely related to Acanthocepola krusensternii. The complete mitochondrial sequence of C. schlegelii will enrich the mitochondrial genome database and provide useful resources for population genetics and evolution analyses.

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1–1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases. [ABSTRACT FROM AUTHOR]

Blood DNA extraction is one of the vital steps in various DNA-based analyses. However, one of the challenges is processing clotted blood samples. Oftentimes these protocols are time consuming and use hazardous organic solvents. Many researchers have also resorted to commercial kits to circumvent the need to optimise different methods. However, manual methods may need to be employed due to cost consideration. Therefore, this study was conducted to assess various extraction methods to determine the best method that can provide DNA that has high quality, integrity, and purity, and the most time and costefficient method. Samples of coagulated and un-coagulated blood were collected and extracted using four published manual methods and a commercial kit. Analysis of the extracted DNA’s quality and quantity was done using gel electrophoresis and biophotometer. Among the methods compared, DNA extracted using a method proposed by Moradi et al. (2014) [1] was the best as it yields the best DNA in terms of quantity and purity, and the most time and cost effective. [ABSTRACT FROM AUTHOR]

Ribonuclease (RNase) H2 is involved in the removal of ribonucleotides embedded in genomic DNA. Eukaryotic RNase H2 is a heterotrimer consisting of the catalytic A subunit (RH2A) and the accessory B and C subunits. This study aimed to compare the cellular activities of wild-type ribonuclease (RNase) H2 and its variants with a mutation causing neuroinflammatory autoimmune disease, Aicardi–Goutières syndrome (AGS). We first analyzed cellular RNase H2 activity and ribonucleotide content in the genomic DNA of RH2A-knockout (KO) mouse fibroblast NIH3T3 cells after transfection with a transient expression plasmid encoding mouse wild-type RH2A. From 4 h after transfection, the RNase H2 activity increased and the amount of ribonucleotides decreased, as compared with the corresponding non-transfected RH2A-KO cells. This demonstrated the rapidness of ribonucleotide turnover in mammalian genomic DNA and the importance of continuous expression of RNase H2 to maintain the ribonucleotide amount low. Next, we expressed mouse RH2A variants with a mutation corresponding to a human AGS-causing mutation in RH2A-KO NIH3T3 cells. Neither increase in RNase H2 activity nor decrease in ribonucleotide amount was observed for G37S; however, both conditions were observed for N213I and R293H. This corresponded with our previous results on the activity of recombinant human RNase H2 variants. [ABSTRACT FROM AUTHOR]

ObjectiveTo satisfy the demands of laboratory quality control and proficiency testing for the detection of enteroinvasive Escherichia coli (EIEC), a stable EIEC reference material was developed with independent intellectual property rights, good stability and clear background genomic information.MethodsNGS Technique was used to sequence the whole genome of enteroinvasive Escherichia coli (CMCC 44840), bioinformatics analysis was performed to identify the species, serotype, MLST type and virulence genes; EIEC lyophilized samples with 103 CFU were prepared by freeze-drying method ; The homogeneity test was performed according to CNAS-GL017, and the result were statistically analyzed by one-way ANOVA; The samples were stored at -20, 4, 25 and 37 ℃, respectively, and their storage stability and transport stability were evaluated; Five kinds of food matrix samples were used to verify the applicability of reference materials, and three laboratories were organized for collaborative calibration.ResultsThe genome size of CMCC 44840 was 4.96 Mb, the GC content was 50.7%, and the genome contained 5424 coding genes. This strain was identified as the species of Escherichia coli, and the serotype and the MLST type were predicted to be O28ac: H7 and ST311, respectively, and it carried ipaH, virB, virF and other virulence genes. The homogeneity test result of the EIEC lyophilized sample was F=1.79, which met the homogeneity requirements of reference materials. The EIEC samples were still 103 CFU when storing at -20 ℃ and 4 ℃ for 60 d and storing at 25 ℃ and 4 ℃ for 7 d, and decreased on the third day at 37 ℃. EIEC were detected in 20 different food matrix samples with EIEC lyophilized samples after enrichment. The samples were 103 CFU determined by three collaborative calibration laboratories, and there were no significant differences among these laboratories (F=0.59).ConclusionThe EIEC reference material prepared in this study has clear genome sequence information. The homogeneity and stability meet the requirements and have good applicability, which can meet the requirements of quality control and capability verification in food testing laboratory.