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Forensic Science International May 2024We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA...
We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.
Topics: Blood Stains; Humans; DNA; Specimen Handling; Nanopores; Sequence Analysis, DNA; DNA Degradation, Necrotic; Time Factors; DNA Fragmentation; DNA Fingerprinting
PubMed: 38581825
DOI: 10.1016/j.forsciint.2024.112010 -
Molecular Phylogenetics and Evolution Jul 2024In the species groups related to Diphasiastrum multispicatum and D. veitchii, hybridization was investigated in samples from northern and southern Vietnam and the island...
In the species groups related to Diphasiastrum multispicatum and D. veitchii, hybridization was investigated in samples from northern and southern Vietnam and the island of Taiwan, including available herbarium specimens from southeast Asia. The accessions were analyzed using flow cytometry (living material only), Sanger sequencing and multiplexed inter-simple sequence repeat genotyping by sequencing. We detected two cases of ancient hybridization involving different combinations of parental species; both led via subsequent duplication to tetraploid taxa. A cross D. multispicatum × D. veitchii from Malaysia represents D. wightianum, a tetraploid taxon according to reported DNA content measurements of dried material (genome formulas MM, VV and MMVV, respectively). The second case involves D. veitchii and an unknown diploid parent (genome formula XX). Three hybridogenous taxa (genome formulas VVX, VVXX, VVVX) were discernable by a combination of flow cytometry and molecular data. Taxon I (VVX, three clones found on Taiwan island) is apparently triploid. Taxon II represents another genetically diverse and sexual tetraploid species (VVXX) and can be assigned to D. yueshanense, described from Taiwan island but occurring as well in mainland China and Vietnam. Taxon III is as well most likely tetraploid (VVVX) and represented by at least one, more likely two, clones from Taiwan island. Taxa I and III are presumably asexual and new to science. Two independently inherited nuclear markers recombine only within, not between these hybrids, pointing towards reproductive isolation. We present an evolutionary scheme which explains the origin of the hybrids and the evolution of new and fully sexual species by hybridization and subsequent allopolyploidization in flat-branched clubmosses.
Topics: Hybridization, Genetic; Taiwan; Vietnam; Phylogeny; Lycopodiaceae; DNA, Plant; Microsatellite Repeats; Sequence Analysis, DNA; Islands; Evolution, Molecular; Genome, Plant; Flow Cytometry
PubMed: 38561082
DOI: 10.1016/j.ympev.2024.108067 -
Zhongguo Dang Dai Er Ke Za Zhi =... Mar 2024To evaluate the incidence rate of Duchenne muscular dystrophy (DMD) in the male newborns in the Ningxia region and establish a critical threshold for screening DMD in...
OBJECTIVES
To evaluate the incidence rate of Duchenne muscular dystrophy (DMD) in the male newborns in the Ningxia region and establish a critical threshold for screening DMD in newborns to distinguish between the normal population and affected individuals.
METHODS
A total of 10 000 male newborns were screened using immunofluorescence analysis of creatine kinase isoenzyme concentrations in heel spot dried blood specimens. Newborns with the concentrations higher than the critical threshold were recalled for serum creatine kinase measurements. Genetic testing was performed to confirm diagnosis in cases showing abnormalities.
RESULTS
Among the screened 10 000 male newborns, two were confirmed to have DMD through genetic testing, resulting in a preliminary estimated incidence rate of 1/5 000 for male newborns in the Ningxia region. The critical threshold for creatine kinase isoenzyme concentration in newborns in this region was determined to be 468.57 ng/mL.
CONCLUSIONS
Screening for DMD in newborns is feasible in the Ningxia region. Early screening, diagnosis, and treatment of DMD can improve the quality of life for affected individuals and help families make informed decisions regarding further pregnancies.
Topics: Humans; Male; Infant, Newborn; Muscular Dystrophy, Duchenne; Isoenzymes; Quality of Life; Neonatal Screening; Creatine Kinase
PubMed: 38557377
DOI: 10.7499/j.issn.1008-8830.2309151 -
Molecular Genetics and Metabolism... Jun 2024Each year thousands of babies are born with rare genetic disorders not identified by current NBS panels, due to programs which are not yet optimal. Next-generation...
Each year thousands of babies are born with rare genetic disorders not identified by current NBS panels, due to programs which are not yet optimal. Next-generation sequencing technologies have the potential to overcome many NBS drawbacks and provide large amounts of molecular data, broadening the number of diseases investigated. Here, we design and set up an NGS-based approach to evaluate the feasibility of NGS from dried blood spot starting from 34 DBSs. After assessing gDNA yield and integrity, libraries were performed using three target enrichment approaches, sequenced on NS500 platform, and analyzed on commercial platform. Specifically, we focus on virtual gene panels related to highly actionable neonatal/pediatric disorders. WES show that amount and quality of DBS-extracted gDNA are suitable for high-throughput sequencing. We obtain 500-1500 ng for each specimen, 1.7-1.8 260/280 wavelength, and DIN of 7 resulting DNA integrity, on par with traditional venous blood collection. A high read depth with 94.3% coverage uniformity is achieved for all samples. Data results on mean coverage are comparable among the different workflows tested and demonstrate that DBS from newborn collected at birth is a suitable material for the developing of gNBS programs.
PubMed: 38544910
DOI: 10.1016/j.ymgmr.2024.101074 -
Clinica Chimica Acta; International... Apr 2024Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to...
BACKGROUND
Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to traditional sampling methods. The objective of this study was to assess the suitability of 30 μL VAMS for the measurement of endogenous steroid hormones.
METHODS
A novel LC-MS/MS method was developed for the quantification of 18 analytes in VAMS samples, including main endogenous free steroids and phase II metabolites of androgens. The method underwent validation in accordance with ISO/IEC 17025:2017 and World Anti-Doping Agency (WADA) requirements. Subsequently, it was applied to authentic VAMS samples obtained from 20 healthy volunteers to assess the stability of target analytes under varying storage conditions.
RESULTS
The validation protocol assessed method's selectivity, matrix effect, extraction recovery, quantitative performance, carry-over and robustness. The analysis of authentic samples demonstrated the satisfactory stability of monitored steroids in VAMS stored at room temperature, 4 °C, -20 °C and -80 °C for up to 100 days and subjected to up to 3 freezing-thawing cycles.
CONCLUSIONS
The validated LC-MS/MS method demonstrated its suitability for the measurement of steroids in dried blood VAMS. The observed stability of steroidal compounds suggests promising prospects for future applications of VAMS, both in anti-doping contexts and clinical research.
Topics: Humans; Androgens; Blood Specimen Collection; Chromatography, Liquid; Doping in Sports; Dried Blood Spot Testing; Liquid Chromatography-Mass Spectrometry; Steroids; Tandem Mass Spectrometry
PubMed: 38537673
DOI: 10.1016/j.cca.2024.117890 -
Data in Brief Jun 2024This dataset presents a comprehensive collection of images representing both dried and live samples from eight distinct Thai cannabis classes. The dataset includes a...
This dataset presents a comprehensive collection of images representing both dried and live samples from eight distinct Thai cannabis classes. The dataset includes a total of 14,094 images, with images depicting dried and healthy specimens. These images serve as a valuable resource for researchers engaged in botanical exploration, machine learning, and computer vision studies. Additionally, the dataset facilitates investigations into the medicinal properties of Thai cannabis. Interdisciplinary collaboration is encouraged, providing opportunities for innovative insights spanning biology, horticulture, and data science. Beyond fundamental research, this dataset holds practical implications for agriculture, technology development, and disease prevention, offering insights into both dried and live states of Thai cannabis plants across various strains.
PubMed: 38516281
DOI: 10.1016/j.dib.2024.110292 -
Clinical and Experimental Dental... Apr 2024The use of silver fluoride followed by stannous fluoride was designed for the treatment of open carious lesions in primary molars in dental outreach programs. However,...
OBJECTIVES
The use of silver fluoride followed by stannous fluoride was designed for the treatment of open carious lesions in primary molars in dental outreach programs. However, during the COVID-19 pandemic when aerosol-producing procedures were inadvisable, one dental location started using it as the first stage in a two-visit restorative procedure for carious primary molars. If the gap between the fluoride application and the restoration placement stages was around 3-5 weeks it was noticed that a black friable crust appeared on the caries surface. To investigate further a normally discarded crust from one patient was retrieved and sent for analysis.
MATERIALS AND METHODS
Two techniques suitable for identification and preliminary analysis of material of unknown composition, scanning electron microscopy and energy dispersive spectroscopy (EDS) were used. The only preparation was that the specimen was dried and coated beforehand.
RESULTS AND CONCLUSIONS
This preliminary examination showed two unexpected findings. The first was that the crust surface indicated a possible dentine derivation as it was covered with reasonably evenly spaced holes. In addition, the EDS spectrum showed it to be, at least, partially mineralized. The second unexpected finding was that the surface was coated with electron-dense particles. The size of the particles and the EDS spectrum pointed to the likelihood of the majority of them being nanosilver. These unexpected findings suggest a possible new direction for research.
Topics: Humans; Tin Fluorides; Pandemics; Dental Caries; Molar; Fluorides; Silver Compounds
PubMed: 38506304
DOI: 10.1002/cre2.838 -
Plant Disease Mar 2024In February 2022, leaf zonate spot disease afflicted Aloe vera L. in Yunnan, China, endangering the $39 billion industry with 0.33ha under cultivation (Wan 2015). The...
In February 2022, leaf zonate spot disease afflicted Aloe vera L. in Yunnan, China, endangering the $39 billion industry with 0.33ha under cultivation (Wan 2015). The disease manifested with watery spots progressing into oval or circular necrosis lesions, characterized by a dark center surrounded by a gray-brown zone. In the late stage of the disease, lesions regress in size and several small dark picnidia dots appeared on the gray-brown zone. The disease incidence ranged from 10% to 15% in three commercial plantations. If left uncontrolled, the disease could diminish the commercial value of Aloe vera plants. Eighteen symptomatic leaf samples underwent morphological and genetic identification. The samples were carefully washed with distilled water and 1×1 cm2 sections of tissue were excised using a sterile scalpel. The sections underwent surface-disinfection with 3% NaOCl for 3 min and 75% ethanol for 30 s. After three sterile water rinses the sections were air-dried. Subsequently, they were transferred to potato dextrose agar (PDA) before being incubated at 25 ℃ in the dark. Of the 18 samples, eight produced the colonies with similar morphological characteristics, named LH7. Isolate LH7 had downy to woolly aerial mycelia, initially pinkish white on the surface, and gradually turned greenish-olivaceous from the middle, and eventually turned dark brown to black after seven days. The fungus formed arthric chains in the aerial mycelium on PDA but did not produce conidiomata. The conidia, which occurred in arthric chains were 5.50-9.9 × 4.08-7.51 μm (mean 7.09× 5.26 μm, n=50) in size, cylindrical, brown, and 0-1 septate. To ascertain LH7's pathogenicity, three healthy one-year old aloe plants were surface-sanitized with a 1% aqueous chlorine solution, rinsed with sterile water, and dried. Three leaves from each plant were punctuated and inoculated using conidial suspension (100 μl of 1x 106 conidial mL-1), while three control plants were inoculated with sterile distilled water. The pathogenicity tests were repeated twice. The inoculated plants were kept at 25 ℃ with a 12-hour light/12-hour dark cycle. After seven days, symptoms observed in the field appeared in the plants, while no disease occurred in the control plants. After 21 days, conidiomata formed on the inoculated leaves, averaging 116.92 μm (n=20) in diameter. These conidiomata were globose to subglobose, and brown to sub-brown. The fungus was successfully re-isolated from symptomatic tissue and the resulting colonies were morphologically consistent with isolate LH7. Based on the characteristics, the fungus was identified as Neoscytalidium dimidiatum (Philips et al. 2013). The specimen was deposited in China Center for Type Culture Collection ( CCTCC AF 2024001). This identification was confirmed through sequencing of ITS gene region of rDNA using ITS1/ITS4 (Imran et al. 2022). The sequence was submitted into GenBank database (ON878059). BLAST analysis of the LH7's ITS amplicon showed 100% similarity with that of JN093303.1. A phylogenetic tree constructed using the maximum likelihood method revealed that ON878059 was clustered with JN093303.1. Previous studies have documented that pathogens such as Colletotrichum gloeosporioides (Penz.), Fusarium spp. and Rhizopus oryzae can also cause diseases in A. vera in China (Zhou et al. 2008; Ding et al. 2015). Additinonally, Cladosporium sphaerospermum, Pseudopestalotiopsis theae, and Lasiodiplodia theobromae have been identified as causal agents of aloe leaf spot diseases in India, Bangladesh and Malaysia (Avasthi et al. 2016; Ahmmed et al. 2022; Khoo et al. 2022). To our knowledge, this is the first report of N. dimidiatum causing leaf necrosis of aloe in China. Vigilant surveillance and disease control measures are imperative to mitigate potential losses in this region.
PubMed: 38499972
DOI: 10.1094/PDIS-09-23-1911-PDN -
Acta Parasitologica Mar 2024The present work aims to expand the knowledge of the digenean species Prosogonotrema bilabiatum (Sclerodistomidae), a parasite of Chaetodipterus faber (Acanthuriformes)...
Integrative Taxonomy of Prosogonotrema bilabiatum Vigueras, 1940 (Digenea: Sclerodistomidae): A Parasite in Atlantic Spadefish Chaetodipterus faber (Broussonet, 1782) (Acanthuriformes: Ephippidae) from Brazil.
OBJECTIVES
The present work aims to expand the knowledge of the digenean species Prosogonotrema bilabiatum (Sclerodistomidae), a parasite of Chaetodipterus faber (Acanthuriformes) from Brazil, with an integrative taxonomic approach, using light microscopy, scanning electron microscopy, histology, and molecular biology.
METHODS
Forty-one digenean specimens were stained with hydrochloric carmine for morphological studies. Eleven parasites were dehydrated through a graded ethanol series, critical point dried with carbon dioxide, and coated with gold for scanning electron microscopy analysis. Four specimens were processed following histological routine and stained with hematoxylin and eosin and Gomori trichrome. DNA extracted was amplified using 28S partial primer D1-D3. Maximum likelihood and Bayesian inference were performed for phylogenetic analysis.
RESULTS
Morphometric and morphological data of the specimens studied ranged in accordance as observed in previous descriptions of the species. Observations from scanning electron microscopy and histology corroborated with those observed in stained whole mounts. Molecular analysis showed that specimens of P. bilabiatum from Brazil clustered with another two sequences of this species from different hosts and localities, with a high node support value.
CONCLUSIONS
The integrative taxonomic approach allowed to record and describe new characteristics of P. bilabiatum related to the tegument, the structure and the arrangement of its tissues. The use of molecular markers confirmed that specimens identified as P. bilabiatum from different hosts and localities are all conspecific. Further studies, mainly molecular with less conserved genetic markers, should be carried out to better understand the phylogenetic relationships of Prosogonotrema with Hemiuroidea.
Topics: Animals; Brazil; Fish Diseases; Trematoda; Phylogeny; Microscopy, Electron, Scanning; Trematode Infections; Fishes; DNA, Helminth; RNA, Ribosomal, 28S
PubMed: 38472688
DOI: 10.1007/s11686-024-00825-y -
Journal of Clinical Microbiology Apr 2024Scaling up of newer innovations that address the limitations of the dried blood spot and the logistics of plasma monitoring is needed. We employed a multi-site,...
UNLABELLED
Scaling up of newer innovations that address the limitations of the dried blood spot and the logistics of plasma monitoring is needed. We employed a multi-site, cross-sectional assessment of the plasma separation card (PSC) on blood specimens collected from all consenting adults, assenting young and pediatric patients living with HIV from 10 primary healthcare clinics in South Africa. Venous blood for EDTA-plasma samples was collected and analyzed according to the standard of care assay, while collected capillary blood for the PSC samples was analyzed using the Roche COBAS AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 Test at the National Reference laboratories. McNemar tests assessed the differences in concordance between the centrifuged plasma and dried plasma spots. The usability of PSC by blood spotting, PSC preparation, and pre-analytical work was assessed by collecting seven-point Likert-scale data from healthcare and laboratory workers. We enrolled 538 patients, mostly adults [ = 515, 95.7% (95% CI: 93.7%-97.1%)] and females [ = 322, 64.2% (95% CI: 60.0%-68.1%)]. Overall, 536 paired samples were collected using both PSC- and EDTA-plasma diagnostics, and 502 paired PSC- and EDTA-plasma samples assessed. Concordance between the paired samples was obtained for 446 samples. Analysis of these 446 paired samples at 1,000 copies per milliliter threshold yielded an overall sensitivity of 87.5% [95% CI: 73.2%-95.8%] and specificity of 99.3% [95% CI: 97.9%-99.8%]. Laboratory staff reported technical difficulties in most tasks. The usability of the PSC by healthcare workers was favorable. For policymakers to consider PSC scale-up for viral load monitoring, technical challenges around using PSC at the clinic and laboratory level need to be addressed.
IMPORTANCE
Findings from this manuscript emphasize the reliability of the plasma separation card (PSC), a novel diagnostic method that can be implemented in healthcare facilities in resource-constrained settings. The agreement of the PSC with the standard of care EDTA plasma for viral load monitoring is high. Since the findings showed that these tests were highly specific, we recommend a scale-up of PSC in South Africa for diagnosis of treatment failure.
Topics: Adult; Female; Humans; Child; Sensitivity and Specificity; HIV-1; Viral Load; South Africa; Cross-Sectional Studies; Edetic Acid; Reproducibility of Results; HIV Infections; RNA, Viral
PubMed: 38470024
DOI: 10.1128/jcm.01649-23