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Scientific Reports Jun 2024Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person's skin's contact with an object or another person. Many...
Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person's skin's contact with an object or another person. Many factors influence touch DNA transfer, including the "destination" substrate's surface. The latter's physicochemical characteristics (wettability, roughness, surface energy, etc.) will impact touch DNA deposition and persistence on a substrate. We selected a representative panel of substrates from objects found at crime scenes (glass, polystyrene, tiles, raw wood, etc.) to investigate the impact of these characteristics on touch DNA deposition and detection. These were shown to impact cell deposition, morphology, retention, and subsequent touch DNA genetic analysis. Interestingly, cell-derived fragments found within keratinocyte cells and fingermarks using in vitro touch DNA models could be successfully detected whichever the substrates' physicochemistry by targeting cellular proteins and carbohydrates for two months, indoors and outdoors. However, swabbing and genetic analyses of such mock traces from different substrates produced informative profiles mainly for substrates with the highest surface free energy and therefore the most hydrophilic. The substrates' intrinsic characteristics need to be considered to better understand both the transfer and persistence of biological traces, as well as their detection and collection, which require an appropriate methodology and sampling device to get informative genetic profiles.
Topics: Humans; DNA; Touch; Surface Properties; Skin; Keratinocytes; DNA Fingerprinting
PubMed: 38858407
DOI: 10.1038/s41598-024-63911-1 -
Frontiers in Microbiology 2024, a wild plant in southern Africa, is utilized in traditional medicine for various ailments, leading to its endangerment and listing on the Red List of South African...
, a wild plant in southern Africa, is utilized in traditional medicine for various ailments, leading to its endangerment and listing on the Red List of South African Plants. To date, there have been no reports on bacterial endophytes from this plant, their classes of secondary metabolites, and potential medicinal properties. This study presents (i) taxonomic characterization of bacterial endophytes in leaf and root tissues using 16S rRNA, (ii) bacterial isolation, morphological, and phylogenetic characterization, (iii) bacterial growth, metabolite extraction, and LC-MS-based metabolite fingerprinting, and (iv) antimicrobial testing of bacterial crude extracts. Next-generation sequencing yielded 693 and 2,459 DNA read counts for the rhizomes and leaves, respectively, detecting phyla including Proteobacteria, Bacteroidota, Gemmatimonadota, Actinobacteriota, Verrucomicrobiota, Dependentiae, Firmicutes, and Armatimonodata. At the genus level, , , , and Ralstonia were the most dominant in both leaves and rhizomes. From root tissues, four bacterial isolates were selected, and 16S rRNA-based phylogenetic characterization identified two closely related sp. (strain BNWU4 and 5), BNWU2, and BNWU1. The ethyl acetate:chloroform (1:1 v/v) organic extract from each isolate exhibited antimicrobial activity against all selected bacterial pathogens. Strain BNWU5 displayed the highest activity, with minimum inhibitory concentrations ranging from 62.5 μg/mL to 250 μg/mL against diarrhoeagenic , , , antibiotic-resistant , , , and . LC-MS analysis of the crude extract revealed common antimicrobial metabolites produced by all isolates, including Phenoxomethylpenicilloyl (penicilloyl V), cis-11-Eicosenamide, 3-Hydroxy-3-phenacyloxindole, and 9-Octadecenamide.
PubMed: 38855763
DOI: 10.3389/fmicb.2024.1383854 -
Journal of Forensic Sciences Jul 2024The subject of inter- and intra-laboratory inconsistency was recently raised in a commentary by Itiel Dror. We re-visit an inter-laboratory trial, with which some of the...
The subject of inter- and intra-laboratory inconsistency was recently raised in a commentary by Itiel Dror. We re-visit an inter-laboratory trial, with which some of the authors of this current discussion were associated, to diagnose the causes of any differences in the likelihood ratios (LRs) assigned using probabilistic genotyping software. Some of the variation was due to different decisions that would be made on a case-by-case basis, some due to laboratory policy and would hence differ between laboratories, and the final and smallest part was the run-to-run difference caused by the Monte Carlo aspect of the software used. However, the net variation in LRs was considerable. We believe that most laboratories will self-diagnose the cause of their difference from the majority answer and in some, but not all instances will take corrective action. An inter-laboratory exercise consisting of raw data files for relatively straightforward mixtures, such as two mixtures of three or four persons, would allow laboratories to calibrate their procedures and findings.
Topics: Humans; Likelihood Functions; Software; Monte Carlo Method; DNA Fingerprinting; Genotype; Laboratories; Decision Making; Forensic Genetics
PubMed: 38853374
DOI: 10.1111/1556-4029.15558 -
The Brazilian Journal of Infectious... 2024C. difficile has been increasingly reported as a cause of gastrointestinal disease in children, ranging from mild self-limiting diarrhea to severe conditions such as...
Molecular epidemiology and antimicrobial resistance in Clostridioides difficile strains isolated from children and adolescents in a tertiary referral pediatric hospital in Fortaleza, Brazil.
BACKGROUND
C. difficile has been increasingly reported as a cause of gastrointestinal disease in children, ranging from mild self-limiting diarrhea to severe conditions such as pseudomembranous colitis and toxic megacolon. Only two pediatric research groups reported the presence of C. difficile infection in Brazilian children, but no previous research has examined C. difficile infection among children in northeastern Brazil. This prospective cross-sectional study investigated the molecular epidemiology and antimicrobial resistance of C. difficile strains isolated from children and adolescents with diarrhea referred to a tertiary pediatric hospital in Brazil while exploring the associated risk factors.
RESULTS
Toxin positivity or C. difficile isolation was found in 30.4 % (17/56) samples. C. difficile was isolated from 35 % (6/17) samples. Four toxigenic strains were identified (tpi+, tcdA+, tcdB+, cdtB-, without tcdC deletions) belonging to PCR ribotypes and PFGE-pulsotypes: 046 (new pulsotype 1174), 106 (NAP11), 002 (new pulsotype 1274), 012 (new pulsotype NML-1235). Two of the six isolates belonging to ribotypes 143 and 133 were non-toxigenic. All toxigenic strains were sensitive to metronidazole and vancomycin. Regarding the clinical manifestation, diarrhea lasted an average of 11 days, ranging from 3 to 50 days and was often associated with mucus and/or blood. All six patients from whom the C. difficile was isolated had a chronic disease diagnosis, with these comorbidities as the main risk factors.
CONCLUSION
Our study enhances our understanding of the present epidemiological landscape of C. difficile-associated diarrhea (CDI) among children in northeastern Brazil, reveling a substantial CDI frequency of 30.4 %, with toxigenic strains detected in 76.4 % of cases, highlighting a higher prevalence compared to earlier Brazilian studies. In the globalized world, an understanding of disease-generating strains, the associated risk factors, clinical manifestation, and antimicrobial sensitivity has fundamental epidemiological importance and draws attention to preventive measures, allowing for more decisive action.
Topics: Humans; Clostridioides difficile; Child; Adolescent; Female; Male; Brazil; Cross-Sectional Studies; Prospective Studies; Tertiary Care Centers; Child, Preschool; Anti-Bacterial Agents; Clostridium Infections; Microbial Sensitivity Tests; Risk Factors; Infant; Hospitals, Pediatric; Molecular Epidemiology; Diarrhea; Ribotyping; Drug Resistance, Bacterial
PubMed: 38843868
DOI: 10.1016/j.bjid.2024.103767 -
Forensic Science International. Genetics Jul 2024Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently...
Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently release the DNA from hydroxyapatite. In trabecular bone where soft tissues are preserved, we assume that full dissolution of the bone powder is not required to release the DNA from collagen. To investigate this issue, research was performed on 45 Second World War diaphysis (compact bone)-epiphysis (trabecular bone) femur pairs, each processed with a full dissolution (FD) and partial dissolution (PD) extraction method. DNA quality and quantity were assessed using qPCR PowerQuant analyses, and autosomal STRs were typed to confirm the authenticity of isolated DNA. Our results support different mechanisms of DNA preservation in compact and trabecular bone because FD method was more efficient than PD method only in compact bone, and no difference in DNA yield was observed in trabecular bone, showing no need for full dissolution of the bone powder when trabecular bone tissue is processed. In addition, a significant difference in DNA yield was observed between compact and trabecular bone when PD was applied, with more DNA extracted from trabecular bone than compact bone. High suitability of trabecular bone processed with PD method is also supported by the similar quantities of DNA isolated by FD method when applied to both compact and trabecular bone. Additionally similar quantities of DNA were isolated when compact bone was extracted with FD method and trabecular bone was extracted with PD method. Processing trabecular bone with PD method in routine identification of skeletonized human remains shortens the extraction procedure and simplifies the grinding process.
Topics: Humans; DNA; Cancellous Bone; Microsatellite Repeats; Femur; DNA Fingerprinting; Polymerase Chain Reaction; Male; Real-Time Polymerase Chain Reaction
PubMed: 38833778
DOI: 10.1016/j.fsigen.2024.103067 -
Forensic Science International. Genetics Jul 2024SE33 or ACTBP2 is the most polymorphic locus in many national DNA databases and in the commercial STR kits used to type both crime scene samples and reference samples to...
SE33 or ACTBP2 is the most polymorphic locus in many national DNA databases and in the commercial STR kits used to type both crime scene samples and reference samples to populate these databases. We describe the molecular reason for a three band pattern of SE33 seen in several samples. A SNP in the flanking SE33 region causes the binding of the unlabelled D3S1358 primer. As a result, a "chimeric" PCR product of the labelled SE33 primer and the D3S1358 primer is generated that is smaller than the regular SE33 amplicon. We call this "Type 3 three band pattern" as the genetic base differs from the Type 1 three band pattern caused by somatic mosaicism and the Type 2 that results from copy number variation.
Topics: Humans; Microsatellite Repeats; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; DNA Fingerprinting; DNA Primers; DNA Copy Number Variations
PubMed: 38833777
DOI: 10.1016/j.fsigen.2024.103064 -
Forensic Science International. Genetics Jul 2024In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures....
In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution.
Topics: Humans; Polymorphism, Single Nucleotide; RNA, Messenger; Female; Semen; Cervix Mucus; Saliva; High-Throughput Nucleotide Sequencing; Male; Body Fluids; DNA Fingerprinting; Skin; Menstruation; Forensic Genetics; Tissue Donors; Sequence Analysis, RNA
PubMed: 38833776
DOI: 10.1016/j.fsigen.2024.103066 -
Clinical Genetics Jun 2024SERPINA11 is a hitherto poorly characterised gene belonging to Clade A of the SERPIN superfamily, with unknown expression pattern and functional significance. We report...
SERPINA11 is a hitherto poorly characterised gene belonging to Clade A of the SERPIN superfamily, with unknown expression pattern and functional significance. We report a perinatal lethal phenotype in two foetuses from the same family associated with a biallelic loss of function variant in SERPINA11, and provide functional evidence to support its candidature as a Mendelian disorder. The SERPINA11 variant-associated foetal phenotype is characterised by gross and histopathological features of extracellular matrix disruption. Western blot and immunofluorescence analyses revealed SERPINA11 expression in multiple mouse tissues, with pronounced expression in the bronchiolar epithelium. We observed a significant decrease in SERPINA11 immunofluorescence in the affected foetal lung compared with a healthy gestation-matched foetus. Protein expression data from HEK293T cell lines following site-directed mutagenesis support the loss of function nature of the variant. Transcriptome analysis from the affected foetal liver indicated the possibility of reduced SERPINA11 transcript abundance. This novel serpinopathy appears to be a consequence of the loss of inhibition of serine proteases involved in extracellular matrix remodelling, revealing SERPINA11 as a protease inhibitor critical for embryonic development.
PubMed: 38831697
DOI: 10.1111/cge.14564 -
Neuro-oncology Jun 2024Formalin-fixed, paraffin-embedded (FFPE) tissue slides are routinely used in cancer diagnosis, clinical decision-making, and stored in biobanks, but their utilization in...
BACKGROUND
Formalin-fixed, paraffin-embedded (FFPE) tissue slides are routinely used in cancer diagnosis, clinical decision-making, and stored in biobanks, but their utilization in Raman spectroscopy-based studies has been limited due to the background coming from embedding media.
METHODS
Spontaneous Raman spectroscopy was used for molecular fingerprinting of FFPE tissue from 46 patient samples with known methylation subtypes. Spectra were used to construct tumor/non-tumor, IDH1WT/IDH1mut, and methylation-subtype classifiers. Support vector machine and random forest were used to identify the most discriminatory Raman frequencies. Stimulated Raman spectroscopy was used to validate the frequencies identified. Mass spectrometry of glioma cell lines and TCGA were used to validate the biological findings.
RESULTS
Here we develop APOLLO (rAman-based PathOLogy of maLignant glioma) - a computational workflow that predicts different subtypes of glioma from spontaneous Raman spectra of FFPE tissue slides. Our novel APOLLO platform distinguishes tumors from nontumor tissue and identifies novel Raman peaks corresponding to DNA and proteins that are more intense in the tumor. APOLLO differentiates isocitrate dehydrogenase 1 mutant (IDH1mut) from wildtype (IDH1WT) tumors and identifies cholesterol ester levels to be highly abundant in IDHmut glioma. Moreover, APOLLO achieves high discriminative power between finer, clinically relevant glioma methylation subtypes, distinguishing between the CpG island hypermethylated phenotype (G-CIMP)-high and G-CIMP-low molecular phenotypes within the IDH1mut types.
CONCLUSIONS
Our results demonstrate the potential of label-free Raman spectroscopy to classify glioma subtypes from FFPE slides and to extract meaningful biological information thus opening the door for future applications on these archived tissues in other cancers.
PubMed: 38828478
DOI: 10.1093/neuonc/noae101 -
Forensic Science International Jul 2024A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR... (Comparative Study)
Comparative Study
A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR typing. This unexpected parity in DNA preservation between the petrous bone, a renowned source of endogenous DNA in skeletal remains, and the epiphyses of metacarpals, which are porous and susceptible to taphonomic changes, is surprising. In this study, we introduced ATR-FTIR spectroscopy as an approach to unravel the correlation between bone molecular structure and DNA preservation. Metacarpals and petrous bones with same taphonomic history were sampled and prepared for DNA analyses. While one portion of the sample was used for DNA analysis, the other underwent ATR-FTIR spectroscopic examination. The normalized spectra and FTIR indices between the epiphyses of metacarpals and petrous bones were compared. Because the taphonomic history of the remains used is relatively short and stable, the ATR-FTIR spectroscopy unveiled subtle structural differences between the two bone types. Petrous bones exhibited higher mineralization, whereas epiphyses contained more organic matter. The unexpected preservation of DNA in the epiphyses of metacarpals can likely be attributed to the presence of soft tissue remnants within the trabeculae. Here observed differences in the molecular structure of bones indicate there are different mechanisms enabling DNA preservation in skeletal tissues.
Topics: Humans; Spectroscopy, Fourier Transform Infrared; Petrous Bone; Epiphyses; DNA; Metacarpal Bones; DNA Fingerprinting; Microsatellite Repeats; World War II
PubMed: 38821024
DOI: 10.1016/j.forsciint.2024.112076