Inference of forensic body fluids/tissues based on mitochondrial DNA copy number: a preliminary study.

The inference of body fluids and tissues is critical in reconstructing crime scenes and inferring criminal behaviors. Nevertheless, present methods are incompatible with conventional DNA genotyping, and additional testing might result in excessive consumption of forensic scene materials. This study aims to investigate the feasibility of distinguishing common body fluids/tissues through the difference in mitochondrial DNA copy number (mtDNAcn). Four types of body fluids/tissues were analyzed in this study - hair, saliva, semen, and skeletal muscle. MtDNAcn was estimated by dividing the read counts of mitochondrial DNA to that of nuclear DNA (RRmt/nu). Results indicated that there were significant differences in RRmt/nu between different body fluids/tissues. Specifically, hair samples exhibited the highest RRmt/nu (log10RRmt/nu: 4.3 ± 0.28), while semen samples showed the lowest RRmt/nu (log10RRmt/nu: -0.1 ± 0.28). RRmt/nu values for DNA samples without extraction were notably higher (approximately 2.9 times) than those obtained after extraction. However, no significant difference in RRmt/nu was observed between various age and gender groups. Hierarchical clustering and Kmeans clustering analyses showed that body fluids/tissues of the same type clustered closely to each other and could be inferred with high accuracy. In conclusion, this study demonstrated that the simultaneous detection of nuclear and mitochondrial DNA made it possible to perform conventional DNA analyses and body fluid/tissue inference at the same time, thus killing two birds with one stone. Furthermore, mtDNAcn has the potential to serve as a novel and promising biomarker for the identification of body fluids/tissues.

Somatic GNA11/GNAQ variants in a cohort of Chinese children with phakomatosis pigmentovascularis.

Importance: Postzygotic mutations in the GNAQ/GNA11 genes, which encode the G-protein nucleotide binding protein alpha subunits, have been identified in patients with phakomatosis pigmentovascularis (PPV). However, little is known about the Chinese population. Objective: To identify pathogenic mutations in pediatric patients with PPV within the Chinese population. Methods: We performed whole-exome sequencing (WES) using skin lesion tissues from pediatric patients diagnosed with PPV. Additionally, ultradeep-targeted sequencing was conducted to validate the somatic mutations. A genotype-phenotype correlation was analyzed by integrating data from previous reports with the findings of the present study. Results: Thirteen patients were enrolled, all diagnosed with the cesioflammea type of PPV, except for one patient with an unclassifiable type. We identified somatic GNA11 c.547C>T (p.R183C) variant in seven patients and GNAQ c.548G>A (p.R183Q) in four patients, with low allelic fractions ranging from 2.1% to 8.6% through ultradeep sequencing. Besides, a GNAQ c.548G>A (p.R183Q) variant was detected through targeted sequencing in one of two patients who did not exhibit detectable variants via WES. The genotype-phenotype correlation analysis, involving 15 patients with a GNA11 variant and 10 with a GNAQ variant, revealed that facial capillary malformation (87% vs. 50%, P = 0.075) and ocular melanocytosis (80% vs. 40%, P = 0.087) appeared to be more frequent in patients with GNA11 mutation compared to those with GNAQ mutations. All four patients diagnosed with cesiomarmorata type or overlapping cesioflammea and cesiomarmorata type PPV carried the GNA11 variant. Interpretation: Our study demonstrated that the majority of PPV patients in the Chinese population carried a postzygotic variant of GNAQ/GNA11, thus further confirming the pathogenic role of GNAQ/GNA11 mosaicism in the development of PPV cesioflammea type.

Improved individual identification in DNA mixtures of unrelated or related contributors through massively parallel sequencing.

DNA mixtures are a common sample type in forensic genetics, and we typically assume that contributors to the mixture are unrelated when calculating the likelihood ratio (LR). However, scenarios involving mixtures with related contributors, such as in family murder or incest cases, can also be encountered. Compared to the mixtures with unrelated contributors, the kinship within the mixture would bring additional challenges for the inference of the number of contributors (NOC) and the construction of probabilistic genotyping models. To evaluate the influence of potential kinship on the individual identification of the person of interest (POI), we conducted simulations of two-person (2 P) and three-person (3 P) DNA mixtures containing unrelated or related contributors (parent-child, full-sibling, and uncle-nephew) at different mixing ratios (for 2 P: 1:1, 4:1, 9:1, and 19:1; for 3 P: 1:1:1, 2:1:1, 5:4:1, and 10:5:1), and performed massively parallel sequencing (MPS) using MGIEasy Signature Identification Library Prep Kit on MGI platform. In addition, in silico simulations of mixtures with unrelated and related contributors were also performed. In this study, we evaluated 1): the MPS performance; 2) the influence of multiple genetic markers on determining the presence of related contributors and inferring the NOC within the mixture; 3) the probability distribution of MAC (maximum allele count) and TAC (total allele count) based on in silico mixture profiles; 4) trends in LR values with and without considering kinship in mixtures with related and unrelated contributors; 5) trends in LR values with length- and sequence-based STR genotypes. Results indicated that multiple numbers and types of genetic markers positively influenced kinship and NOC inference in a mixture. The LR values of POI were strongly dependent on the mixing ratio. Non- and correct-kinship hypotheses essentially did not affect the individual identification of the major POI; the correct kinship hypothesis yielded more conservative LR values; the incorrect kinship hypothesis did not necessarily lead to the failure of POI individual identification. However, it is noteworthy that these considerations could lead to uncertain outcomes in the identification of minor contributors. Compared to length-based STR genotyping, using sequence-based STR genotype increases the individual identification power of the POI, concurrently improving the accuracy of mixing ratio inference using EuroForMix. In conclusion, the MGIEasy Signature Identification Library Prep kit demonstrated robust individual identification power, which is a viable MPS panel for forensic DNA mixture interpretations, whether involving unrelated or related contributors.

Metavalently bonded tellurides: the essence of improved thermoelectric performance in elemental Te.

Elemental Te is important for semiconductor applications including thermoelectric energy conversion. Introducing dopants such as As, Sb, and Bi has been proven critical for improving its thermoelectric performance. However, the remarkably low solubility of these elements in Te raises questions about the mechanism with which these dopants can improve the thermoelectric properties. Indeed, these dopants overwhelmingly form precipitates rather than dissolve in the Te lattice. To distinguish the role of doping and precipitation on the properties, we have developed a correlative method to locally determine the structure-property relationship for an individual matrix or precipitate. We reveal that the conspicuous enhancement of electrical conductivity and power factor of bulk Te stems from the dopant-induced metavalently bonded telluride precipitates. These precipitates form electrically beneficial interfaces with the Te matrix. A quantum-mechanical-derived map uncovers more candidates for advancing Te thermoelectrics. This unconventional doping scenario adds another recipe to the design options for thermoelectrics and opens interesting pathways for microstructure design.

Integrative lncRNA, circRNA, and mRNA analysis reveals expression profiles of six forensic body fluids/tissue.

RNAs have attracted much attention in forensic body fluid/tissue identification (BFID) due to their tissue-specific expression characteristics. Among RNAs, long RNAs (e.g., mRNA) have a higher probability of containing more polymorphic sites that can be used to assign the specific donor of the body fluid/tissue. However, few studies have characterized their overall profiles in forensic science. In this study, we sequenced the transcriptomes of 30 samples from venous blood, menstrual blood, semen, saliva, vaginal secretion, and skin tissue, obtaining a comprehensive picture of mRNA, lncRNA, and circRNA profiles. A total of 90,305 mRNAs, 102,906 lncRNAs (including 19,549 novel lncRNAs), and 40,204 circRNAs were detected. RNA type distribution, length distribution, and expression distribution were presented according to their annotation and expression level, and many novel body fluid/tissue-specific RNA markers were identified. Furthermore, the cognate relations among the three RNAs were analyzed according to gene annotations. Finally, SNPs and InDels from RNA transcripts were genotyped, and 21,611 multi-SNP and 4,471 multi-InDel transcriptomic microhaplotypes (tMHs) were identified. These results provide a comprehensive understanding of transcriptome profiles, which could provide new avenues for tracing the origin of the body fluid/tissue and identifying an individual.

Metatranscriptomic characterization of six types of forensic samples and its potential application to body fluid/tissue identification: A pilot study.

Microorganisms are potential markers for identifying body fluids (venous and menstrual blood, semen, saliva, and vaginal secretion) and skin tissue in forensic genetics. Existing published studies have mainly focused on investigating microbial DNA by 16 S rRNA gene sequencing or metagenome shotgun sequencing. We rarely find microbial RNA level investigations on common forensic body fluid/tissue. Therefore, the use of metatranscriptomics to characterize common forensic body fluids/tissue has not been explored in detail, and the potential application of metatranscriptomics in forensic science remains unknown. Here, we performed 30 metatranscriptome analyses on six types of common forensic sample from healthy volunteers by massively parallel sequencing. After quality control and host RNA filtering, a total of 345,300 unigenes were assembled from clean reads. Four kingdoms, 137 phyla, 267 classes, 488 orders, 985 families, 2052 genera, and 4690 species were annotated across all samples. Alpha- and beta-diversity and differential analysis were also performed. As a result, the saliva and skin groups demonstrated high alpha diversity (Simpson index), while the venous blood group exhibited the lowest diversity despite a high Chao1 index. Specifically, we discussed potential microorganism contamination and the "core microbiome," which may be of special interest to forensic researchers. In addition, we implemented and evaluated artificial neural network (ANN), random forest (RF), and support vector machine (SVM) models for forensic body fluid/tissue identification (BFID) using genus- and species-level metatranscriptome profiles. The ANN and RF prediction models discriminated six forensic body fluids/tissue, demonstrating that the microbial RNA-based method could be applied to BFID. Unlike metagenomic research, metatranscriptomic analysis can provide information about active microbial communities; thus, it may have greater potential to become a powerful tool in forensic science for microbial-based individual identification. This study represents the first attempt to explore the application potential of metatranscriptome profiles in forensic science. Our findings help deepen our understanding of the microorganism community structure at the RNA level and are beneficial for other forensic applications of metatranscriptomics.

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine.

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

Easykin: a flexible and user-friendly online tool for forensic kinship testing and missing person identification.

In forensic kinship testing and missing person identification, it is a fundamental question to choose the most informative reference relatives, select appropriate genotyping systems, and evaluate the weight of evidence comprehensively. Despite that several useful tools have been developed, they have not addressed these questions satisfactorily. In this paper, we develop a flexible and user-friendly online tool, Easykin, to address the aforementioned issues. It has some promising features: (i) Pedigrees can be constructed easily and presented intuitively with just a few mouse clicks. (ii) System power can be estimated before testing based on certain set of markers and reference relatives. (iii) The pruning function of EasyKin enables users to choose appropriate subsets of available references. (iv) Parameters at a specific LR for a single case may ease evidence interpretation. (v) The user interface (UI) is an HTML-based dashboard, which is friendly to both professional and non-professional users and can be used anytime and anywhere. Here, we presented three common cases as examples to demonstrate how kinship testing and missing person identification can be improved with EasyKin. In conclusion, this tool provides a one-stop solution for forensic use, that is, instructing users to choose appropriate kits and reference relatives before testing, calculating LR in the testing, and providing parameters for data interpretation after testing. EasyKin is freely available at https://forensicsysu.shinyapps.io/EasyKin/ .

Does postcholecystectomy increase the risk of colorectal cancer?

With the increasing number of cholecystectomy and the high proportion of colorectal cancer in malignant tumors, the question of whether cholecystectomy is a risk factor for colorectal disease has been widely concerned. After reviewing the literature at home and abroad, the authors will summarize the research progress of the correlation between the occurrence of colorectal tumors after cholecystectomy, in order to provide help for the prevention and treatment of colorectal tumors.

Evaluation of Detection Efficiency for Trio Full Sibling Testing.

OBJECTIVES: To study the detection efficiency of trio full sibling with another known full sibling reference added under different number of autosomal STR typing systems. METHODS: Based on 43 detection systems consisting of 13 to 55 representative autosomal STR loci, 10 000 true families (full sibling group) and 10 000 false families (unrelated individual group) were randomly simulated. The full sibling index (FSI) was calculated based on the method of family reconstruction. The cumulative sibling relationship index (CFSI) of 0.000 1 and 10 000 were used as the evaluation thresholds, and the detection efficiency parameters were calculated and compared with the identification of the duo full sibling testing. RESULTS: With the increasing number of STR loci, the error rate and inability of judgement rate gradually decreased; the sensitivity, specificity, correct rate of judgment and other parameters gradually increased, and the system efficiency gradually improved. Under the same detection system, trio full sibling testing showed higher sensitivity, specificity, system efficiency and lower inability of judgement rate compared with duo full sibling testing. When the system efficiency was higher than 0.85 and inability of judgement rate was less than 0.01%, at least 20 STRs should be detected for trio full sibling testing, which was less than 29 STRs required by duo full sibling testing. CONCLUSIONS: The detection efficiency of trio full sibling testing is superior to that of duo full sibling testing with the same detection system, which is an effective identification scheme for laboratories with inadequate detection systems or for materials with limited conditions.

Comparison of three massively parallel sequencing platforms for single nucleotide polymorphism (SNP) genotyping in forensic genetics.

Three MPS platforms are being used in forensic genetic analysis, i.e., MiSeq FGx, Ion S5 XL, and MGISEQ-2000. However, few studies compared their performance. In this study, we sequenced 83 common SNPs of 71 samples using the ForenSeq™ DNA Signature Prep Kit on MiSeq FGx, the Precision ID Identity Panel on Ion S5 XL, and the MGIEasy Signature Identification Library Prep Kit on MGISEQ-2000 and then the performance was compared. Results showed that the MiSeq FGx had the highest sequence quality but the lowest sequencing depth and allele balance. Discordant genotypes were observed at six SNPs, which may be caused by variants at primer binding regions, indel errors, or misalignments. Besides, two kinds of background noises, allele-specific miscalled reads (ASMR) and allele-nonspecific miscalled reads (ANMR), were characterized. MGISEQ-2000 showed the highest level of ASMR while Ion S5 XL had the highest level of ANMR. Site- and genotype-dependent miscalled patterns were observed at several SNPs on Ion S5 XL and MGISEQ-2000, but few on MiSeq FGx. In conclusion, the three MPS platforms perform differently with respect to sequencing quality, sequencing depth, allele balance, concordance, and background noise. These findings may be useful for data comparison, mixture deconvolution, and heteroplasmy analysis in forensic genetics.

Whole Mitochondrial Genome Detection and Analysis of Two- to Four-Generation Maternal Pedigrees Using a New Massively Parallel Sequencing Panel.

Mitochondrial DNA (mtDNA) is an effective genetic marker in forensic practice, especially for aged bones and hair shafts. Detection of the whole mitochondrial genome (mtGenome) using traditional Sanger-type sequencing is laborious and time-consuming. Additionally, its ability to distinguish point heteroplasmy (PHP) and length heteroplasmy (LHP) is limited. The application of massively parallel sequencing in mtDNA detection helps researchers to study the mtGenome in-depth. The ForenSeq mtDNA Whole Genome Kit, which contains a total of 245 short amplicons, is one of the multiplex library preparation kits for the mtGenome. We used this system to detect the mtGenome in the blood samples and hair shafts of thirty-three individuals from eight two-generation pedigrees, one three-generation pedigree, and one four-generation pedigree. High-quality sequencing results were obtained. Ten unique mtGenome haplotypes were observed in the mothers from the ten pedigrees. A total of 26 PHPs were observed using the interpretation threshold of 6%. Eleven types of LHPs in six regions were evaluated in detail. When considering homoplasmic variants only, consistent mtGenome haplotypes were observed between the twice-sequenced libraries and between the blood and hair shafts from the same individual and among maternal relatives in the pedigrees. Four inherited PHPs were observed, and the remainder were de novo/disappearing PHPs in the pedigrees. Our results demonstrate the effective capability of the ForenSeq mtDNA Whole Genome Kit to generate the complete mtGenome in blood and hair shafts, as well as the complexity of mtDNA haplotype comparisons between different types of maternal relatives when heteroplasmy is considered.

Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression.

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic effects of Zadi-5 against depression have been indicated in previously reported clinical studies, the identity and impact of the active pharmaceutical compounds present in the drug have not been fully elucidated. This study used network pharmacology to predict the drug composition and identify the therapeutically active compounds in Zadi-5 pills. Here, we established a rat model of chronic unpredicted mild stress (CUMS) and conducted an open field test (OFT), Morris water maze (MWM) analysis, and sucrose consumption test (SCT) to investigate the potential therapeutic efficacy of Zadi-5 in depression. This study aimed to demonstrate Zadi-5's therapeutic effects for depression and predict the critical pathway of the action of Zadi-5 against the disorder. The vertical and horizontal scores (OFT), SCT, and zone crossing numbers of the fluoxetine (positive control) and Zadi-5 groups were significantly higher (P < 0.05) than those of the CUMS group rats without treatment. According to the results of network pharmacology analysis, the PI3K-AKT pathway was found to be essential for the antidepressant effect of Zadi-5.

Strong charge carrier scattering at grain boundaries of PbTe caused by the collapse of metavalent bonding.

Grain boundaries (GBs) play a significant role in controlling the transport of mass, heat and charge. To unravel the mechanisms underpinning the charge carrier scattering at GBs, correlative microscopy combined with local transport measurements is realized. For the PbTe material, the strength of carrier scattering at GBs depends on its misorientation angle. A concomitant change in the barrier height is observed, significantly increasing from low- to high-angle GBs. Atom probe tomography measurements reveal a disruption of metavalent bonding (MVB) at the dislocation cores of low-angle GBs, as evidenced by the abrupt change in bond-rupture behavior. In contrast, MVB is completely destroyed at high-angle GBs, presumably due to the increased Peierls distortion. The collapse of MVB is accompanied by a breakdown of the dielectric screening, which explains the enlarged GB barrier height. These findings correlate charge carrier scattering with bonding locally, promising new avenues for the design of advanced functional materials.

DNA and protein analyses of hair in forensic genetics.

Hair is one of the most common pieces of biological evidence found at a crime scene and plays an essential role in forensic investigation. Hairs, especially non-follicular hairs, are usually found at various crime scenes, either by natural shedding or by forcible shedding. However, the genetic material in hairs is usually highly degraded, which makes forensic analysis difficult. As a result, the value of hair has not been fully exploited in forensic investigations and trials. In recent years, with advances in molecular biology, forensic analysis of hair has achieved remarkable strides and provided crucial clues in numerous cases. This article reviews recent developments in DNA and protein analysis of hair and attempts to provide a comprehensive solution to improve forensic hair analysis.

A Comprehensive Characterization of Small RNA Profiles by Massively Parallel Sequencing in Six Forensic Body Fluids/Tissue.

Body fluids/tissue identification (BFID) is an essential procedure in forensic practice, and RNA profiling has become one of the most important methods. Small non-coding RNAs, being expressed in high copy numbers and resistant to degradation, have great potential in BFID but have not been comprehensively characterized in common forensic stains. In this study, the miRNA, piRNA, snoRNA, and snRNA were sequenced in 30 forensic relevant samples (menstrual blood, saliva, semen, skin, venous blood, and vaginal secretion) using the BGI platform. Based on small RNA profiles, relative specific markers (RSM) and absolute specific markers (ASM) were defined, which can be used to identify a specific body fluid/tissue out of two or six, respectively. A total of 5204 small RNAs were discovered including 1394 miRNAs (including 236 novel miRNA), 3157 piRNAs, 636 snoRNAs, and 17 snRNAs. RSMs for 15 pairwise body fluid/tissue groups were discovered by differential RNA analysis. In addition, 90 ASMs that were specifically expressed in a certain type of body fluid/tissue were screened, among them, snoRNAs were reported first in forensic genetics. In brief, our study deepened the understanding of small RNA profiles in forensic stains and offered potential BFID markers that can be applied in different forensic scenarios.

Fermented Dairy Food Intake and Risk of Colorectal Cancer: A Systematic Review and Meta-Analysis.

It was highly controversial whether fermented dairy foods protect against colorectal cancer (CRC) because of conflicting results from current human epidemiologic studies; we therefore conducted this meta-analysis based on the case-control and cohort studies to estimate the holistic analyses. Finally, a total of seven case-control studies and ten cohort studies comprising a total of >20,000 cases were incorporated in the quantitative synthesis. Specifically, statistical evidence of significantly decreasing CRC risk in case-control studies was found to be associated with cheese intake (OR = 0.89, 95% CI = 0.82-0.97). In a subgroup analysis, cheese intake was correlated with lower colon cancer (OR = 0.89, 95% CI = 0.79-1.00) and rectal cancer (OR = 0.86, 95% CI = 0.74-1.00) risk in case-control studies. Furthermore, we also found that the higher intake of yogurt may lower the risk of rectal cancer (OR = 0.75, 95% CI = 0.65-0.88) in cohort studies. The consumption of fermented dairy foods may be relevant to decrease CRC risk in this meta-analysis. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021269798, CRD42021269798.

Ethical issues of the research and practice in forensic genetics.

Forensic genetics mainly uses human biological samples as the objects, solves the identification of biological materials related to law by detecting genetic information, provides clues for investigation and evidences for trial, thus facing many ethical issues. This paper put forward the ethical principles in forensic genetics research and practice, and discussed the ethical issues in sample collection, forensic DNA phenotyping, forensic genetic genealogy analysis, forensic DNA database development, paternity and kinship testing, and research data sharing. We suggest that specific ethical requirements should be formulated, the ethical review system should be established for forensic genetics and ethical training for practitioners should be strengthened.

Pairwise kinship testing with microhaplotypes: Can advancements be made in kinship inference with these markers?

Kinship testing based on genetic relatedness is one of the major tasks in forensic genetics. Although short tandem repeats (STRs) are the "gold standard" biomarkers for relationship testing, microhaplotypes (MHs) have also emerged as viable options for kinship elucidation. In this work, the kinship testing efficiency of 54 highly polymorphic MHs was studied in two extended families consisting of parent-offspring, full siblings, grandparent-grandchildren, uncle/aunt-nephew/nieces, and first cousins. In addition, ten-thousand pairs of different degrees of relationships were simulated using various datasets including 54 MHs, 27 STRs plus 94 single nucleotide polymorphisms (SNPs) that were included in the ForenSeq DNA Signature Prep Kit (ForenSeq), 54 MHs plus loci in ForenSeq, and different subsets of 417-published MHs. The panels' system effectiveness in the kinship analysis were accessed by likelihood ratio distributions. The results showed that 54 MHs could be used in first-degree relationship testing with high reliability. The effectiveness of 54 MHs was slightly lower than ForenSeq but only by a narrow margin. Both 54 MHs and ForenSeq were not sufficient for distant relationship testing, and approximately 200 microhaplotypes with an average expected heterozygosity (He) = 0.79 were enough to determine second-degree relationships, but a panel of 417 MHs with an average He = 0.72 was not sufficient to first cousins testing according to the simulation analysis. In conclusion, 54 MHs could be used to serve as supplement markers for kinship testing; and well-established STR markers plus well-performing microhaplotype markers may become collective tools in forensic applications, though an enlarged pool of forensic markers is needed for distant relationship testing.