Temporal Assessment of Protein Stability in Dried Blood Spots.

The use of protein biomarkers in blood for clinical settings is limited by the cost and accessibility of traditional venipuncture sampling. The dried blood spot (DBS) technique offers a less invasive and more accessible alternative. However, protein stability in DBS has not been well evaluated. Herein, we deployed a quantitative LC-MS/MS system to construct proteomic atlases of whole blood, DBSs, plasma, and blood cells. Approximately 4% of detected proteins' abundance was significantly altered during blood drying into blood spots, with overwhelming disturbances in cytoplasmic fraction. We also reported a novel finding suggesting a decrease in the level of membrane/cytoskeletal proteins (SLC4A1, RHAG, DSC1, DSP, and JUP) and an increase in the level of proteins (ATG3, SEC14L4, and NRBP1) related to intracellular trafficking. Furthermore, we identified 19 temporally dynamic proteins in DBS samples stored at room temperature for up to 6 months. There were three declined cytoskeleton-related proteins (RDX, SH3BGRL3, and MYH9) and four elevated proteins (XPO7, RAN, SLC2A1, and SLC29A1) involved in cytoplasmic transport as representatives. The instability was governed predominantly by hydrophilic proteins and enhanced significantly with an increasing storage time. Our analyses provide comprehensive knowledge of both short- and long-term storage stability of DBS proteins, forming the foundation for the widespread use of DBS in clinical proteomics and other analytical applications.

Developmental Validation of the Microreader 23HS Plex ID System: A Novel Supplementary Non-CODIS STR Multiplex Assay for Forensic Application.

A novel supplementary non-CODIS STR multiplex assay designated as the "Microreader 23HS Plex ID System" was developed. The Microreader 23HS Plex ID System enables simultaneous profiling of 23 STR loci and the amelogenin locus. The majority of these loci are non-CODIS STRs (D4S2408, D9S2157, D20S161, D3S2459, D18S1364, D13S305, D1S2142, D19S400, D6S1017, D7S1517, D2S1776, D2S1360, D3S1744, D16S3391, D3S1545, D11S4463, D20S85, D1S549, D10S2325, D21S2055), with the exception of three CODIS STRs (D2S441, D12S391, and D22S1045). Followed the recommendations of Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese validation standards, a comprehensive set of validation studies were conducted, encompassing PCR conditions, stutter ratio and peak height balance, sensitivity, precision and accuracy, reproducibility, species specificity, inhibition, as well as mixture testing. The results demonstrated that the Microreader 23HS Plex ID System is a reliable and robust assay, with well-balanced peak heights, high precision and accuracy, species specificity, and resistance to common inhibitors. The sensitivity of the assay was determined to be 0.125 ng of template DNA. In mixture study, all minor alleles were detected in two-sample mixtures across various ratios (1:19, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 4:1, 9:1, and 19:1). In population study, a total of 500 unrelated individuals of Han ethnicity from East China were genotyped. The allele frequencies and forensic population genetic parameters were calculated, with a cumulative random match probability of 7.757 × 10-27, and a total power of discrimination exceeding 0.999,999,999,999,999,999,999,999,99. In conclusion, the Microreader 23HS Plex ID System shows promise as a valuable supplementary tool for forensic applications, particularly in addressing complex kinship testing and challenges posed by STR mutation.

Investigation into temporal changes in the human bloodstain lipidome.

Bloodstains are crucial pieces of physical evidences found at violent crime scenes, providing valuable information for reconstructing forensic cases. However, there is limited data on how bloodstain lipidomes change over time after deposition. Hence, we deployed a high-throughput high-performance liquid chromatography-mass spectrometry (HPLC-MS) approach to construct lipidomic atlases of bloodstains, whole blood, plasma, and blood cells from 15 healthy adults. A time-course analysis was also performed on bloodstains deposited for up to 6 months at room temperature (~ 25°C). The molecular levels of 60 out of 400 detected lipid species differed dramatically between bloodstain and whole blood samples, with major disturbances observed in membrane glycerophospholipids. More than half of these lipids were prevalent in the cellular and plasmic fractions; approximately 27% and 10% of the identified lipids were uniquely derived from blood cells and plasma, respectively. Furthermore, a subset of 65 temporally dynamic lipid species arose across the 6-month room-temperature deposition period, with decreased triacylglycerols (TAGs) and increased lysophosphatidylcholines (LPCs) as representatives, accounting for approximately 8% of the total investigated lipids. The instability of lipids increased linearly with time, with the most variability observed in the first 10 days. This study sheds light on the impact of air-drying bloodstains on blood components at room temperature and provides a list of potential bloodstain lipid markers for determining the age of bloodstains.

Technical strategy for monozygotic twin discrimination by single-nucleotide variants.

Monozygotic (MZ) twins are theoretically genetically identical. Although they are revealed to accumulate mutations after the zygote splits, discriminating between twin genomes remains a formidable challenge in the field of forensic genetics. Single-nucleotide variants (SNVs) are responsible for a substantial portion of genetic variation, thus potentially serving as promising biomarkers for the identification of MZ twins. In this study, we sequenced the whole genome of a pair of female MZ twins when they were 27 and 33 years old to approximately 30 × coverage using peripheral blood on an Illumina NovaSeq 6000 Sequencing System. Potentially discordant SNVs supported by whole-genome sequencing were validated extensively by amplicon-based targeted deep sequencing and Sanger sequencing. In total, we found nine bona fide post-twinning SNVs, all of which were identified in the younger genomes and found in the older genomes. None of the SNVs occurred within coding exons, three of which were observed in introns, supported by whole-exome sequencing results. A double-blind test was employed, and the reliability of MZ twin discrimination by discordant SNVs was endorsed. All SNVs were successfully detected when input DNA amounts decreased to 0.25 ng, and reliable detection was limited to seven SNVs below 0.075 ng input. This comprehensive analysis confirms that SNVs could serve as cost-effective biomarkers for MZ twin discrimination.

Epigenetic regulation of human-specific gene expression in the prefrontal cortex.

BACKGROUND: Changes in gene expression levels during brain development are thought to have played an important role in the evolution of human cognition. With the advent of high-throughput sequencing technologies, changes in brain developmental expression patterns, as well as human-specific brain gene expression, have been characterized. However, interpreting the origin of evolutionarily advanced cognition in human brains requires a deeper understanding of the regulation of gene expression, including the epigenomic context, along the primate genome. Here, we used chromatin immunoprecipitation sequencing (ChIP-seq) to measure the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), both of which are associated with transcriptional activation in the prefrontal cortex of humans, chimpanzees, and rhesus macaques. RESULTS: We found a discrete functional association, in which H3K4me3HP gain was significantly associated with myelination assembly and signaling transmission, while H3K4me3HP loss played a vital role in synaptic activity. Moreover, H3K27acHP gain was enriched in interneuron and oligodendrocyte markers, and H3K27acHP loss was enriched in CA1 pyramidal neuron markers. Using strand-specific RNA sequencing (ssRNA-seq), we first demonstrated that approximately 7 and 2% of human-specific expressed genes were epigenetically marked by H3K4me3HP and H3K27acHP, respectively, providing robust support for causal involvement of histones in gene expression. We also revealed the co-activation role of epigenetic modification and transcription factors in human-specific transcriptome evolution. Mechanistically, histone-modifying enzymes at least partially contribute to an epigenetic disturbance among primates, especially for the H3K27ac epigenomic marker. In line with this, peaks enriched in the macaque lineage were found to be driven by upregulated acetyl enzymes. CONCLUSIONS: Our results comprehensively elucidated a causal species-specific gene-histone-enzyme landscape in the prefrontal cortex and highlighted the regulatory interaction that drove transcriptional activation.