Integrated Bioinformatics Analysis for the Identification of Key Molecules and Pathways in the Hippocampus of Rats After Traumatic Brain Injury.

High-throughput and bioinformatics technology have been broadly applied to demonstrate the key molecules involved in traumatic brain injury (TBI), while no study has integrated the available TBI-related datasets for analysis. In this study, four available expression datasets of fluid percussion injury (FPI) and sham samples from the hippocampus of rats were analysed. A total of 248 differentially expressed genes (DEGs) and 10 differentially expressed microRNAs (DEMIs) were identified. Then, functional annotation was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Most of the DEGs were enriched for the term inflammatory immune response. The MCODE plug-in in the Cytoscape software was applied to build a protein-protein interaction (PPI) network, and 18 hub genes were demonstrated to be enriched in the cell cycle pathway. Besides, time sequence (3 h, 6 h, 12 h, 24 h, and 48 h) profile analysis was performed using short time-series expression miner (STEM). The significantly expressed genes were assigned into 24 pattern clusters with four significant uptrend clusters. Four DEGs, Fcgr2a, Bcl2a1, Cxcl16, and Gbp2, were found to be differentially expressed at all time-points. Fifty-three DEGs and eight DEMIs were identified to form a miRNA-mRNA negative regulatory network using miRWalk3.0 and Cytoscape. Moreover, the mRNA levels of eight hub genes were validated by qRT-PCR. These DEGs, DEMIs, and time-dependent expression patterns facilitate our knowledge of the molecular mechanisms underlying the process of TBI in the hippocampus of rats and have the potential to improve the diagnosis and treatment of TBI.

Rapidly mutating Y-STRs study in Chinese Yi population.

Y-chromosomal short tandem repeats (Y-STRs) have been widely used in forensic analysis and population genetics. With low to moderate mutation rates, conventional Y-STR panels, including commercially available Y-STR kits, enable the identification of male pedigrees but typically fail to differentiate related male individuals. The introduction of rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) with higher mutation rates (µ > 10-2) has been demonstrated to increase the discrimination capacity of unrelated men and the differentiation rate of related men compared with standard Y-STRs. To date, several studies have been performed worldwide. Here, 260 father-son pairs from Chinese Yi population were investigated, and 18.8% of them were differentiated with the 13 RM Y-STR markers, which was close to the theoretical estimate of 19.5% based on the mutation rates of these markers. Among the 57 mutations observed, repeat gains were more common than repeat losses (1.48:1), and one-step mutations were more common than two-step mutations (27.5:1). Locus-specific mutation rates ranged from < 3.85 × 10-3 (95% CI 0.00-1.41 × 10-2) to 3.85 × 10-2 (95% CI 1.86 × 10-2-6.96 × 10-2), with an average mutation rate of 1.46 × 10-2 (95% CI 1.11 × 10-2-1.89 × 10-2). Furthermore, we combined the father-son pair data from the present study with the data from the previous studies, generating an overall mutation rate of 1.70 × 10-2. The high differentiation rate obtained in the present study indicates the suitability of RM Y-STRs to distinguish paternal lineages in Chinese Yi population.

Population genetic analysis of a 21-plex DIP panel in seven Chinese ethnic populations.

A 21-plex DIP (deletion and insertion polymorphism) panel has a good performance in analyzing biogeographic ancestry in three main global population groups (European, African, and Asian). This panel was used to investigate 450 unrelated individuals in seven Chinese ethnic groups (Han, Dong, Miao, Zhuang, Uyghur, Tibetan, and Mongolian). Allele frequencies were calculated. Mho (mean value of observed heterozygosity) ranged from 0.3019 to 0.4367, MHe (mean value of expected heterozygosity) ranged from 0.31175 to 0.38341, CMP (combined matching probability) ranged from 3.5834E-07 to 2.5985E-06, CDP (combined power of discrimination) ranged from 0.99999740150 to 0.99999964166, and CPE (combined power of paternity exclusion) ranged from 0.85884504 to 0.97949131. The results suggested the potential of the panel for individual identification and paternity testing in Chinese populations. Pairwise genetic differences Fst values (-0.00091 to 0.05873) and the analysis by STRUCTURE indicated that Chinese populations have good internal consistency.

miR-212-5p attenuates ferroptotic neuronal death after traumatic brain injury by targeting Ptgs2.

Ferroptosis, a newly discovered form of iron-dependent regulated cell death, has been implicated in traumatic brain injury (TBI). MiR-212-5p has previously been reported to be downregulated in extracellular vesicles following TBI. To investigate whether miR-212-5p is involved in the ferroptotic neuronal death in TBI mice, we first examined the accumulation of malondialdehyde (MDA) and ferrous ion, and the expression of ferroptosis-related molecules at 6 h, 12 h, 24 h, 48 h and 72 h following controlled cortical impact (CCI) in mice. There was a significant upregulation in the expression of Gpx4 and Acsl4 at 6 h, Slc7a11 from 12 h to 72 h, and Nox2 and Sat1 from 6 h to 72 h post injury. Similarly, an upregulation in the expression of Gpx4 at 6 h, Nox2 from 6 h to 72 h, xCT from 12 h to 72 h, and Sat1 at 72 h after CCI was observed at the protein level. Interestingly, MDA and ferrous ion were increased whereas miR-212-5p was decreased in the CCI group compared to the sham group. Furthermore, we found that overexpression of miR-212-5p attenuated ferroptosis while downregulation of miR-212-5p promoted ferroptotic cell death partially by targeting prostaglandin-endoperoxide synthase-2 (Ptgs2) in HT-22 and Neuro-2a cell lines. In addition, administration of miR-212-5p in CCI mice significantly improved learning and spatial memory. Collectively, these findings indicate that miR-212-5p may protect against ferroptotic neuronal death in CCI mice partially by targeting Ptgs2.

Circular Ribonucleic Acid Expression Profile in Mouse Cortex after Traumatic Brain Injury.

Traumatic brain injury (TBI) causes high rates of worldwide death and morbidity because of the complex secondary injury cascade. Circular ribonucleic acid (RNA) (circRNA), a type of RNA that forms a covalently closed continuous loop, may be involved in the regulation of secondary injury because it is expressed widely in the brain and contributes to a large class of post-transcriptional regulators. Deep RNA sequencing (RNA-seq) and bioinformatic analysis were performed to investigate the expression profile and function of circRNAs in the mouse cortex after controlled cortical impact (CCI). A total of 19,794 circRNAs were identified, and 1315 were annotated in circBase. There were 191 filtered differentially expressed circRNAs (98 for up-regulated and 93 for down-regulated). The gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that inflammation, cell death, and repair of damage were the main biological processes and molecular mechanisms related to altered circRNAs. The pathway-circRNA interaction network revealed three core circRNAs and five corepathways related to TBI. The circRNA-messenger RNA (mRNA) interaction network and competitive endogenous RNA (ceRNA) analysis suggested potential microRNA (miRNA) sponges and target mRNAs. In addition to five optimal circRNA-miRNA-mRNA pairs were analyzed, circRNA_16895-miRNA myosin-10 (Myo 10) was predicted to regulate fragment crystallizable gamma receptors (FcγR)-mediated phagocytosis pathway. Four circRNAs were selected for quantitative real-time polymerase chain reaction analysis to validate the sequencing data. Our results provide promising functions of circRNAs aberrantly expressed in TBI to explore molecular mechanisms and potential therapeutic targets for its therapy.

Genotyping polymorphic microhaplotype markers through the Illumina® MiSeq platform for forensics.

Microhaplotype markers are emerging forensic genetic markers that have received broad attention in forensics and may supplement existing genetic marker panels. Short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs) are the general genetic markers at present. Stutter and the high mutation rate of STR markers and the low polymorphism of SNP markers obstruct the solving of certain cases. Kidd proposed microhaplotype markers that encompass 2-4 SNPs. In this study, we screened microhaplotype loci through three criteria, and chose the Illumina® MiSeq platform to sequence the new markers. A new nomenclature was proposed and Perl-based tool FLfinder was designed to genotype the microhaplotype marker. After counting the number of haplotypes in samples that were sequenced and calculating common forensic parameters, 13 loci with high polymorphism were reported. Twelve of the 13 loci had an average allele coverage ratio (ACR) of 0.72 to 0.92. Structure analysis showed that 2504 samples (1000 genome project) could be divided into 5 groupings of populations, and each one representing a continental origin. The finding indicates that microhaplotype markers could be used for individual identification and ancestry inference, and a new choice is provided for forensic practice in the future.

Microhaplotype identified and performed in genetic investigation using PCR-SSCP.

The recently introduced concept of microhaplotype loci has attracted attention in forensics. Previous studies estimated the allele frequencies generally through obtaining genotypic data on the individual SNPs from a larger set of unrelated individuals then phasing microhaplotypes by statistical and computational techniques. Determining phase for a single new individual requires the larger set of individuals to have been genotyped previously. Rare microhaplotypes possessed only by the target individual or microhaplotypes private to a specific population not previously studied are unlikely to be accurately phased using data sets of SNPs. Thus, there is a demand for an approach that could directly determine a gain single individual's precise microhaplotype information. In the present study, we introduced potential approaches of single chain sequencing based Massively Parallel Sequencing Technology (MiSeq) and PCR based Single Strand Conformational Polymorphism (SSCP) technology which was simple, accurate, and cost-effective. The results indicated that microhaplotypes contain much more polymorphic information than divided SNPs per locus (average heterozygosity of microhaplotype 0.61 VS SNPs 0.41). When microhaplotype allele frequencies were compared among five Chinese ethnic populations, significantly different distributions were found between the Han and Uyghur populations. Further analysis of pairwise Fst values and analysis of molecular variance (AMOVA), showed significant population differentiation between the Uyghur and other populations.

Screening and confirmation of microRNA markers for distinguishing between menstrual and peripheral blood.

The identification of menstrual blood (MB) and peripheral blood (PB) left at a crime scene is crucial for crime reconstruction, especially in sexual assaults. MicroRNAs (miRNAs), a class of non-protein coding molecules, have been demonstrated to be a viable tool for body fluid identification in forensic casework. Several groups have searched for miRNAs that are specific to different body fluids. Blood has been studied the most extensively. However, menstrual blood was only involved in five studies, and the results confirming the presence of specific miRNAs could not be reproduced in other studies. In this study, we attempted to screen new markers that can differentiate between menstrual blood and peripheral blood by using Exiqon's miRCURY™ LNA Array. Five miRNAs were selected based on the microarray results, namely, miR-141-3p, miR-373-3p, miR-497-5p, miR-143-5p, and miR-136-5p, whose expression levels exhibited 27.95-, 17.96-, 16.74-, 10.14-, and 9.21-fold changes, respectively, compared to the level in peripheral blood. Two classic quantitative methods, TaqMan hydrolysis probes (TaqMan for short) and SYBR Green fluorochrome (SYBR Green for short), were applied in the confirmation step to study the impact of different quantitative methods on the results. Three miRNAs (miR-141-3p, miR-497-5p, and miR-143-5p) were confirmed by TaqMan and one (miR-141-3p) by SYBR Green. Furthermore, bioinformatic methods were applied to interpret the candidate miRNAs. Our results established a multi-step procedure for body fluid identification and showed that the choice of quantitative method is important when miRNAs are used to identify the origin of blood samples.

Modulation of A-type K+ channels by the short-chain cobrotoxin through the protein kinase C-delta isoform decreases membrane excitability in dorsal root ganglion neurons.

A-type K(+) channels are crucial in controlling neuronal excitability, and their regulation in sensory neurons may alter pain sensation. In this study, we identified the functional role of cobrotoxin, the short-chain α-neurotoxin isolated from Naja atra venom, which acts in the regulation of the transient A-type K(+) currents (IA) and membrane excitability in dorsal root ganglion (DRG) neurons via the activation of the muscarinic M3 receptor (M3R). Our results showed that cobrotoxin increased IA in a concentration-dependent manner, whereas the sustained delayed rectifier K(+) currents (IDR) were not affected. Cobrotoxin did not affect the activation of IA markedly, however, it shifted the inactivation curve significantly in the depolarizing direction. The cobrotoxin-induced IA response was blocked by the M3R-selective antagonists DAU-5884 and 4-DAMP. An siRNA targeting the M3R in small DRG neurons abolished the cobrotoxin-induced IA increase. In addition, dialysis of the cells with the novel protein kinase C-delta isoform (PKC-δ) inhibitor δv1-1 or an siRNA targeting PKC-δ abolished the cobrotoxin-induced IA response, whereas inhibition of PKA or classic PKC activity elicited no such effects. Moreover, we observed a significant decrease in the firing rate of the neuronal action potential induced by M3R activation. Pretreatment of the cells with 4-aminopyridine, a selective blocker of IA, abolished this effect. Taken together, these results suggest that the short-chain cobrotoxin selectively enhances IA via a novel PKC-δ-dependent pathway. This effect occurred via the activation of M3R and might contribute to its neuronal hypoexcitability in small DRG neurons.