A preliminary study characterizing temporal changes in soil bacterial communities after dismembered bones were buried.

Determining the burial time of skeletal remains is one of the most important issues of forensic medicine. We speculated that the microbiome of gravesoil may be a promising method to infer burial time by virtue of time-dependent. As we know, forensic scientists have established various models to predict the postmortem interval of a decedent based on the changes in body and soil microbiome communities. However, limited data are available on the burial time prediction for bones, especially dismembered bones. In this exploratory study, we initially conducted 16S rRNA amplicon high-throughput sequencing on the burial soil of 10 porcine femurs within a 120-day period and analyzed the changes in soil microbial communities. Compared with the control soil, a higher Shannon index in the microbial diversity of burial soil containing bones was observed. Correlation analysis identified 61 time-related bacterial families and the best subset selection method obtained best subset, containing Thermomonosporaceae, Clostridiaceae, 0319-A21, and Oxalobacteraceae, which were used to construct a simplified multiple linear regression model with a mean absolute error (MAE) of 56.69 accumulated degree day (ADD). An additional random forest model was established based on indicators for the minimum cross-validation error of Thermomonosporaceae, Clostridiaceae, 0319-A21, Oxalobacteraceae, and Syntrophobacteraceae, with an MAE of 55.65 ADD. The produced empirical data in this pilot study provided the evidence of feasibility that the microbial successional changes of burial soil will predict the burial time of dismembered bones and may also expand the current knowledge of the effects of bone burial on soil bacterial communities.

Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.

Design and Investigation of a Dynamic Auto-Adjusting Ejector for the MED-TVC Desalination System Driven by Solar Energy.

Ejectors have been widely used in multi-effect distillation, thermal vapor compression (MED-TVC) desalination systems due to their simple structures and low energy consumption. However, traditional fixed geometry ejectors fail to operate under unstable working conditions driven by solar energy. Herein, a dynamic auto-adjusting ejector, equipped with a needle at the nozzle throat, is proposed to improve the ejector's performance under changeable operating conditions. A two-dimensional computational fluid dynamics (CFD) model is built to analyze the performance and flow field of the ejector. It is found that the achievable entrainment ratio gradually increases as the needle approaches the nozzle, and the entrainment ratio of the ejector is relatively stable, varying slightly between 1.1-1.2 when the primary pressure changes from 2.5 to 4 bar. Besides, the performance comparison between the proposed ejector and the traditional ejector is studied under the same primary pressure range. The entrainment ratio of the designed ejector was 1.6 times higher than that of the conventional ejector at a primary pressure of 2.5 bar. Furthermore, the average entrainment ratio of the designed ejector is 1.14, as compared to 0.84 for the traditional ejector. Overall, the proposed auto-adjusting ejector could be potentially used in MED-TVC desalination systems under variable conditions.

Inhibition of miR-483-5p improves the proliferation, invasion and inflammatory response of triple-negative breast cancer cells by targeting SOCS3.

microRNAs (miRs) have been indicated to serve oncogenic or tumor suppressor roles. However, the role of miR-483-5p in breast cancer and its associated molecular mechanisms remain unclear. In the present study, compared with adjacent normal tissues and MCF-10a cells, the expression level of miR-483-5p was upregulated in triple-negative breast cancer (TNBC) tissues and TNBC cell lines. Bioinformatic analysis and luciferase reporter assay confirmed the presence of miR-483-5p binding sites in the 3'-untranslated region of suppressor of cytokine signaling 3 (SOCS3). In addition, the expression level of SOCS3 protein in TNBC tissues was markedly lower compared with in adjacent tissues, and miR-483-5p expression was negatively correlated with SOCS3 expression in TNBC tissues. Cell proliferation and flow cytometry assays indicated that knockdown of miR-483-5p inhibited the proliferation and promoted apoptosis in the TNBC cell line BT-549. This effect was markedly attenuated by SOCS3 small interfering (si)RNA transfection. Additionally, wound healing and Transwell assays demonstrated that SOCS3 siRNA reversed the inhibitory effects of miR-483-5p inhibitor on the migration and invasion of BT-549 cells. Moreover, the decrease in miR-483-5p expression significantly reduced the secretion of TNF-α, IL-6, IL-1ß and monocyte chemoattractant protein-1 in BT-549 cells, while SOCS3 siRNA could partially reverse this effect. Additionally, SOCS3 overexpression reversed the effects of miR-483-5p mimic on the proliferation, migration, invasion and inflammation of BT-549 cells. Taken together, these data demonstrated that the inhibition of miR-483-5p could inhibit the proliferation, migration, invasion and inflammatory response, while promoting the apoptosis of TNBC cells by negatively regulating SOCS3. miR-483-5p may be a potential target for TNBC therapy.

Development of a multiplex methylation-sensitive restriction enzyme-based SNP typing system for deconvolution of semen-containing mixtures.

The identification of mixed stains has always been a difficult problem in personal identification in the forensic field. In recent years, tissue-specific methylation sites have proven to be very stable biomarkers for distinguishing tissue origin. However, it is still challenging to perform tissue source identification and individual identification simultaneously. In this study, we developed a method that uses tissue-specific methylation markers combined with single-nucleotide polymorphism (SNP) markers to detect semen from mixed biofluids and to identify individuals simultaneously. Semen-specific CpG markers were chosen from the literature and further validated utilizing methylation-sensitive restriction endonuclease (MSRE) combined with PCR technology. The neighboring SNP markers were searched in the flanking sequence of the target CpG within 400 bp, and SNP typing was then carried out through a single-base extension reaction followed by capillary electrophoresis. Eventually, a method of MSRE combined with SNaPshot that could detect 12 compound CpG-SNP markers was developed. Using this system, 10 ng of total DNA and DNA mixture with semen content up to 25% could be typed successfully. Moreover, the cumulative discrimination power of the system in the northern Chinese Han population is 0.9998. This study provides a valuable strategy for forensic practice to perform tissue origin and individual identification from mixed stains simultaneously.

Predicting the postmortem interval of burial cadavers based on microbial community succession.

Previous studies have demonstrated that microbial community succession during the decomposition of cadavers could be used to estimate the postmortem interval (PMI). However, the vast majority of the existing studies focused on exposed cadavers. In fact, burial cadavers are common scenarios for forensic investigations. In this study, the microbial communities from gravesoil, rectum and skin of burial SD rat cadavers during decomposition were characterized using 16S rRNA gene high-throughput sequencing. We predicted PMI based on the microbial community succession. Obvious differences in microbial community structures were observed between different stages of decomposition. Later decay stages had a lower alpha diversity compared to earlier decay stages. Significant linear relationships between similarities of the microbial communities and postmortem intervals were observed, manifesting regular succession over the course of decomposition. Furthermore, we combined random forest models with postmortem microbial features to predict PMI. The model explained 86.83%, 84.55% and 81.67% of the variation in the microbial community, with a mean absolute error of 1.82, 2.06 and 2.13 days within 60 days of decomposition for gravesoil, rectum and skin of burial cadavers, respectively. Overall, our results suggested that postmortem microbial community data could serve as a potential forensic tool to estimate accurate PMI of burial cadavers.

Predicting human age by detecting DNA methylation status in hair.

Age prediction is of great importance for criminal investigation and judicial expertise. DNA methylation status is considered a promising method to infer tissue age by virtue of age-dependent changes on methylation sites. In recent years, forensic scientists have established various models to predict the chronological age of blood, saliva, and semen based on DNA methylation status. However, hair-inferred age has not been studied in the field of forensic science. In this study, we measured the methylation statuses of potential age-related CpG sites by using the multiplex methylation SNaPshot method. A total of 10 CpG sites from the LAG3, SCGN, ELOVL2, KLF14, C1orf132, SLC12A5, GRIA2, and PDE4C genes were found to be tightly associated with age in hair follicles. A correlation coefficient above 0.7 was found for four CpG sites (cg24724428 and Chr6:11044628 in ELOVL2, cg25148589 in GRIA2, and cg07547549 in SLC12A5). Among four age-prediction models, the multiple linear regression model consisting of 10 CpG sites provided the best-fitting results, with a median absolute deviation of 3.68 years. It is feasible to obtain both human identification and age information from a single scalp hair follicle. No significant differences in methylation degree were found between different sexes, hair types, or hair colors. In conclusion, we established a method to evaluate chronological age by assessing DNA methylation status in hair follicles.

DIP-microhaplotypes: new markers for detection of unbalanced DNA mixtures.

The identification of a suspect in a degraded and unbalanced DNA mixture has been a challenge for the standard short tandem repeat polymorphisms (STR) typing. Several methods have been introduced to solve this problem, such as DIP-STR, DIP-SNP, and SNP-STR markers. In this study, we proposed DIP-microhaplotype (deletion/insertion linked a chain of SNPs) as a kind of new genetic marker to type the unbalanced and degraded DNA mixture. We established the detection method with ten DIP-microhaplotype markers including 26 SNPs using allele-specific multiplex PCR followed by SNaPshot assay. This novel compound marker allows us to detect the minor DNA with a sensitivity of 1:100 to 1:1000 in a DNA mixture of any gender. Most of the DIP-microhaplotype markers had a relatively high probability of informative alleles with an average informative value (I value) of 0.308. In all, we proposed DIP-microhaplotype as a novel type of DNA marker for the detection of minor contributor from unbalanced DNA mixtures. Due to their inherent shorter length, higher polymorphism, and sensitivity, DIP-microhaplotypes are promising markers for the examination of the degraded and unbalanced mixtures in forensic stains or clinical chimeras.

A new set of DIP-SNP markers for detection of unbalanced and degraded DNA mixtures.

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP-SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP-SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP-SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP-SNPs in a Chinese Han population. The DIP-SNPs were capable of detecting the minor contributor's allele in home-made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 -10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP-SNPs may serve as a valuable tool in detection of UDM in forensic medicine.

Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct.

The large-scale identification of putative alternative promoters study shows more than 52% of human genes are regulated by alternative promoters. The human myc-associated zinc finger protein (SAF/MAZ) gene have SAF-1 and SAF-3 variants transcripted from two transcription start sites (TSSs). By using SAF/MAZ promoter as a model, we set up an approach to probe how the alternative promoters are regulated in real time. We have constructed the bichromatic fluorescent reporter driven by SAF/MAZ 5'-proximal promoter plasmids from which transactivation status of SAF-1 and SAF-3 alternative promoter could be monitored by EGFP and DsRed expression respectively. The results showed that the SAF-3 expression is regulated by alternative promoters. When the bichromatic fluorescent reporter was driven by -1692/+277 or -1401/+277 SAF/MAZ promoter the dominant expression of SAF-3 would be observed in comparison with SAF-1 expression. We also identified that Elk-1 is an inhibitory transcription factor for SAF-3 expression. The temporal diversity of SAF-1 and SAF-3 expressions can be observed via bichromatic fluorescent reporters. These imply that the bichromatic fluorescent reporter driven by alternative promoter construct might be a useful tool for decoding the temporal regulatory repertoire of alternative promoter in human genes.

Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress.

Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS). Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER) stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress.

The influence of associative effects on the in vitro-estimated utilizable crude protein (uCP) of feeds for ruminants.

Nineteen feed mixtures, formulated with 16 single feeds, were used to study the influence of associative effects on utilizable crude protein (uCP) of feed mixtures. The in vitro incubation technique of Zhao and Lebzien (2000) was used for uCP determination. It was found that the in vitro-determined uCP (D-uCP) was significantly higher than the weighted uCP (W-uCP) of feed mixtures and there was a significant regressive relationship between W-uCP (x) [g x kg(-1)DM] and D-uCP (y) [g x kg(-1)DM]: y=(0.94 +/- 0.23)x + (18.78 +/- 35.58), r2=0.49, n=19, p < 0.01. It was concluded that there exist significant associative effects of feed mixtures on uCP. In formulation of rations for ruminants the D-uCP should be used instead of W-uCP. Because of the low regression coefficient of the equation above, the D-uCP cannot be estimated from the W-uCP.