Age Estimation Based on DNA Methylation and Its Application Prospects in Forensic Medicine.

With the improvement of DNA methylation detection techniques, studies on age-related methylation sites have found more age-specific ones across tissues, which improves the sensitivity and accuracy of age estimation. In addition, the establishment of various statistical models also provides a new direction for the age estimation of tissues from different sources. This review summarizes the related studies of age estimation based on DNA methylation from the aspects of detection technology, age-related cytosine phosphate guanine site and model selection in recent years.

Cross-Platform Application of DNA Methylation Age Estimation Model in Eastern Chinese Han Population.

OBJECTIVES: To evaluate the forensic application value of an age estimation model based on DNA methylation in eastern Chinese Han population, and to provide a theoretical basis for exploring age estimation models suitable for different detection platforms. METHODS: According to the 6 age-related methylation sites in the published blood DNA methylation age estimation models of Chinese Han population, the DNA methylation level of 48 samples was detected by pyrosequencing and next-generation sequencing (NGS). After submitting DNA methylation levels to the age estimation model, the DNA methylation ages were predicted and compared with their real ages. RESULTS: The 6 DNA methylation sites in both detection techniques were age-related, with an R2 of 0.85 and a median absolute deviation (MAD) of 4.81 years when using pyrosequencing；with an R2 of 0.84 and MAD of 4.41 years when using NGS. CONCLUSIONS: The blood DNA methylation age estimation model can be used under pyrosequencing and multi-purpose regional methylation enrichment sequencing technology based on NGS and it can accurately estimate the age.

Proteomic Difference Analysis of Whole Blood and Bloodstains.

OBJECTIVES: To study the changes of protein levels in peripheral blood after it dried. METHODS: The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed. RESULTS: A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains. CONCLUSIONS: The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

Efficiency Analysis of Uncle-Nephew Relationship Identification by Increasing STR Markers and Adding Reference Samples.

OBJECTIVES: To estimate the system efficiency of uncle-nephew relationship identification by increasing STR markers and adding reference samples based on the test results of simulated data and real samples, so as to provide references for selecting the appropriate number of STRs and reference samples for uncle-nephew relationship identification. METHODS: Five common models of uncle-nephew relationship identification were constructed by adding different reference samples. In each model, the likelihood ratio (LR) for 10 000 pairs of uncle-nephew relationships and 10 000 pairs of unrelated individuals were simulated by detecting 19, 39 or 55 STRs, and the system efficiency at different thresholds was simulated. The samples of the Han population in Zhejiang were collected, and 55 autosomal STRs were obtained by using SiFaSTRTM 23plex kit, Goldeneye® DNA ID 22NC kit and AGCU 21+1 PCR amplification kit. When 19, 39 and 55 STRs were detected, the LR of each model and system efficiency under different thresholds were calculated and compared with the simulation results. RESULTS: Under the same detection system, the calculated results of simulated data and corresponding true samples were basically consistent. In the same model, there was a positive correlation between the system efficiency of uncle-nephew relationship identification and the number of STRs detected. Moreover, the system efficiency of introducing relatives was higher than identifying only two individuals. The order of preference for the introduction of relatives was the full sibling (or mother) of the uncle and the full sibling (or mother) of the nephew. CONCLUSIONS: The system efficiency of uncle-nephew relationship identification could be improved by increasing the number of STRs and introducing known relatives, which would provide the basis for selecting the most appropriate detection system and reference individuals in actual cases.

Effects of Peripheral Blood Different Pretreatment Methods and Preservation Time on RNA Quality.

OBJECTIVES: To evaluate the effects of different pretreatment methods and preservation time on RNA quality of peripheral blood samples, and to optimize the preservation method of peripheral blood samples. METHODS: Eight pretreatment methods were used to preprocess the peripheral blood from 3 healthy unrelated individuals and the treated samples were stored at -80 ℃. Total RNA of samples was extracted using Quick-RNATM Miniprep Plus kit. DNA/RNA ShieldTM was added to peripheral blood and total RNA was extracted after preservation at -80 ℃ for 0, 5, 10, 15, 30 and 60 days, respectively. The concentration, purity and integrity of RNA were determined. Statistical analyses were performed by SPSS 22.0 software to compare the differences in RNA yield, purity and integrity among the eight pretreatment methods. RESULTS: In terms of purity, leukocyte pretreated with RNAlaterTM and directly cryopreservation peripheral blood showed the worst purity. The other six methods showed better purity. In terms of yield, blood cells with DNA/RNA ShieldTM came out with the highest yield, followed by peripheral blood with DNA/RNA ShieldTM. In terms of integrity, peripheral blood preserved in PAXgene Blood RNA tube method had the best integrity. Except for peripheral blood pretreated with DNA/RNA ShieldTM and blood cells pretreated with DNA/RNA shieldTM, the other five methods had statistical differences when compared to the method by keeping peripheral blood in PAXgene Blood RNA tube. The purity of RNA stored at six-time gradients ranged from 1.815 to 1.952. With the increase of storage time, RNA yield decreased from 4.516 ng to 1.039 ng, and RNA integrity decreased from 8.533 to 7.150. CONCLUSIONS: According to the results of total RNA's yield, purity and integrity, peripheral blood pretreated with DNA/RNA ShieldTM was the best pretreatment method. After the pretreatment, samples can be preserved for up to 60 days in low temperature.

The influence of locally applied granulocyte-colony stimulating factor on osteoporotic bone.

OBJECTIVES: The compromised capacity of bone healing in osteoporotic population renders a serious concern of patients and clinicians. This study aimed to investigate the influence of G-CSF on bone reconstruction using an osteoporotic animal model. MATERIALS AND METHODS: Sixty skeletal mature female Spraque-Dawley rats underwent bilateral ovariectomy (OVX) and were assigned into three groups (n = 20). Three months after OVX, defects of 5 mm in cranial and 2 mm in femur were surgically created on all the animals. The defects were left unfilled, filled with gelatin sponge (GS), or filled with granulocyte-colony stimulating factor (G-CSF) infused GS. Specimens were retrieved for histomorphometric and micro-CT analyses at weeks 1, 4, 8, and 12 after surgery. RESULTS: At early stage of week 1 to week 8, the histomorphometric and micro-CT analysis demonstrated more advanced bone formation in femur in the control group; by week 12, all groups achieved cortical closure. In cranial bone, more advanced bone formation was exhibited in G-CSF-treated group at both early and late stages, although this observation was not statistically significant. CONCLUSIONS: The results indicated that in osteoporotic bone, G-CSF may advance bone healing in cranial bone where spontaneous bone formation was insufficient.

Fluorescence-Enhanced Sensing of Hypochlorous Acid Based on 2-Pyridylthiazole Unit.

Hypochlorous acid, being one of reactive oxygen species (ROS), is essential to protect the body against invasion of pathogens. Excess of hypochlorous acid (HOCl) is believed to be in tight connection with various inflammation-related diseases. It remains a challenge to detect the ROS in physiological conditions (aqueous buffer and neutral pH) with selectivity. In the presented paper, we have synthesized a ferrocence-modified pyridylthiazole derivatives, 1,4-di{5-[(4'-ferrocenyl-2'-(4"-pyridyl)]thiazinyl}benzene (DFPT). Only HOCl could turn-on the fluorescence of DFPT with enhanced emission at 465 nm. Compared to the other reported HOCl sensors, DFPT could selectively detect HOCl with rapid response (< 60 s) in the aqueous buffer (pH = 7.0). The detection limit at pH = 7.0 was 0.7 µM according to the titration experiment.

Skeletal site-specific response to ovariectomy in a rat model: change in bone density and microarchitecture.

OBJECTIVES: Ovariectomized (OVX) rat model has been widely used in osteoporosis-related studies. However, the discrepancies in age and skeletal sites being investigated make it difficult to compare the results from different studies. The purpose of this study was to provide information of systemic skeletal site-specific changes in a stable OVX rat model. MATERIAL AND METHODS: Thirty-three 6-month Spraque-Dawley female rats were used. Fifteen rats underwent ovariectomy, and fifteen received sham surgery. Three animals without any surgery were sacrificed at week 0 to serve as baseline. Three animals in the OVX and sham group, respectively, were euthanized at week 2, 4, 12, 24 and 36 post-surgery. Ten bone sites, including parietal bone, interparietal bone, maxilla, mandible, humerus, ulna, femur, tibia, lumber vertebra, and ilium, were subjected to micro-CT. RESULTS: Overall, long bones, lumber vertebra, and ilium showed similar trend of bone loss post-OVX, with tibia and femur suffered the most bone loss and spine the least (decreased by 75.0%, 70.4% and 36.6% in bone mineral density BMD at week 36 from base line, respectively). Upon OVX, jaw bones and cranial bones only showed a minor reduction in BMD (decreased by 1~3% from baseline) at week 36. Significant deterioration of trabecular structure was detected in long bones, lumber vertebra, and ilium post-OVX, while jaw bones remained relatively stable. CONCLUSIONS: This study for the first time assessed the systemic site-specific bone loss and microarchitecture changes in OVX rat model. It provided valuable information for selecting bone site and observation time in osteoporosis-related study.

Healing of osteotomy sites applying either piezosurgery or two conventional saw blades: a pilot study in rabbits.

PURPOSE: The purpose of this study was to compare bone healing of experimental osteotomies applying either piezosurgery or two different oscillating saw blades in a rabbit model. METHODS: The 16 rabbits were randomly assigned into four groups to comply with observation periods of one, two, three and five weeks. In all animals, four osteotomy lines were performed on the left and right nasal bone using a conventional saw blade, a novel saw blade and piezosurgery. RESULTS: All three osteotomy techniques revealed an advanced gap healing starting after one week. The most pronounced new bone formation took place between two and three weeks, whereby piezoelectric surgery revealed a tendency to faster bone formation and remodelling. Yet, there were no significant differences between the three modalities. CONCLUSIONS: The use of a novel as well as the piezoelectric bone-cutting instrument revealed advanced bone healing with a favourable surgical performance compared to a traditional saw.

Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae.

The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.

Effect of Granulocyte-Colony Stimulating Factor on Endothelial Cells and Osteoblasts.

OBJECTIVES: Some animal studies showed that granulocyte-colony stimulating factor (G-CSF) provides beneficial environment for bone healing. It has been well documented that endothelial cells and osteoblasts play critical roles in multiple phases of bone healing. However, the biological effects of G-CSF on these cells remain controversial. This study aimed to investigate the influence of G-CSF at various concentrations on endothelial cells and osteoblasts. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) were treated with G-CSF at 1000, 100, 10, and 0 ng/mL, respectively. The capacity of cell proliferation, migration, and tube formation of HUVECs was evaluated at 72, 8, and 6 hours after treatment, respectively. The capacity of proliferation, differentiation, and mineralization of hOBs was evaluated at 24 hours, 72 hours, and 21 days after treatment, respectively. RESULTS: HUVECs treated with 100 and 1000 ng/mL G-CSF showed a significantly higher value comparing with controls in migration assay (p < 0.001, p < 0.01, resp.); the group treated with 1000 ng/mL G-CSF showed a significantly lower value on tube formation. No significant difference was detected in groups of hOBs. CONCLUSIONS: G-CSF showed favorable effects only on the migration of HUVECs, and no direct influence was found on hOBs.

Wound healing of osteotomy defects prepared with piezo or conventional surgical instruments: a pilot study in rabbits.

AIM: The aim of the present study was to evaluate and compare the wound-healing process following osteotomies performed with either conventional rotary burs or piezoelectric surgery in a rabbit model. METHODS: Two types of osteotomy window defects of the nasal cavities were prepared on the nasal bone of 16 adult New Zealand white rabbits with either a conventional rotary bur or piezo surgery. The defects were covered with a resorbable membrane. Four animals were killed at 1, 2, 3, and 5 weeks after the surgical procedure, respectively. Histological and morphometric evaluations were performed to assess the volumetric density of various tissue components: the blood clot, vascularized structures, provisional matrix, osteoid, mineralized bone, bone debris, residual tissue, and old bone. RESULTS: Significantly more bone debris was found at 1 week in the conventionally-prepared defects compared to the piezo surgically-prepared defects. At 2 and 3 weeks, a newly-formed hard tissue bridge, mainly composed of woven bone, was seen; however, no statistically-significant differences were observed. At 5 weeks, the defects were completely filled with newly-formed bone. CONCLUSION: The defects prepared by piezo surgery showed a significantly decreased proportion of bone debris at 1 week, compared to conventional rotary bur defect.

Effect of ovariectomy on stimulating intracortical remodeling in rats.

OBJECTIVE: Technically primates and dogs represent ideal models to investigate diseases characterized by abnormal intracortical remodeling. High expenses and ethical issues, however, restrict the use of those animals in research. Rodent models have been used as alternatives instead, but their value is limited, if none, because these animals lack intracortical bone remodeling. This study aimed at investigating the effect of ovariectomy onto the stimulation of intracortical remodeling in rat mandibles. MATERIALS AND METHODS: Sixteen 12-week-old Spraque-Dawly (SD) female rats were randomly assigned into two groups, receiving either ovariectomy or sham operation. All the rats were sacrificed 18 weeks postoperatively. The entire mandibles were harvested for microcomputed tomography (micro-CT) and histomorphometric assessments. RESULTS: Micro-CT examination showed significantly decreased bone mineral density (0.95 ± 0.01 versus 1.01 ± 0.02 g/cm(3), P < 0.001) and bone volume (65.78 ± 5.45 versus 87.41 ± 4.12%, P < 0.001) in ovariectomy group. Histomorphometric assessment detected a sixfold increased intracortical bone remodeling as well as an increased bone modeling in mandibles of ovariectomized rats. CONCLUSION: For the first time, to the authors' knowledge, it was detected that ovariectomy stimulates intracortical remodeling in rat mandibles. This animal model might be of use to study various bone diseases associated with an abnormal intracortical remodeling process.

Responses of distraction regenerate to high-frequency traction at a rapid rate.

BACKGROUND: Continuous traction is capable of creating an optimal biological environment for bone healing which may finally compensate for the rapid distraction rate in distraction osteogenesis. This study was designed to investigate the response of distraction callus to continuous distraction at a rapid rate using a rabbit model of mandibular lengthening. METHODS: Thirty adult New Zealand white rabbits were randomly assigned to the intermittent (1 step/d) or continuous distraction (8 steps/s) group, with 15 in each. After osteotomy, manual-driven or autodriven distractor was adapted accordingly. The distraction was activated at a rate of 3.0 mm/d for 4 days. Five rabbits in each group were killed at week 2, week 4, and week 12 of consolidation, respectively. Plain radiography, microcomputed tomography, and histology examinations were used to evaluate the bone regeneration status. RESULTS: Plain radiographs and histologic studies demonstrated more advanced bone healing in continuous distraction group than that in intermittent distraction group at all the examined time points. Quantitative microcomputed tomography analysis showed significantly higher bone volume in continuous distraction group at week 2 (p < 0.01) and week 4 (p < 0.05) of consolidation. CONCLUSIONS: Continuous traction by autodriven distractor could be a promising clinical alternative to shorten the treatment course of distraction osteogenesis. Further studies to test its clinical potential using large animals that have similar metabolic rate and muscular resistance with human being are necessary.

Comparison of gene expression of tissue inhibitor of matrix metalloproteinase-1 between continuous and intermittent distraction osteogenesis: a quantitative study on rabbits.

BACKGROUND: Distraction osteogenesis is a controlled surgical procedure that initiates a regenerative process and uses mechanical strain to enhance the biological responses of the injured tissues to create new bone. To explore the effect of high-frequency mechanical traction on the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), we compared the gene expression of TIMP-1 between continuous and intermittent distraction osteogenesis using a rabbit model of mandibular lengthening. MATERIALS AND METHODS: Forty adult New Zealand white rabbits were randomly assigned to the intermittent and continuous distraction groups. A unilateral mandibular osteotomy was performed and a custom-designed manual-driven or auto-driven distractor was bridged over the osteotomy segments. Animals were sacrificed at day-6, day-10, day-14 and day-21 after osteotomy. Samples were examined with real-time polymerase chain reaction (PCR). RESULTS: Real-time PCR examination showed significantly higher mRNA levels of TIMP-1 under continuous distraction than that under intermittent distraction at day-6 and day-10. No significant differences were found at day-14 and day-21. CONCLUSION: High-frequency traction provides a good mechanical environment for accelerating bone formation by up-regulating TIMP-1.