Detection of RAGE expression and its application to diabetic wound age estimation.

With the prevalence of diabetes, it is becoming important to analyze the diabetic wound age in forensic practice. The present study investigated the time-dependent expression of receptor for advanced glycation end products (RAGE) during diabetic wound healing in mice and its applicability to wound age determination by immunohistochemistry, double immunofluorescence, and Western blotting. After an incision was created in genetically diabetic db/db mice and control mice, mice were killed at posttraumatic intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. Compared with control mice, diabetic mice showed the delayed wound healing. In control and diabetic wound specimens, RAGE immunoreactivity was observed in a small number of polymorphonuclear cells (PMNs), a number of macrophages, and fibroblasts. Morphometrically, the positive ratios of RAGE in macrophages or fibroblasts considerably increased in diabetic wounds during late repair, which exceeded 60% at 7 and 10 days post-injury. There were no control wound specimens to show a ratio of >60% in macrophages or fibroblasts. By Western blotting analysis, the ratios of RAGE to GAPDH were >1.4 in all diabetic wound samples from 7 to 10 days post-injury, which were >1.8 at 10 days after injury. By comparison, no control wound specimens indicated a ratio of >1.4. In conclusion, the expression of RAGE is upregulated and temporally distributed in macrophages and fibroblasts during diabetic wound healing, which might be closely involved in prolonged inflammation and deficient healing. Moreover, RAGE is promising as a useful marker for diabetic wound age determination.

The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

Renal Hypodysplasia/Aplasia 3 Caused by a Rare Variant of GREB1L With Incomplete Penetrance in a Chinese Family.

Renal agenesis represents the most severe form of congenital anomalies of the kidney and urinary tract. Bilateral renal agenesis is almost invariably fatal at birth and has high genetic heterogeneity. Here we report on a Chinese family with two pregnancies affected by a prenatal form of bilateral renal agenesis. Trio-WES was conducted to explore the underlying genetic cause and identified a novel nonsense variant (c .2621G>A: p. Trp874Ter) in the GREB1L gene. Based on previous research, pathogenic mutations in GREB1L can cause renal hypodysplasia/aplasia-3 (RHDA3) with autosomal dominant inheritance. Sanger sequencing performed on the family members revealed that the variant was vertically transmitted from the maternal grandfather through the unaffected mother to the two affected fetuses, fully demonstrating the incomplete dominance of the disease. Our study extends the mutational spectrum associated with RHDA3 and contributes to a more general understanding for the complex genetic inheritance of GREB1L.

[Comparative analysis between diatom nitric acid digestion method and plankton 16S rDNA PCR method].

OBJECTIVE: To compare and explore the application value of diatom nitric acid digestion method and plankton 16S rDNA PCR method for drowning identification. METHODS: Forty drowning cases from 2010 to 2011 were collected from Department of Forensic Medicine of Wenzhou Medical University. Samples including lung, kidney, liver and field water from each case were tested with diatom nitric acid digestion method and plankton 16S rDNA PCR method, respectively. The Diatom nitric acid digestion method and plankton 16S rDNA PCR method required 20 g and 2 g of each organ, and 15 mL and 1.5 mL of field water, respectively. The inspection time and detection rate were compared between the two methods. RESULTS: Diatom nitric acid digestion method mainly detected two species of diatoms, Centriae and Pennatae, while plankton 16S rDNA PCR method amplified a length of 162 bp band. The average inspection time of each case of the Diatom nitric acid digestion method was (95.30 +/- 2.78) min less than (325.33 +/- 14.18) min of plankton 16S rDNA PCR method (P < 0.05). The detection rates of two methods for field water and lung were both 100%. For liver and kidney, the detection rate of plankton 16S rDNA PCR method was both 80%, higher than 40% and 30% of diatom nitric acid digestion method (P < 0.05), respectively. CONCLUSION: The laboratory testing method needs to be appropriately selected according to the specific circumstances in the forensic appraisal of drowning. Compared with diatom nitric acid digestion method, plankton 16S rDNA PCR method has practice values with such advantages as less quantity of samples, huge information and high specificity.

Enhancing potassium ions adsorption on mesoporous carbon spheres with abundant internal surface via engineering sulfur doping sites towards superior rate capability.

Mesoporous carbon spheres (MCSs) show great potential for using as high-performance anodes in potassium-ion batteries (PIBs). Design and synthesis of MCSs with suitable multiscale structures and heteroatom doping or co-doping in MCSs are successfully employed to optimize the ion and electron transportation, however, it is still a challenge to explore MCS-based anodes with satisfactory potassium storage performance. In this work, we report novel S-doped MCS samples with abundant internal surfaces for potassium storage. The S doping sites are controlled during the synthesis, and the effect of different doping sites on the potassium storage is systematically studied. It is found that S doping between the carbon layers enlarges interlayer spacing and facilitates potassium ion adsorption. Consequently, the optimized sample shows an excellent rate capability of 144 mAh/g at 5.0 A/g, and a high reversible specific capacity of 325 mAh/g after 100 cycles at 0.1 A/g with a capacity retention of 91.2%. The important role of element doping sites on ion adsorption and ion storage performance is confirmed by theoretical investigations. Controlling the doping sites in MCSs provides a new approach to designing high-performance electrodes for energy storage and conversion applications.

A Thermally Stable Protein EPP1 of Corn Borer Ostrinia furnacalis Regulates Hemocytic Encapsulation.

Encapsulation is a vital cellular immune reaction of host insects against endoparasitoids; however, how encapsulation is regulated is still unclear. Utilizing a cell line, SYSU-OfHem C, derived from larval hemocytes of the Asian corn borer Ostrinia furnacalis to assay for encapsulation response, an encapsulation-promoting protein (OfEPP1) was isolated from the plasma of O. furnacalis larvae. OfEPP1 is a novel secretory protein, which exists only in O. furnacalis to date. The OfEpp1 gene is intronless and encodes a protein containing several groups of short repetitive sequences and a high proportion of proline residues (18.3%). OfEPP1 is a thermally stable protein that is mainly expressed in fat bodies, and its accumulation could be induced by the injection of foreign objects (Sephadex beads). Eukaryotically expressed recombinant OfEPP1 promoted hemocytes to encapsulate Sephadex beads, while prokaryotically expressed protein did not, indicating that posttranscriptional modification affects the function of OfEPP1. The encapsulation-promoting function of OfEPP1 could be neutralized by the addition of polyclonal antibodies against OfEPP1 or disrupted by the injection of dsRNA targeting OfEpp1. Eukaryotically expressed OfEPP1 promoted the aggregation, but not spreading, of both granulocytes and plasmatocytes. Immunocytochemistry analysis showed that eukaryotically expressed OfEPP1 could bind to the surface of hemocytes. Therefore, we speculate that OfEPP1 possibly promotes hemocytic encapsulation by binding to the surface of hemocytes as a ligand to induce their aggregation. This study provides evidence clarifying the mechanism of encapsulation in insects.

[A discussion on the concentration assay for hemihydrate gypsum in plaster of paris bandage-viscose form].

This essay is to present an improvement on the concentration assay for hemihydrate gypsum in plaster of Paris bandage-Viscose form.

[Pathomorphologic study of the fibrous cap and fatty core of coronary arthrosclerotic plaque in sudden coronary death].

OBJECTIVE: The differences in the thickness of fibrous cap and the percentage of fatty core of the coronary atherosclerotic plaques between sudden coronary death (SCD) group and the control group were investigated. METHODS: Sixty-four autopsy cases were divided into SCD and control groups. Samples were taken from the most severely damaged portions of the coronary atherosclerotic plaques, sectioned, stained with HE, and the percentage of examined by light microscopy for morphologic changes and structural alternations. Image analysis system was adopted to compare the thickness of fibrous cap and percentage of fatty core in the whole plaque between the two groups, and allthe data were analyzed and calculated with SPSS 11.5 statistic software. RESULTS: There were 15 grade III and 21 grade IV atherosclerotic cases found in the SCD group, while there were 16 and 12 found in the control group, respectively. Although no significant differences on the severity of atherosclerosis were found between the two groups (P > 0.05), there were significant differences on the thickness of the fibrous cap and the percentage of fatty core found between the two groups (P < 0.01). CONCLUSION: Our study indicates that there are significant differences in the thickness of fibrous cap and the percentage of fatty core in atherosclerosis plaques between the SCD group and the control group. These observed differences may be helpful for morphological diagnosis of SCD.

[Monocyte chemotactic protein-1 expression in coronary atherosclerosis plaque of sudden coronary death patients].

OBJECTIVE: To investigate the expression of monocyte chemotactic protein 1 (MCP-1) in coronary atherosclerosis plaque of sudden coronary death (SCD) patients and the relationship between MCP-1 expression and SCD. METHODS: Autopsy heart samples (n = 90) collected during 2001 - 2003 were divided to SCD group (n = 36) and 2 control groups: control group I, non-SCD CHD (n = 28), control group II, non-cardiac death (n = 26). The immuno-histochemistry SABC techniques (R, positive MCP-1 cell area/totl area) and computerized images analysis (A) were performed to detect the expression of MCP-1 in different groups. RESULTS: R and A in plaques are significant higher in SCD group than control group I and II (0.1264 +/- 0.013 vs 0.0269 +/- 0.0110 and 0.0267 +/- 0.0100, P = 0.04), (0.4534 +/- 0.083 vs 0.2303 +/- 0.040 and 0.2158 +/- 0.0400, P = 0.00), and similar between control group I and control group II. CONCLUSIONS: MCP-1 expression is increased in coronary atherosclerosis plaque of SCD patients.

Curcumin alleviates brain edema by lowering AQP4 expression levels in a rat model of hypoxia-hypercapnia-induced brain damage.

The present study aimed to investigate the therapeutic effects of curcumin (CU) against brain edema in a rat model of hypoxia-hypercapnia (HH)-induced brain damage (HHBD). Male Sprague-Dawley rats were divided into five groups, including a control group and four treatment groups. The rats in the control group were raised under normal laboratory conditions and were injected with water, whereas the rats in the treatment groups were exposed to a low O2/high CO2 environment simulating HH conditions, and were injected with water, CU, dimethyl sulfoxide (solvent control) or monosialoganglioside GM1. After 2 weeks, the morphological characteristics of the brain tissues were analyzed using optical and electron microscopy. In addition, aquaporin (AQP)-4 protein expression levels in brain tissue samples were analyzed using streptavidin-biotin complex immunohistochemistry and western blotting, and mRNA expression levels were detected using reverse transcription-quantitative polymerase chain reaction. Severe brain edema, tissue structure disruption and increased AQP4 expression levels were detected in the brain tissues of the HH rats. Conversely, the rats treated with CU or GM1 exhibited attenuated HHBD-induced brain edema and tissue structure disruption, and decreased mRNA and protein expression levels of AQP4. The results of the present study suggested that CU treatment was able to attenuate HHBD-induced brain edema by downregulating the expression levels of AQP4 in a rat model. Therefore, CU may be considered a potential therapeutic drug for the treatment of patients with brain edema.

Activation of α7nAChR Promotes Diabetic Wound Healing by Suppressing AGE-Induced TNF-α Production.

Diabetes frequently presents accumulation of advanced glycation end products (AGEs), which might induce excessive TNF-α production from macrophages to cause impaired wound healing. Recent studies have shown that activation of α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages efficiently suppressed TNF-α synthesis. The aim of this study was to investigate the accumulation of AGEs in the wounds and determine whether PNU282987, an α7nAChR agonist, can improve wound repair by inhibiting AGE-mediated TNF-α production in a streptozotocin (STZ)-induced diabetic mouse model. Animals were assigned into four groups: wounded control group, wounded diabetic group, wounded diabetic group treated intraperitoneally with PNU282987, or wounded diabetic group treated intraperitoneally with vehicle. Compared with the non-diabetic control mice, the diabetic mice exhibited delayed wound healing that was characterized by elevated accumulation of AGEs, increased TNF-α level and macrophage infiltration, and decreased fibroblast number and collagen deposition at the late stage of repair. Besides, macrophages of diabetic wounds showed expression of α7nAChR. During late repair, PNU282987 treatment of diabetic mice significantly reduced the level of TNF-α, accelerated wound healing, and elevated fibroblast number and collagen deposition. To investigate the cellular mechanism of these observations, RAW 264.7 cells, a macrophage cell line, were incubated with AGEs in the presence or absence of PNU282987. TNF-α production from AGE-stimulated macrophages was significantly decreased by PNU282987 in a dose-dependent manner. Furthermore, PNU282987 significantly inhibited AGE-induced nuclear factor-κB (NF-κB) activation and receptor for AGE (RAGE) expression. These results strongly suggest that activating α7nAChR can promote diabetic wound healing by suppressing AGE-induced TNF-α production, which may be closely associated with the blockage of NF-κB activation in macrophages.

Curcumin inhibits apoptosis and brain edema induced by hypoxia-hypercapnia brain damage in rat models.

Curcumin, extracted from South Asian spice turmeric, has been determined to have the promising ability in antioxidation and anti-inflammation. However, the effect of curcumin on treating brain damage has been not reported. In this article, the aim was to evaluate the effect of curcumin on cell apoptosis in rats exposed to hypoxia-hypercapnia and explore the therapeutic potential of curcumin in hypoxia-hypercapnia brain damage (HHBD). Sprague Dawley rats were randomly assigned into 3 groups: control group, hypoxia-hypercapnia group and curcumin group. The Fas/FasL expressions in HHBD rats treated by curcumin were measured by immunohistochemical staining and western blotting. The pathological changes of brain cells were observed by transmission electron microscope. Rats with HHBD showed significant increase of Fas/FasL expression and ultrastructural changes in brain tissue cells. Curcumin intervention effectively reversed the Fas/FasL-mediated apoptosis and HHBD-induced brain edema. Curcumin may be a potential therapeutic alternative for HHBD.

Analysis of Forensic Autopsy in 120 Cases of Medical Disputes Among Different Levels of Institutional Settings.

Despite advances in medical science, the causes of death can sometimes only be determined by pathologists after a complete autopsy. Few studies have investigated the importance of forensic autopsy in medically disputed cases among different levels of institutional settings. Our study aimed to analyze forensic autopsy in 120 cases of medical disputes among five levels of institutional settings between 2001 and 2012 in Wenzhou, China. The results showed an overall concordance rate of 55%. Of the 39% of clinically missed diagnosis, cardiovascular pathology comprises 55.32%, while respiratory pathology accounts for the remaining 44. 68%. Factors that increase the likelihood of missed diagnoses were private clinics, community settings, and county hospitals. These results support that autopsy remains an important tool in establishing causes of death in medically disputed case, which may directly determine or exclude the fault of medical care and therefore in helping in resolving these cases.

[Progression of the manner of cardiomyocyte death and its potential significance in forensic medicine].

The manner of cell death is a hotspot of medical researchers. Apoptosis and necrosis were considered as two manners of cell death in the past. But recently a new manner of cell death--oncosis is gradually accepted by the pathologists. Oncosis is different from apoptosis in morphologic, mechanism and the role in cardiovascular diseases. In this paper, the progression of the research about manner of the cardiomyocyte death and its significance in forensic medicine in recent years was reviewed.

A continuous cell line, SYSU-OfHe-C, from hemocytes of Ostrinia furnacalis possesses immune ability depending on the presence of larval plasma.

A continuous cell line, SYSU-OfHe-C, from larval hemocytes of corn borer, Ostrinia furnacalis was established. With increasing passages, the cells grew increasingly faster, and approximately 45% of the cells were in division at passage 55. The culture was mainly composed of two types of cells, granulocytes and plasmatocytes, which showed different division and proliferation behaviors, but possessed similar phagocytic ability. Its spreading ability was significantly weaker than that of hemocytes from naïve larva; however, it could be promoted by larval plasma. Furthermore, its encapsulation ability was also promoted by larval plasma to form multilayer capsules on Sephadex A-25 beads. Finally, the expression of several immune-related genes was verified after provocation by microbes or Sephadex beads. These results indicated that the cell line possessed immune ability depending on the presence of plasma of naïve larvae and are beneficial to studies of insect cellular systems.

Time-dependent expression and distribution of Egr-1 during skeletal muscle wound healing in rats.

Recent studies have shown that early growth response factor-1 (Egr-1) plays an important role in regulation of inflammation and tissue repair, but little is known about its expression after trauma to skeletal muscles. A preliminary study on time-dependent expression and distribution of Egr-1 was performed by immunohistochemistry, immunofluorescence and Western blotting during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 45 Sprague-Dawley male rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days and 21 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. In the uninjured controls, Egr-1 positive staining was observed in the sarcoplasm and nuclei of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells (PMNs), a number of mononuclear cells (MNCs), fibroblastic cells (FBCs) and regenerated multinucleated myotubes showed positive reaction for Egr-1 in contused zones. By morphometric analysis, an increase in Egr-1 expression was verified at inflammatory phase after contusion, which reached a peak in the regenerated phase overlapping with the fibrotic phase during skeletal muscle wound healing. The expression tendency was further confirmed by Western blotting assay. By immunofluorescent staining for co-localization, the Egr-1-positive MNCs and FBCs in wounds were identified as macrophages and myofibroblasts. The results demonstrate that the expression of Egr-1 is up-regulated and temporally distributed in certain cell types after trauma to skeletal muscles, which may be closely involved in inflammatory response, fibrotic repair and muscle regeneration during skeletal muscle wound healing.