Alpha-kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway

BACKGROUND: Alpha-kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1ß in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1ß and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined. METHODS: Immunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1ß, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1ß and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD. RESULTS: The heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68+ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1ß in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1ß, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA. CONCLUSIONS: Our data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom / 法医学杂志

OBJECTIVES@#To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method.@*METHODS@#The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed.@*RESULTS@#When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05).@*CONCLUSIONS@#The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Application of Quantitative Analysis of Diatoms in Lung Tissue for the Diagnosis of Drowning of Experimental Animals / 法医学杂志

Objective To discuss the application value of diatom examination in lung tissue for the forensic diagnosis of drowning. Methods The experimental animals were divided randomly into drowning, postmortem submergence and dying on land group. Diatoms in lung tissue and drowning fluid were analyzed quantitatively by microwave digestion-vacuum filtration-automated scanning electron microscopy diatom examination method. The ratios of content of diatoms in lung tissue and drowning fluid （CL/CD ratio） were recorded. Results The CL/CD ratios of experimental rabbits in the drowning group （5.82±3.50） were much higher than that of postmortem submergence group （0.47±0.35）; the CL/CD ratios of different parts of the lung lobes of experimental pigs in the drowning group were higher than that of postmortem submergence group （P<0.05）; in seawater, brackish water, river fresh water and lake fresh water, the CL/CD ratios of experimental pigs in the drowning group were higher than that of postmortem submergence group （P<0.05）. In animal experiments, all the cases with CL/CD ratio >1.6 were from drowning group. Conclusion CL/CD ratio is an indicator with good application prospects in the diagnosis of drowning.

Performed titanium locking plate versus nickel-titanium memory alloy embracing fixator in the treatment of multiple rib fractures / 中国胸心血管外科临床杂志

@#Objective     To compare clinical outcomes between the performed titanium locking plate and nickel-titanium memory alloy embracing fixator for the treatment of multiple rib fractures, and to select a better internal fixator for multiple rib fractures. Methods     A total of 206 consecutive patients with multiple rib fractures were admitted to Department of Cardiothoracic Surgery in Beijing Luhe Hospital of Capital Medical University from October 2011 to September 2016. According to different treatment strategies, the patients were divided into 2 groups: a performed titanium locking plate group (a titanium plate group, n=105) and a nickel-titanium memory alloy embracing fixator group (an embracing fixator group, n=101). There were 82 males and 23 females with a mean age of 46.5±9.7 years ranging from 23 to 65 years in the titanium plate group, and 83 males and 18 females with a mean age of 44.7±10.3 years ranging from 19 to 63 years in the embracing fixator group. The preoperative data, curative outcomes, visual analogue scale (VAS) and postoperative complications were compared between the two groups. Results     There was no statistical difference in the preoperative data between the two groups, and all patients successfully completed the operation. Compared with the embracing fixator group, the incision length and operation time were shorter, intraoperative bleeding and VAS score were less, and curative outcome was better in the titanium plate group. Conclusion     The performed titanium locking plate has a great advantage in the clinic, which can be preferred.

Research Progress on Determination of Drowning Site / 法医学杂志

The bodies found in water are one of the most common types in forensic practice. The dis-covery site of the body is often not the drowning site. However, the determination of drowning site is vital for the identification of victim. Inorganic particles and planktons, such as granular impurities, diatoms and bacteria, are valuable markers for the diagnosis of drowning. By comparing the granular impurities and planktons in tissues and suspicious drowning mediums, the drowning site can be concluded based on their similarity of types and distribution, which has practical applied value. In this paper, the research progress on determination of drowning site is summarized to provide reference for the peers.

Clinicopathological features of gastric mixed adenoneuroendocrine carcinoma / 中国肿瘤临床

Objective:To explore the clinicopathologic characteristics of gastric mixed adenoneuroendocrine carcinoma (MANEC). Methods:From January 2011 to December 2016, the clinical and pathological data of 35 patients with gastric MANEC who were diagnosed and surgically treated in Tianjin Medical University Cancer Institute and Hospital were analyzed retrospectively. Results:The average age of the 32 men and 3 women in this study was 61.6±7.5 years. Tumor locations were as follows:17 related to gastric cardia, 9 related to gastric body, 8 related to gastric antrum, and 1 related to gastric stump. Clinical symptoms were non-specific and the diagnosis relies on post-operative pathological examination. Using the histological microscope, the affected structures were detected in neuroendocrine systems and tissue linings. Immunohistochemical staining showed that carcinoembryonic antigen (CEA) and cytokeratins 8 and 18 (CK8/18) were expressed in 32 and 33 adenocarcinoma-related cases, respectively, whereas synaptophysin (Syn) and chromogranin A (CgA) were revealed in 33 and 27 neuroendocrine-related cases, respectively. Al patients received surgical resection. A total of 17 incidents of death were reported at three years after the operation, and most of the patients were at clinical stageⅢorⅣ. Conclusion:Gastric MANEC is a rare neoplasm and is often diagnosed at its advanced stage and mainly occurs in the aged population. Neuroendocrine structures and glandular tissues are the most frequent location of such condition. Diagnosis relies on both immunohistochemical and histological examinations. Surgical resection is the most effective treatment, but the prognosis of this condition remains poor.

Value of specific 16S rDNA fragment of algae in diagnosis of drowning: an experiment with rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for amplifying specific 16S rDNA fragment of algae related with drowning and test its value in drowning diagnosis.</p><p><b>METHODS</b>Thirty-five rabbits were randomly divided into 3 the drowning group (n=15), postmortem water immersion group (n=15, subjected to air embolism before seawater immersion), and control group(n=5, with air embolism only). Twenty samples of the liver tissues from human corpses found in water were also used, including 14 diatom-positive and 6 diatom-negative samples identified by microwave digestion-vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM). Seven known species of algae served as the control algae (Melosira sp, Nitzschia sp, Synedra sp, Navicula sp, Microcystis sp, Cyclotella meneghiniana, and Chlorella sp). The total DNA was extracted from the tissues and algae to amplify the specific fragment of algae followed by 8% polyacrylamide gelelectrophoresis and sliver-staining.</p><p><b>RESULTS</b>In the drowning group, algae was detected in the lungs (100%), liver (86%), and kidney (86%); algae was detected in the lungs in 2 rabbits in the postmortem group (13%) and none in the control group. The positivity rates of algae were significantly higher in the drowning group than in the postmortem group (P<0.05). Of the 20 tissue samples from human corps found in water, 15 were found positive for algae, including sample that had been identified as diatom-negative by MD-VF-Auto SEM. All the 7 control algae samples yielded positive results in PCR.</p><p><b>CONCLUSIONS</b>The PCR-based method has a high sensitivity in algae detection for drowning diagnosis and allows simultaneous detection of multiple algae species related with drowning.</p>

Clinical analysis on 9 cases of nervous system disorder caused by Heat stroke / 中国神经精神疾病杂志

Objective To summarize the clinical characteristics , nerve electrophysiological features , imaging manifestations and prognosis of nervous system disorder caused by heat stroke. Methods A retrospective study was un?dertaken on clinical, nerve electrophysiological and imaging characteristics and prognosis of 9 patients who had heat Stroke and received treatment during July and August in 2010 and 2013 at the Second Hospital of Shandong University. Results There were seven males and two females, aged from 47 to 87 years, among 9 patients. All cases are admitted within 6 hours and their Core body temperature were 39.8℃~42.5℃. Patients had disturbance of consciousness and multi?ple organ dysfunction including acute cerebral infarct in 2 cases, peripheral neuropathy in 3 cases, acute respiratory fail?ure in 2 cases, pulmonary infection in 9 cases, disseminated intravascular coagulation in 1 case ,acute myocardial injury in 8 cases, arrhythmia in 3 cases, rhabdomyolysis in 3 cases, acute liver damage in 8 cases, of acute kidney injury in 6 cases, upper gastrointestinal bleeding in 4 cases, metabolic acidosis in 1 case, metabolic alkalosis in 2 cases , electrolyte disorder in 9 cases and quadriplegia in 3 cases. Brain magnetic resonance imaging showed diffuse brain swelling in 6 cas?es and the acute asymmetric lacunar infarction in 2 cases. All patients are treated with cooling , rehydration , anticoagula?tion , reduction of the intracranial pressure and symptomatic treatment. One case died and the condition of remaining cas?es were improved. At one-year follow-up, the symptoms were almost cured in 2 cases, significantly improved in 4 cases with disability degree in grade 1~2, moderately improved in 1 case with disability degree in grade 3, remained unchanged in 1 case with disability degree of grade 5. Conclusion Heat stroke can cause conscious disorder and multiple organ fail?ure .The central nervous system lesions are often presented with diffuse swelling or/and lacunar infarction and presented with peripheral nerve injury in some cases, leaving persistent neurological deficits .

Diatoms in drowned and postmortem immersed rabbits' lungs / 法医学杂志

OBJECTIVE@#To investigate the numbers, sizes and types distribution of diatoms in drowned and postmortem immersed rabbits' lungs.@*METHODS@#Sixty-two rabbits were randomly divided into drowning group (n = 30), postmortem immersion group (n = 30) and land death group (n=2), and the diatoms in each lung lobe were analyzed quantitatively and qualitatively by microwave digestion and scanning electron microscopy.@*RESULTS@#In the drowning group, the diatoms were detected in each lung lobe with Cyclotella and Melosira in the majority. In the postmortem immersion group, Cyclotella was in the majority. And the diatoms weren't detected in some lung lobes in postmortem immersion. There were significant differences in the detection rates of upper lobe of left lung, middle lobe and cardiac lobe of right lung in two groups (P < 0.05).@*CONCLUSION@#Based on the microwave digestion and scanning electron microscopy, the numbers, sizes and types distribution of diatoms in drowned and postmortem immersed rabbits' lungs can be analyzed and used as references for testing theory.

Purification and characterization of 6-phosphogluconate dehydrogenase (6-PGD) from grass carp (Ctenopharyngodon idella) hepatopancreas.

6-Phosphogluconate dehydrogenase (6-PGD, E.C.: 1.1.1.44) was purified and characterized from the hepatopancreas of grass carp (Ctenopharyngodon idella) for the first time. Grass carp represents the second largest aquaculture industry in the world after silver carp, constituting 14.7% of the world aquaculture production, with an average annual increase of 14% in China, mainly as a source of food. The purification procedure involved a single 2’, 5’-ADP-Sepharose 4B affinity chromatographic step by using different elution buffers. The enzyme was purified 309-fold with a specific activity of 5.259 U/mg protein and yield of 68%. The purity and subunit molecular weights of the 6-PGD were checked on SDS-PAGE and purified enzyme showed a single band on the gel. The subunit molecular mass was 57 kDa, with an optimum pH, temperature and ionic strength at 7.96, 50oC and 100 mM Tris-HCl, respectively. The Km values of 6-PGA and NADP+ were 0.019 and 0.0052 mM, respectively, while Vm of 6-PGA and NADP+ was 0.69 U/ml. Dissociation constants (Ki) for 6-PGA and NADP+ were 2.05 and 0.12 mM, respectively. NADPH inhibited the enzyme in a competitive manner and its Ki value was 0.032 mM. The Cu2+, Zn2+, Cd2+ and Al3+ showed inhibitory effects on the enzyme with IC50 values of 0.293, 0.099, 0.045 and 1.526 mM, respectively. All tested metals inhibited the enzyme in a competitive manner, indicating that these metals might be toxic even at low concentrations for the 6-PGD. As the fish is one of valuable foodstuff of animal sources for human consumption, under certain environmental conditions, metal ions accumulated in fish up to a lethal concentration may be harmful for human health. Therefore, it is impending to reduce the concentration of metal ions in contaminated lakes and rivers for fishery and also for human health.

Production of marker-free and RSV-resistant transgenic rice using a twin T-DNA system and RNAi.

A twin T-DNA system is a convenient strategy for creating selectable marker-free transgenic plants. The standard transformation plasmid, pCAMBIA 1300, was modified into a binary vector consisting of two separate T-DNAs, one of which contained the hygromycin phosphotransferase (hpt) marker gene. Using this binary vector, we constructed two vectors that expressed inverted-repeat (IR) structures targeting the rice stripe virus (RSV) coat protein (CP) gene and the special-disease protein (SP) gene. Transgenic rice lines were obtained via Agrobacterium-mediated transformation. Seven independent clones harbouring both the hpt marker gene and the target genes (RSV CP or SP) were obtained in the primary transformants of pDTRSVCP and pDTRSVSP, respectively. The segregation frequencies of the target gene and the marker gene in the T1 plants were 8.72% for pDTRSVCP and 12.33% for pDTRSVSP. Two of the pDTRSVCP lines and three pDTRSVSP lines harbouring the homozygous target gene, but not the hpt gene, were strongly resistant to RSV. A molecular analysis of the resistant transgenic plants confirmed the stable integration and expression of the target genes. The resistant transgenic plants displayed lower levels of the transgene transcripts and specific small interfering RNAs, suggesting that RNAi induced the viral resistance.

Progress in application of microbeam X-ray fluorescence spectroscopy in forensic science / 法医学杂志

Microbeam X-ray fluorescence (micro-XRF) spectrometry has been raised as an analytical technique of microbeam during the recent years. With its advantages of high sensitivity, small sample requirement, high testing accuracy and non-destruction, the technique is widely utilized in forensic science. This review bases on recent researches at home and abroad, describes its applications including identification of gunshot residue, visualization of fingerprints, discrimination of drug source, production process, and other material evidences of analysis in crime scene. Thanks to the advances in technology, intelligent and portable micro-XRF equipment has appeared to be applied. It is believed that it may be more popular and frequent in administration of forensic science in the near future.

Functional polymorphisms in CYP2C19 & CYP3A5 genes associated with decreased susceptibility for paediatric tuberculosis.

Background & objectives: Tuberculosis (TB) bacilli ingested by macrophages evade host immune responses by multiple mechanisms including the inhibition of apoptosis. As the cytochrome-P-450 system (CYP) contributes to apoptosis it has been suggested that genetic variation in CYP may be associated with susceptibility to TB infection. This study was carried out to evaluate cytochrome P-450 polymorphisms in Chinese Han children and to investigate the effect of these polymorphisms in paediatric TB. Methods: Frequencies for the CYP2C19, CYP3A4, CYP3A5 and CYP2E1 mutated alleles and genotypes were compared between 142 Chinese paediatric TB patients and 150 non-infected controls by real time PCR genotyping on peripheral leukocyte DNA. Results: CYP2C19 (636 G>A, rs4986893) A allele and AG genotype were associated with decreased susceptibility to TB (P = 0.006, OR= 0.33, 95% CI: 0.15-0.76; and P = 0.005, OR =0.31, 95% CI: 0.14-0.72 respectively), as were the CYP3A5 (6986A>G, rs776746) G allele and particularly homozygous GG (recessive mode) genotype (P = 0.004, OR=0.61, 95% CI: 0.43-0.85; and P=0.002, OR=0.47, 95% CI: 0.29-0.76). Interpretation & conclusions: The data suggested that CYP2C19 and CYP3A5 polymorphisms affect susceptibility to paediatric TB. Further studies are indicated to confirm and elucidate these observations.

Determination of acid-resistant silicic particles in lung by micro-XRF and its application in diagnosis of drowning / 法医学杂志

OBJECTIVE@#Using microbeam X-ray fluorescence (Micro-XRF) analyzer for determination of acid-resistant silicic particles in lung, and to explore its potential application in diagnosis of drowning.@*METHODS@#Thirty two white rabbits were divided randomly into drowning group (n=12), post-mortem immersion group (n=10) and control group (n=10). Lungs and water sample were collected for determination of area concentration of acid-resistant silicic particles using Micro-XRF method.@*RESULTS@#The area concentration of acid-resistant silicic particles for the drowning water sample was 4.4 mm2/mL. For the lungs of drowning group, the post-mortem immersion group and the control group, the determined average values were (25.30 +/- 10.95) mm2/g, (1.68 +/- 0.63) mm2/g and (1.65 +/- 0.85) mm2/g, respectively, with a statistically significant difference between the drowning group and the other two groups.@*CONCLUSION@#The area concentration of acid-resistant silicic particles in lungs may be used as an indicator of drowning. The method is highly sensitive and rapid. It provides a potential application in drowning diagnosis.