Ziqin Zhao

Autophagy is involved in traumatic brain injury-induced cell death and contributes to functional outcome deficits in mice

Previous data demonstrate that traumatic brain injury (TBI) activates autophagy, and increases microtubule-associated protein 1 light chain 3 (LC3) immunostaining mainly in neurons. However, the role of autophagy in traumatic brain damage remains elusive. The aim of the present study was to investigate the autophagic mechanisms participating in traumatic brain injury. The autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (BFA) were administered with a single i.c.v. injection before TBI. We first examined the protein levels of Beclin-1 and LC3 II, which have been found to promote autophagy previously. Immunoblotting analysis showed that 3-MA pretreatment reduced post-TBI Beclin-1 and LC3-II levels, and maintained p62/SQSTM1 (p62) levels. In addition, double immunolabeling showed that the increased punctate LC3-II dots colocalizing with Propidium Iodide (PI)-stained nuclei at 24 h after injury, were partially inhibited by 3-MA pretreatment. Furthermore, inhibition of autophagy could reduce TBI-induced cell injury assessed with i.p. injection of PI and lesion volume, and attenuate behavioral outcome evaluated by motor test and Morris water maze. The neuroprotective effects were associated with an inhibition on TBI-induced up-regulation of LC3, Beclin-1, cathepsin B, caspase-3 and the Beclin-1/Bcl-2 ratio. Taken together, these data imply that the autophagy pathway is involved in the pathophysiologic responses after TBI, and inhibition of this pathway may help attenuate traumatic damage and functional outcome deficits.

Anaphylactic deaths in Maryland (United States) and Shanghai (China): A review of forensic autopsy cases from 2004 to 2006

Anaphylaxis is a life threatening allergic reaction that is rapid in onset and may cause sudden death. A retrospective study was performed using the database from the Office of the Chief Medical Examiner (OCME) in the State of Maryland and the Department of Forensic Medicine in Shanghai Medical College (DFM-SMC) to examine the etiology and forensic investigation findings of anaphylactic deaths from 2004 to 2006. Details of the medical history, agent responsible for the allergic reaction, death scene investigation and postmortem examination findings were reviewed for all cases. A total of 28 cases of anaphylactic death were identified for the study period, 17 from Maryland and 11 from Shanghai. Of the 17 Maryland cases, 6 (35%) involved allergic reaction to food, 5 (25%) to drugs, 2 to bee stings, 1 to hair dye, and 3 to unknown allergens. Investigation revealed that 9 of 17 cases had a history of asthma and 8 had previous allergic reactions to certain foods and / or drugs. In Shanghai, all 11 deaths resulted from anaphylactic reaction to antibiotics, 10 of which occurred in clinics illegally operated by unlicensed physicians. The interval between the onset of symptoms and death ranged from less than 1 min to 3 days after initially contacting the allergen. In the majority (68%) of cases, death occurred within 8h after the onset of symptoms. Postmortem findings were relatively non-specific, and included pharyngeal/laryngeal edema 14/28 (50%), mucus plugging in the airways 11/28 (39%), and pulmonary congestion and edema 28/28 (100%). Serum tryptase concentrations were measured in 14 cases in Maryland and the concentration ranged from 3.3 ng/ml to 200 ng/ml. There were significant differences with regards to allergen type and the circumstances of death between these two regions. Inappropriate use of antibiotics and illegal medical practices were the main causes of identified anaphylactic death in Shanghai. In Maryland anaphylactic deaths were mainly caused by food reactions. This study indicates that the postmortem diagnosis of anaphylactic death is usually based on exclusion and circumstantial evidence. Knowledge of the patients history and circumstances of death is of major importance for the forensic pathologist when investigating suspected anaphylactic death.

Effects of RNA interference-mediated gene silencing of JMJD2A on human breast cancer cell line MDA-MB-231 in vitro

Previous data demonstrate that JMJD2A is a cancer-associated gene and may be involved in human breast cancer by demethylation of H3K9me3. The aim of this study was to investigate depressive effects on JMJD2A by transfection with JMJD2A-sepcific siRNA in human breast cancer cell line MDA-MB-231 and effects on cell proliferation, invasion and migration. JMJD2A-specific siRNA was chemically synthesised and transfected into human breast cancer cell line MDA-MB-231. Expression levels of JMJD2A were detected by quantitative real-time PCR and Western blot analysis. Cells proliferation was evaluated by using flow cytometric anlysis and MTT assay. The abilities of invasion and migration were evaluated by cell migration and invasion assay with Boyden chambers. The results showed that the transfection was successful and expression levels of JMJD2A mRNA and protein in siRNA group were both down-regulated. By MTT assay, the mean actual absorbance in siRNA group was significantly lower than that in blank control group (P < 0.05) and negative control group (P < 0.05). In addition, the percentage of cells in G0/G1 phase in siRNA group was significantly more than that in blank control group (P < 0.05) and negative control group (P < 0.05). Furthermore, by cell invasion and migration assay, the decreased number of migrated cells in siRNA group was observed (P < 0.05). These data imply that silencing JMJD2A gene could result in cell cycle change and proliferation inhibition, and lead to suppress tumor cell invasion and migration. It provides a new perspective in understanding the pleiotropic functions of JMJD2A and its contribution to human breast cancer.

Lipoxin A4 attenuates brain damage and downregulates the production of pro-inflammatory cytokines and phosphorylated mitogen-activated protein kinases in a mouse model of traumatic brain injury.

The present study was designed to investigate the effects of lipoxin A4 (LXA4) on traumatic brain injury (TBI) and to analyze the possible mechanism. Outcome parameters consist of blood-brain barrier (BBB) breakdown, brain edema and lesion volume. Using western blot and quantitative real-time PCR, we examined the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and activation of mitogen-activated protein kinases (MAPKs) (including ERK, JNK, p38) following TBI. To investigate the cell types in which the LXA4 receptor (ALXR) staining was localized, brain sections pulsed with ALXR were subjected to immunofluorescence staining with antibodies against cell type-specific antigens. Our findings show that LXA4 decreases BBB permeability, attenuates brain edema, and reduces TBI-induced lesion volume. In addition, LXA4 inhibits TBI-induced elevation of mRNA and protein levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). In the injured cortex at 24h post-TBI, the phosphorylated-ERK (p-ERK) and -JNK (p-JNK), but not -p38 (p-p38) levels were increased. The p-ERK and p-JNK production were attenuated by their respective inhibitors (PD98059 and SP600125), as well as LXA4. Moreover, ALXR was found to label more GFAP positive cells, whereas few CD11b-positive cells were labeled by ALXR within the layers of the injured cortex at 24h post-TBI. The activation of ALXR in astrocytes was partially enhanced by LXA4 treatment. Taken together, these data indicate that TBI activates pro-inflammatory cytokines, the MAPK pathways together with ALXR in astrocytes, and these mechanisms may be exploited by pharmacological interventions.

Poloxamer 188 Attenuates in vitro Traumatic Brain Injury-Induced Mitochondrial and Lysosomal Membrane Permeabilization Damage in Cultured Primary Neurons

Acute membrane damage due to traumatic brain injury (TBI) is a critical precipitating event. However, the subsequent effects of the mechanical trauma, including mitochondrial and lysosomal membrane permeability (MOMP and LMP) remain elusive. The main objective of the current study was to assess the role of a putative membrane-resealing agent poloxamer 188 (P188) in MOMP and LMP in response to a well-defined mechanical insult. Using an in vitro cell shearing device (VCSD), mechanical injury resulted in immediate disruption of membrane integrity in cultured primary neurons, and neurons were treated with P188 or a cathepsin B inhibitor (CBI) after VCSD 10 min. The protective effect of P188 on cultured primary neurons was first detected visually with a light microscope, and measured by MTT assay and LDH assay. The validity of monitoring changes in mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining, and Western blot for cytochrome c and truncated Bid (tBid) in purified mitochondria was also performed. In addition, lysosomal integrity was detected by blotting for cathepsin B and tBid in purified lysosomes. Our results showed post-injury P188 treatment moderated the dissipation of ΔΨm in mitochondria, and inhibited VCSD-induced cytochrome c release from mitochondria as well as cathepsin B from lysosomes. Cathepsin B inhibition (CBI) could also increase cell viability, maintain mitochondrial membrane potential, and repress VCSD-induced release of cytochrome c from mitochondria to cytosol. Both P188 and CBI treatment decreased the cytosolic accumulation of tBid in supernatant of purified lysosomes, and the amount of mitochondrial localized tBid. These data indicate injured neurons have undergone mitochondrial and lysosomal membrane permeability damage, and the mechanism can be exploited with pharmacological interventions. P188s neuroprotection appears to involve a relationship between cathepsin B and tBid-mediated mitochondrial initiation of cell death.

JMJD2A contributes to breast cancer progression through transcriptional repression of the tumor suppressor ARHI

Breast cancer is a worldwide health problem and the leading cause of cancer death among females. We previously identified Jumonji domain containing 2A (JMJD2A) as a critical mediator of breast cancer proliferation, migration and invasion. We now report that JMJD2A could promote breast cancer progression through transcriptional repression of the tumor suppressor aplasia Ras homolog member I (ARHI). Immunohistochemistry was performed to examine protein expressions in 155 cases of breast cancer and 30 non-neoplastic tissues. Spearman correlation analysis was used to analyze the correlation between JMJD2A expression and clinical parameters as well as several tumor regulators in 155 cases of breast cancer. Gene and protein expressions were monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Results from knockdown of JMJD2A, overexpression of JMJD2A, Co-immunoprecipitation (Co-IP) assay, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) elucidated molecular mechanisms of JMJD2A action in breast cancer progression. Furthermore, the effects of ARHI overexpression on JMJD2A-mediated tumor progression were investigated in vitro and in vivo. For in vitro experiments, cell proliferation, wound-healing, migration and invasion were monitored by cell counting, scratch and Boyden Chamber assays. For in vivo experiments, control cells and cells stably expressing JMJD2A alone or together with ARHI were inoculated into mammary fat pads of mice. Tumor volume, tumor weight and metastatic nodules were measured by caliper, electronic balance and nodule counting, respectively. JMJD2A was highly expressed in human breast cancers and positively correlated with tumor progression. Knockdown of JMJD2A increased ARHI expression whereas overexpression of JMJD2A decreased ARHI expression at both protein and mRNA levels. Furthermore, E2Fs and histone deacetylases were involved in the transcriptional repression of ARHI expression by JMJD2A. And the aggressive behavior of JMJD2A in breast cancers could be reversed by re-expression of ARHI in vitro and in vivo. We demonstrated a cancer-promoting effect of JMJD2A and defined a novel molecular pathway contributing to JMJD2A-mediated breast cancer progression.IntroductionBreast cancer is a worldwide health problem and the leading cause of cancer death among females. We previously identified Jumonji domain containing 2A (JMJD2A) as a critical mediator of breast cancer proliferation, migration and invasion. We now report that JMJD2A could promote breast cancer progression through transcriptional repression of the tumor suppressor aplasia Ras homolog member I (ARHI).MethodsImmunohistochemistry was performed to examine protein expressions in 155 cases of breast cancer and 30 non-neoplastic tissues. Spearman correlation analysis was used to analyze the correlation between JMJD2A expression and clinical parameters as well as several tumor regulators in 155 cases of breast cancer. Gene and protein expressions were monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Results from knockdown of JMJD2A, overexpression of JMJD2A, Co-immunoprecipitation (Co-IP) assay, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) elucidated molecular mechanisms of JMJD2A action in breast cancer progression. Furthermore, the effects of ARHI overexpression on JMJD2A-mediated tumor progression were investigated in vitro and in vivo. For in vitro experiments, cell proliferation, wound-healing, migration and invasion were monitored by cell counting, scratch and Boyden Chamber assays. For in vivo experiments, control cells and cells stably expressing JMJD2A alone or together with ARHI were inoculated into mammary fat pads of mice. Tumor volume, tumor weight and metastatic nodules were measured by caliper, electronic balance and nodule counting, respectively.ResultsJMJD2A was highly expressed in human breast cancers and positively correlated with tumor progression. Knockdown of JMJD2A increased ARHI expression whereas overexpression of JMJD2A decreased ARHI expression at both protein and mRNA levels. Furthermore, E2Fs and histone deacetylases were involved in the transcriptional repression of ARHI expression by JMJD2A. And the aggressive behavior of JMJD2A in breast cancers could be reversed by re-expression of ARHI in vitro and in vivo.ConclusionWe demonstrated a cancer-promoting effect of JMJD2A and defined a novel molecular pathway contributing to JMJD2A-mediated breast cancer progression.

Distribution of biventricular disease in arrhythmogenic cardiomyopathy: an autopsy study

Arrhythmogenic cardiomyopathy is a rare cardiomyopathy characterized by fibrofatty replacement primarily of the right ventricular myocardium. It is a major cause of sudden death in the young and in athletes. There are few autopsy studies of the ventricular distribution of the disease. Fifty cases of sudden cardiac death with fibrofatty replacement in either ventricle from a single medical examiners office were studied. Distribution of disease as determined grossly and microscopically was correlated with activity at time of death, race, and presence of inflammation. Extent of disease was right ventricular in 6 cases (12%; age, 25 ± 5 years), biventricular in 25 (50%; age, 36 ± 3 years), and left ventricular in 19 (38%; age, 37 ± 3 years) (P = .13). Inflammation was present in 44% of biventricular arrhythmogenic cardiomyopathy versus 74% of left ventricular arrhythmogenic cardiomyopathy and 83% of right ventricular arrhythmogenic cardiomyopathy (P = .06). Arrhythmogenic cardiomyopathy, when presenting with sudden death, is usually biventricular. There is a trend that univentricular involvement occurs at an earlier age and that right ventricular involvement shows more inflammation, suggesting different stages of disease.

Effects of siRNA-mediated knockdown of jumonji domain containing 2A on proliferation, migration and invasion of the human breast cancer cell line MCF-7

Jumonji domain containing 2A (JMJD2A) is a potential cancer-associated gene that may be involved in human breast cancer. The present study aimed to investigate suppressive effects on the MCF-7 human breast cancer cell line by transfection with JMJD2A-specific siRNA. Quantitative real-time PCR and western blot analysis were used to detect the expression levels of JMJD2A. Flow cytometric (FCM) analysis and WST-8 assay were used to evaluate cell proliferation. Boyden chambers were used in cell migration and invasion assays to evaluate the cell exercise capacity. Expression levels of JMJD2A mRNA and protein in the siRNA group were both downregulated successfully by transfection. FCM results showed that the percentage of cells in the G0/G1 phase in the siRNA group was significantly greater than that in the blank (P<0.05) and negative control groups (P<0.05). Additionally, the mean absorbance in the siRNA group was significantly lower (P<0.05), as observed by WST-8 assay. Moreover, a decreased number of migrated cells in the siRNA group was observed (P<0.05) using a cell migration and invasion assay. These data indicated that knockdown of JMJD2A may cause inhibition of proliferation, migration and invasion of MCF-7 cells. This study provides a new perspective in understanding the molecular mechanisms underlying the progression of breast cancer and offers a potential therapeutic target for breast cancer.

Development of 11 X-STR loci typing system and genetic analysis in Tibetan and Northern Han populations from China

X-chromosomal short tandem repeats (X-STRs) loci are used for forensic practice in recent years which play increasingly important roles in some complex kinship cases. In this paper, a new multiplex polymerase chain reaction (PCR) system which can simultaneously analyze 11 X-STR markers (DXS8378, DXS6795, DXS7132, DXS6803, DXS9898, DXS6801, DXS7133, GATA165B12, HPRTB, DXS8377 and DXS7423) was developed. The samples of 1,605 (742 males and 863 females) unrelated individuals from Tibetan and Northern Han population were successfully analyzed using this multiplex system. A total of 103 alleles for all the loci were observed. Hardy–Weinberg equilibrium tests demonstrated no significant deviation from expected values (P > 0.05) for all of the 11 X-STR loci in the two studied populations. Polymorphism information contents of the loci were 0.3864–0.9013, and powers of discrimination in females of the loci were 0.6317–0.9845. There were no statistically significant differences between Tibetan and Northern Han populations in allele distribution of the 11 X-STR loci, in line with analysis of molecular variance (AMOVA) results. Our work indicates that this multiplex system is useful for forensic analysis for the two populations in China.

Bisulfite genomic sequencing of DNA from dried blood spot microvolume samples

DNA methylation is an important event in epigenetic changes in cells, and a fundamental regulator of gene transcription. Bisulfite genomic sequencing is a powerful technique used in studies of DNA methylation. However, the established procedures often require relatively large amounts of DNA. In everyday practice, samples submitted for analysis might contain very small amounts of poor quality material, as is often the case with forensic stain samples. In this study, we assess a modified, more efficient method of bisulfite genomic sequencing. Genomic DNA extracted from 3-mm dried blood spots using QIAamp micro kit was treated with sodium bisulfite (using EpiTect kit). Subsequent methylation-specific PCR (MSP) followed by DNA sequencing displayed the differentially methylated region of imprinted gene SNRPN. Our results show that this new combination of efficient DNA extraction and bisulfite treatment provides high quality conversion of unmethylated cytosine to uracil for bisulfite genomic sequencing analysis. This reliable method substantially improves the DNA methylation analysis of forensic stain samples.