[Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells].

OBJECTIVE: To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs. RESULTS: The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs. CONCLUSION: lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

[Application of diffusion weighted imaging in accurate diagnosis of middle ear cholesteatoma].

Objective: To evaluate the accuracy of diffusion weighted magnetic resonance imaging (DWI-MRI) combined with high resolution temporal bone CT (HRCT) in the location diagnosis of middle ear cholesteatoma and its value in the postoperative follow-up. Methods: 134 patients with inital cholesteatoma and 22 patients with suspected recurrent cholesteatoma were selected for HRCT, conventional MRI and DWI examination. Based on the intraoperative and pathological diagnosis, DWI and HRCT images were combined to evaluate the consistency between the lesion location and invasion area of the initial cholesteatoma and intraoperative lesions. The results of HRCT and DWI in the diagnosis of recurrent middle ear cholesteatoma were statistically analyzed to evaluate their diagnostic efficacy. Results: The accuracy rate of DWI combined with HRCT was 90.3%.The sensitivity, specificity, positive predictive value and negative predictive value of HRCT and DWI in the diagnosis of recurrent middle ear cholesteatoma were 27.8%, 75.0%, 83.3%, 18.8% and 100%, 75.0%, 94.7% and 100%, respectively, and the Kappa values consistent with the pathological results were 0.024 and 0.843, respectively. Chi-square test confirmed that there were differences in the diagnosis between groups (P<0.001). Conclusions: Combined with the high sensitivity of DWI and the high resolution of HRCT, the accuracy of preoperative positioning of the newly diagnosed cholesteatoma can be improved and surgery strategy can be guided. DWI is also of high diagnostic value for recurrent cholesteatoma in the middle ear.

[Cognitive function and cerebral blood perfusion changes in patients with anti-N-methyl-D-aspartate receptor encephalitis].

Objective: To investigated cognitive and cerebral blood flow changes in a cohort of patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. Methods: Fifteen patients with confirmed anti-NMDAR encephalitis from the First Affiliated Hospital of Anhui Medical University and the First Affiliated Hospital of University of Science and Technology of China between June 2015 and February 2019 were included in the study. Meanwhile, another 15 healthy subjects were selected as controls. All participants underwent neuropsychological tests for assessment of the clinical symptoms and arterial spin labeling (ASL) of magnetic resonance perfusion imaging scan were employed to evaluate cerebral perfusion. Then the relationship between changed cerebral blood perfusion and cognitive function was evaluated. Results: Patients with anti-NMDAR encephalitis showed significantly reduced overall cognitive function (Montreal cognitive assessment (MoCA)) than healthy controls (26.5±2.5 vs 28.6±1.9, P=0.015), they also exhibited poorer performance in immediate memory, recognition, attention, language and executive function than healthy controls (P<0.05). However, no significant differences in clinical symptoms, viability, mental state and mood were observed between the two groups (P>0.05). Patients with anti-NMDAR encephalitis showed significantly reduced total brain cerebral blood flow (CBF) compared with the healthy controls (0.949±0.028 vs 0.953±0.025, P=0.001). Compared with the healthy controls, the patients had decreased CBF in triangular and opercular parts of bilateral inferior frontal gyrus and left central frontal gyrus, however, they showed increased CBF in the left inferior temporal gyrus and left fusiform gyrus (all P<0.05). In NMDAR group, the CBF in left central anterior gyrus was negatively correlated with Stroop's Color Word-word (r=-0.68, P=0.005). The CBF in left spindle gyrus was positively correlated with Stroop's Color Word-word (r=0.62, P=0.015) and completion time of color trail test-A (r=0.81, P<0.001), respectively, however, it was negatively correlated with Chinese Auditory Verbal Learning Test-immediate (r=-0.59, P=0.020) and Verbal Fluency Test-animals (r=-0.58, P=0.024), respectively. Conclusions: Patients with anti-NMDAR encephalitis have prevalent cognitive deficits such as memory, language, attention and executive dysfunction as well as changes in blood perfusion, which may be related to cognitive deficits. Changes in cerebral blood perfusion can be used as indicators for early diagnosis, disease monitoring and prognosis evaluation of anti-NMDAR encephalitis. Improving the cerebral blood flow of patients may promote the recovery of cognition.

Protective Effect of Edaravone Against Aß25-35-Induced Mitochondrial Oxidative Damage in SH-SY5Y Cells.

Amyloid-ß (Aß)-induced oxidative stress plays an important role in the pathogenesis of Alzheimer's disease (AD). Recent studies show that Aß accumulation may lead to mitochondrial oxidative damage. In the present study, we investigated the protective effect of edaravone on mitochondrial damage in SH-SY5Y cells treated with Aß25-35. SH-SY5Y cells were pre-treated with 20, 40 or 80 µM edaravone before treatment with 25 µM Aß25-35. After 24h cell culture, cellular apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), ATP levels and mitochondrial morphology were evaluated. SH-SY5Y cells exposed to Aß25-35 had high levels of apoptosis and ROS; loss of ΔΨm, decreased ATP levels and presence of mitochondrial swelling. However, these effects were significantly inhibited by edaravone pre-treatment. These results indicate that edaravone prevents mitochondria oxidative damage caused by Aß in SH-SY5Y cells, which suggests that it may have potential clinical application in AD therapy.

Functional analysis of female-biased odorant binding protein 6 for volatile and nonvolatile host compounds in Adelphocoris lineolatus (Goeze).

The polyphagous mirid bug Adelphocoris lineolatus relies heavily on olfactory cues to track suitable host plants. Thus, a better understanding of the molecular basis of its olfactory detection could contribute to the development of effective pest management strategies. In the present study, we report the expression profile of the odorant binding protein gene A. lineolatus odorant binding protein 6 (AlinOBP6). Quantitative real-time PCR experiments suggest that AlinOBP6 is female adult antennae-biased. Cellular immunolocalization analyses show that AlinOBP6 is highly expressed in the lymph of both multiporous sensilla basiconica and uniporous sensilla chaetica. A ligand binding analysis showed that recombinant AlinOBP6 not only bound tightly to host plant volatile compounds but also to nonvolatile compounds. Homology modelling and molecular docking analyses confirmed these unusual ligand binding profiles and revealed that the amino acid residues involved in the recognition of volatile and nonvolatile compounds are distinct. The results of our study are the first to suggest that an antenna- and female-biased OBP in an hemipteran insect is expressed in both olfactory and gustatory sensilla as a mechanism to respond to volatile and nonvolatile host compounds. These findings warrant further research into the molecular mechanisms of chemosensation for mirid bugs in responsive to host plant location.

Effects of tumor necrosis factor and E-selectin on coronary artery flow.

OBJECTIVE: The aim of the study is to investigate the correlation between tumor necrosis factor (TNF-α), E-selectin and coronary artery flow following myocardial ischemia-reperfusion model (IR) in Yorkshire pigs. MATERIALS AND METHODS: Establishment of IR model in pigs. Following the injury model, Experiment group was administrated intravenously Shenfu injection solution (SFI, 1 mL/kg). The control group received the same amount of saline. After 30 min of blood reflux, thrombolysis in myocardial infarction frame count (TFC) was recorded following surgery. TNF-α, E-selectin expression was determined by ELISA in the venous sheath, coronary sinus, artery sinus, and proximal segment of the coronary artery. RESULTS: After the blood reflowing, TFC in both groups were upregulated, and TFC increased more than the control group. The difference is statistically significant (p<0.05) at the time of 30 min. TNF-α, E-selectin expression increased after IR. After reperfusion, TNF-α, E-selectin levels further increased and the myocardial injury was aggravated. SFI inhibited inflammation in the experimental group. TNF-α, E-selectin levels at coronary sinus, artery sinus, and distal segment of coronary artery after surgery was positively correlated with TIMI in the experimental group (p<0.05). TNF-α, E-selectin levels significantly increased after reperfusion (p<0.05). CONCLUSIONS: The result demonstrated that TNF-α, E-selectin levels were positively correlated with coronary artery reflow only in the experimental group but not in the control group.

Correlation study between interleukin-6 levels and coronary reflow.

OBJECTIVE: To analyze the correlation between local interleukin-6 (IL-6) levels in different parts of blood vessel and the record of Thrombolysis in Myocardial Infarction (TIMI) frame count (TFC) after myocardial ischemia-reperfusion (IR) model. MATERIALS AND METHODS: Establishment of IR model in Yorkshire pigs, the pigs were divided into two groups (n=6). Experiment group pigs were administrated with Shenfu injection (SF) intravenously (1 mL/kg), control group was given saline injection. The blood reflowed after 30 min. TIMI was recorded to evaluate the coronary blood flow and myocardial perfusion. IL-6 levels in venous sheath, coronary sinus, artery sinus, and proximal coronary artery were determined by ELISA. RESULTS: The records of TIMI in experimental group were higher than that in control group. The difference was statistically significant (p < 0.05). The level of IL-6 increased obviously compared with control group after reperfusion (p < 0.05). Shenfu injection reduced the level of IL-6. IL-6 level at the coronary sinus was positively correlated with TIMI in experimental group (p = 0.03, R2 = 0.97) but not in control group. CONCLUSIONS: IL-6 levels were significantly increased after reperfusion, which aggravated myocardial injury. IL-6 may be associated with coronary reflow, but further study is needed.

Case Report Identification of a novel SLC45A2 mutation in albinism by targeted next-generation sequencing.

Albinism is a diverse group of hypopigmentary disorders caused by multiple-genetic defects. The genetic diagnosis of patients affected with albinism by Sanger sequencing is often complex, expensive, and time-consuming. In this study, we performed targeted next-generation sequencing to screen for 16 genes in a patient with albinism, and identified 21 genetic variants, including 19 known single nucleotide polymorphisms, one novel missense mutation (c.1456 G>A), and one disease-causing mutation (c.478 G>C). The novel mutation was not observed in 100 controls, and was predicted to be a damaging mutation by SIFT and Polyphen. Thus, we identified a novel mutation in SLC45A2 in a Chinese family, expanding the mutational spectrum of albinism. Our results also demonstrate that targeted next-generation sequencing is an effective genetic test for albinism.

Altered expression of metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in cervical disc herniation patients.

The aim of the current study was to examine matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in patients with cervical disc herniation (CDH). A total of 127 specimens from CDH patients undergoing posterior spinal surgery were obtained for the case group, which was divided into three subgroups: lateral protrusion (N = 102), median protrusion (N = 18), and paramedian protrusion (N = 7). Another 55 specimens from subjects who had cervical spine trauma and underwent spinal canal decompression were obtained for the control group. Routine hematoxylin and eosin staining was performed for pathological diagnosis. Immunohistochemical (IHC) analysis was used to determine MMP-2 and TIMP-2 expression. Under light microscopy, MMP-2-positive cells presented brown-yellow or dark brown staining in the cell membrane or cytoplasm. MMP-2 expression in the case group was significantly higher than that in controls (P < 0.05). Furthermore, MMP-2 expression in the lateral and median protrusion groups was significantly higher compared to that in the paramedian protrusion group (both P < 0.05), while there was no apparent difference in MMP-2 expression between the lateral and median protrusion groups (P > 0.05). IHC results showed that TIMP-2 expression in cases was significantly lower than that in controls (P < 0.05). Spearman correlation analysis indicated that MMP- 2 was negatively correlated with TIMP-2 expression (r = -0.418, P < 0.001). In conclusion, MMP-2 expression increased, whereas TIMP- 2 expression decreased in CDH patients, suggesting that MMP-2 and TIMP-2 expression may contribute to CDH development.

Phosphatidylinositol 3 kinase/protein kinase B is responsible for the protection of paeoniflorin upon H2O2-induced neural progenitor cell injury.

Promoting neural stem/progenitor cell (NSC/NPC) survival in the pro-apoptotic environment is critical to stem cell replacement for neurodegenerative disease therapy. Paeoniflorin (PF), one of the principal bioactive components in Paeoniae Radix, has been used widely in central nervous system (CNS) diseases treatment and serves as an antioxidant to protect neurons against oxidative stress. The present study investigated the protective effects of PF on NPC injury induced by hydrogen peroxide (H2O2). After challenge with 200 µM H2O2 for 2h, loss of cell viability and excessive apoptotic cell death were observed in cultured NPC, PF treatment conferred protective effects against the loss of cellular viability in a concentration-dependent manner. PF pretreatment also inhibited NPC apoptosis induced by H2O2 by reversing the decreased level of Procaspase-3 and balancing Bcl-2 and Bax expression. Furthermore, PF-mediated NPC protection was associated with an increase in phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt-1) phosphorylation in a time- and concentration-dependent manner. Selective inhibition of PI3K using LY294002 abolished PF-mediated phosphorylation of Akt-1 and NPC protection upon oxidative stress. These data suggest that PF-mediated NPC protection on H2O2 injury is reliant on the activation of the PI3K/Akt-1 pathway, giving insight to an essential role of PF in NPC protection.

Tumor necrosis factor (TNF)-α upregulates progesterone receptor-A by activating the NF-κB signaling pathway in human decidua after labor onset.

To date, the relationship between inflammatory cytokines and progesterone receptors (PRs) has been little studied, although both mediate the mechanism of parturition in human deciduas. Thus, the aim of study was to investigate the role of an inflammatory cytokine, tumor necrosis factor (TNF)-α, in regulating progesterone withdrawal in decidua at human parturition. TNF-α levels and PR isoforms were compared in intrauterine deciduas from women who were in labor (IL, n = 10) or who were not in labor (NIL, n = 10). Nuclear factor-kappaB (NF-κB) signaling and PR status were analyzed in NIL deciduas after TNF-α stimulation. Immunohistochemistry, western blotting, ELISA and reverse transcription-polymerase chain reaction (RT-PCR) were used to localize and quantitate protein and mRNA expression. TNF-α immunostaining, protein levels, PR-A/PR-B ratio and COX-2 level were significantly higher in IL deciduas (all P < 0.05). NF-κB was activated by TNF-α after 24 h stimulation in a dose-dependent manner, and was significantly inactivated by the NF-κB inhibitor panepoxydone, which was associated with decreased PR-A and COX-2 expression (P < 0.05) in not in labor deciduas. In conclusion, TNF-α may have an important role in regulating progesterone withdrawal in human decidua following labor onset.

Cloning, expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner).

A cDNA clone coding for general odorant-binding protein2 was isolated from the antenna of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. Results of sequencing and structural analyses showed that the full-length of GOBP2Harm was 636 bp, possessing 162 amino acid residues and a signal peptide of 21 amino acids. Its predicted molecular weight and isoelectric point were 18.2 kDa and 5.21, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, typical of insect's OBPs. Northern blot showed that GOBP2Harm is specifically expressed in the antenna of Helicoverpa armigera at similar levels in both sexes. In order to obtain sufficient GOBP2 for further determining its biochemical and physiological properties, a bacterical expression vector of GOBP2 was constructed and successfully expressed. The protein was obtained mainly as insoluble inclusion bodies, that, however, could be solubilized and refolded. The rGOBP2 was purified by affinity chromatography and gel filtration. The rGOBP2 was shown to cross-react with an anti-GOBP antiserum from Antheraea polyphemus. Finally, polyclonal antibodies against GOBP2Harm were used to mark the distribution of the protein in olfactory sensilla and were tested by immuno-electron microscopy. In the male, GOBP2Harm is mainly expressed in sensilla basiconica, while in the female, it is equally expressed in sensilla basiconica and in sensilla trichodea.

Liver disease associated with duck hepatitis B virus infection of domestic ducks.

The liver disease associated with duck hepatitis B viremia was investigated in naturally infected ducks from Chi-tung county in China and in both naturally and experimentally infected ducks from the United States. Liver and serum specimens of adult Chinese ducks were examined for duck hepatitis B virus (DHBV) DNA by dot and gel blot hybridization. DHBV was found in serum and (in episomal form only) in livers of 6 of 11 birds exhibiting various degrees of chronic hepatitis. In 1 bird with hepatocellular carcinoma, DHBV DNA was detected at the limit of assay sensitivity and in another not at all, contrasting with findings in humans and woodchucks. In work with California Pekin and Khaki Campbell ducks, known amounts of DHBV were injected into the egg 10 days before, or into ducklings 1 day after, hatching and the livers were examined 6 weeks later. The majority of the injected ducklings had viremia detectable by hybridization 1 or 2 weeks after injection. The presence but not the amount of viremia correlated with incidence and degree of hepatitis, determined under code. The most severe instances of hepatitis, all in Pekin ducks, resembled the hepatitis in adult Chinese ducks of Chi-tung county. Severe and moderate hepatitis were found only in indoor-caged injected animals with viremia and in some uninjected birds without viremia that had been kept in outdoor flocks. The latter hepatitis, as some hepatitis in adult Chinese ducks, may not be related to DHBV. Mild and insignificant hepatitis were also found in injected and noninjected ducklings, some of which had the vertically transmitted spontaneous viremia previously described. The good correlation of experimentally induced viremia with incidence and severity of hepatitis in the Pekin duckling provides a simple, rapid, and relatively inexpensive model to study the relation of lesions to hepatitis B family infection in nonprimates.