A bismuth-based double-network hydrogel-mediated synergistic photothermal-chemodynamic therapy for accelerated wound healing.

Multidrug-resistant bacterial infections present a significant challenge to wound healing. Non-antibiotic approaches such as photothermal therapy (PTT) and chemodynamic therapy (CDT) are promising but have suboptimal anti-bacterial efficacy. Herein, we developed a green bismuth-based double-network hydrogel (Bi@P-Cu) as a PTT/CDT synergistic platform for accelerated drug-resistant bacteria-infected wound healing. Bismuth (Bi) nanoparticles fabricated using a microwave method were used as a highly efficient and biocompatible PTT agent while the integration of a small amount of CDT agent Cu2+ endowed the hydrogel with excellent mechanical and self-healing properties, markedly increased photothermal efficiency, promoted cell migration ability, and negligible toxicity. Importantly, PTT enhanced the production of hydroxyl radicals in CDT and the destruction of bacterial cell membranes, which in turn enhanced the thermal sensitivity of bacteria. This synergistic anti-bacterial effect, together with the demonstrated capability to promote angiogenesis and anti-inflammation as well as enhanced fibroblast proliferation, led to accelerated wound healing in a full-thickness mouse model of resistant bacterial infection. This study provides an effective and safe strategy to eliminate drug-resistant bacteria and accelerate wound healing through green, non-antibiotic, double-network hydrogel-mediated synergistic PTT and CDT.

Flavonoid extracted from Epimedium attenuate cGAS-STING-mediated diseases by targeting the formation of functional STING signalosome.

Hyperactivation of the cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway has been shown to be associated with the development of a variety of inflammatory diseases, and the discovery of an inhibitor of the cGAS-STING signalling pathway holds great promise in the therapeutic interventions. Epimedium flavonoid (EF), a major active ingredient isolated from the medicinal plant Epimedium, has been reported to have good anti-inflammatory activity, but its exact mechanism of action remains unclear. In the present study, we found that EF in mouse bone marrow-derived macrophages (BMDMs), THP-1 (Tohoku Hospital Pediatrics-1) as well as in human peripheral blood mononuclear cells (hPBMC) inhibited the activation of the cGAS-STING signalling pathway, which subsequently led to a decrease in the expression of type I interferon (IFN-ß, CXCL10 and ISG15) and pro-inflammatory cytokines (IL-6 and TNF-α). Mechanistically, EF does not affect STING oligomerization, but inhibits the formation of functional STING signalosome by attenuating the interaction of interferon regulatory factor 3 (IRF3) with STING and TANK-binding kinase 1 (TBK1). Importantly, in vivo experiments, EF has shown promising therapeutic effects on inflammatory diseases mediated by the cGAS-STING pathway, which include the agonist model induced by DMXAA stimulation, the autoimmune inflammatory disease model induced by three prime repair exonuclease 1 (Trex1) deficiency, and the non-alcoholic steatohepatitis (NASH) model induced by a pathogenic amino acid and choline deficiency diet (MCD). To summarize, our study suggests that EF is a potent potential inhibitor component of the cGAS-STING signalling pathway for the treatment of inflammatory diseases mediated by the cGAS-STING signalling pathway.

The fecal microbiota of gravidas with fetal growth restriction newborns characterized by metagenomic sequencing.

BACKGROUND: Fetal growth restriction (FGR) is a complex obstetric complication with various causes and of great harm. However, the specific pathogenesis of FGR is unclear, which limits its effective treatment. Gut microbiota dysbiosis was found to be important in pathogenesis of various diseases. However, its role in FGR development remains unclear and needs to be clarified. METHODS: In our case-control study, we recruited eight FGR and eight control female participants and collected their fecal samples in third trimester before delivery. We performed metagenomic sequencing and bioinformatic analysis to compare the gut microbiota composition and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between the two groups. RESULTS: Our results showed that totally 20 gut microbes were significantly different between two groups (p<0•05), and the correlation analysis found that g__Roseomonas and g__unclassified_f__Propionibacteriaceae were significantly positive correlated with both maternal body mass index (BMI) before delivery, placental weight, and neonatal birth weight (BW) percentile (all p<0•05), while g__Marinisporobacter and g__Sphingomonas were significantly negative correlated with both neonatal BMI and neonatal BW percentile (all p<0•05). Through KEGG pathway analysis, we found that the abundance of the Nitrogen metabolism pathway decreased significantly (p<0•05) whereas the abundance of the Amoebiasis pathway increased significantly in the FGR group (p<0•05). CONCLUSION: In this study, we demonstrated that the occurrence of FGR is associated with the change of gut microbiota of pregnant women.

MXene-based composites as an electrochemical sensor for ultrasensitive determination of ofloxacin.

Sensitive determination of ofloxacin (OFL) is very essential for human health and environmental protection. Here, a novel composite of gold nanoparticles(nAu)@MXene(Ti3C2Tx)/poly-p-aminobenzene sulfonic acid (PABSA) was fabricated on the surface of glassy carbon electrode (GCE) and used to sensitively determine OFL. The results of experiments showed that the obtained nAu@Ti3C2Tx/PABSA/GCE electrode could be used as an electrochemical sensor to directly detect ofloxacin (OFL) by differential pulse voltammetry (DPV). Under the optimal conditions, the proposed electrode displayed a broader linear range and a lower detection limit (LOD) for OFL determination when it was compared to those similar sensors. The linear range was from 5.0 × 10-8 to 5.0 × 10-4 mol/L and the LOD was 3.7 × 10-8 mol/L (S/N = 3). The nAu@Ti3C2Tx/PABSA/GCE electrode also showed good selectivity, repeatability, and reproducibility. Finally, the proposed electrode was used to detect OFL in commercial samples by the standard addition method. The obtained recovery was from 97.3% and 105.7% showing its potential applications in actual sample analysis.

Tunable mechanical behavior of collagen-based films: A comparison of celluloses in different geometries.

Collagen (Col) films were reinforced by celluloses in different geometries: microcrystalline cellulose (MCC), cellulosic fines (CF), cellulose nanofiber (CNF) and cellulose nanocrystals (CNC). The reinforcement mechanisms were investigated by the elastoplasticity and fracture appearance. Compared with the fracture stress of collagen film (67.5 MPa), the Col-CNF films effectively borne the stress (95.8 MPa) by intercrystalline fracture, ascribing the abundant hydrogen bonding and mechanical locking between cellulose and collagen. The toughness of Col-CF films was increased by the interfibrillar slippage of CF and pull-off of CF within the matrix, improving the strain-to-break from 8.37% to 12.13%. The films added with MCC and CNC weaken the mechanical behavior, due to the defects and lack of mechanical locking. Besides, the effects of celluloses' geometries on the thickness, density, water-tightness, thermal stability, crystallinity and FTIR of films were also investigated. These provide the evidence that the geometries of fillers diversely improve the behaviors of collagen film offering strategies for the film with adjustable mechanical properties.

Four methods to analyze H3K27M mutation in diffuse midline gliomas.

The histone H3 K27M mutation has been frequently reported in the majority of diffuse midline gliomas, which is considered as a prognostic and predictive biomarker. A number of different methods and platforms including pyrosequencing (PSQ), sanger sequencing, immunohistochemistry (IHC), Mass array and NGS (Next Generation Sequencing) have been used to detect H3K27M mutation in diffuse midline gliomas. However, controversy remains about the most appropriate method to use for analyzing H3K27M status. The H3K27 M mutation status of a total of 50 diffuse midline gliomas was examined using PSQ, sanger sequencing, IHC and Mass array in parallel. Using PSQ as a recommended standard method, the sensitivity, specificity and correlation with the other assays were calculated. Among 50 diffuse midline glioma cases, the H3K27M mutation was positive in 64 %, 66 %, 62 % and 62 % of the cases by PSQ, IHC, sanger sequencing and mass array, respectively. The sensitivity and specificity of IHC were 100 % and 94.4 %, respectively. The sensitivity and specificity of sanger sequencing and mass array were both 96.9 % and 100 %, respectively. This study demonstrated that IHC is an effective and rapid detection method for routine use in pathology laboratories for the identification of H3K27M mutation. A combination of IHC and sanger sequencing assays can provide 100 % sensitivity and specificity for the prediction of H3K27M status.

LncRNA SNHG4 promotes the increased growth of endometrial tissue outside the uterine cavity via regulating c-Met mediated by miR-148a-3p.

Long noncoding RNAs (lncRNAs) modulate endometriosis. The current study investigated the mechanisms and effects of SNHG4 on endometriosis. The qRT-PCR was conducted to examine the miR-148a-3p and SNHG4 expressions in endometriosis tissues. The 5-ethynyl-2'-deoxyuridine incorporation assay and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay were used to measure the rate of cell proliferation. The association between miR-148a-3p, SNHG4 and c-Met was confirmed via bioinformatical approach and luciferase reporter gene assay. Also, the function of SNHG4 on the growth of endometriotic lesions was investigated in vivo. The SNHG4 expression was considerably upregulated in endometriosis tissues, whereas the level of miR-148a-3p expression was reduced. In addition, SNHG4 can be considered as ceRNAs that bind miR-148a-3p and rise the proliferation activity of HESCs by downregulating miR-148a-3p. Furthermore, silencing SNHG4 could downregulate the c-Met level by enhancing miR-148a-3p expression, and finally inhibiting endometriosis development in vivo. LncRNA SNHG4 promotes the increased growth of endometrial tissue outside the uterine cavity via regulating c-Met mediated by miR-148a-3p, which may be used as diagnostic biomarker as well as molecular target in the treatment of endometriosis.

A top-down approach to improve collagen film's performance: The comparisons of macro, micro and nano sized fibers.

The severe reduction of mechanical strength of collagen once it is extracted or dissociated from animal tissues and no additional crosslinking approaches are conducted, impede its application in biodegradable and edible food packaging. Here, for the first time, high pressure homogenization (HPH) was used to prepare diverse sized fibers and the related fibers-composed films' performance were investigated. These fibers have a diversity of effects on film performance. The films prepared with smaller sized fibers had a more uniform and denser structure. The mechanical and the water barrier properties of the films improved significantly as the fiber size decreased. No obvious change in FTIR and thermal properties suggests that the improved film performance is mainly attributed to the physical entanglement and non-covalent bonds. Given the forementioned benefits of the films, control of fiber size can be a potential industrial approach for producing collagenous materials in edible food packaging.

Increased interleukin 1α and interleukin 1ß expression is involved in the progression of periapical lesions in primary teeth.

BACKGROUND: Interleukin 1 (IL-1) is involved in bone resorption. However, the role of IL-1 in periapical lesions characterized by periapical bone destruction in primary teeth has not yet been fully elucidated. This study aimed to detect the distribution and expression of IL-1 in periapical lesions in primary teeth and assess the relationship between the cytokines and the degree of inflammatory cell infiltration. METHODS: A total of 106 chronic periapical lesions in primary teeth were harvested. Haematoxylin and eosin (H&E) staining was used to determine the histological type and the inflammatory cell infiltration grade (mild, moderate, and severe), and immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution and expression of IL-1α and IL-1ß. RESULTS: Of the 106 chronic periapical lesion samples, there were 85 cases of periapical granuloma, accounting for 80.19% of the total samples, and 21 cases of radicular cysts, accounting for 19.81%; no cases of abscess were detected. Immunohistochemistry results showed that both IL-1α and IL-1ß were expressed in periapical granulomas and cysts. ELISA results showed that IL-1α and IL-1ß levels were higher in the periapical granuloma group than in the radicular cyst and normal control groups (P < 0.05). In the periapical granuloma group, IL-1α and IL-1ß were detected at higher levels in the severe inflammatory cell infiltration subgroup than in the mild-inflammatory cell infiltration subgroup (P < 0.05), and IL-1ß expression was also higher in the moderate inflammatory cell infiltration subgroup than in the mild inflammatory cell infiltration subgroup (P < 0.01). A significant positive correlation was observed between the protein expression levels of IL-1α and IL-1ß and the inflammation grade in periapical granulomas from primary teeth (P < 0.05). CONCLUSION: Expression levels of the cytokines IL-1α and IL-1ß in periapical granulomas from primary teeth increased with increasing inflammatory severity and appeared to be a contributing factor to the progression of periapical lesions.

Effect of Dandelion Extracts on the Proliferation of Ovarian Granulosa Cells and Expression of Hormone Receptors.

BACKGROUND: In the current society, infertility related to age has become a social problem. The in vitro fertilization (IVF) success rate in women with poor ovarian response (POR) is very low. Dandelion extract T-1 (DE-T1) is an effective component of the extract from the leaves and stems of Taraxacum officinale, which is one of the medicines used in some patients with POR, but its molecular mechanism remains unclear. METHODS: Following IVF, ovarian granulosa cells (GCs) of sixty patients were extracted and divided into normal ovarian response (NOR) and POR groups. GCs were cultured in a dose-dependent and time-dependent manner with DE-T1, proliferation of GCs was determined by Cell Counting Kit-8 assay, and mRNA levels of insulin-like growth factor 1 receptor (IGF-1R), luteotropic hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), LHR, and CYP19A1 (aromatase) were determined by quantitative polymerase chain reaction. Progesterone and estradiol (E2) concentrations were determined by enzyme-linked immunosorbent assay. RESULTS: The cell viability gradually increased with the progressive increase in the DE-T1 concentration. Compared with the control group (without DE-T1), the mRNA expressions of FSHR, LHR, IGF-1R, and CYP19A1 were upregulated after the addition of DE-T1, especially in the 2.5% DE-T1 group (P < 0.01). The expression of IGF-1R was upregulated approximately 25 times (24.97 ± 4.02 times) in the POR group with 2.5% DE-T1. E2 and progesterone levels increased with the increasing DE-T1 concentration. There were highly significant differences in the E2 and progesterone secretion between the NOR and POR groups (P < 0.01). CONCLUSION: DE-T1 may promote steroid hormone synthesis by promoting GC proliferation and upregulating GC receptor expression, thereby improving ovarian endocrine function.

Lamin A/C might be involved in the EMT signalling pathway.

We have previously reported a heterogeneous expression pattern of the nuclear membrane protein lamin A/C in low- and high-Gleason score (GS) prostate cancer (PC) tissues, and we have now found that this change is not associated with LMNA mutations. This expression pattern appears to be similar to the process of epithelial to mesenchymal transition (EMT) or to that of mesenchymal to epithelial transition (MET). The role of lamin A/C in EMT or MET in PC remains unclear. Therefore, we first investigated the expression levels of and the associations between lamin A/C and several common EMT markers, such as E-cadherin, N-cadherin, ß-catenin, snail, slug and vimentin in PC tissues with different GS values and in different cell lines with varying invasion abilities. Our results suggest that lamin A/C might constitute a type of epithelial marker that better signifies EMT and MET in PC tissue, since a decrease in lamin A/C expression in GS 4 + 5 cases is likely associated with the EMT process, while the re-expression of lamin A/C in GS 5 + 4 cases is likely linked with MET. The detailed GS better exhibited the changes in lamin A/C and the EMT markers examined. Lamin A/C overexpression or knockdown had an impact on EMT biomarkers in a cell model by direct regulation of ß-catenin. Hence, we suggest that lamin A/C might serve as a reliable epithelial biomarker for the distinction of PC cell differentiation and might also be a fundamental factor in the occurrence of EMT or MET in PC.

Claudin-7 indirectly regulates the integrin/FAK signaling pathway in human colon cancer tissue.

The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in regulating the integrin/focal adhesion kinase (FAK)/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, between colon cancer and adjacent normal tissues. Quantitative real-time reverse transcription-PCR and immunohistochemistry were utilized to verify the results of mRNA and protein expression, respectively. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway components, and the STRING database was used to analyze protein-protein interaction pairs among these proteins. Meta-analysis of expression microarrays in The Cancer Genome Atlas (TCGA) database was used to identify significant correlations between Cldn-7 and components of predicted genes in the integrin/FAK/ERK signaling pathway. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples, and the observed changes for all proteins except Cldn-7 were in agreement with changes in the corresponding mRNA levels. Cldn-7 might indirectly regulate MAPK3 via KRT8 due to KRT8 co-expression with MAPK3 or CLDN7. Our bioinformatics methods supported the hypothesis that Cldn-7 does not directly regulate any genes in the integrin/FAK/ERK signaling pathway. These factors may participate in a common network that regulates cancer progression in which the MAPK pathway serves as the central node.

DJ-1/PARK7, But Not Its L166P Mutant Linked to Autosomal Recessive Parkinsonism, Modulates the Transcriptional Activity of the Orphan Nuclear Receptor Nurr1 In Vitro and In Vivo.

Although mutations of DJ-1 have been linked to autosomal recessive Parkinsonism for years, its physiological function and the pathological mechanism of its mutants are not well understood. We report for the first time that exogenous application of DJ-1, but not its L166P mutant, enhances the nuclear translocation and the transcriptional activity of Nurr1, a transcription factor essential for dopaminergic neuron development and maturation, both in vitro and in vivo. Knockdown of DJ-1 attenuates Nurr1 activity. Further investigation showed that signaling of Raf/MEK/ERK MAPKs is involved in this regulatory process and that activation induced by exogenous DJ-1 is antagonized by U0126, an ERK pathway inhibitor, indicating that DJ-1 modulates Nurr1 activity via the Raf/MEK/ERK pathway. Our findings shed light on the novel function of DJ-1 to enhance Nurr1 activity and provide the first insight into the molecular mechanism by which DJ-1 enhances Nurr1 activity.

Biodistribution of PAMAM dendrimer conjugated magnetic nanoparticles in mice.

Fluorescein-loaded magnetic nanoparticles (FMNPs) have been increasingly utilized in nanomedicine due to their unique properties. In this study, polyamidoamine (PAMAM) dendrimer was used to modify the FMNPs through bifunctional polyethylene glycol linker. The obtained PAMAM modified magnetic nanoparticles (PFMNPs) were characterized by transmission electron microscope, thermogravimetric analysis, zeta potential titration, and fourier transform infrared spectroscopy. The effect of PAMAM conjugation on the biodistribution of FMNPs and PFMNPs were investigated by confocal laser scanning microscopy and inductively coupled plasma atomic emission spectrometry, respectively. It was revealed that PAMAM conjugation resulted in a lower uptake of FMNPs in the lung and less aggregation in the liver, whereas a higher uptake in brain and testis. Furthermore, the serum biochemical and the hematological analysis indicated the PFMNPs caused no significant changes in enzymes reflective of inflammatory response or organ toxicity.

Preliminary molecular analysis of bacterial composition in periapical lesions with primary endodontic infections of deciduous teeth.

BACKGROUND: The bacterial composition of periapical lesions in deciduous teeth has not been well documented. This study was designed to explore the bacterial compositions, especially the dominant bacteria in periapical lesions using 16S rRNA sequencing. METHODS: Tissue samples were collected from 11 periapical lesions in deciduous teeth with primary endodontic infections. DNA was extracted from each sample and analyzed using 16S rRNA cloning and sequencing for the identification of bacteria. RESULTS: All DNA samples were positive for 16S rRNA gene PCR. One hundred and fifty-one phylotypes from 810 clones were identified to eight phyla, and each sample contained an average of 25.9 phylotypes. In addition, 59 phylotypes were detected in more than two samples, and Fusobacterium (F.) nucleatum (8/11), Dialister (D.) invisus (8/11), Campylobacter (C.) gracilis (7/11), Escherichia (E.) coli DH1 (6/11), Aggregatibacter (A.) segnis (6/11), and Streptococcus (S.) mitis (6/11) were the most prevalent species. Furthermore, 45 as-yet-uncultivated phylotypes were also identified. CONCLUSIONS: Chronic periapical lesions in deciduous teeth contained polymicrobial infections. F. nucleatum, D. invisus, C. gracilis, E. coli DH1, A. segnis, and S. mitis were the most prevalent species detected by 16S rRNA sequencing.

Subthalamic hGAD65 gene therapy and striatum TH gene transfer in a Parkinson's disease rat model.

The aim of the present study is to detect a combination method to utilize gene therapy for the treatment of Parkinson's disease (PD). Here, a PD rat model is used for the in vivo gene therapy of a recombinant adeno-associated virus (AAV2) containing a human glutamic acid decarboxylase 65 (rAAV2-hGAD65) gene delivered to the subthalamic nucleus (STN). This is combined with the ex vivo gene delivery of tyrosine hydroxylase (TH) by fibroblasts injected into the striatum. After the treatment, the rotation behavior was improved with the greatest efficacy in the combination group. The results of immunohistochemistry showed that hGAD65 gene delivery by AAV2 successfully led to phenotypic changes of neurons in STN. And the levels of glutamic acid and GABA in the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr) were obviously lower than the control groups. However, hGAD65 gene transfer did not effectively protect surviving dopaminergic neurons in the SNc and VTA. This study suggests that subthalamic hGAD65 gene therapy and combined with TH gene therapy can alleviate symptoms of the PD model rats, independent of the protection the DA neurons from death.

Microbial diversity in failed endodontic root-filled teeth.

BACKGROUND: Persistent/secondary infections of human root canals play an important role in the failure of endodontic treatment. This study used 16S rRNA sequencing to assess microbial diversity in root-filled teeth associated with failed endodontic treatment. METHODS: DNA was extracted from 15 teeth with persistent intraradicular infections, and the 16S rRNA of all present bacteria were amplified by PCR, followed by cloning and sequencing of the 16S rRNA amplicons. RESULTS: All sample extracts were positive for PCR amplification using the universal 16S rRNA gene primers. Negative control reactions yielded no amplicons. Sixty-five phylotypes belonging to seven phyla were identified from 760 clones; a mean of 9.4 phylotypes were detected in each sample (range 3 - 15). Twenty-eight phylotypes were detected in more than one sample, revealing a high inter-sample variability. Parvimonas micra (60%, 9/15), Solobacterium moore (47%, 7/15), Dialister invisus (33%, 5/15), Enterococcus faecalis (33%, 5/15), Filifactor alocis (27%, 4/15), and Fusobacterium nucleatum (27%, 4/15) were the prevalent species. Nineteen as-yet-uncultivated phylotypes were identified, comprising a substantial proportion of the bacteria in many cases. CONCLUSIONS: Persistent intraradicular infections were present in all root-filled teeth associated with failed endodontic treatment. The current observations reveal new candidate endodontic pathogens, including as-yet-uncultivated bacteria and phylotypes that may participate in the mixed infections associated with post-treatment apical periodontitis.

DJ-1 upregulates tyrosine hydroxylase gene expression by activating its transcriptional factor Nurr1 via the ERK1/2 pathway.

Loss-of-function DJ-1 mutations have been linked to autosomal recessive early-onset Parikinsonism. However, the putative function of DJ-1 is not completely understood. Previous studies indicate that DJ-1 overexpression results in upregulation of the tyrosine hydroxylase gene. The mechanism by which DJ-1 affects tyrosine hydroxylase expression remains elusive. In the present study, we show that DJ-1 overexpression induces ERK1/2 activation, along with increased tyrosine hydroxylase expression. The L166P DJ-1 mutant, which has been identified as being responsible for familial Parkinsonism, did not have this effect. Moreover, suppression of ERK1/2 phosphorylation by the pharmacological inhibitor U0126 partially abolished the regulating effect of DJ-1 on tyrosine hydroxylase. Nurr1, a transcriptional factor for tyrosine hydroxylase, can be phosphorylated by ERK1/2 and translocate to the nucleus, where it is activated. Thus, we measured nuclear translocation of Nurr1. Confocal microscopy and Western blotting revealed that Nurr1 translocated to the nucleus and was activated by overexpression of wild-type DJ-1, but not of its L166P mutant. Knockdown of Nurr1 gene expression abolished the effect on tyrosine hydroxylase induced by DJ-1. Taken together, these data suggest that DJ-1 upregulates tyrosine hydroxylase expression by activating its transcription factor Nurr1 via the ERK1/2 pathway.

Loss of PINK1 function decreases PP2A activity and promotes autophagy in dopaminergic cells and a murine model.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of recessive PD. Autophagy, a pathway for clearance of protein aggregates or impaired organelles, is a newly identified mechanism for PD development. However, it is still unclear what molecules regulate autophagy in PINK1-silenced cells. Here we report that autophagosome formation is promoted in the early phase in response to PINK1 gene silencing by lentivirus transfer vectors expressed in mouse striatum. Reduced PP2A activity and increased phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in PINK1-knockdown dopaminergic cells and striatum tissues. Treatment with C2-ceramide (an agonist of PP2A) reduced autophagy levels in PINK1-silenced MN9D cells, which suggests that PP2A plays an important role in the PINK1-knockdown-induced autophagic pathway. Furthermore, phosphorylation of Bcl-2 at S87 increased in PINK1-silenced cells and was negatively regulated by additional treatment with C2-ceramide, which indicates that Bcl-2 may be downstream of PP2A inactivation in response to PINK1 dysfunction. Immunoprecipitation also revealed dissociation of the Bcl-2/Beclin1 complex in PINK1-silenced cells, which was reversed by additional treatment with C2-ceramide, and correlated with changes in level of autophagy and S87 phosphorylation of Bcl-2. Finally, Western blots for cleaved caspase-9 and flow cytometry results for active caspase-3 revealed that PP2A inactivation is involved in the protective effect of autophagy on PINK1-silenced cells. Our findings show that downregulation of PP2A activity in PINK1-silenced cells promotes the protective effect of autophagy through phosphorylation of Bcl-2 at S87 and blockage of the caspase pathway. These results may have implications for identifying the mechanism of PD.

Silencing of PINK1 induces mitophagy via mitochondrial permeability transition in dopaminergic MN9D cells.

Accumulation of dysfunctional Mitochondria has been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in PTEN-induced kinase 1 (PINK1), which encodes a putative mitochondrial serine/threonine kinase, have been identified in early-onset forms of PD. Recent data showed that the loss of PINK1 function led to oxidative stress, mitochondrial damage and autophagic elimination of damaged mitochondria. But the precise mechanism of autophagy induced by loss of PINK1 is unclear. In this study, we found that in mouse dopaminergic MN9D cells, down-regulation of PINK1 by RNA interference resulted in induction of mitochondrial autophagy (mitophagy), abnormal mitochondrial morphology, partial loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS). Mitophagy in these cells was associated with the up-regulation of autophagy activator Beclin 1 and opening of mitochondrial permeability transition (MPT) pore. These findings suggest that PINK1 may regulate mitophagy through controlling MPT pore opening and general autophagy regulators.