At more advanced ages (16–19 months), cognitive deficits progressed in conjunction with resting connectivity abnormalities; furthermore, hypometabolism, Aβ plaque accumulation, reduction of CSF Aβ1-42 concentrations, and hippocampal atrophy  were detectable at this stage. The present results emphasize the early impact of Aβ on brain connectivity and support a framework in which persistent Aβ aggregation itself is sufficient to impose memory circuits dysfunction, which propagates to adjacent brain networks at later stages.Imaging biomarkers are frequently proposed as endpoints for clinical trials targeting brain amyloidosis in Alzheimer's disease (AD); however, the specific impact of amyloid-β (Aβ) aggregation on biomarker abnormalities remains elusive in AD. Using the McGill-R-Thy1-APP transgenic rat as a model of selective Aβ pathology, we characterized the longitudinal progression of abnormalities in biomarkers commonly used in AD research. Middle-aged (9–11 months) transgenic animals  displayed mild spatial memory impairments and disrupted cingulate network connectivity measured by resting-state fMRI, even in the absence of hypometabolism (measured with PET [SIGNIFICANCE STATEMENT The present study proposes a “back translation” of the Alzheimer pathological cascade concept from human to animals. We used the same set of Alzheimer imaging biomarkers typically used in large human cohorts and assessed their progression over time in a transgenic rat model, which allows for a finer spatial resolution not attainable with mice. Using this translational platform, we demonstrated that amyloid-β pathology recapitulates an Alzheimer-like profile of biomarker abnormalities even in the absence of other hallmarks of the disease such as neurofibrillary tangles and widespread neuronal losses. Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) aggregates in various conformations , the occ18F]NAV4694  both reveal AD-specific regional patterns of synaptic dysfunction and neurodegeneration , progressive synaptic dysfunction , and cognitive impairment  compared with wild-type (WT) animals.To test the hypothesis that Aβ aggregation can itself lead to declines in large-scale brain connectivity, metabolism, and cognitive function, we conducted a longitudinal, multimodal biomarker study using the McGill-R-Thy1-APP Tg rat model of AD-like Aβ pathology, which expresses hAPP with Swedish and Indiana mutations. This model displays progressive Aβ aggregation and cognitive deficits, but no NFT inclusions or widespread cell death . ConsideAll procedures described here were performed in accordance with the Canadian Council on Animal Care guidelines and were approved by the McGill University Animal Care Ethics Committee.A sample of 26 rats  was used for this project. Tg McGill-R-Thy1-APP rats  express hAPP751 with the Swedish and Indiana mutations under the control of the murine Thy1.2 promoter, resulting in accumulation of Aβ peptides starting 1 week postnatally, which progress to extracellular plaques at 6–10 months of age in homozygous animals. By 12 months, mature plaques are thioflavin S positive . Homozygad libitum access to food and water. All animals underwent the procedures described below twice: once at a baseline time point (aged 9–11 months old) and one follow-up (16–19 months); imaging modalities of each animal for each time point were acquired within 2–6 weeks.All rats were housed at the Douglas Mental Health University Institute animal facility on a 12/12 h light/darkness cycle and had 18F]NAV4694 for imaging Aβ and [18F]FDG for imaging glucose metabolism. For [18F]NAV4694 scans, anesthesia was first induced using 5% isoflurane in 0.5 l/min oxygen and then maintained throughout the procedure with 2% isoflurane. A 9 min transmission scan using a rotating [57Co] point source was followed by a bolus injection of the radiotracer in the tail vein , concomitant with the beginning of the emission scan, which lasted for 60 min in list mode. The data were then reframed into 27 sequential time frames of increasing durations . For [18F]FDG, tracer injection was done in the tail vein of awake animals (12.7 ± 1.1 MBq in 200 μl), which were anesthetized  50 min later to perform a 20 min emission scan (in a single static time frame) and a 9 min transmission scan. Breathing rate was monitored throughout both scanning procedures; temperature was monitored using a rectal thermometer and maintained at 37 ± 1°C using an electric blanket. Images for both tracers were reconstructed using a maximum a posteriori (MAP) algorithm (voxel size: 0.6 · 0.6 · 1.2 mm) and corrected for scatter, dead time, and decay.PET acquisition was performed using a CTI Concorde R4 microPET for small animals  and two radiotracers: [www.bic.mni.mcgill.ca/ServicesSoftware) were used for image processing and analysis. Image processing steps are summarized in 18F]NAV4694, the binding potential (BPND) was calculated for each voxel using the simplified reference tissue method at the voxel-level  images were generated by normalizing the tissue radioactivity image using the pons as a reference tissue. Each resulting parametric image was first coregistered to the individual animal's sMRI (see below) using six degrees of freedom (rigid body transformation), then nonlinearly transformed to a standardized rat brain space created from the WT Wistar rats used in the present study to account for differences in brain morphology.MINC tools  equipped with Avance III electronics and the 500V/300A B-GA12S2 gradient upgrade with a standard 40 mm quadrature volumetric transceiver. Animals were anesthetized with a 1% isoflurane/medical air mixture. A constant 37°C air flow was used to keep the animals warm.Structural imaging was obtained using the Bruker standard 3D-True Fast Imaging with Steady State Precession pulse sequence . To remove banding artifacts, a root-mean-square image of eight phase advance (angles of 0–315 degrees in increments of 45) acquisitions was obtained. Each TrueFISP phase angle acquisition was acquired as follows: slices oriented in the rostrocaudal axis, FOV of 36 × 36 × 36 mm with a matrix of 180 × 180 × 180, TE/TR of 2.5/5.0 ms, NEX of 2, flip angle of 30°, and a bandwidth of 50 kHz; no accelerations were used. The resulting image is an average of 16 acquisitions with an isotropic 200 μm resolution and was acquired in a total scanning time of 46 min 30 s. Hippocampal volumes were measured using manual segmentation performed by an experimenter blinded to the group conditions and normalized by the intracranial volume.The rs-fMRI acquisitions were completed immediately after the anatomical scans using the standard Bruker 2D-Spin Echo, Echo Planar pulse sequence (2D-SE-EPI) and the following parameters: slices oriented in the rostrocaudal axis, FOV of 25.6 × 25.6 mm with a matrix of 64 × 64 and 32 slices of 1.0 mm for a final resolution of 400 × 400 × 1000 μm, interslice distance of 1.0 mm, TE/TR of 15/2000 ms, flip angle of 70°, bandwidth of 300 kHz, 4 dummy scans to establish steady state, and 450 repetitions for a total scan time of 15 min. A partial-FT acceleration factor of 1.34 (16 overscans) was used, with standard fat suppression and 5 standard saturation slices to isolate the brain volume; the fifth saturation band was used over highly fatty throat areas. Finally, the standard EPI navigator was used, along with automatic ghost correction and automatic trajectory adjustment.https://afni.nimh.nih.gov), the dynamic functional images were corrected for slice time and motion and then band-pass filtered between 0.01 and 0.15 Hz. Connectivity maps were generated by correlating with a seed point in the cingulate cortex, a component of the rat's default mode network (The first four volumes (8 s) were discarded to account for transient drift. Using AFNI   over 4 c1-42 (Fujirebio Europe), with calibrators from 62.5–4000 pg/ml.Under 5% isoflurane anesthesia, 100–150 μl of CSF was collected from each rat through direct puncture of the cisterna magna. Concentrations of Aβ1-42 in the CSF samples were measured using a multiplex xMAP Luminex platform with the ELISA kit INNOTEST β-AMYLOIDm phosphate buffer (PB), pH 7.4. The brains were removed, divided into hemispheres, postfixed in 4% paraformaldehyde in 0.1 m PB for 24 h at 4°C, and then equilibrated in a solution of 30% sucrose in 0.1 m PB. Coronal sections of 40 μm thickness were obtained using a freezing sledge microtome . Free-floating sections  were collected in PBS containing 10 mm Na2HPO4, 150 mm NaCl, and 2.7 mm KCl and processed for immunohistochemistry. Brain sections were incubated first in McSA1 (MediMabs), a mouse monoclonal antibody detecting human Aβ  before transcardiac perfusion with cold 0.1 human Aβ , and the18F]NAV4694, [18F]FDG and rs-fMRI), group effects were estimated using a voxel-level general linear model. Resulting t-statistical maps were corrected for multiple comparisons using a random field theory-based approach , no significant group difference in [18F]NAV4694 binding was found at that age. Similarly, glucose metabolism as measured by [18F]FDG uptake was not significantly altered in younger Tg animals. Group contrast for rs-fMRI showed clusters of significantly lower cingulate connectivity in the Tg group centered on the orbital cortex and the thalamus  = 6.528, p < 0.0001), as well as higher connectivity with the dorsal hippocampus and sensorimotor cortical areas , especially in the dorsal hippocampus see b, no sigal areas .k = 113.34 mm3, peak t(17) = 6.659, p < 0.0001), in which the ratio of BPND in the Tg group compared with the nonspecific binding observed in WT animals was 1.734 ± 0.428. A second cluster covering the dorsal hippocampi, the caudal piriform cortex, and amygdala  = 7.854, p < 0.0001) showed an average ratio of 1.767 ± 0.467 (a–d).At the follow-up time point (16–19 months of age), all three imaging outcomes showed significant group differences . For PET ± 0.467 a–d.18F]FDG PET hypometabolism revealed 2 symmetrical clusters of significant differences where the WT group had higher uptake than Tg animals (t > 3.58), located in the ventral orbital, secondary motor, cingulate, prelimbic, barrel, and entorhinal cortices  = 6.46, p < 0.0001; average SUVr of 1.449 ± 0.157 for WT and 1.343 ± 0.14 for Tg; right hemisphere: k = 117.48 mm3, peak t(17) = 6.403, p < 0.0001; SUVr of 1.484 ± 0.123 for WT and 1.369 ± 0.099 for Tg). A third, median cluster covered the ventral thalamus and medial geniculate, as well as the hippocampal genus and the inferior colliculi  = 4.417, p = 0.0004), where the WT group had an average SUVr of 1.448 ± 0.121 compared with 1.343 ± 0.112 for the Tg group (e–h).Group contrast of NAV4694 BPND reaching differences of up to 47.44% in the parietal association and retrosplenial cortices, 91.22% in the caudal entorhinal cortex, and 94.08% in the basal forebrain and olfactory bulb (a). Conversely, [18F]FDG uptake decreases (b) were observed throughout the brain in Tg animals, with the highest reductions located in frontal (38%) and parietal (37%) cortices as well as the cerebellum (36%). Together, these findings suggest that Aβ deposition per se is sufficient to cause abnormalities in glucose metabolism and connectivity.In terms of age effect, the Tg but not the WT group showed a progressive increase of fibrillary Aβ, with NAV4694 binding in addition to decreased CSF Aβ1-42 concentrations. Compared with WT controls, the Tg animals showed progressive functional decline, reflected by reduced resting brain connectivity and glucose metabolism, as well as spatial memory impairments measured by the MWM task.In summary, we have shown in this longitudinal study that Aβ aggregates secondary to the expression of mutated hAPP in the rat brain  are sufficient to cause specific brain injury quantifiable using the same biomarkers that are used as outcome measures in AD clinical studies. In aged McGill-R-Thy1-APP rats, amyloidosis was observed by increased FDG PET can be induced by an injury in a remote brain region , which underlines the translational value of the present observations. Mild brain atrophy measured with sMRI indicated a modest but significant effect of Aβ on volumetry limited to the hippocampus, which is the only structure where cell death is observed in McGill-R-Thy1-APP rats (The basal Aβulations d, which APP rats . ResilieAPP rats , whereasAPP rats .Our findings indicate that longitudinal biomarker acquisitions in rodents recapitulate large-scale observational and interventional studies in humans, specifically in prodromal and early stages of the disease. This is the first longitudinal, multiparametric study using a robust rat model of Aβ pathology illustrating progressive abnormalities in AD biomarkers. With only a single transgene insertion site per allele, this rat model has minimal genetic invasion compared with other animal models, yet was able to reproduce a biomarker profile closely analogous to that of human disease. Based on the present observations, we propose that biomarker abnormalities as a function of Aβ pathology are more evident at the level of large-scale brain network connectivity and regional brain metabolism measurements than at that of brain atrophy or memory impairment measurements.


Pseudomonas protegens H78, which was isolated from the rhizosphere of oilseed rape in Shanghai, can produce a large array of antibiotics with a broad spectrum of activities. Here, we report the annotated complete genome sequence of P. protegens H78.The plant growth–promoting rhizobacterium  Pseudomonas protegens H78 can produce a set of antibiotics and siderophores, including pyoluteorin (Plt), 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (Prn), orfamide, P. fluorescens insecticidal toxin, hydrogen cyanide, pyoverdine, and pyochelin , giving 150-fold coverage of the genome, were assembled into three scaffolds and 14 contigs. In the PacBio RSII sequencing, a total of 132,587 subreads  were P. protegens H78 contains one circular chromosome of 7,032,394 bp with a G+C content of 63.4%; no plasmid was detected. The H78 complete genome was annotated using Prokka version 1.8 (P. protegens Pf-5 (P. protegens species can produce a specific spectrum of antibiotic compounds, including Plt, DAPG, and Prn, and has been isolated from the P. fluorescens species because of its obvious difference from other fluorescent Pseudomonas species (P. protegens species also exhibit a certain degree of strain-specific diversity (The complete genome of P. protegens H78 has been deposited in GenBank under the accession number CP013184.The complete genome of


Autosomal recessive defects of either the B1, E1, M1 or S1 subunit of the Adaptor Protein complex-4 (AP4) are characterized by developmental delay, severe intellectual disability, spasticity, and occasionally mild to moderate microcephaly of essentially postnatal onset.AP4M1, causing the replacement of an arginine by a stop codon at position 338 of the protein (p.Arg338X). The premature stop codon truncates the Mu homology domain of AP4M1, with predicted loss of function. Exome analysis also showed heterozygous variants in three genes, ATR, MCPH1 and BLM, which are known causes of autosomal recessive primary microcephaly.We report on a patient with severe microcephaly of prenatal onset, and progressive spasticity, developmental delay, and severe intellectual deficiency. Exome sequencing showed a homozygous mutation in Our findings expand the AP4M1 phenotype to severe microcephaly of prenatal onset, and more generally suggest that the AP4 defect might share mechanisms of prenatal neuronal depletion with other genetic defects of brain development causing congenital, primary microcephaly.The online version of this article (doi:10.1186/s12881-017-0412-9) contains supplementary material, which is available to authorized users. ASPM or CEP152, are involved in cell cycle control . Alterations of these processes may lead to isolated primary microcephaly  or primary microcephaly with dwarfism  [Primary microcephalies are a rare, genetically and clinically heterogeneous group of disorders that result from insufficient production of mature neurons during neurogenesis. Known causal genes, e.g. ndromes) .AP4M1 have been associated with severe intellectual disability, progressive spastic paraplegia, and inability to walk, in six unrelated families [AP4M1 is a component of the Adaptor Protein Complex-4 (AP4). Homozygous mutations in families . The AP4AP4M1 mutation, short stature, and severe, congenital microcephaly that initially mimicked MCPH.We here report on a patient with an The patient was a male infant, first born after a 37 weeks gestation to second-cousin, asymptomatic parents of Turkish origin. Both parents had unremarkable medical histories, a normal head size and a normal intellect. They reported no paresis, and showed no spasticity nor hyperreflexia. The pregnancy was unremarkable, with no report of alcohol use or substance abuse, nor infection. Severe microcephaly was noted at birth, with a head circumference (HC) of 29 cm (−4.3SD), a length of 45 cm and a weight of 2.31 kg. Microcephaly progressed, with a HC of 41.5 cm at 1.1 year (−4.1SD), 43 cm at 3yo (−4.5SD), and 44 cm at 5.2 years (−4.7SD). The patient also had short stature, with a length at −3.5SD and a weight at −2.6SD. No dysmorphia was present except for clinodactily of the fifth fingers. Global hypotonia was present in the first months of life. Hypertonia was first noted at age 9 months, and progressed, with a bilateral Babinski sign. Partial complex epilepsy appeared at age 2 years and was treated successfully with valproate and lamotrigine. Motor development was severely impaired. The patient sat without support at age 1 year, crawled at 2 years, and stood with support at age 2.5 years. He walked with support after age 3 years. He had mild clubfeet deformities with flattened arches. Intellectual development was severely impaired. Testing at the age of 4 months revealed a developmental stage corresponding to the age of 3 months, and at the age of 9.5 months corresponding to 5 months . At the age of 8 years he had no words, but showed emotions. MRI at age 2y8m showed a normal cortex, temporo-parietal subcortical atrophy and hippocampal atrophy bilaterally, enlarged lateral ventricles, a thin corpus callosum predominantly in its caudal portion, a normal cerebellum and a normal white matter in FLAIR sequence acquisition  and found heterozygosity of alleles at loci MCPH1,2,3,4,5,7 and 9, making the corresponding genes unlikely to cause microcephaly in the proband. Direct sequencing of the coding exons and exon-intron junctions of genes AP4M1. DNA was amplified using a standard Polymerase Chain Reaction . The PCR product was purified with Exosap-IT (Affymetrix), and sequenced by the company Beckman Coulter Genomics.For whole-exome sequencing, the proband’s genomic DNA was sheared and exonic sequences enriched using Roche SeqCap EZ Human Exome v3.0 (64 Mb) DNA capture. Sequencing was performed on a HiSeq1500 Illumina sequencer at the BRIGHTcore BRussels Interuniversity Genomics High Throughput core . Raw seqATR (c.6109A > G p.Met1996Thr), MCPH1 (c.85C > T p.Ala3Ala) and BLM (c.3967G > A p.Gln1283Gln). Analysis of the rest of the exome data revealed 5 hemizygous, 9 compound heterozygous, and 26 homozygous variants, two of which being encompassed by a 9 Mb homozygous stretch (chr7: 91599825–100494222), consistent with homozygosity by descent (autozygosity) for this chromosomal segment. One of these three variants, located at Chr7:99,703,901, consisted of a homozygous truncating mutation in exon 13 of the AP4M1 gene, c.1170C > T (transcript_uniprot_id O00189) changing the Arginine at position 338 of the polypeptide into a stop codon, p.Arg338X  variants in 68 primary microcephaly-related genes. This showed heterozygous missense variants in three genes: AP4M1 which has 15 exons, and caused the replacement of an arginine by a stop codon at position 338 (p.Arg338X). It is predicted to truncate 41% of the predicted Mu homology domain of the AP4M1 protein. This domain is an important protein-protein interaction module found in proteins involved in endocytosis. It has been shown to interact with the cytosolic tail of transmembrane cargo proteins, through binding to tyrosine-based signals [We investigated a child initially referred for primary microcephaly of prenatal onset. Exome sequencing revealed only one rare (MAF < 0.5%) variant predicted to introduce a premature termination codon. This mutation was located in exon 13 of gene  signals –20. EithAP complexes are important for vesicular trafficking from the trans-golgi network to the plasma membrane , with spATR, MCPH1 and BLM, which are known causes of autosomal recessive primary microcephaly, presumably via their role in DNA damage repair [IER3IP1 is another gene whose defect causes microcephaly via increased apoptosis of neural progenitors [MCPH1, ATR and BLM, together modified the effect of the homozygous AP4M1 mutation, causing increased apoptosis of neural progenitors, and perhaps of other cell types. Of note, our patient also had short stature, a feature reported with homozygous mutations of either MCPH1, ATR, or BLM Additional file 2:Clinical features in reported cases of AP4 defects and in the present proband. (PDF 76 kb)


Drosophila genetic model of neuronal necrosis by calcium overloading through expression of a constitutively opened cation channel mutant. Here, we performed further genetic screens and identified a suppressor of neuronal necrosis, CG17259, which encodes a seryl-tRNA synthetase. We found that loss-of-function (LOF) CG17259 activated eIF2α phosphorylation and subsequent up-regulation of chaperons (Hsp26 and Hsp27) and autophagy. Genetically, down-regulation of eIF2α phosphorylation, Hsp26/Hsp27 or autophagy reduced the protective effect of LOF CG17259, indicating they function downstream of CG17259. The protective effect of these protein degradation pathways indicated activation of a toxic protein during neuronal necrosis. Our data indicated that p53 was likely one such protein, because p53 was accumulated in the necrotic neurons and down-regulation of p53 rescued necrosis. In the SH-SY5Y human cells, tunicamycin (TM), a PERK activator, promoted transcription of hsp27; and necrosis induced by glutamate could be rescued by TM, associated with reduced p53 accumulation. In an ischemic stroke model in rats, p53 protein was also increased, and TM treatment could reduce the p53 accumulation and brain damage.Neuronal necrosis occurs during early phase of ischemic insult. However, our knowledge of neuronal necrosis is still inadequate. To study the mechanism of neuronal necrosis, we previously established a  In treatments of ischemic stroke, prevention of calcium entry by antagonizing the NMDA receptors has been intensively investigated. However, several NMDA receptor antagonists have failed in clinical trials, likely due to their interference with normal functions of the NMDA receptors3. Therefore, identifying key regulators downstream of calcium entry may be desirable for drug targeting.As the second most prevalent disease worldwide, brain ischemia causes massive damage to the cells in the brain. However, there is still much progress needs to be made for neuroprotection in clinics. Cell death in brain ischemia is primarily induced by glutamate through the excessive excitation of NMDA receptors, which leads to the dysregulation of calcium homeostasis4. For neuronal cell death, different insults may activate either apoptotic or necrotic pathways, and these pathways may positively and negatively crosstalk with each other6. For instance, as apoptotic initiators, caspases may have anti-necrotic function through the degradation of the AMPA receptors7. Conversely, as a necrotic trigger, calpain can cleave caspase-38 and apoptosis regulatory proteins, such as apoptosis protease-activating factor-1, Bcl-xL, Bax, Bid and p535. Due to this complexity, identifying key targets that regulate both apoptosis and necrosis is important.In response to an insult, both survival and death signals are activated and their balance determines cell fateDrosophila via neuron-specific expression of a constitutively opened cation channel, the glutamate receptor 1 Lurcher mutant (GluR1Lc)9. Here, we performed genetic screens and identified a new suppressor of neuronal necrosis, CG17259. We found loss-of-function (LOF) CG17259 induced up-regulate small chaperons (Hsp26 and Hsp27) and autophagy. These degradation pathways might coordinate to degrade p53, which was accumulated in necrotic neurons. In mammalian neurons under a necrotic stress or in a rat stroke model, tunicamycin had protective effect, likely through up-regulation of hsp27, similar to the flies. Together, our research provides the genetic evidence for the role of LOF CG17259 in neuronal necrosis, likely through activation of the eIF2α signaling, small chaperones and autophagy to degrade p53.Previously, we have generated a calcium-overload-induced necrosis model in 9. In this Drosophila model, neuronal necrosis was induced by the specific expression of a constitutively open glutamate receptor 1 channel (GluR1Lc) in neurons to overload calcium. The fly contains a neuron-specific promoter Appl-Gal4, UAS-GluR1Lc, and tub-Gal80ts (simplified as “AG” flies). At 18 °C, the AG flies developed normally because Gal4 was suppressed by Gal80ts. When placed at 30 °C, the Gal80ts lost its function and permitted the expression of GluR1Lc, which resulted in calcium overload and neuronal necrosis9. We performed genetic screens using the deficiency lines that cover most of the Drosophila genome (the deficiency kit from Bloomington Drosophila Stock Center). By screening suppressors of the AG fly lethality, we identified nine deficiency lines. Here, the Df(2 L)ED206 line was further investigated ED206, we tested P-element insertion-mediated mutants in this region and found that loss of CG17259 (CG17259KG03126) strongly rescued the AG flies  , its effect on the transcript of CG17259 was confirmed  reduced neuronal necrosis in the larval ventral nerve cord, as inferred from propidium iodide (PI) staining  Fig. . Becausemed Fig. . The CG1ity Fig. . At the ing Fig. . At the ins Fig. . TogetheCG17259 encodes a seryl-tRNA synthetase, an essential enzyme, which catalyzes the ligation of serine to its cognate tRNA, and thereby affects the fundamental building blocks of protein synthesis11. In this respect, CG17259+/− may increase errors in protein synthesis and induce UPR and ER stress. On the ER membrane, three distinct receptors are responsible to UPR, including inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK)12. The IRE1 branch can be quantified by increase of a spliced form of Xbp1 mRNA (Xbp1sp)13. We found that full length Xbp1 (Xbp1FL) was unaltered and the Xbp1sp was undetectable in the CG17259+/− flies  signaling17. Using an in vivo reporter of JNK activation in Drosophila18, the JNK signaling was not activated  that dephosphorylates eIF2α20. The result showed that the loss of Gadd34 (Gadd34e02638) increased eIF2α phosphorylation and suppressed the lethality of AG flies  had the opposite effect  and Hsp27 (Hsp27−/+) were shown. These protein levels in the heterozygous mutant of Hsp26−/+ and Hsp27−/+ were much lower in the than in the wild type (w1118) flies  using the Hsp26 and Hsp27 antibodies. Because the wild type  flies had very low level of p53, we were unable to detect p53 protein after IP with the anti-Hsp26 or anti-hsp27 , both Hsp26 and Hsp27 antibodies could pull down p53  rescued the AG fly , an inhibitor of N-linked glycosylation, known to activate the PERK/eIF2α signalingate Fig. . Howeverate Fig. . Importap27 Fig. . Furtherp27 Fig. , and redp27 Fig. . In addised Fig. . Mdm2 isent Fig. , suggestent Fig. . On the ent Fig. . Togethein vivo, we examined an intraluminal middle cerebral artery occlusion (MCAO) model in rats, in which neuronal necrosis is the predominant form of cell death, especially in the ischemic core32. Six hours after intraperitoneal injection of TM (0.4 mg/kg), we performed permanent focal occlusion33. The infarction volume was assessed by triphenyl-tetrazolium chloride (TTC) staining in the coronally-sectioned brain slices. The defective brain area was not stained by TTC and had a white color34. Our results demonstrated that TM treatment significantly reduced infarct volume  and chaperone-mediated autophagy (CMA). Autophagy requires the formation of autophagosomes and the function of 50. Upregulation of p53 has been linked to neuronal cell death in numerous models of injuries and diseases51, including excitotoxicity52. The absence of p53 protects neurons from a wide variety of toxic insults, including focal ischemia53, ionizing radiation54 and MPTP-induced neurotoxicity55. In response to various types of stress, p53 promotes apoptosis through either transactivation of specific target genes50 or transcription-independent pathways49. As a transcription factor, p53 upregulates proapoptotic genes, such as Bax, Noxa and PUMA56. In addition, p53 can interact with Bcl2 family proteins, such as Bax and Bak, to induce permeabilization of the outer mitochondrial membrane58. Whether p53 is involved in neuronal necrosis is unclear. In support of its involvement in necrosis, p53 may physically interact with cyclophilin D (CypD), a component of the mitochondrial permeability transition pores and trigger the opening of the pores and necrosis59. In addition, the formation of the p53-CypD complex occurs during brain ischemia/reperfusion insult59. Here, we provide the genetic and cell biology evidence indicating that p53 is involved in neuronal necrosis. In SH-SY5Y cells, we showed that p53 was accumulated upon cells treated with glutamate; and this accumulation was prohibited by TM treatment, which enhanced Hsp27 transcription. Similarly, the increased level of p53 in MCAO rat brain was down-regulated by TM treatment. Together, these results indicate conserved function of p53 in neuronal necrosis. In fact, protective effect of TM against neurodegeneration has been widely reported62. The difference is that our study evaluated potential down-stream function of TM to degrade p53 in neuronal necrosis. How does p53 trigger both apoptosis and necrosis? We propose that mild p53 accumulation likely induces apoptosis, whereas the additional accumulation of p53 promotes necrosis. This hypothesis requires further investigation however.Function of p53 in apoptosis has been well documented65. However, p53 also benefits animal survival under hypoxic conditions66. Thus, administration of pifithrins may interfere with the normal function of p53 and thereby produce side effects. An alternative way to target p53 may be to aim to reduce the accumulation of p53. Our research suggests that the promotion of eIF2α signaling may activate endogenous mechanisms  to degrade p53.The inhibition of p53 transcriptional activity by pifithrin α or its mitochondrial targeting by pifithrin μ protects the brain in rodent models of strokeCG17259 RNAi lines, transgenic lines were obtained from the w1118 background using the SympUAST-w vector67 to target −100 to −1 in the 5′-UTR from the translational start site of CG17259. The other RNAi (TRiP) lines were obtained from the Tsinghua Drosophila stock center . All other fly lines were obtained from The Bloomington Drosophila Stock Center .Flies were raised on standard sucrose/cornmeal medium at constant 18 °C or 25 °C with 12 hours light and 12 hours dark cycle. For AG flies were crossed with deficient lines or point mutations or P-element-based mutations or RNAi lines obtained from the Bloomington Drosophila Stock Center, and raised at 18°C. The 3-day-old progeny flies were incubated at 30°C for 13 h, then transferred back to 18°C and after 2 days, their survival rate was recorded.The The following antibodies were used in this study : p53 , actin ; p53 ; eIF2αph ; Hsp26 ; Hsp27; p53 ; LC3 ; Mdm2; p53; ubiquitin .2O and dehydrated for 1 × 10 min in 25, 50, 70, 80, 90% acetone and 2 × 10 min in 100% acetone. The heads were then incubated for 3 × 5 min in propylene oxide and incubated overnight in a 1:1 mixture of propylene oxide and Spurr’s medium. The heads were then immersed in 100% Spurr’s for 2 × 2 h and baked in moulds at 60 °C for 24 h.Heads was collected and fixed overnight on ice in 2.5% glutaraldehyde. The heads were then washed for 3 × 10 min in PB and postfixed on ice for 4 h in 1% osmium tetroxide. The samples were then washed at room temperature 3 × 10 min in HXbp1 full Forward: GCAGGCGCTGAGGGCTGTGCReverse: GGACACACAGTCGTCAGCGCGTCTXbp1 splice Forward: GCAGGCGCTGAGGGCTGTGCReverse: AGACGCGCTGACGACTGTGTGTCC.68.Total RNA was extracted by Trizol reagent (Invitrogen) followed by DNase I treatment using the manufacturer’s standard protocol, then the purity and integrity of total RNA was determined by 1% agarose gel electrophoresis. The concentration of total RNA was measured by Nanodrop. Five μg mRNA was reverse-transcribed into a cDNA library by oligo-dT primer using Revert Aid First Strand cDNA Synthesis Kit (Thermo scientific) based on the manufacturer’s instructions. For qPCR, the final volume of the q-PCR reaction was 25 μl using a Platinum SYBRGreen qPCR SuperMix-UDG Kit (Invitrogen) containing 1μl diluted cDNA sample (1:3). The qPCR was performed in triplicate using the 7500 real time PCR system (ABI). The quantification of target gene was conducted by ΔΔCt methodDrosophila genes:qPCR primer sets for hsp26 Forward: GCTTTCGCTTGTGGATGAACTReverse: CCCAGTCCAAGCTCGTAGATGhsp27 Forward: GGACGGCCATGGAATGATCReverse: GCCCTTGGGCAGGGTATACThsp70-4 Forward: CCAACCAGCTGGCTGACAAReverse: GCACACACCCTCCAGTTCCThsp83 Forward: CCGCAATCCCGATGATATCTReverse: GTCGTTGGTCAGGGATTTGTAGACG17259 Forward: TGACCTCCCCACACGACAAReverse: CCGCATTCCCGATCATCTCGAPDH Forward: CGCAGCGCCATTCTCCTAReverse: GACTGCCGCTTTTTCCTTTTCqPCR primer sets for human gene:Hsp27 Forward: TGACCTCCCCACACGACAAReverse: CCGCATTCCCGATCATCTC.The fly heads were homogenized and lysed in the lysis buffer ). After centrifugation at 13,000 g for 10 min at 4 °C, the supernatant was incubated with Normal IgG antibody  and protein A/G-agarose beads at 4 °C for 2 h. After centrifugation at 13,000 g for 10 min at 4 °C, the supernatant was incubated with the requisite antibody and protein A/G-agarose beads at 4 °C overnight. After washing with washing buffer  6 times, the agarose beads were boiled in 1 × SDS loading buffer.2. We tested for contamination every two months. Different concentrations of TM were used to pretreat these cells. Twenty-four hours later, glutamate (0.3 M) was applied to trigger PNN for four hours. Cell death was quantified by the ATP assay.SH-SY5Y cells  were cultured in DMEM supplemented with 15% (v/v) fetal bovine serum and 1%(v/v) penicillin/streptomycin in a 37 °C incubator with 5% CO69. After 8–11 days in vitro culture, the neurons were treated with 200 μM glutamate for 4 h. Cell death was quantified by the LDH assay.For neuron culture, cortical neurons from 18 day-after-fertilization embryos of mice were collected and cultured following a published protocol70, with the suture-insertion through the left external carotid artery of anesthetized rats. A laser Doppler blood flow monitor was used to ensure successful occlusion (>80% drop from pre-stroke baseline). TM was injected, intraperitoneally, 6 h before the ischemia surgery. The infarct volume was determined 6 h later. The body temperature was maintained with a heated pad, and the infarct volumes were determined by TTC staining  and analyzed using the Image J software.All experiments were approved by the Institutional Animal Care and Use Committee, Peking University; and these experiments were performed in accordance with the relevant guidelines and regulations by the university. For the rat MCAO model, adult male Sprague-Dawley rats , were housed with a 12 h light and 12 h dark cycle. The number of rats per cage was 3 before surgery and they were raised separately after surgery. A permanent MCAO method was used as described previouslyt test with 2-tailed pairing was used to compare two data sets. p < 0.05 is considered statically significant.One-way ANOVA test was used for group comparison, with Tukey’s post hoc test. Student-Supplementary information


Propionibacterium acnes (P. acnes) is an anaerobic, Gram-positive bacteria encountered in inflammatory acne lesions, particularly in the pilosebaceous follicle. P. acnes triggers a strong immune response involving keratinocytes, sebocytes and monocytes, the target cells during acne development. Lipoteicoic acid and peptidoglycan induce the inflammatory reaction, but no P. acnes surface protein interacting with Toll-like receptors has been identified. P. acnes surface proteins have been extracted by lithium stripping and shown to induce CXCL8 production by keratinocytes.P. acnes strains used in this study, CAMP1-TLR2 binding intensity was modulated and appeared to be strong in type IB and II strains, which produced large amounts of CXCL8, whereas most of the type IA1 and IA2 strains presented little or no CAMP1-TLR2 binding and low levels of CXCL8 production. The nucleotide sequence of CAMP factor displays a major polymorphism, defining two distinct genetic groups corresponding to CAMP factor 1 with 14 amino-acid changes from strains phylotyped II with moderate and high levels of CAMP1-TLR2 binding activity, and CAMP factor 1 containing 0, 1 or 2 amino-acid changes from strains phylotyped IA1, IA2, or IB presenting no, weak or moderate CAMP1-TLR2 binding.Far-western blotting identified two surface proteins, of 24.5- and 27.5-kDa in size, specifically recognized by TLR2. These proteins were characterized, by LC-MS/MS, as CAMP factor 1 devoid of its signal peptide sequence, as shown by N-terminal sequencing. Purified CAMP factor 1 induces CXCL8 production by activating the CXCL8 gene promoter, triggering the synthesis of CXCL8 mRNA. Antibodies against TLR2 significantly decreased the CXCL8 response. For the 27 P. acnes virulence, by amplifying the inflammation reaction through direct interaction with TLR2.Our findings indicate that CAMP factor 1 may contribute to  Propionibacterium acnes (P. acnes) is an anaerobic Gram-positive bacterium frequently present in the normal human skin microbiota, where it accumulates preferentially in the pilosebaceaous units in individuals with and without acne , then incubated overnight with 10 ml of human recombinant TLR2  diluted to 0.1 μg/ml in TBS/T, at 4°C, with gentle mixing. Unbound antibodies were removed by washing as described above, and the membrane was incubated with biotinylated human antibodies against TLR2 or TLR4  diluted to 0.1 μg/ml in TBS/T supplemented with 5% BSA, for 20 h at 4°C, with gentle mixing. The membrane was washed to remove unbound antibodies, and the surface protein-TLR2-biotinylated antibody complex was detected by incubation for 1 h at room temperature with HRP (horseradish peroxidase) diluted to 0.5 μg/ml in saturation buffer. Unbound material was removed by washing and peroxidase activity was detected in a chemiluminescence assay .g for 15 minutes at 4°C and the supernatant was used as total protein extract. Protein samples (15 μg), and lysates of human TLR2-transfected 293T cells were fractionated by electrophoresis in a NuPAGE Novex 4–12% Bis-Tris gel  and transferred onto nitrocellulose membranes. Membranes were saturated by incubation for 1 h at room temperature in TBS/T buffer and incubated at 4°C for 18 h with goat polyclonal IgG antibodies against human TLR2 (C-19), diluted to 0.5 μg/ml in TBS supplemented with 5% BSA, and 0.1% Tween-20 . The membranes were washed and bound antibodies were detected by incubation with donkey polyclonal antibodies against goat IgG coupled to peroxidase, diluted 1:2000 in saturation buffer, for 1 h at room temperature. Unbound material was removed by washing and peroxidase activity was detected in a chemiluminescence assay .HaCaT and ThP1 cells were washed in sterile cold PBS and lysed in RIPA buffer containing 50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (pH 8.0). Total extracts were centrifuged at 14,000 x g and 4°C. Protein pellets were suspended in PBS and protein concentration was determined by spectroscopy at 280 nm (NanoDrop).Proteins were fractionated by electrophoresis in denaturing conditions  and detected by Coomassie blue. Proteins from a second gel were transferred onto nitrocellulose membranes and analyzed by Far-western blotting, as described above. Proteins recognized by TLR2 were matched to the stained bands, and excised from the gel. Excised protein bands were incubated for 18 h at 37°C in the presence of elution buffer (1% SDS in PBS), with stirring. The excess of SDS was removed by precipitation of the proteins in cold acetone (vol/vol) and centrifugation for 30 minutes at 14 000 x TM  [Renilla luciferase expression vector. The cells were incubated for 24 h, and were left unstimulated or were stimulated with LiE or proteins of interest eluted from the gel after electrophoretic separation. After 18 h of incubation at 37°C, cells were washed with sterile cold PBS and scraped directly into the lysis buffer provided by the manufacturer . Firefly and Renilla luciferase activities were measured on a MicroLumat Plus LB 96V Luminometer . Results are expressed as a fold-increase relative to the basal activity measured in unstimulated transfected cells.HaCaT cells were cultured in 24-well plates (Corning Costar) with DMEM containing 0.1% FCS without antibiotics. They were then transfected with reporter plasmids containing the CXCL8 promoter (-173 bp) or the NF-κB promoter (5 units) upstream from the firefly luciferase gene in JetPEI France) . Transfe2 and stimulated with whole bacteria (MOI 10), LiE (50 μg/ml), or proteins of interest eluted after electrophoretic separation (50 μg/ml). After stimulation, cells were washed twice with cold sterile PBS and scraped into lysis buffer, according to the manufacturer’s instructions (Macherey-Nagel). Total RNA was isolated with the NucleoSpin® RNA II kit (Machery-Nagel), according to the manufacturer’s instructions. RNA concentration was determined at 260 nm (NanoDrop). It ranged from 110 to 225 ng/μl, and was adjusted to desired concentration. RT-qPCR was performed with the iTaq Universal Sybr Green one-Step kit (BioRad) containing reverse transcriptase (RT) and Taq polymerase. Complementary DNA was generated from 330 ng of total RNA by RT for 10 minutes at 50°C, in a final volume of 10 μl. Standard amplification for quantitative PCR was carried out in the same test tube containing the reaction mixture, and the PCR conditions were as follows: 95°C for 60 s, followed by 40 cycles of 95°C for 15s, 68°C for 60s, and 72°C for 15s, followed by a final stage to obtain a melting curve for 65 to 95°C, at 0.1°C/s over 60 s on a LightCycler Nano (Roche). PCR results were calculated as a fold-increase relative to GADPH gene expression. Threshold cycles (Ct) were determined for the genes studied, from the amplification curves. The amount of RNA in stimulated cells relative to control cells was calculated by the 2ΔCt method. We used the TCTTGGCAGCCTTCCTGATT and TTTCGTGTTGGCGCAGTGT primers for IL-8 and the GCCACATCGCTCAGACAC and GCCCAATACGACCAAATCC primers for GAPDH, which was used as a control.HaCaT cells were grown in six-well plates (Corning Costar) for 24 h with 10% FCS in DMEM, and then for 24 h in 0.1% FCS DMEM. HaCaT cells were then incubated for 5 h at 37°C under an atmosphere containing 5% CO2O/CH3CN solution, to extract the peptides from the gel fragments. Peptide extracts were then dried and dissolved in the initial buffer for chromatographic elution, which consisted of 3% CH3CN and 0.1% HCOOH in water. Peptides were enriched, separated and analyzed with a 6520 Accurate-Mass Q-TOF LC/MS machine equipped with an HPLC-chip cube interface . The fragmentation data were interpreted with Mass Hunter software . For protein identification, MS/MS peak lists were extracted, converted into mzdata.xml format files and compared with the protein database, using the MASCOT Daemon  search engine. The searches were performed with no fixed modification, with variable modifications for methionine oxidation, and with a maximum of one missed cleavage. MS/MS spectra were searched with a mass tolerance of 20 ppm for precursor ions and 0.6 Da for MS/MS fragments. Only peptides matching an individual ion score >56 were considered. Proteins with two or more unique peptides matching the protein sequence were automatically considered as a positive identification.Proteins were separated by electrophoresis in denaturing conditions  and detected by Coomassie blue. Proteins from a second gel were transferred onto nitrocellulose membranes and analyzed by Far-western blotting, as described above. Proteins recognized by TLR2 were matched to stained bands, excised from the gel, reduced by incubation at 50°C for 1 h with 10 mM DTT and then alkylated by incubation for 1 h in the dark with 55 mM iodoacetamide. Fragments were washed several times with water and ammonium carbonate, dehydrated with acetonitrile (ACN) and dried. Trypsin digestion was performed overnight with a dedicated automated system . The gel fragments were then incubated twice, for 15 minutes each, in a HFor N-terminal determination, 20 μg of protein mixture was subjected to electrophoresis and the bands of interest were excised and incubated in a buffer for passive extraction of the protein from the acrylamide gel. After overnight incubation with shaking, the solution was eluted on a ProSorb Filter (Applied Biosystems) to fix the protein on a PVDF disc. The PVDF disc was submitted to microsequencing by Edman degradation (8 cycles of pulsed-liquid chemistry), with a 494 automated protein sequencer (Applied Biosystems).P. acnes strains, we performed PCR with the C1-F and C1-R primers, amplifying a 946 bp fragment, as previously described [2 , 0.5 μl of each primer , 0.2 μl (1 U) of native Taq DNA polymerase , and distilled water. The PCR conditions for amplification were as follows: 95°C for 3 minutes, followed by 35 cycles of 95°C for 1 minute, 60°C for 30 s, and 72°C for 1 minute and a final extension phase at 72°C for 10 minutes. The PCR products (5 μl) were analyzed by electrophoresis in a 2% agarose gel and the amplified nucleotide bands were visualized by UV transillumination of the ethidium bromide-stained gel, with comparison to a 50-bp DNA ladder . Sequencing reactions were performed with ABI PRISM Ready Reaction Terminator cycle sequencing kits (Applied Biosystems) according to the manufacturer’s instructions. Sequencing reactions were performed with the Big Dye V3.1 Terminator Kit  according to the manufacturer’s instructions and analyzed on an ABI PRISM 3100 DNA sequencer (Applied Biosystems). DNA sequences were analyzed with SeqScape ® v2.5 Software from Applied Biosystems .For amplification of the nucleotide sequence of CAMP factor 1 from the 27 escribed . BrieflyP. acnes CAMP factor 1 sequences obtained in this study and other known CAMP sequences. These analyses were carried out with the maximum likelihood method. Multiple sequence alignments were obtained with Mafft version 7 [Phylogenetic analyses were performed to assess the relationships between the ersion 7  and expoersion 7 , for inpersion 7  and Protersion 7  to identersion 7 , implemeersion 7 . SequencAll the sequences determined in this study have been deposited in the GenBank database under the accession numbers listed in t-tests. P < 0.05 was considered significant. Statistical significance is indicated by * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001) and **** (P ≤ 0.0001), respectively.The statistical significance of differences between data from experimental groups was analyzed in paired Student’s S1 FigThe expression of TLR2 was assessed by western blotting, as described in the Materials and Methods. Lanes 2 and 4: 293T cell and ThP1 lysates used as positive controls. Lane 3: HaCaT keratinocyte lysate. Lane 1: MagicMark molecular mass markers. Arrow indicates position of TLR2.(TIF)Click here for additional data file.S2 FigP. acnes surface proteins were extracted from a five-day culture bacterial pellet and separated by electrophoresis in 4–12% NuPAGE LDS BisTris gels (50 μg). The separated proteins were transferred onto nitrocellulose membranes, which were incubated with recombinant TLR2 (0.1 μg/ml). TLR binding activity was detected with specific biotinylated antibodies against TLR2, as described in the Materials and Methods. Lane 1 contains molecular mass markers. Lanes 2 to 22 contain proteins from strains 75150, 22197, 14230, 78910, 16351, 17248, 12513, 22795, 53468, 36862, 41103, 47474, 27647, 27387, 25236, A9, A11, A24, A26, A30, and A44, respectively. Arrows indicate the positions of the 24.5- and the 27.5-kDa bands of interest.(TIF)Click here for additional data file.S3 FigFive-day-old cultures from the 27 strains were used to extract DNA for multiplex-PCR, as described in the Materials and Methods, and the PCR products were analyzed by electrophoresis in a 1.5% agarose gel. Lane 1: 50-bp DNA ladder. Lanes 2 to 28: strains 12513, 14230, 16351, 17248, 22197, 22795, 25236, 27387, 27647, 38862, 41103, 47474, 53468, 75150, 78910, A9, A11, A24, A26, A30, A44, 6919, CHR, PIE, RON, TRI, and GUE, respectively. Letters a, b, c, and e correspond to amplicons of 677, 494, 145, and 351 bp, respectively, as previously described [(TIF)Click here for additional data file.


Similar results were also observed in ASIC3−/− or ASIC3 antagonist (APETx2) injected mice. Using western blotting, we determined that pPKA, pPI3K, and pERK were increased during a dual acidic injection priming period. Nociceptive receptors, such as ASIC3, Nav1.7, and Nav1.8, were upregulated in the dorsal root ganglion (DRG) and spinal cord (SC) of FM mice. Furthermore, pPKA, pPI3K, and pERK were increased in the central thalamus. These aforementioned mechanisms were completely abolished in ASIC3 knockout mice. Electrophysiological results also indicated that acid potentiated Nav currents through ASIC3 and ERK pathway. Our results highlight the crucial role of ASIC3-mediated mechanisms in the treatment of FM-induced mechanical hyperalgesia.Many scientists are seeking better therapies for treating fibromyalgia (FM) pain. We used a mouse model of FM to determine if ASIC3 and its relevant signaling pathway participated in FM pain. We demonstrated that FM-induced mechanical hyperalgesia was attenuated by electroacupuncture (EA). The decrease in fatigue-induced lower motor function in FM mice was also reversed by EA. These EA-based effects were abolished by the opioid receptor antagonist naloxone and the adenosine A1 receptor antagonist rolofylline. Administration of opioid receptor agonist endomorphin (EM) or adenosine A1 receptor agonist N Fibromyalgia (FM) pain, which is a crucial worldwide health problem, is characterized by widespread mechanical pain1371112Acid-sensing ion channel 3 (ASIC3), the most sensitive ion channel for acid sensation, is mainly localized in peripheral sensory neurons, especially the DRG, and typically associated with several pain conditions141618Navs are involved in inflammatory pain, as evidenced by the significant amelioration of pain by sodium channel blockers222326262626Several molecules, such as N-methyl-D-aspartate receptors (NMDARs), ASIC3, TRPV1, calcium channels (Cav), and substance P (SP), have been implicated in mouse models of FM pain1213213233372642Our rational is that EA could reduce FM symptoms through ASIC3 receptor and related mechanisms. We hypothesized that EA could attenuate mechanical hyperalgesia by releasing endomorphin or adenosine to inhibit ASIC3 receptor in this FM mouse model. Our data showed that pPKA/pPI3K-pERK signaling pathway was crucial for FM priming in mice. EA at acupoint ST36 reliably reduced dual acidic saline-induced mechanical hyperalgesia and motor dysfunction via ASIC3-Nav1.7/Nav1.8 signaling pathways. We also determined that mechanical hyperalgesia and nociceptive signaling were abolished in ASIC3 knockout mice. This study provides novel and detailed molecular mechanisms behind the use of EA to treat FM-related mechanical hyperalgesia.6-cyclopentyladenosine (CPA) significantly attenuated mechanical hyperalgesia (Asic3−/−) showed a bilateral decrease in mechanical hyperalgesia , and this symptom was alleviated by EA manipulation . Furthermore, this phenomenon was blocked by naloxone  and rolofylline . Injection of endomorphin (EM)  or N6-cyclopentyladenosine (CPA)  reliably improve motor function. Similar data was also observed in ASIC3−/− or APETx2 injected mice . These data suggest that FM-induced mechanical hyperalgesia and fatigue depend on ASIC3, opioid and adenosine receptors.Similar to our previous publication7ralgesia , n = 10.ralgesia , n = 10.ralgesia , n = 10.p > 0.05, n = 6) or after EA treatment . Interestingly, pPKA expression was increased within this window in FM mice  and rescued by EA . A similar pattern was observed for pPI3K signals . There were no significant differences in pPKC among the 3 groups . pERK levels increased after the first acidic saline injection , which was attenuated by EA treatment . Both Nav1.7 and Nav1.8 were not affected in any group . Next, we assessed the central SC after the first saline injection. ASIC3 was not altered by FM , and this effect was blocked by EA . The same results were observed for pPI3K . There were no significant differences in pPKC levels among the 3 groups . The expression of pERK was increased in the SC after the first acidic saline injection , which was alleviated by EA treatment . Both Nav1.7 and Nav1.8 levels were similar across all groups . We examined if similar mechanisms occurred after a second acidic saline injection. ASIC3 was not changed at 15 min after acid injection in the DRG or SC of FM mice . The FM-induced increase in pPKA was attenuated by EA . A similar result was obtained for pPI3K . No changes in pERK  or nociceptive Nav1.7 and Nav1.8 . EA treatment restored ASIC3 levels . Interestingly, these EA-mediated effects were blocked by naloxone , rolofylline , or not existed in ASIC3−/− mice . Next, we assessed ASIC3-related downstream nociceptive signaling. Western blot results showed that the levels of pPKA, pPI3K, and pPKC were unchanged in all groups ; thus, these molecules are not involved at the time point tested. Based on our previous results, we assessed if pERK-positive neurons were alteredp > 0.05, n = 6). We next examined nociceptive-relevant Nav channels. Nav1.7 was increased in FM mice  and restored to normal levels in the EA group . The EA-mediated Nav1.7 reduction was blocked by opioid, adenosine antagonists, or ASIC3 gene deletion . Similar effects on Nav1.8 protein levels were found . Therefore, we speculate that the ASIC3-Nav1.7/Nav1.8 signaling pathway is responsible for persistent FM-induced hyperalgesia in the peripheral DRG.We examined if ASIC3-related nociceptive proteins were altered in our treatment model. ASIC3 showed a normal distribution in the control group . This increase was alleviated by 2 Hz EA stimulation at acupoint ST36 . Furthermore, naloxone  and rolofylline  blocked the effect of EA on ASIC3, and not existed in ASIC3−/− mice ; thus, EA acts through opioid and adenosine receptors. In addition, we assessed the levels of protein kinases, which are involved in several pathways. Proteins pPKA, pPI3K, and pPKC showed no change in the SC of FM mice on Day 8 after modeling . Downstream of protein kinase, pERK was also unaltered on Day 8 . EA treatment restored Nav1.7 levels in FM mice , and this effect was blocked by naloxone , rolofylline , and ASIC3 gene deletion . Nav1.8 is crucial for FM development; therefore, we assessed if EA altered Nav1.8 expression. Nav1.8 protein signals were distributed in DRG neurons . Nav1.8 levels were ameliorated by EA at 2 Hz, suggesting that EA alleviates FM-mediated nociception . The effects of EA were blocked by naloxone , rolofylline , or ASIC3 gene deletion . These data support the hypothesis that EA acts via opioid and adenosine pathways in the SC.ASIC3 was distributed in the SC of control mice , and restored by EA . The rescue effect of EA on ASIC3 was mediated by opioid , adenosine pathways , or not existed in ASIC3−/− mice . An increase of pPKA was found in the thalamus of FM mice  and rescued by EA . The effect of EA was blocked by opioid , adenosine  antagonists, or ASIC3 gene deletion  Similar data were obtained for pPI3K . Nav1.7 was increased in the thalamus of FM mice  and rescued by EA  via opioid , adenosine , and ASIC3 pathways . A similar pattern was found for Nav1.8  purchased from BioLASCO Co. Ltd, Taipei, Taiwan. The sample size required for an alpha of 0.05 and a power of 80% was eight animals per group. After arrival, the mice were housed under a 12/12 h light/dark cycle, where water and food were available ad libitum. All of the procedures were approved by the Institute of Animal Care and Use Committee of China Medical University (permit No. 2016–061) and they were conducted in accordance with the Guide for the use of Laboratory Animals provided by the National Research Council and the ethical guidelines of the International Association for the Study of Pain. The number of animals used and their suffering were minimized.the opioid antagonist naloxone methiodide   in 100 μl of saline was injected i.p. at a dose of 10 mg/kg. The adenosine A1 receptor antagonist rolofylline (Ro)  in 10 μl of saline was injected i.m. at a dose of 3 mg/kg into acupoint ST36. The opioid agonist endomorphin (EM) , in 100 μl of saline, was administered intraperitoneally (i.p.) at a dose of 10 mg/kg once a day. Alternatively, the adenosine receptor agonist N6-cyclopentyladenosine (CPA)  in 10 μl of saline was administered intramuscularly (i.m.) at a dose of 0.1 mg/kg into acupoint ST36 once a day. A dose of 20 pmole APETx2  was injected into ST36 acupoint under light isoflurane anesthesia (1%).EA was applied using stainless steel needles  that were inserted into the muscle layer to a depth of 2–3 mm at ST36 acupoint. EA was administered immediate after the second injection acid saline every day at the same time (10:00–12:00 AM). A Trio-300 (Japan) stimulator delivered electrical square pulses for 15 min with a 100 μs duration and a 2 Hz frequency. The stimulation amplitude was 1 mA. For pharmacological injection, opioid or adenosine A1 receptor antagonist administration, We injected 20 μL of pH 4.0 acid saline into gastrocnemius muscle (GM) while the mice were anesthetized with isoflurane (1%). The second acid saline injection was performed at day 5 after first injection to successfully induce FM mice. All experiments were performed at room temperature (approximately 25 °C) and the stimuli were applied only when the animals were calm but not sleeping or grooming. Mechanical sensitivity was measured by testing the force of responses to stimulation with three applications of electronic von Frey filaments . Mice were placed on a metal mesh and adapted to the new environment for at least 30 min. The mechanical hyperalgesia of the hindpaw was measured before, 4 h, 1, 5, 6, 8 days after modeling. The FM mice were further euthanized and the L3-L5 DRG neurons, lumbar SC, and brain thalamus were isolated for analysis.The mice were put on a rotating machine with different speeds and durations can be tested. When mice fall down, the sensor can record the falling latency. Mice were placed on an accelerating rotarod apparatus  for 12 trials  with 5-min intervals between trials. Each trial lasted for 60 s with a steady speed of 4 rpm. The latency of each mouse falling from the rod was recorded for each trial. On day 4, mice underwent 4 rpm and increase the speeds with a 3 rpm increase with a 10 s duration.3VO4, 0.02% NaNO3, and 1 × protease inhibitor cocktail (AMRESCO). The extracted proteins (30 μg per sample according to the BCA protein assay) were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBS-T buffer , incubated with the first antibody in TBS-T and 1% bovine serum albumin, and incubated for 1 h at room temperature. A peroxidase-conjugated anti-rabbit antibody (1:5000) was used as the secondary antibody. The bands were visualized using an enhanced chemiluminescencent substrate kit (PIERCE) with LAS-3000 Fujifilm (Fuji Photo Film Co. Ltd). If appropriate, the image intensities of specific bands were quantified with NIH ImageJ software . The protein ratios were obtained by dividing the target protein intensities by the intensity of α-tubulin in the same sample. The calculated ratios were then adjusted by dividing the ratios from the same comparison group relative to the control.L3-L5 DRG, lumbar SC, and thalamus tissues were excised to extract proteins. The total proteins were prepared by homogenizing the DRG, SC, and thalamus in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 250 mM NaCl, 1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM NaMice were anesthetized with isoflurane and then perfused transcardially with 4% paraformaldehyde. The tissue samples were cryprotected with 30% sucrose. The tissues were cut to a thickness of 15 μm and were post-fixed briefly with 4% paraformaldehyde and then incubated with blocking solution containing 3% BSA, 0.1% Triton X-100, and 0.02% sodium azide in PBS for 2 h at room temperature. After blocking, the sections were incubated at 4 °C overnight with the primary antibodies prepared in blocking solution. The secondary antibody was goat anti-rabbit (1:500) antibody . We incubated the slices with fluorescence-conjugated secondary antibodies or avidin-biotin horseradish peroxidase complex (1 h), washed them three times with 0.1 M Tris buffer (5 min each), and then developed them in diaminobenzidine tetrahydrochloride (1–2 min), before washing three times with 0.1 M Tris buffer (5 min each). Finally, the sections were incubated with 0.1 M Tris buffer to stop the reaction. The slides were mounted with cover slips and visualized by using a CKX41 microscope with an Olympus U-RFLT50 Power Supply Unit .2, 2 CaCl2, 10 glucose, and 20 Hepes, adjusted to pH 7.4 with NaOH. ACSF solutions were applied by use of gravity. The recording electrodes were filled with (in mM) 100 KCl, 2 Na2-ATP, 0.3 Na3-GTP, 10 EGTA, 5 MgCl2, and 40 Hepes, adjusted to pH 7.4 with KOH. The pH 5.0 ACSF was titrated by 2-[N-morpholino]ethanesulfonic acid (MES). For Nav current recording, the internal solution contained (in mM) 10 NaCl, 110 CsCl, 20 tetraethylammonium-Cl, 2.5 MgCl2, 5 EGTA, 3 Mg2+-ATP, and 5 HEPES, adjusted to pH 7.2 with CsOH. The external solution contained (in mM) 100 NaCl, 5 CsCl, 30 tetraethylammonium-Cl, 1.8 CaCl2, 1 MgCl2, 0.1 CdCl2, 25 glucose, 5 4-aminopyridine, and 5 HEPES, adjusted to pH 7.4 with HCl. Osmolarity was adjusted to 300 mosm. Voltage-gated sodium channel currents were evoked by a 50 ms test pulse from −70 to 0 mV. All recordings were obtained at room temperature (25 °C) and completed within 24 h after plating. Salicylic acid (SA) was prepared from a 1-M stock solution (in 100% ethanol) to a final concentration of 500 μM in ACSF. U0126 was from Tocris-Cookson .L3–L5 DRGs were isolated from mice at 8 days after FM injection. For ASIC3 currents recording (checked by SA inhibition), recording cells were superfused in artificial cerebrospinal fluid (ACSF) containing (in mM) 130 NaCl, 5 KCl, 1 MgClp < 0.05 was considered significantly different.All of the data were expressed as the mean ± standard error. Significant differences between groups were tested using ANOVA, followed by a post hoc Tukey’s test. How to cite this article: Yen, L.-T. et al. Targeting ASIC3 for Relieving Mice Fibromyalgia Pain: Roles of Electroacupuncture, Opioid, and Adenosine. Sci. Rep.7, 46663; doi: 10.1038/srep46663 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


However, the signaling machinery mediating neuronal activity-induced gene expression, especially the rapid transcription of immediate-early genes (IEGs) remains unclear. Cyclin-dependent kinases (Cdks) are a family of serine/threonine kinases that have been firmly established as key regulators of transcription processes underling coordinated cell cycle entry and sequential progression in nearly all types of proliferative cells. Cdk7 is a subunit of transcriptional initiation factor II-H (TFIIH) and the only known Cdk-activating kinase (CAK) in metazoans. Recent studies using a novel Cdk7 specific covalent inhibitor, THZ1, revealed important roles of Cdk7 in transcription regulation in cancer cells. However, whether Cdk7 plays a role in the regulation of transcription in neurons remains unknown. In this study, we present evidence demonstrating that, in post-mitotic neurons, Cdk7 activity is positively correlated with neuronal activities in cultured primary neurons, acute hippocampal slices and in the brain. Cdk7 inhibition by THZ1 significantly suppressed mRNA levels of IEGs, selectively impaired long-lasting synaptic plasticity induced by 4 trains of high frequency stimulation (HFS) and prevented the formation of long-term memories.In the brain,  They interact with cyclins, the regulatory subunits providing domains essential for enzymatic activity and substrate specificity of Cdks  approved by the Southeast University Animal Care and Use Committee.Cortical cultures were prepared from postnatal day 1 (P1) pups as previously described . Debris was removed by centrifugation at 14,000 g (4°C) for 10 min. Protein samples were separated on SDS-PAGE and electrotransfered to a PVDF membrane. Membranes were blocked with 5% dry milk for 1 h and incubated with primary antibodies in TBST overnight at 4°C. After washing and incubation with secondary antibodies for 1 h, membranes were visualized with the chemiluminescence substrate (Thermo Fisher Scientific) and detected by ImageQuant™ LAS 4000 . Primary antibodies were used as follows: anti-CDK7 , anti-RNAPII CTD repeat YSPTSPS , anti-RNAPII CTD repeat YSPTSPS , anti-RNAPII CTD repeat YSPTSPS , anti-tubulin . Secondary antibodies were used as follows: goat anti-rabbit , goat anti-mouse . Preparation of CA1 tissue punch lysates for SDS-PAGE: hippocampi slices were isolated and were allowed for recovery at 28°C for at least 2 h in a submersion chamber perfused with 95% O2/5% CO2 saturated artificial cerebrospinal fluid (ACSF). Slices of hippocampi were pre-treated with THZ1 for 4 h before stimulations. After early-phase long-term potentiation (E-LTP) or late-phase LTP (L-LTP) stimulation, CA1 areas were removed for preparing protein samples and SDS-PAGE assay.Proteins were extracted from mice cortex using lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na−ΔΔCt method in which the target RNA was adjusted to that of Gapdh. Equations were used as follows: ΔCt = Ct Target − Ct Gapdh, ΔΔCt = ΔCt Treat − ΔCt Ctrl. mRNA levels of drug treatment groups were normalized to the mRNA levels of control groups that received equal duration of treatment with appropriate vehicles .Total RNA was isolated from mice cortical sections or cultured cortical neurons using Trizol reagent (Sangon Biotech) according to the manufacturer’s instructions. Total RNA (1 μg) of each sample was reverse transcribed into cDNA by using the Hiscript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech). Then the cDNA were amplified by using a SYBR Green Master Mix Kit (Vazyme Biotech) in the CFX96 real-time PCR system (Bio-Rad). The amplify cycle was used as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s, 59°C for 1 min. The target genes expression levels were analyzed using the 2Npas4, forward: CTGCATCTACACTCGCAAGG, reverse: GCCACAATGTCTTCAAGCTCT; cFos, forward: ATGGGCTCTCCTGTCAACACAC, reverse: ATGGCTGTCACCGTGGGGATAAAG; Arc, forward: TACCGTTAGCCCCTATGCCATC, reverse: TGATATTGCTGAGCCTCAACTG; Nr4a1, forward: AGCCCAGGACCGCGTGACC, reverse: GGCAGCTGGTAGAGGAAGGTG; Egr1, forward: GGGAGCCGAGCGAACAA, reverse: CATTATTCAGAGCGATGTCAGAA; c-Jun, forward: TCCACGGCCAACATGCT, reverse: CCACTGTTAACGTGGTTCATGAC; Homer1a, forward: GAAGTCGCAGGAGAAGATG, reverse: TGATTGCTGAATTGAATGTGTACC; Gapdh, forward: AGGTCGGTGTGAACGGATTTG, reverse: TGTAGACCATGTAGTTGAGGTCA.The primers for target genes were used as follows: 2/5% CO2. All slices were sagittaled to 360 μm. ACSF contained (in mM): 120.0 NaCl, 3.0 KCl, 1.0 NaH2PO4, 26.0 NaHCO3, 11.0 D-glucose, 1.2 MgSO4 and 2.0 CaCl2. The slices were recovered at 28°C for at least 2 h before a single slice was transferred to a submersion chamber perfused with 95% O2/5% CO2 saturated ACSF. Synaptic transmission was evoked by stimulation at 0.067 Hz (for field recordings) of Schaffer collaterals and recorded with glass pipettes (3–5 MΩ) filled with ACSF. For field recording: E-LTP was induced by two trains of high-frequency stimulation  stimulation, the interval between each train was 10 s. L-LTP was induced by four trains of HFS  stimulation, the interval between each train was 5 min. All data acquisition and analysis were done using pCLAMP 10.2 (Axon Instruments). In all electrophysiological experiments, n represents the number of neurons or slices, and normally only one or two slices per animal were used.Standard procedures for brain slices were described as previous study  was aimed at the left lateral ventricle using the following coordinates: anteroposterior (AP) relative to Bregma, −0.5 mm; lateral (L) to midline, 1.0 mm; ventral (V) from the skull surface, −2.0 mm. Prior to tests, mice were recovered for a week and handled for 5 min a day for last 3 days. For the novel object recognition task, the mice were individually placed and accustomed to a square open field (50 cm × 50 cm × 50 cm) for 10 min for 4 days. THZ1-hydrochloride  and equal volume of DMSO (1 μl) were injected with 0.5 μl/min flow via the cannula controlled by a pump  and the mice were awake. Three hours later, mice were exposed to two identical objects (object 1 and object 2) for 10 min, mice were returned to the field that now contained, the familiar object (object 2) and a novel object which was different in the size and color 2 h (for short term memory (STM)) or 24 h (for long term memory (LTM)) later. The mice were independent cohorts for STM and LTM. Time spent interacting with each object was manually analyzed in a blind manner. Discrimination index was calculated as % time with object 1/(time interaction with object 1 + time interaction with object 2) and novel object/(time interaction with novel object + time interaction with object 2). THZ1-hydrochloride (20 μg per mouse dissolved in DMSO at the concentration of 40 mM) and equal volume of DMSO (1 μl) were injected with 0.5 μl/min flow via the cannula controlled by a pump to allow the mice awake. Three hours later, each mouse was placed in a fear conditioning chamber and allowed to explore it for 3 min before the delivery of a 40 s tone (80 dB) which was immediately accompanied with a 2 s foot shock (0.7 mA) in the last 2 s of the tone, 40 s and 120 s later the second and third pairs of tone and foot shock stimulation were given. Mice were removed from the testing chambers 10 s after the third shock. During the cued test, the mouse was placed in a new chamber with different contextual cues for 2 h (for STM) or 24 h (for LTM) later. The mice were independent cohorts for STM and LTM. After a 3-min acclimation, the same tone used for training was presented without shocks, and freezing behavior was monitored for 4 min. Freezing behavior was analyzed for the percent of freezing time during the cued fear conditioning test. Percentage of freezing time was quantified using automated motion detection software Superfcs .post hoc tests or by Student’s t-test, wherever applicable. All data analyzed were normally distributed. Results with P values of less than 0.05 were regarded as statistically significant, and * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 in the graphs.All the averaged data were stated as mean ± SEM. Statistical significance was assessed by one-way ANOVA with Bonferroni First, we examined the expression pattern of Cdk7 in the brain at different ages in mice. Quantitative RT-PCR results showed similar levels of Cdk7 mRNA at embryonic day 14.5 (E14.5) and postnatal day 0 (P0), which gradually decreased to about 40% at the adult ages , DIV 5, DIV 9 and DIV 15 by Western blot , are essential to consolidate newly acquired memory . So far, we have demonstrated the critical role of Cdk7 in activity-driven gene expression of IEGs. Next, to address the question whether Cdk7-regulated transcription underlies the forms of synaptic plasticity that require gene expression, we performed electrophysiological recordings in the CA1 region of the hippocampal Schaffer collateral pathway. In this pathway, two distinct phases of LTP, namely E-LTP and L-LTP, exist and are thought to be important for STM and LTM, respectively  protein family, binds acetylated histones and regulates general transcription in many cell types. Similar to the effects of THZ1-mediated Cdk7 inhibition examined in this study, Brd4 inhibitor Jq1 significantly prevented IEG transcription, synaptic modifications and LTM in mice (Korb et al., CDK7 inhibitor THZ1 has been proposed as potential treatment for several types of cancer (Chipumuro et al., GH: performed the experiments, analysis and interpretation of data, revising the article; XY: performed the experiments, analysis and interpretation of data; GW, JQ, RM and ZW: performed the experiments, analysis of data; ZZ: conception and design, interpretation of data, designed the study, wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BRAFV600 kinase approved in more than 90 countries, based on results obtained primarily in Caucasian patients. Limited data are available regarding the efficacy and safety of vemurafenib in Asian patients.Melanoma is a rare, deadly disease without effective treatment options in China. Vemurafenib is a selective inhibitor of oncogenic BRAFV600 mutation–positive unresectable or metastatic melanoma. The study included two cohorts: a pharmacokinetic cohort (n = 20) and an expansion cohort (n = 26).This phase I study investigated the pharmacokinetics, efficacy, and tolerability of vemurafenib (960 mg twice daily) in Chinese patients with After 21 days of dosing, vemurafenib demonstrated marked accumulation and relatively constant steady-state exposure over the dosing period. Confirmed best overall response rate was 52.2% (95% CI 37.0–67.1%). Median progression-free survival was 8.3 months (95% CI 5.7–10.9%); median overall survival was 13.5 months (95% CI 12.2%–not estimable). The most common adverse events were dermatitis acneiform, arthralgia, diarrhea, blood cholesterol level increase, blood bilirubin level increase, melanocytic nevus, and alopecia. A total of nine grade 3 or 4 adverse events were reported in seven patients (15.2%).Overall, vemurafenib showed a favorable benefit-risk profile among Chinese patients. Pharmacokinetics, safety, and efficacy were generally consistent with those reported in Caucasian patients.NCT01910181. Registered 29 July 2013, prospectively registered.ClinicalTrials.gov identification: The online version of this article (10.1186/s12885-018-4336-3) contains supplementary material, which is available to authorized users. Melanoma is relatively rare in China, with a reported incidence of 0.6 per 100,000 persons in 2012 [Several new treatment options for metastatic melanoma have emerged in the past 5 years, including monoclonal antibodies targeting the programmed cell death 1 receptor and cytotoxic T-lymphocyte–associated antigen 4 and small-molecule inhibitors of BRAF and MEK. However, clinical trials for these agents have been conducted almost exclusively in Caucasian populations. Consequently, these agents are largely considered investigational in China . DespiteBRAF mutations, resulting in constitutive BRAF kinase activity [BRAF mutation is approximately 25% [BRAF kinase. In the pivotal phase III study (BRIM-3), vemurafenib significantly improved progression-free survival (PFS) and OS compared with dacarbazine in patients with BRAFV600-mutated metastatic melanoma [BRAFV600 mutation–positive unresectable or metastatic melanoma.Approximately 50% of melanomas in Caucasian patients harbor activity . Among Ctely 25% . Vemurafmelanoma , 10. Mormelanoma , and limThis open-label, multicenter, phase I study enrolled two cohorts: a pharmacokinetic cohort and an expansion cohort. For the pharmacokinetic cohort, patients received treatment in three periods after a 28-day screening period. In period A (days 1–21), patients received oral vemurafenib 960 mg twice daily. Patients fasted overnight for at least 8 h before and at least 4 h after the morning dose on days 1 and 21. Light snacks  were allowed in the 4-h post-dose period. All other doses were administered either 1 h before or 2 h after a meal. Only the morning dose was administered on day 21. In period B (days 22–27), patients temporarily discontinued vemurafenib to enable characterization of the elimination profile. In period C (day 28 onwards), patients continued to receive vemurafenib 960 mg twice daily until disease progression, unacceptable toxicity, withdrawal of consent, or discontinuation for any other reason.Enrollment in the expansion cohort was initiated after completion of recruitment into the pharmacokinetic cohort. In the expansion cohort, all patients received vemurafenib 960 mg twice daily until progressive disease, unacceptable toxicity, withdrawal of consent, or discontinuation for any other reason. Dose modifications (temporary interruption or dose reductions) were allowed within both cohorts for management of symptomatic adverse events (AEs).ClinicalTrials.gov  on July 29, 2013. The trial was designed jointly by the corresponding author and the sponsor (F. Hoffmann-La Roche Ltd). Data were collected by the site investigators and were retained and analyzed by the sponsor. All authors had full access to the data.This study was conducted in accordance with International Congress on Harmonisation guidelines for Good Clinical Practice, the principles of the Declaration of Helsinki, and local regulations. All patients provided written informed consent. This trial was registered prospectively at BRAFV600 mutation by cobas® BRAF V600 Mutation Test  were eligible. Other key inclusion criteria included measurable disease according to Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1); Eastern Cooperative Oncology Group performance status 0–1; adequate hematologic, renal, and liver function; life expectancy > 3 months; and absence of active central nervous system metastases. Patients could be treatment-naive or have previously received systemic therapy (excluding BRAF or MEK inhibitors).Chinese patients ≥18 years of age with histologically confirmed metastatic melanoma (unresectable stage IIIC or stage IV) positive for max); time to maximal concentration (tmax); area under the concentration-time curve from 0 to 8 hours (AUC0-8h), AUC0-12h, and AUC0-168h; trough concentration (Ctrough); accumulation ratio (defined as AUC0-8h on day 21/AUC0-8h on day 1); terminal elimination rate constant (Kel); and terminal half-life (t½). The schedule of assessments is in the Additional file The primary objective was to evaluate the pharmacokinetics of vemurafenib 960 mg twice daily in Chinese patients, including maximal concentration  according to RECIST v1.1, confirmed by repeat assessment at least 4 weeks after criteria were first met, PFS, OS, and duration of response (DOR). Tumor assessments (computed tomography or magnetic resonance imaging) were obtained at screening, day 1 of cycle 3, and every 2 cycles thereafter  until documented disease progression. DOR, PFS, and OS were estimated using the Kaplan-Meier method.Safety assessments consisted of monitoring and recording of AEs, serious AEs, and nonserious AEs of special interest; protocol-specified laboratory assessments; vital signs; and other protocol-specified tests. AEs were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. Patients were followed for safety for 28 days after the last dose of study medication or until resolution of drug-related AEs. All patients who received at least one dose of vemurafenib were to be followed for evaluation of cutaneous squamous cell carcinoma (cuSCC) until 6 months after discontinuation of study treatment, or until death, withdrawal of consent, or loss to follow-up, whichever occurred first.The pharmacokinetic analysis population included all patients who provided sufficient pharmacokinetic data to obtain at least one of the primary pharmacokinetic variables. The safety population included all patients who received at least one dose of vemurafenib, and the tumour response population included all patients.No formal sample size calculation was performed. The sample size of 20 patients in the pharmacokinetic cohort was chosen to characterize the vemurafenib pharmacokinetic profile with consideration of interpatient variability. With 20 patients enrolled in the pharmacokinetic cohort, at least 10 patients would be expected to complete the pharmacokinetic portion of the study. A total of 45 patients (20 in the pharmacokinetic cohort and 25 in the expansion cohort) would give a Clopper-Pearson 95% CI of 26–56% for BORR, assuming the target BORR (confirmed) is 40%.n = 28), patient withdrawal (n = 2), or other reasons (n = 2). The median follow-up duration was 11.3 months (range 3.3–16.0).Forty-six patients were enrolled between August 17, 2013, and January 24, 2014 at two centers in China. Twenty patients were enrolled in the pharmacokinetic cohort and 26 were enrolled in the expansion cohort. Baseline demographics and disease characteristics are shown in Table ½ and volume of distribution because Kel could not be reliably estimated owing to insufficient concentration timepoint data during the elimination phase.All patients in the pharmacokinetic cohort were included in the pharmacokinetic analysis population; one patient had treatment interruption on day 14 of cycle 1 and was excluded from pharmacokinetic analysis for subsequent time points. Additionally, one patient was excluded from analysis of tmax at approximately 5 h . Interpatient variability in AUC and Cmax was relatively high, and was more pronounced after the first dose than after multiple doses (Table max was 1 h (range 0.0–5.0) after the morning dose on day 21. During the 168-h drug holiday after the morning dose on day 21, vemurafenib plasma concentrations decreased, with a mean terminal t½ of approximately 35.6 h. After 21 days of vemurafenib 960 mg twice-daily dosing, use of vemurafenib resulted in extensive accumulation (≈18-fold) and relatively constant steady-state exposure throughout the dosing interval  were included in the efficacy analysis population. At clinical cut-off, one patient (2%) had confirmed complete response and 23 patients (50%) had confirmed partial response  had experienced a PFS event (disease progression or death). Kaplan-Meier–estimated median PFS was 8.3 months (95% CI 5.7–10.9) (Table n = 46) received at least one dose of study medication and were included in the safety analysis. The median duration of treatment was 7.9 months (range 1.8–16.0).All patients  are reported in Table 0-8h and Cmax in Chinese patients were 39% and 44% higher, respectively, than those in predominantly Caucasian populations. More importantly, at steady state (day 15 in the previous Caucasian pharmacokinetic study and day 21 in the current study), mean AUC0-8h and Cmax (day 21) were 28% and 26% higher, respectively, than in predominantly Caucasian populations (day 15) [To date, this is the largest study to evaluate the efficacy and safety of vemurafenib in an Asian population. Vemurafenib demonstrated marked accumulation and relatively constant steady-state exposure in Chinese patients, consistent with previous observations in predominantly Caucasian populations , 12. Com(day 15) .trough) from days 15 to 21, although it is unclear whether these increases in concentration were caused by continuous accumulation or intrapatient variability. While a lower average body weight is frequently identified as a possible reason for increased drug exposure in Asians relative to Caucasians, body weight or body mass index has not been identified as a covariant for vemurafenib pharmacokinetic parameters from population pharmacokinetic analysis of data from phase I, II, and III studies [These differences in exposure may be partially explained by increases in vemurafenib trough concentrations , actual incidences of these skin disorders in Asian patients must be confirmed in larger patient populations.In the BRIM-3 study, grade 3 cuSCC and grade 3 keratoacanthoma occurred in 19% (65 of 337) and 10% of patients (34 of 337) in the vemurafenib arm, respectively , and onlIn conclusion, vemurafenib pharmacokinetics, efficacy, and safety in Chinese patients were generally consistent with those observed in predominantly Caucasian populations, despite slightly higher vemurafenib exposure in Chinese patients. Overall, vemurafenib showed a favorable benefit/risk profile among Chinese patients.Additional file 1:Schedule of assessments. (DOCX 19 kb)Additional file 2:Figure S1. Vemurafenib Ctrough concentrations (mean ± SD) after day 28 in the pharmacokinetics and expansion cohorts. SD standard deviation, CV coefficient of variation. (PDF 71 kb)Additional file 3:Figure S2. Kaplan-Meier plots of (A) progression-free survival (PFS) and (B) overall survival (OS). (PDF 80 kb)Additional file 4:Table S1. Comparison of pharmacokinetic parameters between study YO28390 (Chinese patients) and study NP25163 (predominantly Caucasian patients). (DOCX 19 kb)Additional file 5:Table S2. Comparison of efficacy between study YO28390 (Chinese patients) and the BRIM-2 and BRIM-3 studies (predominantly Caucasian patients). (DOCX 18 kb)Additional file 6:Table S3. Comparison of safety between study YO28390 (Chinese patients) and the BRIM-3 study (predominantly Caucasian patients). (DOCX 18 kb)


Mimiviridae family are large DNA viruses that infect Acanthamoeba cells. Here, we report the genome sequences of two new Mimiviridae family members, isolated from water samples from Shirakoma Pond and the mouth of the Arakawa River in Japan, with nearly complete genome sizes of 1,182,849 and 1,182,801 bp, respectively.Members of the  Mimiviridae have very large icosahedral particles of 750 nm in diameter, including dense surface fibers, and encode the largest genomes of around 1.2 Mb in size  and M. kasaii (1.2 µg) was prepared from PYG culture media including viral particles using NucleoSpin Tissue XS (Macherey-Nagel GmbH and Co. KG) according to the manufacturer’s protocol. A DNA library for sequencing was prepared using a TruSeq Nano DNA LT library prep kit , and sequencing was performed on the HiSeq 2500 platform . Edena software was used to assemble 58,319,134 reads into nine contigs with an average length of 131,428 nucleotides (nt) for M. shirakomae, and 71,243,710 reads into eight contigs with an average length of 147,850 nt for M. kasaii. The maximum contig lengths for M. shirakomae and M. kasaii were 680,266 nt and 908,954 nt, respectively. The total length of the nine contigs in M. shirakomae was 1,182,849 nt, and the total length of the eight contigs in M. kasaii was 1,182,801 nt. Prediction of the coding regions of these genomes was conducted using Prodigal version 2.6.3, and prediction of tRNAs was conducted using tRNAScan-SE version 1.23, according to the manufacturers’ protocols. The prediction of gene function was conducted using NCBI BLASTp in the NCBI NR and COG databases.Water samples were collected from the pond and the mouth of the river. After mud was removed by filtration , samples were further filtered . Filtered samples were supplemented with 4% rice media ( cloning , genomicMimivirus strains have genomes of around 1,182 kb, which are approximately equal to the genome sizes of other Mimiviridae lineage A members (–M. shirakomae and M. kasaii most closely resemble M. bombay (Mimiviridae.Genome analysis showed that these  members –9. TheseM. shirakomae and M. kasaii have been deposited in DDBJ/ENA/GenBank under the accession numbers AP017645 and AP017644, respectively.The nearly complete genomic sequences of