The CEBPB+ glioblastoma subcluster specifically drives the formation of M2 tumor-associated macrophages to promote malignancy growth.

Rationale: The heterogeneity of tumor cells within the glioblastoma (GBM) microenvironment presents a complex challenge in curbing GBM progression. Understanding the specific mechanisms of interaction between different GBM cell subclusters and non-tumor cells is crucial. Methods: In this study, we utilized a comprehensive approach integrating glioma single-cell and spatial transcriptomics. This allowed us to examine the molecular interactions and spatial localization within GBM, focusing on a specific tumor cell subcluster, GBM subcluster 6, and M2-type tumor-associated macrophages (M2 TAMs). Results: Our analysis revealed a significant correlation between a specific tumor cell subcluster, GBM cluster 6, and M2-type TAMs. Further in vitro and in vivo experiments demonstrated the specific regulatory role of the CEBPB transcriptional network in GBM subcluster 6, which governs its tumorigenicity, recruitment of M2 TAMs, and polarization. This regulation involves molecules such as MCP1 for macrophage recruitment and the SPP1-Integrin αvß1-Akt signaling pathway for M2 polarization. Conclusion: Our findings not only deepen our understanding of the formation of M2 TAMs, particularly highlighting the differential roles played by heterogeneous cells within GBM in this process, but also provided new insights for effectively controlling the malignant progression of GBM.

Inactivation of the DNA-sensing pathway facilitates oncolytic herpes simplex virus inhibition of pancreatic ductal adenocarcinoma growth.

Oncolytic viruses are a new class of therapeutic agents for the treatment of cancer that have shown promising results in clinical trials. Oncolytic virus-mediated tumor rejection is highly dependent on viral replication in tumor cells to induce cell death. However, the antiviral immune response of tumor cells limits the replication capacity of oncolytic viruses. We hypothesized that inhibition of the antiviral immune response in infected cells would enhance the antitumor effect. Here, we confirmed that ablation of the key adaptor protein of cellular immunity, STING, significantly suppressed the antiviral immune response and promoted oncolytic herpes simplex virus-1 (oHSV1) proliferation in tumor cells. In a murine pancreatic ductal adenocarcinoma (PDAC) model, oHSV1 enhanced tumor suppression and prolonged the survival of mice in the absence of STING. On this basis, we further found that the TBK1 inhibitor can also significantly enhance the tumor-control ability of oHSV1. Our studies provide a novel strategy for oncolytic virus therapy by inhibiting the intrinsic antiviral response in solid tumors to improve antitumor efficacy.

Combinatorial Application of Papain and CD66B for Isolating Glioma- Associated Neutrophils.

BACKGROUND: Stromal cells in the tumor microenvironment play crucial roles in glioma development. Current methods for isolating tumor-associated stromal cells (such as neutrophils) are inefficient due to the conflict between tissue dissociation and cell surface protein protection, which hampers the research on patient-derived stromal cells. Our study aims to establish a novel method for isolating glioma-associated neutrophils (GANs). METHODS: To observe neutrophil-like polymorphonuclear cells, we performed Hematoxylin-Eosin staining on glioma tissues. For isolating single cells from glioma tissues, we evaluated the efficiency of tissue dissociation with FastPrep Grinder-mediated homogenization or proteases (trypsin or papain) digestion. To definite specific markers of GANs, fluorescence-activated cell sorting (FACS) and immunofluorescence staining were performed. FACS and Ficoll were performed for the separation of neutrophils from glioma tissue-derived single-cell or whole blood pool. To identify the isolated neutrophils, FACS and RT-PCR were carried out. RESULTS: Neutrophil-like cells were abundant in high-grade glioma tissues. Among the three tissue dissociation methods, papain digestion produced a 5.1-fold and 1.7-fold more living cells from glioma mass than physical trituration and trypsin digestion, respectively, and it preserved over 97% of neutrophil surface protein markers. CD66B could be adopted as a unique neutrophil surface protein marker for FACS sorting in glioma. Glioma-derived CD66B+ cells specifically expressed neutrophil marker genes. CONCLUSION: A combination of papain-mediated tissue dissociation and CD66B-mediated FACS sorting is an effective novel method for the isolation of GANs from glioma tissues.

Identification of Neural Progenitor Cell-associated Chemoradiotherapy Resistance Gene Set (ARL4C, MSN, TNFAIP6) for Prognosis of Glioma.

BACKGROUND: Glioma is the most common malignant intracranial tumor with high lethality. Despite surgery combined with chemoradiotherapy, the prognosis for patients with glioma remains poor. This is primarily due to acquired chemoradiotherapy resistance. Therefore, to improve the prognosis of glioma, further study into the mechanism of chemoradiotherapy resistance is needed. OBJECTIVE: This study aimed to (1) evaluate the prognosis of patients with glioma by using a prognostic risk score model constructed by chemoradiotherapy resistance genes, (2) provide new targets and directions for precise treatment of glioma, and (3) discuss the tumor heterogeneity of tumor cells. METHODS: According to therapy class and overall survival (OS), we identified 53 genes associated with glioma chemoradiotherapy resistance in The Cancer Genome Atlas Glioblastoma (TCGA GBM) database. Considering the important role of chemoradiotherapy resistance-related genes in the prognosis of glioma, we preliminarily screened and identified vital prognostic factors among these genes by using the Cox regression model of absolute contraction and selection operators in the TCGA GBM lower-grade glioma (TCGA GBMLGG) dataset. Next, the heterogeneity of the chemoradiotherapy resistance-associated genes in different glioma cells was revealed by single-cell sequencing in the GSE117891 cohort. RESULTS: A prognostic risk score model consisting of three genes (ARL4C, MSN, TNFAIP6) was constructed. The expression of this model was high in glioma neural progenitor cells (NPCs) and low in glioma oligodendrocytes. The OS rates were significantly lower in the high- vs. low-risk group. CONCLUSION: Our 3 gene risk score complements the current glioma diagnosis and provides a novel insight into chemoradiotherapy resistance mechanisms for the prognosis of patients with glioma.

[Corrigendum] Ouabain suppresses the growth and migration abilities of glioma U­87MG cells through inhibiting the Akt/mTOR signaling pathway and downregulating the expression of HIF­1α.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that several pairings of panels in Fig. 5, as shown on p. 5599, were strikingly similar. After having examined their original data, the authors realized that they uploaded some images incorrectly during the process of compiling this figure, and that there were duplicated data panels in this figure. However, the authors were able to consult their original data, and had access to the correct images. The revised version of Fig. 5, showing the correct data for the Akt/Control, p­Akt/Control, mTOR/0.05 µM Ouabain, HIF­1α/0.05 µM Ouabain and Akt/0.5 µM Ouabain experiments, is shown opposite. Note that the replacement of the erroneous data does not affect either the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 17: 5595­5600, 2018; DOI: 10.3892/mmr.2018.8587].

Glucagon-like peptide-1 receptor agonist Exendin-4 improves neurological outcomes by attenuating TBI- induced inflammatory responses and MAPK activation in rats.

Traumatic brain injury (TBI) can be exacerbated and prolonged for months or even years by chronic inflammatory processes with long-term consequences on neurodegeneration and neurological impairment. However, there are no clear pharmacological therapies of benefit to manage neurological dysfunctions, which, relating to the molecular mechanisms underlying the behavioral deficits after TBI, have yet to be fully identified. Recently, a glucagon-like peptide 1 (GLP-1) agonist, Exendin-4, was approved not only for the treatment of type 2 diabetes mellitus, but it also played a neurotrophic role in various CNS neurological diseases. In this study, we evaluated the neuroprotective effects of Exendin-4 on neurological outcome, cerebral blood flow, neurodegeneration, and inflammatory responses by utilizing a cortical contusion impact injury (CCI) model in rats. We found that TBI rats displayed neurological impairments, neurodegeneration, reduction of cerebral blood flow, and inflammatory responses, while Exendin-4 promoted neurological, cognitive, and cerebral blood flow recovery and attenuated neural degeneration and inflammatory cytokines after TBI. Furthermore, Exendin-4 treatment significantly diminished the TBI-induced overexpression of TNFα and IL-1ß, as well as phosphorylation of p38 and ERK1/2. These data suggest a strong beneficial action of the glucagon-like peptide-1 receptor agonist Exendin-4 in improving neurological outcomes by attenuating inflammatory responses induced by traumatic brain injury, which is of therapeutic potential for TBI.

Bloodletting Puncture at Hand Twelve Jing-Well Points Improves Neurological Recovery by Ameliorating Acute Traumatic Brain Injury-Induced Coagulopathy in Mice.

Traumatic brain injury (TBI) contributes to hypocoagulopathy associated with prolonged bleeding and hemorrhagic progression. Bloodletting puncture therapy at hand twelve Jing-well points (BL-HTWP) has been applied as a first aid measure in various emergent neurological diseases, but the detailed mechanisms of the modulation between the central nervous system and systemic circulation after acute TBI in rodents remain unclear. To investigate whether BL-HTWP stimulation modulates hypocoagulable state and exerts neuroprotective effect, experimental TBI model of mice was produced by the controlled cortical impactor (CCI), and treatment with BL-HTWP was immediately made after CCI. Then, the effects of BL-HTWP on the neurological function, cerebral perfusion state, coagulable state, and cerebrovascular histopathology post-acute TBI were determined, respectively. Results showed that BL-HTWP treatment attenuated cerebral hypoperfusion and improve neurological recovery post-acute TBI. Furthermore, BL-HTWP stimulation reversed acute TBI-induced hypocoagulable state, reduced vasogenic edema and cytotoxic edema by regulating multiple hallmarks of coagulopathy in TBI. Therefore, we conclude for the first time that hypocoagulopathic state occurs after acute experimental TBI, and the neuroprotective effect of BL-HTWP relies on, at least in part, the modulation of hypocoagulable state. BL-HTWP therapy may be a promising strategy for acute severe TBI in the future.

[Effect of bloodletting acupuncture at twelve jing-well points of hand on microcirculatory disturbance in mice with traumatic brain injury].

OBJECTIVE: To observe the effect of bloodletting acupuncture at twelve jing-well points of hand on microcirculatory disturbance in mice with traumatic brain injury (TBI), and to explore the protective effect of bloodletting therapy on TBI. METHODS: Sixty clean adult male C57BL/6J mice were randomly divided into a sham-operation group, a model group and a treatment group, 20 mice in each group. The TBI model was established by using electronic controlled cerebral cortex impact instrument in the model group and the treatment group. The mice in the treatment group were treated with bloodletting acupuncture at bilateral "Shaoshang" (LU 11), "Shangyang" (LI 1), "Zhongchong" (PC 9), "Guanchong" (TE 1), "Shaochong" (HT 9) and "Shaoze" (SI 1) immediately after trauma. The mice in the sham-operation group only opened the bone window but did not receive the strike. The regional cerebral blood flow (rCBF) was monitored by laser speckle contrast analysis (LASCA) using a PeriCam PSI System before trauma, immediately after trauma and 1, 2, 12, 24, 48, 72 h after trauma. The brain water content was measured by wet-dry weight method 24 h after trauma. The severity of functional impairment at 2, 12, 24, 48 and 72 h after trauma was evaluated by modified neurological scale scores (mNSS). RESULTS: ① 2 h after trauma, the mNSS in the model group and treatment group were >7 points, suggesting the successful establishment of model; compared with the sham-operation group, the mNSS was increased significantly from 12 to 72 h after trauma in the model group ( all P<0.01), but the mNSS in the treatment group was significantly lower than that in the model group from 2 to 24 h after trauma (P<0.01, P<0.05). ② Compared with the sham-operation group, rCBF in the model group was decreased significantly immediately after trauma (P<0.01), and the rCBF in the model group was lower than that in the sham-operation group from 1 to 72 h after trauma ( all P<0.01); rCBF in the treatment group began to rise and was significantly higher than that in the model group 1-2 h after trauma (P<0.01); 12-48 h after trauma, the increasing of rCBF in the two groups tended to be gentle until 72 h after injury, and rCBF in the model group was decreased while that in the treatment group continued to rise and was higher than that in the model group (P<0.01). ③ 24 h after trauma, the brain water content in the model group was significantly higher than that in the sham-operation group (P<0.01), and brain water content in the treatment group was significantly lower than that in the model group (P<0.01). CONCLUSION: The bloodletting acupuncture at twelve jing-well points of hand could improve microcirculation disturbance, increase microcirculation perfusion, alleviate secondary brain edema and promote the recovery of nerve function in mice with TBI.

Casein Kinase 2 Interacting Protein-1 Suppresses Glioma Cell Proliferation via Regulating the AKT/GSK3ß/ß-Catenin Pathway.

OBJECTIVE: Casein kinase 2 interacting protein-1 (CKIP-1) has exhibited multiple functions in regulating cell proliferation, apoptosis, differentiation, and cytoskeleton. CKIP-1 also plays an important role as a critical regulator in tumorigenesis. The aim of this study is to further examine the function of CKIP-1 in glioma cells. METHODS: The expression level of CKIP-1 protein was determined in gliomas tissues and cell lines by immunohistochemistry stain and western blotting while the association of CKIP-1 expression with prognosis was analyzed by Kaplan-Meier method and compared by log-rank test. CKIP-1 was overexpressed or silenced in gliomas cell lines. CCK-8, colony formation assay, and BrdU incorporation assay were used to determine cell proliferation and DNA synthesis. Cell cycle and apoptosis rate were determined with fluorescence-activated cell sorting (FACS) method. Then, expression of key members in AKT/GSK3ß/ß-catenin pathway was detected by western blot analysis. RESULTS: In the present study, we reported new evidence that CKIP-1 was reversely associated with the proliferation of glioma cells and survival in glioma patients. Additionally, the overexpressed CKIP-1 significantly inhibited glioma cell proliferation. Further experiments revealed that CKIP-1 functioned through its antiproliferative and proapoptotic activity in glioma cells. Importantly, mechanistic investigations suggested that CKIP-1 sharply suppressed the activity of AKT by inhibiting the phosphorylation, markedly downregulated the phosphorylated GSK3ß at Ser9, and promoted ß-catenin degradation. CONCLUSIONS: Overall, our results provided new insights into the clinical significance and molecular mechanism of CKIP-1 in glioma, which indicated CKIP1 might function as a therapeutic target for clinical treatment of glioma.

An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury.

BACKGROUND: Traumatic spinal cord injury (SCI) causes neuronal death, demyelination, axonal degeneration, inflammation, glial scar formation, and cystic cavitation resulting in interruption of neural signaling and loss of nerve function. Multifactorial targeted therapy is a promising strategy for SCI. METHODS: The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for minimally invasive, localized, and sustained intrathecal protein delivery. The physical and biological characteristics of HAMC-KAFAK/BDNF hydrogel were measured in vitro. SCI model was performed in rats and HAMC-KAFAK/BDNF hydrogel was injected into the injured site of spinal cord. The neuronal regeneration effect was evaluated by inflammatory cytokine levels, behavioral test and histological analysis at 8 weeks post operation. RESULTS: HAMC-KAFAK/BDNF hydrogel showed minimally swelling property and sustained release of the KAFAK and BDNF. HAMC-KAFAK/BDNF hydrogel significantly improved the proliferation of PC12 cells in vitro without cytotoxicity. Significant recovery in both neurological function and nerve tissue morphology in SCI rats were observed in HAMC-KAFAK/BDNF group. HAMC-KAFAK/BDNF group showed significant reduction in proinflammatory cytokines expression and cystic cavitation, decreased glial scar formation, and improved neuronal survival in the rat SCI model compared to HAMC group and SCI group. CONCLUSION: The HAMC-KAFAK/BDNF hydrogel promotes functional recovery of rats with spinal cord injury by regulating inflammatory cytokine levels and improving axonal regeneration.

iTRAQ-based secretome reveals that SiO2 induces the polarization of RAW264.7 macrophages by activation of the NOD-RIP2-NF-κB signaling pathway.

Silicosis is characterized by inflammation and pulmonary fibrosis due to long-term inhalation of crystalline silica (SiO2). To clarify the role of macrophage polarization in the inflammatory response of silicosis, we used iTRAQ-coupled 2D LC-MS/MS to study the change in the secretome in RAW264.7 macrophages. We successfully screened 330 differentially expressed proteins, including 120 proteins with upregulated expression and 210 proteins with down-regulated expression (p < 0.05). Bioinformatics analysis showed that the differentially expressed proteins were mainly involved in biological processes, such as oxidative stress, mitochondrial damage, apoptosis and acute inflammatory response. In particular, the expression levels of mitochondrial apoptosis-related proteins, such as AKT1, BAX, HSPD1, TNF, CASP8 and DAP, were increased after SiO2 exposure. Taken together, our study indicated that SiO2 could induce macrophage polarization by activation of the NOD-RIP2-NF-κB signaling pathway in RAW264.7 macrophages. This may represent a potential mechanism in the development of silicosis.

Ouabain suppresses the growth and migration abilities of glioma U­87MG cells through inhibiting the Akt/mTOR signaling pathway and downregulating the expression of HIF­1α.

Glioma is one of the most malignant forms of brain tumor, and has been of persistent concern due to its high recurrence and mortality rates, and limited therapeutic options. As a cardiac glycoside, ouabain has widespread applications in congestive heart diseases due to its positive cardiac inotropic effect by inhibiting Na+/K+­ATPase. Previous studies have demonstrated that ouabain has antitumor activity in several types of human tumor, including glioma. However, the exact underlying mechanism remains to be elucidated. The purpose of present study was to elucidate the effect of ouabain on human glioma cell apoptosis and investigate the exact mechanism. U­87MG cells were treated with various concentrations of ouabain for 24 h, following which cell viability and survival rate were assessed using a 3­(4,5-dimethylthiazol-2­yl)­2,5­diphenyltetrazolium bromide assay. The dynamic changes and cell motility were observed using digital holographic microscopy. Additionally, western blot analysis and high­content screening assays were used to detect the protein expression levels of phosphorylated (p­)Akt, mammalian target of rapamycin (mTOR), p­mTOR and hypoxia­inducible factor (HIF)­1α, respectively. Compared with the control group, ouabain suppressed U­87MG cell survival, and attenuated cell motility in a dose­dependent manner (P<0.01). The downregulation of p­Akt, mTOR, p­mTOR and HIF­1α were observed following treatment with 2.5 and 25 µmol/l of ouabain. These results suggested that ouabain exerted suppressive effects on tumor cell growth and motility, leading to cell death via regulating the intracellular Akt/mTOR signaling pathway and inhibiting the expression of HIF­1α in glioma cells. The present study examined the mechanism underlying the antitumor property of ouabain, providing a novel potential therapeutic agent for glioma treatment.

iTRAQ-Based Quantitative Proteomics Reveals the New Evidence Base for Traumatic Brain Injury Treated with Targeted Temperature Management.

This study aimed to investigate the effects of targeted temperature management (TTM) modulation on traumatic brain injury (TBI) and the involved mechanisms using quantitative proteomics technology. SH-SY5Y and HT-22 cells were subjected to moderate stretch injury using the cell injury controller (CIC), followed by incubation at TTM (mild hypothermia, 32°C), or normothermia (37°C). The real-time morphological changes, cell cycle phase distribution, death, and cell viability were evaluated. Moderate TBI was produced by the controlled cortical impactor (CCI), and the effects of TTM on the neurological damage, neurodegeneration, cerebrovascular histopathology, and behavioral outcome were determined in vivo. Results showed that TTM treatment prevented TBI-induced neuronal necrosis in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, reduced cortical lesion volume and neuronal loss, attenuated cerebrovascular histopathological damage, brain edema, and improved behavioral outcome. Using an iTRAQ proteomics approach, proteins that were significantly associated with TTM in experimental TBI were identified. Importantly, changes in four candidate molecules (plasminogen [PLG], antithrombin III [AT III], fibrinogen gamma chain [FGG], transthyretin [TTR]) were verified using TBI rat brain tissues and TBI human cerebrospinal fluid (CSF) samples. This study is one of the first to investigate the neuroprotective effects of TTM on the proteome of human and experimental models of TBI, providing an overall landscape of the TBI brain proteome and a scientific foundation for further assessment of candidate molecules associated with TTM for the promotion of reparative strategies post-TBI.

Hypoxia-inducible factor-1 alpha is involved in RIP-induced necroptosis caused by in vitro and in vivo ischemic brain injury.

Necroptosis, a novel type of programmed cell death, is involved in stroke-induced ischemic brain injury. Although studies have sought to explore the mechanisms of necroptosis, its signaling pathway has not yet to be completely elucidated. Thus, we used oxygen-glucose deprivation (OGD) and middle cerebral artery occlusion (MCAO) models mimicking ischemic stroke (IS) conditions to investigate mechanisms of necroptosis. We found that OGD and MCAO induced cell death, local brain ischemia and neurological deficit, while zVAD-fmk (zVAD, an apoptotic inhibitor), GSK'872 (a receptor interacting protein kinase-3 (RIP3) inhibitor), and combined treatment alleviated cell death and ischemic brain injury. Moreover, OGD and MCAO upregulated protein expression of the triggers of necroptosis: receptor interacting protein kinase-1 (RIP1), RIP3 and mixed lineage kinase domain-like protein (MLKL). The upregulation of these proteins was inhibited by GSK'872, combination treatments and RIP3 siRNA but not zVAD treatment. Intriguingly, hypoxia-inducible factor-1 alpha (HIF-1α), an important transcriptional factor under hypoxic conditions, was upregulated by OGD and MCAO. Similar to their inhibitory effects on aforementioned proteins upregulation, GSK'872, combination treatments and RIP3 siRNA decreased HIF-1α protein level. These findings indicate that necroptosis contributes to ischemic brain injury induced by OGD and MCAO and implicate HIF-1α, RIP1, RIP3, and MLKL in necroptosis.

Hepatitis B virus X induces inflammation and cancer in mice liver through dysregulation of cytoskeletal remodeling and lipid metabolism.

Hepatitis B virus X protein (HBx) participates in the occurrence and development processes of hepatocellular carcinoma (HCC) as a multifunctional regulation factor. However, the underlying molecular mechanism remains obscure. Here, we describe the use of p21HBx/+ mouse and SILAM (Stable Isotope Labeling in Mammals) strategy to define the pathological mechanisms for the occurrence and development of HBx induced liver cancer. We systematically compared a series of proteome samples from regular mice, 12- and 24-month old p21HBx/+ mice representing the inflammation and HCC stages of liver disease respectively and their nontransgenic wild-type (WT) littermates. Totally we identified 22 and 97 differentially expressed proteins out of a total of 2473 quantified proteins. Bioinformatics analysis suggested that the lipid metabolism and CDC42-induced cytoskeleton remodeling pathways were strongly activated by the HBx transgene. Interestingly, the protein-protein interaction MS study revealed that HBx directly interacted with multiple proteins in these two pathways. The same effect of up-regulation of cytoskeleton and lipid metabolism related proteins, including CDC42, CFL1, PPARγ and ADFP, was also observed in the Huh-7 cells transfected with HBx. More importantly, CFL1 and ADFP were specifically accumulated in HBV-associated HCC (HBV-HCC) patient samples, and their expression levels were positively correlated with the severity of HBV-related liver disease. These results provide evidence that HBx induces the dysregulation of cytoskeleton remodeling and lipid metabolism and leads to the occurrence and development of liver cancer. The CFL1 and ADFP might be served as potential biomarkers for prognosis and diagnosis of HBV-HCC.

Neuroprotective effects of bloodletting at Jing points combined with mild induced hypothermia in acute severe traumatic brain injury.

Bloodletting at Jing points has been used to treat coma in traditional Chinese medicine. Mild induced hypothermia has also been shown to have neuroprotective effects. However, the therapeutic effects of bloodletting at Jing points and mild induced hypothermia alone are limited. Therefore, we investigated whether combined treatment might have clinical effectiveness for the treatment of acute severe traumatic brain injury. Using a rat model of traumatic brain injury, combined treatment substantially alleviated cerebral edema and blood-brain barrier dysfunction. Furthermore, neurological function was ameliorated, and cellular necrosis and the inflammatory response were lessened. These findings suggest that the combined effects of bloodletting at Jing points (20 µL, twice a day, for 2 days) and mild induced hypothermia (6 hours) are better than their individual effects alone. Their combined application may have marked neuroprotective effects in the clinical treatment of acute severe traumatic brain injury.

[Progress and application of phosphoproteomics in the proteomics of liver pathological and physiological state].

The phosphorylation is one of most common protein post-translational modifications. The protein phosphorylation plays important roles in the life through the reversible process of phosphorylation and dephosphorylation by kinases and phosphatases. Systematical analysis of the phosphorylation state of proteins would greatly help to reveal the mystery of the life. Recently, with the development of mass spectrometer, bioinformatics sortwares and enrichment methods of phosphopeptides, phosphorylation stduy of orgnism proteins by mass spectrometer has become mature gradually. Liver is one of the most important metabolic and immune organs. In-depth study of protein phosphorylation in liver is of great importance to reveal its function. And booming phosphoproteomics has been applied into the study of liver, which has deepened the knowledge of molecular mechnism of its physiology and pathology states. Here, we review the recent progress on the research and development of phosphoproteomics and their application in liver proteomics study.

[Feeding mouse with stable isotope labelled with amino acid].

The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6J mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues' labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥ 95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.

First proteomic exploration of protein-encoding genes on chromosome 1 in human liver, stomach, and colon.

The launch of the Chromosome-Centric Human Proteome Project provides an opportunity to gain insight into the human proteome. The Chinese Human Chromosome Proteome Consortium has initiated proteomic exploration of protein-encoding genes on human chromosomes 1, 8, and 20. Collaboration within the consortium has generated a comprehensive proteome data set using normal and carcinomatous tissues from human liver, stomach, and colon and 13 cell lines originating in these organs. We identified 12,101 proteins (59.8% coverage against Swiss-Prot human entries) with a protein false discovery rate of less than 1%. On chromosome 1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot entries, were identified; however, 805 proteins remain unidentified, suggesting that analysis of more diverse samples using more advanced proteomic technologies is required. Genes encoding the unidentified proteins were concentrated in seven blocks, located at p36, q12-21, and q42-44, partly consistent with correlation of these blocks with cancers of the liver, stomach, and colon. Combined transcriptome, proteome, and cofunctionality analyses confirmed 23 coexpression clusters containing 165 genes. Biological information, including chromosome structure, GC content, and protein coexpression pattern was analyzed using multilayered, circular visualization and tabular visualization. Details of data analysis and updates are available in the Chinese Chromosome-Centric Human Proteome Database ( http://proteomeview.hupo.org.cn/chromosome/ ).