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Abstract Objective To investigate the prognostic significance of CXCL8 in cervical cancer and its effect on immune response based on bioinformatics method. Methods This study employs the HPA database to investigate CXCL8 expression in normal human tissues.The TIMER2.0 database is utilized to analyze CXCL8 expression across various types of cancer.Utilizing the TCGA database, we analyze the correlation between CXCL8 and the overall survival (OS) and progression-free interval (PFI) of patients with various tumors using R 4.3.2.Additionally, its association with immune checkpoint-related genes across various types of cancer is examined.We further analyze the association between CXCL8 expression and the expression of LAG3, CTLA4, PDCD1, and CD274 in cervical cancer.The TIMER database is used to study the association between CXCL8 and the extent of Tumor-Infiltrating Immune Cells (TIICs) infiltration.Enrichment analysis of genes related to CXCL8 is conducted using the LinkedOmics database. Result CXCL8 is found in a wide range of normal human tissues.In the majority of tumor tissues, CXCL8 expression is elevated compared to their normal counterparts.There is a significant correlation between CXCL8 expression and the overall survival (OS) and progression-free interval (PFI) of patients with various tumors.CXCL8 expression is associated with the expression of diverse immune checkpoint-related genes and the extent of Tumor-Infiltrating Immune Cells (TIICs) infiltration.Genes related to CXCL8 participate in diverse immune-related processes in cervical cancer. Conclusion CXCL8 plays a role in modulating immune infiltration, thereby influencing the prognosis of patients with various tumors, particularly those with cervical cancer.CXCL8 could potentially act as a biomarker for forecasting the prognosis and immune response of patients with tumors.

Abstract Septic lung injury is strongly associated with polarization of M1 macrophages and excessive cytokine release. Fibroblast growth factor (FGF) signaling plays a role in both processes. However, the impact of FGF2 deficiency on macrophage polarization and septic acute lung injury remains unclear. To investigate this, we obtained macrophages from FGF2 knockout mice and examined their polarization and inflammatory cytokine expression. We also eliminated endogenous macrophages using clodronate liposomes and administered FGF2 knockout or WT macrophages intravenously in conjunction with cecal ligation and puncture (CLP) surgery to induce sepsis. In vitro analysis by flow cytometry and real-time PCR analysis demonstrated that FGF2 deficiency resulted in increased expression of M1 markers (iNOS and CD86) and inflammatory cytokines (CXCL1, IL1β, and IL6), especially after LPS stimulation. Additionally, immunofluorescence demonstrated increased nuclear translocation of p65 NF-κB in FGF2 knockout macrophages and RNA-seq analysis showed enrichment of differentially expressed genes in the IL17 and TNFα inflammatory signaling pathways. Furthermore, in vivo experiments revealed that depletion of FGF2 in macrophages worsened sepsis-induced lung inflammation, lung vascular leak, and lung histological injury, accompanied by an increase in CD86-positive cells and apoptosis. Our study suggests that FGF2 deficiency in macrophages plays a critical role in the pathogenesis of septic ALI, possibly because of the enhanced M1 macrophage polarization and production of proinflammatory cytokines. These findings provide empirical evidence for potential therapeutic interventions targeting FGF2 signaling to modulate the polarization of M1 and M2 macrophages in the management of sepsis-induced acute lung injury.

Abstract Background To explore the role of GAB2 in pan-cancer based on bioinformatics analysis. Methods Based on TCGA and GTEx databases, we used TIMER2.0 online analysis tool and R language to analyze the expression of GAB2 in pan-cancer. We used Kaplan–Meier Plotter to analyze the relationship between GAB2 and OS and RFS in pan-cancer. We utilized the CPTAC database to examine the expression of phosphorylated GAB2 in pan-cancer. We investigated the effects of mutation features on the occurrence and development of human cancers by cBioPortal and COSMIC. Using the database, we conducted an analysis of molecular compounds that have the potential to interact with GAB2 through molecular docking. Moreover, we use the TIMER to explore the relationship between GAB2 and immune cell infiltration, and draw relevant heatmaps by R language. Results GAB2 was abnormally expressed in various tumors and was associated with prognosis. There were differences in the expression of GAB2 phosphorylation in tumor tissues and corresponding normal tissues among different types of tumors. GAB2 interacts with Docetaxel and was associated with immune cell infiltration in various tumors. Conclusion GAB2 participates in regulating immune infiltration and affects the prognosis of patients. GAB2 may serve as a potential tumor marker.

Summary: Protein turnover is an important mechanism to maintain proteostasis. Long-lived proteins (LLPs) are vulnerable to lose their function due to time-accumulated damages. In this study we employed in vivo stable isotope labeling in mice from birth to postnatal day 89. Quantitative proteomics analysis of ten tissues and plasma identified 2113 LLPs, including widespread and tissue-specific ones. Interestingly, a significant percentage of LLPs was detected in plasma, implying a potential link to age-related cardiovascular diseases. LLPs identified in brains were related to neurodegenerative diseases. In addition, the relative quantification of DNA-derived deoxynucleosides from the same tissues provided information about cellular DNA renewal and showed good correlation with LLPs in the brain. The combined data reveal tissue-specific maps of mouse LLPs that may be involved in pathology due to a low renewal rate and an increased risk of damage. Tissue-derived peripheral LLPs hold promise as biomarkers for aging and age-related diseases.

Abstract Imaging the crustal structure and state of the Himalayan Orogenic Belt (HOB) is of great significance for understanding crustal deformation and its tectonic response in the collision front of the Indian‐Eurasian plates. To avoid the influence from the anomalies underneath the north‐south trending rifts, a three‐dimensional electrical resistivity model across the central HOB was obtained between the Dingjie‐Shenzha and Yadong‐Gulu rifts. No lower‐crustal conductor was found in the southern Lhasa Terrane, and the lower crust with a moderate resistivity value of approximately 100 Ωm is interpreted to be the residue of previous partial melting beneath the Gangdese porphyry copper deposits. The middle crust between the Main Himalayan Thrust and Southern Tibet Detachment System is electrically resistive to the north of the north Himalayan gneiss domes (NHGD) belt and conductive to the south. The middle crust in the northern Himalayas may have undergone partial melting and crystallization during southward migration and duplexing. There is a strong deep‐shallow coupling relationship in the central HOB, where an increase in the underthrusting angle of the Indian crust has resulted in a southward turn of the NHGD belt.

Many software suites and spectral libraries have been developed for DIA proteomics data analysis. Here, the authors create benchmark data sets to evaluate four commonly used software tools combined with seven spectral libraries in both global proteomics and phosphoproteomics analysis.

Summary: Creatine kinase (CK) is an essential metabolic enzyme mediating creatine/phosphocreatine interconversion and shuttle to replenish ATP for energy needs. Ablation of CK causes a deficiency in energy supply that eventually results in reduced muscle burst activity and neurological disorders in mice. Besides the well-established role of CK in energy-buffering, the mechanism underlying the non-metabolic function of CK is poorly understood. Here we demonstrate that creatine kinase brain-type (CKB) may function as a protein kinase to regulate BCAR1 Y327 phosphorylation that enhances the association between BCAR1 and RBBP4. Then the complex of BCAR1 and RPPB4 binds to the promoter region of DNA damage repair gene RAD51 and activates its transcription by modulating histone H4K16 acetylation to ultimately promote DNA damage repair. These findings reveal the possible role of CKB independently of its metabolic function and depict the potential pathway of CKB-BCAR1-RBBP4 operating in DNA damage repair.

Summary: Cyclic GMP-AMP synthase (cGAS), as the major DNA sensor, initiates DNA-stimulated innate immune responses and is essential for a healthy immune system. Although some regulators of cGAS have been reported, it still remains largely unclear how cGAS is precisely and dynamically regulated and how many potential regulators govern cGAS. Here we carry out proximity labeling of cGAS with TurboID in cells and identify a number of potential cGAS-interacting or -adjacent proteins. Deubiquitinase OTUD3, one candidate identified in cytosolic cGAS-DNA complex, is further validated to not only stabilize cGAS but also enhance cGAS enzymatic activity, which eventually promotes anti-DNA virus immune response. We show that OTUD3 can directly bind DNA and is recruited to the cytosolic DNA complex, increasing its association with cGAS. Our findings reveal OTUD3 as a versatile cGAS regulator and find one more layer of regulatory mechanism in DNA-stimulated innate immune responses.

Abstract Necroptosis is a form of regulated necrotic cell death that promotes inflammation. In cells undergoing necroptosis, activated RIPK1 kinase mediates the formation of RIPK1/RIPK3/MLKL complex to promote MLKL oligomerization and execution of necroptosis. RIPK1 kinase activity also promotes cell-autonomous activation of proinflammatory cytokine production in necroptosis. However, the signaling pathways downstream of RIPK1 kinase in necroptosis and how RIPK1 kinase activation controls inflammatory response induced by necroptosis are still largely unknown. Here, we quantitatively measured the temporal dynamics of over 7000 confident phosphorylation-sites during necroptosis using mass spectrometry. Our study defined a RIPK1-dependent phosphorylation pattern in late necroptosis that is associated with a proinflammatory component marked by p-S473 TRIM28. We show that the activation of p38 MAPK mediated by oligomerized MLKL promotes the phosphorylation of S473 TRIM28, which in turn mediates inflammation during late necroptosis. Taken together, our study illustrates a mechanism by which p38 MAPK may be activated by oligomerized MLKL to promote inflammation in necroptosis.

G51D mutation of α-synuclein (α-syn) causes a subset of familial Parkinson’s disease that is characterized by an early onset and rapid progression of the disease. Here, the authors present the cryo-EM structure of full-length G51D α-syn fibrils that is distinct from other known α-syn fibril structures, and they show that G51D fibrils can cross-seed wild-type (WT) α-syn and that these cross-seeded WT fibrils replicate the G51D fibril structure.

Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer’s disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD. Notably, phosphorylation of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD.

Abstract Background Bone tissue engineering is a new concept bringing hope for the repair of large bone defects, which remains a major clinical challenge. The formation of vascularized bone is key for bone tissue engineering. Growth of specialized blood vessels termed type H is associated with bone formation. In vivo and in vitro studies have shown that low level laser therapy (LLLT) promotes angiogenesis, fracture healing, and osteogenic differentiation of stem cells by increasing reactive oxygen species (ROS). However, whether LLLT can couple angiogenesis and osteogenesis, and the underlying mechanisms during bone formation, remains largely unknown. Methods Mouse bone marrow mesenchymal stem cells (BMSCs) combined with biphasic calcium phosphate (BCP) grafts were implanted into C57BL/6 mice to evaluate the effects of LLLT on the specialized vessel subtypes and bone regeneration in vivo. Furthermore, human BMSCs and human umbilical vein endothelial cells (HUVECs) were co-cultured in vitro. The effects of LLLT on cell proliferation, angiogenesis, and osteogenesis were assessed. Results LLLT promoted the formation of blood vessels, collagen fibers, and bone tissue and also increased CD31hiEMCNhi-expressing type H vessels in mBMSC/BCP grafts implanted in mice. LLLT significantly increased both osteogenesis and angiogenesis, as well as related gene expression (HIF-1α, VEGF, TGF-β) of grafts in vivo and of co-cultured BMSCs/HUVECs in vitro. An increase or decrease of ROS induced by H2O2 or Vitamin C, respectively, resulted in an increase or decrease of HIF-1α, and a subsequent increase and decrease of VEGF and TGF-β in the co-culture system. The ROS accumulation induced by LLLT in the co-culture system was significantly decreased when HIF-1α was inhibited with DMBPA and was followed by decreased expression of VEGF and TGF-β. Conclusions LLLT enhanced vascularized bone regeneration by coupling angiogenesis and osteogenesis. ROS/HIF-1α was necessary for these effects of LLLT. LLLT triggered a ROS-dependent increase of HIF-1α, VEGF, and TGF-β and resulted in subsequent formation of type H vessels and osteogenic differentiation of mesenchymal stem cells. As ROS also was a target of HIF-1α, there may be a positive feedback loop between ROS and HIF-1α, which further amplified HIF-1α induction via the LLLT-mediated ROS increase. This study provided new insight into the effects of LLLT on vascularization and bone regeneration in bone tissue engineering.

Summary: Mammalian cyclic dinucleotide 2′3′-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2′3′-cGAMP photoaffinity probes to capture 2′3′-cGAMP-binding or 2′3′-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC–based quantitative MS. Further validation is also executed using photoaffinity probes to demonstrate the direct interaction of 2′3′-cGAMP with purified target proteins in vitro or endogenous target proteins in 293T cells.For complete details on the use and execution of this profile, please refer to Hou et al. (2021).

Nerve injury induces profound and complex changes at molecular and cellular levels, leading to axonal self-destruction as well as immune and inflammatory responses that may further promote neurodegeneration. To better understand how neural injury changes the proteome within the injured nerve, we set up a mouse model of sciatic nerve injury (SNI) and conducted an unbiased, quantitative proteomic study followed by biochemical assays to confirm some of the changed proteins. Among them, the protein levels of ADP-dependent glucokinase (ADPGK) were significantly increased in the injured sciatic nerve. Further examination indicated that ADPGK was specifically expressed and upregulated in macrophages but not neurons or Schwann cells upon injury. Furthermore, culturing immortalized bone marrow-derived macrophages (iBMDMs) in vitro with the conditioned media from transected axons of mouse dorsal root ganglion (DRG) neurons induced ADPGK upregulation in iBMDMs, suggesting that injured axons could promote ADPGK expression in macrophages non-cell autonomously. Finally, we showed that overexpression of ADPGK per se did not activate macrophages but promoted the phagocytotic activity of lipopolysaccharides (LPS)-treated macrophages. Together, this proteomic analysis reveals interesting changes of many proteins within the injured nerve and our data identify ADPGK as an important in vivo booster of injury-induced macrophage phagocytosis.

Post-translational protein modifications can affect lifespan and aging but age-dependent ubiquitylation changes have not yet been systematically characterized. Here, the authors analyze age-related proteome and ubiquitylome dynamics in Drosophila and identify increasing H2A ubiquitylation as a conserved aging marker.

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

Summary: Data-independent acquisition mass spectrometry (DIA-MS) is a powerful technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning. Leveraging this DIA hybrid library substantially deepens the coverage of three transmembrane protein families (G protein-coupled receptors, ion channels, and transporters) in mouse brain tissues with increases in protein identification of 37%–87% and peptide identification of 58%–161%. Moreover, of the 412 novel GPCR peptides exclusively identified with the DIA hybrid library strategy, 53.6% were validated as present in mouse brain tissues based on orthogonal experimental measurement. : Analytical Chemistry; Biological Sciences; Classification of Proteins; Proteomics Subject Areas: Analytical Chemistry, Biological Sciences, Classification of Proteins, Proteomics

Epigenetic alteration has been implicated in aging. However, the mechanism by which epigenetic change impacts aging remains to be understood. H3K27me3, a highly conserved histone modification signifying transcriptional repression, is marked and maintained by Polycomb Repressive Complexes (PRCs). Here, we explore the mechanism by which age-modulated increase of H3K27me3 impacts adult lifespan. Using Drosophila, we reveal that aging leads to loss of fidelity in epigenetic marking and drift of H3K27me3 and consequential reduction in the expression of glycolytic genes with negative effects on energy production and redox state. We show that a reduction of H3K27me3 by PRCs-deficiency promotes glycolysis and healthy lifespan. While perturbing glycolysis diminishes the pro-lifespan benefits mediated by PRCs-deficiency, transgenic increase of glycolytic genes in wild-type animals extends longevity. Together, we propose that epigenetic drift of H3K27me3 is one of the molecular mechanisms that contribute to aging and that stimulation of glycolysis promotes metabolic health and longevity.

Multiple sclerosis (MS), a common neurodegenerative disease of the CNS, is characterized by the loss of oligodendrocytes and demyelination. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine implicated in MS, can activate necroptosis, a necrotic cell death pathway regulated by RIPK1 and RIPK3 under caspase-8-deficient conditions. Here, we demonstrate defective caspase-8 activation, as well as activation of RIPK1, RIPK3, and MLKL, the hallmark mediators of necroptosis, in the cortical lesions of human MS pathological samples. Furthermore, we show that MS pathological samples are characterized by an increased insoluble proteome in common with other neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). Finally, we show that necroptosis mediates oligodendrocyte degeneration induced by TNF-α and that inhibition of RIPK1 protects against oligodendrocyte cell death in two animal models of MS and in culture. Our findings demonstrate that necroptosis is involved in MS and suggest that targeting RIPK1 may represent a therapeutic strategy for MS.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (Xbp1) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional Xbp1 mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated Ire1α deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon Ire1α deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, Ire1α deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure.

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the gold standard for quantitative mass spectrometry analysis. The simultaneous processing of a mixture of labeled and unlabeled samples allows a sensitive and accurate comparative analysis between the respective proteomes. Here, we describe a cost-effective feeding protocol based on a newly developed (15)N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from (15)N-labeled vs. (14)N-unlabeled mice was examined by mass spectrometry and differences in the expression of glyoxalase-1 (GLO1) and histidine triad nucleotide binding protein 2 (Hint2) proteins were correlated with the animals' psychopathological behaviors for methodological validation and proof of concept, respectively. Additionally, phenotyping unraveled an antidepressant-like effect of the incorporation of the stable isotope (15)N into the proteome of highly anxious mice. This novel phenomenon is of considerable relevance to the metabolic labeling method and could provide an opportunity for the discovery of candidate proteins involved in depression-like behavior. The newly developed (15)N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry.