The prognostic value of serum C-reactive protein-bound serum amyloid A in early-stage lung cancer

Background:Elevated levels of serum C-reactive protein(CRP) have been reported to have prognostic significance in lung cancer patients.This study aimed to further identify CRP-bound components as prognostic markers for lung cancer and validate their prognostic value.Methods:CRP-bound components obtained from the serum samples from lung cancer patients or healthy controls were analyzed by differential proteomics analysis.CRP-bound serum amyloid A(CRP-SAA) was evaluated by coimmunoprecipitation(IP).Serum samples from two independent cohorts with lung cancer(retrospective cohort,242patients;prospective cohort,222 patients) and healthy controls(159 subjects) were used to evaluate the prognostic value of CRP-SAA by enzyme-linked immunosorbent assay.Results:CRP-SAA was identified specifically in serum samples from lung cancer patients by proteomic analysis.CRP binding to SAA was confirmed by co-IP in serum samples from lung cancer patients and cell culture media.The level of CRP-SAA was significantly higher in patients than in healthy controls(0.37 ± 0.58 vs.0.03 ± 0.04,P < 0.001).Elevated CRP-SAA levels were significantly associated with severe clinical features of lung cancer.The elevation of CRPSAA was associated with lower survival rates for both the retrospective(hazard ration[HR]= 2.181,95%confidence interval[CI]= 1.641-2.897,P < 0.001) and the prospective cohorts(HR = 2.744,95%CI = 1.810-4.161,P < 0.001).Multivariate Cox analysis showed that CRP-SAA was an independent prognostic marker for lung cancer.Remarkably,in stages l-ll patients,only CRP-SAA,not total SAA or CRP,showed significant association with overall survival in two cohorts.Moreover,univariate and multivariate Cox analyses also showed that only CRP-SAA could be used as an independent prognostic marker for early-stage lung cancer patients.Conclusion:CRP-SAA could be a better prognostic marker for lung cancer than total SAA or CRP,especially in earlystage patients.

Global characterization of modifications to the charge isomers of IgG antibody

Posttranslational modifications of antibody products affect their stability,charge distribution,and drug activity and are thus a critical quality attribute.The comprehensive mapping of antibody modifications and different charge isomers(CIs)is of utmost importance,but is challenging.We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity.The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection,followed by stepwise structural characterization at three levels.First,the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach;this showed differences in glycoforms and deamidation status.Second,at the peptide level,common modifications of oxidation,deamidation,and glycosylation were identified.Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs.In total,10 N-glycoforms were detected by peptide mapping.Finally,an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides.Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms.The results revealed that sialic acid modification is a critical factor accounting for charge heterogeneity,which is otherwise missed in peptide mapping and intact molecular weight analyses.This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

Prediction of risk of cardiovascular events in patients with mild to moderate coronary artery lesions using naive Bayesian networks

Background This prospective study integrated multiple clinical indexes and inflammatory markers associated with coronary atherosclerotic vulnerable plaque to establish a risk prediction model that can evaluate a patient with certain risk factors for the likelihood of the occurrence of a coronary heart disease event within one year. Methods This study enrolled in 2686 patients with mild to moderate coronary artery lesions. Eighty-five indexes were recorded, included baseline clinical data, laboratory studies, and procedural characteristics. During the 1-year follow-up, 233 events occurred, five patients died, four patients suffered a nonfatal myocardial infarction, four patients underwent revascularization, and 220 patients were readmitted for angina pectoris. The Risk Estimation Model and the Simplified Model were conducted using Bayesian networks and compared with the Single Factor Models. Results The area under the curve was 0.88 for the Bayesian Model and 0.85 for the Simplified Model, while the Single Factor Model had a maximum area under the curve of 0.65. Conclusion The new models can be used to assess the short-term risk of individual coronary heart disease events and may assist in guiding preventive care.

Recent advances in CRISPR-based genome editing technology and its applications in cardiovascular research

The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats(CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein(Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases(CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.

Development and Validation of a Simoa Assay for Determination of Recombinant Batroxobin in Human Serum

Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies.Therefore,a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101.In this study,a sensitive bioanalytical method was developed and validated,using a Quanterix single molecular array(Simoa)assay.Moreover,to thoroughly assess the platform,enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed,and their performance was compared with that of this novel technology platform.The assay was validated in compliance with the current guidelines.Measurements with the Simoa assay were precise and accurate,presenting a valid assay range from 6.55 to 4000 pg/mL.The intra-and inter-run accuracy and precision were within-19.3%to 15.3%and 5.5%to 17.0%,respectively.S3101 was stable in human serum for 280 days at-20℃and-70℃,for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature(22℃-28℃),respectively,and after five and two freeze-thaw cycles at-70℃and-20oC,respectively.The Simoa assay also demonstrated sufficient dilution linearity,assay sensitivity,and parallelism for quantifying S3101 in human serum.The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.

Sustained Akt signaling in articular chondrocytes causes osteoarthritis via oxidative stress-induced senescence in mice

Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are unclear. In this study, we found activated Akt signaling in human OA cartilage as well as in a mouse OA model with surgical destabilization of the medial meniscus.Genetic mouse models mimicking sustained Akt signaling in articular chondrocytes via PTEN deficiency driven by either Col2a1-Cre or Col2a1-Cre^(ERT2) developed OA, whereas restriction of Akt signaling reversed the OA phenotypes in PTEN-deficient mice.Mechanistically, prolonged activation of Akt signaling caused an accumulation of reactive oxygen species and triggered chondrocyte senescence as well as a senescence-associated secretory phenotype, whereas chronic administration of the antioxidant N-acetylcysteine suppressed chondrocyte senescence and mitigated OA progression in PTEN-deficient mice. Therefore,inhibition of Akt signaling by PTEN is required for the maintenance of articular cartilage. Disrupted Akt signaling in articular chondrocytes triggers oxidative stress-induced chondrocyte senescence and causes OA.

Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3-10 and 4-7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5-9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.

Toll-like receptor 5-mediated signaling enhances liver regeneration in mice

Background:Toll-like receptor 5(TLR5)-mediated pathways play critical roles in regulating the hepatic immune response and show hepatoprotective effects in mouse models of hepatic diseases.However,the role of TLR5 in experimental models of liver regeneration has not been reported.This study aimed to investigate the role of TLR5 in partial hepatectomy(PHx)-induced liver regeneration.Methods:We performed 2/3 PHx in wild-type(WT)mice,TLR5 knockout mice,or TLR5 agonist CBLB502 treated mice,as a model of liver regeneration.Bacterial flagellin content was measured with ELISA,and hepatic TLR5 expression was determined with quantitative PCR analyses and flow cytometry.To study the effects of TLR5 on hepatocyte proliferation,we analyzed bromodeoxyuridine(BrdU)incorporation and proliferating cell nuclear antigen(PCNA)expression with immunohistochemistry(IHC)staining.The effects of TLR5 during the priming phase of liver regeneration were examined with quantitative PCR analyses of immediate early gene mRNA levels,and with Western blotting analysis of hepatic NF-κB and STAT3 activation.Cytokine and growth factor production after PHx were detected with real-time PCR and cytometric bead array(CBA)assays.Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx.Results:The bacterial flagellin content in the serum and liver increased,and the hepatic TLR5 expression was significantly up-regulated in WT mice after PHx.TLR5-deficient mice exhibited diminished numbers of BrdU-and PCNA-positive cells,suppressed immediate early gene expression,and decreased cytokine and growth factor production.Moreover,PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5–/–mice,as compared with WT mice.Consistently,the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation,which was correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver.Furthermore,Tlr5–/–mice displayed significantly lower hepatic lipid concentrations and smaller Oil Red O positive areas than those in control mice after PHx.Conclusions:We reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx.Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest the potential of TLR5 agonist to promote liver regeneration.

An overview of human protein databases and their application to functional proteomics in health and disease

Functional proteomics can be defined as a strategy to couple proteomic information with biochemical and physiological analyses with the aim of understanding better the functions of proteins in normal and diseased organs.In recent years,a variety of publicly available bioinformatics databases have been developed to support protein-related information management and biological knowledge discovery.In addition to being used to annotate the proteome,these resources also offer the opportunity to develop global approaches to the study of the functional role of proteins both in health and disease.Here,we present a comprehensive review of the major human protein bioinformatics databases.We conclude this review by discussing a few examples that illustrate the importance of these databases in functional proteomics research.

Pathological features of COVID-19-associated lung injury:a preliminary proteomics report based on clinical samples

The COVID-19 pandemic has emerged as a global health emergency due to its association with severe pneumonia and relative high mortality.However,the molecular characteristics and pathological features underlying COVID-19 pneumonia remain largely unknown.To characterize molecular mechanisms underlying COVID-19 pathogenesis in the lung tissue using a proteomic approach,fresh lung tissues were obtained from newly deceased patients with COVID-19 pneumonia.After virus inactivation,a quantitative proteomic approach combined with bioinformatics analysis was used to detect proteomic changes in the SARS-CoV-2-infected lung tissues.We identified significant differentially expressed proteins involved in a variety of fundamental biological processes including cellular metabolism,blood coagulation,immune response,angiogenesis,and cell microenvironment regulation.Several inflammatory factors were upregulated,which was possibly caused by the activation of NF-κB signaling.Extensive dysregulation of the lung proteome in response to SARS-CoV-2 infection was discovered.Our results systematically outlined the molecular pathological features in terms of the lung response to SARS-CoV-2 infection,and provided the scientific basis for the therapeutic target that is urgently needed to control the COVID-19 pandemic.

Proteomics provides individualized options of precision medicine for patients with gastric cancer

While precision medicine driven by genome sequencing has revolutionized cancer care,such as lung cancer,its impact on gastric cancer(GC)has been minimal.GC patients are routinely treated with chemotherapy,but only a fraction of them receive the clinical benefit.There is an urgent need to develop biomarkers or algorithms to select chemo-sensitive patients or apply targeted therapy.Here,we carried out retrospective analyses of 1,020 formalin-fixed,paraffin-embedded GC surgical resection samples from 5 hospitals and developed a mass spectrometry-based workflow for proteomic subtyping of GC.We identified two proteomic subtypes:the chemo-sensitive group(CSG)and the chemo-insensitive group(CIG)in the discovery set.The 5-year overall survival of CSG was significantly improved in patients who had received adjuvant chemotherapy after surgery compared with those who received surgery only(64.2%vs.49.6%;Cox P-value=0.002),whereas no such improvement was observed in CIG(50.0%vs.58.6%;Cox P-value=0.495).We validated these results in an independent validation set.Further,differential proteome analysis uncovered 9 FDA-approved drugs that may be applicable for targeted therapy of GC.A prospective study is warranted to test these findings for future GC patient care.

A new photolabeling probe for efficient enrichment and deep profiling of cell surface membrane proteome by mass spectrometry

The cell surface membrane proteome is a class of proteins encoded by ~25% of all protein-coding genes in living organisms and plays a key role in mediating communication between the cells and their surrounding environment. However, most cell surface membrane proteins(CSMPs) are naturally expressed at very low levels compared with intracellular proteins. The difficulties in their purification with high specificity further hinder the understanding of their structure and function. In this study, we developed a new photolabeling probe to achieve efficient tagging and facile enrichment of the CSMPs. The probe is composed of a lipid tail for cell surface localization, a polyethylene glycol(PEG) spacer for increased water solubility, two 4-(N-maleimido)benzophenone(MBP) groups for UV-active tagging of the CSMPs, and a biotin tag for subsequent isolation. Application of this photolabeling probe resulted in the successful enrichment and identification of 3098 annotated CSMPs in HT22 cells with close to 70% selectivity. The proposed photolabeling probe and enrichment strategy were demonstrated to be a powerful method for deep cell surface proteome profiling, representing one of the largest groups of current drug targets.

Proteome-wide epitope mapping identifies a resource of antibodies for SARS-CoV-2 detection and neutralization

Dear Editor,Since December 2019,the coronavirus disease 2019(COVID-19)caused by the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)has become a worldwide pandemic.1 Significant efforts have been made to generate antibodies to help study COVID-19 pathogenesis,perform diagnostic testing,and develop treatment to neutralize SARS-CoV-2 activity.However,the specific sequence of amino acids recognized and bound by an antibody,the"epitope"is unknown for most antibodies.In this study,we developed a high-throughput epitope mapping platform using a peptide-based SARS-CoV-2 proteome microarray.2 to detect the binding epitopes of 57 commercial antibodies to ORFlab,nudeocapsid(N);spike(S),envelop(E),membrane(M),ORF3a,ORF6,ORF7a,and ORF8 proteins(Supplementary Table S1).

Proteomic Stratification of Prognosis and Treatment Options for Small Cell Lung Cancer

Small cell lung cancer(SCLC)is a highly malignant and heterogeneous cancer with limited therapeutic options and prognosis prediction models.Here,we analyzed formalin-fixed,paraffin-embedded(FFPE)samples of surgical resections by proteomic profiling,and stratified SCLC into three proteomic subtypes(S-I,S-II,and S-III)with distinct clinical outcomes and chemotherapy responses.The proteomic subtyping was an independent prognostic factor and performed better than current tumor–node–metastasis or Veterans Administration Lung Study Group staging methods.The subtyping results could be further validated using FFPE biopsy samples from an independent cohort,extending the analysis to both surgical and biopsy samples.The signatures of the S-II subtype in particular suggested potential benefits from immunotherapy.Differentially overexpressed proteins in S-III,the worst prognostic subtype,allowed us to nominate potential therapeutic targets,indicating that patient selection may bring new hope for previously failed clinical trials.Finally,analysis of an independent cohort of SCLC patients who had received immunotherapy validated the prediction that the S-II patients had better progression-free survival and overall survival after first-line immunotherapy.Collectively,our study provides the rationale for future clinical investigations to validate the current findings for more accurate prognosis prediction and precise treatments.

Integrative analysis of tumor stemness and immune microenvironment deciphers novel molecular subtypes in hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is a highly heterogeneous tumor,with dynamic equilibrium and complex interplay between its intricate tumor nature and ambient tumor immune microenvironment(TIME).1 Elegant research has indicated that cancer stem cells,a small subset of neoplastic cells confined within dedicated niches,display stem cell-like properties and interact with cells in TIME,thereby imparting an indelible impact on stemness regulation,tumor heterogeneity,and cancer cell plasticity.2 Previous taxonomies solely from the perspective of stemness or TIME may introduce some degree of bias in the comprehension of HCC carcinogenesis,3,4 and thus it is of paramount importance to systematically consider tumor stemness and TIME as a whole to truly portray the biological landscape of HCC.

Resident to exhausted CD4^(+)T cell ratio is associated with the prognosis of gastric cancer

Gastric cancer(GC)ranks fifth for cancer incidence and fourth for mortality globally.1 Clinical outcomes have varied among patients receiving similar treatments at the same stage,suggesting the current prognostic tools remain somewhat flawed.2,3 single-cell analysis of GC data allowed us to dissect transcriptional programs underlying lymphocyte residency and exhaustion.

At a glance:Proteomics in China

Proteomics is a new science that focuses on the comprehensive analysis of proteins in intact organisms or in molecule machineries, organelles, cells, tissues, or organs. It has become an important area of interests in life sciences and has propelled the rapid development of cutting-edge biotechnology in the 21st century.

Lifeomics leads the age of grand discoveries

When our knowledge of a field accumulates to a certain level,we are bound to see the rise of one or more great scientists.They will make a series of grand discoveries/breakthroughs and push the discipline into an 'age of grand discoveries'.Mathematics,geography,physics and chemistry have all experienced their ages of grand discoveries;and in life sciences,the age of grand discoveries has appeared countless times since the 16th century.Thanks to the ever-changing development of molecular biology over the past 50 years,contemporary life science is once again approaching its breaking point and the trigger for this is most likely to be 'lifeomics'.At the end of the 20th century,genomics wrote out the 'script of life';proteomics decoded the script;and RNAomics,glycomics and metabolomics came into bloom.These 'omics',with their unique epistemology and methodology,quickly became the thrust of life sciences,pushing the discipline to new high.Lifeomics,which encompasses all omics,has taken shape and is now signalling the dawn of a new era,the age of grand discoveries.

Antigenically dominant proteins within the human liver mitochondrial proteome identified by monoclonal antibodies

Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning. Antibodies that recognized mitochondrial proteins were identified by screening human liver cDNA expression libraries. In this study, we identified 6 major antigens that were recognized by at least 2 different monoclonal antibodies (mAbs). The proteins that were antigenically dominant were: acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase), aldehyde dehydrogenase 1 family member A1, carbamoyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), enoyl coenzyme A hydratase 1, and hydroxysteroid (11-beta) dehydrogenase 1. We also determined the subcellular localizations of these enzymes within the mitochondria using immunohistocytochemistry. We believe that these well-characterized antibodies will provide a valuable resource for the Human Liver Proteome Project (HLPP), and will make studies aimed at investigating liver mitochondrial function far easier to perform in future. Our results provide strong evidence that, (i) depletion of dominant proteins from liver mitochondrial samples is possible and, (ii) the approaches adopted in this study can be used to explore or validate protein-protein interactions in this important organelle.

Hydrogen sulfide-linked persulfidation of ABI4 controls ABA responses through the transactivation of MAPKKK18 in Arabidopsis

Hydrogen sulfide(H2S)is a signaling molecule that regulates plant hormone and stress responses.The phytohormone abscisic acid(ABA)plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1(DES1)and the production of H2S in guard cells.However,it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals.In this study,we discovered that ABSCISIC ACID INSENSITIVE 4(ABI4)acts downstream of DES1 in the control of ABA responses in Arabidopsis.ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA,and loss of DES1 function impairs this process.Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination,seedling establishment,and stomatal closure,which are abolished in the ABI4Cys250Ala mutated variant.Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA.Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18(MAPKKK18),which propagates the MAPK signaling cascade induced by ABA.Furthermore,the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18,and ABI4 can bind the DES1 promoter,forming a regulatory loop.Taken together,these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.