Targeting Smurf1 to block PDK1-Akt signaling in KRAS-mutated colorectal cancer.

The phosphoinositide 3-kinase (PI3K)-Akt axis is one of the most frequently activated pathways and is demonstrated as a therapeutic target in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC). Targeting the PI3K-Akt pathway has been a challenging undertaking through the decades. Here we unveiled an essential role of E3 ligase SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated phosphoinositide-dependent protein kinase 1 (PDK1) neddylation in PI3K-Akt signaling and tumorigenesis. Upon growth factor stimulation, Smurf1 immediately triggers PDK1 neddylation and the poly-neural precursor cell expressed developmentally downregulated protein 8 (poly-Nedd8) chains recruit methyltransferase SET domain bifurcated histone lysine methyltransferase 1 (SETDB1). The cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS) consisting of PDK1, Smurf1 and SETDB1 directs Akt membrane attachment and T308 phosphorylation. Smurf1 deficiency dramatically reduces CRC tumorigenesis in a genetic mouse model. Furthermore, we developed a highly selective Smurf1 degrader, Smurf1-antagonizing repressor of tumor 1, which exhibits efficient PDK1-Akt blockade and potent tumor suppression alone or combined with PDK1 inhibitor in KRAS-mutated CRC. The findings presented here unveil previously unrecognized roles of PDK1 neddylation and offer a potential strategy for targeting the PI3K-Akt pathway and KRAS mutant cancer therapy.

The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis.

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.

Dhx38 is required for the maintenance and differentiation of erythro-myeloid progenitors and hematopoietic stem cells by alternative splicing.

Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.

OncoSplicing: an updated database for clinically relevant alternative splicing in 33 human cancers.

Alternative splicing (AS) represents a crucial method in mRNA level to regulate gene expression and contributes to the protein complexity. Abnormal splicing has been reported to play roles in several diseases, including cancers. We developed the OncoSplicing database for visualization of survival-associated and differential alternative splicing in 2019. Here, we provide an updated version of OncoSplicing for an integrative view of clinically relevant alternative splicing based on 122 423 AS events across 33 cancers in the TCGA SpliceSeq project and 238 558 AS events across 32 cancers in the TCGA SplAdder project. The new version of the database contains several useful features, such as annotation of alternative splicing-associated transcripts, survival analysis based on median and optimal cut-offs, differential analysis between TCGA tumour samples and adjacent normal samples or GTEx normal samples, pan-cancer views of alternative splicing, splicing differences and results of Cox'PH regression, identification of clinical indicator-relevant and cancer-specific splicing events, and downloadable splicing data in the SplAdder project. Overall, the substantially updated version of OncoSplicing (www.oncosplicing.com) is a user-friendly and registration-free database for browsing and searching clinically relevant alternative splicing in human cancers.

Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP-MS.

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma-mass spectrometry (ICP-MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP-MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.

Sensitive N-Glycopeptide Profiling of Single and Rare Cells Using an Isobaric Labeling Strategy without Enrichment.

Single-cell omics is critical in revealing population heterogeneity, discovering unique features of individual cells, and identifying minority subpopulations of interest. As one of the major post-translational modifications, protein N-glycosylation plays crucial roles in various important biological processes. Elucidation of the variation in N-glycosylation patterns at single-cell resolution may largely facilitate the understanding of their key roles in the tumor microenvironment and immune therapy. However, comprehensive N-glycoproteome profiling for single cells has not been achieved due to the extremely limited sample amount and incompatibility with the available enrichment strategies. Here, we have developed an isobaric labeling-based carrier strategy for highly sensitive intact N-glycopeptide profiling for single cells or a small number of rare cells without enrichment. Isobaric labeling has unique multiplexing properties, by which the "total" signal from all channels triggers MS/MS fragmentation for N-glycopeptide identification, while the reporter ions provide quantitative information. In our strategy, a carrier channel using N-glycopeptides obtained from bulk-cell samples significantly improved the "total" signal of N-glycopeptides and, therefore, promoted the first quantitative analysis of averagely 260 N-glycopeptides from single HeLa cells. We further applied this strategy to study the regional heterogeneity of N-glycosylation of microglia in mouse brain and discovered region-specific N-glycoproteome patterns and cell subtypes. In conclusion, the glycocarrier strategy provides an attractive solution for sensitive and quantitative N-glycopeptide profiling of single/rare cells that cannot be enriched by traditional workflows.

Comprehensive characterization of alternative splicing in renal cell carcinoma.

Irregular splicing was associated with tumor formation and progression in renal cell carcinoma (RCC) and many other cancers. By using splicing data in the TCGA SpliceSeq database, RCC subtype classification was performed and splicing features and their correlations with clinical course, genetic variants, splicing factors, pathways activation and immune heterogeneity were systemically analyzed. In this research, alternative splicing was found useful for classifying RCC subtypes. Splicing inefficiency with upregulated intron retention and cassette exon was associated with advanced conditions and unfavorable overall survival of patients with RCC. Splicing characteristics like splice site strength, guanine and cytosine content and exon length may be important factors disrupting splicing balance in RCC. Other than cis-acting and trans-acting regulation, alternative splicing also differed in races and tissue types and is also affected by mutation conditions, pathway settings and the response to environmental changes. Severe irregular splicing in tumor not only indicated terrible intra-cellular homeostasis, but also changed the activity of cancer-associated pathways by different splicing effects including isoforms switching and expression regulation. Moreover, irregular splicing and splicing-associated antigens were involved in immune reprograming and formation of immunosuppressive tumor microenvironment. Overall, we have described several clinical and molecular features in RCC splicing subtypes, which may be important for patient management and targeting treatment.

Role of human papillomavirus and associated viruses in bladder cancer: An updated review.

Bladder cancer (BC) is a complex disease affecting the urinary system and is regulated by several carcinogenic factors. Viral infection is one such factor that has attracted extensive attention in BC. Human papillomavirus (HPV) is the most common sexually transmitted infection, and although multiple researchers have explored the role of HPV in BC, a consensus has not yet been reached. In addition, HPV-associated viruses (e.g., human immunodeficiency virus, herpes simplex virus, BK virus, and JC virus) appear to be responsible for the occurrence and progression of BC. This study systematically reviews the relationship between HPV-associated viruses and BC to elucidate the role of these viruses in the onset and progression of BC. In addition, the study aims to provide a greater insight into the biology of HPV-associated viruses, and assess potential strategies for treating virus-induced BC. The study additionally focuses on the rapid development of oncolytic viruses that provide a potentially novel option for the treatment of BC.

Prpf31 is essential for the survival and differentiation of retinal progenitor cells by modulating alternative splicing.

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.

Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance.

Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here, through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore, we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn (D213N) mutation (OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.

Synthesis of Surface-Functionalized Molybdenum Disulfide Nanomaterials for Efficient Adsorption and Deep Profiling of the Human Plasma Proteome by Data-Independent Acquisition.

Blood is one of the most important clinical samples for protein biomarker discovery, as it provides rich physiological and pathological information and is easy to obtain with low invasiveness. However, the discovery of protein biomarkers in the blood by mass spectrometry (MS)-based proteomic strategies has been shown to be highly challenging due to the particularly large concentration range of proteins and the strong interference by the high-abundant proteins in the blood. Therefore, developing sensitive methods for low-abundant biomarker protein identification is a key issue that has received great attention. Here, we report the synthesis and characterization of surface-functionalized magnetic molybdenum disulfide (MoS2) for the large-scale adsorption of low-abundant plasma proteins and deep profiling by MS. MoS2 nanomaterials resulted in the coverage of more than 3400 proteins (including a single-peptide hit) in a single LC-MS analysis without peptide prefractionation using pooled plasma samples, which were five times more than those obtained by the direct analysis of the plasma proteome. A detection limit in the low ng L-1 range was obtained, which is rare compared with previous reports.

Novel Two-Dimensional MoS2-Ti4+ Nanomaterial for Efficient Enrichment of Phosphopeptides and Large-Scale Identification of Histidine Phosphorylation by Mass Spectrometry.

Due to its key roles in regulating the occurrence and development of cancer, protein histidine phosphorylation has been increasingly recognized as an important form of post-translational modification in recent years. However, large-scale analysis of histidine phosphorylation is much more challenging than that of serine/threonine or tyrosine phosphorylation, mainly because of its acid lability. In this study, MoS2-Ti4+ nanomaterials were synthesized using a solvothermal method and taking advantage of the electrostatic adsorption between MoS2 nanosheets and Ti4+. The MoS2-Ti4+ nanomaterials have the advantage of the combined affinity of Ti4+ and Mo toward phosphorylation under medium acidic conditions (pH = 3), which is crucial for preventing hydrolysis and loss of histidine phosphorylation during enrichment. The feasibility of using the MoS2-Ti4+ nanomaterial for phosphopeptide enrichment was demonstrated using mixtures of ß-casein and bovine serum albumin (BSA). Further evaluation revealed that the MoS2-Ti4+ nanomaterial is capable of enriching synthetic histidine phosphopeptides from 1000 times excess tryptic-digested HeLa cell lysate. Application of the MoS2-Ti4+ nanomaterials for large-scale phosphopeptide enrichment results in the identification of 10 345 serine, threonine, and tyrosine phosphosites and the successful mapping of 159 histidine phosphosites in HeLa cell lysates, therefore indicating great potential for deciphering the vital biological roles of protein (histidine) phosphorylation.

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

Integrated Analysis of Genetic Abnormalities of the Histone Lysine Methyltransferases in Prostate Cancer.

BACKGROUND The histone methyltransferase (HMT) family includes histone lysine methyltransferases (HKMTs) and histone/protein arginine methyltransferases (PRMTs). The role of HMT gene variants in prostate cancer remains unknown. Therefore, this study aimed to evaluate HMT gene variants in the pathogenesis and prognosis of human prostate cancer, using in vitro cell studies and bioinformatics analysis. MATERIAL AND METHODS Integrative bioinformatics analysis of the expression of 51 HMT genes in human prostate cancer was based on datasets from the Cancer Genome Atlas (TCGA). Correlation and regression analysis were used to identify critical HMTs in prostate cancer. Kaplan-Meier and the area under the receiver operating characteristics curve (AUROC) were performed to evaluate the function of the HMTs on prognosis. Gene expression and function of 22Rv1 human prostate carcinoma cells were studied. RESULTS The HMT genes identified to have a role in the pathogenesis of prostate cancer included the EZH2, SETD5, PRDM12, NSD1, SETD6, SMYD1, and the WHSC1L1 gene. The EZH2, SETD5, and SMYD1 genes were selected as a prognostic panel, with the SUV420H2 HMT gene. SETD2, NSD1, and ASH1L were identified as critical genes in the development of castration-resistant prostate cancer (CRPC), similar to mixed-lineage leukemia (MLL) complex family members. Knockdown of the SETD5 gene in 22Rv1 prostate carcinoma cells in vitro inhibited cancer cell growth and migration. CONCLUSIONS HMT gene variants may have a role in the pathogenesis of prostate cancer. Future studies may determine the role of HMT genes as prognostic biomarkers in patients with prostate cancer.

Two-Dimensional MoS2-Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides.

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS2/Au-NP (gold nanoparticle)-L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS2 nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g-1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 µg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics.

Efficacy and safety of PDE5-Is and α-1 blockers for treating lower ureteric stones or LUTS: a meta-analysis of RCTs.

BACKGROUND: Lower ureteric stones and lower urinary tract symptoms are common in urology.Drug treatment is one of standard therapy,but the efficacy was controversial.Thus we aimed to investigate the efficacy and safety of monotherapy or combination therapy of adrenoceptor1 blockers and phosphodiesterase5 inhibitors for treatment. METHODS: Randomized controlled trials up to November 2016 were retrieved from PubMed, the Cochrane Library, Web of Science and Embase. A total of 17 studies were included. We analyzed data through random or fixed effect models. The heterogeneity between studies was assessed by the I2 test statistic. RESULTS: As for lower ureter stones, our analysis demonstrated tadalafil had a significantly lower incidence of abnormal ejaculation than adrenoceptor1 blockers (2.31 95%CI 0.22to0.84, P = 0.01),while combination therapy had a higher expulsion rate (2.49 95%CI 1.44to4.29, P = 0.001) and shorter expulsion time (- 1.98 95%CI -3.08to0.88, P = 0.0004) than tamsulosin. As for lower urinary tract symptoms, our analysis indicated adrenoceptor1 blockers was more effective than phosphodiesterase5 inhibitors on decreasing International Prostate Symptom Score (1.96 95%CI 0.03to3.89, P = 0.05) and Post-Void Residual (9.41 95%CI 1.40to14.41, P = 0.02) and phosphodiesterase5 inhibitors showed a greater effect than adrenoceptor1 blockers on improving Erectile Dysfunction (2.23 95%CI 1.24to3.22, P<0.0001).Combination therapy had a significantly better effect on International Prostate Symptom Score (1.47 95%CI 1.25to1.69, P<0.0001), Maximum flow rate (0.87 95%CI 0.71to1.04, P<0.0001), Post-Void Residual (10.74 95%CI 3.53to17.96,P = 0.004) and Quality of life (0.59 95%CI 0.22to0.97, P = 0.002) but was associated with higher incidences of adverse events (3.40 95%CI 1.82to6.36, P = 0.0001) than adrenoceptor1 blockers. Combination therapy had a significantly better effect on International Prostate Symptom Score (4.19 95%CI 3.34to5.04, P<0.0001), Maximum flow rate (1.86 95%CI 1.32to2.39, P<0.0001), Post-Void Residual (22.58 95%CI 9.13to36.04, P = 0.001) and Quality of life (0.68 95%CI 0.37to1.00, P<0.0001) without higher incidences of adverse events than PDE5-Is. CONCLUSIONS: In conclusion, this meta-analysis suggested combination therapy had a best efficacy of therapy for lower ureteric stones or lower urinary tract symptoms correlated with benign prostatic hyperplasia than monotherapy. Adrenoceptor1 blockers was more effective than phosphodiesterase5 inhibitors on International Prostate Symptom Score and Post-Void Residual. Both monotherapy and combination therapy were safe.

Covalent organic framework-coated magnetic graphene as a novel support for trypsin immobilization.

Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268 µg mg-1). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2 min. For the digestion of proteins extracted from Amygdalus pedunculata, a total of 2833 protein groups were identified, which was slightly more than those obtained by 12 h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis. Graphical abstract Covalent organic frameworks-coated magnetic graphene was prepared as novel carrier for highly efficient tryptic immobilization.

Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer.

Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively. Among the above MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed MPs after verifying with the stringent spectra match and isobaric and single amino acid variants filtering. Functional investigation of these 47 MPs revealed that 11 MPs were testis-specific proteins and 7 MPs were involved in spermatogenesis process. Therefore, we concluded that higher scanning speed and resolution of HF might be factors for improving the low-abundance MP identification in future C-HPP studies. All mass-spectrometry data from this study have been deposited in the ProteomeXchange with identifier PXD004092.

TOND1 confers tolerance to nitrogen deficiency in rice.

Nitrogen (N), the most important mineral nutrient for plants, is critical to agricultural production systems. N deficiency severely affects rice growth and decreases rice yields. However, excessive use of N fertilizer has caused severe pollution to agricultural and ecological environments. The necessity of breeding of crops that require lower input of N fertilizer has been recognized. Here we identified a major quantitative trait locus on chromosome 12, Tolerance Of Nitrogen Deficiency 1 (TOND1), that confers tolerance to N deficiency in the indica cultivar Teqing. Sequence verification of 75 indica and 75 japonica cultivars from 18 countries and regions demonstrated that only 27.3% of cultivars (41 indica cultivars) contain TOND1, whereas 72.7% of cultivars, including the remaining 34 indica cultivars and all 75 japonica cultivars, do not harbor the TOND1 allele. Over-expression of TOND1 increased the tolerance to N deficiency in the TOND1-deficient rice cultivars. The identification of TOND1 provides a molecular basis for breeding rice varieties with improved grain yield despite decreased input of N fertilizers.

Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content.