Ludovic C. Gillet
Jun 1, 2012; 11:O111.016717-O111.016717
Research

Linn Fagerberg
Feb 1, 2014; 13:397-406
Research

Haiyong Wang
Math. Comp. 93 (), 2861-2884.
Abstract, references and article information

Katherine Baker, Lehel Banjai and Mariya Ptashnyk
Math. Comp. 93 (), 2711-2743.
Abstract, references and article information

T. De Ryck and S. Mishra
Math. Comp. 93 (), 2643-2677.
Abstract, references and article information

Constantin Carle and Marlis Hochbruck
Math. Comp. 93 (), 2611-2641.
Abstract, references and article information

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15–25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host–pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence.

Visual Abstract

Toma Keser
Dec 29, 2020; 0:RA120.002433v1-mcp.RA120.002433
Research

Litong Nie
Dec 1, 2020; 19:2015-2029
Research

David Durán
Nov 19, 2020; 0:RA120.002276v1-mcp.RA120.002276
Research

Xinting Zhang
Dec 8, 2020; 0:RA120.002306v1-mcp.RA120.002306
Research

Colin T. McDowell
Nov 24, 2020; 0:RA120.002256v1-mcp.RA120.002256
Research

Daniel J. Geiszler
Dec 1, 2020; 0:TIR120.002216v1-mcp.TIR120.002216
Technological Innovation and Resources

Johannes Griss
Dec 1, 2020; 19:2115-2124
Technological Innovation and Resources

Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates, limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use, and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user-defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percentage of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates, and (v) localization of aggregates (cytosol, perinuclear, or nuclear). A data table of aggregate information on a per cell basis, as well as a summary table, is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules, and inclusion bodies caused by huntingtin polyglutamine expansion.

Montgomery Blencowe
Dec 23, 2020; 0:jlr.RA120000713v1-jlr.RA120000713
Research Articles

Genome-wide association studies (GWAS) have implicated ~380 genetic loci for plasma lipid regulation. However, these loci only explain 17-27% of the trait variance and a comprehensive understanding of the molecular mechanisms has not been achieved. In this study, we utilized an integrative genomics approach leveraging diverse genomic data from human populations to investigate whether genetic variants associated with various plasma lipid traits, namely total cholesterol (TC), high and low density lipoprotein cholesterol (HDL and LDL), and triglycerides (TG), from GWAS were concentrated on specific parts of tissue-specific gene regulatory networks. In addition to the expected lipid metabolism pathways, gene subnetworks involved in ‘interferon signaling’, ‘autoimmune/immune activation’, ‘visual transduction’, and ‘protein catabolism’ were significantly associated with all lipid traits. Additionally, we detected trait-specific subnetworks, including cadherin-associated subnetworks for LDL, glutathione metabolism for HDL, valine, leucine and isoleucine biosynthesis for TC, and insulin signaling and complement pathways for TG. Finally, utilizing gene-gene relations revealed by tissue-specific gene regulatory networks, we detected both known (e.g. APOH, APOA4, and ABCA1) and novel (e.g. F2 in adipose tissue) key regulator genes in these lipid-associated subnetworks. Knockdown of the F2 gene (Coagulation Factor II, Thrombin) in 3T3-L1 and C3H10T1/2 adipocytes reduced gene expression of Abcb11, Apoa5, Apof, Fabp1, Lipc, and Cd36, reduced intracellular adipocyte lipid content, and increased extracellular lipid content, supporting a link between adipose thrombin and lipid regulation. Our results shed light on the complex mechanisms underlying lipid metabolism and highlight potential novel targets for lipid regulation and lipid-associated diseases.

The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.

Ion mobility brings an additional dimension of separation to LC–MS, improving identification of peptides and proteins in complex mixtures. A recently introduced timsTOF mass spectrometer (Bruker) couples trapped ion mobility separation to TOF mass analysis. With the parallel accumulation serial fragmentation (PASEF) method, the timsTOF platform achieves promising results, yet analysis of the data generated on this platform represents a major bottleneck. Currently, MaxQuant and PEAKS are most used to analyze these data. However, because of the high complexity of timsTOF PASEF data, both require substantial time to perform even standard tryptic searches. Advanced searches (e.g. with many variable modifications, semi- or non-enzymatic searches, or open searches for post-translational modification discovery) are practically impossible. We have extended our fast peptide identification tool MSFragger to support timsTOF PASEF data, and developed a label-free quantification tool, IonQuant, for fast and accurate 4-D feature extraction and quantification. Using a HeLa data set published by Meier et al. (2018), we demonstrate that MSFragger identifies significantly (~30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (~10% more peptides). IonQuant outperforms both in terms of number of quantified proteins while maintaining good quantification precision and accuracy. Runtime tests show that MSFragger and IonQuant can fully process a typical two-hour PASEF run in under 70 min on a typical desktop (6 CPU cores, 32 GB RAM), significantly faster than other tools. Finally, through semi-enzymatic searching, we significantly increase the number of identified peptides. Within these semi-tryptic identifications, we report evidence of gas-phase fragmentation before MS/MS analysis.

In-depth coverage of proteomic analysis could enhance our understanding to the mechanism of the protein functions. Unfortunately, many highly hydrophobic proteins and low-abundance proteins, which play critical roles in signaling networks, are easily lost during sample preparation, mainly attributed to the fact that very few extractants can simultaneously satisfy the requirements on strong solubilizing ability to membrane proteins and good enzyme compatibility. Thus, it is urgent to screen out ideal extractant from the huge compound libraries in a fast and effective way. Herein, by investigating the interior mechanism of extractants on the membrane proteins solubilization and trypsin compatibility, a molecular dynamics simulation system was established as complement to the experimental procedure to narrow down the scope of candidates for proteomics analysis. The simulation data shows that the van der Waals interaction between cation group of ionic liquid and membrane protein is the dominant factor in determining protein solubilization. In combination with the experimental data, 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) is on the shortlist for the suitable candidates from comprehensive aspects. Inspired by the advantages of C12Im-Cl, an ionic liquid-based filter-aided sample preparation (i-FASP) method was developed. Using this strategy, over 3,300 proteins were confidently identified from 10³ HeLa cells (~100 ng proteins) in a single run, an improvement of 53% over the conventional FASP method. Then the i-FASP method was further successfully applied to the label-free relative quantitation of human liver cancer and para-carcinoma tissues with obviously improved accuracy, reproducibility and coverage than the commonly used urea-based FASP method. The above results demonstrated that the i-FASP method could be performed as a versatile tool for the in-depth coverage proteomic analysis of biological samples.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including actin dynamics and focal adhesion kinase (FAK) activity and one resistant cell line showed a strong migration phenotype. Pharmacological inhibition of the kinases FAK by Defactinib and of CHEK1 by Rabusertib showed a statistically significant sensitization to radiation in radioresistant PDAC cells. Together, the presented data map a comprehensive molecular network of radiation-induced signaling, improves the understanding of radioresistance and provides avenues for developing radiotherapeutic strategies.

High-speed analysis of large (prote)omics sample sets at the rate of thousands or millions of samples per day on a single platform has been a challenge since the beginning of proteomics. For many years, ESI-based MS methods have dominated proteomics because of their high sensitivity and great depth in analyzing complex proteomes. However, despite improvements in speed, ESI-based MS methods are fundamentally limited by their sample introduction, which excludes off-line sample preparation/fractionation because of the time required to switch between individual samples/sample fractions, and therefore being dependent on the speed of on-line sample preparation methods such as liquid chromatography. Laser-based ionization methods have the advantage of moving from one sample to the next without these limitations, being mainly restricted by the speed of modern sample stages, i.e. 10 ms or less between samples. This speed matches the data acquisition speed of modern high-performing mass spectrometers whereas the pulse repetition rate of the lasers (>1 kHz) provides a sufficient number of desorption/ionization events for successful ion signal detection from each sample at the above speed of the sample stages. Other advantages of laser-based ionization methods include the generally higher tolerance to sample additives and contamination compared with ESI MS, and the contact-less and pulsed nature of the laser used for desorption, reducing the risk of cross-contamination. Furthermore, new developments in MALDI have expanded its analytical capabilities, now being able to fully exploit high-performing hybrid mass analyzers and their strengths in sensitivity and MS/MS analysis by generating an ESI-like stable yield of multiply charged analyte ions. Thus, these new developments and the intrinsically high speed of laser-based methods now provide a good basis for tackling extreme sample analysis speed in the omics.

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.

Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.

We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the β-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies.

This article has been withdrawn by the authors. We discovered an error after this manuscript was published as a Paper in Press. Specifically, we learned that the structures of glycans presented for the PD-L1 peptide were drawn and labeled incorrectly. We wish to withdraw this article and submit a corrected version for review.

Intact glycopeptide identification has long been known as a key and challenging barrier to the comprehensive and accurate understanding the role of glycosylation in an organism. Intact glycopeptide analysis is a blossoming field that has received increasing attention in recent years. Mass spectrometry (MS)-based strategies and relative software tools are major drivers that have greatly facilitated the analysis of intact glycopeptides, particularly intact N-glycopeptides. This manuscript provides a systematic review of the intact glycopeptide identification process using mass spectrometry data generated in shotgun proteomic experiments, which typically focus on N-glycopeptide analysis. Particular attention is paid to the software tools that have been recently developed in the last decade for the interpretation and quality control of glycopeptide spectra acquired using different MS strategies. The review also provides information about the characteristics and applications of these software tools, discusses their advantages and disadvantages, and concludes with a discussion of outstanding tools.

This review covers recent developments in glycosaminoglycan (GAG) analysis via mass spectrometry (MS). GAGs participate in a variety of biological functions, including cellular communication, wound healing, and anticoagulation, and are important targets for structural characterization. GAGs exhibit a diverse range of structural features due to the variety of O- and N-sulfation modifications and uronic acid C-5 epimerization that can occur, making their analysis a challenging target. Mass spectrometry approaches to the structure assignment of GAGs have been widely investigated, and new methodologies remain the subject of development. Advances in sample preparation, tandem MS techniques (MS/MS), on-line separations and automated analysis software have advanced the field of GAG analysis. These recent developments have led to remarkable improvements in the precision and time efficiency for the structural characterization of GAGs.

Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and post-translational modifications (PTMs) leading to the pathogenesis of ESCC remains unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and post-translational modification (PTM) pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock which has seen a rapid rise in prevalence throughout Western Europe in recent years. Following ingestion of metacercariae (parasite cysts) by the mammalian host, newly-excysted juveniles (NEJs) emerge and invade the duodenal submucosa which causes significant pathology in heavy infections. The immature larvae then migrate upwards, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients and to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognise antigens from other helminths commonly found as co-infections with rumen fluke.

The Rhizobium-legume symbiosis is a beneficial interaction in which the bacterium converts atmospheric nitrogen into ammonia and delivers it to the plant in exchange for carbon compounds. This symbiosis implies the adaptation of bacteria to live inside host plant cells. In this work we apply RP-LC-MS/MS and  iTRAQ techniques to study the proteomic profile of endosymbiotic cells (bacteroids) induced by Rhizobium leguminosarum bv viciae strain UPM791 in legume nodules. Nitrogenase subunits, tricarboxylic acid cycle enzymes, and stress response proteins are amongst the most abundant from over one thousand rhizobial proteins identified in pea (Pisum sativum) bacteroids. Comparative analysis of bacteroids induced in pea and in lentil (Lens culinaris)nodules revealed the existence of a significant host-specific differential response affecting dozens of bacterial proteins, including stress-related proteins, transcriptional regulators, and proteins involved in the carbon and nitrogen metabolisms. A mutant affected in one of these proteins, homologous to a GntR-like transcriptional regulator, showed a symbiotic performance significantly  impaired in symbiosis with pea, but not with lentil plants. Analysis of the proteomes of bacteroids isolated from both hosts also revealed the presence of different sets of plant-derived nodule-specific cysteine rich (NCR) peptides, indicating that the endosymbiotic bacteria find a host-specific cocktail of chemical stressors inside the nodule. By studying variations of the bacterial response to different plant cell environments we will be able to identify specific limitations imposed by the host that might give us clues for the improvement of rhizobial performance.

The early detection of pancreatic ductal adenocarcinoma is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers CA19-9 and sTRA are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and non-cancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry approach was utilized to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of PDAC patients represented by tissue microarrays and whole tissue sections. Orthogonally, these same tissues were characterized by multi-round immunofluorescence which defined expression of CA19-9 and sTRA as well as other lectins towards carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated bi-antennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9 expressing tissues tended to be bi-, tri- and tetra-antennary structures with both core and terminal fucose residues and bisecting N-acetylglucosamines. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored tri- and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-IHC and IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

Open searching has proven to be an effective strategy for identifying both known and unknown modifications in shotgun proteomics experiments. Rather than being limited to a small set of user-specified modifications, open searches identify peptides with any mass shift that may correspond to a single modification or a combination of several modifications. Here we present PTM-Shepherd, a bioinformatics tool that automates characterization of PTM profiles detected in open searches based on attributes such as amino acid localization, fragmentation spectra similarity, retention time shifts, and relative modification rates. PTM-Shepherd can also perform multi-experiment comparisons for studying changes in modification profiles, e.g. in data generated in different laboratories or under different conditions. We demonstrate how PTM-Shepherd improves the analysis of data from formalin-fixed paraffin-embedded samples, detects extreme underalkylation of cysteine in some datasets, discovers an artefactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multi-center proteomics profiling study.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in mature medaka testis. As for the interaction with TGFβ-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid (Y2H) screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired Y2H assay. Co-immunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastrutural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (TGFβ and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA which may be fundamentally conserved across phyla during sexual differentiation.

Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases and some changes appear to be disease-specific, thus it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers and enable better understanding of AGP’s role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96 well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography based solid-phase extraction and analyzed by RP-LC-ESI-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in 3 time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N-glycans on AGP’s third and AGP1’s fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.

This study evaluates the diagnostic utility of PET/MRI for primary, locoregional, and nodal head and neck squamous cell carcinoma (HNSCC) through systematic review and metaanalysis. Methods: A systematic search was conducted using PubMed and Scopus to identify studies on the diagnostic accuracy of PET/MRI for HNSCC. The search included specific terms and excluded nonhybrid PET/MRI studies, and those with a sample size of fewer than 10 patients were excluded. Results: In total, 15 studies encompassing 638 patients were found addressing the diagnostic test accuracy for PET/MRI within the chosen subject domain. Squamous cell carcinoma of the nasopharynx was the most observed HNSCC subtype (n = 198). The metaanalysis included 12 studies, with pooled sensitivity and specificity values of 93% and 95% per patient for primary disease evaluation, 93% and 96% for locoregional evaluation, and 89% and 98% per lesion for nodal disease detection, respectively. An examination of a subset of studies comparing PET/MRI against PET/CT or MRI alone for evaluating nodal and locoregional HNSCC found that PET/MRI may offer slightly higher accuracy than other modalities. However, this difference was not statistically significant. Conclusion: PET/MRI has excellent potential for identifying primary, locoregional, and nodal HNSCC.

PITTSBURGH, Feb. 22, 2024 — Ansys and Intel Foundry have collaborated to provide multiphysics signoff solutions for Intel’s innovative 2.5D chip assembly technology, which uses EMIB technology to connect the […]

The post Ansys, Intel Foundry Collaborate on Multiphysics Analysis Solution for EMIB 2.5D Assembly Tech appeared first on HPCwire.

Teacher-evaluation reforms in places like New Mexico, Tennessee, Denver, and the District of Columbia have paid off, says the National Council on Teacher Quality.

The Minnesota Star Tribune review found that similar to traditional district schools, the highest performing charters generally served wealthier families.

Jennifer L. Zamanian
May 2, 2012; 32:6391-6410
Neurobiology of Disease

Emerging research in nonhuman animals implicates cerebellar projections to the ventral tegmental area (VTA) in appetitive behaviors, but these circuits have not been characterized in humans. Here, we mapped cerebello-VTA white matter connectivity in a cohort of men and women using probabilistic tractography on diffusion imaging data from the Human Connectome Project. We uncovered the topographical organization of these connections by separately tracking from parcels of cerebellar lobule VI, crus I/II, vermis, paravermis, and cerebrocerebellum. Results revealed that connections between the cerebellum and VTA predominantly originate in the right cerebellar hemisphere, interposed nucleus, and paravermal cortex and terminate mostly ipsilaterally. Paravermal crus I sends the most connections to the VTA compared with other lobules. We discovered a mediolateral gradient of connectivity, such that the medial cerebellum has the highest connectivity with the VTA. Individual differences in microstructure were associated with measures of negative affect and social functioning. By splitting the tracts into quarters, we found that the socioaffective effects were driven by the third quarter of the tract, corresponding to the point at which the fibers leave the deep nuclei. Taken together, we produced detailed maps of cerebello-VTA structural connectivity for the first time in humans and established their relevance for trait differences in socioaffective regulation.

GABAergic inhibitory interneurons comprise many subtypes that differ in their molecular, anatomical, and functional properties. In mouse visual cortex, they also differ in their modulation with an animal’s behavioral state, and this state modulation can be predicted from the first principal component (PC) of the gene expression matrix. Here, we ask whether this link between transcriptome and state-dependent processing generalizes across species. To this end, we analysed seven single-cell and single-nucleus RNA sequencing datasets from mouse, human, songbird, and turtle forebrains. Despite homology at the level of cell types, we found clear differences between transcriptomic PCs, with greater dissimilarities between evolutionarily distant species. These dissimilarities arise from two factors: divergence in gene expression within homologous cell types and divergence in cell-type abundance. We also compare the expression of cholinergic receptors, which are thought to causally link transcriptome and state modulation. Several cholinergic receptors predictive of state modulation in mouse interneurons are differentially expressed between species. Circuit modelling and mathematical analyses suggest conditions under which these expression differences could translate into functional differences.

Estimating the direction of functional connectivity (FC) can help further elucidate complex brain function. However, the estimation of directed FC at the voxel level in fMRI data, and evaluating its performance, has yet to be done. We therefore developed a novel directed seed-based connectivity analysis (SCA) method based on normalized pairwise Granger causality that provides greater detail and accuracy over ROI-based methods. We evaluated its performance against 145 cortical retrograde tracer injections in male and female marmosets that were used as ground truth cellular connectivity on a voxel-by-voxel basis. The receiver operating characteristic (ROC) curve was calculated for each injection, and we achieved area under the ROC curve of 0.95 for undirected and 0.942 for directed SCA in the case of high cell count threshold. This indicates that SCA can reliably estimate the strong cellular connections between voxels in fMRI data. We then used our directed SCA method to analyze the human default mode network (DMN) and found that dlPFC (dorsolateral prefrontal cortex) and temporal lobe were separated from other DMN regions, forming part of the language-network that works together with the core DMN regions. We also found that the cerebellum (Crus I-II) was strongly targeted by the posterior parietal cortices and dlPFC, but reciprocal connections were not observed. Thus, the cerebellum may not be a part of, but instead a target of, the DMN and language-network. Summarily, our novel directed SCA method, visualized with a new functional flat mapping technique, opens a new paradigm for whole-brain functional analysis.

New York- FAO Director-General José Graziano da Silva and the President of the UN Security Council (UNSC), Ambassador Ismael Gaspar Martins, have concurred upon the importance of using FAO’s regular [...]