Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells.

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside-out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin-like protein PPLP2 (here termed P-EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle-resident proteins is yet unknown. Here, we used high-resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P-EVs and that exocytosis of the P-EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress-related proteins, we identified novel components of OBs and P-EVs, including vesicle-trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

The Plasmodium falciparum CCCH zinc finger protein MD3 regulates male gametocytogenesis through its interaction with RNA-binding proteins.

The transmission of malaria parasites to mosquitoes is dependent on the formation of gametocytes. Once fully matured, gametocytes are able to transform into gametes in the mosquito's midgut, a process accompanied with their egress from the enveloping erythrocyte. Gametocyte maturation and gametogenesis require a well-coordinated gene expression program that involves a wide spectrum of regulatory proteins, ranging from histone modifiers to transcription factors to RNA-binding proteins. Here, we investigated the role of the CCCH zinc finger protein MD3 in Plasmodium falciparum gametocytogenesis. MD3 was originally identified as an epigenetically regulated protein of immature gametocytes and recently shown to be involved in male development in a barcode-based screen in P. berghei. We report that MD3 is mainly present in the cytoplasm of immature male P. falciparum gametocytes. Parasites deficient of MD3 are impaired in gametocyte maturation and male gametocytogenesis. BioID analysis in combination with co-immunoprecipitation assays unveiled an interaction network of MD3 with RNA-binding proteins like PABP1 and ALBA3, with translational initiators, regulators and repressors like elF4G, PUF1, NOT1 and CITH, and with further regulators of gametocytogenesis, including ZNF4, MD1 and GD1. We conclude that MD3 is part of a regulator complex crucial for post-transcriptional fine-tuning of male gametocytogenesis.

Gut Pathobiont-Derived Outer Membrane Vesicles Drive Liver Inflammation and Fibrosis in PSC-IBD.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC), often associated with inflammatory bowel disease (IBD), presents a multifactorial etiology involving genetic, immunological, and environmental factors. Gut dysbiosis and bacterial translocation have been implicated in PSC-IBD, yet the precise mechanisms underlying their pathogenesis remain elusive. Here, we describe the role of gut pathobionts in promoting liver inflammation and fibrosis due to the release of bacterial outer membrane vesicles (OMVs). METHODS: Preclinical mouse models in addition to ductal organoids were used to acquire mechanistic data. A proof-of-concept study including serum and liver biopsies of a patient cohort of PSC (n=22), PSC-IBD (n=45) and control individuals (n=27) was performed to detect OMVs in the systemic circulation and liver. RESULTS: In both, preclinical model systems and in human PSC-IBD patients, the translocation of OMVs to the liver correlated with enhanced bacterial sensing and accumulation of the NLRP3 inflammasome. Using ductal organoids, we were able to precisely attribute the pro-inflammatory and pro-fibrogenic properties of OMVs to signaling pathways dependent on TLR4 and NLRP3-GSDMD. The immunostimulatory potential of OMVs could be confirmed in macrophages and hepatic stellate cells. Furthermore, when we administered gut pathobiont-derived OMVs to Mdr2-/- mice, we observed a significant enhancement in liver inflammation and fibrosis. In a translational approach, we substantiated the presence of OMVs in the systemic circulation and hepatic regions of severe fibrosis using a PSC-IBD patient cohort. CONCLUSION: This study demonstrates the contribution of gut pathobionts in releasing OMVs that traverse the mucosal barrier, and thus, promote liver inflammation and fibrosis in PSC-IBD. OMVs might represent a critical new environmental factor that interacts with other disease factors to cause inflammation and thus define potential new targets for fibrosis therapy.

HowDirty: An R package to evaluate molecular contaminants in LC-MS experiments.

Contaminants derived from consumables, reagents, and sample handling often negatively affect LC-MS data acquisition. In proteomics experiments, they can markedly reduce identification performance, reproducibility, and quantitative robustness. Here, we introduce a data analysis workflow combining MS1 feature extraction in Skyline with HowDirty, an R-markdown-based tool, that automatically generates an interactive report on the molecular contaminant level in LC-MS data sets. To facilitate the interpretation of the results, the HTML report is self-contained and self-explanatory, including plots that can be easily interpreted. The R package HowDirty is available from https://github.com/DavidGZ1/HowDirty. To demonstrate a showcase scenario for the application of HowDirty, we assessed the impact of ultrafiltration units from different providers on sample purity after filter-assisted sample preparation (FASP) digestion. This allowed us to select the filter units with the lowest contamination risk. Notably, the filter units with the lowest contaminant levels showed higher reproducibility regarding the number of peptides and proteins identified. Overall, HowDirty enables the efficient evaluation of sample quality covering a wide range of common contaminant groups that typically impair LC-MS analyses, facilitating corrective or preventive actions to minimize instrument downtime.

Ionmob: a Python package for prediction of peptide collisional cross-section values.

MOTIVATION: Including ion mobility separation (IMS) into mass spectrometry proteomics experiments is useful to improve coverage and throughput. Many IMS devices enable linking experimentally derived mobility of an ion to its collisional cross-section (CCS), a highly reproducible physicochemical property dependent on the ion's mass, charge and conformation in the gas phase. Thus, known peptide ion mobilities can be used to tailor acquisition methods or to refine database search results. The large space of potential peptide sequences, driven also by posttranslational modifications of amino acids, motivates an in silico predictor for peptide CCS. Recent studies explored the general performance of varying machine-learning techniques, however, the workflow engineering part was of secondary importance. For the sake of applicability, such a tool should be generic, data driven, and offer the possibility to be easily adapted to individual workflows for experimental design and data processing. RESULTS: We created ionmob, a Python-based framework for data preparation, training, and prediction of collisional cross-section values of peptides. It is easily customizable and includes a set of pretrained, ready-to-use models and preprocessing routines for training and inference. Using a set of ≈21 000 unique phosphorylated peptides and ≈17 000 MHC ligand sequences and charge state pairs, we expand upon the space of peptides that can be integrated into CCS prediction. Lastly, we investigate the applicability of in silico predicted CCS to increase confidence in identified peptides by applying methods of re-scoring and demonstrate that predicted CCS values complement existing predictors for that task. AVAILABILITY AND IMPLEMENTATION: The Python package is available at github: https://github.com/theGreatHerrLebert/ionmob.

A Systematic Review of Lipid-Focused Cardiovascular Disease Research: Trends and Opportunities.

Lipids are important modifiers of protein function, particularly as parts of lipoproteins, which transport lipophilic substances and mediate cellular uptake of circulating lipids. As such, lipids are of particular interest as blood biological markers for cardiovascular disease (CVD) as well as for conditions linked to CVD such as atherosclerosis, diabetes mellitus, obesity and dietary states. Notably, lipid research is particularly well developed in the context of CVD because of the relevance and multiple causes and risk factors of CVD. The advent of methods for high-throughput screening of biological molecules has recently resulted in the generation of lipidomic profiles that allow monitoring of lipid compositions in biological samples in an untargeted manner. These and other earlier advances in biomedical research have shaped the knowledge we have about lipids in CVD. To evaluate the knowledge acquired on the multiple biological functions of lipids in CVD and the trends in their research, we collected a dataset of references from the PubMed database of biomedical literature focused on plasma lipids and CVD in human and mouse. Using annotations from these records, we were able to categorize significant associations between lipids and particular types of research approaches, distinguish non-biological lipids used as markers, identify differential research between human and mouse models, and detect the increasingly mechanistic nature of the results in this field. Using known associations between lipids and proteins that metabolize or transport them, we constructed a comprehensive lipid-protein network, which we used to highlight proteins strongly connected to lipids found in the CVD-lipid literature. Our approach points to a series of proteins for which lipid-focused research would bring insights into CVD, including Prostaglandin G/H synthase 2 (PTGS2, a.k.a. COX2) and Acylglycerol kinase (AGK). In this review, we summarize our findings, putting them in a historical perspective of the evolution of lipid research in CVD.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Adaptive Mechanisms of Somatostatin-Positive Interneurons after Traumatic Brain Injury through a Switch of α Subunits in L-Type Voltage-Gated Calcium Channels.

Unilateral traumatic brain injury (TBI) causes cortical dysfunctions spreading to the primarily undamaged hemisphere. This phenomenon, called transhemispheric diaschisis, is mediated by an imbalance of glutamatergic versus GABAergic neurotransmission. This study investigated the role of GABAergic, somatostatin-positive (SST) interneurons in the contralateral hemisphere 72 h after unilateral TBI. The brain injury was induced to the primary motor/somatosensory cortex of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice at postnatal days 19-21 under anesthesia in vivo. Single GFP+ interneurons of the undamaged, contralateral cortex were isolated by fluorescence-activated cell sorting and analyzed by mass spectrometry. TBI caused a switch of 2 α subunits of pore-forming L-type voltage-gated calcium channels (VGCC) in GABAergic interneurons, an increased expression of CaV1.3, and simultaneous ablation of CaV1.2. This switch was associated with 1) increased excitability of single SST interneurons in patch-clamp recordings and (2) a recovery from early network hyperactivity in the contralateral hemisphere in microelectrode array recordings of acute slices. The electrophysiological changes were sensitive to pharmacological blockade of CaV1.3 (isradipine, 100 nM). These data identify a switch of 2 α subunits of VGCCs in SST interneurons early after TBI as a mechanism to counterbalance post-traumatic hyperexcitability.

Multiomic analysis of papillary thyroid cancers identifies BAIAP2L1-BRAF fusion and requirement of TRIM25, PDE5A and PKCδ for tumorigenesis.

BACKGROUND: Papillary thyroid carcinoma (PTC) is one of the most common forms of thyroid cancer with a cure rate of over 90% after surgery. However, aggressive forms may still occur, and personalized therapeutic strategies are increasingly required. METHODS: We performed integrated genomic and proteomic analysis of PTC tumor samples from patients who did not harbor BRAF or RAS mutations. We validate the analysis and present in-depth molecular analysis of the identified genetic rearrangement by employing biochemical and cell biological assays. Finally, we employ 3D spheroid models, loss of function studies and chemical inhibitors to target the hitherto upregulated factors. The data are analysed with appropriate statistical tests which are mentioned in the legends section. RESULTS: In a 23-year-old patient with thyroiditis, we identified a novel rearrangement leading to a BAIAP2L1-BRAF fusion that transforms immortalized human thyroid cells in a kinase and CC-domain dependent manner. Moreover, quantitative proteomic analysis of the same patient samples revealed the upregulation of several proteins including the Ubiquitin E3 ligase TRIM25, PDE5A, and PKCδ. Further, in a cohort of PTC patients, we observed higher expression of TRIM25 and PKCδ in the tumor and metastatic lesions, when compared to the matched normal tissue. Inhibition of TRIM25, PDE5A and PKCδ with small molecules or RNA interference affected not only viability and proliferation of BAIAP2L1-BRAF transformed cells, but also the viability, growth and invasion of corresponding 3D spheroid cultures. CONCLUSIONS: Apart from unveiling a novel oncogenic BRAF fusion in PTCs, our data may open a novel avenue of therapeutic targeting in human PTCs.

Quantitative proteomics analysis reveals core and variable tick salivary proteins at the tick-vertebrate host interface.

Few studies have examined tick proteomes, how they adapt to their environment, and their roles in the parasite-host interactions that drive tick infestation and pathogen transmission. Here we used a proteomics approach to screen for biologically and immunologically relevant proteins acting at the tick-host interface during tick feeding and, as proof of principle, measured host antibody responses to some of the discovered candidates. We used a label-free quantitative proteomic workflow to study salivary proteomes of (i) wild Ixodes ricinus ticks fed on different hosts, (ii) wild or laboratory ticks fed on the same host, and (iii) adult ticks cofed with nymphs. Our results reveal high and stable expression of several protease inhibitors and other tick-specific proteins under different feeding conditions. Most pathways functionally enriched in sialoproteomes were related to proteolysis, endopeptidase, and amine-binding activities. The generated catalogue of tick salivary proteins enabled the selection of six candidate secreted immunogenic peptides for rabbit immunizations, three of which induced strong and durable antigen-specific antibody responses in rabbits. Furthermore, rabbits exposed to ticks mounted immune responses against the candidate peptides/proteins, confirming their expression at the tick-vertebrate interface. Our approach provides insights into tick adaptation strategies to different feeding conditions and promising candidates for developing antitick vaccines or markers of exposure of vertebrate hosts to tick bites.

GABAA Receptor-Stabilizing Protein Ubqln1 Affects Hyperexcitability and Epileptogenesis after Traumatic Brain Injury and in a Model of In Vitro Epilepsy in Mice.

Posttraumatic epilepsy (PTE) is a major public health concern and strongly contributes to human epilepsy cases worldwide. However, an effective treatment and prevention remains a matter of intense research. The present study provides new insights into the gamma aminobutyric acid A (GABAA)-stabilizing protein ubiquilin-1 (ubqln1) and its regulation in mouse models of traumatic brain injury (TBI) and in vitro epilepsy. We performed label-free quantification on isolated cortical GABAergic interneurons from GAD67-GFP mice that received unilateral TBI and discovered reduced expression of ubqln1 24 h post-TBI. To investigate the link between this regulation and the development of epileptiform activity, we further studied ubqln1 expression in hippocampal and cortical slices. Epileptiform events were evoked pharmacologically in acute brain slices by administration of picrotoxin (PTX, 50 µM) and kainic acid (KA, 500 nM) and recorded in the hippocampal CA1 subfield using Multi-electrode Arrays (MEA). Interestingly, quantitative Western blots revealed significant decreases in ubqln1 expression 1-7 h after seizure induction that could be restored by application of the non-selective monoamine oxidase inhibitor nialamide (NM, 10 µM). In picrotoxin-dependent dose-response relationships, NM administration alleviated the frequency and peak amplitude of seizure-like events (SLEs). These findings indicate a role of the monoamine transmitter systems and ubqln1 for cortical network activity during posttraumatic epileptogenesis.

OpenTIMS, TimsPy, and TimsR: Open and Easy Access to timsTOF Raw Data.

The Bruker timsTOF Pro is an instrument that couples trapped ion mobility spectrometry (TIMS) to high-resolution time-of-flight (TOF) mass spectrometry (MS). For proteomics, lipidomics, and metabolomics applications, the instrument is typically interfaced with a liquid chromatography (LC) system. The resulting LC-TIMS-MS data sets are, in general, several gigabytes in size and are stored in the proprietary Bruker Tims data format (TDF). The raw data can be accessed using proprietary binaries in C, C++, and Python on Windows and Linux operating systems. Here we introduce a suite of computer programs for data accession, including OpenTIMS, TimsR, and TimsPy. OpenTIMS is a C++ library capable of reading Bruker TDF files. It opens up Bruker's proprietary codebase. TimsPy and TimsR build on top of OpenTIMS, enabling swift and user-friendly data access to the raw data with Python and R. Both programs are available under a GPL3 license on all major platforms, extending the possibility to interact with timsTOF data to macOS. Additionally, OpenTIMS is capable of translating Bruker data into HDF5 files that can be easily analyzed from Python with the vaex module. OpenTIMS and TimsPy therefore provide easy and quick access to Bruker timsTOF raw data.

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in replication and evaluated the reproducibility and precision of the workflow for the quantification of ATIs. Applying the method to analyze different wheat species (i.e., common wheat, spelt, durum wheat, emmer, and einkorn) and comparing the results to published data, we validated inter-laboratory and cross-methodology reproducibility of ATI quantification, which is essential in the context of large-scale breeding projects. Additionally, we applied our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of same varieties grown at different locations. Finally, we explored the potential of combining QconCAT-based absolute quantification with DIA-based LFQ proteome analysis for the generation of new hypotheses or assay development.

Correction: Synaptic phospholipids as a new target for cortical hyperexcitability and E/I balance in psychiatric disorders.

Following the publication of this article the authors noted that Torfi Sigurdsson's name was misspelled. Instead of Sigrudsson it should be Sigurdsson. The PDF and HTML versions of the paper have been modified accordingly. The authors would like to apologise for this error and the inconvenience this may have caused.

Structural and mechanistic insights into the interaction of the circadian transcription factor BMAL1 with the KIX domain of the CREB-binding protein.

The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1's C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537 Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.

Fungicide resistance toward fludioxonil conferred by overexpression of the phosphatase gene MoPTP2 in Magnaporthe oryzae.

The fungicide fludioxonil causes hyperactivation of the Hog1p MAPK within the high-osmolarity glycerol signaling pathway essential for osmoregulation in pathogenic fungi. The molecular regulation of MoHog1p phosphorylation is not completely understood in pathogenic fungi. Thus, we identified and characterized the putative MoHog1p-interacting phosphatase gene MoPTP2 in the filamentous rice pathogen Magnaporthe oryzae. We found overexpression of MoPTP2 conferred fludioxonil resistance in M. oryzae, whereas the 'loss of function' mutant ΔMoptp2 was more susceptible toward the fungicide. Additionally, quantitative phosphoproteome profiling of MoHog1p phosphorylation revealed lower phosphorylation levels of MoHog1p in the MoPtp2p overexpression mutant compared to the wild-type strain, whereas MoHog1p phosphorylation increased in the ΔMoptp2 mutant. Furthermore, we identified a set of MoHog1p-dependent genes regulated by the MoPtp2p expression level. Our results indicate that the phosphatase MoPtp2p is involved in the regulation of MoHog1p phosphorylation and that overexpression of the gene MoPTP2 is a novel molecular mechanism of fungicide resistance.

Naphthoquinones as Covalent Reversible Inhibitors of Cysteine Proteases-Studies on Inhibition Mechanism and Kinetics.

The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action.

New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain.

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the SNAr addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (Ki = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the SNAr reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation.

The role of TCF3 as potential master regulator in blastemal Wilms tumors.

Wilms tumors are the most common type of pediatric kidney tumors. While the overall prognosis for patients is favorable, especially tumors that exhibit a blastemal subtype after preoperative chemotherapy have a poor prognosis. For an improved risk assessment and therapy stratification, it is essential to identify the driving factors that are distinctive for this aggressive subtype. In our study, we compared gene expression profiles of 33 tumor biopsies (17 blastemal and 16 other tumors) after neoadjuvant chemotherapy. The analysis of this dataset using the Regulator Gene Association Enrichment algorithm successfully identified several biomarkers and associated molecular mechanisms that distinguish between blastemal and nonblastemal Wilms tumors. Specifically, regulators involved in embryonic development and epigenetic processes like chromatin remodeling and histone modification play an essential role in blastemal tumors. In this context, we especially identified TCF3 as the central regulatory element. Furthermore, the comparison of ChIP-Seq data of Wilms tumor cell cultures from a blastemal mouse xenograft and a stromal tumor provided further evidence that the chromatin states of blastemal cells share characteristics with embryonic stem cells that are not present in the stromal tumor cell line. These stem-cell like characteristics could potentially add to the increased malignancy and chemoresistance of the blastemal subtype. Along with TCF3, we detected several additional biomarkers that are distinctive for blastemal Wilms tumors after neoadjuvant chemotherapy and that may provide leads for new therapeutic regimens.

Enhancing Sensitivity of Microflow-Based Bottom-Up Proteomics through Postcolumn Solvent Addition.

The introduction of more sensitive mass spectrometers allows researchers to adapt front-end liquid chromatography (LC) to individual needs for the analysis of complex proteomes. Where absolute sensitivity is not paramount, it is advantageous to switch from a highly sensitive nanoflow-LC setup, the de facto standard platform in mass-spectrometry (MS)-based discovery proteomics, to a more robust, high-throughput-compatible microflow or conventional-flow setup. To enhance the microflow-LC-MS electrospray process of complex proteomic samples, we tested the effects of different solvents, including 2-propanol, methanol, and acetonitrile, pure or as mixture with dimethyl sulfoxide, which were added postcolumn to the eluting sample. Postcolumn addition of organic solvents strongly enhanced the electrospray efficiency in microflow-LC-MS experiments and improved the sensitivity across the entire gradient and for early eluting peptides by up to 10-fold. Postcolumn solvent addition did not negatively affect chromatographic performance and resulted in an overall 28-36% increase in identifications at both the protein and peptide levels. The presented microflow-LC-MS workflow, including postcolumn solvent addition, can be easily adopted on any LC-MS/MS platform.