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ABSTRACT Campylobacter jejuni and Arcobacter butzleri are microaerobic food-borne human gastrointestinal pathogens that mainly cause diarrheal disease. These related species of the Campylobacteria class face variable atmospheric environments during infection and transmission, ranging from nearly anaerobic to aerobic conditions. Consequently, their lifestyles require that both pathogens need to adjust their metabolism and respiration to the changing oxygen concentrations of the colonization sites. Our transcriptomic and proteomic studies revealed that C. jejuni and A. butzleri, lacking a Campylobacteria-specific regulatory protein, C. jejuni Cj1608, or a homolog, A. butzleri Abu0127, are unable to reprogram tricarboxylic acid cycle or respiration pathways, respectively, to produce ATP efficiently and, in consequence, adjust growth to changing oxygen supply. We propose that these Campylobacteria energy and metabolism regulators (CemRs) are long-sought transcription factors controlling the metabolic shift related to oxygen availability, essential for these bacteria’s survival and adaptation to the niches they inhabit. Besides their significant universal role in Campylobacteria, CemRs, as pleiotropic regulators, control the transcription of many genes, often specific to the species, under microaerophilic conditions and in response to oxidative stress.IMPORTANCEC. jejuni and A. butzleri are closely related pathogens that infect the human gastrointestinal tract. In order to infect humans successfully, they need to change their metabolism as nutrient and respiratory conditions change. A regulator called CemR has been identified, which helps them adapt their metabolism to changing conditions, particularly oxygen availability in the gastrointestinal tract so that they can produce enough energy for survival and spread. Without CemR, these bacteria, as well as a related species, Helicobacter pylori, produce less energy, grow more slowly, or, in the case of C. jejuni, do not grow at all. Furthermore, CemR is a global regulator that controls the synthesis of many genes in each species, potentially allowing them to adapt to their ecological niches as well as establish infection. Therefore, the identification of CemR opens new possibilities for studying the pathogenicity of C. jejuni and A. butzleri.

ABSTRACTEscherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics.IMPORTANCEThe study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.

BackgroundNewer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems.MethodsPrimary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry.ResultsWe observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response.ConclusionOur results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

A new strain of xanthan-degrading bacteria identified as Cohnella sp. has been isolated from a xanthan thickener for food production. The strain was able to utilize xanthan as the only carbon source and to reduce the viscosity of xanthan-containing medium during cultivation. Comparative analysis of the secretomes of Cohnella sp. after growth on different media led to the identification of a xanthanase designated as CspXan9, which was isolated after recombinant production in Escherichia coli. CspXan9 could efficiently degrade the β-1,4-glucan backbone of xanthan after previous removal of pyruvylated mannose residues from the ends of the native xanthan side chains by xanthan lyase treatment (XLT-xanthan). Compared with xanthanase from Paenibacillus nanensis, xanthanase CspXan9 had a different module composition at the N- and C-terminal ends. The main putative oligosaccharides released from XLT-xanthan by CspXan9 cleavage were tetrasaccharides and octasaccharides. To explore the functions of the N- and C-terminal regions of the enzyme, truncated variants lacking some of the non-catalytic modules (CspXan9-C, CspXan9-N, CspXan9-C-N) were produced. Enzyme assays with the purified deletion derivatives, which all contained the catalytic glycoside hydrolase family 9 (GH9) module, demonstrated substantially reduced specific activity on XLT-xanthan of CspXan9-C-N compared with full-length CspXan9. The C-terminal module of CspXan9 was found to represent a novel carbohydrate-binding module of family CBM66 with binding affinity for XLT-xanthan, as was shown by native affinity polyacrylamide gel electrophoresis in the presence of various polysaccharides. The only previously known binding function of a CBM66 member is exo-type binding to the non-reducing fructose ends of the β-fructan polysaccharides inulin and levan.

IntroductionBacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown.MethodsTo gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC–MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment.ResultsParticle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions.ConclusionOur study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

Abstract The gastric human pathogen Helicobacter pylori has developed mechanisms to combat stress factors, including reactive oxygen species (ROS). Here, we present a comprehensive study on the redox switch protein HP1021 regulon combining transcriptomic, proteomic and DNA-protein interactions analyses. Our results indicate that HP1021 modulates H. pylori’s response to oxidative stress. HP1021 controls the transcription of 497 genes, including 407 genes related to response to oxidative stress. 79 proteins are differently expressed in the HP1021 deletion mutant. HP1021 controls typical ROS response pathways (katA, rocF) and less canonical ones, particularly DNA uptake and central carbohydrate metabolism. HP1021 is a molecular regulator of competence in H. pylori, as HP1021-dependent repression of the comB DNA uptake genes is relieved under oxidative conditions, increasing natural competence. Furthermore, HP1021 controls glucose consumption by directly regulating the gluP transporter and has an important impact on maintaining the energetic balance in the cell.

Abstract The Arabidopsis thaliana Receptor-Like Protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum. Here we identify the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02. However, RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein. Moreover, some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide instead of the folded protein, suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants. Surprisingly, RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads. RLP30 expression in N. tabacum results in quantitatively lower susceptibility to bacterial, fungal and oomycete pathogens, thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms.

Overlapping genes are widely prevalent; however, their expression and consequences are poorly understood. Here, we describe and functionally characterize a novel zyx-1 overlapping gene, azyx-1, with distinct regulatory functions in Caenorhabditis elegans. We observed conservation of alternative open reading frames (ORFs) overlapping the 5' region of zyxin family members in several animal species, and find shared sites of azyx-1 and zyxin proteoform expression in C. elegans. In line with a standard ribosome scanning model, our results support cis regulation of zyx-1 long isoform(s) by upstream initiating azyx-1a. Moreover, we report on a rare observation of trans regulation of zyx-1 by azyx-1, with evidence of increased ZYX-1 upon azyx-1 overexpression. Our results suggest a dual role for azyx-1 in influencing zyx-1 proteoform heterogeneity and highlight its impact on C. elegans muscular integrity and locomotion.

ABSTRACT Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

This study further characterizes the non-inbred Titan (also known as DU6) mouse line, which could be a useful model for obesity research.

Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking—in part due to the difficulty in assessing migration using high‐throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the ‘identification of Migration control genes by differential Expression’. MigExpress exploits the combination of in‐depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration‐associated genes by their differential expression in slow‐ versus fast‐migrating cells. We applied MigExpress to investigate non‐small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration.

The apple fruit (Malus domestica L. Borkh) is one of the most popular fruits worldwide. Beyond their beneficial properties, apples contain proteins that trigger allergic reactions in susceptible consumers. Mal d1 to d4 are allergens present in a variety of different isoforms in apples. In this study, we used proteomics to quantify all four Mal d proteins in 52 apple genotypes with varying allergenic potentials. A total of 195, 17, 14, and 18 peptides were found to be related to Mal d1, d2, d3, and d4 proteins, respectively of which 25 different Mal d proteins could be unambiguously identified. The allergenic potential of the Mal d isoforms was characterized by comparing the isoform abundance with the allergenic score of genotypes from oral challenge tests. The detected Mal d peptides presumably have different IgE binding properties and could be used as potential molecular markers to discriminate between hypoallergenic and hyperallergenic cultivars.

Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. This study aimed to determine the impact of high oxygen MAP (70% O2, 30% CO2, red and white meats) and oxygen-free MAP (70% N2, 30% CO2, also white meat and seafood) on preventing the growth of spoiling photobacteria on meat. Growth of Photobacterium carnosum and P. phosphoreum was monitored in a meat simulation media under different gas mixtures of nitrogen, oxygen, and carbon dioxide, and samples were taken during exponential growth for a comparative proteomic analysis. Growth under air atmosphere appears optimal, particularly for P. carnosum. Enhanced protein accumulation affected energy metabolism, respiration, oxygen consuming reactions, and lipid usage. However, all the other atmospheres show some degree of growth reduction. An increase in oxygen concentration leads to an increase in enzymes counteracting oxidative stress for both species and enhancement of heme utilization and iron-sulfur cluster assembly proteins for P. phosphoreum. Absence of oxygen appears to switch the metabolism toward fermentative pathways where either ribose (P. phosphoreum) or glycogen (P. carnosum) appear to be the preferred substrates. Additionally, it promotes the use of alternative electron donors/acceptors, mainly formate and nitrate/nitrite. Stress response is manifested as an enhanced accumulation of enzymes that is able to produce ammonia (e.g., carbonic anhydrase, hydroxylamine reductase) and regulate osmotic stress. Our results suggest that photobacteria do not sense the environmental levels of carbon dioxide, but rather adapt to their own anaerobic metabolism. The regulation in presence of carbon dioxide is limited and strain-specific under anaerobic conditions. However, when oxygen at air-like concentration (21%) is present together with carbon dioxide (30%), the oxidative stress appears enhanced compared to air conditions (very low carbon dioxide), as explained if both gases have a synergistic effect. This is further supported by the increase in oxygen concentration in the presence of carbon dioxide. The atmosphere is able to fully inhibit P. carnosum, heavily reduce P. phosphoreum growth in vitro, and trigger diversification of energy production with higher energetic cost, highlighting the importance of concomitant bacteria for their growth on raw meat under said atmosphere.

Abstract Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data‐independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichiacoli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non‐metabolic stresses, and non‐planktonic states. The resulting high‐quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low‐abundant proteins under various metabolic limitations, the non‐specificity of catabolic enzymes upregulated under carbon limitation, the lack of large‐scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain‐dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi‐omics studies of gene regulation and metabolic control in E. coli.

As one of the major greenhouse gas (GHG) emitters that has not seen significant emission reductions in the previous decades, the transportation sector requires special attention from policymakers. Policy decisions, thereby need to be supported by traffic emission assessments. Estimations of traffic emissions often rely on huge amounts of actual traffic data whose availability is limited, hampering the transferability of the estimation approaches in time and space. Here, we propose a high-resolution estimation of traffic emissions, which is based entirely on open data, such as the road network and points of interest derived from OpenStreetMap (OSM). We estimated the annual average daily GHG emissions from individual motor traffic for the OSM road network in Berlin by combining the estimated Annual Average Daily Traffic Volume (AADTV) with respective emission factors. The AADTV was calculated by simulating car trips with the open routing engine Openrouteservice, weighted by activity functions based on statistics of the German Mobility Panel. Our estimated total annual GHG emissions were 7.3 million t CO2 equivalent. The highest emissions were estimated for the motorways and major roads connecting the city center with the outskirts. The application of the approach to Berlin showed that the method could reflect the traffic pattern. As the input data is freely available, the approach can be applied to other study areas within Germany with little additional effort.

ABSTRACT Microbes that can recycle one-carbon (C1) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO2 and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model acetogen Clostridium autoethanogenum grown autotrophically on three gas mixtures (CO, CO+H2, or CO+CO2+H2). We detect the prioritization of proteome allocation for C1 fixation and the significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are likely relevant in vivo for CO oxidation, H2 metabolism, and ethanol production. The integration of proteomic and metabolic flux data demonstrated that enzymes catalyze high fluxes with high concentrations and high in vivo catalytic rates. We show that flux adjustments were dominantly accompanied by changing enzyme catalytic rates rather than concentrations. IMPORTANCE Acetogen bacteria are important for maintaining biosustainability as they can recycle gaseous C1 waste feedstocks (e.g., industrial waste gases and syngas from gasified biomass or municipal solid waste) into fuels and chemicals. Notably, the acetogen Clostridium autoethanogenum is being used as a cell factory in industrial-scale gas fermentation. Here, we perform reliable absolute proteome quantification for the first time in an acetogen. This is important as our work advances both rational metabolic engineering of acetogen cell factories and accurate in silico reconstruction of their phenotypes. Furthermore, this absolute proteomics data set serves as a reference toward a better systems-level understanding of the ancient metabolism of acetogens.

The substrate specificity of phosphoprotein phosphatases PP1 and PP2A depends on their catalytic and regulatory subunits. Using proteomics approaches, the authors here provide insights into the sequence specificity of the catalytic subunits and their distinct contributions to PP1 and PP2A selectivity.

Summary: The existence of overlapping genes (OLGs) with significant coding overlaps revolutionizes our understanding of genomic complexity. We report two exceptionally long (957 nt and 1536 nt), evolutionarily novel, translated antisense open reading frames (ORFs) embedded within annotated genes in the pathogenic Gram-negative bacterium Pseudomonas aeruginosa. Both OLG pairs show sequence features consistent with being genes and transcriptional signals in RNA sequencing. Translation of both OLGs was confirmed by ribosome profiling and mass spectrometry. Quantitative proteomics of samples taken during different phases of growth revealed regulation of protein abundances, implying biological functionality. Both OLGs are taxonomically restricted, and likely arose by overprinting within the genus. Evidence for purifying selection further supports functionality. The OLGs reported here, designated olg1 and olg2, are the longest yet proposed in prokaryotes and are among the best attested in terms of translation and evolutionary constraint. These results highlight a potentially large unexplored dimension of prokaryotic genomes.

Large-scale mapping activities can benefit from the vastly increasing availability of earth observation (EO) data, especially when combined with volunteered geographical information (VGI) using machine learning (ML). High-resolution maps of inland surface water bodies are important for water supply and natural disaster mitigation as well as for monitoring, managing, and preserving landscapes and ecosystems. In this paper, we propose an automatic surface water mapping workflow by training a deep residual neural network (ResNet) based on OpenStreetMap (OSM) data and Sentinel-2 multispectral data, where the Simple Non-Iterative Clustering (SNIC) superpixel algorithm was employed for generating object-based training samples. As a case study, we produced an open surface water layer for Germany using a national ResNet model at a 10 m spatial resolution, which was then harmonized with OSM data for final surface water products. Moreover, we evaluated the mapping accuracy of our open water products via conducting expert validation campaigns, and comparing to existing water products, namely the WasserBLIcK and Global Surface Water Layer (GSWL). Using 4,600 validation samples in Germany, the proposed model (ResNet+SNIC) achieved an overall accuracy of 86.32% and competitive detection rates over the WasserBLIcK (87.47%) and GSWL (98.61%). This study provides comprehensive insights into how to best explore the synergy of VGI and ML of EO data in a large-scale surface water mapping task.

Traffic prediction is a topic of increasing importance for research and applications in the domain of routing and navigation. Unfortunately, open data are rarely available for this purpose. To overcome this, the authors explored the possibility of using geo-tagged social media data (Twitter), land-use and land-cover point of interest data (from OpenStreetMap) and an adapted betweenness centrality measure as feature spaces to predict the traffic congestion of eleven world cities. The presented framework and workflow are termed as SocialMedia2Traffic. Traffic congestion was predicted at four tile spatial resolutions and compared with Uber Movement data. The overall precision of the forecast for highly traffic-congested regions was approximately 81%. Different data processing steps including ways to aggregate data points, different proxies and machine learning approaches were compared. The lack of a universal definition on a global scale to classify road segments by speed bins into different traffic congestion classes has been identified to be a major limitation of the transferability of the framework. Overall, SocialMedia2Traffic further improves the usability of the tested feature space for traffic prediction. A further benefit is the agnostic nature of the social media platform’s approach.

Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation thereof are poorly described. This study focuses on carbohydrate active enzymes, including glycosyl transferases (GT) and glycoside hydrolases (GH), and their roles in the formation and potential degradation of O2-substituted (1,3)-β-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, β-glucan, and D-glucose concentrations during the exponential, stationary, and early death phases. While the viscosity reached its maximum during the stationary phase and subsequently decreased, the β-glucan concentration only increased to a plateau. Results were correlated with secretome and proteome data to identify involved enzymes and pathways. The suggested pathway for β-glucan biosynthesis involved a β-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the β-glucan concentration did not decrease, most likely due to missing extracellular carbohydrate active enzymes. In addition, an operon was discovered containing known moonlighting genes, all of which were detected in both proteome and secretome samples.

Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.

Summary: Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

Besides intrinsic and extrinsic factors such as antagonism for organic substrates or temperature, the storage atmosphere of meat has a high influence on the development of its initial microbiota. Specific modified atmospheres (MAs) selectively suppress growth of aerobic and anaerobic bacteria, thus reshaping the initial microbiota. As some microorganisms are more tolerant to MA, they overgrow competitors and produce metabolites that cause rejection of the product. In order to elucidate responses to different MA by means of metabolic adaptation and competition for organic substrates on meat, the typical representative meat spoilage bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were chosen. Bacteria were grown in sterile glass bottles filled with a meat simulation medium, which was aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth of bacteria during incubation at 25°C and stirring at 120 rpm was monitored over 48 h and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to oxygen and carbon dioxide. Both Leuconostoc subsp. were intrinsically tolerant to MA, exhibiting no proteomic regulation of enzymes, whereas the other species provide a set of metabolic adaptation mechanism, enabling higher resistance to the detrimental effects of MA. Those mechanisms comprise: enhanced oxidative stress reduction, adjustment of the pyruvate metabolism and catabolic oxygen consumption in response to oxygen and intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition in response to carbon dioxide. We further evaluated the potential of industrial used MA to inhibit specific bacterial spoilage. No bacterial inhibition is predicted for 30%_CO2/70%_O2 for the analyzed species, whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101. Furthermore, species-specific metabolic pathways enabling different and preferential carbon source utilization were identified, which enable non-competitive coexistence of respective bacteria on meat, resulting in synergistic spoilage. In conclusion, this study gives mechanistically explanations of their acknowledged status as typical spoilage organisms on MAP meats.

Abstract Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH‐MS is a specific variant of data‐independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH‐MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH‐MS data, a strategy based on peptide‐centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH‐MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH‐MS data using peptide‐centric scoring. Furthermore, concepts on how to improve SWATH‐MS data acquisition, potential trade‐offs of parameter settings and alternative data analysis strategies are discussed.

Cockayne syndrome (CS) is a developmental disorder with symptoms that are typical for the aging body, including subcutaneous fat loss, alopecia, and cataracts. Here, we show that in the cells of CS patients, RNA polymerase I transcription and the processing of the pre-rRNA are disturbed, leading to an accumulation of the 18S-E intermediate. The mature 18S rRNA level is reduced, and isolated ribosomes lack specific ribosomal proteins of the small 40S subunit. Ribosomal proteins are susceptible to unfolding and the CS cell proteome is heat-sensitive, indicating misfolded proteins and an error-prone translation process in CS cells. Pharmaceutical chaperones restored impaired cellular proliferation. Therefore, we provide evidence for severe protein synthesis malfunction, which together with a loss of proteostasis constitutes the underlying pathophysiology in CS.

Water kefir is a slightly alcoholic and traditionally fermented beverage, which is prepared from sucrose, water, kefir grains, and dried or fresh fruits (e.g., figs). Lactobacillus (L.) nagelii, L. hordei, and Saccharomyces (S.) cerevisiae are predominant and stable lactic acid bacteria and yeasts, respectively, isolated from water kefir consortia. The growth of L. nagelii and L. hordei are improved in the presence of S. cerevisiae. In this work we demonstrate that quantitative comparative proteomics enables the investigation of interactions between LAB and yeast to predict real-time metabolic exchange in water kefir. It revealed 73 differentially expressed (DE) in L. nagelii TMW 1.1827 in the presence of S. cerevisiae. The presence of the yeast induced changes in the changes in the carbohydrate metabolism of L. nagelii and affected reactions involved in NAD+/NADH homeostasis. Furthermore, the DE enzymes involved in amino acid biosynthesis or catabolism predict that S. cerevisiae releases glutamine, histidine, methionine, and arginine, which are subsequently used by L. nagelii to ensure its survival in the water kefir consortium. In co-culture with S. cerevisiae, L. nagelii profits from riboflavin, most likely secreted by the yeast. The reaction of L. nagelii to the presence of S. cerevisiae differs from that one of the previously studied L. hordei, which displays 233 differentially expressed proteins, changes in citrate metabolism and an antidromic strategy for NAD+/NADH homeostasis. So far, aggregation promotion factors, i.e., formation of a specific glucan and bifunctional enzymes were only detected in L. hordei.

Minor prenylated hop compounds have been attracting increasing attention due to their promising anticarcinogenic properties. Even after intensive purification from natural raw extracts, allocating certain activities to single compounds or complex interactions of the main compound with remaining impurities in very low concentration is difficult. In this study, dose-dependent antiproliferative and cytotoxic effects of the promising xanthohumol (XN) analogue xanthohumol C (XNC) were evaluated and compared to XN and a XN-enriched hop extract (XF). It was demonstrated that the cell growth inhibition of human breast cancer cell line (MCF-7) significantly increases after being treated with XNC compared to XN and XF. Based on label-free data-dependent acquisition proteomics, physiological influences on the proteome of MCF-7 cells were analyzed. Different modes of action between XNC and XN treated MCF-7 cells could be postulated. XNC causes ER stress and seems to be involved in cell-cell adhesion, whereas XN influences cell cycles and DNA replication as well as type I interferon signaling pathway. The results demonstrate the utility of using quantitative proteomics for bioactivity screenings of minor hop compounds and underscore the importance of isolating highly pure compounds into their distinct forms to analyze their different and possibly synergistic activities and modes of action.

Public urban green spaces are important for the urban quality of life. Still, comprehensive open data sets on urban green spaces are not available for most cities. As open and globally available data sets, the potential of Sentinel-2 satellite imagery and OpenStreetMap (OSM) data for urban green space mapping is high but limited due to their respective uncertainties. Sentinel-2 imagery cannot distinguish public from private green spaces and its spatial resolution of 10 m fails to capture fine-grained urban structures, while in OSM green spaces are not mapped consistently and with the same level of completeness everywhere. To address these limitations, we propose to fuse these data sets under explicit consideration of their uncertainties. The Sentinel-2 derived Normalized Difference Vegetation Index was fused with OSM data using the Dempster–Shafer theory to enhance the detection of small vegetated areas. The distinction between public and private green spaces was achieved using a Bayesian hierarchical model and OSM data. The analysis was performed based on land use parcels derived from OSM data and tested for the city of Dresden, Germany. The overall accuracy of the final map of public urban green spaces was 95% and was mainly influenced by the uncertainty of the public accessibility model.

Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei) is one of the dominating lactic acid bacteria within the water kefir consortium, being highly adapted to survive in this environment, while producing high molecular weight dextrans from sucrose. In this work, we extensively studied the physiological response of L. hordei TMW 1.1822 to sucrose compared to glucose, applying label-free, quantitative proteomics of cell lysates and exoproteomes. This revealed the differential expression of 53 proteins within cellular proteomes, mostly associated with carbohydrate uptake and metabolism. Supported by growth experiments, this suggests that L. hordei TMW 1.1822 favors fructose over other sugars. The dextransucrase was expressed irrespectively of the present carbon source, while it was significantly more released in the presence of sucrose (log2FC = 3.09), being among the most abundant proteins within exoproteomes of sucrose-treated cells. Still, L. hordei TMW 1.1822 expressed other sucrose active enzymes, predictively competing with the dextransucrase reaction. While osmolysis appeared to be unlikely, sucrose led to increased release of a multitude of cytoplasmic proteins, suggesting that biofilm formation in L. hordei is not only composed of a polysaccharide matrix but is also of proteinaceous nature. Therefore, our study highlights the intrinsic adaptation of water kefir-borne L. hordei to sucrose-rich habitats and provides fundamental knowledge for its use as a starter culture in plant-based food fermentations with in situ dextran formation.

Lactobacillus (L.) sakei belongs to the dominating lactic acid bacteria in indigenous meat fermentations, while diverse strains of this species have also been isolated from plant fermentations. We could recently show, that L. sakei TMW 1.411 produces a high molecular weight dextran from sucrose, indicating its potential use as a dextran forming starter culture. However, the general physiological response of L. sakei to sucrose as carbohydrate source has not been investigated yet, especially upon simultaneous dextran formation. To address this lack of knowledge, we sequenced the genome of L. sakei TMW 1.411 and performed a label-free, quantitative proteomics approach to investigate the sucrose-induced changes in the proteomic profile of this strain in comparison to its proteomic response to glucose. In total, 21 proteins were found to be differentially expressed at the applied significance criteria (FDR ≤ 0.01). Among these, 14 were associated with the carbohydrate metabolism including several enzymes, which enable sucrose and fructose uptake, as well as, their subsequent intracellular metabolization, respectively. The plasmid-encoded, extracellular dextransucrase of L. sakei TMW 1.411 was expressed at high levels irrespective of the present carbohydrate and was predominantly responsible for sucrose consumption in growth experiments using sucrose as sole carbohydrate source, while the released fructose from the dextransucrase reaction was more preferably taken up and intracellularly metabolized than sucrose. Genomic comparisons revealed, that operons coding for uptake and intracellular metabolism of sucrose and fructose are chromosomally conserved among L. sakei, while plasmid-located dextransucrase genes are present only in few strains. In accordance with these findings, all 59 different L. sakei strains of our strain collection were able to grow on sucrose as sole carbohydrate source, while eight of them exhibited a mucous phenotype on agar plates indicating dextran formation from sucrose. Our study therefore highlights the intrinsic adaption of L. sakei to plant environments, where sucrose is abundant, and provides fundamental knowledge regarding the use of L. sakei as starter culture for sucrose-based food fermentation processes with in-situ dextran formation.

Background & Aims: Antibiotic (ABx) therapy is associated with increased risk for Crohn’s disease but underlying mechanisms are unknown. We observed high fecal serine protease activity (PA) to be a frequent side effect of ABx therapy. The aim of the present study was to unravel whether this rise in large intestinal PA may promote colitis development via detrimental effects on the large intestinal barrier. Methods: Transwell experiments were used to assess the impact of high PA in ABx-treated patients or vancomycin/metronidazole-treated mice on the epithelial barrier. Serine protease profiling was performed using liquid chromatography–mass spectrometry/mass spectrometry analysis. The impact of high large intestinal PA on the intestinal barrier in wild-type and interleukin (IL)10-/- mice and on colitis development in IL10-/- mice was investigated using vancomycin/metronidazole with or without oral serine protease inhibitor (AEBSF) treatment. Results: The ABx-induced, high large intestinal PA was caused by significantly increased levels of pancreatic proteases and impaired epithelial barrier integrity. In wild-type mice, the rise in PA caused a transient increase in intestinal permeability but did not affect susceptibility to chemically induced acute colitis. In IL10-/- mice, increased PA caused a consistent impairment of the intestinal barrier associated with inflammatory activation in the large intestinal tissue. In the long term, the vancomycin/metronidazole-induced lasting increase in PA aggravated colitis development in IL10-/- mice. Conclusions: High large intestinal PA is a frequent adverse effect of ABx therapy, which is detrimental to the large intestinal barrier and may contribute to the development of chronic intestinal inflammation in susceptible individuals. Keywords: Epithelial Barrier, Serine Proteases, Gut Microbiota, Inflammatory Bowel Diseases

Abstract Cells react to nutritional cues in changing environments via the integrated action of signaling, transcriptional, and metabolic networks. Mechanistic insight into signaling processes is often complicated because ubiquitous feedback loops obscure causal relationships. Consequently, the endogenous inputs of many nutrient signaling pathways remain unknown. Recent advances for system‐wide experimental data generation have facilitated the quantification of signaling systems, but the integration of multi‐level dynamic data remains challenging. Here, we co‐designed dynamic experiments and a probabilistic, model‐based method to infer causal relationships between metabolism, signaling, and gene regulation. We analyzed the dynamic regulation of nitrogen metabolism by the target of rapamycin complex 1 (TORC1) pathway in budding yeast. Dynamic transcriptomic, proteomic, and metabolomic measurements along shifts in nitrogen quality yielded a consistent dataset that demonstrated extensive re‐wiring of cellular networks during adaptation. Our inference method identified putative downstream targets of TORC1 and putative metabolic inputs of TORC1, including the hypothesized glutamine signal. The work provides a basis for further mechanistic studies of nitrogen metabolism and a general computational framework to study cellular processes.

Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in CD pathogenesis, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent Nε-(γ-glutamyl)-lysine isopeptide crosslinks resulting in TG2−gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear. To gain more insight into the molecular structures of TG2-gluten peptide complexes, we used different proteomics software tools that enable the comprehensive identification of isopeptides. Thus, 34 different isopeptides involving 20 TG2 lysine residues were identified in a model system, only six of which were previously known. Additionally, 36 isopeptides of TG2-TG2 multimers were detected. Experiments with different TG2-gluten peptide molar ratios revealed the most preferred lysine residues involved in isopeptide crosslinking. Expanding the model system to three gluten peptides with more glutamine residues allowed the localization of the preferred glutamine crosslinking sites. These new insights into the structure of TG2-gluten peptide complexes may help clarify the role of extracellular TG2 in CD autoimmunity and in other inflammatory diseases.

People share data in different ways. Many of them contribute on a voluntary basis, while others are unaware of their contribution. They have differing intentions, collaborate in different ways, and they contribute data about differing aspects. Shared Data Sources have been explored individually in the literature, in particular OpenStreetMap and Twitter, and some types of Shared Data Sources have widely been studied, such as Volunteered Geographic Information (VGI), Ambient Geographic Information (AGI), and Public Participation Geographic Information Systems (PPGIS). A thorough and systematic discussion of Shared Data Sources in their entirety is, however, still missing. For the purpose of establishing such a discussion, we introduce in this article a schema consisting of a number of dimensions for characterizing socially produced, maintained, and used ‘Shared Data Sources,’ as well as corresponding visualization techniques. Both the schema and the visualization techniques allow for a common characterization in order to set individual data sources into context and to identify clusters of Shared Data Sources with common characteristics. Among others, this makes possible choosing suitable Shared Data Sources for a given task and gaining an understanding of how to interpret them by drawing parallels between several Shared Data Sources.

Abstract As a frequent post‐translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino‐acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol‐3‐phosphate dehydrogenase Gpd1 and phosphofructose‐1‐kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes.