High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

An Introduction to Analytical Challenges, Approaches, and Applications in Mass Spectrometry-Based Secretomics.

The active release of proteins into the extracellular space and the proteolytic cleavage of cell surface proteins are key processes that coordinate and fine-tune a multitude of physiological functions. The entirety of proteins that fulfill these extracellular tasks are referred to as the secretome and are of special interest for the investigation of biomarkers of disease states and physiological processes related to cell-cell communication. LC-MS-based proteomics approaches are a valuable tool for the comprehensive and unbiased characterization of this important subproteome. This review discusses procedures, opportunities, and limitations of mass spectrometry-based secretomics to better understand and navigate the complex analytical landscape for studying protein secretion in biomedical science.

An optimized quantitative proteomics method establishes the cell type-resolved mouse brain secretome.

To understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we miniaturized secretome analysis by developing the "high-performance secretome protein enrichment with click sugars" (hiSPECS) method. To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPS-induced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons, and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion and brain function and to identify cell type-specific biomarkers for CNS diseases.

Interval-Based Secretomics Unravels Acute-Phase Response in Hepatocyte Model Systems.

Mass spectrometry-based secretomics approaches frequently utilize serum-free culture conditions to circumvent serum-induced interference and to increase analytical depth. However, this can negatively affect a wide range of cellular functions and cell viability. These effects become particularly apparent when investigating transcriptionally regulated secretion events and feedback-loops in response to perturbations that require 48 h or more to fully manifest. We present an "interval-based" secretomics workflow, which determines protein secretion rates in short serum-free time windows. Relative quantification using tandem mass tags enables precise monitoring of time-dependent changes. We applied this approach to determine temporal profiles of protein secretion in the hepatocyte model cell lines HepG2 and HepaRG after stimulation of the acute-phase response (APR) by the cytokines IL1b and IL6. While the popular hepatocarcinoma cell line HepG2 showed an incomplete APR, secretion patterns derived from differentiated HepaRG cells recapitulated the expected APR more comprehensively. For several APR response proteins, substantial secretion was only observed after 72 h, a time window at which cell fitness is substantially impaired under serum-free cell culture conditions. The interval-based secretomics approach enabled the first comprehensive analysis of time-dependent secretion of liver cell models in response to these proinflammatory cytokines. The extended time range facilitated the observation of distinct chronological phases and cytokine-dependent secretion phenotypes of the APR. IL1b directed the APR toward pathogen defense over three distinct phases-chemotaxis, effector, clearance-while IL6 directed the APR toward regeneration. Protein shedding on the cell surface was pronounced upon IL1b stimulation, and small molecule inhibition of ADAM and matrix metalloproteases identified induced as well as constitutive shedding events. Inhibition of ADAM proteases with TAPI-0 resulted in reduced shedding of the sorting receptor SORT1, and an attenuated cytokine response suggesting a direct link between cell surface shedding and cytokine secretion rates.

Mass spectrometry-based proteomics approaches to interrogate skeletal muscle adaptations to exercise.

Acute exercise and chronic exercise training elicit beneficial whole-body changes in physiology that ultimately depend on profound alterations to the dynamics of tissue-specific proteins. Since the work accomplished during exercise owes predominantly to skeletal muscle, it has received the majority of interest from exercise scientists that attempt to unravel adaptive mechanisms accounting for salutary metabolic effects and performance improvements that arise from training. Contemporary scientists are also beginning to use mass spectrometry-based proteomics, which is emerging as a powerful approach to interrogate the muscle protein signature in a more comprehensive manner. Collectively, these technologies facilitate the analysis of skeletal muscle protein dynamics from several viewpoints, including changes to intracellular proteins (expression proteomics), secreted proteins (secretomics), post-translational modifications as well as fiber-, cell-, and organelle-specific changes. This review aims to highlight recent literature that has leveraged new workflows and advances in mass spectrometry-based proteomics to further our understanding of training-related changes in skeletal muscle. We call attention to untapped areas in skeletal muscle proteomics research relating to exercise training and metabolism, as well as basic points of contention when applying mass spectrometry-based analyses, particularly in the study of human biology. We further encourage researchers to couple the hypothesis-generating and descriptive nature of omics data with functional analyses that propel our understanding of the complex adaptive responses in skeletal muscle that occur with acute and chronic exercise.

Multiomics of Colorectal Cancer Organoids Reveals Putative Mediators of Cancer Progression Resulting from SMAD4 Inactivation.

The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Although loss of SMAD4 is a key event in CRC progression, the resulting changes in biological processes in advanced disease and metastasis are not fully understood. Here, we applied a multiomics approach to a CRC organoid model that faithfully reflects the metastasis-supporting effects of SMAD4 inactivation. We show that loss of SMAD4 results in decreased differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFß, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by SMAD4-mutant organoids─DKK3─reduces the antitumor effects of natural killer cells (NK cells). Altogether, our data provide new insights into the role of SMAD4 perturbation in advanced CRC.

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors that are directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified which are mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development.

C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR-Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR-Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16.

Foodborne pathogens in the omics era.

Outbreaks and deaths related to Foodborne Diseases (FBD) occur constantly in the world, as a result of the consumption of contaminated foodstuffs with pathogens such as Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Salmonella spp, Clostridium spp. and Campylobacter spp. The purpose of this review is to discuss the main omic techniques applied in foodborne pathogen and to demonstrate their functionalities through the food chain and to guarantee the food safety. The main techniques presented are genomic, transcriptomic, secretomic, proteomic, and metabolomic, which together, in the field of food and nutrition, are known as "Foodomics." This review had highlighted the potential of omics to integrate variables that contribute to food safety and to enable us to understand their application on foodborne diseases. The appropriate use of these techniques had driven the definition of critical parameters to achieve successful results in the improvement of consumers health, costs and to obtain safe and high-quality products.

Identification of Copper-Containing Oxidoreductases in the Secretomes of Three Colletotrichum Species with a Focus on Copper Radical Oxidases for the Biocatalytic Production of Fatty Aldehydes.

Copper radical alcohol oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens, are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum, and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in the absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29 to 52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11, AA12, AA13, and AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris, and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavor and fragrance ingredients. Copper radical alcohol oxidases (CRO-AlcOx) have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial applications to alleviate various costs pertaining to biocatalyst production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative stress conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, and lytic polysaccharide monooxygenases [LPMOs]) usually assigned to "plant cell wall degradation," despite the absence of any plant-biomass mimicking compound. However, in these conditions, only small amounts of CRO-AlcOx were secreted, pointing out recombinant expression as the most promising path for their biocatalytic application.

Embryo sHLA-G secretion is related to pregnancy rate.

SummaryHLA-G expression has been detected in early preimplantation embryos and it has been postulated that a relationship between embryonic expression of this factor and successful pregnancy may exist. Forty-six patients were prospectively selected from our centre 'Unidad de Reproducción Humana, Hospital Universitario de Canarias' for conducting this study. In all cases, metaphase II (MII) oocytes were fertilized using intracytoplasmic sperm injection 2-4 h after retrieval. Embryos were cultured individually in 20 µl droplets of G-1 medium (VitroLife) under oil at 37°C and a 6% CO2 environment. Fertilization was assessed at 18 h postinsemination and all oocytes fertilized were passed into a new culture plaque individually in 300 µl culture medium until day 3 of culture. The culture medium was examined for the expression and secretion of sHLA-G with a sandwich enzyme-linked immunosorbent assay kit (BioVendor, Heidelberg, Germany) according to the manufacturer's instructions. We found statistical significance between higher levels of sHLA-G secretion and pregnancy rate. When both groups were compared there was no difference in embryo quality of transferred embryos, but a significant difference in the number of oocytes and the embryo quality of the cohort existed that was greater in the pregnant group. A standardized sHLA-G assay with a specifically defined range and standard units provides a non-invasive method to identify the most competent embryos for transfer.

Secretomics to Discover Regulators in Diseases.

Secretory proteins play important roles in the cross-talk of individual functional units, including cells. Since secretory proteins are essential for signal transduction, they are closely related with disease development, including metabolic and neural diseases. In metabolic diseases, adipokines, myokines, and hepatokines are secreted from respective organs under specific environmental conditions, and play roles in glucose homeostasis, angiogenesis, and inflammation. In neural diseases, astrocytes and microglia cells secrete cytokines and chemokines that play roles in neurotoxic and neuroprotective responses. Mass spectrometry-based secretome profiling is a powerful strategy to identify and characterize secretory proteins. This strategy involves stepwise processes such as the collection of conditioned medium (CM) containing secretome proteins and concentration of the CM, peptide preparation, mass analysis, database search, and filtering of secretory proteins; each step requires certain conditions to obtain reliable results. Proteomic analysis of extracellular vesicles has become a new research focus for understanding the additional extracellular functions of intracellular proteins. Here, we provide a review of the insights obtained from secretome analyses with regard to disease mechanisms, and highlight the future prospects of this technology. Continued research in this field is expected to provide valuable information on cell-to-cell communication and uncover new pathological mechanisms.

Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.

Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.

Adapting extracellular matrix proteomics for clinical studies on cardiac remodeling post-myocardial infarction.

Following myocardial infarction (MI), the left ventricle (LV) undergoes a series of cardiac wound healing responses that involve stimulation of robust inflammation to clear necrotic myocytes and tissue debris and induction of extracellular matrix (ECM) protein synthesis to generate a scar. Proteomic strategies provide us with a means to index the ECM proteins expressed in the LV, quantify amounts, determine functions, and explore interactions. This review will focus on the efforts taken in the proteomics research field that have expanded our understanding of post-MI LV remodeling, concentrating on the strengths and limitations of different proteomic approaches to glean information that is specific to ECM turnover in the post-MI setting. We will discuss how recent advances in sample preparation and labeling protocols increase our successes at detecting components of the cardiac ECM proteome. We will summarize how proteomic approaches, focusing on the ECM compartment, have progressed over time to current gel-free methods using decellularized fractions or labeling strategies that will be useful for clinical applications. This review will provide an overview of how cardiac ECM proteomics has evolved over the last decade and will provide insight into future directions that will drive forward our understanding of cardiac ECM turnover in the post-MI LV.

In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy.

Pterygium is a triangular shaped ocular fibrous surface lesion growing from conjunctiva towards central cornea, causing ocular irritation, astigmatism, and visual disturbance. The condition is characterized by epithelial proliferation, fibrovascular growth, chronic inflammation, and prominent extracellular matrix remodeling. Studies have suggested that aberrant extracellular proteins secreted by fibroblasts lead to abnormal matrix production and tissue invasion contributing to the development of the disease. In this study, secreted proteins collected from paired pterygium and conjunctival fibroblasts in vitro were identified and quantified by LC-MS iTRAQ-based analysis, in which 433 proteins common to all samples were identified. Among these proteins, 48.0% (208) were classified as classically secreted proteins, 17.1% (74) were exported out of the cells via non-classical secretion pathways, and 31.2% (135) were exosome proteins. A minority (3.7%) was not previously known to be secreted, or might be contaminants. 31 and 27 proteins were found up- or down-regulated in the conditioned media of pterygium fibroblasts relative to the media of control cells, respectively. Molecular function analysis showed that these proteins either belonged to catalytic proteins, structural molecules or were involved with receptor activities and protein binding. Further pathway analysis revealed that these proteins were involved in ECM-receptor interaction, focal adhesion, cancer-related, p53 signaling, complement and coagulation, and TGF-beta signaling pathways. These molecules identified may serve as extracellular ligands to activate intracellular pathways, possibly serving as potential therapeutic targets.

Proteomic analysis of secreted proteins by human bronchial epithelial cells in response to cadmium toxicity.

For years, many studies have been conducted to investigate the intracellular response of cells challenged with toxic metal(s), yet, the corresponding secretome responses, especially in human lung cells, are largely unexplored. Here, we provide a secretome analysis of human bronchial epithelial cells (BEAS-2B) treated with cadmium chloride (CdCl2 ), with the aim of identifying secreted proteins in response to Cd toxicity. Proteins from control and spent media were separated by two-dimensional electrophoresis and visualized by silver staining. Differentially-secreted proteins were identified by MALDI-TOF-MS analysis and database searching. We characterized, for the first time, the extracellular proteome changes of BEAS-2B dosed with Cd. Our results unveiled that Cd treatment led to the marked upregulation of molecular chaperones, antioxidant enzymes, enzymes associated with glutathione metabolic process, proteins involved in cellular energy metabolism, as well as tumor-suppressors. Pretreatment of cells with the thiol antioxidant glutathione before Cd treatment effectively abrogated the secretion of these proteins and prevented cell death. Taken together, our results demonstrate that Cd causes oxidative stress-induced cytotoxicity; and the differentially-secreted protein signatures could be considered as targets for potential use as extracellular biomarkers upon Cd exposure.

Hepatic stellate cells and extracellular matrix in hepatocellular carcinoma: more complicated than ever.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer death. Recent epidemiological data indicate that the mortality rate of HCC will double over the next decades in the USA and Europe. Liver cancer progresses in a large percentage of cases during the clinical course of chronic fibro-inflammatory liver diseases leading to cirrhosis. Therefore, HCC development is regarded as the result of different environmental risk factors each involving different genetic, epigenetic- and chromosomal alterations and gene mutations. During tumour progression, the malignant hepatocytes and the activated hepatic stellate cells are accompanied by cancer-associated fibroblasts, myofibroblasts and immune cells generally called tumour stromal cells. This new and dynamic milieu further enhances the responsiveness of tumour cells towards soluble mediators secreted by tumour stromal cells, thus directly affecting the malignant hepatocytes. This results in altered molecular pathways with cell proliferation as the most important mechanism of liver cancer progression. Given this contextual complexity, it is of utmost importance to characterize the molecular pathogenesis of HCC, and to identify the dominant pathways/drivers and aberrant signalling pathways. This will allow an effective therapy for HCC that should combine strategies affecting both cancer and the tumour stromal cells. This review provides an overview of the recent challenges and issues regarding hepatic stellate cells, extracellular matrix dynamics, liver fibrosis/cirrhosis and therapy, tumour microenvironment and HCC.

Effect of an endothelial regulatory module on plasma proteomics in exercising horses.

Elite performing exercise requires an intricate modulation of the blood pressure to support the working muscles with oxygen. We have previously identified a genomic regulatory module that associates with differences in blood pressures of importance for elite performance in racehorses. This study aimed to determine the effect of the regulatory module on the protein repertoire. We sampled plasma from 12 Coldblooded trotters divided into two endothelial regulatory module haplotype groups, a sub-elite performing haplotype (SPH) and an elite performing haplotype (EPH), each at rest and exercise. The haplotype groups and their interaction were interrogated in two analyses, i) individual paired ratio analysis for identifying differentially abundant proteins of exercise (DAPE) and interaction (DAPI) between haplotype and exercise, and ii) unpaired ratio analysis for identifying differentially abundant protein of haplotype (DAPH). The proteomics analyses revealed a widespread change in plasma protein content during exercise, with a decreased tendency in protein abundance that is mainly related to lung function, tissue fluids, metabolism, calcium ion pathway and cellular energy metabolism. Furthermore, we provide the first investigation of the proteome variation due to the interaction between exercise and related blood pressure haplotypes, which this difference was related to a faster switch to the lipoprotein and lipid metabolism during exercise for EPH. The molecular signatures identified in the present study contribute to an improved understanding of exercise-related blood pressure regulation.

Elucidating Thermothielavioides terrestris secretome changes for improved saccharification of mild steam-pretreated spruce.

BACKGROUND: The efficient use of softwood in biorefineries is hampered by its recalcitrance to enzymatic saccharification. In the present study, the fungus Thermothielavioides terrestris LPH172 was cultivated on three steam-pretreated spruce materials (STEX180°C/auto, STEX210°C/auto, and STEX210°C/H2SO4), characterized by different hemicellulose content and structure, as well as on untreated biomass. The aim of the study was to map substrate-induced changes in the secretome of T. terrestris grown on differently treated spruce materials and to evaluate the hydrolytic efficiency of the secretome as supplement for a commercial enzyme mixture. RESULTS: The cultivation of T. terrestris was monitored by endo-cellulase, endo-xylanase, endo-mannanase, laccase, and peroxidase activity measurements. Proteomic analysis was performed on the secretomes induced by the spruce materials to map the differences in enzyme production. Growth of T. terrestris on STEX180°C/auto and STEX210°C/auto induced higher expression level of mannanases and mannosidases of the GH5_7 CAZy family compared to cultivation on the other materials. Cultivation on untreated biomass led to overexpression of GH47, GH76, and several hemicellulose debranching enzymes compared to the cultivation on the pretreated materials. T. terrestris grown on untreated, STEX180°C/auto and STEX210°C/auto induced three arabinofuranosidases of the GH43 and GH62 families; while growth on STEX210°C/H2SO4 induced a GH51 arabinofuranosidase and a GH115 glucuronidase. All secretomes contained five lytic polysaccharide monooxygenases of the AA9 family. Supplementation of Celluclast® + Novozym188 with the secretome obtained by growing the fungus grown on STEX180°C/auto achieved a twofold higher release of mannose from spruce steam-pretreated with acetic acid as catalyst, compared to the commercial enzyme cocktail alone. CONCLUSIONS: Minor changes in the structure and composition of spruce affect the composition of fungal secretomes, with differences in some classes explaining an increased hydrolytic efficiency. As demonstrated here, saccharification of spruce biomass with commercial enzyme cocktails can be further enhanced by supplementation with tailor-made secretomes.

Culturing Conditions Dictate the Composition and Pathways Enrichment of Human and Rat Perirenal Adipose-Derived Stromal Cells' Secretomes.

Understanding the impact of various culturing strategies on the secretome composition of adipose-derived stromal cells (ASC) enhances their therapeutic potential. This study investigated changes in the secretome of perirenal ASC (prASC) under different conditions: normoxia, cytokine exposure, high glucose, hypoxia, and hypoxia with high glucose. Using mass spectrometry and enrichment clustering analysis, we found that normoxia enriched pathways related to extracellular matrix (ECM) organization, platelet degranulation, and insulin-like growth factor (IGF) transport and uptake. Cytokine exposure influenced metabolism, vascular development, and protein processing pathways. High glucose affected the immune system, metabolic processes, and IGF transport and uptake. Hypoxia impacted immune and metabolic processes and protein processing. Combined hypoxia and high glucose influenced the immune system, IGF transport and uptake, and ECM organization. Our findings highlight the potential of manipulating culturing conditions to produce secretomes with distinct protein and functional profiles, tailoring therapeutic strategies accordingly.