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Endophytic fungi, as plant symbionts, produce an elaborate array of enzymes for efficient disintegration of lignocellulosic biomass into constituent monomeric sugars, making them novel source of lignocellulolytic CAZymes with immense potential in future biorefineries. The present study reports lignocellulolytic enzymes production potential of an endophytic halotolerant Penicillium oxalicum strain isolated from Citrus limon, under submerged and solid-state fermentation (SmF & SSF, respectively), in the presence and absence of salt (1 M NaCl). The comparative QTOF–LC/MS-based exoproteome analysis of the culture extracts unveiled differential expression of CAZymes, with the higher abundance of GH6 and GH7 family cellobiohydrolase in the presence of 1 M salt. The strain improvement program, employing cyclic mutagenesis and diploidization, was utilized to develop hyper-cellulase producing mutant strains of P. oxalicum. The enzyme production of the developed strain (POx-M35) was further enhanced through statistical optimization of the culture conditions utilizing glucose mix disaccharides (GMDs) as an inducer. This optimization process resulted in the lignocellulolytic cocktail that contained high titers (U/mL) of endoglucanase (EG) (146.16), cellobiohydrolase (CBHI) (6.99), β-glucosidase (β-G) (26.21), xylanase (336.05) and FPase (2.02 U/mL), which were 5.47-, 5.54-, 8.55-, 4.96-, and 4.39-fold higher when compared to the enzyme titers obtained in wild HP1, respectively. Furthermore, the lignocellulolytic cocktails designed by blending secretome produced by mutant POx-M35 with xylanases (GH10 and GH11) derived from Malbranchea cinnamomea resulted in efficient hydrolysis of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw. This study underscores the potential of bioprospecting novel fungus and developing an improved strain for optimized production and constitution of lignocellulolytic cocktails that can be an important determinant in advancing biomass conversion technologies. [ABSTRACT FROM AUTHOR]

Aureobasidium pullulans LB83 is a versatile biocatalyst that produces a plethora of bioactive products thriving on a variety of feedstocks under the varying culture conditions. In our last study using this microorganism, we found cellulase activity (FPase, 2.27 U/ml; CMCase, 7.42 U/ml) and other plant cell wall degrading enzyme activities grown on sugarcane bagasse and soybean meal as carbon source and nitrogen, respectively. In the present study, we provide insights on the secretome analysis of this enzymatic cocktail. The secretome analysis of A. pullulans LB83 by Liquid Chromatography coupled to Mass Spectroscopy (LC-MS/MS) revealed 38 classes of Carbohydrate Active enZymes (CAZymes) of a total of 464 identified proteins. These CAZymes consisted of 21 glycoside hydrolases (55.26%), 12 glycoside hydrolases harboring carbohydrate-binding module (31.58%), 4 carbohydrate esterases (10.53%) and one glycosyl transferase (2.63%). To the best of our knowledge, this is the first report on the secretome analysis of A. pullulans LB83. [ABSTRACT FROM AUTHOR]

Tigilanol tiglate (TT, also known as EBC-46) is a novel, plant-derived diterpene ester possessing anticancer and wound-healing properties. Here, we show that TT-evoked PKC-dependent S985 phosphorylation of the tyrosine kinase MET leads to subsequent degradation of tyrosine phosphorylated p-Y1003 and p-Y1234/5 MET species. PKC inhibition with BIM-1 blocked S985 phosphorylation of MET and led to MET cell surface accumulation. Treatment with metalloproteinase inhibitors prevented MET-ECD release into cell culture media, which was also blocked by PKC inhibitors. Furthermore, unbiased secretome analysis, performed using TMT-technology, identified additional targets of TT-dependent release of cell surface proteins from H357 head and neck cancer cells. We confirm that the MET co-signalling receptor syndecan-1 was cleaved from the cell surface in response to TT treatment. This was accompanied by rapid cleavage of the cellular junction adhesion protein Nectin-1 and the nerve growth factor receptor NGFRp75/TNFR16. These findings, that TT is a novel negative regulator of protumorigenic c-MET and NGFRp75/TNFR16 signalling, as well as regulating Nectin-1-mediated cell adhesion, further contribute to our understanding of the mode of action and efficacy of TT in the treatment of solid tumours. [ABSTRACT FROM AUTHOR]

Abstract Background Valorizing waste residues is crucial to reaching sustainable development goals and shifting from a linear fossil-based economy to a circular economy. Fungal cell factories, due to their versatility and robustness, are instrumental in driving the bio-transformation of waste residues. The present work isolated a potent strain, i.e., Aspergillus fumigatus (ZS_AF), from an ancient Złoty Stok gold mine, which showcased distinctive capabilities for efficient hydrolytic enzyme production from lignocellulosic wastes. Results The present study optimized hydrolytic enzyme production (cellulases, xylanases, and β-glucosidases) from pine sawdust (PSD) via solid-state fermentation using Aspergillus fumigatus (ZS_AF). The optimization, using response surface methodology (RSM), produced a twofold increase with maximal yields of 119.41 IU/gds for CMCase, 1232.23 IU/gds for xylanase, 63.19 IU/gds for β-glucosidase, and 31.08 IU/gds for FPase. The secretome profiling validated the pivotal role of carbohydrate-active enzymes (CAZymes) and auxiliary enzymes in biomass valorization. A total of 77% of carbohydrate-active enzymes (CAZymes) were constituted by glycoside hydrolases (66%), carbohydrate esterases (9%), auxiliary activities (3%), and polysaccharide lyases (3%). The saccharification of pretreated wheat straw and PSD generated high reducing sugar yields of 675.36 mg/g and 410.15 mg/g, respectively. Conclusion These findings highlight the significance of an efficient, synergistic, and cost-effective arsenal of fungal enzymes for lignocellulosic waste valorization and their potential to contribute to waste-to-wealth creation through solid-waste management. The utilization of Aspergillus fumigatus (ZS_AF) from an unconventional origin and optimization strategies embodies an innovative approach that holds the potential to propel current waste valorization methods forward, directing the paradigm toward improved efficiency and sustainability.

Background: Valorizing waste residues is crucial to reaching sustainable development goals and shifting from a linear fossil-based economy to a circular economy. Fungal cell factories, due to their versatility and robustness, are instrumental in driving the bio-transformation of waste residues. The present work isolated a potent strain, i.e., Aspergillus fumigatus (ZS_AF), from an ancient Złoty Stok gold mine, which showcased distinctive capabilities for efficient hydrolytic enzyme production from lignocellulosic wastes. Results: The present study optimized hydrolytic enzyme production (cellulases, xylanases, and β-glucosidases) from pine sawdust (PSD) via solid-state fermentation using Aspergillus fumigatus (ZS_AF). The optimization, using response surface methodology (RSM), produced a twofold increase with maximal yields of 119.41 IU/gds for CMCase, 1232.23 IU/gds for xylanase, 63.19 IU/gds for β-glucosidase, and 31.08 IU/gds for FPase. The secretome profiling validated the pivotal role of carbohydrate-active enzymes (CAZymes) and auxiliary enzymes in biomass valorization. A total of 77% of carbohydrate-active enzymes (CAZymes) were constituted by glycoside hydrolases (66%), carbohydrate esterases (9%), auxiliary activities (3%), and polysaccharide lyases (3%). The saccharification of pretreated wheat straw and PSD generated high reducing sugar yields of 675.36 mg/g and 410.15 mg/g, respectively. Conclusion: These findings highlight the significance of an efficient, synergistic, and cost-effective arsenal of fungal enzymes for lignocellulosic waste valorization and their potential to contribute to waste-to-wealth creation through solid-waste management. The utilization of Aspergillus fumigatus (ZS_AF) from an unconventional origin and optimization strategies embodies an innovative approach that holds the potential to propel current waste valorization methods forward, directing the paradigm toward improved efficiency and sustainability. [ABSTRACT FROM AUTHOR]

Tigilanol tiglate (TT, also known as EBC-46) is a novel, plant-derived diterpene ester possessing anticancer and wound-healing properties. Here, we show that TT-evoked PKC-dependent S985 phosphorylation of the tyrosine kinase MET leads to subsequent degradation of tyrosine phosphorylated p-Y1003 and p-Y1234/5 MET species. PKC inhibition with BIM-1 blocked S985 phosphorylation of MET and led to MET cell surface accumulation. Treatment with metalloproteinase inhibitors prevented MET-ECD release into cell culture media, which was also blocked by PKC inhibitors. Furthermore, unbiased secretome analysis, performed using TMT-technology, identified additional targets of TT-dependent release of cell surface proteins from H357 head and neck cancer cells. We confirm that the MET co-signalling receptor syndecan-1 was cleaved from the cell surface in response to TT treatment. This was accompanied by rapid cleavage of the cellular junction adhesion protein Nectin-1 and the nerve growth factor receptor NGFRp75/TNFR16. These findings, that TT is a novel negative regulator of protumorigenic c-MET and NGFRp75/TNFR16 signalling, as well as regulating Nectin-1-mediated cell adhesion, further contribute to our understanding of the mode of action and efficacy of TT in the treatment of solid tumours.

The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum. Both the wild-type strain and a strain that produced and secreted a recombinant β-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production.

Background: Breast cancer is the leading cause of frequent malignancy and morbidity among women across the globe, with an increment of 0.5% incidences every year. The deleterious effects of traditional treatment on off-target surrounding cells make it difficult to win the battle against breast cancer. Hence, an advancement in the therapeutic approach is crucial. Nanotechnology is one of the emerging methods for precise, targeted, and efficient drug delivery in cells. The previous study has demonstrated the cytotoxic effect of Ipomoea turpethum extract on breast cancer cells delivered via NIPAAM-VP-AA nanoparticles (NVA-IT). Manipulating the tumor microenvironment (TME) to inhibit cancer progression, invasion, and metastasis seems to be very insightful for researchers these days. With the help of secretome analysis of breast cancer cells after treatment with NVA-IT, we have tried to find out the possible TME manipulation achieved to favor a better prognosis of the disease.Method: MCF-7 and MDA MB-231 cells were treated with the IC50 value of NVA-IT, and the medium was separated from the cells after 24 h of the treatment. Nano LCMS/MS analysis was performed to identify the secretory proteins in the media. Further bioinformatics tools like GENT2, GSCA, GeneCodis 4, and STRING were used to identify the key proteins and their interactions.Result: From the nano LCMS/MS analysis, 70 differentially expressed secretory proteins in MCF-7 and 191 in MDA MB-231 were identified in the cell’s media. Fifteen key target proteins were filtered using bioinformatics analysis, and the interaction of proteins involved in vesicular trafficking, cell cycle checkpoints, and oxidative stress-related proteins was prominent.Conclusion: This study concluded that I. turpethum extract-loaded NIPAAM-VP-AA nanoparticles alter the secretory proteins constituting the TME to cease cancer cell growth and metastasis.

ABSTRACT The soft rot pathogen Janthinobacterium agaricidamnosum causes devastating damage to button mushrooms (Agaricus bisporus), one of the most cultivated and commercially relevant mushrooms. We previously discovered that this pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin, however, could not solely explain the rapid decay of mushroom fruiting bodies, indicating that J. agaricidamnosum implements a more sophisticated infection strategy. In this study, we show that secretion systems play a crucial role in soft rot disease. By mining the genome of J. agaricidamnosum, we identified gene clusters encoding a type I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs). We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays implicated both in soft rot disease. Furthermore, through a combination of comparative secretome analysis and activity-guided fractionation, we identified a number of secreted lytic enzymes responsible for mushroom damage. Our findings regarding the contribution of secretion systems to the disease process expand the current knowledge of bacterial soft rot pathogens and represent a significant stride toward identifying targets for their disarmament with secretion system inhibitors. IMPORTANCE The button mushroom (Agaricus bisporus) is the most popular edible mushroom in the Western world. However, mushroom crops can fall victim to serious bacterial diseases that are a major threat to the mushroom industry, among them being soft rot disease caused by Janthinobacterium agaricidamnosum. Here, we show that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due to degradative enzymes and putative effector proteins secreted via the type II secretion system (T2SS) and the type III secretion system (T3SS), respectively. The ability to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants, which establishes that secretion systems are key factors in mushroom soft rot disease. This insight is of both ecological and agricultural relevance by shedding light on the disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves as a starting point for the development of secretion system inhibitors to control disease progression.

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting., (© 2024 The Author(s). PROTEOMICS published by Wiley‐VCH GmbH.)

Acute Myeloid Leukemia (AML) is a heterogeneous disease with highest mortality compared to other types of leukemia. There is a need to find the gene abnormalities and mechanisms behind them due to their heterogenic nature. The present study is aimed to understand genes, pathways and biomarker proteins influenced by transcriptomic deregulation due to AML. Differentially expressed gene (DEG), protein-protein interaction network, gene ontology, KEGG pathway, variant analysis and secretome analyses were performed using different AML RNAseq datasets. A total of 655 DEGs including 291 up-regulated and 364 down-regulated genes, which were satisfied with a fold change of 1.5 were identified. Top hub genes for AML were identified as TP53, PTPRC and AKT1. This integrative bioinformatics approach revealed the deregulation of T Cell Receptor (TCR) pathway and altered immune response related genes. The survival analysis revealed the associated deregulation of multiple TCR pathway related genes. Variant analysis identified the benign and likely benign nature of many important target genes and markers screened, which were found to have an important role in the progression of AML. DEGs and secretome analysis found out a set of seven molecules represents potential biomarkers for AML. In vitro analytical validation showed overexpression pattern of CD109 and LRP12 in AML cell line and HL-60 cells than the normal human bone marrow-derived stromal cell line HS-5. Here we report first time for CD109 and LRP12 as a possible biomarkers for the diagnostic significance. Amino acid substitutions detected by variant analysis and deregulation of immune checkpoint molecules revealed their role in reducing immune response and inability to fight cancer cells. In conclusion, this study highlights the possibility of new biomarkers for AML and the mechanism of decrease in immune response due to the downregulation of co-stimulatory immune molecules, which needs further clinical validation investigations.

Macrophomina phaseolina is a global devastating necrotrophic fungal pathogen. It causes charcoal rot disease in more than 500 host plants including major food crops, pulse crops, fiber crops, and oil crops. Despite having the whole-genome sequence of M. phaseolina, understanding the M. phaseolina genome-based plant–pathogen interactions is limited in the absence of direct experimental proof of secretion. Thus, it is essential to understand the host–microbe interaction and the disease pathogenesis, which can ensure global agricultural crop production and security. An in silico–predicted secretome of M. phaseolina is unable to represent the actual secretome. We could identify 117 proteins present in the secretome of M. phaseolina using liquid chromatography–electrospray ionization–tandem mass spectrometry. Data are available via ProteomeXchange with identifier PXD032749. An array of putative virulence factors of M. phaseolina were identified in the present study using solid-state culture. Similar virulence factors have been reported in other plant pathogenic fungi also. Among the secretory fungal proteins with positive economic impacts, lignocellulolytic enzymes are of prime importance. Further, we validated our results by detecting the cell wall–degrading enzymes xylanase, endoglucanase, and amylase in the secretome of M. phaseolina. The present study may provide a better understanding about the necrotrophic fungi M. phaseolina, which modulate the host plant defense barriers using secretory proteins. [ABSTRACT FROM AUTHOR]

Macrophomina phaseolina is a global devastating necrotrophic fungal pathogen. It causes charcoal rot disease in more than 500 host plants including major food crops, pulse crops, fiber crops, and oil crops. Despite having the whole-genome sequence of M. phaseolina, understanding the M. phaseolina genome-based plant–pathogen interactions is limited in the absence of direct experimental proof of secretion. Thus, it is essential to understand the host–microbe interaction and the disease pathogenesis, which can ensure global agricultural crop production and security. An in silico–predicted secretome of M. phaseolina is unable to represent the actual secretome. We could identify 117 proteins present in the secretome of M. phaseolina using liquid chromatography–electrospray ionization–tandem mass spectrometry. Data are available via ProteomeXchange with identifier PXD032749. An array of putative virulence factors of M. phaseolina were identified in the present study using solid-state culture. Similar virulence factors have been reported in other plant pathogenic fungi also. Among the secretory fungal proteins with positive economic impacts, lignocellulolytic enzymes are of prime importance. Further, we validated our results by detecting the cell wall–degrading enzymes xylanase, endoglucanase, and amylase in the secretome of M. phaseolina. The present study may provide a better understanding about the necrotrophic fungi M. phaseolina, which modulate the host plant defense barriers using secretory proteins.

The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta , gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFβ family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFβ signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFβ effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFβ targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis. In conclusion our data strongly suggest that TGFβ -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications. [ABSTRACT FROM AUTHOR]

The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta, gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFβ family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFβ signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFβ effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFβ targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis.In conclusion our data strongly suggest that TGFβ -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications.

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. Key points: • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis. [ABSTRACT FROM AUTHOR]

The type 6 secretion system (T6SS) of Gram-negative bacteria (GNB) has implications for bacterial competition, virulence, and survival. For the broad host range pathogen, Pectobacterium brasiliense 1692, T6SS-mediated competition occurs in a tissue-specific manner. However, no other roles have been investigated. The aim of this study was to identify T6SS-associated proteins under virulence inducing conditions. We used Bastion tools to predict 1479 Pbr 1692 secreted proteins. Sixteen percent of these overlap between type 1–4 secretion systems (T1SS-T4SS) and T6SS. Using label-free quantitative mass spectrometry of Pbr 1692 T6SS active and T6SS inactive strains' secretomes cultured in minimal media supplemented with host extract, 49 T6SS-associated proteins with varied gene ontology predicted functions were identified. We report 19 and 30 T6SS primary substrates and differentially secreted proteins, respectively, in T6SS mutants versus wild type strains. Of the total 49 T6SS-associated proteins presented in this study, 25 were also predicted using the BastionX platform as T6SS exclusive and shared substrates with T1SS-T4SS. This work provides a list of Pbr 1692 T6SS secreted effector candidates. These include a potential antibacterial toxin HNH endonuclease and several predicted virulence proteins, including plant cell wall degrading enzymes. A preliminary basis for potential crosstalk between GNB secretion systems is also highlighted. [ABSTRACT FROM AUTHOR]

The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum . Both the wild-type strain and a strain that produced and secreted a recombinant β-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Balasubramanian, Køhler, Jers, Jensen and Mijakovic.)

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa. [ABSTRACT FROM AUTHOR]

Background: Breast cancer is the leading cause of frequent malignancy and morbidity among women across the globe, with an increment of 0.5% incidences every year. The deleterious effects of traditional treatment on off-target surrounding cells make it difficult to win the battle against breast cancer. Hence, an advancement in the therapeutic approach is crucial. Nanotechnology is one of the emerging methods for precise, targeted, and efficient drug delivery in cells. The previous study has demonstrated the cytotoxic effect of Ipomoea turpethum extract on breast cancer cells delivered via NIPAAM-VP-AA nanoparticles (NVA-IT). Manipulating the tumor microenvironment (TME) to inhibit cancer progression, invasion, and metastasis seems to be very insightful for researchers these days. With the help of secretome analysis of breast cancer cells after treatment with NVA-IT, we have tried to find out the possible TME manipulation achieved to favor a better prognosis of the disease. Method: MCF-7 and MDA MB-231 cells were treated with the IC 50 value of NVA-IT, and the medium was separated from the cells after 24 h of the treatment. Nano LCMS/MS analysis was performed to identify the secretory proteins in the media. Further bioinformatics tools like GENT2, GSCA, GeneCodis 4, and STRING were used to identify the key proteins and their interactions. Result: From the nano LCMS/MS analysis, 70 differentially expressed secretory proteins in MCF-7 and 191 in MDA MB-231 were identified in the cell's media. Fifteen key target proteins were filtered using bioinformatics analysis, and the interaction of proteins involved in vesicular trafficking, cell cycle checkpoints, and oxidative stress-related proteins was prominent. Conclusion: This study concluded that I. turpethum extract-loaded NIPAAM-VP-AA nanoparticles alter the secretory proteins constituting the TME to cease cancer cell growth and metastasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Swami, Mughees, Mangangcha, Kauser and Wajid.)

Laccases are copper-containing enzymes belonging to the family of multicopper oxidases (MCOs). All MCOs use molecular oxygen to oxidize a wide range of organic compounds by radical catalysis. One of the key fundamental properties of laccases is having high or low redox potentials depending on the active site organization. Several experimental studies have been done to rationalize the high and low redox potential laccases (LRPL), however, molecular understanding is still lacking. In this work, we explored the proteomic profile of laccases produced in the fungal cultures, specifically induced with lignocellulosic biomass such as rice straw. This study was undertaken to explain the differences in the high redox and low redox potential values of different laccases using in-silico approaches. Proteomic profiling and structural and sequence analysis revealed a low level of similarity among them. Docking analyses and molecular dynamics simulation analysis revealed that high redox potential laccases (HRPL) are having good binding affinity compared to low or medium redox potential laccases (MRPL). The stability of these complexes was further analyzed based on reactive distances, active site volume comparison and a number of tunnel formations that were observed to be significantly higher for HRPL. Our results indicate that the number of tunnel formations calculated from the simulation's trajectories and available water molecules at the T3 site directly correlates with the laccases' redox potentials. This study will be helpful and provide valuable inputs for the designing of new laccases to improve lignin degradation.Communicated by Ramaswamy H. Sarma.

Arabinoxylan, one of the major non‐starch polysaccharides of the endosperm cell wall of barley, has a negative impact on the filterability of wort and beer. The addition of microbial xylanase would be an effective strategy to degrade arabinoxylan. In this study, the secretome of Trichoderma reesei CICC 41495 cultivated under submerged fermentation with wheat bran as a carbon source was characterised by two‐dimensional electrophoresis combined with MALDI‐TOF/TOF tandem mass spectrometry. A total of 24 proteins were identified in the culture supernatant, 23 of which were involved in non‐starch polysaccharide degradation. The proteins belonged to 10 different families of glycoside hydrolases, including xyloglucanase, cellobiohydrolase I, cellobiohydrolase II, endoglucanase II, endo‐1,4‐β‐glucanase VII, β‐glucosidase, endo‐1,4‐β‐xylanase I and II, endo‐1,4‐β‐xylanase III, and α‐l‐arabinofuranosidase. Of these, four were correlated with arabinoxylan degradation. Supplementation of the secretome loading of 0.14 mg protein/g malt to mash resulted in the total degradation of high molecular weight arabinoxylan, a 90% increase in filtration rate and a 7.9% decrease in viscosity. The results suggest that T. reesei produces a tailored xylanolytic enzyme system that efficiently hydrolysed high molecular weight arabinoxylan. Accordingly, the secretome of this fungi has potential value in the brewing industry. © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

The soft rot pathogen Janthinobacterium agaricidamnosum causes devastating damage to button mushrooms ( Agaricus bisporus ), one of the most cultivated and commercially relevant mushrooms. We previously discovered that this pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin, however, could not solely explain the rapid decay of mushroom fruiting bodies, indicating that J. agaricidamnosum implements a more sophisticated infection strategy. In this study, we show that secretion systems play a crucial role in soft rot disease. By mining the genome of J. agaricidamnosum , we identified gene clusters encoding a type I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs). We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays implicated both in soft rot disease. Furthermore, through a combination of comparative secretome analysis and activity-guided fractionation, we identified a number of secreted lytic enzymes responsible for mushroom damage. Our findings regarding the contribution of secretion systems to the disease process expand the current knowledge of bacterial soft rot pathogens and represent a significant stride toward identifying targets for their disarmament with secretion system inhibitors. IMPORTANCE The button mushroom ( Agaricus bisporus ) is the most popular edible mushroom in the Western world. However, mushroom crops can fall victim to serious bacterial diseases that are a major threat to the mushroom industry, among them being soft rot disease caused by Janthinobacterium agaricidamnosum . Here, we show that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due to degradative enzymes and putative effector proteins secreted via the type II secretion system (T2SS) and the type III secretion system (T3SS), respectively. The ability to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants, which establishes that secretion systems are key factors in mushroom soft rot disease. This insight is of both ecological and agricultural relevance by shedding light on the disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves as a starting point for the development of secretion system inhibitors to control disease progression., Competing Interests: The authors declare no conflict of interest.

This study reports thermophilic fungus Malbranchea flava as a potent source of xylanase and xylan-debranching accessory enzymes. M. flava produced high levels of xylanase on sorghum straw containing solidified culture medium. The optimization of culture conditions for production of hemicellulases was carried out using one factor at a time approach and Box-Behnken design of experiments with casein (%), inoculum age (h) and inoculum level (ml) as process variables and xylanase, β-xylosidase, acetyl esterases and arabinofuranosidase as response variables. The results showed that casein concentration between 3.0 and 3.5 %, inoculum age (56-60 h) and inoculum level (2-2.5 ml) resulted in production of 16,978, 10.0, 67.7 and 3.8 (U/gds) of xylanase, β-xylosidase, acetyl esterase and α- l-arabinofuranosidase, respectively. Under optimized conditions M. flava produced eight functionally diverse xylanases with distinct substrate specificity against different xylan types. The peptide mass fingerprinting of 2-D gel electrophoresis resolved proteins indicated to the presence of cellobiose dehydrogenase and glycosyl hydrolases suggesting the potential of this strain in oxidative and classical cellulase-mediated hydrolysis of lignocellulosics. Addition of xylanase (300 U/g substrate) during saccharification (at 15 % substrate loading) of different pretreated (acid/alkali) substrates (cotton stalks, wheat straw, rice straw, carrot grass) by commercial cellulase (NS28066) resulted in 9-36 % increase in saccharification and subsequent fermentation to ethanol when compared to experiment with commercial enzyme only. High ethanol level 46 (g/l) was achieved with acid pretreated cotton stalk when M. flava xylanase was supplemented as compared to 39 (g/l) with xylanase without xylanase addition. [ABSTRACT FROM AUTHOR]

[Display omitted] • Comparative genome wide sequence analysis of Aspergillus strains. • Developing hyper lignocellulolytic strain of A. allahabadii. • Secretome profiling identifies up-regulated GH and transcriptional factor in mutant. • Enzyme cocktail of mutant MAN 40 and CBH from R. emersonii for efficient hydrolysis. • In situ addition of cellulase for valorization of waste lignin stream in biogas. The study was aimed at developing lignocellulolytic strain capable of efficient hydrolysis of mild alkali deacetylated (MAD) rice straw. The valorisation of lignin rich black liquor obtained during pre-treatment of rice straw into biogas was also evaluated. Study reports highly proficient cellulolytic Aspergillus allahabadii strain harbouring a spectrum of CAZymes based on comparative genome wide analysis that was subjected to strain breeding for developing a hyper producing strain. The secretome analysis showed up-modulation and several folds increase in the CAZyme activities in the culture extracts of the developed strain MAN 40 when compared to parent. The cellulolytic cocktail of the developed strain showed 1.52 folds higher saccharification of MAD rice straw when compared to Cellic CTec 3. Moreover, in-situ addition of cellulases derived from developed strains resulted in ∼3.7 folds higher methane production during anaerobic digestion of mixture of lignin rich black liquor and differently treated rice straw. [ABSTRACT FROM AUTHOR]

Trichoderma spp can colonize host plants protecting them from pathogen infection. This association is accomplished by an exchange of molecules secreted by both the host plant and symbiotic fungus. In the present work, using LC-MS/MS analysis, our research group described a list of proteins secreted by T. harzianum TR274 during association with common bean (Phaseolus vulgaris). The main goal is to describe candidate proteins critical for the formerly mentioned association and understand mechanisms related to hosting plant tissue's colonization and modulation of bean plant defense response or metabolism. T. harzianum TR 274 could colonize common bean roots, increasing the transcript levels of defense-related genes (chit, glu1, and pal). Secretomes were obtained from culture filtrates harvested after the growth of T. harzianum TR 274 in the presence (Secretome of co-cultivation-SCC) or absence (Secretome of fungus condition-SFC) of the host plant. We identified 124 and 190 fungal proteins in SCC and SFC, respectively. A total of 30 proteins were detected only in SCC, and 94 were detected in both secretomes (SCC and SFC). The host plant presence decreases the number and diversity of proteins secreted by T. harzianum. The main functional categories represented are glycoside hydrolases, proteases, oxidoreductases, and SSCPs (Small soluble cysteine-rich secreted proteins). Our results provide a list of protein candidates for functional gene studies and validate their role in interactions between T. harzianum and P. vulgaris. Therefore, it contributes to understanding the main mechanisms of T. harzianum host plant colonization and the modulation of defense response. • Proteases, small cysteine rich proteins and glycoside hydrolases were the main activities detected in Trichoderma harzianum TR274 secretome in the presence of Phaseolus vulgaris. • Enzymes previously described as presenting a potential role in T. harzianum /host plants interactions were constitutively produced. • Effector-like proteins were identified. [ABSTRACT FROM AUTHOR]

Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism , whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25 °C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25 °C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn 2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions ( vp1/mnp3 > vp2/vp3/mnp1/mnp2/mnp6 > mnp4/mnp5 ). Decreasing or increasing the incubation temperature (to 10 °C or 37 °C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10 °C treatment, while many of them were alternatively upregulated (often 6 h after the thermal shock) and downregulated (12 h) at 37 °C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion. [ABSTRACT FROM AUTHOR]

Acute Myeloid Leukemia (AML) is a heterogeneous disease with highest mortality compared to other types of leukemia. There is a need to find the gene abnormalities and mechanisms behind them due to their heterogenic nature. The present study is aimed to understand genes, pathways and biomarker proteins influenced by transcriptomic deregulation due to AML. Differentially expressed gene (DEG), protein-protein interaction network, gene ontology, KEGG pathway, variant analysis and secretome analyses were performed using different AML RNAseq datasets. A total of 655 DEGs including 291 up-regulated and 364 down-regulated genes, which were satisfied with a fold change of 1.5 were identified. Top hub genes for AML were identified as TP53, PTPRC and AKT1. This integrative bioinformatics approach revealed the deregulation of T Cell Receptor (TCR) pathway and altered immune response related genes. The survival analysis revealed the associated deregulation of multiple TCR pathway related genes. Variant analysis identified the benign and likely benign nature of many important target genes and markers screened, which were found to have an important role in the progression of AML. DEGs and secretome analysis found out a set of seven molecules represents potential biomarkers for AML. In vitro analytical validation showed overexpression pattern of CD109 and LRP12 in AML cell line and HL-60 cells than the normal human bone marrow-derived stromal cell line HS-5. Here we report first time for CD109 and LRP12 as a possible biomarkers for the diagnostic significance. Amino acid substitutions detected by variant analysis and deregulation of immune checkpoint molecules revealed their role in reducing immune response and inability to fight cancer cells. In conclusion, this study highlights the possibility of new biomarkers for AML and the mechanism of decrease in immune response due to the downregulation of co-stimulatory immune molecules, which needs further clinical validation investigations., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Microglia have recently been established as key regulators of brain development. However, their role in neuronal subtype specification remains largely unknown. Using three different co-culture setups, we show that microglia-secreted factors enhance dopaminergic differentiation of somatic and induced pluripotent stem cell-derived human neural stem cells (NSCs). The effect was consistent across different NSC and microglial cell lines and was independent of prior microglial activation, although restricted to microglia of embryonic origin. We provide evidence that the effect is mediated through reduced cell proliferation and decreased apoptosis and necrosis orchestrated in a sequential manner during the differentiation process. tumor necrosis factor alpha, interleukin-1β, and insulinlike growth factor 1 are identified as key mediators of the effect and shown to directly increase dopaminergic differentiation of human NSCs. These findings demonstrate a positive effect of microglia on dopaminergic neurogenesis and may provide new insights into inductive and protective factors that can stimulate in vitro derivation of dopaminergic neurons., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In this chapter we describe in detail how to prepare a sample containing the complete set of secretion products from primary adipocytes, which are suitable for comprehensive and sensitive secretome analysis. The underlying protocol should be seen as starting point guiding through critical steps of the complex workflow of preperation of secretomes. For diverse research questions and in the context of different sample types used, the protocol has to be carefully adjusted in order to approximate to the real secretome.

In situ regeneration of lacrimal gland (LG) tissue would be a promising approach to curatively treat dry eye disease (DED). Mesenchymal stem cells (MSC) exhibit therapeutic effects in a variety of pathological conditions and our group recently reported that their number increases in regenerating mouse LG. Since the therapeutic effects are suggested to arise from secreted trophic factors, the application of MSC-secreted proteins seems to be a promising approach to induce/enhance LG regeneration. Therefore, this study aims to optimize the isolation of murine LG-MSC and analyze their secretome to investigate its potential for LG epithelial cell survival in vitro. For optimization, LG-MSC were isolated by an explant technique or cell sorting and their secretome was investigated under normal and inflammatory conditions. Results showed that the secretome of MSC had beneficial effects on the viability of ethanol-damaged LG epithelial cells. Additional, Lipocalin-2, prosaposin, ras GTPase-activating protein-binding protein 1 (Rac1) and signal transducer and activator of transcription 1 (STAT1), proteins that were up-regulated under inflammatory conditions, further improved the cell survival of ethanol-damaged LG epithelial cells. Interestingly, recovery of cell viability was highest, when the cells were incubated with STAT1. Summarizing, this study identified promising proteins for further studies on LG regeneration. Unlabelled Image • Lacrimal gland specific MSC could be obtained by explant culture and FACS. • MSC conditioned medium contained proteins with therapeutic effects. • Lipocalin-2, Prosaposin, Rac1 and STAT1 were found in MSC conditioned medium. • Lipocalin-2, Prosaposin, Rac1, STAT1 enhanced the survival of epithelial cells. • STAT1 enhanced lacrimal gland epithelial cell survival the most. [ABSTRACT FROM AUTHOR]

Background: Thermophilic bacilli in both aerobic or facultative anaerobic forms have been isolated for over a hundred years from different mesophilic or thermophilic environments as they are potential source of bioactive secondary metabolites. But the taxonomic resolution in the Bacillus genus at species or at strain level is very challenging for the insufficient divergence of the 16S rRNA genes. One such recurring problem is among Bacillus anthracis , B . cereus and B . thuringiensis . The disease-causing B. anthracis strains have their characteristic virulence factors coded in two well-known plasmids, namely pXO1 (toxin genes) and pXO2 (capsule genes)., Objective: The present study aimed at the molecular and genomic characterization of a recently reported thermophilic and environmental isolate of B. anthracis , strain PFAB2., Methods: We performed comparative genomics between the PFAB2 genome and different strains of B. anthracis , along with closely related B. cereus strains., Results: The pangenomic analysis suggests that the PFAB2 genome harbors no complete prophage genes. Cluster analysis of Bray-Kurtis similarity resemblance matrix revealed that gene content of PFAB2 is more closely related to other environmental strains of B. anthracis . The secretome analysis and the in vitro and in vivo pathogenesis experiments corroborate the avirulent phenotype of this strain. The most probable explanation for this phenotype is the apparent absence of plasmids harboring genes for capsule biosynthesis and toxins secretion in the draft genome. Additional features of PFAB2 are good spore-forming and germinating capabilities and rapid replication ability., Conclusion: The high replication rate in a wide range of temperatures and culture media, the non-pathogenicity, the good spore forming capability and its genomic similarity to the Ames strain together make PFAB2 an interesting model strain for the study of the pathogenic evolution of B . anthracis ., (© 2019 Bentham Science Publishers.)

Cell microenvironment consists of various types of cells which communicate with each other by vast number of secreted proteins. An unbiased profiling of these secreted proteins on a global scale is often critical for understanding the intercellular signaling in an autocrine or paracrine manner. Mass spectrometry-based proteomics has become one of the most popular technology for characterization of the secreted proteins. In this chapter, we discuss the standard workflow for secreted proteins characterization, including harvesting secreted proteins from conditioned media, digesting the obtained proteins, liquid chromatography-mass spectrometry analysis, and downstream data analysis.

Multi-drug resistance (MDR) remains a great obstacle to effective chemotherapy for gastric cancer. A number of secreted glycoproteins have been reported to be involved in the development of MDR in gastric cancer. However, whether glycosylation of secreted glycoproteins changes during MDR of gastric cancer is unclear. Our present work manifested that N-glycosites and site-specific glycoforms of secreted proteins in drug-resistant cell lines were distinctly different from those in the parental cell line for the first time. Further characterization highlighted the significance of some aberrantly glycosylated secretory proteins in MDR, suggesting that manipulating the glycosylation of specific glycoproteins could be a potential target for overcoming multi-drug resistance in gastric cancer., Competing Interests: The authors declare no Conflicts of Interest.

Liver cancer typically occurs with a background of chronic fibrosis, characterized by the accumulation of myofibroblast-like cells. We performed 2D-PAGE-based comparative analyses with the aim to identify proteins expressed in human hepatocellular carcinoma (HCC) tissue but not in neighboring healthy liver tissue, and to make out which cell types are responsible for the expression of proteins most characteristic for HCC. LC-MS/MS analysis of the most striking spots identified proteins that were mainly related to myofibroblast-like cells. To gain more insights into the role of these cells in their contribution to HCC, we isolated myofibroblast-like cells as well as hepatocytes, both derived from HCC tissues, and subjected them to proteome profiling based on shotgun experiments. Comparative analysis, also referring to proteome profiles of other cell types previously investigated by us, pointed again to a marked contribution of myofibroblast-like cells to HCC. Intriguingly, secretome analysis of these cells identified several growth factors that may act as tumor promoters and several proteins that have been described as potential biomarkers for HCC including dickkopf-1, connective tissue growth factor, and CXCL1. Other biomarker candidates presently identified in the secretome of myofibroblasts, including lipocalin-1 and pappalysin-1, may be selected for future clinical validation. The identification of myofibroblast-like cells as important source of tumor-promoters may open new avenues to therapeutic intervention by targeting these stroma cells in addition to the cancer cells., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)