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Background: Prostate Cancer (PCa) represents the second leading cause of cancer-related death in men. Prostate-specific antigen (PSA) is currently used for PCa screening but because of its low specificity and sensitivity new diagnostic tools are required. Methods: In this work, 32 formerly N-glycosylated peptides were quantified by PRM in 163 serum samples (79 from PCa patients and 84 from individuals affected by benign prostatic hyperplasia (BPH)) in two technical replicates. These potential biomarker candidates were prioritized through a multi-stage biomarker discovery pipeline articulated in: discovery, LC-PRM assay development and verification phases. Because of the well-established involvement of glycoproteins in cancer development and progression, the proteomic analysis was focused on glycoproteins enriched by TiO2 strategy. Results: Machine learning algorithms have been applied to the combined matrix comprising proteomic and clinical variables, resulting in a predictive model based on six proteomic variables (LAMB1, LAMP2, LUM, TFRC, NCAM1, GPLD1) and five clinical variables (prostate dimension, proPSA, free-PSA, total-PSA, free/total-PSA). Conclusions: A predictive model combining proteomic and clinical variables able to distinguish PCa from BPH with an AUC of 0.82 was developed. This model outperformed PSA alone which, on the same sample set, was able to discriminate PCa from BPH with an AUC of 0.74. Data are available via ProteomeXchange with identifier PXD035935.

Atopic Dermatitis (AD) is a common inflammatory skin disease characterized by skin and systemic inflammation, and barrier dysfunction. Herein, we investigate the proteomic profile of AD skin barrier to identify a unique signature with an easy-performed sampling approach. We enrolled 8 moderate-to-severe AD patients and 8 age- and gender-matched healthy controls. Swabs were obtained from non-lesional skin of retroauricular area and antecubital fold. Peptide mixtures obtained through protein precipitation and in-solution digestion were analysed using NanoLC-MS/MS. Label-free quantification and statistical analysis were conducted in MaxQuant and Perseus. Bioinformatics analysis was performed using Gene Ontology and STRING. We identified 908 proteins and 35 differentially expressed proteins were selected (fold change 2, FDR

The insulin receptor (IR) gene undergoes differential splicing generating two IR isoforms, IR-A and IR-B. The roles of IR-A in cancer and of IR-B in metabolic regulation are well known but the molecular mechanisms responsible for their different biological effects are poorly understood. We aimed to identify different or similar protein substrates and signaling linked to each IR isoforms. We employed mouse fibroblasts lacking IGF1R gene and expressing exclusively either IR-A or IR-B. By proteomic analysis a total of 2530 proteins were identified and quantified. Proteins and pathways mostly associated with insulin-activated IR-A were involved in cancer, stemness and interferon signaling. Instead, proteins and pathways associated with insulin-stimulated IR-B-expressing cells were mostly involved in metabolic or tumor suppressive functions. These results show that IR-A and IR-B recruit partially different multiprotein complexes in response to insulin, suggesting partially different functions of IR isoforms in physiology and in disease.

The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF-β ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra-epithelial non-canonical Activin A signaling inhibits cell proliferation in a Smad-independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non-canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors.

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

Although the concepts of somatic cell reprogramming and human‐induced pluripotent stem cells (hiPSCs) generation have undergone several analyses to validate the usefulness of these cells in research and clinic, it remains still controversial whether the hiPSCs are equivalent to human embryonic stem cells (hESCs), pointing to the need of further characterization for a more comprehensive understanding of pluripotency. Most of the experimental evidence comes from the transcriptome analysis, while a little is available on protein data, and even less is known about the post‐translational modifications. Here, we report a combined strategy of mass spectrometry and gene expression profiling for proteogenomic analysis of reprogrammed and embryonic stem cells. The data obtained through this integrated, multi‐“omics” approach indicate that a small, but still significant, number of distinct pathways is enriched in reprogrammed versus embryonic stem cells, supporting the view that pluripotency is an extremely complex, multifaceted phenomenon, with peculiarities that are characteristic of each cell type.

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.

Aberrant glycosylation has long been known to be associated with cancer, since it is involved in key mechanisms such as tumour onset, development and progression. This review will focus on protein glycosylation studies in cells, tissue, urine and serum in the context of prostate cancer. A dedicated section will cover the glycoforms of prostate specific antigen, the molecule that, despite some important limitations, is routinely tested for helping prostate cancer diagnosis. Our aim is to provide readers with an overview of mass spectrometry-based glycoproteomics of prostate cancer. From this perspective, the first part of this review will illustrate the main strategies for glycopeptide enrichment and mass spectrometric analysis. The molecular information obtained by glycoproteomic analysis performed by mass spectrometry has led to new insights into the mechanism linking aberrant glycosylation to cancer cell proliferation, migration and immunoescape.

This contribution is the result of our progressive engagement to develop and to apply a top-down liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (LC-MALDI-TOF) analysis for the histone post-translational modifications (PTMs) and variants characterization, mainly in order to provide comprehensive and fast results. The histone post-translational modifications and the differential expression of the histone variants play an essential role both in the DNA packaging mechanism in chromosomes and in the regulation of gene expression in different cellular processes, also in response to molecular agents of environmental origin. This epigenetic mechanism is widely studied in different field such as cellular differentiation, development and in the understanding of mechanisms underlying diseases. The characterization of histone PTMs has traditionally performed by antibodies-based assay, but immunological methods have significant limits, and today systems that use mass spectrometry are increasingly employed. We evaluated an in-source decay (ISD) analysis for the histone investigation on human lymphoblastoid cells, and by this approach, we were able to identify and quantify several PTMs such as the di-methylation in the lysine 20 and the acetylation in the lysine 16 in H4 and the mono-methylation, di-methylation and trimethylations at K9 of the histone H3.1. Moreover, we detected and quantified in the same H2B spectrum the prevalent H2B 1C/2E type but also the minor H2B 1D, 1M and 1B/1L/1N, 1O/2F, 1J/1K variants. In this work, we show that MALDI-ISD represents an excellent methodology to obtain global information on histone PTMs and variants from cells in culture, with rapidity and simplicity of execution. Finally, this is a useful approach to get label-free relative quantitative data of histone variants and PTMs.

Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.

MS-based proteomics is expanding its role as a routine tool for biological discovery. Nevertheless, the task of accurately and precisely quantifying thousands of analytes in a single experiment remains challenging. In this study, the diagnostic accuracy of three popular data-dependent methods for protein relative quantification (label-free [LF], dimethyl labelling [DML] and tandem mass tags [TMT]) has been assessed using a mixed species proteome (three species) and five experimental replicates per condition. Data were produced using a quadrupole-Orbitrap mass spectrometer and analysed using a single platform (the MaxQuant/Perseus software suite). The whole comparative analysis was repeated three times over a period of 6 months, in order to assess the consistency of the reported findings. As expected, label-based methods reproducibly provided a lower false positives rate, whereas TMT and LF performed similarly, and significantly better than DML, in terms of proteome coverage using the same instrument time. Although parameters like proteome coverage and precision were consistent in between replicates, other parameters like sensitivity, intended as the capacity of correctly classifying true positives (regulated proteins), were found to be less reproducible, especially at challenging fold-changes (1.5). Collectively, data suggest that an increased interest in data reproducibility would be desirable in the quantitative proteomics field.

SUMMARYHuman heart regeneration is one of the most critical unmet clinical needs at a global level1. Muscular regeneration is hampered both by the limited renewing capacity of adult cardiomyocytes2-4 and the onset of cardiac fibrosis5,6, resulting in reduced compliance of the tissue. Primate have proven to be ideal models for pluripotent stem cell strategies for heart regeneration, but unravelling specific approaches to drive cell migration to the site of injury and inhibition of subsequent fibrosis have been elusive. Herein, by combining human cardiac progenitor lineage tracing and single-cell transcriptomics in injured non-human primate heart bio-mimics, we uncover the coordinated muscular regeneration of the primate heart via directed migration of human ventricular progenitors to sites of injury, subsequent fibroblast repulsion targeting fibrosis, and ultimate functional replacement of damaged cardiac muscle by differentiation and electromechanical integration. Single-cell RNAseq captured distinct modes of action, uncovering chemoattraction mediated by CXCL12/CXCR4 signalling and fibroblast repulsion regulated by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Moreover, transplantation of human cardiac progenitors into hypo-immunogenic CAG-LEA29Y transgenic porcine hearts following injury proved their chemotactic response and their ability to generate a remuscularized scar without the risk of arrhythmogenesis in vivo. Our study demonstrates that inherent developmental programs within cardiac progenitors are sequentially activated in the context of disease, allowing the cells to sense and counteract injury. As such, they may represent an ideal bio-therapeutic for functional heart rejuvenation.

21q22.2-3 deletion is the most common copy number alteration in prostate cancer (PCa). The genomic rearrangement results in the androgen-dependent de novo expression of ETS-related gene (ERG) in prostate cancer cells, a condition promoting tumor progression to advanced stages of the disease. Interestingly, ERG expression characterizes 5-30% of tumor precursor lesions - High Grade Prostatic Intraepithelial Neoplasia (HGPIN) - where its role remains unclear. Here, by combining organoids technology with Click-chemistry coupled Mass Spectrometry, we demonstrate a prominent role of ERG in remodeling the protein secretome of prostate progenitors. Functionally, by lowering autocrine Wnt-4 signaling, ERG represses canonical Wnt pathway in prostate progenitors, and, in turn, promotes the accumulation of DNA double strand breaks via Gsk3β-dependent degradation of the tumor suppressor Nkx3.1. On the other hand, by shaping extracellular paracrine signals, ERG strengthens the pro-oxidative transcriptional signature of inflammatory macrophages, which we demonstrate to infiltrate pre-malignant ERG positive prostate lesions. These findings highlight previously unrecognized functions of ERG in undermining adult prostate progenitor niche through cell autonomous and non-autonomous mechanisms. Overall, by supporting the survival and proliferation of prostate progenitors in the absence of growth stimuli and promoting the accumulation of DNA damage through destabilization of Nkx3.1, ERG could orchestrate the prelude to neoplastic transformation.

Glycopeptide enrichment can be a strategy to allow the detection of peptides belonging to low abundance proteins in complex matrixes such as blood serum or plasma. Though several glycopeptide enrichment protocols have shown excellent sensitivities in this respect, few reports have demonstrated the applicability of these methods to relatively large sample cohorts. In this work, a fast protocol based on TiO2 enrichment and highly sensitive mass spectrometric analysis by Selected Reaction Monitoring (SRM) has been applied to a cohort of serum samples from prostate cancer and benign prostatic hyperplasia patients in order to detect low abundance proteins in a single LC-MS/MS analysis in nanoscale format, without immunodepletion or peptide fractionation. A peptide library of over 700 formerly N-glycosylated peptides was created by data dependent analysis. Then, 16 medium to low abundance proteins were selected for detection by single injection LC-MS/MS based on selected-reaction monitoring. Results demonstrated the consistent detection of the low-level proteins under investigation. Following label-free quantification, four proteins (Adipocyte plasma membrane-associated protein, Periostin, Cathepsin D and Lysosome-associated membrane glycoprotein 2) were found significantly increased in prostate cancer sera compared to the control group. Graphical abstract ᅟ.

[Image: see text] A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10111a–g) was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOX-2 isozyme, indicating that 2,3,4-trisubstituted 2,3-dihydrothiazoles are a promising scaffold for the inhibition of hCA isozymes and of hCOX-2 enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enzyme families. EMAC10111g resulted as the best performing compound toward both enzyme families and exhibited preferential activity toward hCA XII and hCOX-2 isozymes.

A series of N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids (EMAC8000a–m) was designed and synthesized with the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumor associated hCA XII. Moreover, resolution of EMAC8000d racemic mixture led to the isolation of the levorotatory eutomer exhibiting an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine activity and selectivity toward hCA isozymes. These results can provide further indication for the design and optimization of selective hCA inhibitors.

Neurons are extraordinarily large and highly polarized cells that require rapid and efficient communication between cell bodies and axons over long distances. In peripheral neurons, transcripts are transported along axons to growth cones, where they are rapidly translated in response to extrinsic signals. While studying Tp53inp2, a transcript highly expressed and enriched in sympathetic neuron axons, we unexpectedly discovered that Tp53inp2 is not translated. Instead, the transcript supports axon growth in a coding-independent manner. Increasing evidence indicates that mRNAs may function independently of their coding capacity; for example, acting as a scaffold for functionally related proteins. The Tp53inp2 transcript interacts with the nerve growth factor (NGF) receptor TrkA, regulating TrkA endocytosis and signaling. Deletion of Tp53inp2 inhibits axon growth in vivo, and the defects are rescued by a non-translatable form of the transcript. Tp53inp2 is an atypical mRNA that regulates axon growth by enhancing NGF-TrkA signaling in a translation-independent manner.

Ochrobactrum anthropi is a Gram-negative rod belonging to the Brucellaceae family, able to colonize a variety of environments, and actually reported as a human opportunistic pathogen. Despite its low virulence, the bacterium causes a growing number of hospital-acquired infections mainly, but not exclusively, in immunocompromised patients. The aim of this study was to obtain an overview of the global proteome changes occurring in O. anthropi in response to different growth temperatures, in order to achieve a major understanding of the mechanisms by which the bacterium adapts to different habitats and to identify some potential virulence factors. Combined quantitative mass spectrometry-based proteomics and bioinformatics approaches were carried out on two O. anthropi strains grown at temperatures miming soil/plants habitat (25°C) and human host environment (37°C), respectively. Proteomic analysis led to the identification of over 150 differentially expressed proteins in both strains, out of over 1200 total protein identifications. Among them, proteins responsible for heat shock response (DnaK, GrpE), motility (FliC, FlgG, FlgE), and putative virulence factors (TolB) were identified. The study represents the first quantitative proteomic analysis of O. anthropi performed by high-resolution quantitative mass spectrometry.

The ability of tandem mass spectrometry to determine the primary structure of proteolytic peptides can be exploited to trace back the organisms from which the corresponding proteins were extracted. This information can be important when food products, such as protein powders, can be supplemented with lower-quality starting materials. In order to dissect the origin of proteinaceous material composing a given unknown mixture, a two-step database search strategy for bottom-up nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) data was implemented. A single nanoLC-MS/MS analysis was sufficient not only to determine the qualitative composition of the mixtures under examination, but also to assess the relative percent composition of the various proteomes, if dedicated calibration curves were previously generated. The approach of two-step database search for qualitative analysis and proteome total ion current (pTIC) calculation for quantitative analysis was applied to several binary and ternary mixtures which mimic the composition of milk replacers typically used in calf feeding.

Fhit protein is lost in cancers of most, perhaps all, cancer types; when restored, it can induce apoptosis and suppress tumorigenicity, as shown in vitro and in mouse tumor models in vivo. Following protein cross-linking and proteomics analyses, we characterized a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes the heat-shock chaperonin pair, HSP60/10, which is likely involved in importing Fhit into the mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin, in electron transport chain complex III. Overexpression of Fhit protein in Fhit-deficient cancer cells modulates the production of intracellular reactive oxygen species, causing increased ROS, following peroxide treatment, with subsequent increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape ROS overproduction and ROS-induced apoptosis, likely carrying oxidative damage. Thus, characterization of Fhit-interacting proteins has identified direct effectors of a Fhit-mediated apoptotic signal pathway that is lost in many cancers. This is of translational interest considering the very recent emphasis in a number of high-profile publications, concerning the role of oxidative phosphorylation in the treatment of human cancers, and especially cancer stem cells that rely upon oxidative phosphorylation for survival. Additionally, we have shown that cells from a Fhit-deficient lung cancer cell line, are sensitive to killing by exposure to atovaquone, thought to act as a selective oxidative phosphorylation inhibitor by targeting the CoQ10 dependence of the mitochondrial complex III, while the Fhit-expressing sister clone is resistant to this treatment.

[Image: see text] A series of isatin–dihydropyrazole hybrids have been synthesized in order to assess their potential as anticancer agents. In particular, 12 compounds were evaluated for their antiproliferative activity toward A549, IGR39, U87, MDA-MB-231, MCF-7, BT474, BxPC-3, SKOV-3, and H1299 cell lines, and human foreskin fibroblasts. Four compounds exhibited interesting antiproliferative activity and were further examined to determine their EC(50) values toward a panel of selected tumor cell lines. The best compounds were then investigated for their induced mechanism of cell death. Preliminary structure–activity relationship indicates that the presence of a substituent such as a chlorine atom or a methyl moiety in position 5 of the isatin nucleus is beneficial for the antitumor activity. EMAC4001 proved the most promising compound within the studied series with EC(50) values ranging from 0.01 to 0.38 μM.

Glycopeptide enrichment can be a strategy to allow the detection of peptides belonging to low abundance proteins in complex matrixes such as blood serum or plasma. Though several glycopeptide enrichment protocols have shown excellent sensitivities in this respect, few reports have demonstrated the applicability of these methods to relatively large sample cohorts. In this work, a fast protocol based on TiO

Neurons modulate gene expression in response to extrinsic signals to enable brain development, cognition, and learning and to process stimuli that regulate systemic physiological functions. This signal-to-gene communication is facilitated by posttranslational modifications such as S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, but few other nuclear targets of S-nitrosylation have been identified to date. We used S-nitrosothiol resin-assisted capture on NO donor-treated nuclear extracts from rat cortical neurons and identified 614 S-nitrosylated nuclear proteins. Of these, 131 proteins have not previously been shown to be S-nitrosylated in any system, and 555 are previously unidentified targets of S-nitrosylation in neurons. The sites of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines were found at 33% of these sites. In addition, lysine motifs were necessary for promoting the S-nitrosylation of HDAC2 and methyl-CpG binding protein 3 (MBD3). Moreover, S-nitrosylation of the histone-binding protein RBBP7 was necessary for dendritogenesis of cortical neurons in culture. Together, our findings characterize S-nitrosylated nuclear proteins in neurons and identify S-nitrosylation motifs that may be shared with other targets of NO signaling.

[Image: see text] A small library of psoralen carboxylic acids and their corresponding benzenesulfonamide derivatives were designed and synthesized to evaluate their activity and selectivity toward tumor associated human carbonic anhydrase (hCA) isoforms IX and XII. Both psoralen acids and sulfonamides exhibited potent inhibition of IX and XII isozymes in the nanomolar concentration range. However, psoralen acids resulted as the most selective in comparison with the corresponding benzenesulfonamide derivatives. Our data indicate that the psoralen scaffold is a promising starting point for the design of highly selective tumor associated hCA inhibitors.

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte-secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3-L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin-1 and -2, and matrix metalloproteinase-11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3-L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.