We employed laser microdissection to selectively harvest histology-resolved tumors and stroma from formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. Peptide digests from the LMD-enriched HNSCC tissue were analyzed by quantitative mass-spectrometry-based proteomics using a data independent analysis approach. In paired samples, excellent proteome coverage was achieved, having quantified 6668 proteins with a median quantitative coefficient of variation under 10%. Significant differences in relevant functional pathways between the tumor and the stroma regions were observed. Extracellular matrix (ECM) was identified as a major component enriched in the stroma, including many cancer-associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from a very low starting input in a scalable format. Correlating such results with clinical features or disease progression will likely enable the identification of novel targets for disease classification and interventions.

High throughput proteomics is gaining increasing traction as it facilitates screening of large sample cohorts required in clinical research and systems biology studies. Recent developments in mass spectrometry-based proteomics resulted in improved hardware and software providing deep proteome coverage, robustness, and scale accessible to a wide range of laboratories. Here, we benchmark dia-PASEF, a data-independent acquisition scheme that integrates trapped ion mobility with high scan speed, with a high-resolution time-of-flight mass analyzer (timsTOF HT) for the deep proteome analysis of a human cell line applying short 5-minute gradients. To show intra-and interlaboratory reproducibility, we performed a multi-laboratory study including 11 sites. We demonstrate that on average 7,072 protein groups and 99,835 peptides were identified in human chronic myelogenous leukemia cells on the timsTOF HT with low variation. Our results underline that dia-PASEF data acquisition combined with reproducible chromatography enables high robustness and data consistency across instruments and laboratories, which is a prerequisite for translational biomedical insights. ### Competing Interest Statement SKS, JRK, CM, PS, LA, RS, GK, TK, LH, DA, TM, and DH are employees of Bruker, manufacturer of the instrumentation used in this work. SJ is an employee of PreOmics GmbH. All remaining authors declare no further competing interests.

N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.

N-glycoproteomic analyses provide valuable resources for investigation of cancer mechanisms, biomarkers, and therapeutic targets. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 display hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells.

Introduction: Probiotics have variable microbiological composition, which results in distinct by-product profiles and consequent beneficial probiotic action for animals. Objective: it aimed to characterize the microorganisms present in kefir samples and those stabilized in commercial rations for consumption in fish farming and to evaluate the result in a zootechnical profile. Methodology: The microbial quantification process of kefir grown in milk, brown sugar and feed mixed with grains and fermented solutions was carried out by decimal dilutions, plated in De Man Rogosa and Sharpe (MRS) medium for Lactobacillus, "Brain Heart Infusion", (BHI) for global bacterial count, glucose Sabouraud for yeasts and the NMP technique for coliforms at 35ºC and 45ºC and EMB for Escherichia coli. For the identification of the genus and species, mass spectrometry (Mald Tof) was used. Treatments with milk, grains and water with brown sugar were used, with kefir in the dose of 4% of the different forms for each kilogram of feed. Results: These treatments indicate changes in the results that are statistically significant in relation to the total size, partial size and width. As for weight, it showed statistically significant results only after 15 days of treatment. Conclusion: The experimental conditions used and the proportion of 4% of the feed did not influence the parameters of the zootechnical profile of the animals.

SARS-CoV-2 infection poses a global health crisis. In parallel with the ongoing world effort to identify therapeutic solutions, there is a critical need for improvement in the prognosis of COVID-19. Here, we report plasma proteome fingerprinting that predict high (hospitalized) and low-risk (outpatients) cases of COVID-19 identified by a platform that combines machine learning with matrix-assisted laser desorption ionization mass spectrometry analysis. Sample preparation, MS, and data analysis parameters were optimized to achieve an overall accuracy of 92%, sensitivity of 93%, and specificity of 92% in dataset without feature selection. We identified two distinct regions in the MALDI-TOF profile belonging to the same proteoforms. A combination of SDS-PAGE and quantitative bottom-up proteomic analysis allowed the identification of intact and truncated forms of serum amyloid A-1 and A-2 proteins, both already described as biomarkers for viral infections in the acute phase. Unbiased discrimination of high- and low-risk COVID-19 patients using a technology that is currently in clinical use may have a prompt application in the noninvasive prognosis of COVID-19. Further validation will consolidate its clinical utility.

Ethnopharmacological relevance: Species Cissus gongylodes has been used in the traditional medicine in South America and India for the treatment of urolithiasis, biliary and inflammatory problems without any scientific evidence. Aim of the study: This work was developed to investigate for the first time the anti-inflammatory and antiurolithiatic activities of leaf decoction of C. gongylodes. Materials and methods: Decoction was subjected to anti-inflammatory evaluation by the in vivo assay of ear oedema and quantification of the main mediators of inflammation PGE(2) and LTB4, and the cytokine TNF-alpha. The decoction's anti-urolithiatic activity was determined by different in vitro assays to evaluate the inhibition and dissolution of the most prevalent types of kidney stones: calcium oxalate (CaOx) and struvite. Diffusion in gel technique and fresh urine of a patient with renal stone were used to investigate the inhibition and dissolution of CaOx, respectively, and the single diffusion gel growth technique was used to evaluate the inhibition and dissolution of struvite crystals. The decoction was chemically characterized by UHPLC-ESI-HRMS analysis. Results: Decoction showed in vivo anti-inflammatory activity by potent decreasing the level of both the main mediators of inflammation and dose-dependent in vitro anti-urolithiatic action by inhibition and dissolution of both type of crystals, CaOx and struvite. Conclusions: Results obtained corroborate the reports of the traditional use of the decoction of Cissus gongylodes. Besides, it showed multi-target mechanisms actions, inhibition of the main inflammatory pathways, and inhibition/dissolution of the most prevalent types of crystals on urolithiasis. These actions make the decoction a promissory source to the development of new and more efficient drugs.

This work aimed includes performing the sclerotia chemical profile and evaluates their biological effects on mutagenesis, oxidative stress, cancer, and malaria. A chemical profile was determined by ultraperformance liquid chromatography mass spectrometry (UHPLC-HRMS) analysis dereplicating norditerpenoid dilactone, sclerolide, and other compounds. The GI50 values to cancer cells (19.8 to 277.6 µg/mL) were higher than normal (16.05 µg/mL), meaning high cytotoxicity. Regarding the oxidative stress, the results showed that the all AcOET fraction concentrations tested on IMR90 noncancer cell increased reactive oxygen species (ROS) production in more intense way (by fivefold) than in tested cancer cells. The in vivo study showed an increase of the following biomarkers (by 296.00%): % DNA in comet tail in peripheral blood and liver cells; micronucleated erythrocytes and colon cells and lipid serum peroxidation. These results indicate the sclerotia as genotoxic and mutagenic agent and its contamination may lead to fungal toxic effects with a risk to human health.

Absorption and accumulation of bioavailable cyanobacterial metabolites (including cyanotoxins) are likely in fish after senescence and the rupturing of cells during bloom episodes. We determined the toxicity of cyanopeptides identified from two strains of Microcystis (M. panniformis MIRS-04 and M. aeruginosa NPDC-01) in a freshwater tropical fish, Astyanax altiparanae (yellowtail tetra, lambari). Aqueous extracts of both Microcystis strains were prepared in order to simulate realistic fish exposure to these substances in a freshwater environment. Both strains were selected because previous assays evidenced the presence of microcystins (MCs) in MIRS-04 and lack of cyanotoxins in NPDC-01. Identification of cyanobacterial secondary metabolites was performed by LC-HR-QTOF-MS and quantification of the MC-LR was carried out by LC-QqQ-MS/MS. MIRS-04 produces the MCs MC-LR, MC-LY and MC-HilR as well as micropeptins B, 973, 959 and k139. NPCD-01 biosynthetizes microginins FR1, FR2/FR4 and SD-755, but does not produce MCs. Larval fish survival and changes in morphology were assessed for 96 h exposure to aqueous extracts of both strains at environmentally relevant concentrations from 0.1 to 0.5 mg (dry weight)/mL, corresponding to 0.15 to 0.74 μg/mL of MC-LR (considering dried amounts of MIRS-04 for comparison). Fish mortality increased with concentration and time of exposure for both strains of Microcystis. The frequencies of morphological abnormalities increased with concentration in both strains, and included abdominal and pericardial oedema, and spinal curvature. Results demonstrate that toxicity was not solely caused by MCs, other classes of cyanobacterial secondary metabolites contributed to the observed toxicity.

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family.In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M-1 cm(-1). MpBBI inhibits strongly trypsin with K (i) in the 10(-10) M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

Mass spectrometry imaging (MSI) of animal tissues has become an important tool for in situ molecular analyses and biomarker studies in several clinical areas, but there are few applications in parasitological studies. Leishmaniasis is a neglected tropical disease, and experimental mouse models have been essential to evaluate pathological and immunological processes and to develop diagnostic methods. Herein we have employed MALDI MSI to examine peptides and low molecular weight proteins (2 to 20 kDa) differentially expressed in the liver during visceral leishmaniasis in mice models. We analyzed liver sections of Balb/c mice infected with Leishmania infantum using the SCiLS Lab software for statistical analysis, which facilitated data interpretation and thus highlighted several key proteins and/or peptides. We proposed a decision tree classification for visceral leishmaniasis with distinct phases of the disease, which are named here as healthy, acute infection and chronic infection. Among others, the ion of m/z 4963 was the most important to identify acute infection and was tentatively identified as Thymosin β4. This peptide was previously established as a recovery factor in the human liver and might participate in the response of mice to Leishmania infection. This preliminary investigation shows the potential of MALDI MSI to complement classical compound selective imaging techniques and to explore new features not yet recognized by these approaches.

Candida haemulonii is now considered a complex of two species and one variety: C. haemulonii sensu stricto, Candida duobushaemulonii and the variety C. haemulonii var. vulnera. Identification (ID) of these species is relevant for epidemiological purposes and for therapeutic management, but the different phenotypic commercial systems are unable to provide correct species ID for these emergent pathogens. Hence, we evaluated the MALDI-TOF MS performance for the ID of C. haemulonii species, analyzing isolates/strains of C. haemulonii complex species, Candida pseudohaemulonii and Candida auris by two commercial platforms, their databases and softwares. To differentiate C. haemulonii sensu sctricto from the variety vulnera, we used the ClinProToolsTM models and a single-peak analysis with the software FlexAnalysisTM. The BiotyperTM database gave 100% correct species ID for C. haemulonii sensu stricto, C. pseudohaemulonii and C. auris, with 69% of correct species ID for C. duobushaemulonii. Vitek MSTM IVD database gave 100% correct species ID for C. haemulonii sensu stricto, misidentifying all C. duobushaemulonii and C. pseudohaemulonii as C. haemulonii, being unable to identify C. auris. The Vitek MSTM RUO database needed to be upgraded with in-house SuperSpectra to discriminate C. haemulonii sensu stricto, C. duobushaemulonii, C. pseudohaemulonii, and C. auris strains/isolates. The generic algorithm model from ClinProToolsTM software showed recognition capability of 100% and cross validation of 98.02% for the discrimination of C. haemulonii sensu stricto from the variety vulnera. Single-peak analysis showed that the peaks 5670, 6878, or 13750 m/z can distinguish C. haemulonii sensu stricto from the variety vulnera.

The human tissue kallikreins (KLK1–KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.

Candida haemulonli is now considered a complex of two species and one variety: C. haemulonii sensu strict, Candida duobushaemulonii and the variety C. haemulonii var. vulnera. Identification (ID) of these species is relevant for epidemiological purposes and for therapeutic management, but the different phenotypic commercial systems are unable to provide correct species ID for these emergent pathogens. Hence, we evaluated the MALDI-TOF MS performance for the ID of C. haemulonli species, analyzing isolates/strains of C. haemulonli complex species, Candida pseudohaemulonii and Candida auris by two commercial platforms, their databases and softwares. To differentiate C. haemulonli sensu sctricto from the variety vulnera, we used the ClinProTools (TM) models and a single-peak analysis with the software FlexAnalysis (TM). The Biotyper (TM) database gave 100% correct species ID for C. haemulonii sensu strict, C. pseudohaemulonii and C. auris, with 69% of correct species ID for C. duobushaemulonii. Vitek MS (TM) IVD database gave 100% correct species ID for C. haemulonii sensu stricto, misidentifying all C. duobushaemulonii and C. pseudohaemulonii as C. haemulonii, being unable to identify C. auris. The Vitek MS (TM) RUO database needed to be upgraded with in-house SuperSpectra to discriminate C. haemulonii sensu stricto, C. duobushaemulonii, C. pseudohaemulonii, and C. auris strains/isolates. The generic algorithm model from ClinProTools (TM) software showed recognition capability of 100% and cross validation of 98.02% for the discrimination of C. haemulonli sensu stricto from the variety vulnera. Single-peak analysis showed that the peaks 5670, 6878, or 13750 m/z can distinguish C. haemulonli sensu stricto from the variety vulnera.

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50 degrees C and 8.5, respectively. The enzyme was thermostable until 55 degrees C and its activity was strongly inhibited by the classic trypsin inhibitors N-rho-tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using N-alpha-benzoyl-DL-arginine rho-nitroanilide hydrochloride (BApNA) as substrate, the apparent k(m) value of the purified trypsin was 0.38 mM, k(cat) value was 3.14s(-1), and k(cat)/k(m) was 8.26s(-1) mM(-1). The catalytic proficiency of the purified enzyme was 2.75 x 10(12) M-1 showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93 kcal mol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. (C) 2016 Elsevier B.V. All rights reserved.

The human tissue kallik every tissue of the hum events. Some kallikreins ple, epithelial disorders our group have made ef molecules, and all the in tion. Here we describe f against KLK1 and KLK2. of exosite-type inhibitor specific substrate. The family of the tissue kallikrein and kallikrein-related pept dases (KLKs), encoded by the largest contiguous cluster of proteas genes in the human genome, consists of 15 homologous, single chain, secreted serine proteases. They possess at least 30 sequence identity and the mature proteins are approximately o 25–30 kDa, with similar structural characteristics and trypsin or chymotrypsin-like specificities. KLKs are synthesized a zymogens, secreted and then proteolytically processed to generat the active form. Proteolytic activity of KLKs is also regulated b complex formation with endogenous plasma and tissue inhibitor such as a2-macroglobulin and serpins, and/or inactivation throug self-fragmentation. All KLKs have different expression pattern and physiological roles in different tissues with diverse function and substrates. Based on KLK1–kininogen interaction, the crysta structures of KLKs complexes and the functional data define thre distinct regions in the protease domain as important to recognitio http://dx.doi.org/10.1016/j.bmcl.2016.01.039 0960-894X/ 2016 Published by Elsevier Ltd. ⇑ Corresponding author. Tel.: +55 11 2320 6238. E-mail address: luciano.puzer@ufabc.edu.br (L. Puzer). y Both authors contributed equally to this work. 1

The present study describes the synthesis of non-isocyanate poly(azo-carbonate-urethane)s and characterization by FT-IR, NMR, MALDI, GPC and TG. Biocompatible properties were investigated by the liver, kidney and colon histological analyses.

This article reports the three biflavonoids isolated from the fruit pericarp of Garcinia brasiliensis Mart. (Clusiaceae): Morelloflavone-4```-glycoside (compound 1), (±)-Fukugiside (compound 2), and Morelloflavone (compound 3). Structural modifications by acylation and alkylation reactions were performed on the natural biflavonoid (±) morelloflavone to obtain three semisynthetic compounds: Morelloflavone-7,4`,7``,3```,4```-penta–O–acetyl (compound 4), Morelloflavone-7,4`,7``,3```,4```-penta-O-methyl (compound 5), and Morelloflavone-7,4`,7``,3```,4```-penta-O-butanoyl (compound 6). The inhibitory effects of these naturally isolated biflavonoid-type compounds and three semisynthetic derivatives on the activity of the cysteine proteases papain and cruzain, and on the serine protease trypsin were investigated. The potential inhibitory IC50 of natural bioflavonoids compounds 1, 2, and 3 were 11.0 ± 3.0, 23.0 ± 4.0, and 10.5 ± 0.3 µM, respectively, for papain; 0.86 ± 0.12, 106 ± 7, and 3.8 ± 0.1, 50 ± 2, 119.5 ± 5, and 9.6 ± 1.0 µM, respectively, for cruzain. On the other hand, the semisynthetic biflavonoids compounds 4, 5, and 6 were more efficient in the inhibition of enzyme activity with IC50 values 0.60 ± 0.02 µM (papain) for biflavonoids compound 4, 1.64 ± 0.11 µM (trypsin) for biflavonoids compound 5, and 8.1 ± 0.6 µM (cruzain) for biflavonoids compound 6. Compound 4 is more active owing to the carbonyl group in the structure; perhaps, this modification could favor a higher nucleophilic attack by serine and cysteine proteases. However, the semisynthetic compound 5 (IC50 = 15.4 ± 0.7 µM for papain), which has no carbonyl group in structure, was less active in the inhibition. Interestingly, structure–activity relationships (SARs) were confirmed by flexible docking simulations. Likewise, the potential usefulness of natural compound 1 as an antioxidant compound was strengthened by our results concerning the antiproteolytic activity. Key words: Garcinia brasiliensis, biflavonoids, proteases, antiproteolytic activity.