The Protein-Rich Fraction from Spirulina platensis Exerts Neuroprotection in Hemiparkinsonian Rats by Decreasing Brain Inflammatory-Related Enzymes and Glial Fibrillary Acidic Protein Expressions.

Spirulina platensis is a cyanobacterium with high protein content and presenting neuroprotective effects. Now, we studied a protein-enriched fraction (SPF), on behavior, neurochemical and immunohistochemical (IHC) assays in hemiparkinsonian rats, distributed into the groups: SO (sham-operated), 6-hydroxydopamine (6-OHDA), and 6-OHDA (treated with SPF, 5 and 10 mg/kg, p.o., 15 days). Afterward, animals were subjected to behavioral tests and euthanized, and brain areas used for neurochemical and IHC assays. SPF partly reversed the changes in the apomorphine-induced rotations, open field and forced swim tests, and also the decrease in striatal dopamine and 3,4-dihydroxyphenylacetic acid contents seen in hemiparkinsonian rats. Furthermore, SPF reduced brain oxidative stress and increased striatal expressions of tyrosine hydroxylase and dopamine transporter and significantly reduced hippocampal inducible nitric oxide synthase, cyclooxygenase-2 and glial fibrillary acidic protein expressions. The data suggest that the protein fraction from S. platensis, through its brain anti-inflammatory and antioxidative actions, exerts neuroprotective effects that could benefit patients affected by neurodegenerative diseases, like Parkinson's disease.

The essential anti-angiogenic strategies in cartilage engineering and osteoarthritic cartilage repair.

In the cartilage matrix, complex interactions occur between angiogenic and anti-angiogenic components, growth factors, and environmental stressors to maintain a proper cartilage phenotype that allows for effective load bearing and force distribution. However, as seen in both degenerative disease and tissue engineering, cartilage can lose its vascular resistance. This vascularization then leads to matrix breakdown, chondrocyte apoptosis, and ossification. Research has shown that articular cartilage inflammation leads to compromised joint function and decreased clinical potential for regeneration. Unfortunately, few articles comprehensively summarize what we have learned from previous investigations. In this review, we summarize our current understanding of the factors that stabilize chondrocytes to prevent terminal differentiation and applications of these factors to rescue the cartilage phenotype during cartilage engineering and osteoarthritis treatment. Inhibiting vascularization will allow for enhanced phenotypic stability so that we are able to develop more stable implants for cartilage repair and regeneration.

Effect of Detergents on Activity and Magnesium-Dependent Properties of Different Isoforms of Na+,K+-ATPase in the Crude Membrane Fraction of Rat Cerebral Cortex.

We studied the effect of various detergents (Tween-20, Triton X-100, and sodium deoxycholate) on activity and magnesium-dependent properties of Na+,K+-ATPase of the crude membrane fraction of rat cerebral cortex. All studied detergents significantly increased activity of the studied enzyme in a concentration-dependent manner. Sodium deoxycholate provided significantly higher values Na+,K+-ATPase activity (by ≈50%) than Triton X-100 and Tween-20. In the presence of Triton X-100, a changed pattern of the dependence of enzyme activity on the concentration of magnesium ions in the incubation solution was noted. Separate measurement of activities of Na+,K+-ATPase isoforms made it possible to assume that changes in magnesium-dependent properties are due to the predominant effect of Triton X-100 on ouabain-sensitive α2- and α3-isoforms.

Integrated analytical workflow for chromatographic profiling and metabolite annotation of a cytotoxic Phorbas amaranthus extract.

Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.

Enzyme-Digested Peptides Derived from Lates calcarifer Enhance Wound Healing after Surgical Incision in a Murine Model.

Surgical wounds are common injuries of skin and tissues and usually become a clinical problem. Until now, various synthetic and natural peptides have been widely explored as potential drug candidates for wound healing. Inhibition of the TNF-α signaling pathway and promotion of angiogenesis are suggested to be involved in their effects. Angiogenesis at the wound site is one of the essential requisites for rapid healing. In the present study, a novel peptide extract derived from the natural source Lates calcarifer, commonly known as sea bass or barramundi, was evaluated for its wound healing property. The specific acidic and enzymatic approaches were employed for producing sea bass extract containing small size peptides (molecular weight ranging from 1 kD to 5 kD). The cytotoxicity of the extract was examined in HaCaT and NIH3T3. After this, the effects of enzyme digested peptide extracts of sea bass on wound healing in mice were investigated. The peptide extracts (660 and 1320 mg/kg/day) and control protein (1320 mg/kg/day) was orally given to the wounded mice, respectively, for 12 days. The surgical method was improved by implanting a silicone ring at the wound site. The ring avoided the contracting effect in murine wounds, making it more closely related to a clinical condition. The results showed promising improvement at the wound site in mice. Sea bass peptide extracts accelerated the wound healing process and enhanced the microvessel formation at the wound site. The remarkable effects of this novel sea bass peptide extract in healing traumatic injuries revealed a new option for developing wound management.

1 H NMR-based metabolomics of paired esophageal tumor tissues and serum samples identifies specific serum biomarkers for esophageal cancer.

Serum metabolites of healthy controls and esophageal cancer (EC) patients have previously been compared to predict cancer-specific profiles. However, the association between metabolic alterations in serum samples and esophageal tissues in EC patients remains unclear. Here, we analyzed 50 pairs of EC tissues and distant noncancerous tissues, together with patient-matched serum samples, using 1 H NMR spectroscopy and pattern recognition algorithms. EC patients could be differentiated from the controls based on the metabolic profiles at tissue and serum levels. Some overlapping discriminatory metabolites, including valine, alanine, glucose, acetate, citrate, succinate and glutamate, were identified in both matrices. These results suggested deregulation of metabolic pathways, and potentially revealed the links between EC and several metabolic pathways, such as the tricarboxylic acid cycle, glutaminolysis, short-chain fatty acid metabolism, lipometabolism and pyruvate metabolism. Perturbation of the pyruvate metabolism was most strongly associated with EC progression. Consequently, an optimal serum metabolite biomarker panel comprising acetate and pyruvate was developed, as these two metabolites are involved in pyruvate metabolism, and changes in their serum levels were significantly correlated with alterations in the levels of some other esophageal tissue metabolites. In comparison with individual biomarkers, this panel exhibited better diagnostic efficiency for EC, with an AUC of 0.948 in the test set, and a good predictive ability of 82.5% in the validation set. Analysis of key genes related to pyruvate metabolism in EC patients revealed patterns corresponding to the changes in serum pyruvate and acetate levels. These correlation analyses demonstrate that there were distinct metabolic characteristics and pathway aberrations in the esophageal tumor tissue and in the serum. Changes in the serum metabolic signatures could reflect the alterations in the esophageal tumor profile, thereby emphasizing the importance of distinct serum metabolic profiles as potential noninvasive biomarkers for EC.

Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography.

BACKGROUND: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. METHODS: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. RESULTS: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 µM-4 mM and 0.2 µM-0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 µM and 0.26 µM respectively whilst their limits of quantification were 1.14 µM and 0.88 µM respectively. CONCLUSION: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Evaluation of the impact of a rat small intestinal extract on the digestion of four different functional fibers.

The degree of digestion, modulated by rat small intestinal extract on different functional fibers was investigated. In general, inulin-type fructans and fructooligosaccharides were the most resistant to the enzymatic digestion. Results evidenced the high-resistance of fructosyl-fructose bonds. This fits well with the concept of prebiotic carbohydrates. However, the mixture of melibiose, manninotriose and verbascotetraose (α-GOS) from peas, with a considerably lower molecular weight (0.6 kDa) than the fructans studied, were highly digested (61.2%). Interestingly, the Gal-(1 → 6)-Gal bonds present into the manninotriose and verbascotetraose were more prone to be hydrolyzed than Gal-(1 → 6)-Glc (melibiose). However, when melibiose was the only disaccharide present in the reaction mixture, the hydrolysis was also high (67.7%). The use of small intestinal enzymatic preparations is a realistic approximation to evaluate the digestion of different carbohydrates, thus, showing that recognized non-digestible carbohydrates can also be partially digested.

Prostaglandin E2, Produced by Mast Cells in Colon Tissues From Patients With Irritable Bowel Syndrome, Contributes to Visceral Hypersensitivity in Mice.

BACKGROUND AND AIMS: Visceral hypersensitivity is common in patients with irritable bowel syndrome (IBS). We investigated whether inflammatory molecules, such as histamine and proteases, activate prostaglandin-endoperoxide synthase 2 (also called COX2) to increase the synthesis of prostaglandin E2 (PGE2) by mast cells, which activates the receptor PTGER2 (also called EP2) in the dorsal root ganglia to promote visceral hypersensitivity. METHODS: We used an enzyme-linked immunosorbent assay to measure levels of spontaneous release of molecules from mast cells in colonic mucosa from patients with IBS with diarrhea (IBS-D; 18 women and 5 men; aged 28-60 years), healthy individuals (controls, n = 24), mice, and rats. We measured visceromotor responses to colorectal distension in rodents after intracolonic administration of colon biopsy supernatants, histamine, PGE2, a small interfering RNA against EP2, or an agonist of F2R like trypsin receptor 1 (F2RL1, also called protease-activated receptor 2 [PAR2]). We investigated the role of COX2, produced by mast cells, in mediation of visceral hypersensitivity using mice with the Y385F substitution in Ptgs2 (Ptgs2Y385F mice), mast cell-deficient (W/WV) mice, and W/WV mice given injections of mast cells derived from wild-type or Ptgs2Y385F mice. RESULTS: Colon biopsies from patients with IBS-D had increased levels of PGE2, based on enzyme-linked immunosorbent assay, and COX2 messenger RNA and protein, compared with control biopsies. Immunohistochemistry showed that most of the COX2 was in mast cells. Intracolonic infusions of rats with IBS-D biopsy supernatants generated a 3- to 4-fold increase in visceromotor responses to colorectal distension; this was associated with significant increases in PGE2, histamine, and tryptase in the colonic mucosa. These increases were prevented by a mast cell stabilizer, COX2 inhibitor, or knockdown of EP2. Intracolonic administration of supernatants from biopsies of patients with IBS-D failed to induce visceral hypersensitivity or increase the level of PGE2 in W/WV and Ptgs2Y385Fmice. Reconstitution of mast cells in W/WV mice restored the visceral hypersensitivity response. CONCLUSIONS: Abnormal synthesis of PGE2 by colonic mast cells appears to induce visceral hypersensitivity in patients with IBS-D.

Metabolic changes in mice cardiac tissue after low-dose irradiation revealed by 1H NMR spectroscopy.

Ionizing radiation may cause cardiotoxicity not only at high, but even at low (considered as harmless) doses, yet the molecular mechanisms of the heart's response to low doses are not clear. In this work, we used high-resolution nuclear magnetic resonance (NMR) spectroscopy to detect the early and late effects of radiation on the metabolism of murine hearts. The hearts of C57Bl/6NCrl female mice were irradiated in vivo with single 0.2 Gy or 2 Gy doses using 6 MV photons, then tissues were collected 48 h and 20 weeks after exposure. The most distinct changes in the profile of polar metabolites were detected 48 h after irradiation with 2 Gy, and included increased levels of pantothenate and glutamate as well as decreased levels of alanine, malonate, acetylcarnitine, glycine and adenosine. Significant effects of the 2 Gy dose were also observed 20 weeks after irradiation and included decreased levels of glutamine and acetylcarnitine when compared with age-matched controls. Moreover, several differences were observed between hearts irradiated with 2 Gy and analyzed either 48 h or 20 weeks after the exposure, which included changes in levels of acetylcarnitine, alanine, glycine, glutamate, glutamine, formate, myo-inositol and trimethylamine. No statistically significant effects induced by the 0.2 Gy dose were observed 20 weeks after irradiation. In general, radiation-affected compounds were associated with energy metabolism, fatty acid beta-oxidation, oxidative stress and damage to cell structures. At the same time, radiation-related effects were not detected at the level of tissue histology, which indicated a higher sensitivity of metabolomics-based tests for cardiac tissue response to radiation.

Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts.

Parkinson's disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies.

Dysregulation of inflammatory cytokines and inhibition of VEGFA in the human umbilical cord are associated with negative pregnancy outcomes.

INTRODUCTION: Cytokines and vascular endothelial growth factors (VEGF) are involved in all aspects of pregnancy: from placentation, through fetal development, parturition and neonatal well-being. Umbilical cord inflammatory cytokines and/or VEGF have not been well studied with respect to dysregulation associated with disorders of pregnancy or maternal/neonatal outcomes. METHODS: Here we have used multiplex ELISA to screen umbilical cord lysates (comprising cord blood, endothelia and Wharton's jelly, n = 380), for levels of IFN-γ, IL1-ß, IL-6, IL-8, IL-10, TNF-α and VEGFs A, C and D and associations with 46 ICD9/10 codes encompassing obstetric, maternal and neonatal variables. RESULTS: No significant differences were observed for IFNγ, VEGFC or VEGFD with any clinical outcomes. The cytokines IL1-ß, IL-6, IL-8, IL-10, and TNF-α showed varying levels of induction and suppression with primarily fetal-placental and neonatal complications. The largest number of significant differences between umbilical cytokines and clinical outcomes were observed for chorioamnionitis (IL1-ß, IL-6, IL-8, TNF-α), and meconium passage during birth (IL1-ß, IL-6, IL-8) where significant pro-inflammatory responses occurred and sex differences in IL-8 expression were noted. In contrast, gonococcal infection showed suppressed immune response significantly lowering IL1-ß, IL-6, IL-8, IL-10 and TNF-α. For 12/46 negative pregnancy outcomes, strong suppression of VEGFA occurred. DISCUSSION: Angiogenic and inflammatory changes in the umbilical cord could be detrimental by increasing vascular permeability in the umbilical artery or vein and/or altering vascular tone, either of which would alter blood flow affecting delivery and removal of compounds. Further elucidation of inflammatory responses in the umbilical cord may provide mechanistic understanding of adverse pregnancy outcomes.

A rapid, safe, and quantitative in vitro assay for measurement of uracil-DNA glycosylase activity.

Base excision repair (BER) is a frontline repair mechanism that operates through the G1 phase of the cell cycle, which ensures the genome integrity by repairing thousands of DNA lesions due to endogenous and exogenous agents. Its correct functioning is fundamental for cell viability and the health of the organism. Uracil is one of the most prevalent lesions that appears in DNA arising by spontaneous or enzymatic deamination of cytosine or misincorporation of the deoxyuridine 5'-triphosphate nucleotide (dUTP) in place of deoxythymidine 5'-triphosphate (dTTP) during DNA replication. In the first pathway, the uracil will preferentially pair with adenine, leading to C:G → T:A transition. When uracil in DNA arises from misincorporation of dUTP instead of dTTP, this process will result in A:U pairs. Organisms counteract the mutagenic effects of uracil in DNA using the BER repair system, which is mediated by a member of the uracil-DNA glycosylase (UDG) superfamily. Several assays evaluating the in vitro BER enzyme activity have been described so far. Some of these measure the BER activity by an oligonucleotide incision assay using radiolabeled duplex oligo. Others use circular double-stranded DNA substrates containing a defined lesion. The novelty of our method resides in its rapidity and safety (radioactive free detection) as well as in the possibility of having a reliable quantitative determination of UDG activity in both cell and tissue extracts. We also demonstrated the effectiveness of our method in assessing UDG activity in cell lines with a reduced DNA repair capacity and in different kinds of tissues. KEY MESSAGES: • Base excision repair is a fundamental repair mechanism ensuring the genome integrity. • Uracil is one of the most prevalent lesions that appears in DNA. • The mutagenic effects of uracil in DNA are mitigated by the uracil-DNA glycosylase. • Several assays evaluating the in vitro BER activity have been described so far. • A safe and quantitative assay evaluating the in vitro UDG activity is required.

In Vitro Analysis of Cartilage Regeneration Using a Collagen Type I Hydrogel (CaReS) in the Bovine Cartilage Punch Model.

OBJECTIVE: Limitations of matrix-assisted autologous chondrocyte implantation to regenerate functional hyaline cartilage demand a better understanding of the underlying cellular/molecular processes. Thus, the regenerative capacity of a clinically approved hydrogel collagen type I implant was tested in a standardized bovine cartilage punch model. METHODS: Cartilage rings (outer diameter 6 mm; inner defect diameter 2 mm) were prepared from the bovine trochlear groove. Collagen implants (± bovine chondrocytes) were placed inside the cartilage rings and cultured up to 12 weeks. Cartilage-implant constructs were analyzed by histology (hematoxylin/eosin; safranin O), immunohistology (aggrecan, collagens 1 and 2), and for protein content, RNA expression, and implant push-out force. RESULTS: Cartilage-implant constructs revealed vital morphology, preserved matrix integrity throughout culture, progressive, but slight proteoglycan loss from the "host" cartilage or its surface and decreasing proteoglycan release into the culture supernatant. In contrast, collagen 2 and 1 content of cartilage and cartilage-implant interface was approximately constant over time. Cell-free and cell-loaded implants showed (1) cell migration onto/into the implant, (2) progressive deposition of aggrecan and constant levels of collagens 1 and 2, (3) progressively increased mRNA levels for aggrecan and collagen 2, and (4) significantly augmented push-out forces over time. Cell-loaded implants displayed a significantly earlier and more long-lasting deposition of aggrecan, as well as tendentially higher push-out forces. CONCLUSION: Preserved tissue integrity and progressively increasing cartilage differentiation and push-out forces for up to 12 weeks of cultivation suggest initial cartilage regeneration and lateral bonding of the implant in this in vitro model for cartilage replacement materials.

Optimizing adipose tissue extract isolation with stirred suspension culture.

OBJECTIVES: Adherent culture which is used to collect adipose tissue extract (ATE) previously brings the problem of inhomogeneity and non-repeatability. Here we aim to extract ATE with stirred suspension culture to speed up the extraction process, stabilize the yield, and improve consistent potency metrics of ATE. MATERIALS AND METHODS: ATE was collected with adherent culture (ATE-A) and stirred suspension culture (ATE-S) separately. Protein yield and composition were detected by SDS-PAGE, while cytokines in ATE were determined with ELISA. The adipogenic and angiogenic potential of ATE were compared by western blot and qPCR. In addition, haematoxylin and eosin staining and lactate dehydrogenase (LDH) activity assays were used to analyze the cell viability of adipose tissue cultured with different methods. RESULTS: The yield of ATE-S was consistent while ATE-A varied notably. Characterization of the protein composition and exosome-like vesicles (ELVs) indicated no significant difference between ATE-S and ATE-A. The concentrations of cytokines (VEGF, bFGF, and IL-6) showed no significant difference, while IGF in ATE-S was higher than that in ATE-A. ATE-S showed upregulated adipogenic and angiogenic potential compared to ATE-A. Morever, stirred suspension culture decreased the LDH activity of ATE while increased the number of viable adipocytes and reduced adipose tissue necrosis. CONCLUSION: Compared with adherent culture, stirred suspension culture is a reliable, time- and labor-saving method to collect ATE, which might be used to improve the downstream applications of ATE.

Patient-derived frontotemporal lobar degeneration brain extracts induce formation and spreading of TDP-43 pathology in vivo.

The stereotypical distribution of TAR DNA-binding 43 protein (TDP-43) aggregates in frontotemporal lobar degeneration (FTLD-TDP) suggests that pathological TDP-43 spreads throughout the brain via cell-to-cell transmission and correlates with disease progression, but no in vivo experimental data support this hypothesis. We first develop a doxycycline-inducible cell line expressing GFP-tagged cytoplasmic TDP-43 protein (iGFP-NLSm) as a cell-based system to screen and identify seeding activity of human brain-derived pathological TDP-43 isolated from sporadic FTLD-TDP and familial cases with Granulin (FTLD-TDP-GRN) or C9orf72 repeat expansion mutations (FTLD-TDP-C9+). We demonstrate that intracerebral injections of biologically active pathogenic FTLD-TDP seeds into transgenic mice expressing cytoplasmic human TDP-43 (lines CamKIIa-hTDP-43NLSm, rNLS8, and CamKIIa-208) and non-transgenic mice led to the induction of de-novo TDP-43 pathology. Moreover, TDP-43 pathology progressively spreads throughout the brain in a time-dependent manner via the neuroanatomic connectome. Our study suggests that the progression of FTLD-TDP reflects the templated cell-to-cell transneuronal spread of pathological TDP-43.

Preparation of Joint Extracts.

Since mice are widely used to establish rheumatoid arthritis models, assessment of the pathogenesis of local arthritis is fundamental. Proteins are the most diverse group of biologically important molecules and are essential for cellular structure and function. The first step in pathogenesis-related protein analysis is joint tissue extraction. Unlike other large rodents, obtaining synovium from model mice is challenging, since it is so small and fragile. In this chapter, methods for harvesting synovium through a quadriceps approach and preparing protein extracts are introduced.

Combined proteomic and functional analysis reveals rich sources of protein diversity in skin mucus and venom from the Scorpaena plumieri fish.

The biological activities observed upon envenomation by Scorpaena plumieri could be linked to both the venom and the skin mucus. Through a proteomic/functional approach we analyzed protein composition and biological activities of the venom and skin mucus. We identified 885 proteins: 722 in the Venomous Apparatus extracts (Sp-VAe) and 391 in the Skin Mucus extract (Sp-SMe), with 494 found exclusively in Sp-VAe, being named S. plumieri Venom Proteins (Sp-VP), while 228 were found in both extracts. The majority of the many proteins identified were not directly related to the biological activities reported here. Nevertheless, some were classified as toxins/potentially interesting molecules: lectins, proteases and protease inhibitors were detected in both extracts, while the pore-forming toxin and hyaluronidase were associated with Sp-VP. Proteolytic and anti-microbial activities were linked to both extracts, while the main toxic activities - cardiovascular, inflammatory, hemolytic and nociceptive - were elicited only by Sp-VAe. Our study provided a clear picture on the composition of the skin mucus and the venom. We also show that the classic effects observed upon envenomation are produced by molecules from the venomous gland. Our results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins. SIGNIFICANCE: In this study a large number of proteins - including classical and non-classical toxins - were identified in the venomous apparatus and the skin mucus extracts of the Scorpaena plumieri fish through shotgun proteomic approach. It was shown that the toxic effects observed upon envenomation are elicited by molecules originated from the venomous gland. These results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins - so scarcely explored when compared to the venoms and bioactive components of terrestrial animals. Data are available via ProteomeXchange with identifier PXD009983.

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes.

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates' molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.