Inhibition Of Proteolysis Research Articles

Jejunal mucosa proteomics unravel metabolic adaptive processes to mild chronic heat stress in dairy cows

Climate change affects the duration and intensity of heat waves during summer months and jeopardizes animal health and welfare. High ambient temperatures cause heat stress in dairy cows resulting in a reduction of milk yield, feed intake, and alterations in gut barrier function. The objectives of this study were to investigate the mucosal amino acid, glucose and lactate metabolism, as well as the proteomic response of the small intestine in heat stressed (HS) Holstein dairy cows. Cows of the HS group (n = 5) were exposed for 4 days to 28 °C (THI = 76) in a climate chamber. Percentage decrease in daily ad libitum intake of HS cows was calculated to provide isocaloric energy intake to pair-fed control cows kept at 15 °C (THI = 60) for 4 days. The metabolite, mRNA and proteomic analyses revealed that HS induced incorrect protein folding, cellular destabilization, increased proteolytic degradation and protein kinase inhibitor activity, reduced glycolysis, and activation of NF-κB signaling, uronate cycling, pentose phosphate pathway, fatty acid and amino acid catabolism, mitochondrial respiration, ATPase activity and the antioxidative defence system. Our results highlight adaptive metabolic and immune mechanisms attempting to maintain the biological function in the small intestine of heat-stressed dairy cows.

Perineuronal Net Dynamics in the Pathophysiology of Epilepsy.

Perineuronal nets (PNNs) are condensed extracellular matrix (ECM) assemblies of polyanionic chondroitin sulfate proteoglycans, hyaluronan, and tenascins that primarily wrap around GABAergic parvalbumin (PV) interneurons. During development, PNN formation terminates the critical period of neuroplasticity, a process that can be reversed by experimental disruption of PNNs. Perineuronal nets also regulate the intrinsic properties of the enclosed PV neurons thereby maintaining their inhibitory activity. Recent studies have implicated PNNs in central nervous system diseases as well as PV neuron dysfunction; consequently, they have further been associated with altered inhibition, particularly in the genesis of epilepsy. A wide range of seizure presentations in human and rodent models exhibit ECM remodeling with PNN disruption due to elevated protease activity. Inhibition of PNN proteolysis reduces seizure activity suggesting that PNN degrading enzymes may be potential novel therapeutic targets.

Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering.

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that additional crosslinking of fibrin gels using bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) slows the degradation rate of fibrin. In this study, we aimed to further improve the longevity of these PEG-fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. Here, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

Phosphodiesterase 4 inhibition restrains muscle proteolysis in diabetic rats by activating PKA and EPAC/Akt effectors and inhibiting FoxO factors

AimThere is growing evidence about the ability of cyclic adenosine monophosphate (cAMP) signaling and nonselective phosphodiesterase (PDE) inhibitors on mitigate muscle atrophy. PDE4 accounts for the major cAMP hydrolyzing activity in skeletal muscles, therefore advances are necessary about the consequences of treatment with PDE4 inhibitors on protein breakdown in atrophied muscles. We postulated that rolipram (selective PDE4 inhibitor) may activate cAMP downstream effectors, inhibiting proteolytic systems in skeletal muscles of diabetic rats. Main methodsStreptozotocin-induced diabetic rats were treated with 2 mg/kg rolipram for 3 days. Changes in the levels of components belonging to the proteolytic machineries in soleus and extensor digitorum longus (EDL) muscles were investigated, as well as cAMP effectors. Key findingsTreatment of diabetic rats with rolipram decreased the levels of atrogin-1 and MuRF-1 in soleus and EDL, and reduced the activities of calpains and caspase-3; these findings partially explains the low ubiquitin conjugates levels and the decreased proteasome activity. The inhibition of muscle proteolysis may be occurring due to phosphorylation and inhibition of forkhead box O (FoxO) factors, probably as a consequence of the increased cAMP levels, followed by the activation of PKA and Akt effectors. Akt activation may be associated with the increased levels of exchange protein directly activated by cAMP (EPAC). As a result, rolipram treatment spared muscle mass in diabetic rats. SignificanceThe antiproteolytic responses associated with PDE4 inhibition may be helpful to motivate future investigations about the repositioning of PDE4 inhibitors for the treatment of muscle wasting conditions.

N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 Mpro Dimer

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug’s properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug’s P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.

Digestive Inflammation: Role of Proteolytic Dysregulation.

Dysregulation of the proteolytic balance is often associated with diseases. Serine proteases and matrix metalloproteases are involved in a multitude of biological processes and notably in the inflammatory response. Within the framework of digestive inflammation, several studies have stressed the role of serine proteases and matrix metalloproteases (MMPs) as key actors in its pathogenesis and pointed to the unbalance between these proteases and their respective inhibitors. Substantial efforts have been made in developing new inhibitors, some of which have reached clinical trial phases, notwithstanding that unwanted side effects remain a major issue. However, studies on the proteolytic imbalance and inhibitors conception are directed toward host serine/MMPs proteases revealing a hitherto overlooked factor, the potential contribution of their bacterial counterpart. In this review, we highlight the role of proteolytic imbalance in human digestive inflammation focusing on serine proteases and MMPs and their respective inhibitors considering both host and bacterial origin.

SARS-CoV-2 Protease-Dependent Human Cell Infection Induces Immunological Response Imbalance and Neutrophils “Proteolytic Storm”. Proteolysis Inhibitors can Prevent and Treat Covid-19

Based on Chinese CDCP report on COVID-19, 14% of patients presented severe disease and 5% critical conditions. The average case-fatality rate was 2.3%, but mortality was as high as 49% in patients with critical illness. Serious life threatening thromboembolic complications have been found in 71•4% of non survivors and micro/macro angiopathic coagulopathy has been found, at autopsy also, with highly increased neutrophil number, fibrinogen, concentrations of D-dimer and FDPs and NETs, ATIII decrease and normal number of platelets. A cytokine storm and interaction between inflammation and coagulation has been advocated as explanation of hypercoagulability. It has been shown that SARS-CoV-2 infection of alveolar cells is driven by the S-protein by engaging ACE2 and TMPRSS2 cell receptors. whose activation depends on the activity of various host proteases. Full inhibition of SARS-CoV-2 entry was observed when serine proteases inhibitor camostat mesylate was coupled with Cathepsin B/L inhibitor E-64d. In addition multiple proteases are involved in host immune response against viral invasion and immunopathology related to imbalanced immune activation. In this paper it’s hypothesised that the severity of Covid-19 is induced by recruitment of innate responder neutrophils, which release proteases and NETs inducing endothelial damage and imbalance of the four major proteolytic cascades (coagulation, complement, fibrinolysis and kallikrein) with prevalence of activators over inhibitors and consequent thrombotic complications. Platelets adhesion to damaged endothelium and vWFVIII multimers presence, due to loss of ADAMTS13, contributes to hypercoagulability state. Human plasma or serine protease inhibitors like aprotinin can help to control neutrophil induced “proteolytic storm”. The goal of this paper is to support the view that, in SARS-CoV-2 infection, proteases have a key role and exceeding imbalanced neutrophil innate “unfriendly fire” response can be identified as the trigger of a “proteolytic storm”, responsible for subsequent well known hypercoagulation and “cytokine storm” and human plasma, in adequate volumes, together with serine proteases inhibitors can be an effective therapeutic strategy.

Repurposing approved drugs for cancer therapy.

BackgroundMany drugs approved for other indications can control the growth of tumor cells and limit adverse events (AE).Data sourcesLiterature searches with keywords ‘repurposing and cancer’ books, websites: https://clinicaltrials.gov/, for drug structures: https://pubchem.ncbi.nlm.nih.gov/Areas of agreementIntroducing approved drugs, such as those developed to treat diabetes (Metformin) or inflammation (Thalidomide), identified to have cytostatic activity, can enhance chemotherapy or even replace more cytotoxic drugs. Also, anti-inflammatory compounds, cytokines and inhibitors of proteolysis can be used to control the side effects of chemo- and immuno-therapies or as second-line treatments for tumors resistant to kinase inhibitors (KI). Drugs specifically developed for cancer therapy, such as interferons (IFN), the tyrosine KI abivertinib TKI (tyrosine kinase inhibitor) and interleukin-6 (IL-6) receptor inhibitors, may help control symptoms of Covid-19.Areas of controversyBetter knowledge of mechanisms of drug activities is essential for repurposing. Chemotherapies induce ER stress and enhance mutation rates and chromosome alterations, leading to resistance that cannot always be related to mutations in the target gene. Metformin, thalidomide and cytokines (IFN, tumor necrosis factor (TNF), interleukin-2 (IL-2) and others) have pleiomorphic activities, some of which can enhance tumorigenesis. The small and fragile patient pools available for clinical trials can cloud the data on the usefulness of cotreatments.Growing pointsBetter understanding of drug metabolism and mechanisms should aid in repurposing drugs for primary, adjuvant and adjunct treatments.Areas timely for developing researchOptimizing drug combinations, reducing cytotoxicity of chemotherapeutics and controlling associated inflammation.

Elevated inflammatory gene expression in intervertebral disc tissues in mice with ADAM8 inactivated

We found ADAM8 enzymatic activity elevated in degenerative human intervertebral disc (IVD). Here, we examined the discs in ADAM8-inactivation mice that carry a mutation preventing self-activation of the enzyme. Surprisingly, elevated gene expression for inflammatory markers (Cxcl1, IL6) was observed in injured discs of ADAM8 mutant mice, along with elevated expression of type 2 collagen gene (Col2a1), compared with wild type controls. Injured annulus fibrosus of mutant and wild type mice contained a higher proportion of large collagen fibers compared with intact discs, as documented by microscopic examination under circular polarized light. In the intact IVDs, Adam8EQ mouse AF contained lower proportion of yellow (intermediate) fiber than WT mice. This suggests that ADAM8 may regulate inflammation and collagen fiber assembly. The seemingly contradictory findings of elevated inflammatory markers in mutant mice and excessive ADAM8 activity in human degenerative discs suggest that ADAM8 may interact with other enzymatic and pro-inflammatory processes needed for tissue maintenance and repair. As a future therapeutic intervention to retard intervertebral disc degeneration, partial inhibition of ADAM8 proteolysis may be more desirable than complete inactivation of this enzyme.

Antiplasmodial activity of Hyptis pectinata extract and its analog against 3D7 Plasmodium falciparum through caseinolytic protease proteolytic (ClpP) inhibition

Antiplasmodial activity of Hyptis pectinata extract and its analog against 3D7 Plasmodium falciparum through caseinolytic protease proteolytic (ClpP) inhibition

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation

The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the Δ5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP, exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus’s recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.

Potential of medicinal plant compounds to targeting Tau protein in the therapy of Alzheimer’s disease– A review

Alzheimer’s disease (AD) is a devastative neurodegenerative disorder with complex etiology. AD is characterized by blood-brain barrier disruption, oxidative stress, mitochondrial impairment, neuro- inflammation, hypo-metabolism; it decreases in acetylcholine levels and a reduction of cerebral blood flow. It is also not solely the end-product of aberrantly processed, misfolded, and aggregated oligomeric amyloid- beta peptides but hyper phosphorylated Tau (tubulin binding protein) which formed senile plaque and intracellular neurofibrillary tangles respectively. However, despite the long-term and worldwide effort for a more effective therapy, the only available treatment is a symptomatic use of acetylcholinesterase inhibitors and memantine. Then, many researchers focused their attention to modulate amyloid-beta peptides. These therapeutic approaches as well as those based on cholinergic or amyloid theory have not brought the desired benefits yet. Thus, the main features related with the Tau pathology found in AD are Tau phosphorylation and aggregation. Based on the biochemically diverse range of pathological Tau protein, a number of approaches have been proposed to develop new potential therapeutics like inhibition of Tau phosphorylation, proteolysis and aggregation; promotion of intra- and extracellular Tau clearance and stabilization of microtubules (MTs). Medicinal plants have been used in different systems of medicine and exhibited their powerful roles in the management and cure of memory disorders. This review paper discusses the potential of medicinal plant molecules to targeting Tau protein in Alzheimer’s disease therapy.
 

Performance of MDP-based system in eroded and carious dentin associated with proteolytic inhibitors: 18-Month exploratory study

The aim of this study was to explore the impact of the interaction between an MDP-based universal adhesive system in etch-and-rinse mode and two proteolytic inhibitors on the longevity of restorations bonded to artificially-affected-dentin substrates. 90 sound human third molars were randomly distributed into three groups according to the substrate: N-no challenges-control (stored in artificial saliva), ACD-artificial caries dentin (6 h DE + 18 h-RE/5 days + 48 h RE) and ERO-artificial erosion dentin (3 × 5 min/5 days with orange juice). They were further redistributed according to dentin pretreatment: W- water (control), CHX-2% digluconate chlorhexidine and E64- 5 μM E64-Trans-Epoxysuccinyl-L-Leucylamido-(4-guanidino) butane, which resulted in the following 9 groups (n = 10): N–W, N-CHX, N-E64, ACD-W, ACD-CHX, ACD-E64, ERO-W, ERO-CHX and ERO-E64. All specimens were restored with Adper Single Bond Universal (Etch-and-rinse mode)/Filtek Z250. Sticks (0.64 mm2) were obtained and subjected to microtensile test (μTBS) in a universal testing machine at 0.5 mm/min for 7-days, 6 and 18-month analyses. Failure modes were classified using optical microscopy (40X). Data were statistically analyzed by three-way ANOVA and Tukey tests (p < 0.05). All individual factors (p < 0.0001) and interaction between factors were statistically significant (substrate X pretreatment (p = 0.00093); substrate X time (p = 0.01035) and pretreatment X time (p = 0.0035). Caries-affected substrate was the most compromised one, disregarding the pretreatment. CHX was mostly affected compared with E64 up to 18 months, possibly due to its calcium-dependent mechanism.

Antitrichomonal activity and docking analysis of thiazole derivatives as TvMP50 protease inhibitors.

Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the most prevalent non-viral sexually transmitted infection that affects over 170 million people worldwide. The only type of drug recommended for the therapeutic control of trichomoniasis is the 5-nitroimidazoles, although there have been reports of some undesirable side effects and clinical resistance. Hence, the need for the search for new tricomonicidal agents is necessary. In a previous work, we demonstrated that two 2-amino-4-aryl thiazole derivatives (ATZ-1 and ATZ-2) possess a portent antigiardial effect. In the current paper, we investigated the in vitro antitrichomonal activity of these thiazole compounds. Both ATZ-1 and ATZ-2 reduced the viability and growth of parasites in a dose-dependent manner, with an IC50 value of 0.15 μg/mL and 0.18 μg/mL, respectively. Furthermore, both thiazole compounds were able to decrease the proteolytic activity in T. vaginalis trophozoites compared with untreated parasites. Interestingly, a full proteolytic inhibition profile was observed in the 50-kDa region which was associated with the decreased expression of the gene that codes for the trichomonad protease TvMP50. The docking simulations predicted strong interactions of the thiazole compounds in the TvMP50 protease's active site, suggesting a possible role as protease inhibitors. Our results demonstrate the potential of 2-amino-4-aryl thiazole derivatives as trichomonicidal compounds and could be, mechanistically, involved in the inhibition of key trichomonad proteases.

Transcriptional characterization of conjunctival melanoma identifies the cellular tumor microenvironment and prognostic gene signatures

This study characterizes the transcriptome and the cellular tumor microenvironment (TME) of conjunctival melanoma (CM) and identifies prognostically relevant biomarkers. 12 formalin-fixed and paraffin-embedded CM were analyzed by MACE RNA sequencing, including six cases each with good or poor clinical outcome, the latter being defined by local recurrence and/or systemic metastases. Eight healthy conjunctival specimens served as controls. The TME of CM, as determined by bioinformatic cell type enrichment analysis, was characterized by the enrichment of melanocytes, pericytes and especially various immune cell types, such as plasmacytoid dendritic cells, natural killer T cells, B cells and mast cells. Differentially expressed genes between CM and control were mainly involved in inhibition of apoptosis, proteolysis and response to growth factors. POU3F3, BIRC5 and 7 were among the top expressed genes associated with inhibition of apoptosis. 20 genes, among them CENPK, INHA, USP33, CASP3, SNORA73B, AAR2, SNRNP48 and GPN1, were identified as prognostically relevant factors reaching high classification accuracy (area under the curve: 1.0). The present study provides new insights into the TME and the transcriptional profile of CM and additionally identifies new prognostic biomarkers. These results add new diagnostic tools and may lead to new options of targeted therapy for CM.

5-(4H)-Oxazolones and Their Benzamides as Potential Bioactive Small Molecules.

The five membered heterocyclic oxazole group plays an important role in drug discovery. Oxazolones present a wide range of biological activities. In this article the synthesis of 4-substituted-2-phenyloxazol-5(4H)-ones from the appropriate substituted aldehydes via an Erlenmeyer–Plochl reaction is reported. Subsequently, the corresponding benzamides were produced via a nucleophilic attack of a secondary amine on the oxazolone ring applying microwave irradiation. The compounds are obtained in good yields up to 94% and their structures were confirmed using IR, 1H-NMR, 13C-NMR and LC/MS data. The in vitro anti-lipid peroxidation activity and inhibitory activity against lipoxygenase and trypsin induced proteolysis of the novel derivatives were studied. Inhibition of carrageenin-induced paw edema (CPE) and nociception was also determined for compounds 4a and 4c. Oxazolones 2a and 2c strongly inhibit lipid peroxidation, followed by oxazolones 2b and 2d with an average inhibition of 86.5%. The most potent lipoxygenase inhibitor was the bisbenzamide derivative 4c, with IC50 41 μM. The benzamides 3c, 4a–4e and 5c were strong inhibitors of proteolysis. The replacement of the thienyl moiety by a phenyl group does not favor the protection. Compound 4c inhibited nociception higher than 4a. The replacement of thienyl groups by phenyl ring led to reduced biological activity. Docking studies of the most potent LOX inhibitor highlight interactions through allosteric mechanism. All the potent derivatives present good oral bioavailability.

Magnolol inhibits myotube atrophy induced by cancer cachexia through myostatin signaling pathway in vitro.

Cancer cachexia is a complex and multifactorial syndrome that influences about 50-80% of cancer patients and may lead to 20% of cancer deaths and muscle atrophy is the key characteristic of the syndrome. Recentresearches have shown that myostatin is a negative regulator in the growth and differentiation of skeletal muscle. Herein, C2C12 cancer cachexia model was established with C26 conditioned culture medium (CCM), then treated with magnolol to evaluate the pharmacological activity of magnolol in myotube atrophy. Our results demonstrated that magnolol inhibited the activity of myostatin promotor and the myostatin signaling pathway. In C2C12 cancer cachexia model, magnolol decreased myostatin expression, inhibited the phosphorylation of SMAD2/3 activated by C26 conditioned culture medium (CCM), and elevated the phosphorylation of FOXO3a lowered by CCM. Myosin heavy chain (MyHC), myogenin (MyoG), and myogenic differentiation (MyoD), as three common myotube markers in C2C12 myotube, were decreased by CCM, which could be effectivelyreversed by magnolol via activation of AKT/mTOR-regulated protein synthesis and inhibition of ubiquitin-mediated proteolysis. This study reveals that magnolol inhibits myotube atrophy induced by CCM by increasing protein synthesis and decreasing ubiquitin-mediated proteolysis, so that magnolol is a promising leading compound in treating muscle atrophy induced by cancer cachexia.

Profile of a 10-MDP-based universal adhesive system associated with chlorhexidine: Dentin bond strength and in situ zymography performance

The incorporation of functional monomers and proteolytic inhibitors into adhesive systems have shown to be promising strategies to improve the longevity of adhesive restorations. The aim of this study was to evaluate the long-term bonding performance and anti-gelatinolytic effect of a 10-MDP-based universal adhesive system applied in combination with 2% chlorhexidine digluconate (CHX). For that, this study assessed the resin-dentin bond strength and the in situ gelatinolytic activity profile at the adhesive interface at initial and after 6 month of storage. One hundred and two sound human third molars were prepared and randomly divided into 3 groups according to the adhesive strategy: SB (two-step etch-and-rinse adhesive, Adper Single Bond 2, 10-MDP-free control group); SU-ER (Adper Single Bond Universal, 10-MDP containing universal adhesive applied on etch-and-rinse mode); and SU-SE (SU applied on self-etching mode). The groups were subdivided into two according to the dentin pretreatment: W - water or CHX- 2% chlorhexidine digluconate aqueous solution (SB–W; SB-CHX; SU-ER-W; SU-ER-CHX; SU-SE-W; SU-SE-CHX) and subsequently restored according to the manufacturer's instructions. Bond strength (n = 12) was assessed by a microtensile test (μTBS) (500N/0.5 mm/min) after 24h or after 6 months of storage. In situ zymography was performed to evaluate anti-gelatinolytic activity (n = 5). Resin-dentin samples were incubated with fluorescein-conjugated gelatin for 24 h at 37 °C and analyzed by confocal laser scanning microscopy. Fluorescence indicating gelatinolytic activity at hybrid layer zone and adjacencies was quantified using Image J. Data was analyzed by three-way ANOVA and Tukey post-hoc tests (p < 0.05). Results: SU-SE showed the highest bond strength values, while similar results were observed for SU-ER and SB. No statistical significant differences were observed between pretreatment (CHX vs. W) or storage time (initial vs. 6 months of aging). For in situ zymography, fluorescence was detected in all groups and CHX pre-treatment was able to inhibit the gelatinolytic activity in all conditions. The 10-MDP-based universal adhesive system in self-etching mode was the strategy that showed the best bonding performance irrespective of its combination with chlorhexidine. Pre-treatment with CHX did not impair the bond strength when used in combination with 10-MDP and it may promote collagen stability overtime.

Proteolytic parameter changes in the plasma of patients with bladder cancer – depending on tumor stage

Abstract Bladder cancer (BC) is a worldwide common disease with a high mortality rate. Recognizing the dynamic changes in plasma that proteases and their inhibitors undergo might be valuable in understanding the carcinogenesis of invasive bladder cancer and in identifying BC patients with poor prognosis. This study aims to determine the activity of the proteolytic enzyme system and their inhibitors in patients with BC. In this paper, the total proteolytic activity, the activity of matrix metalloproteases (MMPs) and serine proteases was analyzed by the method of caseinolytic activity. For detection of activity of some inhibitors of proteolysis, we used the unified method for determining the activity of alpha-1-antitrypsin (α1A) and alpha-2-Macroglobulin (α2M) in human plasma. The level of medium-mass molecules (MMM) was assessed spectrophotometrically by applying the Nikolaichik method. The activity of MMPs was elevated in all groups of patients. Moreover, the activity of serine proteases was found to be enhanced in patients with Stage I, III and IV BC, while the activity of α1A was up by 1.4 and 1.3 times in patients with Stage I and Stage IV. The most significant increase was observed with regard to the activity of α2M in patients with I and III stages of BC. In addition, the level of MMM correlated with cancer stage. Indeed, the highest increase in the activity of protease inhibitors was observed in Stage I bladder cancer patients, which might signify their protective role at the onset of the bladder carcinogenesis. In contrast, significant growth in activity of α2M in patients with III stage of BC may point at a compensatory mechanism that inhibits tumor growth.