Elastase Activity Research Articles

Genomic and phenotypic characterization of Pseudomonas aeruginosa isolates from two Mexican cystic fibrosis attention centers.

Thirty-nine clinical isolates of Pseudomonas aeruginosa collected from 11 cystic fibrosis (CF) patients at two CF attention centers over 10 years were subjected to whole genome sequencing (WGS). Phenotypic tests (i.e., elastase, motility, biofilm, growth rate, and antibiotic susceptibility) were performed to correlate results. A single strain of P. aeruginosa was found to persist over time in longitudinal isolates. No transmission between patients or centers was observed. A tendency to lack genes related to pyoverdine, flagellum, pili, and O-antigen was observed, whereas those related to biofilm, phenazine, and pyochelin were conserved among isolates. In a patient with a 10-year follow-up, a single strain of P. aeruginosa persisted and showed a gradual decrease in elastase activity and growth rate, demonstrating an adaptive phenotype.IMPORTANCEThis study investigates the genomic and phenotypic characteristics of Pseudomonas aeruginosa isolates from Mexican cystic fibrosis (CF) patients, an underrepresented group in CF research. To our knowledge, it is the first to use whole genome sequencing (WGS) to study longitudinally collected P. aeruginosa isolates from this population, evaluating both genomic features and clonal relationships. Remarkably, the study includes samples from one patient over 10 years, offering an extended observation time compared to existing literature. Unlike similar studies, which often lack phenotypic testing, this research incorporates various virulence-related phenotypic assays, enhancing our understanding of gene-to-phenotype correlations. Two potential mechanisms for the loss of elastolytic activity were identified. Furthermore, we conduct an in-depth mobilome analysis, an area that remains largely unexplored in CF contexts. Whole genome sequencing data are publicly available through the NCBI SRA database, facilitating further re-analysis for studies on P. aeruginosa in CF, as well as epidemiological and population structure research.

Computational Approach to Identifying New Chemical Entities as Elastase Inhibitors with Potential Antiaging Effects.

In the aging process, skin morphology might be affected by wrinkle formation due to the loss of elasticity and resilience of connective tissues linked to the cleavage of elastin by the enzymatic activity of elastase. Little information is available about the structural requirements to efficiently inhibit elastase 1 (EC 3.4.21.36) expressed in skin keratinocytes. In this study, a structure-based approach led to the identification to the pharmacophoric hypotheses that described the main structural requirements for binding to porcine pancreatic elastase as a valuable tool for the development of skin therapeutic agents due to its similarity with human elastase 1. The obtained models were subsequently refined through the application of computational alanine-scanning mutagenesis to evaluate the effect of single residues on the binding affinity and protein stability; in turn, molecular dynamic simulations were carried out; these procedures led to a simplified model bearing few essential features, enabling a reliable collection of chemical features for their interactions with elastase. Then, a virtual screening campaign on the in-house library of synthetic compounds led to the identification of a nonpeptide-based inhibitor (IC50 = 60.4 µM) belonging to the class of N-substituted-1H-benzimidazol-2-yl]thio]acetamides, which might be further exploited to obtain more efficient ligands of elastase for therapeutic applications.

Tangeretin inhibits airway inflammatory responses by reducing early growth response 1 (EGR1) expression in mice exposed to cigarette smoke and lipopolysaccharide

BackgroundTangeretin, a natural polymethoxyflavone compound, possesses potent anti-inflammatory activity that improves respiratory inflammation in chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms underlying the anti-COPD effects of tangeretin remain unclear. In this study, we aimed to investigate the key molecular mechanisms by which tangeretin suppresses COPD-related inflammatory responses. MethodsWe conducted the investigation in phorbol-12-myristate-13-acetate (PMA)-stimulated human airway epithelial cells (in vitro) and cigarette smoke (CS)/lipopolysaccharide (LPS)-exposed mice (in vivo). ResultsTangeretin decreased the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and mucin 5AC (MUC5AC), by suppressing early growth response 1 (EGR1) expression in vitro. Tangeretin and EGR1 small interfering ribonucleic acid (siRNA) combination showed a synergistic reduction in MUC5AC and TNF-α secretion. Tangeretin administration significantly inhibited the levels of reactive oxygen species (ROS) production, elastase activity, TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1) secretion, and macrophage and neutrophil numbers in the bronchoalveolar lavage fluid of CS/LPS-exposed mice. Tangeretin also prevented CS/LPS-induced abnormal pathological changes and excessive MUC5AC and EGR1 expression in lung tissue. ConclusionComprehensively, tangeretin inhibits the lung inflammatory response associated with COPD by reducing EGR1 expression in PMA-induced human epithelial cells and in a CS/LPS-stimulated mouse model. This study shows that tangeretin has anti-COPD properties and can be a promising alternative (or complementary) treatment for inflammatory lung disease.

Alpha-1 antitrypsin deficiency associated with increased risks of skin cancer, leukemia, and hepatic cancer: A nationwide cohort study.

α1-Antitrypsin deficiency is characterized by elevated elastase activity and excessive elastin degradation, which may impact cancer development and progression. We tested the hypothesis that individuals with α1-antitrypsin deficiency have increased susceptibility to cancer in the Danish population. In a nationwide nested study, we identified 2702 individuals with α1-antitrypsin deficiency and 26,750 control subjects without α1-antitrypsin deficiency matched on age, sex, and municipality. We recorded admissions due to cancer as outcomes during a median follow-up of 62 years. Individuals with α1-antitrypsin deficiency versus control subjects had an increased hazard of skin cancer (2.18, 95%CI: 1.81-2.63), leukemia (1.76, 1.12-2.79), liver cancer (3.91, 2.23-6.85), and cancer overall (1.25, 1.13-1.38). Corresponding hazard ratios when the entire Danish population was used as control group were 3.02 (2.55-3.58), 1.83 (1.19-2.81), 4.46 (2.74-7.28), and 1.45 (1.31-1.59). When the analysis was stratified according to comorbidities, the hazard for skin cancer was higher in those with chronic obstructive pulmonary disease (COPD) (3.59, 2.60-4.95) and skin disease (2.93, 2.19-3.92) but remained elevated in those without any of these diseases. Hazards for skin cancer in individuals with α1-antitrypsin deficiency were similar when stratified by liver cirrhosis and ischemic heart disease (ps for interaction: ≥0.76). Hazards for liver cancer in individuals with α1-antitrypsin deficiency versus control subjects were similar when stratified according to liver cirrhosis, COPD, skin disease, and ischemic heart disease (ps for interaction: ≥0.13). Individuals with α1-antitrypsin deficiency have increased risks of skin cancer, leukemia, and liver cancer in the Danish population.

Green-synthesized silver nanoparticles from Zataria multiflora as a promising strategy to target quorum sensing and biofilms in Pseudomonasaeruginosa

The global challenge to human health is significantly heightened by the resistance of harmful bacteria to antimicrobial treatments. Given the limited advancement in developing new antimicrobial medications, exploring innovative strategies is imperative to tackle the challenge of resistance to multiple drugs. Furthermore, there is a growing emphasis on the environmentally friendly synthesis of nanoparticles with potent medicinal attributes, specifically those targeting virulence, to combat the rise of multidrug resistance. Focusing on the inhibition of virulence factors and biofilms influenced by quorum sensing has become a promising and novel strategy in the development of anti-infective drugs. An aqueous extract of Zataria multiflora leaves was used to create green-synthesized silver nanoparticles, or AgNPs. X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and UV–visible absorption spectroscopy were used to characterize the AgNPs. The impact of AgNPs on the virulence factors and biofilms of Pseudomonas aeruginosa PAO1, mediated by quorum sensing, was assessed at concentrations below the minimum inhibitory concentration (sub-MIC). Sub-MIC concentrations of Green-synthesized AgNPs inhibited various P. aeruginosa virulence factors, including bacterial motility (89 % inhibition), pyocyanin production (81.48 % inhibition), pyoverdin production (55.80 % inhibition), elastase activity (87.43 % inhibition), exoprotease activity (75.60 % inhibition), and rhamnolipid production (71.28 % inhibition). Additionally, these AgNPs demonstrated 80 % inhibition of P. aeruginosa biofilms. The in vitro efficacy of green-synthesized AgNPs against P. aeruginosa can be utilized for the creation of alternative therapeutic agents for managing bacterial infections, particularly for topical application in cases such as wound infections. Additionally, they can be used for surface coating to inhibit the attachment of bacteria to medical devices.

Evaluation of the Effectiveness of Using the CFTR Modulator Ivacaftor/Lumacaftor in Children with Cystic Fibrosis in the Sverdlovsk Region (Prospective Cohort Study)

Background. A modern achievement in the treatment of cystic fibrosis has been the discovery of small molecules that restore the processes of synthesis, transport to the membrane, and the work of the defective CFTR protein. Drugs whose action is aimed at restoring the function of the CFTR protein are called CFTR modulators. The aim of the study is the conduction of a comprehensive assessment of the effectiveness of CFTR modulator ivacaftor/lumacaftor therapy in children with cystic fibrosis based on the study of clinical, laboratory, and instrumental data. Methods. CFTR modulator ivacaftor/lumacaftor was received for 12 months by 23 children of the main group aged 2–17 years, who are homozygous carriers of the F508del mutation of the CFTR gene in the absence of the L467F complex allele. A control examination of the main group was conducted at the start of the study and then every 3 months for 12 months of drug administration. Results. The condition of the patients in the observation groups at the start of the study was assessed as moderate to severe, the bronchopulmonary process was in the stage of incomplete clinical remission. In the main group, the level of aspartate aminotransferase significantly decreased, in the control, on the contrary, there was a tendency for this indicator to increase. The level of alkaline phosphatase (alkaline phosphatase) decreased and reached normal in the main group; the relative risk of hyperphosphatasemia during treatment decreased by 5 times (0.187 (0.063–0.557)). The level of gamma-glutamyltranspeptidase (gamma-GTP) and the number of patients with elevated GGTP decreased (24.65 and 19.69 IU/l, respectively; 61 and 13%). In the control group, the level of gamma-GTP increased and the alkaline phosphatase did not normalize. The indicators of forced exhalation volume in the first second and forced vital capacity of the lungs were initially lower in the main group, after 12 months the differences remained. There was a tendency to decrease the frequency of severe pancreatic insufficiency in the main group by 17.3%, and no changes in the control group. Sweat chlorides tended to decrease in the main group in the absence of reaching the standard values. Conclusion. The usage of the CFTR modulator ivacaftor/lumacaftor showed a greater clinical effect in improving the functioning of the digestive organs: a decrease in the risk of hyperphosphatasemia and a significant decrease in the number of patients with high levels of gamma-GTP were established, a stable level of liver enzyme activity was noted, and a tendency to increase the activity of pancreatic elastase was revealed. The tendency to decrease the indicators of sweat chlorides is shown. There were no changes in the indicators of respiratory function.

Towards the Use of Lichens as a Source of Bioactive Substances for Topical Applications.

The increasing incidence of dermatological diseases prompts the search for new natural methods of treatments, and lichens, with their special symbiotic structure, are a little-known and promising source of biologically active substances. Seven lichen species, Cladonia unicialis (L.) Weber ex F.H. Wigg. (Cladoniaceae), Evernia prunastri (L.) Ach. (Parmeliaceae), Hypogymnia physodes (L.) Nyl. (Parmaliaceae), Parmelia sulcata (Taylor) (Parmeliaceae), Physcia adscendens (Fr.) H. Olivier (Physciaceae), Pseudoevernia furfuracea (L.) Zopf (Parmeliaceae), and Xanthoria parietina (L.) Th. Fr. (Teloschistaceae), were used in our experiment. We identified different metabolites in the acetone extracts of all the lichen species. Based on the high-performance liquid chromatography analysis, the content of lichen substances in the extracts was evaluated. The impact of the individual lichen-specific reference substances, compared to the lichen extracts, on the viability of keratinocytes (HaCaT cell line) and fibroblasts (BJ cell line) and on the activity of selected skin-related enzymes was investigated. Our results revealed that only emodin anthrone at a concentration of 200 mg/L was cytotoxic to keratinocytes and fibroblasts in both cell viability assays. In turn, the C. uncialis extract was only cytotoxic to keratinocytes when used at the same concentration. The other tested treatments showed a positive effect on cell viability and no cytotoxicity or indeterminate cytotoxicity (shown in only one of the tests). Elastase and collagenase activities were inhibited by most of the lichen extracts. In turn, the individual lichen compounds (with the exception of evernic acid) generally had an undesirable stimulatory effect on hyaluronidase and collagenase activity. In addition, almost all the tested compounds and extracts showed anti-inflammatory activity. This suggests that some lichen compounds hold promise as potential ingredients in dermatological and skincare products, but their safety and efficacy require further study. The high cytotoxicity of emodin anthrone highlights its potential use in the treatment of hyperproliferative skin diseases such as psoriasis.

Modification of Bacillus sp. IMV B-7883 Elastase Activity by the Hetero-Metallic Carboxylatogermanates/Stannates

In recent years, some researchers have found that although many Gram-negative and Gram-positive bacteria secrete elastase, the bacterial forms of elastase have either a low activity or harmful effects. Therefore, further research is needed in isolating and screening microorganisms that produce a high level of elastase activity. Previously we selected strain Bacillus sp. IMV B-7883, which exhibits fairly high elastase activity. To increase its activity, we chose one of the well-known approaches, in particular, the use of a number of coordination compounds capable to influence elastase activity. In this regard, the purpose of this work was to study the effect of such coordination compounds as hetero-metallic carboxylatogermanates/stannates on the elastase activity of Bacillus sp. IMV B-7883. Methods. The object of the investigation was the strain of Bacillus sp., deposited in the Ukrainian Collection of Microorganisms under the number IMV B-7883, isolated from soil. The culture was grown under conditions of submerged cultivation at 28 °С, with a mixing speed of the nutrient medium of 244 rpm for three to six days (72–144 hours). We used an enzyme purified from the supernatant of the culture liquid by precipitation with 90% ammonium sulfate, with further fractionation on neutral and charged carriers. Elastase activity was determined colorimetrically by the intensity of the color of the solution upon enzymatic hydrolysis of elastin stained with Congo red. As modifiers of enzyme activity, hetero-metallic carboxylatogermanates/stannates were used. Results. Of the 15 studied in this work coordination compounds presented by hetero-metallic carboxylatogermanates/stannates, only 1 [Ba(H2O)6][Ge2(OH)2(C6H8O7)2]·nH2O, n=2 and 3 [Ni (H2O)6][Ge2(OH)2(C6H8O7)2]·nH2O, n=4, depending on the concentration used and incubation time, increase the elastase activity by only 3–5%. All other compounds have an inhibitory effect. Conclusions. Obtained data on the inhibitory effect of hetero-metallic carboxylatogermanates/stannates on the elastase activity of Bacillus sp. IMV B-7883 provide new information which may help in solving the issue of the mechanism of interaction between enzymes and complex chemical molecules.

Abstract P222: O-GlcNAcylation induces aortic stiffness in aging female rats via CD47-SIRPα -1 signaling

A reduction in aortic compliance serves as an early indicator of incipient vascular disease. While extracellular matrix modifications, such as elastin fragmentation, are the major mediators of aortic stiffness, the mechanisms that underlie these phenotypes remain to be elucidated. O-GlcNAc, the attachment of β-N-acetylglucosamine to serine and threonine residues of proteins, is a highly dynamic and widespread post-translational modification. This modification plays a pivotal role in modulating the function, activity, localization, and stability of target proteins across nuclear, cytoplasmic, and mitochondrial compartments. Primarily involved in intracellular signaling and transcriptional regulation, O-GlcNAc responds to nutrient availability and cell stress. However, chronic O-GlcNAc is consistently linked to vascular impairments. Indeed, our preliminary data has demonstrated that O-GlcNAc is elevated in aorta of Fisher-334 female at 16 months of age (16MO) than 3MO and pharmacological induction of O-GlcNAc (TMG 100 uM 24h) in 3MO rats can cause aortic stiffness via increased elastase activity and elastin fragmentation. However, how this inherently intracellular cellular signaling could impact aorta remodeling is unknown. CD47 (Cluster of Differentiation 47), also known as integrin-associated protein, is a transmembrane protein that acts as a “do not eat me” signal to immune cells. RNAseq data from our lab suggested that TMG (1 uM, 24 h) increased CD47 expression in vascular smooth muscle cells (n =3, p=0.08). Therefore, we hypothesized that CD47 could be an important downstream mediator of O-GlcNAc-induced aortic stiffness. Our findings revealed that CD47 inhibitors, [(RRx 10 uM) and SEN 177 (100 nM)] effectively mitigated the aortic stiffness and elastase activity (Ctrl 2302±38; TMG 4058±328; RRx 1776±121; Sen177 3032±50 A.U.) induced by TMG, suggesting that CD47 is a novel mechanism of O-GlcNAc-induced aortic stiffness. As aging is a major risk factor for aortic stiffness, we also revealed that CD47 was increased in the aorta of 16MO rats compared to 3MO controls (0.1±0.03 vs. 0.3±0.06). Potential ligands for CD47 include thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). We observed that in 16MO rats, circulating SIRPα is increased, but not TSP1, suggesting a receptor-ligand interaction that is applicable in aging. Overall, these data reveal a previously unidentified and significant role for CD47 in O-GlcNAc-induced aortic stiffness.

Study on interaction with high-abundant blood proteins and identification of low-abundant proteins to 5-phenyl-1-(p-tolyl)-1 H-1,2,3-triazole by serum proteomics

In this study, a comprehensive investigation was undertaken to elucidate a simple triazole compound, 5-phenyl-1-(p-tolyl)-1 H-1,2,3-triazole (PPTT), its interactions with high-abundant proteins and identification of low-abundant proteins by serum proteomics. Employing a combination of spectroscopic techniques and computational chemistry, the interactions between PPTT and three high-abundance blood globular proteins, namely human serum albumin (HSA), human immunoglobulin G (HIgG), and hemoglobin (BHb), were explored, thereby ascertaining their binding constants and thermodynamic parameters at the molecular level. Subsequently, based on the differential proteomics, utilizing two-dimensional gel electrophoresis (2-DE) in conjunction with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), the research team isolated and identified differentially expressed low-abundance proteins in human blood serum samples following exposure to PPTT. The results showed that there were twenty highly expressed proteins identified from blood serum samples intervened by PPTT. Combining bioinformatics techniques, these proteins were classified, providing preliminary insights like preproprotein or precursors inhibiting the activity of elastase, defending and regulating the immune system, carrying lipid, and other functions into their biological functionalities. One of the differential proteins, apolipoprotein A-1 (ApoA-1) protein, was selected as a possible target to explore the mechanism of action of PPTT intervention on the related signaling pathways involved in human hepatocellular carcinomas(Hep G2) cells. These research findings offer scientifically sound guidance for further in-depth exploration, development, and application of the 1,2,3-triazole compound.

Volatile profile of Astragalus sinicus L. flowers by GC×GC-TOF-MS and evaluation of nutraceutical and nutricosmetic potential of the flower extracts

The aroma property and nutraceutical capacity are two crucial characteristics for the acceptance of edible flowers. The volatile profile of the Chinese milk vetch flowers (CMVF) was investigated using HS-SPME-GC × GC-TOF-MS for the first time. A total of 279 compounds were identified by the two-dimensional gas chromatography technology, including 46 alcohols, 45 aldehydes, 54 esters, 39 ketones, 66 hydrocarbons, 9 phenols, and 20 other compounds, with pentadecanal (14.35%) revealed to be the major volatile component. Meanwhile, The five CMVF extracts were evaluated for their phytochemical composition and biological activity. The antioxidant activity assay indicated that the ethanol extract of CMVF revealed the highest scavenging capacity against DPPH radicals with IC50 of 384 μg/mL, the ethyl acetate extracts of CMVF revealed the strongest scavenging capacity against ABTS·+ (IC50 = 140.83 μg/mL) and ·OH (IC50 = 14.25 μg/mL). In addition, the water extracts of CMVF inhibited tyrosinase activity through mixed type and the alcohol extracts of CMVF inhibited elastase activity through competitive type. The Pearson correlation analysis showed that the bioactivity of the CMVF extracts were significantly impacted by TPC (total phenolic content) and TFC (total flavonoids content). These results suggested that CMVF extracts exhibited great potential for functional foods and cosmetics utilization due to its promising nutraceutical and nutricosmetic properties.

Gasdermin D deficiency attenuates development of ascending aortic dissections in a novel mouse model.

Thoracic aortic dissection (TAD) is a silent killer. Approximately two-thirds of the cases occur in the ascending aorta (i.e. type A dissection) and majority of them are unrelated to genetic mutations. However, animal models of spontaneous type A dissection are not widely available. In the present study, a novel mouse TAD model was created. Further, the role of gasdermin D (GSDMD) in TAD development was evaluated. TADs were created by treating ascending aorta of adult mice (C57BL/6J) with active elastase (40.0 U/ml) and β-aminopropionitrile (Act E+BAPN). The temporal progress of the TAD pathology was rigorously characterized by histological evaluation and scanning electron microscopy, while potential mechanisms explored with bulk RNA sequencing of specimens collected at multiple timepoints. With this novel TAD model, further experiments were performed with Gsdmd -/- mice to evaluate its impact on TAD formation. The ascending aorta challenged with Act E+BAPN developed pathology characterized by an early onset of intimomedial tears (complete penetration) and intramural hematoma, followed by progressive medial loss and aortic dilation. Ingenuity Pathway Analysis and functional annotation of differentially expressed genes suggested that a unique inflammatory micro-environment, rather than general inflammation, promoted the onset of TADs by specifically recruiting neutrophils to the aortic wall, while the pathology at the advanced stage was driven by T-cell mediated immune injury. Gsdmd -/- attenuated medial loss, adventitial fibrosis, and dilation of TADs. This protective effect was associated with a reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive cells and T-cells in TADs. A novel mouse TAD model was created in the ascending aorta. It produces a unique microenvironment to activate different immune cell subsets, promoting onset and subsequent remodeling of TADs. Consistently, Gsdmd -/- attenuates TAD development, with modulation of cell death and T-cell response likely acting as the underlying mechanism.

Curcumin loaded starch-based aerogels interfere with quorum sensing regulated virulence functions and biofilm of bacterial pathogens

Over the last two decades, the sharp escalation of antimicrobial resistance (AMR) has emerged as a formidable threat to human health. This pressing situation demands innovative interventions to combat infectious diseases. Aerogels, characterized by their nanostructured composition and high porosity, present a promising avenue. In this study, we manufactured curcumin loaded starch-based aerogels (CSA) and evaluated their impact on the quorum sensing (QS) mechanism in Gram-negative bacteria. Fourier-transform infrared (FTIR) analysis highlighted the presence of cellulose hydroxyl groups engaged in hydrogen bond formation. Thermogravimetric analysis (TGA) revealed that over 50 % of the initial mass was lost when CSA underwent heating to 350 °C. Microscopic examination showcased a uniform and compact pattern, suggesting reduced pore distribution. The elemental composition analysis indicated that carbon and oxygen constituted 23.00 % and 77.00 % of the weight, respectively. The presence of CSA resulted in over 30.4 % inhibition of violacein pigment production. Furthermore, CSA modulated pyocyanin production, pyoverdin production, LasB elastase activity, and rhamnolipid production by 34.4 %, 31.07 %, 22.7 %, and 19.7 %, respectively. The total exoproteases, cell surface hydrophobicity and exopolysaccharide production in E. coli, L. monocytogenes, S. marcescens, and P. aeruginosa, experienced a significant decrease at sub-MICs. Production of biofilms as well as the mature biofilms in test bacteria were reduced in dose-dependent manner significantly. Due to their antibiofilm and anti-quorum sensing properties, these CSAs could prove to be functional biomaterials with versatile applications, particularly in the food industry.

ShSPI Inhibits Thrombosis Formation and Ischemic Stroke In Vivo.

Thrombotic diseases, emerging as a global public health hazard with high mortality and disability rates, pose a significant threat to human health and longevity. Although current antithrombotic therapies are effective in treating these conditions, they often carry a substantial risk of bleeding, highlighting the urgent need for safer therapeutic alternatives. Recent evidence has increasingly pointed to a connection between elastase activity and thrombosis. In the current study, we investigated the antithrombotic effects of ShSPI, an elastase inhibitor peptide derived from the venom of Scolopendra hainanum. Results showed that ShSPI significantly attenuated carrageenan-induced thrombosis in vivo. Furthermore, ShSPI effectively inhibited the carrageenan-induced decrease in serum superoxide dismutase (SOD) activity and increase in prothrombin time, fibrinogen level, and endothelial nitric oxide synthase (eNOS) activity. In addition, ShSPI reduced intracerebral thrombosis and improved functional outcomes following ischemic stroke in a transient middle cerebral artery occlusion (tMCAO) mouse model. Collectively, these findings suggest that ShSPI is a promising candidate for the development of novel thrombotic therapies.

ESSENTIAL OIL CONTENTS AND BIOLOGICAL ACTIVITIES OF THYMUS CANOVIRIDIS JALAS AND THYMUS SIPYLEUS BOISS.

Objective: Members of the Lamiaceae family are considered to be major sources of bioactive therapeutic agents. Many of them are important medicinal and aromatic plants used in traditional and modern medicine and in the food, cosmetic and pharmaceutical industries. The aim of this study was to investigate in detail the biological activities and chemical composition of the essential oils of Thymus canoviridis Jalas and Thymus sipyleus Boiss. species belonging to the genus Thymus, one of the most important genera of the Lamiaceae family. Material and Method: The essential oil content of the species was determined by GC-MS. Antioxidant activities of the essential oils were determined using lipid peroxidation, DPPH free radical, ABTS cation radical and CUPRAC methods. In addition, cytotoxic activities against breast cancer (MCF-7) and colon cancer (HT-29) cell lines and anticholinesterase (against AChE and BChE enzymes), urease, tyrosinase, elastase, collagenase and angiotensin converting enzyme inhibition activities were determined. Result and Discussion: When the essential oil composition of T. sipyleus was analyzed, the major compounds were 1,8-cineole (eucalyptol) (18.16%), camphor (15.08%) and endo-borneol (11.63%), while T. canoviridis was found to be rich in carvacrol (72.88%). T. canoviridis showed high antioxidant activity in lipid peroxidation (IC50: 45.72±0.12 μg/ml), ABTS (IC50: 6.12±0.03 μg/ml) and CUPRAC (IC50: 5.31±0.01 μg/ml) methods. The selectivities of T. canoviridis and T. sipyleus species against MCF-7 cell line were 4.39 and 6.81, respectively. In the enzyme inhibition studies, both Thymus species showed moderate inhibition activity against BChE enzyme (Inhibition%: 57.88±1.14, 39.21±0.89, respectively). In addition, T. sipyleus showed moderate inhibition of elastase enzyme (Inhibition%: 25.33±0.79). When the results are evaluated in general, it can be said that T. canoviridis essential oil with its rich carvacrol content and high cytotoxic and antioxidant activity can be preferred as a safer and natural option instead of synthetic preservatives in food, pharmaceutical and cosmetic industries to extend shelf life and ensure food safety.

Neutrophil extracellular traps characterize caseating granulomas

Tuberculosis (TB) remains one of the top 10 causes of death worldwide and still poses a serious challenge to public health. Recent attention to neutrophils has uncovered unexplored areas demanding further investigation. Therefore, the aim of this study was to determine neutrophil activation and circulatory neutrophil extracellular trap (NET) formation in various types of TB. Sera from TB patients (n = 91) and healthy controls (NHD; n = 38) were analyzed for NE-DNA and MPO–DNA complexes, cell-free DNA (cfDNA), and protease activity (elastase). We show that these NET parameters were increased in TB sera. Importantly, NET formation and NE activity were elevated in TB patients with extensive tissue damage when compared to those with minor damage and in patients with relapse, compared to new cases. We discuss the importance of balancing NET formation to prevent tissue damage or even relapse and argue to analyze circulating NET parameters to monitor the risk of disease relapse. To investigate the tissues for NETs and to find the source of the circulating NET degradation products, we collected sections of granulomas in lung and lymph node biopsies. Samples from other diseases with granulomas, including sarcoidosis (SARC) and apical periodontitis (AP), served as controls. Whereas NET formation characterizes the caseating granulomas, both caseating and non-caseating granulomas harbor DNA with unusual conformation. As TB is associated with hypercoagulation and thromboembolism, we further imaged the pulmonary vessels of TB patients and detected vascular occlusions with neutrophil aggregates. This highlights the dual role of neutrophils in the pathology of TB.

Comparative Characteristics of Inflammatory Markers in Alzheimer’s Disease in Males and Females

Background: the role of the sex factor in the differences between the manifestations of normality and pathology is not limited to the phenomenon of sexual dimorphism. It is known that the prevalence of certain diseases in males and females is different, in particular, there is a multiple increase in the incidence of dementia in females compared to males in Alzheimer’s disease (AD). Taking into account the role of neuroinflammation in the pathogenesis of neurodegenerative diseases, there is reason to assume gender differences in inflammation indicators at different stages of dementia in AD. The aim of the study was to conduct comparative analysis of indicators of the inflammatory system in the blood plasma of males and females at different stages of Alzheimer’s disease. Patients, control group and methods: a total of 210 patients with AD (101 males and 109 females) aged 49 to 94 years (average age 72.3 ± 8.2) with varying degrees of dementia severity, i.e. mild, moderate, severe, were examined. In peripheral blood plasma, the enzymatic activity of leukocyte elastase (LE) and the functional activity of α1-proteinase inhibitor (α1-PI) were determined by the spectrophotometric method, and the level of C-reactive protein (CRP) and IL-6 were determined by the enzyme-linked immunosorbent method (ELISA). The control group consisted of 52 healthy people, who did not differ from the patients in age and gender. Results and discussion: in the blood of patients with varying severity of dementia in AD, a statistically significant increase in α1-PI activity was observed compared to controls (p < 0.0001), regardless of gender. For all subgroups of patients with AD, the indicators of LE enzymatic activity were within the control range or beyond its lower limit. Low LE activity was observed in males compared to females both in the general group and in moderate dementia (p = 0.005105, p = 0.028672, respectively). In severe dementia, a significant decrease in LE activity compared to the controls did not depend on gender. Low LE activity in the blood of patients with AD, along with elevated levels of other inflammatory markers, may reflect a critical violation of the permeability of the blood-brain barrier and/or functional exhaustion of neutrophils due to a long-term inflammatory process. In males, compared with females, an increase in the level of the pro-inflammatory cytokine IL-6 was detected in the general group and in moderate dementia (p = 0.021238, p = 0.027894, respectively). A highly significant increase in CRP levels was only detected in males in subgroups with different severity of dementia. CRP levels in males were significantly higher than in females at the stage of moderate and severe dementia (p = 0.000906, p = 0.000049, respectively). Conclusion: distinctive features of inflammatory markers spectrum were identified, depending on gender and severity of dementia in AD. These results can be used to develop sex-specific preventive or therapeutic strategies for patients with mild cognitive impairment to determine risk and resistance to developing dementia.

Synovial fibroblasts from children with oligoarticular juvenile idiopathic arthritis induce migration and prolong viability of neutrophils.

Little is known of the processes that trigger neutrophil activation in the joint of patients with oligoarticular juvenile idiopathic arthritis (oJIA), and if synovial fibroblasts (S-Fib) play an important role in the activation. Therefore, we aimed to investigate whether S-Fib derived from oJIA patients drive neutrophil activation. Synovial fluid (SF) was collected from patients with oJIA. S-Fib were isolated from the SF of n = 7 patients through passaging. Subsequently, the S-Fib were primed or not with 20% of pooled SF. Supernatants were used to study migration of neutrophils in a transwell system. Additionally, the influence of S-Fib on neutrophils were studied in co-cultures. Phenotype and viability were assessed by flow cytometry. Neutrophil function was tested through the production of reactive oxygen species (ROS), and supernatants were tested for myeloperoxidase (MPO) release and elastase activity. Supernatants of S-Fib induced neutrophil migration (n = 5, p = 0.0491), which was further pronounced using supernatants from SF-primed S-Fib (p = 0.0063). Additionally, co-culture between SF-primed S-Fib and neutrophils resulted in prolonged viability (n = 5, p = 0.0094), with little effect on activation markers, e.g., CD11b. Conversely, co-culture did not induce functional alterations (n = 4), such as production of ROS (p > 0.1570), release of MPO (p > 0.4934) or elastase activity (p > 0.0904). Finally, supernatant stimulation did not replicate the results of prolonged viability (p = 0.9102), suggesting a role of cell-contact. S-Fib from patients with oJIA induce migration of neutrophils via soluble mediators and, in addition, S-Fib prolong neutrophil viability in a cell-contact dependent manner. These mechanisms could be important for accumulation of neutrophils during arthritis.

Molecular characterization of Pseudomonas aeruginosa from diabetic foot infections in Tunisia.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

Green synthesis of silver nanoparticles using food supplement from Avena sativa L., and their antioxidant, antiglycation, and anti-aging activities: In vitro and in silico studies

Food supplement of Avena sativa L. dry extract (AsDE) is produced with green leaves of wild oats and it is commonly used in patients with cognitive disorders and muscle deficits. These symptoms and pathologies can be promoted and/or aggravated by oxidative stress and protein glycation. In order to prevent or treat these disorders, new formulations that could enhance and benefit AsDE have been developed and the formation of metallic nanoparticles by green synthesis has shown to be an alternative. Thus, this study aimed to prepare silver nanoparticles (AgNPs) by green synthesis using AsDE and characterize these AgNPs. Besides, it aimed to evaluate in vitro antioxidant and antiglycation activities of AsDE and AgNPS, and analyze in vitro and in silico inhibitory action on aging enzymes (collagenase, elastase, and tyrosinase). UV–visible spectroscopy, Zeta potential and scanning transmission electron microscopy were used to characterize AgNPs. Antioxidant activity was determined by DPPH free radical scavenging, ferric ion reducing power (FRAP), lipid peroxidation inhibition, ABTS radical scavenging, and oxygen radical absorption capacity (ORAC). Molecular docking analyses were performed to evaluate interactions between the major compounds of AsDE and aging enzymes or DNA. Antioxidant tests demonstrated antioxidant activity of 19.67 % for AsDE and 42.98 % for AgNPs in DPPH test; and 115.67 µM Trolox Equivalent (TE)/g for AsDE and 554.33 µM TE/g for AgNPs in FRAP test. In addition, AsDE and AgNPs inhibited 25.40 % and 11.33 % of lipid peroxidation, respectively, and presented 255.22 µM TE/g for AsDE and 317.44 µM TE/g for AgNPs in ABTS test. AsDE and AgNPs exhibited antioxidant potential observed in ORAC assay. In addition, AsDE presented inhibitory action on collagenase, elastase and tyrosinase activities (78.23 %, 68.14 % and 62.27 % inhibition, respectively). Antiglycation tests demonstrated that bovine serum albumin exposed to ribose and treated with AsDE at 0.5 mg/mL exhibited the highest percentage of free amino groups (45.21 %), while samples treated with AgNPs showed 35.41 % free amino groups. On the other hand, AgNPs exhibited the highest percentage of inhibition of advanced glycation end-products formation (85.75 %), which did not differ from aminoguanidine, a known antiglycation agent. Besides, relative electrophoretic mobility assay demonstrated that samples treated with AsDE at 0.1 mg/mL or AgNPs showed antiglycation activity comparable to aminoguanidine. The in silico assays showed interactions between the major compounds of AsDE and aging enzymes or DNA. Therefore, results represent an important indicator for the development and discovery of new nanostructured pharmaceutical and cosmetic formulations using plants and their bioactive compounds.