Essential oil and plant extract of oregano as agents influencing the virulence factors of Candida albicans.

Candida albicans, a polymorphic yeast, is a physiological component of the human and animal commensal microbiome. It is an etiological factor of candidiasis, which is treated by azole antifungals. Growing resistance to azoles is a reason to look for other alternative treatment options. The pharmacotherapeutic use of plant extracts and essential oils has become increasingly important. In our experiment, C. albicans showed susceptibility to four observed plant extracts and essential oils from peppermint ( Mentha piperita), thyme ( Thymus vulgaris), sage ( Salvia officinalis), and oregano ( Origanum vulgare). Oregano plant extract and essential oil showed the highest antifungal activity, at MIC values of 4.9 mg/mL and 0.4 mg/mL respectively. Therefore, it was subjected to further research on the influence of virulence factors - biofilm formation, extracellular phospholipase production and germ tube formation. Oregano plant extract and essential oil showed an inhibitory effect on the observed C. albicans virulence factors at relatively low concentrations. The extract inhibited the adherence of cells at MIC 12.5 mg/mL and essential oil at MIC 0.25 mg/mL. Degradation of the formed biofilm was detected at MIC 14.1 mg/mL for plant extract and at MIC 0.4 mg/mL for essential oil. Extracellular phospholipase production was most effectively inhibited by the essential oil. In particular, the number of isolates with intensive extracellular phospholipase production decreased significantly. Of the 12 isolates intensively producing extracellular phospholipase, only 1 isolate (4.5%) retained intense production. Essential oil caused up to a 100 % reduction in germ tubes formation and plant extract reduced their formation depending on the concentration as follows: 2.6% (0.8 mg/mL), 21.2 % (6.25 mg/mL), and 64.5 % (12.5 mg/mL) compared to the control.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 µM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.

Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

Selective inhibition of neurogenic, but not agonist-induced contractions by phospholipase A2 inhibitors points to presynaptic phospholipase A2 functions in contractile neurotransmission to human prostate smooth muscle.

BACKGROUND: Phospholipases A2 (PLA2 ) may be involved in α1 -adrenergic contraction by formation of thromboxane A2 in different smooth muscle types. However, whether this mechanism occurs with α1 -adrenergic contractions of the prostate, is still unknown. While α1 -adrenoceptor antagonists are the first line option for medical treatment of voiding symptoms in benign prostatic hyperplasia (BPH), improvements are limited, probably by nonadrenergic contractions including thromboxane A2 . Here, we examined effects of PLA2 inhibitors on contractions of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy. Contractions were induced by electric field stimulation (EFS) and by α1 -adrenergic agonists in an organ bath, after application of the cytosolic PLA2 inhibitors ASB14780 and AACOCF3, the secretory PLA2 inhibitor YM26734, the leukotriene receptor antagonist montelukast, or of solvent to controls. RESULTS: Frequency-dependent contractions of human prostate tissues induced by EFS were inhibited by 25% at 8 Hz, 38% at 16 Hz and 37% at 32 Hz by ASB14780 (1 µM), and by 32% at 16 Hz and 22% at 32 Hz by AACOCF3 (10 µM). None of both inhibitors affected contractions induced by noradrenaline, phenylephrine or methoxamine. YM26734 (3 µM) and montelukast (0.3 and 1 µM) neither affected EFS-induced contractions, nor contractions by α1 -adrenergic agonists, while all contractions were substantially inhibited by silodosin (100 nM). CONCLUSIONS: Our findings suggest presynaptic PLA2 functions in prostate smooth muscle contraction, while contractions induced by α1 -adrenergic agonists occur PLA2 -independent. Lacking sensitivity to montelukast excludes an involvement of PLA2 -derived leukotrienes in promotion of contractile neurotransmission.

PLAAT1 deficiency alleviates high-fat diet-induced hepatic lipid accumulation in mice.

The phospholipase A and acyltransferase (PLAAT) family is composed of three isoforms in mice (PLAAT1, 3, and 5), all of which function as phospholipid-metabolizing enzymes exhibiting phospholipase A1 /A2 and acyltransferase activities. Plaat3-deficient (Plaat3-/- ) mice were previously reported to show lean phenotype and remarkable hepatic fat accumulation under high-fat diet (HFD) feeding, while Plaat1-/- mice have not been analyzed. In the present study, we generated Plaat1-/- mice and investigated the effects of PLAAT1 deficiency on HFD-induced obesity, hepatic lipid accumulation, and insulin resistance. After HFD treatment, PLAAT1 deficiency caused a lower body weight gain compared to wild-type mice. Plaat1-/- mice also showed reduced liver weight with negligible hepatic lipid accumulation. In accordance with these findings, PLAAT1 deficiency improved HFD-induced hepatic dysfunction and lipid metabolism disorders. Lipidomics analysis in the liver revealed that in Plaat1-/- mice, the levels of various glycerophospholipids tended to increase, while all classes of lysophospholipids examined tended to decrease, suggesting that PLAAT1 functions as phospholipase A1 /A2 in the liver. Interestingly, the HFD treatment of wild-type mice significantly increased the mRNA level of PLAAT1 in the liver. Furthermore, the deficiency did not appear to elevate the risk of insulin resistance in contrast to PLAAT3 deficiency. These results suggested that the suppression of PLAAT1 improves HFD-induced overweight and concomitant hepatic lipid accumulation.

Hypoxia-induced GPCPD1 depalmitoylation triggers mitophagy via regulating PRKN-mediated ubiquitination of VDAC1.

Mitophagy, which selectively eliminates the dysfunctional and excess mitochondria by autophagy, is crucial for cellular homeostasis under stresses such as hypoxia. Dysregulation of mitophagy has been increasingly linked to many disorders including neurodegenerative disease and cancer. Triple-negative breast cancer (TNBC), a highly aggressive breast cancer subtype, is reported to be characterized by hypoxia. However, the role of mitophagy in hypoxic TNBC as well as the underlying molecular mechanism is largely unexplored. Here, we identified GPCPD1 (glycerophosphocholine phosphodiesterase 1), a key enzyme in choline metabolism, as an essential mediator in hypoxia-induced mitophagy. Under the hypoxic condition, we found that GPCPD1 was depalmitoylated by LYPLA1, which facilitated the relocating of GPCPD1 to the outer mitochondrial membrane (OMM). Mitochondria-localized GPCPD1 could bind to VDAC1, the substrate for PRKN/PARKIN-dependent ubiquitination, thus interfering with the oligomerization of VDAC1. The increased monomer of VDAC1 provided more anchor sites to recruit PRKN-mediated polyubiquitination, which consequently triggered mitophagy. In addition, we found that GPCPD1-mediated mitophagy exerted a promotive effect on tumor growth and metastasis in TNBC both in vitro and in vivo. We further determined that GPCPD1 could serve as an independent prognostic indicator in TNBC. In conclusion, our study provides important insights into a mechanistic understanding of hypoxia-induced mitophagy and elucidates that GPCPD1 could act as a potential target for the future development of novel therapy for TNBC patients.Abbreviations: ACTB: actin beta; 5-aza: 5-azacytidine; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; ChIP: chromatin immunoprecipitation; co-IP: co-immunoprecipitation; CQ: chloroquine; CsA: cyclosporine; DOX: doxorubicin; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GPCPD1: glycerophosphocholine phosphodiesterase 1; HAM: hydroxylamine; HIF1A: hypoxia inducible factor 1 subunit alpha; HRE: hypoxia response element; IF: immunofluorescence; LB: lysis buffer; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; LC-MS: liquid chromatography-mass spectrometry; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; MDA231: MDA-MB-231; MDA468: MDA-MB-468; MFN1: mitofusin 1; MFN2: mitofusin 2; MKI67: marker of proliferation Ki-67; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; OS: overall survival; PalmB: palmostatin B; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; SDS: sodium dodecyl sulfate; TOMM20: translocase of outer mitochondrial membrane 20; TNBC: triple-negative breast cancer; VBIT-4: VDAC inhibitor; VDAC1: voltage dependent anion channel 1; WT: wild type.

ß-Sitosterol could serve as a dual inhibitor of Trypanosoma congolense sialidase and phospholipase A2: in vitro kinetic analyses and molecular dynamic simulations.

The involvement of Trypanosoma congolense sialidase alongside phospholipase A2 has been widely accepted as the major contributing factor to anemia during African animal trypanosomiasis. The enzymes aid the parasite in scavenging sialic acid and fatty acids necessary for survival in the infected host, but there are no specific drug candidates against the two enzymes. This study investigated the inhibitory effects of ß-sitosterol on the partially purified T. congolense sialidase and phospholipase A2. Purification of the enzymes using DEAE cellulose column led to fractions with highest specific activities of 8016.41 and 39.26 µmol/min/mg for sialidase and phospholipase A2, respectively. Inhibition kinetics studies showed that ß-sitosterol is non-competitive and an uncompetitive inhibitor of sialidase and phospholipase A2 with inhibition binding constants of 0.368 and 0.549 µM, respectively. Molecular docking of the compound revealed binding energies of - 8.0 and - 8.6 kcal/mol against the sialidase and phospholipase A2, respectively. Furthermore, 100 ns molecular dynamics simulation using GROMACS revealed stable interaction of ß-sitosterol with both enzymes. Hydrogen bond interactions between the ligand and Glu284 and Leu102 residues of the sialidase and phospholipase A2, respectively, were found to be the major stabilizing forces. In conclusion, ß-sitosterol could serve as a dual inhibitor of T. congolense sialidase and phospholipase A2; hence, the compound could be exploited further in the search for newer trypanocides.

Novel Preclinical Study of Galloylquinic Acid Compounds from Copaifera lucens with Potent Antifungal Activity against Vaginal Candidiasis Induced in a Murine Model via Multitarget Modes of Action.

Vaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 µg/mL) as well as minimum fungicidal concentrations (2 to 32 µg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 µg/mL and 32 µg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis. IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

A New Berberine Preparation Protects Pancreatic Islet Cells from Apoptosis Mediated by Inhibition of Phospholipase A2/p38 MAPK Pathway.

We studied an amorphous solid dispersion of berberine with absorption enhancer sodium caprate (Huang-Gui solid dispersion preparations, HGSD). A therapeutic effect of HGSD was revealed in mice with type 2 diabetes mellitus and palmitate-induced injury to MIN6 ß-cells. HGSD treatment (150 mg/kg) improved glucose metabolism and decreased ß-cell apoptosis in diabetic mice. Furthermore, the effective component of HGSD berberine significantly attenuated the palmitate-induced decrease in MIN6 ß-cells viability and insulin secretion. Moreover, molecular docking analysis and Western blotting showed that berberine decreased cell apoptosis and expression of group VIA phospholipase A2 (iPLA2), p38 mitogen-activated protein kinase (p38 MAPK), and caspase-3. These data suggest that HGSD treatment protected ß-cells via inhibiting the iPLA2/p38 MAPK pathway.

Hypoxia-sensitive adjuvant loaded liposomes enhance the antimicrobial activity of azithromycin via phospholipase-triggered releasing for Pseudomonas aeruginosa biofilms eradication.

Robust biofilms and the complex airway environment with thick sputum, local hypoxia and persistent inflammation induce the intractability of chronic pulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). Herein, we proposed a type of antibiotic-adjuvant liposomes (NANO@PS-LPs), co-incorporating azithromycin (AZI), adjuvant (2-nitroimidazole derivative, 6-NIH) and biofilm dispersant (nitric oxide donor, DETA NONOate). NANO@PS-LPs possessing negatively-charged surface and good hydrophilicity could easily penetrate through the sputum layer, then disassembled triggered by overexpressed phospholipase A2 (PLA2) in the microenvironment around biofilms. Nitric oxide produced by DETA NONOate promoted P. aeruginosa biofilms dispersal. 6-NIH was reduced to 2-aminomidazole derivative (6-AIH) under a hypoxic condition, and hence acted as an AZI adjuvant to enhance the antibacterial activity of AZI. It was found that NANO@PS-LPs could significantly eliminate mature P. aeruginosa biofilms, effectively kill dispersed bacteria, inhibit the metabolism of survivors and prevent P. aeruginosa adherence to airway epithelial cells, accordingly restrain recurrent infections. Additionally, NANO@PS-LPs performed a remarkable advantage in killing AZI-resistant P. aeruginosa and removing their biofilms. In summary, NANO@PS-LPs present a potential nano-strategy to treat stubborn pseudomonal pulmonary infections and overcome correlative drug resistance.

Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran.

Pseudomonas aeruginosa is an important nosocomial pathogen with a capacity of resistance to multiple antibiotics and production of various extracellular and cell-associated virulence factors that clearly contribute to its pathogenicity. The objective of this study was to investigate the antibiotic susceptibility, virulence factors, and clonal relationship among clinical isolates of P. aeruginosa. Different clinical specimens from hospitalized patients were investigated for P. aeruginosa. Susceptibility of the isolates was evaluated by disc diffusion and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute (CLSI) guideline. A total of 97 P. aeruginosa isolates were recovered from clinical specimens. The percentage of isolates resistant to antimicrobials was imipenem 25.77%, meropenem 15.46%, gentamicin 16.49%, tobramycin 15.46%, amikacin 16.49%, ciprofloxacin 20.61%, levofloxacin 24.74, ceftazidime 20.61%, piperacillin 15.46%, piperacillin/tazobactam 12.37%, colistin 9.27%, and polymyxin B 11.34%. Of isolates, 87.62% possessed ß-hemolytic activity, 78.35% lecithinase, 59.8% elastase, 37.11% DNase, and 28.86% twitching motility. The frequency of virulence genes in isolates was lasB 82.47%, plcH 82.47%, exoA 58.76%, exoS 56.7%, and pilA 10.3%. ERIC-PCR typing clustered P. aeruginosa isolates to 19 common types (CT1-CT19) containing isolates from different hospitals and 43 single types (ST1-ST43). Colistin and polymyxin B were the most effective agents against the majority of P. aeruginosa isolates, emphasizing the effort to maintain their antibacterial activity as last-line therapy. The frequency of some virulence factors and genes was noticeably high, which is alarming. In addition, more effective strategies and surveillance are necessary to confine and prevent the inter-hospital and/or intra-hospital dissemination of P. aeruginosa between therapeutic centers.

Bisphenol A acts as developmental agonist in Culex quinquefasciatus Say.

Plastic wastes deposited in canals running through Thiruvananthapuram city have created stagnant waters providing breeding sites for mosquitoes. In the present study, plastic waste-derived bisphenol A (BPA) was quantified from four mosquito breeding sites. During summer rain, the concentration of BPA in the stagnant water samples was found to be between 0.86 and 1.14 mg/L, and hence 1 mg/L BPA was considered as the environmentally relevant concentration. In the present study, the effect of BPA on the life cycle and metamorphosis of filarial vector, Culex quinquefasciatus Say was elucidated by rearing larvae in water added with BPA at and above the environmentally relevant concentration viz., 1, 2, and 4 mg/L. The duration required for adult emergence was reduced from 10 to 8.5 days, when the concentration of BPA was increased from 1 to 4 mg/L respectively. Our study revealed that embryonic and larval developments were shortened by BPA treatment. BPA also caused a dose-dependent advancement of 20-hydroxyecdysone (20-E) peaks; phospholipase A2 induction; and upregulation of ecdysone receptor gene, EcRA, and ecdysone inducible gene E75A, which culminated in early pupation. No significant difference in sanguivory and fecundity was observed in adult mosquitoes treated with 1 mg/L of BPA. Our study reveals that BPA is a developmental agonist of C. quinquefasciatus.

Suppressing phosphoinositide-specific phospholipases Cγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.

INTRODUCTION: Phosphoinositide-specific phospholipases C-γ1 (PLC-γ1) signaling has been shown to modulate osteoarthritis (OA) chondrocyte metabolism. However, the role of PLC-γ1 in OA osteoblasts remains unclear. Herein, whether and how PLC-γ1 was involved in mineralization in OA subchondral bone osteoblasts were investigated. METHODS: Primary non-OA and OA osteoblasts of human and rat isolated from the subchondral bone or the calvaria were cultured in vitro, as well as mouse pre-osteoblastic cell line MC3T3-E1 cells. Rat knee OA model was induced by anterior cruciate ligament transection (ACLT), in which bone canal was carried out from the surface of lateral epicondyle of femur using micro-electric drill. Morphological characteristics of subchondral bone structure and articular cartilage were assessed using CT, micro-CT, and Safranin O/Fast green staining, respectively. Mineralization was measured by alizarin red staining. The expression and production of genes involved in osteoblastic phenotype and mineralization were evaluated by qPCR, western blotting, and immunohistochemistry assays, respectively. The inhibitions were performed using inhibitors and ShRNAs. RESULTS: The decreased relative bone density and thickness in the early stage of OA and the increased one in the late stage of OA were observed in subchondral bone of ACLT-rat model. Decreased ALP and OCN levels and absorbance values of ARS content were observed in in vitro osteoblasts isolated from 2 w post-ACLT rat model, as well as IL-1ß-treated (for maintaining and mimicking inflammatory status) human OA and rat osteoblasts. Decreased Atg7 level and LC3BII/I ratio in combination with an increase in the P62 level, was concomitant with decreased ALP and OCN mRNA levels and absorbance values of ARS content in OA or IL-1ß-treated osteoblasts. Specific inhibition of PLC-γ1 by ShRNAs or inhibitor (U73122) elevated ALP and OCN mRNA levels and absorbance values of ARS content accompanied with increased Atg7 level and LC3BII/I ratio in combination with a decrease in the P62 level in OA osteoblasts. Furthermore, the promoting effect of PLC-γ1 inhibition on ALP and OCN mRNA levels and absorbance values of ARS content was reversed by endoplasmic reticulum (ER) stress activator HA15, as well as autophagic inhibitors CQ and 3MA. Injection with PLC-γ1 inhibitor U73122 from the surface of lateral epicondyle of femur reduced aberrant subchondral bone formation and attenuated articular cartilage degeneration in ACLT-rat. CONCLUSION: Aberrant changes of OA subchondral bone structure were concomitant with altered osteoblastic phenotype and mineralization. Impaired autophagy contributed to decreased osteoblastic mineralization in the early stage of OA. PLC-γ1 inhibition promoted osteoblastic mineralization through increasing autophagy in OA osteoblasts, which was partially attributed to suppression of ER stress. Targeting PLC-γ1 in subchondral bone osteoblasts could be more efficacious for OA therapy through treating the bone and cartilage at the same time. In summary, we hypothesize that suppressing PLCγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.

Integrin-mediated human glioblastoma cells adhesion, migration and invasion by native and recombinant phospholipases of Scorpio maurus venom glands.

Integrins are a large family of cell surface receptors mediating the interaction of cells with their microenvironment and they play an important role in glioma biology. In the present work, we reported the anti-tumor effect of Sm-PLGV a phospholipase A2 from Tunisian scorpion venom glands-as well as its recombinant forms expressed in Escherichia coli-through interference with integrin receptor function in malignant glioma cells U87. These phospholipases inhibited in a dose dependent manner the adhesion, migration and invasion onto fibrinogen and fibronectin without any cytotoxicity. We showed that Sm-PLGV and its recombinant constructs blocked U87 migration by reducing their velocity and directional persistence. The inhibitory effect was related to a blockage of the integrins αvß3 and α5ß1 function. Inactivation of the enzymatic activity of Sm-PLGV by chemical modification with p-bromophenacyl bromide did not affect its anti-tumor properties, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in scorpion venom phospholipases A2.

In vitro antiplasmodial activity of phospholipases A2 and a phospholipase homologue isolated from the venom of the snake Bothrops asper.

The antimicrobial and antiparasite activity of phospholipase A(2) (PLA(2)) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A(2) (PLA(2)) (fraction V) and another containing a PLA(2) homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA(2) and its homologue have antiplasmodial potential.

Phospholipases and cationic peptides inhibit Cryptosporidium parvum sporozoite infectivity by parasiticidal and non-parasiticidal mechanisms.

The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, ß-defensin 1, ß-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, ß-defensin 1, and ß-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.

Intraspecies variability in Vipera ammodytes ammodytes venom related to its toxicity and immunogenic potential.

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms.

Ammodytoxin content of Vipera ammodytes ammodytes venom as a prognostic factor for venom immunogenicity.

Venoms are complex mixtures of proteins, peptides and other compounds whose biochemical and biological variability has been clearly demonstrated. These molecules have been used as antigens for immunization of anti-venom-producing animals (horses or sheep). Ammodytoxins (Atx) are potently neurotoxic compounds, and the most toxic compounds isolated so far from the Vipera ammodytes ammodytes (Vaa) venom. Recently we have shown that the level of antibodies specific to Vaa venom's most toxic component, ammodytoxin A (AtxA), (anti-AtxA IgG) in Vaa venom immunized rabbit sera highly correlated to the venom toxicity-neutralization potential of these sera. Here we investigated whether Atx content of Vaa venom could influence the outcome of immunization procedure. The novel ELISA was developed for precise determination of Atx content and Atx was quantified in venom samples used for immunization of rabbits. We clearly showed that animals immunized with the venom containing lower amount of Atx produced sera with significantly lower venom toxicity-neutralizing power and, vice versa, animals immunized with venoms containing higher amount of Atx produced sera with higher venom toxicity-neutralizing ability. Thus, the content of Atx in Vaa venom is a relevant parameter of its suitability in the production of highly protective Vaa anti-venom.

Pharmacological perspectives of wasp venom.

Venoms of several animals have been used to study various physiopathologic processes, and also to offer opportunity to design and develop new therapeutic drugs. We briefly review certain wasp venom components and their biological effects, which may be potential sources of novel pharmacologically active compounds.

Cell surface sialylation and fucosylation are regulated by the cell recognition molecule L1 via PLCgamma and cooperate to modulate embryonic stem cell survival and proliferation.

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown. Using a panel of carbohydrate markers, we have shown that cell surface sialylation and fucosylation are upregulated in L1-transfected embryonic stem cells (L1-ESCs). Consistently, the mRNA levels of sialyltransferase ST6Gal1 and ST3Gal4, and fucosyltransferase FUT9 were significantly increased in L1-transfected ESCs. Activation of L1 signaling promoted cell survival and inhibited cell proliferation. ShRNAs knocking down FUT9, ST6Gal1 and ST3Gal4 blocked these effects. A phospholipase Cgamma (PLCgamma) inhibitor and shRNA reduced ST6Gal1, ST3Gal4 and FUT9 mRNA levels in the L1-ESCs. Thus, embryonic stem cell surface sialylation and fucosylation are regulated via PLCgamma by L1, with which they cooperate to modulate cell survival and proliferation.