OBJECTIVE: To determine whether moxibustion had an anti-inflammatory effect on rheumatoid arthritis (RA) by regulating Annexin 1 expression and interfering with the phospholipaseA2 signaling pathway.
METHODS: Thirty male Sprague-Dawley rats were randomly categorized into five groups (six rats per group): blank control (CON) group, RA model (RA) group, moxibustion (MOX) group, Annexin 1 lentiviral intervention (RNAi-Anxa1) group, and Annexin 1 lentiviral intervention + moxibustion (RNAi-Anxa1 + MOX) group. The rats in the RNAi-Anxa1 and the RNAi-Anxa1 + MOX groups were injected with the lentiviral vector-mediated RNAi-Anxa1 into the rat foot pad. An experimental RA rat model was established by injecting Freund\'s complete adjuvant (FCA) into the RA, MOX, RNAi-Anxa1, and RNAi-Anxa1 + MOX groups. Rats in the MOX and RNAi-Anxa1 + MOX groups received moxibustion treatment. After modeling, using moxibustion \"Shenshu (BL23)\" and \"Zusanli (ST36)\", each point is 5 times, bilateral alternating, once a day, 6 times for a course of treatment, between the courses of rest for a one day. A total of three treatment courses were conducted. Both bilateral pad thicknesses were measured using Vernier calipers on experimental days 1, 7, 14, 21, and 28. The expression of cPLA2α signaling in the synovium of diseased joints was observed using Western blot. The pathology of the rat ankle synovium was observed using hematoxylin-eosin (HE) staining. Interleukin (IL)-1β, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) were detected using enzyme-linked immunosorbent assay.
RESULTS: Moxibustion increased the levels of Annexin 1 and decreased the inflammatory response in rats with RA. After increasing the expression of Annexin 1, the phosphorylated expression of cPLA2α was inhibited, the serum levels of IL-1β, PGE2, and LTB4 decreased, and the level of IL-10 increased. In moxibustion treated RA rats after the Annexin 1 lentiviral intervention, the serum levels of IL-1β, PGE2, LTB4, and IL-10 were almost unchanged.
CONCLUSIONS: Moxibustion enhanced the negative regulation of the cPLA2α signaling pathway, increased the synovial Annexin 1 expression, inhibited the cPLA2α signaling pathway, indirectly inhibited the expression of downstream inflammatory factors, and played a role in reducing inflammation.

Currently, the search for new alternatives to conventional antibiotics to combat bacterial resistance is an urgent task, as many microorganisms threaten human health due to increasing bacterial resistance to traditional medicines. Thus, new molecules such as antimicrobial peptides have emerged as promising alternatives because of their low induction of resistance and broad spectrum of action. In this context, in the past few years, our research group has synthesized and characterized a peptide derived from the C-terminal region of the Lys49 PLA2-like BthTX-I, named p-BthTX-I. After several studies, the peptide (p-BthTX-I)2K was proposed as the molecule with the most considerable biotechnological potential. As such, the present work aimed to evaluate whether the modifications made on the peptide (p-BthTX-I)2K can be applied to other molecules originating from the C-terminal region of PLA2-like Lys49 from snake venoms. The peptides were obtained through the solid-phase peptide synthesis technique, and biochemical and functional characterization was carried out using dichroism techniques, mass spectrometry, antimicrobial activity against ESKAPE strains, hemolytic activity, and permeabilization of lipid vesicles. The antimicrobial activity of the peptides was promising, especially for the peptides (p-AppK)2K and (p-ACL)2K, which demonstrated activity against all strains that were tested, surpassing the model molecule (p-BthTX-I)2K in most cases and maintaining low hemolytic activity. The modifications initially proposed for the (p-BthTX-I)2K peptide were shown to apply to other peptides derived from Lys49 PLA2-like from snake venoms, showing promising results for antimicrobial activity. Future assays comparing the activity of the dimers obtained through this strategy with the monomers of these peptides should be carried out.

Endogenous phospholipase A2 (PLA2) plays an important role in phospholipids degradation during cured meat products manufacturing. The present study was undertaken to reveal more information about the endogenous PLA2 in muscles and its role in degradation of intramuscular phospholipids. With the catalytic domain of pork calcium-independent PLA2 (iPLA2cd), impacts of physic-chemical factors on the activity were investigated and substrate specificity of the enzyme were tested respectively. The optimum temperature and pH of pork iPLA2cd were 40 °C and 7.5, respectively. The iPLA2cd could be stimulated by adequate contents of NaCl and ATP, and inhibited by CaCl2 and NaNO2. For native phospholipids, the iPLA2cd was of a little higher affinity towards phosphatidylcholine (PC) than phosphatidylethanolamine (PE), phosphoserine (PS) and phosphatidylinositol (PI). The iPLA2cd could preferentially hydrolyze peroxidized PC over the native PC. The results would help better understand the degradation of phospholipids and the role played by endogenous enzymes during meat products manufacturing.

Phospholipase A2 (PLA2) is a key enzyme involved in the formation of pro-inflammatory mediators like eicosanoids. Inhibition of PLA2 is regarded as one of the effective methods of controlling inflammation. The present study investigated the binding potentials of three natural compounds, rosmarinic acid (RA), capsaicin (CAP), and curcumin (CUR) by means of in silico and in vitro methods. Our study revealed that RA has relatively better binding affinity and inhibition potentials when compared to the other two molecules. Our ITC experiments were also suggested a slightly better binding energy for the RA. The stoichiometry of the protein ligand complex obtained from one of the ITC experiments suggested the possibilities of binding of a small molecule MCW (degraded product of CUR) on PLA2. Overall study demonstrated that the anti-inflammatory activity of RA, CUR and CAP may be partly due to the inhibition of PLA2.

Vesicle exo- and endocytosis mediate important biological functions, including synaptic transmission. In this issue of Cell Reports Methods, Seong J. An et al. found that the fluorescently tagged C2 domain of phospholipase A2 binds to membrane phosphatidylcholine and thus labels vesicle membrane, allowing for super-resolution and electron microscopic visualization of vesicle trafficking.

This work compared the presence of phospholipase A2 inhibitors (PLIs) in the serum of 19 snake species maintained at Instituto Butantan to better understand the mechanisms of venom resistance in snakes and improve the treatment of snakebite. PLI was isolated from blood of 19 snake species by one-step chromatography and identified in all samples, besides its identity was confirmed through the interaction with both phospholipase A2 and anti-γPLI. These findings highlight the diversity of snake serum PLIs and emphasize the importance of structure-function studies.

Therapeutics with activity specifically at the inflamed sites throughout the gastrointestinal tract (GIT) would be a major advance in our therapeutic approach to inflammatory bowel disease (IBD). We aimed to develop the prodrug approach that can allow such site-specific drug delivery. Currently, using cyclosporine as a drug of choice in IBD is limited to the most severe cases due to substantial systemic toxicities and narrow therapeutic index of this drug. Previously, we synthesized a series of a phospholipid-linker-cyclosporine (PLC) prodrugs designed to exploit the overexpression of phospholipase A2 (PLA2) in the inflamed intestinal tissues, as the prodrug-activating enzyme. Nevertheless, the extent and rate of prodrug activation differed significantly. In this study we applied in-vitro and modern in-silico tools based on molecular dynamics (MD) simulation, to gain insight into the dynamics and mechanisms of the PLC prodrug activation. We aimed to elucidate the reason for the significant activation change between different linker lengths in our prodrug design. Our work reveals that the PLC conjugate with the 12-carbon linker length yields the optimal prodrug activation by PLA2 in comparison to shorter linker length (6-carbons). This optimized length efficiently allows cyclosporine to be released from the prodrug to the active pocket of PLA2. This newly developed mechanistic approach, presented in this study, can be applied for future prodrug optimization to accomplish optimal prodrug activation and drug targeting in various conditions that include overexpression of PLA2.

Snakebite is a neglected disease with a high impact in tropical and subtropical countries. Therapy based on antivenom has limited efficacy in local tissue damage caused by venoms. Phospholipases A2 (PLA2) are enzymes that abundantly occur in snake venoms and induce several systemic and local effects. Furthermore, sulfur compounds such as thioesters have an inhibitory capacity against a snake venom PLA2. Hence, the objective of this work was to obtain a carbodithioate from a thioester with known activity against PLA2 and test its ability to inhibit the same enzyme. Benzyl 4-nitrobenzenecarbodithioate (I) was synthesized, purified, and characterized using as precursor 4-nitrothiobenzoic acid S-benzyl ester (II). Compound I showed inhibition of the enzymatic activity a PLA2 isolated from the venom of the Colombian rattlesnake Crotalus durissus cumanensis with an IC50 of 55.58 μM. This result is comparable with the reported inhibition obtained for II. Computational calculations were performed to support the study, and molecular docking results suggested that compounds I and II interact with the active site residues of the enzyme, impeding the normal catalysis cycle and attachment of the substrate to the active site of the PLA2.

Soil fungi play an important role in the environment decomposing dead organic matter and degrading persistent organic pollutants (POP). The presence of hydrophobic POP in the soil and membrane-lytic substances excreted by competing microorganism to the soil solution is the constant threat to these organisms. To survive in the harsh environment and counteract these hazards the fungal cells have to strictly control the composition of the lipids in their cellular membranes. However, in the case of fungal membranes the correlation between their composition and physical properties is not fully understood. In our studies we applied Langmuir monolayers formed by phospholipids typical to fungal membranes and ergosterol as versatile model membranes. These membranes were characterized by the Langmuir technique, Brewster Angle Microscopy and Grazing Incidence X-ray Diffraction, as well as were exposed to the action of phospholipase A2 treated as a model membrane-lytic protein. We started our studies from the equimolar mixture of phosphatidylethanolamine with phosphatidylcholine and doped this matrix with phosphatidylserine (PS) or phosphatidylinositol (PI). It turned out that the membranes with PS were much more condensed at the mesoscale and periodically organized at the molecular level. Starting from these models we derived two families of model fungal membranes adding to these phospholipid matrices ergosterol. It turned out that the level of ergosterol content is of crucial importance for the model membrane structure and its durability. Changing the ergosterol mole ratio from 0 to 0.5 we defined and described in detail four different 2D crystalline phases.

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.