A soluble recombinant form of human leucocyte antigen-G 6 (srHLA-G6).

Human Leucocyte Antigen-G (HLA-G) is a non classical major histocompatibility complex (MHC) molecule that through RNA splicing can encode seven isoforms which are membrane bound (-G1, -G2, -G3 and -G4) and soluble (-G5, -G6 and -G7). HLA-G is described as important immune suppressor endogenous molecule to favor maternal-fetal tolerance, transplant survival and tumor immune scape. HLA-G shows low protein variability and a unique structural complexity that is related with the expression of different isoforms followed by biochemical processes, such as, proteolytic cleavage, molecular interactions, and protein ubiquitination. Studies with HLA-G have shown difficult to assess the role of the individual isoforms. Thus, the aim of this work was to obtain a HLA-G6 recombinant form. The results indicated the production of high homogeneous preparations of soluble recombinant HLA-G6 (srHLA-G6) with molecular mass 23,603.76 Da, determined by MALD-TOF/TOF. In addition, native and denatured srHLA-G6 were detected by ELISA, using commercial monoclonal antibodies. Finally, we developed a suitable methodology to express srHLA-G6 that could contribute in structural and functional studies involving specific isoforms.

Naphthoquinone derivatives as potential immunomodulators: prospective for COVID-19 treatment.

Inflammation plays a crucial role in COVID-19, and when it becomes dysregulated, it can lead to severe outcomes, including death. Naphthoquinones, a class of cyclic organic compounds widely distributed in nature, have attracted significant interest due to their potential biological benefits. One such naphthoquinone is 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa-1,3-dienyl)-naphthanthene-1,4-dione (3,5,8-TMON), a compound produced by fungi. Despite its structural similarity to shikonin, limited research has been conducted to investigate its biological properties. Therefore, the objective of this study was to evaluate the effects of 3,5,8-TMON and its synthetic derivatives in the context of inflammation induced by lipopolysaccharide (LPS) and SARS-CoV-2 infection in vitro using cell cultures. 3,5,8-TMON was obtained by acid treatment of crude extracts of fermentation medium from Cordyceps sp., and two derivatives were accessed by reaction with phenylhydrazine under different conditions. The results revealed that the crude extract of the fungi (C. Ex) inhibited the activity of transcription factor NF-kB, as well as the production of nitric oxide (NO) and interleukin-6 (IL-6) when LPS induced it in RAW 264.7 cells. This inhibitory effect was observed at effective concentrations of 12.5 and 3.12 µg mL-1. In parallel, 3,5,8-TMON and the new derivatives 3 and 4 demonstrated the ability to decrease IL-6 production while increasing TNF, with a specific effect depending on the concentration. These concentration-dependent agonist and antagonist effects were observed in THP-1 cells. Furthermore, 3,5,8-TMON inhibited IL-6 production at concentrations of 12.5 and 3.12 µg mL-1 in Calu-3 cells during SARS-CoV-2 viral infection. These findings present promising opportunities for further research into the therapeutic potential of this class of naphthoquinone in the management of inflammation and viral infections.

Host macrocyclic acylcarnitines mediate symbiotic interactions between frogs and their skin microbiome.

The host-microbiome associations occurring on the skin of vertebrates significantly influence hosts' health. However, the factors mediating their interactions remain largely unknown. Herein, we used integrated technical and ecological frameworks to investigate the skin metabolites sustaining a beneficial symbiosis between tree frogs and bacteria. We characterize macrocyclic acylcarnitines as the major metabolites secreted by the frogs' skin and trace their origin to an enzymatic unbalance of carnitine palmitoyltransferases. We found that these compounds colocalize with bacteria on the skin surface and are mostly represented by members of the Pseudomonas community. We showed that Pseudomonas sp. MPFS isolated from frogs' skin can exploit acylcarnitines as its sole carbon and nitrogen source, and this metabolic capability is widespread in Pseudomonas. We summarize frogs' multiple mechanisms to filter environmental bacteria and highlight that acylcarnitines likely evolved for another function but were co-opted to provide nutritional benefits to the symbionts.

Intestinal permeability and nutritional status in developmental disorders.

CONTEXT: Autism is a developmental disorder with a possible connection between dietary components and triggering or worsening of symptoms. An altered intestinal permeability might allow absorption of incompletely digested peptides (gluten and casein) that could produce opioid-like activity on the brain, causing significant changes in behavior. OBJECTIVE: To assess the intestinal permeability and nutritional status of participants with developmental disorders to determine if changes in the intestinal mucosal barrier and/or injury to the intercellular junctions have occurred that might justify application of further dietary modifications. DESIGN: To assess intestinal permeability, the research team analyzed participants urine under fasting conditions, using gas chromatography to determine chromatographic peaks. To assess nutritional status, the team determined participants heights and weights and performed a bioelectric bioimpedance examination at least 4 hours after their most recent meal. In addition, the team determined food intake using three diet diaries. They asked participants and caregivers to register each food consumed during 2 nonconsecutive weekdays and 1 weekend day. SETTING: The study occurred at the Ribeirao Preto School of Medicine, Sao Paulo University. PARTICIPANTS: Seven participants aged 9 to 23 years with developmental disorders (the developmental group, DG) completed the study. The research team recruited them through the Association of Friends of the Autistic Persons of Ribeirao Preto in Ribeirao Preto, Brazil. The control group (CG) consisted of nonsmoking healthy volunteers in the general population who were similar in age to the experimental group and did not suffer from diseases that potentially could influence nutritional status and intestinal function. INTERVENTION: To assess intestinal permeability, participants ingested 150 mL of an isosmolar solution of the sugars mannitol (2 g) and lactulose (7.5 g) under fasting conditions and the researchers collected all voided urine over a period of 5 hours. OUTCOME MEASURES: Using chromatographic peaks, the research team quantified the mannitol and lactulose in participants urine by calculating the percentage excreted in relation to the ingested amounts of sugar. This calculation gave them the lactulose-to-mannitol ratio (L/M). To evaluate nutritional status, they used data regarding bioimpedance resistance, heights, and weights to estimate lean mass and body water (in liters). They classified adults and adolescents using the body mass index (BMI). For children (2-10 y), they classified participants height-to-age and weight-to-height ratios. The research team used food intake to examine the macronutrient interval, the mean added sugar consumption, and the quantity of protein, in g/kg weight. RESULTS: Participants with developmental disorders (n = 7) were more likely to be overweight. Their usual diet revealed a high intake of lipids (%) and proteins (g/kg) (compared to reference values) and a high intake of calories (kcal) and carbohydrates (%) (compared to CG) as well as a high intake of food sources that are important contributors of casein and gluten. The DGs (n = 7) mean mannitol excretion was lower, and their L/M higher than the CGs (n = 7) (P < .05). Their increased L/M may indicate atrophy of the intestinal-mucosa surface and/or injury to the intercellular junctions or the effect of some other abnormality. The small number of participants, however, prevented more complex statistical analysis. CONCLUSIONS: Researchers need to complete additional studies to confirm the existence of abnormalities in autistic individuals intestines and to justify the use of dietary restrictions on gluten and casein to improve the symptoms of autism.

Eco-Friendly Pharmaceutical Analysis of Rifaximin in Tablets by HPLC-MS and Microbiological Turbidimetry.

Rifaximin, an antimicrobial used for the treatment of various diseases, lacks analytical methods in official compendia for evaluation of the final product. This paper presents an eco-friendly protocol for rifaximin tablets by high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). The method was completely validated according to the International Conference on Harmonization guidelines and developed following the concept of Quality by Design. The separation was achieved using a C18 column, purified water +0.1% glacial acetic acid and ethyl alcohol, 52:48 (v/v), as mobile phase, 0.9 mL min-1 at 290 nm and ambient room temperature. Mass spectral analyses were performed using electrospray ionization (ESI) ion source and ion trap mass analyzer. The method was linear over the concentration range of 5-50 µg mL-1. The sample was subjected to acidic, basic, neutral, oxidative and photolytic degradation. Degradation products did not interfere in the quantification of the rifaximin, so the method can be considered indicative of stability. Degradation products were also evaluated individually by microbiological method using Escherichia coli. The validated method could be used promisingly as green analytical strategies for detection and quantification of rifaximin in tablets.

Identification of flavonoids in Hymenaea martiana Hayne (Fabaceae) by HPLC-DAD-MSn analysis.

Hymenaea martiana is a native tree known in Brazil as 'jatobá' and used in folk medicine to treat pain and inflammation. The aim of this work was to identify the flavonoids present in the crude ethanolic extract and ethyl acetate fraction using HPLC-DAD and LC-MSn analysis. The ethanolic extract was partitioned to obtain the ethyl acetate fraction. The analysis of astilbin content also was carried out by HPLC analysis. HPLC-DAD-ESI/MSn analysis of the ethanolic extract and ethyl acetate fraction revealed the presence of eleven peaks in the chromatograms, and all these peaks were identified: taxifolin, eucryphin, astilbin and 3 diastereoisomers, engeletin and 2 diastereoisomers, quercitrin and 2,6,3',4'-tetrahydroxy-2-benzylcoumaran-3-one. The ethyl acetate fraction had a higher astilbin concentration (151.87 µg/mL) than the ethanolic extract (40.13 µg/mL). In conclusion, the species could be considered a good source of flavonoids, which can be related to the main chemotaxonomic markers for the genus Hymenaea.

Cleavage of Peptides from Amphibian Skin Revealed by Combining Analysis of Gland Secretion and in Situ MALDI Imaging Mass Spectrometry.

Peptides from skin secretions of amphibians are considered important components of their immune system and also play a relevant role in their defense mechanism against predators. Herein, by using mass spectrometry (MS), we characterize the sequence of 13 peptides from the gland secretion of the hylid tree frog, Boana punctata. Using in situ matrix-assisted laser desorption ionization imaging MS of a transverse section of the skin tissue, we show that some peptides are stored as longer molecules that are cleaved after being secreted, whereas others do not undergo any modification. Sequence comparison with peptides from other Boana species and analysis of the three-dimensional theoretical structure indicate that this cleavage depends on both the presence of a specific sequence motif and the secondary structure. The fact that peptides undergo a rapid cleavage upon secretion suggests that stored and secreted peptides may have distinct roles for anuran survival, including defense against pathogens and predators.

Iontophoresis-stimulated silk fibroin films as a peptide delivery system for wound healing.

Silk fibroin (SF) films containing a peptide, neurotensin (NT), stimulated by iontophoresis were developed aiming to modulate the inflammatory process and prevent the growth of microorganisms typical of wounds. NT-loaded SF films composition shows predominance of ß-sheet structures that conferred adequate mechanical properties, transparency, moderate roughness and low swelling index to fibroin films. Infrared spectroscopy and thermal analysis suggested the presence of non-covalent interactions between NT and fibroin. Using the MALDI imaging technique, it was possible to visualize the homogeneous NT distribution throughout the film surface, in addition to its prolonged release for up to 72 h. In vitro studies in E. coli liposaccharide-stimulated macrophages showed a significant reduction of interleukins production after NT-loaded film application, whereas NT solution did not reduce them. Bi-laminated NT-loaded fibroin films containing silver electrodes provided a burst release of NT when anodic iontophoresis was applied, enabling a rapid onset of drug action. In addition, anodic iontophoresis presented a bacteriostatic effect against gram-positive microorganisms. Different iontophoresis densities, from 0.2 to 0.6 mA/cm2, did not significantly reduce fibroblast viability after 30 min of application. In conclusion, iontophoretic-stimulated peptide-loaded fibroin films could be a promising platform for the treatment of wounds.

Combining in vitro and in silico approaches to evaluate the multifunctional profile of rosmarinic acid from Blechnum brasiliense on targets related to neurodegeneration.

Natural products are important sources of chemical diversity leading to unique scaffolds that can be exploited in the discovery of new drug candidates or chemical probes. In this context, chemical and biological investigation of ferns and lycophytes occurring in Brazil is an approach adopted by our research group aiming at discovering bioactive molecules acting on neurodegeneration targets. In the present study, rosmarinic acid (RA) isolated from Blechnum brasiliense showed an in vitro multifunctional profile characterized by antioxidant effects, and monoamine oxidases (MAO-A and MAO-B) and catechol-O-methyl transferase (COMT) inhibition. RA showed antioxidant effects against hydroxyl (HO(•)) and nitric oxide (NO) radicals (IC50 of 29.4 and 140 µM, respectively), and inhibition of lipid peroxidation (IC50 of 19.6 µM). In addition, RA inhibited MAO-A, MAO-B and COMT enzymes with IC50 values of 50.1, 184.6 and 26.7 µM, respectively. The MAO-A modulation showed a non-time-dependent profile, suggesting a reversible mechanism of inhibition. Structural insights on RA interactions with MAO-A and COMT were investigated by molecular docking. Finally, RA (up to 5 mM) demonstrated no cytotoxicity on polymorphonuclear rat cells. Taken together, our results suggest that RA may be exploited as a template for the development of new antioxidant molecules possessing additional MAO and COMT inhibition effects to be further investigated on in vitro and in vivo models of neurodegenerative diseases.