(S)-ML-SA1 ACTIVATES AUTOPHAGY VIA TRPML1-TFEB PATHWAY.

Autophagic flux plays a crucial role in various diseases. Recently, the lysosomal ion channel TRPML1 has emerged as a promising target in lysosomal storage diseases, such as mucolipidosis. The discovery of mucolipin synthetic agonist-1 (ML-SA1) has expanded our understanding of TRPML1's function and its potential therapeutic uses. However, ML-SA1 is a racemate with limited cellular potency and poor water solubility. In this study, we synthetized rac-ML-SA1, separated the enantiomers by chiral liquid chromatography and determined their absolute configuration by vibrational circular dichroism (VCD). In addition, we focused on investigating the impact of each enantiomer of ML-SA1 on the TRPML1-TFEB axis. Our findings revealed that (S)-ML-SA1 acts as an agonist for TRPML1 at the lysosomal membrane. This activation prompts transcription factor EB (TFEB) to translocate from the cytosol to the nucleus in a dose-dependent manner within live cells. Consequently, this signaling pathway enhances the expression of coordinated lysosomal expression and regulation (CLEAR) genes and activates autophagic flux. Our study presents evidence for the potential use of (S)-ML-SA1 in the development of new therapies for lysosomal storage diseases that target TRPML1.

Postoperative neutrophil-to-lymphocyte ratio variation is associated with chronic subdural hematoma recurrence.

INTRODUCTION: Chronic subdural haematoma (CSDH) is one of the most common neurosurgical pathologies. The recurrence of chronic subdural haematomas is an important concern, considering that elderly patients are the most affected and reoperations in these patients may represent a risk of neurological and clinical complications. In accordance with the inflammatory theory regarding CSDH and its recurrence, we aimed to evaluate the role of an inflammatory marker, neutrophil-to-lymphocyte ratio (NLR), as a risk factor and prognostic variable for CSDH recurrence. METHODS: We performed a cohort study of adult patients operated for post-traumatic CSDH traumatic CSDH between January 2015 and December 2019 in our neurotrauma unit, whose data was retrospectively retrieved. We excluded patients with previous inflammatory or infectious diseases as well as use of anticoagulant/antiplatelet medications. Neutrophil and lymphocyte counts were obtained 24 h preoperatively and 48-72 h postoperatively. The primary endpoint was symptomatic recurrence of CSDH up to 1 year after the surgery. An independent sample was used to validate the findings. RESULTS: The testing sample comprised 160 patients (59.4% male, mean age 69.3 ± 14.3 years, recurrence rate 22.5%). Postoperative neutrophil count and NLR were higher in those who recurred, as well as the neutrophils (median 1.15 vs 0.96, p = 0.022) and NLR (median 1.29 vs 0.79, p = 0.001) postoperative-to-preoperative ratios. Preoperative laboratory parameters or other baseline variables were not associated with recurrence. Postoperative NLR ratio (each additional unit, OR 2.53, 95% CI 1.37-4.67, p = 0.003) was independently associated with recurrence. The best cut-off for the postoperative NLR ratio was 0.995 (AUC-ROC 0.67, sensitivity 63.9%, specificity 76.6%). Postoperative NLR ratio ≥ 1 (i.e. a post-operative NLR that does not decrease compared to the preoperative value) was associated with recurrence (OR 4.59, 95% CI 2.00-10.53, p < 0.001). The validation sample analysis (66 patients) yielded similar results (AUC-ROC 0.728, 95% CI 0.594-0.862, p = 0.002) and similar cut-off (≥ 1.05, sensitivity 77.8%, specificity 66.7%). CONCLUSION: NLR ratio can be a useful parameter for the prediction of post-traumatic CSDH recurrence. This hypothesis was validated in an independent sample and the accuracy was moderate.

The Brazilian octocoral Phyllogorgia dilatata as a source of cytotoxic compounds.

The extensive marine biodiversity has proved to be a promising source of substances with biomedical potential. In this study, the cytotoxicity of the Brazilian octocoral Phyllogorgia dilatata (Gorgoniidae) was evaluated against two tumor cell lines and three bacterial strains. The methanol/dichloromethane crude extract presented no antibacterial activity up to the highest concentration tested (512 µg/mL), however it revealed a noteworthy antiproliferative effect against HCT-116 (80%) and MCF-7 (54%) cell lines at 50 µg/mL. Therefore, guided by the cytotoxic activity, a multistep chemical fractionation of the extract provided the subfraction 5 (PDPH2-5) with IC50 values of 3.18 and 17.80 µg/mL against HCT-116 and MCF-7, respectively. The LC-HRMS/MS analysis of PDPH2-5 showed ions of m/z 219.1742 and 219.1743, characterized as (E,E) and (Z,E) germacrone, after a LC-DAD-SPE/NMR analysis of the hexanic fraction and comparisons of NMR data with the literature. Previously reported assessments to the cytotoxic activity of the (E,E)-diastereoisomer disclosed higher IC50 values than that obtained for the PDPH2-5 fraction, suggesting, herein, a potentiated effect of the diastereoisomeric mixture. Such remark encourage further bioactivity studies with stereoisomer mixtures and reduce the urge for compound isolation.

Integrated analytical workflow for chromatographic profiling and metabolite annotation of a cytotoxic Phorbas amaranthus extract.

Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.

Phosphoenolpyruvate carboxykinase from T. cruzi magnetic beads affinity-based screening assays on crude plant extracts from Brazilian Cerrado.

In T. cruzi, a causative agent of Chagas disease, phosphoenolpyruvate carboxykinase (TcPEPCK) is associated with carbohydrate catabolism. Due to its importance in the metabolism of the parasite, it has become a promising target for the development of new drugs against Chagas disease. Aiming to investigate different approaches for ligands screening, TcPEPCK was immobilized on amine-terminated magnetic beads (TcPEPCK-MB) and kinetically characterized by liquid chromatography tandem mass spectrometry activity assay with a KMapp value of 10 ± 1 µM to oxaloacetate as substrate. Natural products library affords highly diverse molecular frameworks through their secondary metabolites, herein a ligand fishing TcPEPCK-MB assay is described for prospecting ligands in four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora (Vochysiaceae), Diospyros burchellii (Ebenaceae), Anadenanthera falcata (Fabaceae) and Byrsonima coccolobifolia (Malpighiaceae). The chemical characterization of eleven identified ligands was carried out by liquid chromatography tandem high-resolution mass spectrometry experiments. Senecic acid, syneilesinolide A, phytosphingosine and vanillic acid 4-glucopyranoside are herein reported for the first time for Q. grandiflora, D. burchellii, A. falcata, respectively. In addition, the specificity of the assay was observed since only catechin was fished out from the ethanolic extract of B. coccolobifolia leaves, despite the presence of epicatechin epimer.