José Antonio Fagundes Assumpção

Image-based spatial transcriptomics depends on cell segmentation to assign transcripts to individual cells, but how segmentation algorithms perform across tissues with distinct cellular architectures is poorly understood. This study presents the broadest independent benchmark to date of cell segmentation algorithms for spatial transcriptomics, comparing five approaches across ten mouse tissues using a 5,006-gene Xenium panel. To quantify segmentation errors, Co-expression Rejection in Segmentation Purity (CRISP) was developed, an open-source tool available in R and Python that measures cell purity through tissue-specific mutually exclusive marker co-expression without requiring ground truth annotations. This benchmark revealed that segmentation algorithms face a fundamental tradeoff between maximizing transcript capture and maintaining cell purity, and that the severity of this tradeoff is tissue-dependent. Proseg achieved the highest average performance across tissues, though the magnitude of its advantage varies with tissue architecture. Overall, CRISP provides per-tissue performance profiles as a practical resource for algorithm selection.

Objectives: Caffeine is extensively consumed as a psychostimulant drug, acting on A₁and A_2Aadenosine receptors blockade. Chronic exposure to caffeine during gestation and breast‐feeding may be involved in infant rat's behavioral and biochemical alterations. Our goal was to evaluate the effect of chronic caffeine exposure during gestation and breast‐feeding in the functionality of adenosine A₁receptors in infant rats at P14. NTPDase and 5'‐nucleotidase activities were also evaluated.Methods: Mating of adult female Wistar rats was confirmed by presence of sperm in vaginal smears. Rats were divided into three groups on the first day of pregnancy: (1) control: tap water, (2) caffeine: 0.3 g/L until P14, and (3) washout caffeine: caffeine was changed to tap water at P7. Evaluation of nociceptive response was performed at P14 using hot plate (HP) and tail‐flick latency (TFL) tests. A₁receptor involvement was assessed using caffeine agonist (CPA) and antagonist (DPCPX). Enzymatic activities assays were conducted in the spinal cord.Results: Gestational and breastfeeding exposure to caffeine (caffeine and washout groups) did not induce significant alterations in thermal nociceptive thresholds (HP and TF tests). Both caffeine groups did not show analgesic response induced by CPA when compared to the control group at P14, indicating chronic exposure to caffeine in the aforementioned periods inhibits the antinociceptive effects of the systemic A₁receptor agonist administration. No effect was observed upon ectonucleotidase activities.Conclusions: Our results demonstrate that chronic caffeine exposure in gestational and breastfeeding alters A1‐mediated analgesic response in rats.

Background/Aim: Magnetic stimulation is used in the treatment of a diversity of diseases, but a complete understanding of the underlying mechanisms of action requires further investigation. We examined the effect of static magnetic stimulation (SMS) in different cell lines. Materials and Methods: A culture plate holder with attached NeFeB magnets was developed. Different magnetic field intensities and periods were tested in tumoral and non-tumoral cell lines. To verify the cellular responses to SMS, cell viability, cell death, cell cycle and BDNF expression were evaluated. Results: Exposure of SH-SY5Y cells to SMS for 24 hours led to a decrease in cell viability. Analysis 24 h after stimulation revealed a decrease in apoptotic and double-positive cells, associated with an increase in the number of necrotic cells. Conclusion: The effects of SMS on cell viability are cell type-specific, inducing a decrease in cell viability in SH-SY5Y cells. This suggests that SMS may be a potential tool in the treatment of neuronal tumors.

Neuromodulatory techniques have been studied to treat drug addiction or compulsive eating as well as different chronic pain conditions, such as neuropathic

Omega 3-docosahexaenoic acid (DHA) and vitamin E Delta-tocotrienol (Delta-T3) are extensively studied as protective nutrients against cancer development. Little is known about the biological mechanisms targeted by these bioactive molecules on lipid droplet (LD) biogenesis, an important breast cancer aggressiveness marker, and the occurrence of lipophagy in breast cancer cells. The aim of this study was to investigate the effect of DHA, Delta-T3 and DHA plus Delta-T3 co-treatment in LD biogenesis and lipophagy process in triple negative breast cancer cell line MDA-MB-231. Cells were treated with 50 μM DHA and/or 5 μM Delta-T3. Our results demonstrated that DHA can trigger an increase in LD biogenesis and co-treatment with Delta-T3 was able to reduce this LD biogenesis. In addition, we showed that a higher cytoplasmic LD content is associated with a higher breast cancer cells malignance and proliferation. Reduction of cytoplasmic LD content by silencing ADRP (adipose differentiation-related protein), a structural LD protein, also decreased cell proliferation in MDA-MB-231 cells. Treatment with DHA and Delta-T3 alone or co-treatment did not reduce cell viability. Moreover, we showed here that DHA can trigger lipophagy in MDA-MB-231 cells and DHA plus Delta-T3 co-treatment was able to enhance this lipophagy process. Our findings demonstrated that co-treatment with DHA plus Delta-T3 in MDA-MB-231 cells could reduce LD biogenesis and potentiate lipophagy in these cells, possibly having a positive impact to inhibit breast cancer malignancy. Therefore, suitable doses of DHA and Delta-T3 vitamin E isoform supplementation can be a prominent tool in therapeutic treatments against breast cancer.

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The current article describes the synthesis, characterization, and application of a designed hybrid fluorescent BTD–coumarin (2,1,3-benzothiadiazole-coumarin) derivative (named BTD-Lip). The use of BTD-Lip for live-cells staining showed excellent results, and lipid droplets (LDs) could be selectively stained. When compared with the commercially available dye (BODIPY) for LD staining, it was noted that the designed hybrid fluorescence was capable of staining a considerable larger number of LDs in both live and fixed cells (ca. 40% more). The new dye was also tested on live Caenorhabditis elegans (complex model) and showed an impressive selectivity inside the worm, whereas the commercial dye showed no selectivity in the complex model.

The current manuscript describes the use of a heteropolyacid-containing task-specific ionic liquid, supported in imidazolium-based ionic liquids, as the catalyst for an efficient multicomponent synthesis of hexahydroimidazo[1,2-alpha]pyridine derivatives. The reactions conditions were fully optimized and the bridgehead nitrogen heterocycle derivatives could be obtained in just one hour exclusively as a single isomer (trans). Single crystal X-ray analysis confirmed the trans derivative as the only isomer. The mechanism of the reaction was investigated by ESI(+)-MS(/MS) and the use of the elegant charge-tag strategy allowed a catalytic cycle to be proposed for the current transformation and revealed the possibility of at least two reaction pathways. One derivative bearing a coumarin scaffold were synthesized and its fluorescent properties allowed it to be tested as a probe for live cell imaging experiments with a preference for mitochondria.

The implication of inflammation in pathophysiology of several type of cancers has been under intense investigation. Omega-3 fatty acids can modulate inflammation and present anticancer effects, promoting cancer cell death. Pyroptosis is an inflammation related cell death and so far, the function of docosahexaenoic acid (DHA) in pyroptosis cell death has not been described. This study investigated the role of DHA in triggering pyroptosis activation in breast cancer cells. MDA-MB-231 breast cancer cells were supplemented with DHA and inflammation cell death was analyzed. DHA-treated breast cancer cells triggered increased caspase-1and gasdermin D activation, enhanced IL-1β secretion, translocated HMGB1 towards the cytoplasm, and membrane pore formation when compared to untreated cells, suggesting DHA induces pyroptosis programmed cell death in breast cancer cells. Moreover, caspase-1 inhibitor (YVAD) could protect breast cancer cells from DHA-induced pyroptotic cell death. In addition, membrane pore formation showed to be a lysosomal damage and ROS formation-depended event in breast cancer cells. DHA triggered pyroptosis cell death in MDA-MB-231by activating several pyroptosis markers in these cells. This is the first study that shows the effect of DHA triggering pyroptosis programmed cell death in breast cancer cells and it could improve the understanding of the omega-3 supplementation during breast cancer treatment.