Biology subjects

Cardoso, F. C.

Publications and source records attributed to Cardoso, F. C..

3 recordsLinked to original sources

bioRxiv2023

High-content fluorescence bioassay to investigate pore formation, ion channel modulation and cell membrane lysis induced by venoms

Venoms comprise highly evolved bioactive molecules modulating ion channels, receptors, coagulation factors, and the cellular membrane. This array of targets and bioactivity requires high-content bioassays to aid the development of novel envenomation treatments and biotechnological and pharmacological agents. To address this gap in venoms research, we developed a fluorescence-based high-throughput and high-content cellular assay to simultaneously identify common cellular activities produced by venoms: membrane lysis, pore-formation, and ion channel modulation. By combining intracellular calcium with extracellular nucleic acid measurements, we distinguished these venom mechanisms using one cellular assay. We applied our high-content bioassay in three cell types exposed to venom components representing lytic, ion pore-forming or ion channel modulator toxins. Beyond the distinct profiles produced by these three types of action mechanisms, we found that the pore-forming latrotoxin -Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes, while the lytic bee peptide melittin is not selective. Furthermore, evaluation of snake venoms showed that Elapid species induced rapid membrane lysis, while Viper species showed variable to no activity on neuroblastoma cells. These findings demonstrate that our high-content bioassay distinguish clades and interspecific traits and capture the clinical observations at venom level and is capable of differentiate ion pore-forming from membrane lysis and ion channel modulation. We hope our research will accelerate the understanding of venom biology and the diversity of toxins inducing cytotoxic, cardiotoxic and neurotoxic effects and assist in identifying venom components whose properties could benefit humankind.

pharmacology and toxicology↗

bioRxiv2022

Holistic profiling of the venom from the lethal spider Phoneutria nigriventer by combining high-throughput ion channel screens with venomics

Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. In this study, we explored the neuroactivity and molecular diversity P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggests that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

pharmacology and toxicology↗

bioRxiv2022

Characterization of rumen microbiome and metabolome from an oro-esophageal probe and fluid, particulate and fluid-particulate fractions from rumen fistula in Holstein dairy cow

Less invasive rumen sampling methods such as oro-esophageal probes became widely popular to explore the rumen microbiome and metabolome. However, it remains unclear if such methods represent well the rumen contents from fluid and particulate fractions. Herein, we characterized the microbiome and metabolome in rumen content collected by an oro-esophageal probe and fluid, particulate, and the combined fluid-particulate fractions collected by rumen fistula in ten multiparous Holstein dairy cows. The 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Untargeted metabolome was characterized using gas chromatography of a time-of-flight mass spectrometer. Although the pH of oro-esophageal samples was greater than those of fluid, fluid-particulate, and particulate ones, we found no difference in alpha and beta-diversity of their microbiomes. Bacteroidetes, Firmicutes, and Proteobacteria were consistently the top three most abundant phyla representing ~90% of all detected phyla across all samples. The overall metabolome PLS-DA of oro-esophageal samples was similar to the fluid-particulate samples but differed from fluid and particulate. Enrichment analysis pathways revealed few differences between oro-esophageal and fluid-particulate samples, such as the synthesis of unsaturated fatty acids. The results of the current study suggest that oro-esophageal sampling can be a proxy to screen the rumen microbiome with the 16S platform and overall fluid-particulate metabolome for a single-time and diet context. Nonetheless, studies focusing specifically on fluid and particulate metabolomes and specific metabolic pathways should carefully consider the sampling method used. IMPORTANCEThe techniques used to collect the rumen contents (oro-esophageal probe and rumen fistula) suggested potential differences in the populations of rumen microbes, and the implications of these techniques for high throughput studies characterizing the rumen microbiome and metabolome need further elucidation. Ten rumen-fistulated Holstein dairy cows were used to characterize the microbiome and metabolome of samples collected using an oro-esophageal probe and the rumen-fistula fluid, particulate, and fluid-particulate fractions. The results of the current study suggest that oro-esophageal sampling represents well the rumen microbiome and overall fluid-particulate metabolome. However, fluid and particulate metabolomes and specific metabolic pathways across all types of rumen samples differed, indicating that studies focused on the characterization of rumen metabolome variable fractions should carefully consider the sampling method used.

microbiology↗