Mitochondria from the salt-tolerant yeast <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> (halophilic organelles?)

The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na⁺ and 10 mM K⁺; these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca²⁺ or Mg²⁺. However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K⁺ and Na⁺, and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K⁺/Na⁺-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na⁺ or K⁺. In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a ΔΨ due to a low AOX activity. In D. hansenii mitochondria K⁺ and Na⁺ optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.     

Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (K_d = 1.8 nM) and MSA5 (K_d = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective K_d values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.     

The roles of Cdc42 and Rac1 in the formation of plasma membrane protrusions in cancer epithelial HeLa cells

The inducible model of clones generated from the cervical cancer epithelial HeLa cell line has shown the role of DOCK10 as a guanine-nucleotide exchange factor for Rho GTPases Cdc42 and Rac1 and as an inducer of filopodia and plasma membrane (PM) ruffles. In this model, constitutively active (CA) mutants of Cdc42 and Rac1 promote filopodia and ruffles, respectively, as in other models. DOCK9 also induces filopodia and ruffles, although ruffling activity is less prominent. By exploiting this model further, the aim of this work is to provide a more complete understanding of the role of Cdc42 and Rac1, and their interactions with DOCK10 and DOCK9, in regulation of PM protrusions. New clones have been generated from HeLa, including single clones expressing one form of wild-type (WT) or dominant negative (DN) Cdc42 or Rac1, and double clones co-expressing one of them together with either DOCK10 or DOCK9. Expression of WT- and DN-Cdc42 induced filopodia. WT-Cdc42 and, especially, DN-Cdc42 also gave rise to veil protrusions, which were neutralized by DOCK10. Moreover, DN-Cdc42 stimulated the emergence of ruffles, further increased by DOCK10, and WT-Cdc42 also augmented ruffles in presence of DOCK9 and DOCK10. WT-Rac1 greatly increased PM blebbing, as did DN-Rac1 more moderately. In both cases, blebs were enhanced by DOCK10. DN-Rac1 and CA-Rac1 moderately raised filopodia, and DOCK10 and DOCK9 had opposed effects on filopodia (up and down, respectively) in presence of WT-Rac1. As conclusions, we highlight that Cdc42 promotes filopodia regardless of its conformational state, and Rac1 induces blebs in conformations other than CA, especially WT-Rac1, in the inducible HeLa clones. The model could be useful to learn more about the mechanisms underlying PM protrusions.

     

Insights into RNA biology from an atlas of mammalian mRNA-binding proteins

RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.     

Epidemiology of Invasive Fungal Infections in Latin America

The pathogenic role of invasive fungal infections (IFIs) has increased during the past two decades in Latin America and worldwide, and the number of patients at risk has risen dramatically. Working habits and leisure activities have also been a focus of attention by public health officials, as endemic mycoses have provoked a number of outbreaks. An extensive search of medical literature from Latin America suggests that the incidence of IFIs from both endemic and opportunistic fungi has increased. The increase in endemic mycoses is probably related to population changes (migration, tourism, and increased population growth), whereas the increase in opportunistic mycoses may be associated with the greater number of people at risk. In both cases, the early and appropriate use of diagnostic procedures has improved diagnosis and outcome.     

A New Glyptodont (Xenarthra: Cingulata) from the Late Miocene of Argentina: New Clues About the Oldest Extra-Patagonian Radiation in Southern South America

Glyptodonts (Xenarthra, Cingulata) are one of the most amazing Cenozoic South American mammals, with some terminal forms reaching ca. two tons. The Paleogene record of glyptodonts is still poorly known, although some of their diversification is observable in Patagonian Argentina. Since the early and middle Miocene (ca. 19–13 Ma), two large clades can be recognized in South America. One probably has a northern origin (Glyptodontinae), while the other one, called the “austral clade”, is interpreted to have had an austral origin, with the oldest records represented by the “Propalaehoplophorinae” from the late early Miocene of Patagonian Argentina. In this scenario, the extra-Patagonian radiations are still poorly known, despite their importance for understanding the late Miocene and Pliocene diversity. Here, we carry out a comprehensive revision of late Miocene (Chasicoan Stage/Age) glyptodonts of central Argentina (Buenos Aires and San Juan provinces). Our results show that, contrary to what is traditionally assumed, it was a period of very low diversity, with only one species recognized in this region, Kelenkura castroi gen et sp. nov. Our phylogenetic analysis shows that this species represents the sister taxon of the remaining species of the “austral clade”, representing the first branch of the extra-Patagonian radiation. Additionally, K. castroi is the first taxon showing a “fully modern” morphology of the caudal tube.

     

Microscopic origin of gating current fluctuations in a potassium channel voltage sensor

Voltage-dependent ion channels open and close in response to changes in membrane electrical potential due to the motion of their voltage-sensing domains (VSDs). VSD charge displacements within the membrane electric field are observed in electrophysiology experiments as gating currents preceding ionic conduction. The elementary charge motions that give rise to the gating current cannot be observed directly, but appear as discrete current pulses that generate fluctuations in gating current measurements. Here we report direct observation of gating-charge displacements in an atomistic molecular dynamics simulation of the isolated VSD from the KvAP channel in a hydrated lipid bilayer on the timescale (10-μs) expected for elementary gating charge transitions. The results reveal that gating-charge displacements are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1-S3 transmembrane segments in the hydrated interior of the VSD.     

Gene methylation parallelisms between peripheral blood cells and oral mucosa samples in relation to overweight

Epigenetics has an important role in the regulation of metabolic adaptation to environmental modifications. In this sense, the determination of epigenetic changes in non-invasive samples during the development of metabolic diseases could play an important role in the procedures in primary healthcare practice. To help translate the knowledge of epigenetics to public health practice, the present study aims to explore the parallelism of methylation levels between white blood cells and buccal samples in relation to obesity and associated disorders. Blood and buccal swap samples were collected from a subsample of the Spanish cohort of the Food4Me study. Infinium HumanMethylation450 DNA Analysis was carried out for the determination of methylation levels. Standard deviation for β values method and concordance correlation analysis were used to select those CpG which showed best parallelism between samples. A total of 277 CpGs met the criteria and were selected for an enrichment analysis and a correlation analysis with anthropometrical and clinical parameters. From those selected CpGs, four presented high associations with BMI (cg01055691 in GAP43; r = ?0.92 and rho = ?0.84 for blood; r = ?0.89 and rho = ?0.83 for buccal sample), HOMA-IR (cg00095677 in ATP2A3; r = 0.82 and rho = ?0.84 for blood; r = ?0.8 and rho = ?0.83 for buccal sample) and leptin (cg14464133 in ADARB2; r = ?0.9182 and rho = ?0.94 for blood; r = ?0.893 and rho = ?0.79 for buccal sample). These findings demonstrate the potential application of non-invasive buccal samples in the identification of surrogate epigenetic biomarkers and identify methylation sites in GAP43, ATP2A3 and ADARB2 genes as potential targets in relation to overweight management and insulin sensibility.