Biology subjects

Fernandez, N. B.

Publications and source records attributed to Fernandez, N. B..

5 recordsLinked to original sources

bioRxiv2024

Investigating the genetic relationship of Trichosporon asahii isolates using a newly developed microsatellite typing assay

The basidiomycetous yeast-like fungus Trichosporon asahii is increasingly encountered in the clinic and has been known to cause nosocomial infections. Here, we aim to develop a microsatellite typing tool to investigate the genetic relatedness of Trichosporon asahii isolates. Six microsatellite markers were selected from a nanopore long-read sequencing de novo assembled genome of the T. asahii type-strain CBS 2479. PCR reactions amplified three di-, tri-, or tetranucleotide repeats, respectively. All six microsatellite markers were used to analyze 111 T. asahii isolates, including single and related-patient isolates from clinical and environmental sources. Each marker exhibited between 11 and 37 alleles in this population, resulting in 71 different microsatellite genotypes. The Simpsons diversity index ranged from 0.6452 to 0.8280 for the individual markers, and for the combined set of microsatellites 0.9793, indicating the high discriminatory power of the reported panel. In summary, this panel combines high reproducibility and specificity, making it suitable for use in epidemiological studies of T. asahii and in investigations of potential outbreaks caused by this pathogen.

molecular biology↗

bioRxiv2022

RUNX1 is regulated by androgen receptor to promote cancer stem markers and chemotherapy resistance in triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which no effective targeted therapies are available. Growing evidence suggests that chemotherapy-resistant cancer cells with stem-like properties (CSC) may repopulate the tumor. Androgen receptor (AR) is expressed in up to 50% of TNBC, and AR inhibition decreases CSC and tumor initiation. Runt-related transcription factor 1 (RUNX1) correlates with poor prognosis in TNBC and is regulated by AR in prostate cancer. Our group has shown that RUNX1 promotes TNBC cell migration and regulates tumor gene expression. We hypothesized that RUNX1 is regulated by AR and that both may work together in TNBC CSC to promote disease recurrence following chemotherapy. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) experiments in MDA-MB-453 revealed AR binding to RUNX1 regulatory regions. RUNX1 expression is upregulated by dihydrotestosterone (DHT) in MDA-MB-453 and in HCI-009 patient-derived xenograft (PDX) tumors (p<0.05). RUNX1 is increased in a CSC-like experimental model in MDA-MB-453 and SUM-159PT cells (p<0.05). Inhibition of RUNX1 transcriptional activity reduced the expression of CSC markers. Interestingly, RUNX1 inhibition reduced cell viability and enhanced paclitaxel and enzalutamide sensitivity. Targeting RUNX1 may be an attractive strategy to potentiate the anti-tumor effects of AR inhibition, specifically in the slow growing CSC-like populations that resist chemotherapy leading to metastatic disease.

cancer biology↗

bioRxiv2022

Selection of Synthetic Proteins to Modulate the Human Frataxin Function

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreichs Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five selection rounds. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of supercomplex activity. Affi_224 and frataxin showed a KD value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (NMR) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.

biochemistry↗

bioRxiv2021

Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

biochemistry↗

bioRxiv2020

Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 {degrees}C and 52 {degrees}C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by HPLC, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

biochemistry↗