Biology subjects

Briones, C.

Publications and source records attributed to Briones, C..

5 recordsLinked to original sources

bioRxiv2025

Ultrasensitive graphene FET aptasensor for direct attomolar detection of glutamate in human clinical samples

Glutamate, the principal excitatory neurotransmitter in the brain, is crucial for cognition and memory, and its dysregulation is implicated in several neurological disorders, including Alzheimers Disease and epilepsy. However, precise and high-throughput quantification of glutamate in physiological samples remains challenging. Here, we report an ultrasensitive and highly specific glutamate aptamer-based biosensor, or aptasensor, developed through in silico design and microfabrication, followed by in vitro and clinical validation. The biosensor consists of arrays of graphene field-effect transistors functionalized with a novel DNA aptamer, NG-Apt-Glu, designed and characterized computationally and biochemically, revealing two putative glutamate binding sites. The aptasensor detects glutamate in artificial cerebrospinal fluid with a 1 aM detection limit, a wide linear range (1 aM-10 pM), and 24 mV/decade sensitivity, showing strong selectivity against GABA, glutamine, dopamine, and serotonin. To evaluate clinical applicability, glutamate levels were measured in cerebrospinal fluid from patients with Alzheimers Disease, showing a significant increase relative to controls and correlating with neurofilament light chain concentrations, a biomarker of neuronal death. These findings underscore glutamates involvement in Alzheimers pathophysiology and its potential as a biomarker for neurodegeneration. This ultrasensitive graphene-based aptasensor enables point-of-care monitoring, paving the way for early diagnosis and the development of novel therapeutic strategies.

bioengineering↗

bioRxiv2024

Phase Separation Clustering of Poly Ubiquitin Cargos on the Ternary Mixture Lipid Membranes by Synthetically Cross-Linked Ubiquitin Binder Peptides.

Ubiquitylation is involved in various physiological processes in our bodies such as signaling and vesicle trafficking, thus Ubiquitin (UB) is an important medical target system of interest. Polymeric addition of UB allows cargo molecules to be recognized specifically by multivalent binding interaction with UB binding proteins leading to various downstream processes. Recently, implication of protein condensate formation by Ubiquitylated proteins have been reported in many independent UB processes suggesting its potential role in governing the spatial organization of Ubiquitylated cargo proteins. We created modular polymeric UB binder motifs and polymeric UB cargos by synthetic bioconjugation and protein purification. Giant unilamellar vesicles with lipid raft composition were created to reconstitute polymeric UB cargo organization on the membranes. Fluorescence imaging was used to observe the outcome. We found that polymeric UB cargos clustered on the membranes by forming phase separation codomains in interaction with multivalent UB binder conjugate. This phase separation was valency dependent and was strongly correlated to their potency to form protein condensate droplets in solution. Multivalent UB binding interactions showed a general trend toward the formation of phase separated condensates, and the resulting condensates were either in liquid-like or solid-like state depending on the conditions and interactions used. It implies that polymeric UB cargos on the plasma and endosomal membranes may use the codomain phase separation to assist clustering of UB cargos on the membranes for cargo sorting. Our work also shows that model systems of such phase behavior can be created by modular synthetic approach that can potentially be used to further engineer biomimetic interactions in vitro. Graphical abstracts (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/608403v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@eca49dorg.highwire.dtl.DTLVardef@2b64d2org.highwire.dtl.DTLVardef@e82b98org.highwire.dtl.DTLVardef@862558_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Spontaneous transfer of small peripheral peptides between supported lipid bilayer and giant unilamellar vesicles

Vesicular trafficking facilitates material transport between membrane-bound organelles. Membrane protein cargos are trafficked for relocation, recycling, and degradation during various physiological processes. In vitro fusion studies utilized synthetic lipid membranes to study the molecular mechanisms of vesicular trafficking and to develop synthetic materials mimicking the biological membrane trafficking. Various fusogenic conditions which can induce vesicular fusion have been used to establish synthetic systems that can mimic biological systems. Despite these efforts, the mechanisms underlying vesicular trafficking of membrane proteins remain limited and robust in vitro methods that can construct synthetic trafficking systems for membrane proteins between large membranes (>1 m2) are unavailable. Here, we provide data to show the spontaneous transfer of small membrane-bound peptides ([~]4 kD) between a supported lipid bilayer (SLB) and giant unilamellar vesicles (GUVs). We found that the contact between the SLB and GUVs led to the occasional but notable transfer of membrane-bound peptides in a physiological saline buffer condition (pH 7.4, 150 mM NaCl). Quantitative and dynamic time-lapse analyses suggested that the observed exchange occurred through the formation of hemi-fusion stalks between the SLB and GUVs. Larger protein cargos with a size of [~]77 kD could not be transferred between the SLB and GUVs, suggesting that the larger-sized cargos limited diffusion across the hemi-fusion stalk, which was predicted to have a highly curved structure. Our system serves as an example synthetic platform that enables the investigation of small-peptide trafficking between synthetic membranes and reveals hemi-fused lipid bridge formation as a mechanism of peptide transfer. Graphical abstracts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/544237v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f72d8corg.highwire.dtl.DTLVardef@cf7ed5org.highwire.dtl.DTLVardef@8fee0forg.highwire.dtl.DTLVardef@1565484_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Amino acid substitutions associated with treatment failure of hepatitis C virus infection

Despite the high virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV from 220 patients who failed therapy, which are not bona fide resistance-associated substitutions (RAS). They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. Also, they have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value of treatment response are discussed.

microbiology↗

bioRxiv2020

Dissimilar conservation pattern in hepatitis C virus mutant spectra, consensus sequences, and data banks

The influence of quasispecies dynamics on long-term virus diversification in nature is a largely unexplored question. Specifically, whether intra-host nucleotide and amino acid variation in quasispecies fits variation observed in consensus sequences or data bank alignments is unknown. Genome conservation and dynamics simulations are used for the computational design of universal vaccines, therapeutic antibodies and pan-genomic antiviral agents. The expectation is that selection of escape mutants will be limited when mutations at conserved residues are required. This strategy assumes long-term (epidemiologically relevant) conservation but, critically, does not consider short-term (quasispecies-dictated) residue conservation. We have calculated mutant frequencies of individual loci from mutant spectra of hepatitis C virus (HCV) populations passaged in cell culture and from infected patients. Nucleotide or amino acid conservation in consensus sequences of the same populations, or in the Los Alamos HCV data bank did not match residue conservation in mutant spectra. The results relativize the concept of sequence conservation in viral genetics, and suggest that residue invariance in data banks is an insufficient basis for the design of universal viral ligands for clinical purposes. Our calculations suggest relaxed mutational restrictions during quasispecies dynamics, which may contribute to higher calculated short-term than long-term viral evolutionary rates.

microbiology↗