Biology subjects

Sanchez, H.

Publications and source records attributed to Sanchez, H..

9 recordsLinked to original sources

bioRxiv2026

A single cooperative experience increases corticolimbic synaptic density and prosocial behavior

Cooperation is typically studied as an output of the social brain, as opposed to an experience that shapes it. Here, we developed a task in rats that permits interaction while requiring joint action for reward. Cooperation depended on dyad familiarity and visual access. Anterior cingulate cortex (ACC) to basolateral amygdala (BLA) projections were activated during cooperation with a familiar partner, whereas ACC to anterior insula (AI) projections were simultaneously suppressed regardless of familiarity. A single 90 min cooperative experience increased presynaptic marker SV2A, detected by positron emission tomography (PET), in the amygdala and insula one day later. Concurrently, rodents demonstrated heightened attention toward a distressed conspecific. Cooperation is thus an experience that recruits dissociable corticolimbic pathways and changes brain and behavior beyond the encounter itself.

neuroscience↗

bioRxiv2025

Structure of active methyl-CoM reductase, Earth's main methane producer

Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the {beta}-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes.

biochemistry↗

bioRxiv2024

Cell Cycle Regulation has Shaped Budding Yeast Replication Origin Structure and Function

Eukaryotic DNA replication initiates from multiple genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by Origin Recognition Complex (ORC), Cdc6 and Cdt1 via sequential hexamer loading from two opposed ORC binding sites. Cyclin Dependent Kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to G1 phase. Here we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MO intermediate); CDK phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO functions by stabilising ORC at the lower affinity binding sites required for second hexamer loading. Origins comprising two high affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers; these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.

biochemistry↗

bioRxiv2023

Metabolic reprogramming during Candida albicans planktonic-biofilm transition is modulated by the ZCF15 and ZCF26 paralogs

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 specifically impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their direct role in reprogramming cellular metabolism by regulating glycolytic cycle and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling.

microbiology↗

bioRxiv2023

A chromatinized origin reduces ORC mobility and spatially constrains MCM

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

biophysics↗

bioRxiv2023

Assessment of the Glycan-Binding Profile of Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic pathogen that can establish acute and chronic infections in individuals that lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance of P. aeruginosa. Individuals with neutropenia or cystic fibrosis are highly susceptible to P. aeruginosa infection thus underscoring the importance of the host innate immune response. Cell-to-cell contact between host innate immune cells and the pathogen, a first step in phagocytic uptake, is facilitated by simple and complex glycan structures present at the host cell surface. We have previously shown that endogenous polyanionic N-linked glycans localized to the cell surface of phagocytes mediate binding and subsequent phagocytosis of P. aeruginosa. However, the suite of glycans that P. aeruginosa binds to on host phagocytic cells remains poorly characterized. Here we demonstrate, with the use of exogenous N-linked glycans and a glycan array, that P. aeruginosa PAO1 preferentially attaches to a subset of glycans, including a bias towards monosaccharide versus more complex glycan structures. Consistent with these findings, we were able to competitively inhibit bacterial adherence and uptake by the addition of exogenous N-linked mono- and di-saccharide glycans. We discuss of findings in the context of previous reports of P. aeruginosa glycan binding. IMPORTANCEP. aeruginosa binds to a variety of glycans as part of its interaction with host cells, and a number of P. aeruginosa-encoded receptors and target ligands have been described that allow this microbe to bind to such glycans. Here we extend this work by studying the glycans used by P. aeruginosa PAO1 to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that could facilitate host cell-binding by this microbe. This study provides an increased understanding of the glycans bound by P. aeruginosa, and furthermore, provides a useful dataset for future studies of P. aeruginosa-glycan interactions.

microbiology↗

bioRxiv2022

Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation

The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks1. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies2,3. Conversely, single-molecule studies of CMG motion have thus far4-6 relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents7,8. Here, we report the first activation at the single-molecule level of CMG fully reconstituted from purified yeast proteins and the quantification of its motion. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel without melting it, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication.

biophysics↗

bioRxiv2019

A robotic platform for fluidically-linked human body-on-chips experimentation

Here we describe of an Interrogator instrument that uses liquid-handling robotics, a custom software package, and an integrated mobile microscope to enable automated culture, perfusion, medium addition, fluidic linking, sample collection, and in situ microscopic imaging of up to 10 Organ Chips inside a standard tissue culture incubator. The automated Interrogator platform maintained the viability and organ-specific functions of 8 different vascularized, 2-channel, Organ Chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier (BBB), and brain) for 3 weeks in culture when fluidically coupled through their endothelium-lined vascular channels using a common blood substitute medium. When an inulin tracer was perfused through the multi-organ Human Body-on-Chips (HuBoC) fluidic network, quantitative distributions of this tracer could be accurately predicted using a physiologically-based multi-compartmental reduced order (MCRO) in silico model of the experimental system derived from first principles. This automated culture platform enables non-invasive imaging of cells within human Organ Chips and repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling, which should facilitate future HuBoc studies and pharmacokinetics (PK) analysis in vitro.

bioengineering↗

bioRxiv2018

Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function, drug penetration, and antibody shuttling properties

The highly specialized human brain microvascular endothelium forms a selective blood-brain barrier (BBB) with adjacent pericytes and astrocytes that restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip (Organ Chip) model of the BBB lined by induced pluripotent stem cell-derived human brain microvascular endothelium (iPS-BMVEC) interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins, multiple functional efflux pumps, and displays selective transcytosis of peptides and anti-transferrin receptor antibodies previously observed in vivo. This increased level of barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.\n\nThe human blood-brain barrier (BBB) is a unique and selective physiological barrier that controls transport between the blood and the central nervous system (CNS) to maintain homeostasis for optimal brain function. The BBB is composed of brain microvascular endothelial cells (BMVECs) that line the capillaries as well as surrounding extracellular matrix (ECM), pericytes, and astrocytes, which create a microenvironment that is crucial to BBB function1. The brain microvascular endothelium differs from that found in peripheral capillaries based on its complex tight junctions, which restrict paracellular transit and instead, require that transcytosis be used to transport molecules from the blood through the endothelium and into the CNS2. BMVECs also express multiple broad-spectrum efflux pumps on their luminal surface that inhibit uptake of lipophilic molecules, including many drugs, into the brain3,4. The astrocytes and pericytes provide signals that are required for differentiation of the BMVECs5,6, and all three cell types are needed to maintain BBB integrity in vivo as well as in vitro7-9. The BBB is also of major clinical relevance because dysfunction of the BBB associated is observed in many neurological diseases, and the efficacy of drugs designed to treat neurological disorders is often limited by their inability to cross the BBB10. Unfortunately, neither animal models of the BBB nor in vitro cultures of primary or immortalized human BMVECs alone effectively mimic the barrier and transporter functions of the BBB observed in humans11-14. Thus, there is a great need for a human BBB model that could be used to develop new and more effective CNS-targeting therapeutics and delivery technologies as well as advance fundamental and translational research8,9.\n\nDevelopment of human induced pluripotent stem (iPS) cell technology has enabled differentiation of brain-like microvascular endothelial cells (iPS-BMVECs) that exhibit many properties of the human BBB, including well-organized tight junctions, expression of nutrient transporters and polarized efflux transporter activity15,16. The trans-endothelial electrical resistance (TEER) values exhibited by the permeability barrier generated by these human iPS-BMVECs reach physiological levels ([~]3000-5000 {Omega}{middle dot}cm2) within 24-48 h when cultured in Transwell inserts or within a microfluidic organ-on-a-chip (Organ Chip) device15,17-19, a level that is more than an order of magnitude higher than TEER values previously reported in other in vitro human BBB models6,17,20.\n\nHowever, the usefulness of these iPS-BMVEC models for studies on targeted delivery to the CNS is limited because they can only maintain these high TEER levels for [~]2 days, and the expression of efflux pumps in these iPS-BMVECs does not fully mimic those of human brain endothelium in vivo21. Here, we describe the development of an enhanced human BBB model created with microfluidic Organ Chip culture technology22,23 that contains human iPS-BMVECs interfaced with primary human pericytes and astrocytes, and that uses a developmentally-inspired differentiation protocol24-26. The resulting human BBB Chip exhibits physiologically relevant levels of human BBB function for at least one week in vitro, including low barrier permeability and expression of multiple efflux pumps and transporter functions that are required for analysis of drug and therapeutic antibody transport.

bioengineering↗