Biology subjects

Pacheco, S.

Publications and source records attributed to Pacheco, S..

10 recordsLinked to original sources

bioRxiv2025

High Dimensional Proteomic Multiplex Imaging of the Central Nervous System Using the COMET(TM) System

Sequential multiplex methodologies such as Akoya CODEX, Miltenyi MACSima, Rarecyte Orion, and others require modification of the antibodies by conjugation to an oligo or a specific fluorophore which means the use of off-the-shelf reagents is not possible. Modifications of these antibodies are typically performed via reduction chemistry and thus require verification and validation post-modification. Fixed panels are therefore developed due to various limitations including spectral overlap that creates spectral unmixing issues, steric hindrance, harsh antibody removal, and tissue degradation throughout the labeling. As such, a complex interrogation evaluating multiple study hypotheses and/or endpoints requires the development of sequential panels, reconstruction, and realignment of the tissue that necessitate a z-stack strategy. Standardized antibody panels are typically fixed and require substantial validation efforts to modify a single target and thus do not evolve with the pace of research interests. To increase the throughput of profiling cells within the human central nervous system (CNS), we developed and validated a CNS-specific library with an associated analysis platform using the newly developed Lunaphore COMETTM platform. The COMETTM is an automated staining/imaging instrument integrating a reagent deck for staining buffers and off-the-shelf label-free primary antibodies and fluorophore-labeled secondary antibodies, which feed into a circular plate holding up to 4 slides that are automatically imaged in microscope-operated control software. For this study, standard formalin fixed paraffin embedded histology slides are used. However, the COMET is capable of imaging fresh-frozen samples using specialized settings. Our methodologies address an unmet need in the neuroscience field while leveraging prior developmental efforts in the domain of immunology spatial profiling. Cataloging and validating a large series of antibodies on the COMET along with developing CNS autofluorescence management strategies while optimizing standard operating procedures have allowed for the visualization at the subcellular level. Forty analytes can be used to analyze one specimen which has clinical utility in cases in which the CNS can only be sampled by biopsy. CNS biopsies, depending on the anatomical location, can have limited available volume to a degree that requires prioritization and restriction to select analysis. In-depth bioinformatic imaging analysis can be done using standard bioinformatic tools and software such as Visiopharm(R). These results establish a general framework for imaging and quantifying cell populations and networks within the CNS while providing the scientific community with standard operating procedures.

bioinformatics↗

bioRxiv2024

Phosphorylation of α-synuclein fibrils at S129 changes DNAJB1 binding as probed by solid-state NMR

Amyloid fibrils composed of the protein -synuclein (aSyn) are implicated in the pathogenesis of synucleinopathies. These pathological fibrils, characterized by their rigid amyloid cores, also feature flexible intrinsically disordered regions (IDRs) that interact with various cellular components. Due to their solvent exposure and flexibility, the N- and C-termini, IDRs of aSyn fibrils, have been used as targets for immunotherapies and serve as binding sites for many chaperones. Chaperones are known to play a vital role in preventing and reversing amyloid formation in neurodegenerative diseases, but how they recognize misfolded proteins is often poorly understood. DNAJB1 is a co-chaperone that is part of a larger complex formed with Hsp70 and Apg2, which collectively disaggregate amyloid fibrils such as those formed by aSyn, tau, or huntingtin. Although the entire chaperone and co-chaperone complex are required for aggregate disassembly, DNAJB1 directly recognizes these amyloid fibrils. More specifically, DNAJB1 preferentially binds to the C-terminus of wild-type aSyn. However, over 90% of aSyn fibrils in Lewy bodies are phosphorylated at S129. Here, we determine the effect of S129 phosphorylation on DNAJB1-aSyn fibril binding using biochemical assays and solid-state NMR. We show that DNAJB1 preferentially binds aSyn pS129 fibrils and that the aSyn binding site of pS129 and wild-type fibrils is different. These results suggest that pS129 might influence chaperone-mediated degradation efficiency.

biophysics↗

bioRxiv2024

Academic Success and Mental Health: The Paradox of Frontoparietal-Default Mode Network Coupling among Children Facing Poverty

Childhood family income is a powerful predictor of academic achievement and mental health. Here, we ask whether children living in poverty who succeed academically are subsequently protected from, or at risk for, internalizing symptoms. Prior research indicates that children in poverty with better academic performance tend to have higher temporal coupling between the Lateral Frontoparietal Network (LFPN) and Default Mode Network (DMN) than lower-performing children in poverty. An open question is whether higher LFPN-DMN coupling has maladaptive long-term consequences for mental health for this population. In this pre-registered longitudinal study, we analyzed data from 10,829 children (1,931 in poverty) in the ABCD study across four time points (ages 9-13). Higher grades correlated with fewer internalizing symptoms; this association was more pronounced for children below poverty. Longitudinally, LFPN-DMN connectivity correlated positively with internalizing symptoms across both groups and timepoints. Thus, although higher academic performance was associated with better mental health outcomes for all children, the specific pattern of LFPN-DMN connectivity that supports academic resilience among children in poverty may be a risk factor for developing internalizing symptoms. These findings highlight the complex nature of academic resilience in the context of structural inequity. HighlightsO_LIHigh grades linked to fewer internalizing symptoms, especially for kids in poverty. C_LIO_LIHigh LFPN-DMN connectivity predicts higher internalizing symptoms in children. C_LIO_LINeural correlates of academic resilience may predispose children to internalizing. C_LIO_LIChildren below poverty had higher internalizing symptoms. C_LI

neuroscience↗

bioRxiv2022

GRAS-1 is a conserved novel regulator of early meiotic chromosome dynamics in C. elegans

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 serves as a scaffold for a multi-protein complex coordinating the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I.

cell biology↗

bioRxiv2021

Systemic gene therapy with thymosin β4 alleviates glomerular injury in mice

Plasma ultrafiltration in the kidney occurs across glomerular capillaries, which are surrounded by epithelial cells called podocytes. Podocytes have a unique shape maintained by a complex cytoskeleton, which becomes disrupted in glomerular disease resulting in defective filtration and albuminuria. Lack of endogenous thymosin {beta}4 (TB4), an actin sequestering peptide, exacerbates glomerular injury and disrupts the organisation of the podocyte actin cytoskeleton, however, the effect of exogenous TB4 therapy on podocytopathy is unknown. Here, through interrogating single-cell RNA-sequencing data of isolated glomeruli we demonstrate that Adriamycin, a toxin which injures podocytes and leads to leakage of albumin in the urine of mice, results in reduced levels of podocyte TB4. Systemic administration of an adeno-associated virus vector encoding TB4 prevented Adriamycin-induced podocyte loss and albuminuria. Adriamycin injury was associated with disorganisation of the actin cytoskeleton in vitro, which was ameliorated by exogenous TB4. Furthermore, Adriamycin administration in mice was associated with increased prevalence of podocyte vesicles, a mechanism by which albumin may leak into the urine, which was also prevented by TB4. Collectively, we propose that TB4 gene therapy prevents podocyte injury and maintains glomerular filtration via modulation of the podocyte cytoskeleton thus presenting a novel treatment strategy for glomerular disease.

physiology↗

bioRxiv2020

Structural model of the proline-rich domain of huntingtin exon-1 fibrils

Huntingtons disease (HD) is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine (polyQ) domain that increases the propensity of huntingtin exon-1 (HTTex1) to form cross-{beta} fibrils. While the polyQ domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of HTTex1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800 ns long molecular dynamics (MD) simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired EPR DEER distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline II (PPII) helical conformation. The two polyproline (polyP) regions within the PRD stay in a PPII helix for most of the simulation, whereas occasional kinks in the proline rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD. Statement of SignificanceHD is caused by a polyQ expansion in the exon-1 of huntingtin, which results in the formation of fibrillar huntingtin aggregates. Although the polyQ domain is the site of the disease-causing mutation, the PRD domain of HTTex1 is important for fibril toxicity and contains many epitopes of fibril-specific HTTex1 antibodies. Here, we present a structural and dynamic model of the highly dynamic PRD domain using a combination of EPR, solid-state NMR, and MD simulations. This model paves the way for studying known HTTex1 fibril specific binders and designing new ones.

biophysics↗

bioRxiv2019

Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

Despite the interest in characterizing all genomic variation, the presence of large repeats at the breakpoints of many structural variants hinders their analysis. This is especially problematic in the case of inversions, since they are balanced changes without gain or loss of DNA. Here we tested novel linkage-based droplet digital PCR (ddPCR) assays on 20 inversions ranging from 3.1 to 742 kb and flanked by long inverted repeats (IRs) of up to 134 kb. Among these, we validated 13 inversions predicted by different genome-wide techniques. In addition, we have generated new experimental human population information across 95 African, European and East-Asian individuals for 16 of them, including four already known inversions for which there were no high-throughput methods to determine directly the orientation, like the well-characterized 17q21 inversion. Through comparison with previous data, independent replicates and both inversion breakpoints, we have demonstrated that the technique is highly accurate and reproducible. Most of the studied inversions are frequent and widespread across continents, showing a negative correlation with genetic length. Moreover, all except two show clear signs of being recurrent, and the new data allowed us to define more clearly the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, thanks to the generated genotypes, we have been able to check inversion functional effects in multiple tissues, validating gene expression differences reported before for two inversions and finding new candidate associations. Our work therefore provides a tool to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

genomics↗

bioRxiv2019

Reactivation of chromosome signalling induces reversal of the meiotic program

Chromosome movements and programmed DNA double-strand breaks (DSBs) promote homologue pairing and initiate recombination at meiosis onset. Meiotic progression involves checkpoint-controlled termination of these events when all homologue pairs achieve synapsis and form crossover precursors. We show that termination of chromosome movement and DSB formation is reversible and is continuously implemented by the synaptonemal complex (SC), which silences chromosome signals that promote CHK-2 activity. Forced removal of the SC or different meiosis-specific cohesin complexes, which are individually required for SC stability, causes rapid CHK-2-dependent reinstallation of the DSB-formation and chromosome-movement machinery. This nuclear reorganization occurs without transcriptional changes, but requires signalling from HORMA protein HTP-1. Conversely, CHK-2 inactivation causes rapid disassembly of the DSB-formation and chromosome-movement machinery. Thus, nuclear organization is constantly controlled by the level of CHK-2 activity. Our results uncover an unexpected plasticity of the meiotic program and show how chromosome signalling integrates nuclear organization with meiotic progression.

cell biology↗

bioRxiv2018

ATR function is indispensable to allow proper mammalian follicle development.

Mammalian female fertility relies on the proper development of follicles. Right after birth in mouse, oocytes associate with somatic ovarian cells to form follicles. These follicles grow during adult lifetime to produce viable gametes. In this study, we analyzed the role of the ATM and rad3-related (ATR) kinase in mouse oogenesis and folliculogenesis using a hypomorphic mutation of the Atr gene (Murga et al., 2009). Female mice homozygote for this allele have been reported to be sterile. Our data show that female meiotic prophase is not grossly altered when ATR levels are reduced. However, follicle development is majorly compromised since Atr mutant ovaries present a decrease of growing follicles. Comprehensive analysis of follicular cell death and proliferation suggest that wild-type levels of ATR are required to achieve optimal follicular development. Altogether, these findings suggest that reduced ATR expression causes sterility due to defects in follicular progression rather than in meiotic recombination. We discuss the implication of these findings for the use of ATR inhibitors as anti-cancer drugs and its possible side effects on female fertility.

cell biology↗

bioRxiv2017

ATR Is Required To Complete Meiotic Recombination In Mice

Precise execution of recombination during meiosis is essential for forming chromosomally balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Binding of replication protein A (RPA) at resected DSBs fosters association of RAD51 and DMC1, the primary effectors of homology search. It is well appreciated that cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these responses remain poorly understood, particularly in mammals. Here we provide evidence that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

cell biology↗