Biology subjects

Fernandez, B.

Publications and source records attributed to Fernandez, B..

12 recordsLinked to original sources

bioRxiv2025

Single-cell RNA sequencing reveals distinct senotypes and a quiescence-senescence continuum at the transcriptome level following chemotherapy

Quiescence (reversible cell-cycle arrest) and senescence (irreversible arrest) are challenging to distinguish due to a lack of specific biomarkers, yet both arise simultaneously after chemotherapy. While senescence suppresses tumors by limiting proliferation and recruiting the immune system, quiescent cancer cells evade future therapies and may resume proliferation. Here, we pair time-lapse imaging of cell-cycle dynamics with single-cell RNA-sequencing after etoposide treatment to differentiate these states, linking heterogeneous cell-cycle phenotypes to the transcriptomic landscape. We identify diverse senescent types (senotypes) and link them to two arrest pathways - a gradual path arising after a standard mitosis-to-G0 transition, and a rarer but direct path driven by a mitotic slip. Using pseudotime trajectory analysis, we find that senescent phenotypes begin to manifest early and gradually along the first trajectory, even in shallow quiescent cells. These data support a model wherein, following chemotherapy, quiescence and senescence exist on a continuum of cell-cycle withdrawal at a transcriptome-wide level.

cell biology↗

bioRxiv2025

p16 expression confers sensitivity to CDK2 inhibitors

Blocking the cell cycle is a promising avenue for cancer therapy, with Cyclin-Dependent Kinase 2 (CDK2) emerging as a key target. However, in multiple cell types, CDK4/6 activity compensates for CDK2 inhibition and sustains the proliferative program, enabling CDK2 reactivation. Thus, we hypothesized that sensitivity to CDK2 inhibition is linked to the absence of this CDK4/6-mediated compensatory mechanism. Here we show that Cyclin E1-driven ovarian cancers often co-express the tumor suppressor p16, which inhibits CDK4/6. We show that ovarian cancer cells expressing p16 exhibit heightened sensitivity to CDK2 inhibitors and that depletion of p16 renders them significantly more resistant. Multiplexed immunofluorescence of 225 ovarian patient tumors reveals that at least 18% of tumors express high Cyclin E1 and high p16, a group that we expect to be particularly sensitive to CDK2 inhibition. Thus, p16 may be a useful biomarker for identifying the patients most likely to benefit from CDK2 inhibitors.

cancer biology↗

bioRxiv2024

Image Correlation Spectroscopy is a Robust Tool to Quantify Cellular DNA Damage Response

The DNA Damage response (DDR) is both essential and highly complex. Evaluating the DDR is a critical aspect of cell biology. Counting DNA damage foci is one of the most common approaches to study the DDR. Yet, quantification of protein foci suffers from experimental limitations, subjectivity of analysis and is restricted to a handful of the hundreds of DDR proteins. Here we apply image correlation spectroscopy (ICS) to quantify the local clustering at sites of DNA damage directly. We found that ICS outperformed foci counting of traditional DDR markers and enabled quantification of other markers without the complex labeling procedures that are otherwise required. ICS analysis also provided insight into DDR protein recruitment that was previously undetectable. Further expansion incorporating analysis to cell cycle classification demonstrates a rapid, non-biased approach to fully study the DNA damage response within cells. ICS analysis presents an objective, quantitative image analysis technique to study the DNA damage response in unaltered cells that we expect will significantly enhance quantitative DNA damage response research.

cell biology↗

bioRxiv2023

Unraveling the Molecular Complexity of Bicuspid Aortopathy: Lessons from Comparative Proteomics

BackgroundMolecular markers and pathways involved in the etiology and pathophysiology of bicuspid aortopathy are poorly understood. The aim here is to delve into the molecular and cellular mechanisms of the disease and identify potential predictive molecular markers using a well-established isogenic hamster model (T-strain) of bicuspid aortic valve (BAV) and thoracic aortic dilatation (TAD). MethodsComparative quantitative proteomics combined with western blot and morpho-molecular analyses in the ascending aorta of tricuspid aortic valve (TAV) and BAV animals from the T-strain, and TAV animals from a control strain. This strategy allows discriminating between genetic and hemodynamic factors in genetically homogeneous populations. ResultsThe major molecular alteration in the aorta of genetically homogeneous BAV individuals is PI3K/AKT overactivation caused by changes in the EGF, ANGII and TGF-{beta} pathways. PI3K/AKT affects downstream eNOS, MAP2K1/2, NF-{kappa}B, mTOR and WNT pathways. Most of these alterations are seen in independent patient studies with different clinical presentations, but not in TAV hamsters from T-strain that mainly exhibit WNT pathway downregulation. ConclusionsWe identify a combination of defective interconnected molecular pathways, directly linked to the central PI3K/AKT pathway, common to both BAV-associated TAD patients and hamsters. The defects indicate smooth muscle cell shift towards the synthetic phenotype induced by endothelial-to-mesenchymal transition, oxidative stress and inflammation. WNT signaling represent one genetic factor that may cause structural aortic abnormalities and aneurysm predisposition, whereas hemodynamics is the main trigger of molecular alterations, probably determining aortopathy progression. We identify twenty-seven novel potential biomarkers with a high predictive value.

molecular biology↗

bioRxiv2023

A potential patient stratification biomarker for Parkinson's disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

Parkinsos disease (PD) is a common neurodegenerative movement disorder and leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for disease intervention. However, the ability to stratify patients who will benefit from such treatment modalities based on shared etiology is critical for the success of disease-modifying therapies. Ciliary and centrosomal alterations are commonly associated with pathogenic LRRK2 kinase activity and can be detected in many cell types. We previously found centrosomal deficits in immortalized lymphocytes from G2019S-LRRK2 PD patients. Here, to investigate whether such deficits may serve as a potential blood biomarker for PD which is susceptible to LRKK2 inhibitor treatment, we characterized patient-derived cells from distinct PD cohorts. We report centrosomal alterations in peripheral cells from a subset of early-stage idiopathic PD patients which is mitigated by LRRK2 kinase inhibition, supporting a role for aberrant LRRK2 activity in idiopathic PD. Centrosomal defects are detected in R1441G-LRRK2 and G2019S-LRRK2 PD patients and in non-manifesting LRRK2 mutation carriers, indicating that they acumulate prior to a clinical PD diagnosis. They are present in immortalized cells as well as in primary lymphocytes from peripheral blood. These findings indicate that analysis of centrosomal defects as a blood-based patient stratification biomarker may help nominate PD patients who will benefit from LRRK2-related therapeutics. One-sentence summaryPeripheral blood-derived cells can be employed to stratify Parkinsos disease patients most likely to respond to LRRK2-related therapeutics.

neuroscience↗

bioRxiv2023

The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal

Senescence, a state of permanent cell-cycle withdrawal, is difficult to distinguish from quiescence, a transient state of cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether quiescence and senescence are truly distinct states. To address this, we used single-cell time-lapse imaging to distinguish slow-cycling quiescent cells from bona fide senescent cells after chemotherapy treatment, followed immediately by staining for various senescence biomarkers. We found that the staining intensity of multiple senescence biomarkers is graded rather than binary and primarily reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data suggest that quiescence and senescence are not distinct cellular states but rather fall on a continuum of cell-cycle withdrawal, where the intensities of canonical senescence biomarkers reflect the likelihood of cell-cycle re-entry.

systems biology↗

bioRxiv2022

Biomolecular condensates govern PARP inhibitor trapping and present mechanisms of resistance

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a class of cancer drugs that enzymatically inhibit PARP activity at sites of DNA damage. In the context of BRCA mutations, PARPi can be synthetically lethal, presenting ideal genetic targeting. Yet, PARPi function primarily by trapping PARP1 onto sites of DNA damage. How PARPi trap and why some are better trappers remain unknown. Here, we show trapping occurs primarily through a kinetic phenomenon within biomolecular condensates that correlates with PARPi koff. Our results suggest PARP trapping is not the physical stalling of PARP1 on DNA, rather the high probability of PARP re-binding damaged DNA in the absence of other DNA binding protein recruitment. Furthermore, we found recruitment of the DNA binding protein RPA1 correlates to cell line PARPi sensitivity independent of RPA1 expression, demonstrating that condensate recruitment alone can impact efficacy. These results shed new light on how PARPi function, describe how PARPi properties correlate to trapping potency, and suggest previously unknown mechanisms of PARPi resistance.

biophysics↗

bioRxiv2022

A Geometric View of Signal Sensitivity Metrics in multi-echo fMRI

In multi-echo fMRI (ME-fMRI), two metrics have been widely used to measure the performance of various acquisition and analysis approaches. These are temporal SNR (tSNR) and differential contrast-to-noise ratio (dCNR). A key step in ME-fMRI is the weighted combination of the data from multiple echoes, and prior work has examined the dependence of tSNR and dCNR on the choice of weights. However, most studies have focused on only one of these two metrics, and the relationship between the two metrics has not been examined. In this work, we present a geometric view that offers greater insight into the relation between the two metrics and their weight dependence. We identify three major regimes: (1) a tSNR robust regime in which tSNR is robust to the weight selection with most weight variants achieving close to optimal performance, whereas dCNR shows a pronounced dependence on the weights with most variants achieving suboptimal performance; (2) a dCNR robust regime in which dCNR is robust to the weight selection with most weight variants achieving close to optimal performance, while tSNR exhibits a strong dependence on the weights with most variants achieving significantly lower than optimal performance; and (3) a within-type robust regime in which both tSNR and dCNR achieve nearly optimal performance when the form of the weights are variants of their respective optimal weights and exhibit a moderate decrease in performance for other weight variants. Insight into the behavior observed in the different regimes is gained by considering spherical representations of the weight dependence of the components used to form each metric. For multi-echo acquisitions, dCNR is shown to be more directly related than tSNR to measures of CNR and signal-to-noise ratio (SNR) for both task-based and resting-state fMRI scans, respectively.

neuroscience↗

bioRxiv2021

LRRK2 causes centrosomal deficits via phosphorylated Rab10 and RILPL1 at centriolar subdistal appendages

The Parkinsons disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that various LRRK2 risk variants and all currently described regulators of the LRRK2 signaling pathway converge upon causing centrosomal cohesion deficits. The cohesion deficits do not require the presence of RILPL2 or of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendages of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab protein to cause the centrosomal defects. These data reveal a common molecular pathway by which alterations in the LRRK2 kinase activity impact upon centrosome-related events.

neuroscience↗

bioRxiv2021

CRISPR-Cas-mediated tethering recruits the yeast HMR mating-type locus to the nuclear periphery but fails to silence gene expression

To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing phenotype has an unexpected dependence on the structure of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.

synthetic biology↗

bioRxiv2020

Distinct roles for RAB10 and RAB29 in pathogenic LRRK2-mediated endolysosomal trafficking alterations

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinsons disease, and sequence variations are associated with the sporadic form of the disease. LRRK2 phosphorylates a subset of RAB proteins implicated in secretory and recycling trafficking pathways, including RAB8A and RAB10. Another RAB protein, RAB29, has been reported to recruit LRRK2 to the Golgi where it stimulates its kinase activity. Our previous studies revealed that G2019S LRRK2 expression or knockdown of RAB8A deregulate epidermal growth factor receptor (EGFR) trafficking, with a concomitant accumulation of the receptor in a RAB4-positive recycling compartment. Here, we show that the G2019S LRRK2-mediated EGFR deficits are mimicked by knockdown of RAB10 and rescued by expression of active RAB10. By contrast, RAB29 knockdown is without effect, but expression of RAB29 also rescues the pathogenic LRRK2-mediated trafficking deficits independently of Golgi integrity. Our data suggest that G2019S LRRK2 deregulates endolysosomal trafficking by impairing the function of RAB8A and RAB10, whilst RAB29 positively modulates non-Golgi-related trafficking events impaired by pathogenic LRRK2.

cell biology↗

bioRxiv2020

Weight Sensitivity of Temporal SNR Metrics in multi-echo fMRI

PurposeIn multi-echo fMRI (ME-fMRI), various weighting schemes have been proposed for the combination of the data across echoes. Here we introduce a framework that facilitates a deeper understanding of the weight dependence of temporal SNR measures in ME-fMRI. Theory and MethodsWe examine two metrics that have been used to characterize ME-fMRI performance: temporal SNR (tSNR) and multi-echo temporal (metSNR). Both metrics can be described using the generalized Rayleigh quotient (GRQ) and are predicted to be relatively insensitive to the weights when there is a high degree of similarity between a metric-specific matrix in the GRQ numerator and a metricindependent covariance matrix in the GRQ denominator. The application of the GRQ framework to experimental data is demonstrated using a resting-state fMRI dataset acquired with a multi-echo multi-band EPI sequence. ResultsIn the example dataset, similarities between the covariance matrix and the metSNR and tSNR numerator matrices are highest in grey matter (GM) and cerebrospinal fluid (CSF) voxels, respectively. For representative GM and CSF voxels that exhibit high matrix similarity values, the metSNR and tSNR values, respectively, are both within 4% of their optimal values across a range of weighting schemes. However, there is a fundamental tradeoff, with a high degree of weight sensitivity in the tSNR and metSNR metrics for the representative GM and CSF voxels, respectively. Geometric insight into the observed weight dependencies is provided through a graphical interpretation of the GRQ. ConclusionA GRQ framework can provide insight into the factors that determine the weight sensitivity of tSNR and metSNR measures in ME-fMRI.

neuroscience↗