Biology subjects

Gonzalez, M. M.

Publications and source records attributed to Gonzalez, M. M..

4 recordsLinked to original sources

bioRxiv2025

Toxoplasma gondii RAD51 recombinase is required to overcome DNA replication stress and its inactivation leads to bradyzoite differentiation

Toxoplasma gondii is an obligate intracellular parasite with a high replication rate that can lead to DNA replicative stress, in turn associated with the generation of DNA double-strand breaks (DSBs). Cells have two main pathways to repair DSBs: non-homologous end joining and homologous recombination repair (NHEJ and HRR respectively). RAD51 is the key recombinase in the HRR pathway. In this work, we achieved endogenous tagging of the RAD51 gene using the Auxin Inducible Degron (AID) system, to generate the clonal line RH RAD51HA-AID. Here we demonstrate that RAD51 is expressed in replicative tachyzoites and establishes damage foci. Auxin-induced knock-down (KD) affects the correct replication of tachyzoites which show loss of synchronization. The use of the RAD51 inhibitor B02 also affects parasite growth, with an IC50 of 4.8 {micro}M. B02 produced alterations in tachyzoite replication and arrest in the S phase of the cell cycle. Additionally, B02 induced tachyzoite to bradyzoite differentiation showing small cyst-like structures. In conclusion, the HRR pathway is necessary for maintaining proper tachyzoite replication under normal growth conditions, supporting that replicative stress occurs during the cell cycle. Our findings also suggest that DNA replication stress can induce bradyzoite differentiation.

molecular biology↗

bioRxiv2025

Regional susceptibility of PV interneurons in an hAPP-KI mouse model of Alzheimer's disease pathology

Early-stage Alzheimers pathology correlates with disrupted neuronal excitability, which can drive network and cognitive dysfunction even prior to neurodegeneration. However, the emergence and extent of these changes may vary by brain region and cell types situated in those regions. Here we aimed to investigate the effects of AD pathology on different neuron subtypes in both the entorhinal cortex, a region with enhanced pathology in early AD, and the primary visual cortex, a relatively unaffected region in early-stage AD. We designed and employed a semi-automated patch clamp electrophysiology apparatus to record from fast-spiking parvalbumin interneurons and excitatory neurons in these regions, recording from over 150 cells in young adult APP-KI mice. In entorhinal cortex, amyloid overproduction resulted in PV interneuron hypoexcitability, whereas excitatory neurons were concurrently hyperexcitable. Conversely, neurons of either subclass were largely unaffected in the visual cortex. Together, these findings suggest that fast-spiking parvalbumin interneurons in the entorhinal cortex, but not in the visual cortex, play an integral role in AD progression.

neuroscience↗

bioRxiv2025

Ontogeny Dictates Oncogenic Potential, Lineage Hierarchy, and Therapy Response in Pediatric Leukemia

Accumulating evidence links pediatric cancers to prenatal transformation events, yet the influence of the developmental stage on oncogenesis remains elusive. We investigated how hematopoietic stem cell developmental stages affect leukemic transformation, disease progression, and therapy response using a novel, humanized model of NUP98::NSD1-driven pediatric acute myeloid leukemia, that is particularly aggressive with WT1 co-mutations. Fetal-derived hematopoietic stem cells readily transform into leukemia, and WT1 mutations further enhance stemness and alter lineage hierarchy. In contrast, stem cells from later developmental stages become progressively resistant to transformation. Single-cell analyses revealed that fetal-origin leukemia stem cells exhibit greater quiescence and reliance on oxidative phosphorylation than their postnatal counterparts. These differences drive distinct therapeutic responses, despite identical oncogenic mutations. In patients, onco-fetal transcriptional programs correlate with worse outcomes. By targeting key vulnerabilities of fetal-origin leukemia cells, we identified combination therapies that significantly reduce aggressiveness, highlighting the critical role of ontogeny in pediatric cancer treatment.

cancer biology↗

bioRxiv2024

Patch-walking: Coordinated multi-pipette patch clamp for efficiently finding synaptic connections

Significant technical challenges exist when measuring synaptic connections between neurons in living brain tissue. The patch clamping technique, when used to probe for synaptic connections, is manually laborious and time-consuming. To improve its efficiency, we pursued another approach: instead of retracting all patch clamping electrodes after each recording attempt, we cleaned just one of them and reused it to obtain another recording while maintaining the others. With one new patch clamp recording attempt, many new connections can be probed. By placing one pipette in front of the others in this way, one can "walk" across the tissue, termed "patch-walking." We performed 136 patch clamp attempts for two pipettes, achieving 71 successful whole cell recordings (52.2%). Of these, we probed 29 pairs (i.e., 58 bidirectional probed connections) averaging 91 {micro}m intersomatic distance, finding 3 connections. Patch-walking yields 80-92% more probed connections, for experiments with 10-100 cells than the traditional synaptic connection searching method. MotivationRecognizing the manual labor and time-intensive nature of patch clamping when trying to find synaptic connections, we aim to improve its efficiency. We introduce a novel approach, termed "patch-walking," where one patch clamping electrode is cleaned and reused, enabling the exploration of numerous connections with a single recording attempt and improving the efficiency of identifying synaptic connections.

bioengineering↗