Biology subjects

Sullivan, T.

Publications and source records attributed to Sullivan, T..

6 recordsLinked to original sources

bioRxiv2026

Goal directed closed-loop training of network bursts in cortical cultures produces improved performance and error correction over days

Dissociated neural cultures are living systems with no evolutionary history in their current form, which makes them a testbed for asking what goals neural tissue can learn to pursue once it is removed from its evolved body. It remains unclear which features of network activity such a culture can be trained to control, and whether that training is retained from one day to the next. We built an open-source closed-loop platform, the Holistic Agential Learning platform (HAL), that detects a network burst on a high-density microelectrode array, estimates its spatial origin in real time, and applies a minimal reinforcement rule. Here, a burst that originates off the goal side triggers global random stimulation that persists until the next burst. Over days of training, rat cortical cultures (n=10) increased the fraction of bursts that initiated on the goal side. This gain accrued between sessions, with little change within a session, a consolidation-like pattern that we interpret as a statement about timescale while the mechanism remains untested. The burst origin moved toward the goal side, while the burst peak and the repertoire of propagation directions were preserved. Trained cultures also recovered from incorrect states more reliably over days, while the rate at which they entered those states was unchanged, and this recovery effect was the most robust difference from unstimulated controls (n=6). Cultures that began on either side of chance converged to a similar level of performance. Damaged cultures (n=6) with comparable burst statistics showed neither the across-day gain nor the consolidation-like pattern. Training reweighted the activity patterns each culture already had, and we saw no new ones, which we read as a simple form of niche construction in which the tissue keeps a trace of its history that biases what it does next. These results identify burst initiation and error recovery as trainable variables in a novel living system, and they support training the tissue rather than engineering the machine.

bioengineering↗

bioRxiv2026

A single BRCA2 BRC repeat supports viability while multiple repeats ensure resilience under stress

BRC repeats are integral to BRCA2 function and mediate RAD51 loading during homologous recombination (HR). Their number varies across species, but mammals, including mice and humans, harbor eight repeats. Prior studies have suggested functional redundancy among these repeats, but the biological significance of maintaining multiple repeats remains unresolved. Here, we demonstrate that the presence of a single BRC repeat, either BRC2 or BRC4, is sufficient for mouse embryonic stem cell (mESC) viability, RAD51 loading, PARP inhibitor resistance and protection of stalled replication forks. Consistent with these findings, we show that knock-in mice with a single BRC repeat 2 or 4 are viable and exhibit normal growth and fertility. In contrast, embryonic fibroblasts from these mice display genomic instability and impaired RAD51 recruitment. Notably, this defect is rescued under low oxygen culture conditions, which mimics physoxic levels, whereas exposure to oxidative stress impairs RAD51 recruitment in mESCs harboring a single BRC repeat. Together, our findings indicate that while a single BRC repeat is sufficient under physiological conditions, the evolutionary retention of multiple BRC repeats likely ensures robust genome stability under extreme oxidative stress.

genetics↗

bioRxiv2024

Adipogenic differentiation of hematopoietic lineage cells isolated from adipose tissue of humans

We previously discovered some adipocytes in the major white fat depots of mice and humans arise from bone marrow-derived cells of hematopoietic lineage rather than conventional mesenchymal precursors, termed bone marrow-derived adipocytes (BMDA). Here we aimed to determine if hematopoietic lineage cells isolated from adipose tissue and circulation of humans could undergo adipogenic differentiation in vitro, thereby establishing an in vitro model for studies of BMDA. We hypothesized that hematopoietic lineage cells isolated from adipose tissue, but not circulation, of humans would demonstrate adipogenic potential. Participants included younger (20-50 years) and older (>50-75 years) men and women, BMI 20-37 kg/m2. Subcutaneous abdominal adipose tissue biopsies were obtained and stromal cell populations identified by flow cytometry. Sorted cells underwent in vitro cultivation via traditional mesenchymal culture methodology (mesenchymal lineage) or a novel 3D-fibrin clot followed by traditional adherent culture (hematopoietic lineage) for assessment of proliferation and differentiation capacity. We found hematopoietic lineage cells isolated from the adipose tissue stroma, but not the circulation, were capable of proliferation and multilineage (adipogenic and osteogenic) differentiation in vitro. We provide a new investigative tool that can be used to perform translational studies of BMDAs and provide initial evidence that hematopoietic lineage cells isolated from the adipose tissue of humans can undergo hematopoietic-to-mesenchymal transition with multilineage differentiation potential in an in vitro environment.

physiology↗

bioRxiv2024

Identification of a gene expression signature of vascular invasion and recurrence in stage I lung adenocarcinoma via bulk and spatial transcriptomics

Microscopic vascular invasion (VI) is predictive of recurrence and benefit from lobectomy in stage I lung adenocarcinoma (LUAD) but is difficult to assess in resection specimens and cannot be accurately predicted prior to surgery. Thus, new biomarkers are needed to identify this aggressive subset of stage I LUAD tumors. To assess molecular and microenvironment features associated with angioinvasive LUAD we profiled 162 resected stage I tumors with and without VI by RNA-seq and explored spatial patterns of gene expression in a subset of 15 samples by high-resolution spatial transcriptomics (stRNA-seq). Despite the small size of invaded blood vessels, we identified a gene expression signature of VI from the bulk RNA-seq discovery cohort (n=103) and found that it was associated with VI foci, desmoplastic stroma, and high-grade patterns in our stRNA-seq data. We observed a stronger association with high-grade patterns from VI+ compared with VI- tumors. Using the discovery cohort, we developed a transcriptomic predictor of VI, that in an independent validation cohort (n=60) was associated with VI (AUROC=0.86; p=5.42x10-6) and predictive of recurrence-free survival (HR=1.98; p=0.024), even in VI- LUAD (HR=2.76; p=0.003). To determine our VI predictors robustness to intra-tumor heterogeneity we used RNA-seq data from multi-region sampling of stage I LUAD cases in TRACERx, where the predictor scores showed high correlation (R=0.87, p<2.2x10-16) between two randomly sampled regions of the same tumor. Our study suggests that VI-associated gene expression changes are detectable beyond the site of intravasation and can be used to predict the presence of VI. This may enable the prediction of angioinvasive LUAD from biopsy specimens, allowing for more tailored medical and surgical management of stage I LUAD.

cancer biology↗

bioRxiv2023

AVENGERS: Analysis of Variant Effects using Next Generation sequencing to Enhance BRCA2 Stratification

Accurate interpretation of genetic variation is a critical step towards realizing the potential of precision medicine. Sequencing-based genetic tests have uncovered a vast array of BRCA2 sequence variants. Due to limited clinical, familial and/or epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS). To determine the functional impact of VUSs, here we develop AVENGERS: Analysis of Variant Effects using NGs to Enhance BRCA2 Stratification, utilizing CRISPR-Cas9-based saturation genome editing (SGE) in a humanized-mouse embryonic stem cell line. We have categorized nearly all possible missense single nucleotide variants (SNVs) encompassing the C-terminal DNA binding domain of BRCA2. We have generated the function scores for 6270 SNVs, covering 95.5% of possible SNVs in exons 15-26 spanning residues 2479-3216, including 1069 unique missense VUS, with 81% functional and 14% found to be nonfunctional. Our classification aligns strongly with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. Our statistical classifier exhibits 92.2% sensitivity and 96% specificity in distinguishing clinically benign and pathogenic variants recorded in ClinVar. Furthermore, we offer proactive evidence for 617 SNVs being non-functional and 3396 SNVs being functional demonstrated by impact on cell growth and response to DNA damaging drugs like cisplatin and olaparib. This classification serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counselors assessing BRCA2 VUSs in patients.

genetics↗

bioRxiv2019

Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional bioreactor system where cancer cells are grown in Matrigel in modified 6-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3% to 0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional 2D monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

bioengineering↗