Biology subjects

Keith, J.

Publications and source records attributed to Keith, J..

6 recordsLinked to original sources

bioRxiv2025

Liquid Lung Rest During Extracorporeal Life Support in a Porcine Model of Acute Lung Injury

RationaleIn patients with severe lung injury, extracorporeal life support can enable lung rest, but the optimal strategy is unknown. ObjectiveTo compare liquid lung rest versus standard gas rest ventilation in a porcine model of acute lung injury and extracorporeal life support. MethodsTwenty neonatal pigs with lung injury after systemic injection of oleic acid were placed on extracorporeal life support. Pigs were ventilated with standard rest settings (peak inspiratory pressure: 20 cmH2O; end expiratory pressure: 10 cmH2O; rate: 10 breaths/minute) and randomized to receive no additional therapy (gas ventilation) or 5 mL/kg perfluorooctylbromide instilled endotracheally (liquid lung rest). The study continued for 4 hours with hourly pulmonary compliance measurements. After euthanasia, lung samples were taken for histologic analysis. Inflammation was quantified via blood and lung tissue cytokines. Measurements and Main ResultsAll pigs achieved significant lung injury; static compliance decreased by a mean of 54%. After 4 hours of lung rest, static compliance was higher with liquid lung rest compared to gas ventilation (0.88{+/-}0.05 mL/kg/cmH2O versus 0.57{+/-}0.05, p<0.001). Histology revealed 2.1-fold greater airspace in dependent lung regions with liquid lung rest (p<0.001) and a reduced airspace heterogeneity index (LLR: 0.071{+/-}0.004, gas: 0.095{+/-}0.010, p=0.04), suggesting more uniform alveolar recruitment. Cytokine analysis demonstrated a 3.7-fold decrease in tissue interleukin-10 levels with liquid lung rest (p=0.02). ConclusionsLiquid lung rest improved pulmonary compliance, achieved better alveolar recruitment, and decreased tissue interleukin-10 levels compared to standard gas ventilation. This strategy may enhance lung recovery in acute lung injury requiring extracorporeal life support.

physiology↗

bioRxiv2024

C9orf72 expansion creates the unstable folate-sensitive fragile site FRA9A

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions (C9orf72Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown. Here, we show C9orf72Exp in pre-symptomatic and ALS-FTD patient cells and brains cause the folate- sensitive chromosomal fragile site, FRA9A. FRA9A centers on >33kb of C9orf72 as highly-compacted chromatin embedded in an 8.2Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one C9orf72Exp patient contained highly-rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72Exp repeat instability and chromosomal fragility are sensitive to folate-deficiency. Age-dependent repeat instability, chromosomal fragility, and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic C9orf72Exp mice, implicating C9orf72Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/620312v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@154b23borg.highwire.dtl.DTLVardef@1814a6dorg.highwire.dtl.DTLVardef@13d713eorg.highwire.dtl.DTLVardef@d5d994_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Behavioral and neural correlates of diverse conditioned fear responses in male and female rats

Pavlovian fear conditioning is a widely used tool that models associative learning in rodents. For decades the field has used predominantly male rodents and focused on a sole conditioned fear response: freezing. However, recent work from our lab and others has identified darting as a female-biased conditioned response, characterized by an escape-like movement across a fear conditioning chamber. It is also accompanied by a behavioral phenotype: Darters reliably show decreased freezing compared to Non-darters and males and reach higher velocities in response to the foot shock ("shock response"). However, the relationship between shock response and conditioned darting is not known. This study investigated if this link is due to differences in general processing of aversive stimuli between Darters, Non-darters and males. Across a variety of modalities, including corticosterone measures, the acoustic startle test, and sensitivity to thermal pain, Darters were found not to be more reactive or sensitive to aversive stimuli, and, in some cases, they appear less reactive to Non-darters and males. Analyses of cFos activity in regions involved in pain and fear processing following fear conditioning identified discrete patterns of expression among Darters, Non-darters, and males exposed to low and high intensity foot shocks. The results from these studies further our understanding of the differences between Darters, Non-darters and males and highlight the importance of studying individual differences in fear conditioning as indicators of fear state.

neuroscience↗

bioRxiv2023

Single-nucleus multiomic atlas of frontal cortex in amyotrophic lateral sclerosis with a deep learning-based decoding of alternative polyadenylation mechanisms

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two age-related and fatal neurodegenerative disorders that lie on a shared disease spectrum. While both disorders involve complex interactions between neuronal and glial cells, the specific cell-type alterations and their contributions to disease pathophysiology remain incompletely understood. Here, we applied single-nucleus RNA sequencing of the orbitofrontal cortex, a region affected in ALS-FTLD, to map cell-type specific transcriptional signatures in C9orf72-related ALS (with and without FTLD) and sporadic ALS cases. Our findings reveal disease- and cell-type-specific transcriptional changes, with neurons exhibiting the most pronounced alterations, primarily affecting mitochondrial function, protein homeostasis, and chromatin remodeling. A comparison with independent datasets from different cortical regions of C9orf72 and sporadic ALS cases showed concordance in several pathways, with neuronal STMN2 and NEFL showing consistent up-regulation between brain regions and disease subtypes. We also interrogated alternative polyadenylation (APA) as an additional layer of transcriptional regulation, demonstrating that APA events are not correlated with identified gene expression changes. To interpret these events, we developed APA-Net, a deep learning model that integrates transcript sequences with RNA-binding protein expression profiles, revealing cell type-specific patterns of APA regulation. Our atlas illuminates cell type-specific pathomechanisms of ALS/FTLD, providing a valuable resource for further investigation.

neuroscience↗

bioRxiv2022

Sounding the alarm: sex differences in rat ultrasonic vocalizations during Pavlovian fear conditioning and extinction

Pavlovian fear conditioning is a prevalent tool in the study of aversive learning, which is a key component of stress-related psychiatric disorders. Adult rats can exhibit various threat-related behaviors, including freezing, motor responses and ultrasonic vocalizations (USVs). While these responses can all signal aversion, we know little about how they relate to one another. Here we characterize USVs emitted by male and female rats during cued fear acquisition and extinction and assess the relationship between different threat-related behaviors. To probe the effects of aversive stimulus intensity, we exposed the rats to mild (0.3 mA), moderate (0.5 mA) or strong (1 mA) foot shocks. We found that males consistently emitted more alarm calls than females, and male alarm calls were more closely contingent on shock intensity than were female alarm calls. Furthermore, 25 % of males and 45 % of females did not emit alarm calls. Males that made alarm calls had significantly higher levels of freezing than males who did not, while no differences in freezing were observed between female alarm callers and non-callers. Alarm call emission was also affected by the predictability of the shock; when unpaired from a tone cue, both males and females started emitting alarm calls significantly later. Some rats continued to alarm-call during extinction learning (90% of males, 30% of females) and retrieval (65% of males, 20% of females). Collectively these data suggest sex-dependence in how behavioral readouts relate to innate and conditioned threat responses. Importantly, we suggest that the same behaviors can signal sex-dependent features of aversion. Significance statementBehavioral neuroscientists can access various outputs during behavioral tests to draw conclusions about internal states of animals. While freezing is the most common index of rodents feeling threatened, these animals also emit specific ultrasonic vocalizations during aversive situations. Here we record several motor and vocal behaviors to assess how they relate to each other as threat responses, and how such relationships vary across sex. We found robust differences in how much male and female rats engaged in so-called alarm vocalizations. These vocalizations were subject to extinction in both sexes, but correlated with freezing only in males. As the field advances to include more females in preclinical research, it is crucial that we understand how similar-appearing outputs may reflect sex-biased features.

animal behavior and cognition↗

bioRxiv2018

Unc 51-like autophagy-activating kinase (ULK1) mediates clearance of free {alpha}-globin in {beta}-thalassemia

Erythroid maturation is coordinated to maximize the production of hemoglobin A heterotetramers (2{beta}2) and minimize the accumulation of potentially toxic free - or {beta}-globin subunits. In {beta}-thalassemia, mutations in the {beta}-globin gene cause a build-up of free -globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality-control systems mitigate {beta}-thalassemia pathophysiology by degrading toxic free -globin. We show that loss of the Unc 51-like autophagy-activating kinase gene Ulk1 in {beta}-thalassemic mice reduces autophagic clearance of -globin in red cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy gene Atg5 has minimal effects. Systemic treatment with rapamycin to inhibit the ULK1 inhibitor mTORC1 reduces -globin precipitates and lessens pathologies in {beta}-thalassemic mice, but not in those lacking Ulk1. Similarly, rapamycin reduces free -globin accumulation in erythroblasts derived from {beta}-thalassemic patient CD34+ hematopoietic progenitors. Our findings identify a new, drug-regulatable pathway for ameliorating {beta}-thalassemia.\n\nOne Sentence SummaryRapamycin alleviates {beta}-thalassemia by stimulating ULK1-dependent autophagy of toxic free -globin.

cell biology↗