Biology subjects

Graham, A.

Publications and source records attributed to Graham, A..

15 recordsLinked to original sources

bioRxiv2026

RE-1 silencing transcription factor is reduced in endometriosis and uterine deletion in mice alters progesterone responsiveness

Endometriosis is a steroid-dependent gynecologic disease characterized by progesterone (P4) resistance, subfertility/infertility, and pelvic pain; however, the molecular mechanisms underlying impaired P4 responsiveness in endometriosis tissue are not fully understood. RE-1 silencing transcription factor (REST), a transcriptional regulator implicated in steroid hormone signaling, has emerged as a potential mediator of P4 responsiveness. Here, we investigated the role of REST in endometriosis using human tissues and a uterine-specific Rest conditional knockout mouse model. Immunohistochemical analysis of eutopic endometrium and ectopic lesions from patients with endometriosis revealed significantly reduced nuclear REST expression compared with control endometrium, suggesting loss of functional REST in disease. To assess the physiological consequences of REST deficiency, uterine-specific Rest knockout (Rest d/d) mice were generated. Rest d/d females exhibited progressive subfertility and hyper-estrogenic uterine tissue characteristics that displayed a blunted responsiveness to P4 treatment. Loss of Rest selectively altered expression of P4-responsive genes associated with endometriosis pathology, despite preserved P4 receptor expression. Following induction of experimental endometriosis, female mice that developed endometriotic-like lesions using Rest-deficient donor tissue developed significantly larger lesions that were less responsive to P4 treatment compared to lesions induced using control tissue. Mechanical sensitivity was modestly increased in mice receiving Rest-deficient tissue, whereas vaginal hyperalgesia was unaffected. These findings identify loss of nuclear REST as a feature of endometriosis and support a role of REST in subfertility, lesion progression, and blunted response to P4. REST may represent a novel molecular contributor to altered P4 responsiveness and a potential therapeutic target in endometriosis. Significance StatementEndometriosis is a common disease in women characterized by altered steroid hormone signaling, infertility, and pelvic pain. RE-1 silencing transcription factor (REST) is a candidate regulator of steroid hormone signaling in gynecologic disease but a role in endometriosis pathophysiology remains unexplored. To fill this knowledge gap, our study utilizes human endometrial and endometriotic tissues coupled with a conditional knockout mouse model for uterine Rest deficiency. We show that REST is significantly reduced in eutopic and ectopic endometrial tissue from women with endometriosis and that deletion from mouse uterine tissue recapitulates clinical characteristics in women with endometriosis including progesterone resistance, sub-fertility and pelvic pain. These findings will further guide future research to understand impaired steroid signaling in the pathophysiology of endometriosis.

physiology↗

bioRxiv2026

The role of the roof plate for mesencephalic trigeminal neuron

The mesencephalic trigeminal nucleus (MTN) contains the proprioceptive sensory neurons that innervate mechanoreceptors in the jaw closing muscles. In the chick embryo, MTN neurons are the first neurons generated in the mesencephalon. They arise bilaterally adjacent to the roof plate and then extend their axons ventrally before projecting caudally towards the rhombencephalon. MTN axons remain in a mid - dorsoventral position and pioneer the lateral longitudinal fasciculus. Notably, MTN axons never cross the roof plate, raising the question of which mechanisms underlie this restriction. Here, we investigated the effects of tissue transplants on the guidance of MTN axons. We found that both the diencephalon and the notochord exert repulsive effects on MTN axons, which could partially explain their early trajectory. We have also analysed the potential roles of the guidance cues BMP2/4, GDF7, SLIT and NETRIN in MTN axon navigation, both in vivo and in vitro. We found no evidence for a role of BMP2/4 or GDF7 in directing MTN axons. However, SLIT-ROBO signaling was found to play a significant role. SLIT proteins are repulsive guidance cues expressed by roof and floor plate. Loss or reduced expression of ROBO2 led to aberrant axon meandering within the dorsal midbrain. Most axons eventually reoriented posteriorly, and only a small fraction crossed the roof plate. Unexpectedly, in the absence of ROBO2, MTN somata migrated into the roof plate, resulting in the loss of a defined roof plate region. Taken together, these results suggest that SLIT2-ROBO2 signaling not only prevents MTN axons from crossing the roof plate but also maintains MTN cell bodies adjacent to the roof plate. With regards to MTN neuron guidance, we conclude that additional roof plate - derived factors are likely to co-operate with SLIT proteins to prevent crossing of the roof plate. Another possibility could be that SLIT might signal through additional receptors.

developmental biology↗

bioRxiv2025

Inhibition of histone lysine demethylase restores learning and memory in aged mice

Chromatin undergoes dramatic changes during the ageing process. In the brain, these chromatin changes are thought to underlie age-associated deficits in the activity-dependent gene transcription necessary for memory consolidation. Here, we show that the levels of a specific histone post-translational modification (PTM), trimethylation of lysine 4 on histone 3 (H3K4me3) is markedly increased in the hippocampus of aged mice and that the activity-induced increase in H3K4me3 that is observed in response to a learning stimulus in young mice, is severely blunted in the aged hippocampus. H3K4me3 typically marks open, accessible chromatin at the transcriptional start sites (TSSs) of actively transcribed genes. We identify altered H3K4me3 peaks at TSSs and show that ca. 90% of the activity-induced H3K4me3 changes at TSSs are either absent or reduced in the aged hippocampus. To understand the biological significance of these age-associated changes, we screened a library of pharmacological compounds for compounds that can alter H3K4me3 levels in hippocampal neurons. We show that treatment of aged mice with one of these, the LSD1 inhibitor ORY-1001, restored normal learning and memory in object location and recognition tasks. Furthermore, we show that ORY-1001 treatment increased long-term potentiation (LTP), a form of synaptic plasticity deficient in the aged hippocampus. These findings suggest that targeting the epigenetic machinery that regulates activity-dependent gene transcription may represent an avenue for treating age-associated cognitive impairment. HighlightsO_LIH3K4me3 levels are elevated in the naive aged mouse hippocampus C_LIO_LIActivity-dependent H3K4me3 induction is impaired in the aged mouse hippocampus C_LIO_LILSD1 inhibition restores learning and memory in aged mice C_LIO_LILSD1 inhibition restores activity-dependent Arc induction and increases LTP in the aged hippocampus C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/695858v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@fccd92org.highwire.dtl.DTLVardef@11c4d93org.highwire.dtl.DTLVardef@128f8b8org.highwire.dtl.DTLVardef@e1341f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Efficient installation of heterozygous mutations in human pluripotent stem cells using prime editing

The utility of human pluripotent stem cells (hPSCs) is greatly enhanced by the ability to introduce precise, site-specific genetic modifications with minimal off-target effects. Although Cas9 endonuclease is an exceptionally efficient gene-editing tool, its propensity for generating biallelic modifications often limits its capacity for introducing heterozygous variants. Here, we use prime editing (PE) to install heterozygous edits in over ten distinct genetic loci, achieving knock-in efficiencies of up to 40% without the need for subsequent purification or drug selection steps. Moreover, PE enables the precise introduction of heterozygous edits in paralogous genes that are otherwise extremely challenging to achieve using endonuclease-based editing approaches. We also show that PE can be successfully combined with reprogramming to derive heterozygous iPSC clones directly from human fibroblasts and peripheral blood mononuclear cells. Our findings highlight the utility of PE for generating hPSCs with complex edits and represents a powerful platform for modelling disease-associated dominant mutations and gene-dosage effects in an entirely isogenic context.

cell biology↗

bioRxiv2025

ISLET-1 knockdown causes abnormal peripheral axonal growth of mesencephalic trigeminal neurones in the chick embryo

The trigeminal is one of the best characterized sensory systems in amniotes. It comprises two populations of first-order sensory neurons: the trigeminal ganglion (TG) which are peripheral to the central nervous system and the mesencephalic trigeminal nucleus (MTN), the only sensory neurons that lie within the central nervous system in amniotes. Islet-1, a LIM homeodomain transcription factor, which plays essential roles during embryogenesis, contributes to axon pathfinding of sensory neurons in the TG. However, if Islet-1 plays a similar role for the MTN neurons is unknown. To answer whether Islet-1 is as important for axon guidance in these centrally - located sensory neurons as it is for the peripherally-located TG neurons, we investigated the effect of reduced Islet-1 expression on axonal pathfinding in the chick MTN. We employed in ovo electroporation to transfect short interfering RNA for Islet-1 (si-Islet-1) into the dorsal midbrain. Our findings showed that, within the central nervous system, Islet-1 knockdown did not affect axonal growth direction of MTN neurons. However, outside the central nervous system a reduction of Islet-1 led to disorganized axonal growth. We observed an abnormal organisation in the maxillary division of the trigeminal nerve when Islet-1 was reduced in MTN neurons.

developmental biology↗

bioRxiv2025

Structural basis of antiphage defense by an ATPase-associated reverse transcriptase

Reverse transcriptases (RTs) have well-established roles in the replication and spread of retroviruses and retrotransposons. However, recent evidence suggests that RTs have been conscripted by cells for diverse roles in antiviral defense. Here we determine structures of a type I-A retron, which explain how RNA, DNA, RT, HNH-nuclease and four molecules of an SMC-family ATPase assemble into a 364 kDa complex that provides phage defense. We show that phage-encoded nucleases trigger degradation of the retron-associated DNA, leading to disassembly of the retron and activation of the HNH nuclease. The HNH nuclease cleaves tRNASer, stalling protein synthesis and arresting viral replication. Taken together, these data reveal diverse and paradoxical roles for RTs in the perpetuation and elimination of genetic parasites.

biochemistry↗

bioRxiv2024

Baby Open Brains: An Open-Source Repository of Infant Brain Segmentations

Reproducibility of neuroimaging research on infant brain development remains limited due to highly variable protocols and processing approaches. Progress towards reproducible pipelines is limited by a lack of benchmarks such as gold standard brain segmentations. Addressing this core limitation, we constructed the Baby Open Brains (BOBs) Repository, an open source resource comprising manually curated and expert-reviewed infant brain segmentations. Markers and expert reviewers manually segmented anatomical MRI data from 71 infant imaging visits across 51 participants, using both T1w and T2w images per visit. Anatomical images showed dramatic differences in myelination and intensities across the 1 to 9 month age range, emphasizing the need for densely sampled gold standard manual segmentations in these ages. The BOBs repository is publicly available through the Masonic Institute for the Developing Brain (MIDB) Open Data Initiative, which links S3 storage, Datalad for version control, and BrainBox for visualization. This repository represents an open-source paradigm, where new additions and changes can be added, enabling a community-driven resource that will improve over time and extend into new ages and protocols. These manual segmentations and the ongoing repository provide a benchmark for evaluating and improving pipelines dependent upon segmentations in the youngest populations. As such, this repository provides a vitally needed foundation for early-life large-scale studies such as HBCD.

neuroscience↗

bioRxiv2023

Gestational and postnatal age associations for striatal tissue iron deposition in early infancy

Striatal development is crucial for later motor, cognitive, and reward behavior, but age-related change in striatal physiology during the neonatal period remains understudied. An MRI-based measure of tissue iron deposition, T2*, is a non-invasive way to probe striatal physiology neonatally, linked to dopaminergic processing and cognition in children and adults. Striatal subregions have distinct functions that may come online at different time periods in early life. To identify if there are critical periods before or after birth, we measured if striatal iron accrued with gestational age at birth [range=34.57-41.85 weeks] or postnatal age at scan [range=5-64 days], using MRI to probe the T2* signal in N=83 neonates in three striatal subregions. We found iron increased with postnatal age in the pallidum and putamen but not the caudate. No significant relationship between iron and gestational age was observed. Using a subset of infants scanned at preschool age (N=26), we show distributions of iron shift between timepoints. In infants, the pallidum had the least iron of the three regions but had the most by preschool age. Together, this provides evidence of distinct change for striatal subregions, a possible differentiation between motor and cognitive systems, identifying a mechanism that may impact future trajectories. HighlightsO_LINeonatal striatal tissue iron can be measured using the T2* signal from rsfMRI C_LIO_LInT2* changed with postnatal age in the pallidum and putamen but not in the caudate C_LIO_LInT2* did not change with gestational age in any of the three regions C_LIO_LIPatterns of iron deposition (nT2*) among regions shift from infancy to preschool C_LI

neuroscience↗

bioRxiv2023

Endometrial cyclin A2 deficiency is associated with human female infertility and is recapitulated in a conditional knockout mouse model

Proper action of the female sex steroids, 17{beta}-estradiol (E2) and progesterone (P4) on endometrium is essential for fertility. Beyond its role in regulating the cell cycle, cyclin A2 (CCNA2) also mediates E2 and P4 signaling in vitro, but a potential role in modulating steroid action for proper endometrial tissue development and function is unknown. To fill this gap in our knowledge, we examined human endometrial tissue from fertile and infertile women for CCNA2 expression and correlated this with pregnancy outcome. Functional assessment of CCNA2 was validated in vivo using a conditional Ccna2 uterine deficient mouse model while in vitro function was assessed using human cell culture models. We found that CCNA2 expression was significantly reduced in endometrial tissue, specifically the stromal cells, from women undergoing in vitro fertilization who failed to achieve pregnancy. Conditional deletion of Ccna2 from moue uterine tissue recapitulated the inability to achieve successful pregnancy which appears to be due to alterations in the process of decidualization, which was confirmed using in vitro models. From these studies, we conclude that CCNA2 expression during the proliferative/regenerative stage of the menstrual cycle acts as a safeguard allowing for proper steroid responsiveness, decidualization and pregnancy. When CCNA2 expression levels are insufficient there is impaired endometrial responsiveness, aberrant decidualization and loss of pregnancy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/545284v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@150b471org.highwire.dtl.DTLVardef@176a804org.highwire.dtl.DTLVardef@110a47dorg.highwire.dtl.DTLVardef@19f186c_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

BIBSNet: A Deep Learning Baby Image Brain Segmentation Network for MRI Scans

ObjectivesBrain segmentation of infant magnetic resonance (MR) images is vitally important for studying typical and atypical brain development. The infant brain undergoes many changes throughout the first years of postnatal life, making tissue segmentation difficult for most existing algorithms. Here we introduce a deep neural network BIBSNet (Baby and Infant Brain Segmentation Neural Network), an open-source, community-driven model for robust and generalizable brain segmentation leveraging data augmentation and a large sample size of manually annotated images. Experimental DesignIncluded in model training and testing were MR brain images from 90 participants with an age range of 0-8 months (median age 4.6 months). Using the BOBs repository of manually annotated real images along with synthetic segmentation images produced using SynthSeg, the model was trained using a 10-fold procedure. Model performance of segmentations was assessed by comparing BIBSNet, joint label fusion (JLF) inferred segmentation to ground truth segmentations using Dice Similarity Coefficient (DSC). Additionally, MR data along with the FreeSurfer compatible segmentations were processed with the DCAN labs infant-ABCD-BIDS processing pipeline from ground truth, JLF, and BIBSNet to further assess model performance on derivative data, including cortical thickness, resting state connectivity and brain region volumes. Principal ObservationsBIBSNet segmentations outperforms JLF across all regions based on DSC comparisons. Additionally, with processed derived metrics, BIBSNet segmentations outperforms JLF segmentations across nearly all metrics. ConclusionsBIBSNet segmentation shows marked improvement over JLF across all age groups analyzed. The BIBSNet model is 600x faster compared to JLF, produces FreeSurfer-compatible segmentation labels, and can be easily included in other processing pipelines. BIBSNet provides a viable alternative for segmenting the brain in the earliest stages of development.

neuroscience↗

bioRxiv2022

In vivo Safety and Immunoactivity of Oncolytic Jurona Virus in Hepatocellular Carcinoma: A Comprehensive Proteogenomic Analysis

Oncolytic viruses can effectively unwrap a multimodal anti-tumor activity, encompassing a selective tumor cell killing and promoting a systemic anti-tumor immunity, making them a formidable foe against cancer. Among these, several members of the Rhabdoviridae family are particularly attractive as oncolytic agents due to their natural tumor selectivity and non-pathogenicity in humans. In this study, we demonstrated that intratumorally (IT) administration of Jurona virus (JURV), a novel oncolytic Rhabdovirus, induces dynamic tumor regression in human HCC xenograft and syngeneic models. Our data shows that IT injections of JURV trigger the recruitment and activation of cytotoxic T (CTLs) and decrease the tumor-associated macrophages (TAM) infiltration leading to tumor growth delay in both local and distant murine HCC tumors in a syngeneic model. Moreover, when administered concomitantly, JURV and anti-PD-1 therapy profoundly modulate the tumor microenvironment (TME) via enhanced infiltration of CTLs, suggesting that immune checkpoint blockade therapy could potentiate the immunomodulatory effect of JURV and potentially provide durable anti-tumor immunity. Our analysis of the molecular and cellular mechanism of JURV-medicated anti-cancer activity unveiled that JURV and anti-PD-1 antibodies activate different effectors of the immune system but have complementary anti-tumor activities. Furthermore, our results indicate that the abscopal effect induced by JURV is likely mediated by the mechanism regulating the T helper cell responses. Our work supports the further development of JURV as a novel immunovirotherapy platform for hepatocellular carcinoma.

immunology↗

bioRxiv2022

Loss of the repressor REST affects progesterone receptor function and promotes uterine leiomyoma pathogenesis

Uterine Leiomyomas (UL) are benign tumors that arise in the myometrial layer of the uterus. The standard treatment option for UL is hysterectomy, although hormonal therapies such as selective progesterone receptor modulators are often used as temporary treatment options to reduce symptoms or slow the growth of tumors. However, since the pathogenesis of UL is poorly understood and most hormonal therapies are not based on UL-specific, divergent hormone signaling pathways, hallmarks that predict long-term efficacy and safety of pharmacotherapies remain largely undefined. In a previous study, we reported aberrant expression of REST/NRSF target genes activate UL growth due to the near ubiquitous loss of REST. Here, we show that ablation of the Rest gene in mouse uterus leads to UL phenotype and gene expression patterns analogous to UL, including altered estrogen and progesterone signaling pathways. We demonstrate that many of the genes dysregulated in UL harbor cis-regulatory elements bound by REST and progesterone receptor (PGR) adjacent to each other. Crucially, we identify an interaction between REST and PGR in healthy myometrium and present a putative mechanism for the dysregulation of progesterone responsive genes in UL ensuing the loss of REST. Using three Rest conditional knockout mouse lines, we provide a comprehensive picture of the impact loss of REST has in UL pathogenesis and in altering the response of UL to steroid hormones. Significance statementAblation of Rest gene in the mouse uterus, modelling the loss REST in uterine fibroids, results in tumor formation and gene expression patterns analogous to human UL, including altered estrogen and progesterone receptor (PGR) pathways. The current study provides a putative mechanism for the aberrant function of PGR in UL.

physiology↗

bioRxiv2020

Towards Reproducible Brain-Wide Association Studies

Magnetic resonance imaging (MRI) continues to drive many important neuroscientific advances. However, progress in uncovering reproducible associations between individual differences in brain structure/function and behavioral phenotypes (e.g., cognition, mental health) may have been undermined by typical neuroimaging sample sizes (median N=25)1,2. Leveraging the Adolescent Brain Cognitive Development (ABCD) Study3 (N=11,878), we estimated the effect sizes and reproducibility of these brain-wide associations studies (BWAS) as a function of sample size. The very largest, replicable brain-wide associations for univariate and multivariate methods were r=0.14 and r=0.34, respectively. In smaller samples, typical for brain-wide association studies (BWAS), irreproducible, inflated effect sizes were ubiquitous, no matter the method (univariate, multivariate). Until sample sizes started to approach consortium-levels, BWAS were underpowered and statistical errors assured. Multiple factors contribute to replication failures4-6; here, we show that the pairing of small brain-behavioral phenotype effect sizes with sampling variability is a key element in wide-spread BWAS replication failure. Brain-behavioral phenotype associations stabilize and become more reproducible with sample sizes of N{gap}2,000. While investigator-initiated brain-behavior research continues to generate hypotheses and propel innovation, large consortia are needed to usher in a new era of reproducible human brain-wide association studies.

neuroscience↗

bioRxiv2020

Transmembrane Stem Cell Factor Protein Therapeutics EnhanceRevascularization in Ischemia without Mast Cell Activation

Stem cell factor (SCF) is a cytokine that regulates hematopoiesis and other biological processes. While clinical treatments using SCF would be highly beneficial, these have been limited by toxicity related to mast cell activation. Transmembrane SCF (tmSCF) has differential activity from soluble SCF and has not been explored as a therapeutic agent. We created novel therapeutics using tmSCF embedded in proteoliposomes or lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and improved the revascularization in the ischemic hind limb. Proteoliposomal tmSCF preferentially acted on endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization and recruitment to the site of injury. The type of lipid nanocarrier used altered the relative cellular uptake pathways and signaling in a cell type dependent manner. Overall, we found that tmSCF-based therapies can provide therapeutic benefits without off target effects.

bioengineering↗

bioRxiv2018

Replication fork collapse at a protein-DNA roadblock leads to fork reversal, promoted by the RecQ helicase

There are numerous impediments that DNA replication can encounter while copying a genome, including the many proteins that bind DNA. Collapse of the replication fork at a protein roadblock must be dealt with to enable replication to eventually restart; failure to do so efficiently leads to mutation or cell death. Several prospective models have been proposed that process a stalled or collapsed replication fork. This study shows that replication fork reversal (RFR) is the preferred pathway for dealing with a collapsed fork in Escherichia coli, along with exonuclease activity that digests the two nascent DNA strands. RFR moves the Y-shaped replication fork DNA away from the site of the blockage and generates a four-way DNA structure, the Holliday junction (HJ). Direct endo-nuclease activity at the replication fork is either slow or does not occur. The protein that had the greatest effect on HJ processing/RFR was found to be the RecQ helicase. RecG and RuvABC both played a lesser role, but did affect the HJ produced: mutations in these known HJ processing enzymes produced longer-lasting HJ intermediates, and delayed replication restart. The SOS response is not induced by the protein-DNA roadblock under these conditions and so does not affect fork processing.\n\nAuthor SummaryTo transfer genetic material to progeny, a cell must replicate its DNA accurately and completely. If a cell does not respond appropriately to inhibitors of the DNA replication process, genetic mutation and cell death will occur. Previous works have shown that protein-DNA complexes are the greatest source of replication fork stalling and collapse in bacteria. This work examines how the cell deals with replication fork collapse at a persistent protein blockage, at a specific locus on the chromosome of Escherichia coli. Cells were found to process the DNA at the replication fork, moving the branch point away from the site of blockage by replication fork reversal and exonuclease activity. Our data indicate that it is the RecQ helicase that has the main controlling role in this process, and not the proteins RecG and RuvABC, as currently understood. RecQ homologs have been shown to be involved in replication fork processing in eukaryotes and their mutation predisposes humans to genome instability and cancer. Our findings suggest that RecQ proteins could play more important role in replication fork reversal than previously understood, and that this role could be conserved across domains.

microbiology↗