Biology subjects

Cunningham, R.

Publications and source records attributed to Cunningham, R..

7 recordsLinked to original sources

bioRxiv2025

AI for IACUC: Accurate Initial Assessment of Institutional Animal Care and Use Committee Protocols

Every animal protocol requires careful review to ensure humane principles, laws, and policies are followed before any experiments may be performed. Developing protocols and receiving approval can be a lengthy process requiring multiple rounds of feedback and refinement. Artificial Intelligence (AI) has the potential to improve the quality and speed of IACUC protocol reviews by detecting submission errors. We describe an approach to achieve this and show that our approach can be replicated, while addressing issues of ethics and bias, robustness, and trustworthiness1. Replication is addressed by developing a template and demonstrating that it can be reused. Ethics and bias issues are addressed by early, limited use of a large language Model (LLM) to identify errors and rapidly provide useful feedback to the protocol developer. In our approach, subsequent steps still require a review by the IACUC board, which retains ethical responsibility for approval. Robustness and trustworthiness were evaluated by using the template that is replicated for eleven distinct checks and performed on 50 new protocols using two different LLMs. We show that every actual problem in those submissions (within the test parameters) is found by our AI review (i.e., recall=100%), giving IACUC boards confidence in the utility of our system. In addition, precision for our checks is between 80%-100%, with most being 100%, thereby making the system a practical compliment to administrative protocol review for those submitting. Overall, we report F1 scores of between 89-100%.

scientific communication and education↗

bioRxiv2024

Development of a pipeline to evaluate YAP-TEAD inhibitor potential - direct TEAD inhibition represses cancerous growth in a NF2-mutant model of mesothelioma

As the core, tumorigenic downstream effectors of the Hippo signalling pathway, YAP/TAZ and the TEAD family of transcription factors represent attractive targets for drug discovery efforts within cancer research. This is particularly true within the context of pleural mesothelioma, in which there are many recent preclinical developments and clinical trials evaluating the efficacy of TEAD inhibitors. The range of inhibitors have shown great promise, but comparisons of their performances are so far limited. Here we develop a high content pipeline that enables a comparative analysis of currently developed YAP/TAZ-TEAD inhibitors. We take advantage of isogenic cellular models that enable us to examine inhibitor specificity. We identify genetic compensation of the Hippo pathway transcriptional module, with implications for therapeutic targeting, and implement Cell Painting to develop a detailed morphological profiling pipeline that enables further characterisation, quantification, and analysis of off-target effects. Our pipeline is scalable and allows us to establish specificity and comparative potency within cancer relevant assays in a clinically relevant cellular model.

cancer biology↗

bioRxiv2024

Cas9 RNP Physiochemical Analysis for Enhanced CRISPR-AuNP Assembly and Function

CRISPR therapy for hematological disease has proven effective for transplant dependent beta thalassemia and sickle cell anemia, with additional disease targets in sight. The success of these therapies relies on high rates of CRISPR-induced double strand DNA breaks in hematopoietic stem and progenitor cells (HSPC). To achieve these levels, CRISPR complexes are typically delivered by electroporation ex vivo which is toxic to HSPCs. HSPCs are then cultured in stimulating conditions that promote error-prone DNA repair, requiring conditioning with chemotherapy to facilitate engraftment after reinfusion. In vivo delivery by nanocarriers of CRISPR gene editing tools has the potential to mitigate this complexity and toxicity and make this revolutionary therapy globally available. To achieve in vivo delivery, the inherent restriction factors against oligonucleotide delivery into HSPCs, that make ex vivo manipulation including electroporation and stimulation essential, must be overcome. To this end, our group developed a CRISPR carrying gold nanoparticle (CRISPR-AuNP) capable of delivering either Cas9 or Cas12a CRISPRs as ribonucleoprotein complexes (RNP) without compromising HSPC fitness. However, the most commonly used CRISPR, Cas9, demonstrated inconsistent activity in this delivery system, with lower activity relative to Cas12a. Investigation of Cas9 RNP biophysics relative to Cas12a revealed duplex RNA instability during the initial loading onto Au cores, resulting in undetectable Cas9 loading to the particle surface. Here we demonstrate preformation of RNP before loading, coupled with optimization of the loading chemistry and conditions, resulted in 39.6 {+/-} 7.0 Cas9 RNP/AuNP without compromising RNP activity in both in vitro assays and primary human HSPC. The same alterations improved Cas12a RNP/AuNP loading 10-fold over previously reported levels. To achieve particle stability, the reported polyethyleneimine outer coating was altered to include PEGylation and the resulting 2nd generation CRISPR-AuNP demonstrates favorable nanoformulation characteristics for in vivo administration, with a hydrophilic, more neutral nanoparticle surface. Direct treatment of HSPC in vitro showed 72.5 {+/-} 7.37% uptake of 2nd generation CRISPR-AuNP in primary human HSPC, but with endosomal accumulation and low rates of gene editing consistent with low levels of endosomal escape.

bioengineering↗

bioRxiv2023

Oncogenic RAS drives rapid onset cellular plasticity and elicits a tumour-promoting neutrophil response at the inception of preneoplastic development

Oncogenic driver mutations are frequently found in normal tissues, suggesting that additional non-genetic factors are required for tumourigenesis. Phenotypic plasticity is an important gateway to malignancy and inflammation can fuel tumourigenesis, however, little is known about when and how these hallmarks first arise. Using single-cell transcriptomics and in vivo live imaging we have characterised the immediate cell intrinsic and innate immune responses during the first 24 hours following oncogenic Ras activation, in an inducible zebrafish model of HRASG12V-mediated skin tumour initiation. We found that only a subset of basal keratinocytes, but not superficial keratinocytes, are susceptible to RAS-driven phenotypic plasticity. These preneoplastic cells undergo dedifferentiation and partial EMT, resembling malignant cells observed in human squamous cell carcinoma (SCC). Strikingly, the same subset instigates the development of tumour-promoting neutrophils, which in turn enhance preneoplastic cell proliferation. Our findings demonstrate that the effects of oncogenic Ras are primarily determined by the cell of origin and reveal an association between the unlocking of phenotypic plasticity and the onset of tumour-promoting inflammation.

cancer biology↗

bioRxiv2021

The transcription factor EGR2 is indispensable for tissue-specific imprinting of alveolar macrophages in health and tissue repair

Alveolar macrophages are the most abundant macrophages in the healthy lung where they play key roles in homeostasis and immune surveillance against air-borne pathogens. Tissue-specific differentiation and survival of alveolar macrophages relies on niche-derived factors, such as colony stimulating factor 2 (CSF-2) and transforming growth factor beta (TGF-{beta}). However, the nature of the downstream molecular pathways that regulate the identity and function of alveolar macrophages and their response to injury remains poorly understood. Here, we identify that the transcriptional factor EGR2 is an evolutionarily conserved feature of lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by TGF-{beta} and CSF-2 in a PPAR-{gamma}-dependent manner to control alveolar macrophage differentiation. Functionally, EGR2 was dispensable for lipid handling, but crucial for the effective elimination of the respiratory pathogen Streptococcus pneumoniae. Finally, we show that EGR2 is required for repopulation of the alveolar niche following sterile, bleomycin-induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar macrophages are vital for effective tissue repair following injury. Collectively, we demonstrate that EGR2 is an indispensable component of the transcriptional network controlling the identity and function of alveolar macrophages in health and disease. One Sentence SummaryEGR2 controls alveolar macrophage function in health and disease

immunology↗

bioRxiv2019

Structural basis for adhesion G protein-coupled receptor Gpr126 function

Many drugs target the extracellular regions (ECRs) of cell-surface receptors. The large and alternatively-spliced ECRs of adhesion G protein-coupled receptors (aGPCRs) have key functions in diverse biological processes including neurodevelopment, embryogenesis, and tumorigenesis. However, their structures and mechanisms of action remain unclear, hampering drug development. The aGPCR Gpr126/Adgrg6 regulates Schwann cell myelination, ear canal formation, and heart development; and GPR126 mutations cause myelination defects in human. Here, we determine the structure of the complete zebrafish Gpr126 ECR and reveal five domains including a previously-unknown domain. Strikingly, the Gpr126 ECR adopts a closed conformation that is stabilized by an alternatively spliced linker and a conserved calcium-binding site. Alternative splicing regulates ECR conformation and receptor signaling, while mutagenesis of the newly-characterized calcium-binding site abolishes Gpr126 function in vivo. These results demonstrate that Gpr126 ECR utilizes a multi-faceted dynamic approach to regulate receptor function and provide novel insights into ECR-targeted drug design.

biochemistry↗

bioRxiv2018

Can machine learning assess trunk alignment directly from raw video?

BackgroundThe Segmental Assessment of Trunk Control (SATCo) evaluates sitting control at seven separate trunk segments, making a judgement based on their position in space relative to a defined, aligned posture. SATCo is in regular clinical and research use and is a Recommended Instrument for Cerebral Palsy and Spinal Cord Injury-Paediatric by The National Institute of Neurological Disorders and Stroke (US). However, SATCo remains a subjective assessment.\n\nResearch questionThis study tests the feasibility of providing an objective, automated identification of frames containing the aligned, reference trunk posture using deep convolutional neural network (DCNN) analysis of raw high definition and depth (HD+D) images.\n\nMethodsA SATCo was conducted on sixteen healthy male adults and recorded using a Kinect V2. For each of seven segments tested, two different trials were collected (control and no-control) to simulate a range of alignment configurations. For all images, classification of alignment obtained from a trained and validated DCNN was compared to expert clinicians labelling.\n\nResultsUsing leave-one-out testing, at the optimal operating threshold, the DCNN correctly classified individual images (alignment v misaligned) with average precision 92.7{+/-}16% (mean{+/-}SD).\n\nSignificanceThese results show for the first time, automation of a key component of the SATCo test, namely identification of aligned trunk posture directly from raw images (HD+D). This demonstrates the potential of machine learning to provide a fully automated, objective SATCo test to enhance assessment of trunk control in children and adults for research and treatment of various conditions including neurodisability and stroke.

bioengineering↗