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Johnson, K. A.

Publications and source records attributed to Johnson, K. A..

At least 19 recordsLinked to original sources

Single Channel Strongly-Coupled Geometry Surface Coil for Small Mammal Whole Brain fMRI

1Abstract/SummarySingle-channel surface coils remain central to rodent MRI, but conventional circular loop designs face an inherent trade-off between surface and depth sensitivity, limiting whole-brain coverage for applications such as resting-state BOLD fMRI. This work introduces a single-channel strongly-coupled geometry surface coil. It consists of a stop-sign shaped loop inductively overcoupled to a nested, three-turn elongated racetrack spiral designed to improve depth sensitivity and thru-plane coverage while remaining robust to variable sample loading. Benchtop characterization across three phantoms of differing size showed the parallel resonant mode and loaded quality factor changed negligibly with loading. In phantom imaging at 9.4 T, the SCG coil achieved in-plane SNR and temporal SNR comparable to, and at shallow depths exceeding, a commercial Bruker 2x2 receive-only rat brain array, while showing substantially more consistent tSNR across loading conditions. The SCG coil also demonstrated superior thru-plane tSNR over a 20 mm slice range at 3.5 mm depth, approximating the anterior-posterior extent of the rat brain. In vivo resting-state BOLD fMRI in eight rats, acquired with a double asymmetric spin-echo EPI sequence, yielded a default mode network consistent with prior reports and revealed a previously undescribed subcortical network spanning superior/inferior colliculi and cerebellar regions. These results establish the single-channel SCG as a promising foundation for next-generation rodent receive coil arrays, combining loading-independent tuning with extended sensitive coverage suitable for whole-brain functional imaging.

biophysics↗

Large-scale automated detection reveals pervasive sex imbalance in biomedical research

Sex is a critical biological variable that impacts disease risk, progression, and treatment response across virtually every organ system. However, decades of biomedical research have relied primarily on male study subjects, leaving large gaps in our understanding of female-specific disease biology. Quantifying the extent of this imbalance across thousands of disease areas and millions of publicly available biological samples has remained computationally intractable. Here, we present a multimodal computational framework that infers the biological sex of [~]230,000 publicly available human transcriptome samples and links inferred sex labels to disease terms extracted from [~]9,000 associated study records and [~]5,000 publication abstracts to quantify sex imbalance at scale. Applying this approach revealed that the majority of disease terms with the largest research-derived sex imbalance are skewed toward male representation, including areas with no known biological justification for that imbalance. After adjusting for global sex-specific disease prevalence to isolate biologically unjustified imbalance, up to 58% of all disease terms showed male-leaning association. Diseases including glioblastoma, cirrhosis, idiopathic pulmonary fibrosis, and schizophrenia emerged as critically understudied in females despite affecting both sexes comparably. These findings provide a principled, data-driven basis for prioritizing compensatory research efforts and offer a reusable framework for ongoing monitoring of sex representation in the biomedical literature. HighlightsO_LISkewed male and female study subject representation in biomedical research is the result of decades of studies conducted without adequate female representation. C_LIO_LIWe developed an automated, multimodal framework to estimate the sex imbalance across thousands of disease terms using metadata from [~]230,000 transcriptomics samples and their associated [~]9,000 studies and [~]5,000 publications. C_LIO_LIOur approach identifies non-sex-specific disease research areas that have been studied using an unbalanced sex demographic. These areas need compensatory and balanced studies to understand sex differences. C_LI

genomics↗

Transient signaling of free ADP-ribose monitored with an intracellular biosensor

We have developed a biosensor enabling the dynamic, compartmentalized, and longitudinal measurements of intracellular ADP-ribose (ADPR) in live cells. Free ADPR is a critical signaling metabolite derived from nicotinamide adenine dinucleotide (NAD+). As an agonist for Transient Receptor Potential Melastatin 2 (TRPM2), ADPR levels can regulate immune responses during infection, as well as nociception and adjustment of core body temperature. The study of ADPR signaling has been limited, however, by a lack of methods to measure this metabolite in situ. Using the biosensor and its paired non-responsive control, we determine that intracellular ADPR accumulation was transient and tunable. We found that basal concentrations were in the nanomolar range and could be stimulated [~]30-fold to activate TRPM2. We identified that TRPM2 activation, measured by calcium influx, required an intracellular ADPR threshold concentration between 2 - 4 {micro}M at physiological temperature. We observed that the timing of the ADPR rise coincided with TRPM2 activation, thus providing support for ADPR fluctuations being a critically regulated aspect for channel activation. Notably, transient fluctuations of ADPR were not accurately reflected by measurements of intracellular NAD+ loss or calcium levels. Significance StatementWe have developed a unique real-time biosensor for free ADP-ribose that is tuned to physiological concentrations and capable of intracellular measurements in individual cells. Using a calibrated system we determined that concentrations and timing of induced intracellular ADPR aligned with the thermosensitive TRPM2 activity. The data support ADPR as a critical component whose intracellular levels are regulated to control TRPM2 channel opening in cells.

biochemistry↗

Blood-based Transcriptomics Reveal Sex- and Amyloid-Modulated Biology of Plasma pTau217 in Preclinical Alzheimer's Disease

Plasma pTau217, an emerging Alzheimers disease (AD) biomarker, may reflect a synaptic response to {beta}-amyloid (A{beta}) plaques before cortical tangle formation, but its broader biological correlates remain unclear. We sought to identify associations between whole blood gene expression and plasma pTau217, and to determine whether APOE{varepsilon}4, sex, and neocortical A{beta}-PET modify these associations in 724 participants from the Anti-Amyloid Treatment in Asymptomatic Alzheimers and accompanying LEARN studies (A4/LEARN, Agemean(SD)=72.2(4.6); 63%female). Of 20,621 genes tested (1,048 X-linked), none were directly associated with pTau217; one gene was moderated by APOE{varepsilon}4, 1,540 genes by A{beta}-PET, and 772 genes by both A{beta}-PET and sex. Over 100 of these significant associations were X-linked, supporting a role of the X chromosome in AD. Sex interactions were only observed in the presence of elevated A{beta}-PET. Our results underscore the complexity of molecular mechanisms that can be linked to plasma pTau217, particularly in the context of elevated A{beta}-PET.

neuroscience↗

Kinetic mechanism of Renilla luciferase guides induced-fit engineering for improved bioluminescence

Luciferases are widely used bioluminescent reporters, yet the molecular determinants of their catalytic efficiency and light-emission stability remain incompletely understood. Here, we reconstruct the complete catalytic pathway of Renilla luciferase by combining steady-state, transient, and temperature-dependent kinetics with crystallography and molecular simulations. We show that the enzyme is substantially undersaturated with oxygen (Km,O2 = 719 M), causing its true turnover number (kcat = 21.9 s-1) to be systematically underestimated. Concurrently, elevated oxygen drives irreversible enzyme inactivation after ~1,500 turnovers, revealing a fundamental trade-off that limits oxygen-affinity engineering. Instead, the genuine bottleneck of the catalytic cycle is the induced-fit conformational opening of the product-bound enzyme. Selective engineering of this transition step through rational loop grafting yielded a variant AncFT-L14 with enhanced catalytic efficiency and glow-type bioluminescence with substantially slower signal decay in cell lysates. Collectively, our results identify conformational dynamics as a primary tunable determinant of luciferase function. More broadly, this work establishes a mechanistically grounded framework for the development of next-generation bioluminescent tools and for engineering enzymes controlled by dynamically gated ligand exchange.

biochemistry↗

Generative genomics accurately predicts future experimental results

Realizing AIs promise to accelerate biomedical research requires AI models that are both accurate and sufficiently flexible to capture the diversity of real-life experiments. Here, we describe a generative genomics framework for AI-based experimental prediction that mirrors the process of designing and conducting an experiment in the lab or clinic. We created GEM-1 (Generate Expression Model-1), an AI system that effectively models the enormous range of bulk and single-cell gene expression experiments performed by scientists and benchmarked its performance across multiple biological axes. GEM-1s prediction of future gene expression experiments-RNA-seq data deposited in public archives after our training data cutoff-yielded accuracy comparable to the best-possible performance estimated by comparing the results of matched lab experiments. Overall, our approach illustrates the transformative potential of generative genomics for applications ranging from predicting cellular perturbations in vitro to de novo generation of data from large clinical cohorts.

genomics↗

Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing owing to their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems such as Cas12a and SpCas9. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here, we characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Cas12f-MG119-28, which supports robust genome editing in human cells. Through structural, biochemical, and kinetic analyses, we compare Cas12f-MG119-28 with two recently described orthologs, Oscillibacter sp. Cas12f (OsCas12f) and Ruminiclostridium herbifermentans Cas12f (RhCas12f). These orthologs present divergent architectures and regulatory features governing PAM recognition, gRNA binding, dimerization, and DNA cleavage. Notably, Cas12f-MG119-28 achieves efficient R-loop formation via a stable dimer interface and a naturally optimized guide RNA. These discoveries elucidate key mechanistic determinants of Cas12f activity and may offer a framework for engineering compact genome editors with therapeutic potential.

biochemistry↗

Targeted Protein Degradation by KLHDC2 Ligands Identified by High Throughput Screening

Proteolysis targeting chimeras (PROTACs) enable the selective and sub-stoichiometric elimination of pathological proteins, yet only two E3 ligases are routinely used for this purpose. Here, we expand the repertoire of PROTAC compatible E3 ligases by identifying a novel small molecule scaffold targeting the ubiquitin E3 ligase KLHDC2 using a fluorescence polarization-based high throughput screen. We highlight the utility of this ligand with the synthesis of PROTACs capable of potently degrading BRD4 in cells. This work affords additional chemical matter for targeting KLHDC2 and suggests a practical approach for identifying novel E3 binders by high throughput screening.

biochemistry↗

Enhancement of Prednisolone efficacy and safety in Duchenne muscular dystrophy via neutrophil elastase inhibition

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by muscle wasting, and leading to premature death. The pathogenesis of DMD is complex. It is triggered by loss of the cytoskeletal protein dystrophin, which causes muscle fiber instability and damage, followed by chronic inflammation and fibrotic replacement of damaged muscle tissue. Here we investigated the hypothesis that inhibition of neutrophil elastase, which is increased in dystrophic mice and impairs myogenesis, could provide a therapeutic effect in DMD. While neutrophil elastase inhibition, via the orally bioavailable compound Alvelestat, enhances myogenesis both in vitro and in vivo and reduces muscle damage and fibrosis, it does not produce a significant beneficial effect on the functional outcomes. However, when co- administered with the standard of care Prednisolone, Alvelestat dramatically enhances Prednisolone efficacy in a mouse model of DMD. In key functional tests, the performance of mice co-dosed with Alvelestat and Prednisolone is almost three times better than that of mice treated with Prednisolone alone. Additionally, co-administration of Alvelestat and Prednisolone reduces Prednisolones negative effects on muscle mass and resistance to fatigue. These findings are consistent with the finding that Prednisolone induces neutrophil elastase expression in patients affected by DMD. Our data support a beneficial role for neutrophil elastase inhibition in a mouse model of DMD that is also treated with Prednisolone, suggesting that a combinatorial therapy for DMD including a corticosteroid and a neutrophil elastase inhibitor could be trialed in humans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/640472v3_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@f20853org.highwire.dtl.DTLVardef@b7fforg.highwire.dtl.DTLVardef@12fe312org.highwire.dtl.DTLVardef@e4cf0c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

Asymmetric spin echo multi-echo echo planar imaging (ASEME-EPI) sequence for pre-clinical high-field fMRI

In functional magnetic resonance imaging (fMRI) of the blood oxygen level-dependent (BOLD) contrast, gradient-recalled echo (GRE) acquisitions offer high sensitivity but suffer from susceptibility-induced signal loss and lack specificity to microvasculature. In contrast, spin echo (SE) acquisitions provide improved specificity at the cost of reduced sensitivity. This study introduces Asymmetric Spin Echo Multi-Echo Echo Planar Imaging (ASEME-EPI), a technique designed to combine the benefits of both GRE and SE for high-field preclinical fMRI. ASEME-EPI employs a spin echo readout followed by two asymmetric spin echo (ASE) GRE readouts, providing an initial T2-weighted SE image and subsequent T2*-weighted ASE images. A feasibility study for the technique was implemented on a 9.4 T pre-clinical MRI system and tested using a visual stimulation in northern tree shrews. Comparing ASEME-EPI with conventional GRE echo planar imaging (GRE-EPI) and SE echo planar imaging (SE-EPI) acquisitions, results showed that ASEME-EPI achieved BOLD contrast-to-noise ratio (CNR) comparable to GRE-EPI while offering improved specificity in activation maps. ASEME-EPI activation was more confined to the primary visual cortex (V1), unlike GRE-EPI which showed activation extending beyond anatomical boundaries. Additionally, ASEME-EPI demonstrated the ability to recover signal in areas of severe field inhomogeneity where GRE-EPI suffered from signal loss. The performance of ASEME-EPI is attributed to its multi-echo nature, allowing for SNR-optimized combination of echoes, effectively denoising the data. The inclusion of the initial SE also contributes to signal recovery in areas prone to susceptibility artifacts. This feasibility study demonstrates the potential of ASEME-EPI for high-field pre-clinical fMRI, offering a promising compromise between GRE sensitivity and SE specificity while addressing challenges of T2* decay at high field strengths.

biophysics↗

DNA targeting by compact Cas9d and its resurrected ancestor

The type II-A CRISPR effector SpCas9 has gained widespread popularity as an efficient and programmable genome editing tool. However, much remains to be known about novel compact variants that may overcome some limitations of current systems1,2. Recently, alternative CRISPR-Cas systems with highly compact nucleases capable of genome editing in mammalian cells have been discovered through metagenomic analysis of uncultivated microbes, including Cas9d (a type II-D CRISPR-Cas effector)3. Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains and facilitate DNA target recognition. We find that the sgRNA can be truncated by up to [~]25% yet still retain activity in vivo. We also show that despite preferentially targeting an NGG PAM, Cas9d exhibits a unique mechanism for PAM recognition. Finally, we identify the first Cas9d smaller than 800 amino acids exhibiting robust nuclease activity in mammalian cells. Using ancestral sequence reconstruction, we demonstrate that it is possible to generate compact nucleases capable of efficient genome editing by expanding the diversity of Cas9d families. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a molecular blueprint for future rational re-engineering of minimal RNA-guided DNA nucleases.

biochemistry↗

CONE: COntext-specific Network Embedding via Contextualized Graph Attention

Human gene interaction networks, commonly known as interactomes, encode genes functional relationships, which are invaluable knowledge for translational medical research and the mechanistic understanding of complex human diseases. Meanwhile, the advancement of network embedding techniques has inspired recent efforts to identify novel human disease-associated genes using canonical interac-tome embeddings. However, one pivotal challenge that persists stems from the fact that many complex diseases manifest in specific biological contexts, such as tissues or cell types, and many existing interactomes do not encapsulate such information. Here, we propose CONE3, a versatile approach to generate context-specific embeddings from a context-free interactome. The core component of CONE consists of a graph attention network with contextual conditioning, and it is trained in a noise contrastive fashion using contextualized interactome random walks localized around contextual genes. We demonstrate the strong performance of CONE embeddings in identifying disease-associated genes when using known associated biological contexts to the diseases. Furthermore, our approach offers insights into understanding the biological contexts associated with human diseases.

bioinformatics↗

Assessment of behavioral flexibility in mice with conditional deletion of metabotropic glutamate receptor 2 from Emx1-lineage neurons

Convergent lines of evidence from animal models suggest that disrupted metabotropic glutamate receptor 2 (mGlu2) function promotes high levels of drug consumption for a variety of psychoactive drugs including alcohol, opioids, and psychostimulants. In both rodents and humans, impaired behavioral flexibility prior to first drug use correlates with high levels of drug consumption later in life. Thus, we posited that deletion of mGlu2 from brain regions that contribute to behavioral flexibility, including cortical regions, could predispose animals to high levels of drug consumption by impairing behavioral flexibility. To evaluate the role of mGlu2 in behavioral flexibility, we generated mice with a floxed Grm2 allele (Grm2f/f) and selectively disrupted mGlu2 expression in neurons of the Emx1 lineage (primarily telencephalonic projection neurons) by crossing these mice with an Emx1-IRES-Cre driver line. Behavioral flexibility, including sensitivity to change in either outcome value or action-outcome contingency, was evaluated in adult male and female mice trained to press a lever for a food reinforcer. Contrary to our hypothesis, mGlu2 deletion did not facilitate habitual responding assessed by devaluation, contingency degradation, or omission tests. Male Grm2f/f;Emx1-IRES-Cre+/- mice showed modest impairment in reversal learning compared with littermate controls. Finally, we saw a sex-specific effect of mGlu2 deletion on response vigor in male mice trained on a random ratio reinforcement schedule. However, we did not find evidence of a general reduction in motivation in a progressive ratio test. These findings suggest that loss of mGlu2 from cortical circuitry is unlikely to create a predisposition to inflexible behavior that facilitates excessive drug consumption.

neuroscience↗

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an "NGG" protospacer-adjacent motif (PAM) sequence immediately adjacent to the DNA target limits the number of editable genes. To overcome the PAM requirement, a recently developed Cas9 variant, called SpRY-Cas9 was engineered to be "PAMless" (1, 2). However, the molecular mechanisms of how SpRY can recognize all potential PAM sequences and still accurately identify DNA targets have not been investigated. Here, we combined enzyme kinetics, cryo-EM, and single-molecule imaging to determine how SpRY interrogates DNA and recognizes target sites for cleavage. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region of SpRY, which facilitates tight binding to off-target DNA sequences. Once SpRY correctly identifies a target site, nuclease activation occurs [~]1,000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Insights gained from our intermediate structures prompted rationally designed mutants with improved DNA cleavage efficiency. Our findings shed light on the molecular mechanisms of PAMless genome editing with SpRY and provide a framework for the design of future genome editing tools with improved versatility, precision, and efficiency.

biochemistry↗

Super-resolution imaging of neuronal structure with structured illumination microscopy

Super-resolution structured illumination microscopy (SR-SIM) is a method in optical fluorescence microscopy which is suitable for imaging a wide variety of cells and tissues in biological and biomedical research. Typically, SIM methods use high spatial frequency illumination patterns generated by laser interference. This approach provides high resolution but is limited to thin samples such as cultured cells. Using a different strategy for processing the raw data and coarser illumination patterns, we imaged through a 150 {micro}m thick coronal section of a mouse brain expressing GFP in a subset of neurons. The resolution reached 144 nm, an improvement of 1.7 fold beyond conventional widefield imaging.

biophysics↗

Joint representation of molecular networks from multiple species improves gene classification

Network-based machine learning (ML) has the potential for predicting novel genes associated with nearly any health and disease context. However, this approach often uses network information from only the single species under consideration even though networks for most species are noisy and incomplete. While some recent methods have begun addressing this shortcoming by using networks from more than one species, they lack one or more key desirable properties: handling networks from multiple species, incorporating many-to-many orthology information, or generating a network representation that is reusable across different types of and newly-defined prediction tasks. Here, we present GenePlexusZoo, a framework that casts molecular networks from multiple species into a single reusable feature space for network-based ML. We demonstrate that this multi-species network representation improves both gene classification within a single species and knowledge-transfer across species, even in cases where the inter-species correspondence is undetectable based on shared orthologous genes. Thus, GenePlexusZoo enables effectively leveraging the high evolutionary molecular, functional, and phenotypic conservation across species to discover novel genes associated with diverse biological contexts.

bioinformatics↗

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may therefore display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP dependent cellular growth.

cell biology↗

Leveraging public transcriptome data with machine learning to infer pan-body age- and sex-specific molecular phenomena

Age and sex are historically understudied factors in biomedical studies even though many complex traits and diseases vary by these factors in their incidence and presentation. As a result, there are massive gaps in our understanding of genes and molecular mechanisms that underlie sex- and age-associated physiology and disease. Hundreds of thousands of publicly-available human transcriptomes capturing gene expression profiles of tissues across the body and subject to various biomedical and clinical factors present an invaluable, yet untapped, opportunity for bridging these gaps. Here, we present a computational framework that leverages these data to infer genome-wide molecular signatures specific to sex and age groups. As the vast majority of these profiles lack age and sex labels, the core idea of our framework is to use the measured expression data to predict missing age/sex metadata and derive the signatures from the predictive models. We first curated [~]30,000 primary samples associated with age and sex information and profiled using microarray and RNA-seq. Then, we used this dataset to infer sex-biased genes within eleven age groups along the human lifespan and then trained machine learning (ML) models to predict these age groups from gene expression values separately within females and males. Specifically, we trained one-vs-rest logistic regression classifiers with elastic-net regularization to classify transcriptomes into age groups. Dataset-level cross validation shows that these ML classifiers are able to discriminate between age groups in a biologically meaningful way in each sex across technologies. Further, these predictive models capture sex-stratified age-group gene signatures, i.e., the strength and the direction of importance of genes across the genome for each age group in each sex. Enrichment analysis of these gene signatures with prior gene annotations helped in identifying age- and sex-associated multi-tissue and pan-body molecular phenomena (e.g., general immune response, inflammation, metabolism, hormone response). We developed a web-app (http://mlgenesignatures.org/) to visualize our expression dataset, signatures, and enrichment results to make these easily accessible for interested researchers. Overall, we have presented a path for effectively leveraging massive public omics data collections to investigate the molecular basis of age- and sex-differences in physiology and disease. SummaryHundreds of thousands of publicly-available human transcriptomes capturing gene expression profiles of tissues across the body and subject to various biomedical and clinical factors present an invaluable, yet untapped, opportunity for studying age and sex. We first curated [~]30,000 primary microarray and RNA-seq samples. Then, we used this dataset to infer sex-biased genes within eleven age groups along the human lifespan and trained machine learning models to predict these age groups from gene expression values separately within females and males. These predictive models capture sex-stratified age-group gene signatures, i.e., the strength and the direction of importance of every gene in each age group in each sex. Enrichment analysis of these gene signatures with prior gene annotations helped identify age- and sex-associated multi-tissue molecular phenomena. A web-app makes our dataset and results easily visualizable. Overall, we have presented a path for effectively leveraging massive public omics data collections to investigate the molecular basis of age- and sex-differences in physiology and disease.

bioinformatics↗