Biology subjects

Williams, J. C.

Publications and source records attributed to Williams, J. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Meditope-Enabled Chimeric Antigen Receptors Facilitate Plug-and-Play Control of T Cells

Chimeric antigen receptor (CAR) T cells have transformed cancer treatment, yet challenges for achieving broader clinical success remain, including overcoming tumor antigen heterogeneity and limited T cell fitness. To address these challenges and enhance CAR T cell functionality, we leveraged meditope technology, a lock-and-key platform where Fab regions of antibodies are modified to bind a small cyclic peptide termed meditope (meP). We developed a panel of meditope-enabled Fab-based CARs (meCARs), which show selective binding to the meP and comparable activity to traditional single-chain variable fragment (scFv)-based CARs. Focusing on HER2-targeted meCARs for evaluating platform utility, we exploited the modularity of the meditope platform to detect meCAR T cells using meP-fused fluorescent agents, promote meCAR T cell expansion via meP-fused IL-15 cytokine, and broaden tumor antigen targeting through meP-fused antibodies to address tumor heterogeneity. These findings establish the meditope technology as a versatile strategy to augment CAR T cell functionality and overcome key limitations of current CAR-based therapies.

bioengineering↗

bioRxiv2025

Towards Optimizing Target Engagement in Non-Invasive Trigeminal Nerve Stimulation: Anatomical Characterization and Computational Modeling of the Human Trigeminal Nerve

ObjectiveCranial nerve stimulation (CNS) uses electric current to modulate higher-order brain activity and organ function via nerves, including the vagus and trigeminal, with applications in migraine, epilepsy, and pediatric ADHD. The trigeminal nerve is an emerging target for non-invasive neuromodulation due to the superficial trajectory of its branches, the supraorbital (SON), infraorbital (ION), and mental nerves (MN), and the predominantly sensory composition of the SON and ION. However, the parameters and outcomes of trigeminal nerve stimulation (TNS) remain varied. ApproachThis study characterizes the anatomical course, tissue composition, and activation profiles of the SON, ION, and MN using five human donors. CT imaging was utilized to localize each nerves exit foramen and distance to midline. Microdissections quantified nerve circumference and depth relative to the skin surface. Histological analysis described the number of fascicles and fascicular tissue area. Nerve depths were incorporated into computational models to illustrate the activation function across tissue layers, comparing expected nociceptor and nerve trunk activation functions as a measure of neural engagement. Main ResultsThe SON was found to be significantly more superficial than the ION and MN and had a higher nerve-to-connective tissue ratio relative to the MN. Computational modeling demonstrated that the activation function at the depths of nociceptors was orders of magnitude greater than within the main nerve trunks, suggesting preferential recruitment of cutaneous nociceptors, dependent on nociceptor density. SignificanceThe SON presents the most accessible and anatomically favorable target for transcutaneous trigeminal nerve stimulation among the branches examined due to its superficial location. However, preferential activation of low-threshold nociceptors compared to nerve trunks may lead to treatment-limiting off-target side effects, favoring strategies that target fibers of interest within the skin. These findings offer an anatomically informed framework to guide further computational modeling and electrode design for targeted trigeminal neuromodulation.

neuroscience↗

bioRxiv2025

Depth-Resolved Macroscopic Fluorescence Lifetime Imaging via High-Spatial-Frequency Structured Illumination

SignificanceMacroscopic Fluorescence Lifetime Imaging (MFLI) is a powerful, non-invasive imaging modality that offers robust, physiologically relevant contrast largely independent of fluorophore concentration, excitation intensity, and tissue signal attenuation. However, accurately determining the depth of signal origin remains challenging, potentially leading to ambiguity in biological interpretation. Here, we present a novel optical correction method that effectively eliminates surface signal bias, such as that from skin in preclinical imaging, without the need for chemical clearance. This advancement supports the robust applicability of MFLI in translational research. AimEstablishment of a High Spatial Frequency-Fluorescence Lifetime Imaging (HSF-FLI) framework to selectively isolate subsurface fluorescence (deeper signals) from surface fluorescence, while preserving the accuracy of lifetime estimation. ApproachA modulation transfer function (MTF) that relates spatial frequency to penetration depth was derived using Monte Carlo eXtreme (MCX) simulations (for physics-based modeling) and validated with agar-based capillary phantoms on a time-gated ICCD-DMD system. Depth-independent fluorescence was decomposed into surface and subsurface components through structured three-phase sinusoidal illumination, and nonlinear least squares fitting was applied to recover lifetime or lifetime based parameters maps. HSF-FLI was demonstrated in vivo in mouse models bearing tumor xenogratfs and was cross validated with ex vivo measurements. ResultsWe extensively characterized the performance of High Spatial Frequency-Fluorescence Lifetime Imaging (HSF-FLI) through simulations and tissue-mimicking phantoms. The approach was further validated in vivo by assessing drug delivery in preclinical models using MFLI-FRET (Forster Resonance Energy Transfer). ConclusionBy coupling structured illumination with physics-based depth modeling, HSF-FLI delivers accurate, depth-selective lifetime readouts, setting the stage for robust and fast FLI implementation in translational studies.

bioengineering↗

bioRxiv2024

A tale of two n-backs: Diverging associations of dorsolateral prefrontal cortex activation with n-back task performance

BackgroundIn studying the neural correlates of working memory (WM) ability via functional magnetic resonance imaging (fMRI) in health and disease, it is relatively uncommon for investigators to report associations between brain activation and measures of task performance. Additionally, how the choice of WM task impacts observed activation-performance relationships is poorly understood. We sought to illustrate the impact of WM task on brain-behavior correlations using two large, publicly available datasets. MethodsWe conducted between-participants analyses of task-based fMRI data from two publicly available datasets: the Human Connectome Project (HCP; n = 866) and the Queensland Twin Imaging (QTIM) Study (n = 459). Participants performed two distinct variations of the n-back WM task with different stimuli, timings, and response paradigms. Associations between brain activation ([2-back - 0-back] contrast) and task performance (2-back % correct) were investigated separately in each dataset, as well as across datasets, within the dorsolateral prefrontal cortex (dlPFC), medial prefrontal cortex, and whole cortex. ResultsGlobal patterns of activation to task were similar in both datasets. However, opposite associations between activation and task performance were observed in bilateral pre-supplementary motor area and left middle frontal gyrus. Within the dlPFC, HCP participants exhibited a significantly greater activation-performance relationship in bilateral middle frontal gyrus relative to QTIM Study participants. ConclusionsThe observation of diverging activation-performance relationships between two large datasets performing variations of the n-back task serves as a critical reminder for investigators to exercise caution when selecting WM tasks and interpreting neural activation in response to a WM task.

neuroscience↗

bioRxiv2024

Functional Localization of the Human Auditory and Visual Thalamus Using a Thalamic Localizer Functional Magnetic Resonance Imaging Task

Functional magnetic resonance imaging (fMRI) of the auditory and visual sensory systems of the human brain is an active area of investigation in the study of human health and disease. The medial geniculate nucleus (MGN) and lateral geniculate nucleus (LGN) are key thalamic nuclei involved in the processing and relay of auditory and visual information, respectively, and are the subject of blood-oxygen-level-dependent (BOLD) fMRI studies of neural activation and functional connectivity in human participants. However, localization of BOLD fMRI signal originating from neural activity in MGN and LGN remains a technical challenge, due in part to the poor definition of boundaries of these thalamic nuclei in standard T1-weighted and T2-weighted magnetic resonance imaging sequences. Here, we report the development and evaluation of an auditory and visual sensory thalamic localizer (TL) fMRI task that produces participant-specific functionally-defined regions of interest (fROIs) of both MGN and LGN, using 3 Tesla multiband fMRI and a clustered-sparse temporal acquisition sequence, in less than 16 minutes of scan time. We demonstrate the use of MGN and LGN fROIs obtained from the TL fMRI task in standard resting-state functional connectivity (RSFC) fMRI analyses in the same participants. In RSFC analyses, we validated the specificity of MGN and LGN fROIs for signals obtained from primary auditory and visual cortex, respectively, and benchmark their performance against alternative atlas- and segmentation-based localization methods. The TL fMRI task and analysis code (written in Presentation and MATLAB, respectively) have been made freely available to the wider research community.

neuroscience↗

bioRxiv2023

Identification, Characterization, and Mitigation of a Simultaneous Multi-Slice fMRI Signal Artifact: Multiband Artifact Regression in Simultaneous Slices (MARSS)

Simultaneous multi-slice (multiband) acceleration in fMRI has become widespread, but may be affected by novel forms of signal artifact. Here, we demonstrate a previously unreported artifact manifesting as a shared signal between simultaneously acquired slices in all resting-state and task-based multiband fMRI datasets we investigated, including publicly available consortium data from the Human Connectome Project (HCP) and Adolescent Brain Cognitive Development (ABCD) Study. We propose Multiband Artifact Regression in Simultaneous Slices (MARSS), a regression-based detection and correction technique that successfully mitigates this shared signal in unprocessed data. We demonstrate that the signal isolated by MARSS correction is likely non-neural, appearing stronger in neurovasculature than grey matter. Additionally, we evaluate MARSS both against and in tandem with sICA+FIX denoising, which is implemented in HCP resting-state data, to show that MARSS mitigates residual artifact signal that is not modeled by sICA+FIX. MARSS correction leads to study-wide increases in signal-to-noise ratio, decreases in cortical coefficient of variation, and mitigation of systematic artefactual spatial patterns in participant-level task betas. Finally, MARSS correction has substantive effects on second-level t-statistics in analyses of task-evoked activation. We recommend that investigators apply MARSS to multiband fMRI datasets with moderate or higher acceleration factors, in combination with established denoising methods.

neuroscience↗

bioRxiv2023

Single Nucleus RNA Sequencing of Remnant Kidney Biopsies and Urine Cell RNA Sequencing Reveal Cell Specific Markers of Covid-19 Acute Kidney Injury

Acute kidney injury (AKI) in COVID-19 patients is associated with high mortality and morbidity. Critically ill COVID-19 patients are at twice the risk of in-hospital mortality compared to non-COVID AKI patients. We know little about the cell-specific mechanism in the kidney that contributes to worse clinical outcomes in these patients. New generation single cell technologies have the potential to provide insights into physiological states and molecular mechanisms in COVID-AKI. One of the key limitations is that these patients are severely ill posing significant risks in procuring additional biopsy tissue. We recently generated single nucleus RNA-sequencing data using COVID-AKI patient biopsy tissue as part of the human kidney atlas. Here we describe this approach in detail and report deeper comparative analysis of snRNAseq of 4 COVID-AKI, 4 reference, and 6 non-COVID-AKI biopsies. We also generated and analyzed urine transcriptomics data to find overlapping COVID-AKI-enriched genes and their corresponding cell types in the kidney from snRNA-seq data. We identified all major and minor cell types and states by using by using less than a few cubic millimeters of leftover tissue after pathological workup in our approach. Differential expression analysis of COVID-AKI biopsies showed pathways enriched in viral response, WNT signaling, kidney development, and cytokines in several nephron epithelial cells. COVID-AKI profiles showed a much higher proportion of altered TAL cells than non-COVID AKI and the reference samples. In addition to kidney injury and fibrosis markers indicating robust remodeling we found that, 17 genes overlap between urine cell COVID-AKI transcriptome and the snRNA-seq data from COVID-AKI biopsies. A key feature was that several of the distal nephron and collecting system cell types express these markers. Some of these markers have been previously observed in COVID-19 studies suggesting a common mechanism of injury and potentially the kidney as one of the sources of soluble factors with a potential role in disease progression. Translational StatementThe manuscript describes innovation, application and discovery that impact clinical care in kidney disease. First, the approach to maximize use of remnant frozen clinical biopsies to inform on clinically relevant molecular features can augment existing pathological workflow for any frozen tissue without much change in the protocol. Second, this approach is transformational in medical crises such as pandemics where mechanistic insights are needed to evaluate organ injury, targets for drug therapy and diagnostic and prognostic markers. Third, the cell type specific and soluble markers identified and validated can be used for diagnoses or prognoses in AKI due to different etiologies and in multiorgan injury.

genetics↗

bioRxiv2023

Leveraging IFNγ/CD38 regulation to unmask and target leukemia stem cells in acute myelogenous leukemia

Elimination of drug-resistant leukemia stem cells (LSCs) represents a major challenge to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), the presence of CD34 and lack of CD38 expression (CD34posCD38neg) are immunophenotypic features of both LSC-enriched AML blasts and normal hematopoietic stem cells (HSCs). We report that IFN-{gamma} induces CD38 upregulation in LSC-enriched CD34posCD38neg AML blasts, but not in CD34posCD38neg HSCs. To leverage the IFN-{gamma} mediated CD38 up-regulation in LSCs for clinical application, we created a compact, single-chain CD38-CD3-T cell engager (CD38-BIONIC) able to direct T cells against CD38pos blasts. Activated CD4pos and CD8pos T cells not only kill AML blasts but also produce IFN{gamma}, which leads to CD38 expression on CD34posCD38neg LSC-enriched blasts. These cells then become CD38-BIONIC targets. The net result is an immune-mediated killing of both CD38neg and CD38pos AML blasts, which culminates in LSC depletion. Statement of significanceThis work represents a potential advancement in the treatment of AML, as it involves the release of IFN-{gamma} by T cells to induce CD38 expression and thus sensitizing leukemia stem cells, which have been resistant to current treatment regimens, to CD38-directed T cell engagers.

cancer biology↗

bioRxiv2023

Prior vaccination enhances immune responses during SARS-CoV-2 breakthrough infection with early activation of memory T cells followed by production of potent neutralizing antibodies

SARS-CoV-2 infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memory and identify potential mediators of lasting vaccine efficacy, we used high-dimensional flow cytometry and SARS-CoV-2 antigen probes to examine immune responses in longitudinal samples from vaccinated individuals infected during the Omicron wave. These studies revealed heightened Spike-specific responses during infection of vaccinated compared to unvaccinated individuals. Spike-specific CD4 T cells and plasmablasts expanded and CD8 T cells were robustly activated during the first week. In contrast, memory B cell activation, neutralizing antibody production, and primary responses to non-Spike antigens occurred during the second week. Collectively, these data demonstrate the functionality of vaccine-primed immune memory and highlight memory T cells as rapid responders during SARS-CoV-2 infection.

immunology↗

bioRxiv2022

Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes

Protection from SARS-related coronaviruses with spillover potential and SARS-CoV-2 variants could prevent and/or end pandemics. We show that mice immunized with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicit cross-reactive anti-sarbecovirus antibodies against conserved class 1/4 and class 3 RBD epitopes. Monoclonal antibodies (mAbs) identified from initial screening of <10,000 single B-cells secreting IgGs binding two or more sarbecovirus RBDs showed cross-reactive binding and neutralization of SARS-CoV-2 variants and animal sarbecoviruses. Single-particle cryo-EM structures of antibody-spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes and revealed neutralization mechanisms, potentials for intra-spike trimer crosslinking by single IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticles to identify therapeutic pan-sarbecovirus and pan-variant mAbs and to elicit them by vaccination.

immunology↗

bioRxiv2022

A spatially anchored transcriptomic atlas of the human kidney papilla identifies significant immune injury and matrix remodeling in patients with stone disease

Kidney stone disease causes significant morbidity and increases health care utilization. The pathogenesis of stone disease is not completely understood, due in part to the poor characterization of the cellular and molecular makeup of the kidney papilla and its alteration with disease. We deciphered the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease compared to healthy subjects using single nuclear RNA sequencing, spatial transcriptomics and high-resolution large-scale multiplexed 3D and Co-Detection by indexing (CODEX) imaging. In addition to identifying cell types important in papillary physiology, we defined subtypes of immune, stromal and principal cells enriched in the papilla, and characterized an undifferentiated epithelial cell cluster that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncovered a global injury signature involving multiple cell types within the papilla, characterized by immune activation, oxidative stress and extracellular matrix remodeling. The microenvironment of mineral deposition had features of an immune synapse with antigen presenting inflammatory macrophages interacting with T cells, and an immune repertoire ranging from inflammation to fibrosis. The expression of MMP7 and MMP9 was associated with stone disease and mineral deposition, respectively. MMP7 and MMP9 were significantly increased in the urine of patients with CaOx stone disease compared to non-stone formers, and their levels correlated with disease activity in stone formers. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla, and identify potential urinary biomarkers.

genomics↗

bioRxiv2022

Spatially selective stimulation of the pig vagus nerve to modulate target effect versus side effect

Electrical stimulation of the cervical vagus nerve using implanted electrodes (VNS) is FDA-approved for the treatment of drug-resistant epilepsy, treatment-resistant depression, and most recently, chronic ischemic stroke rehabilitation. However, VNS is critically limited by the unwanted stimulation of nearby neck muscles - a result of non-specific stimulation activating motor nerve fibers within the vagus. Prior studies suggested that precise placement of small epineural electrodes can modify VNS therapeutic effects, such as cardiac responses. However, it remains unclear if placement can alter the balance between intended effect and limiting side effect. We used an FDA investigational device exemption approved six-contact epineural cuff to deliver VNS in pigs and quantified how epineural electrode location impacts on- and off-target VNS activation. Detailed post-mortem histology was conducted to understand how the underlying neuroanatomy impacts observed functional responses. Here we report the discovery and characterization of clear neuroanatomy-dependent differences in threshold and saturation for responses related to both effect (change in heart rate) and side effect (neck muscle contractions). The histological and electrophysiological data were used to develop and validate subject-specific computation models of VNS, creating a well-grounded quantitative framework to optimize electrode location-specific activation of nerve fibers governing intended effect versus unwanted side effect.

bioengineering↗

bioRxiv2022

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for [~]9-10 months after the primary 2-dose mRNA vaccine series, as well as for [~]3 months after a 3rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and [~]40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3rd dose. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/481163v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@123d2d9org.highwire.dtl.DTLVardef@e7db82org.highwire.dtl.DTLVardef@1fc73deorg.highwire.dtl.DTLVardef@11b21f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Integrated cytometry with machine learning applied to high-content imaging of human kidney tissue for in-situ cell classification and neighborhood analysis

The human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities such as mesoscale and highly multiplexed fluorescence microscopy are increasingly applied to human kidney tissue to create single cell resolution datasets that are both spatially large and multi-dimensional. These single cell resolution high-content imaging datasets have a great potential to uncover the complex spatial organization and cellular make-up of the human kidney. Tissue cytometry is a novel approach used for quantitative analysis of imaging data, but the scale and complexity of such datasets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEAs integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses for hyperdimensional large-scale imaging datasets. These novel capabilities enable the analysis of mesoscale two and three-dimensional multiplexed human kidney imaging datasets (such as CODEX and 3D confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney based on labels, spatial association and their microenvironment or neighborhood membership. VTEA provides integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complement other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.

physiology↗

bioRxiv2021

mRNA Vaccination Induces Durable Immune Memory to SARS-CoV-2 with Continued Evolution to Variants of Concern

SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antibody and cellular immune responses in SARS-CoV-2 naive and recovered individuals from pre-vaccine baseline to 6 months post-mRNA vaccination. Antibody and neutralizing titers decayed from peak levels but remained detectable in all subjects at 6 months post-vaccination. Functional memory B cell responses, including those specific for the receptor binding domain (RBD) of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants, were also efficiently generated by mRNA vaccination and continued to increase in frequency between 3 and 6 months post-vaccination. Notably, most memory B cells induced by mRNA vaccines were capable of cross-binding variants of concern, and B cell receptor sequencing revealed significantly more hypermutation in these RBD variant-binding clones compared to clones that exclusively bound wild-type RBD. Moreover, the percent of variant cross-binding memory B cells was higher in vaccinees than individuals who recovered from mild COVID-19. mRNA vaccination also generated antigen-specific CD8+ T cells and durable memory CD4+ T cells in most individuals, with early CD4+ T cell responses correlating with humoral immunity at later timepoints. These findings demonstrate robust, multi-component humoral and cellular immune memory to SARS-CoV-2 and current variants of concern for at least 6 months after mRNA vaccination. Finally, we observed that boosting of pre-existing immunity with mRNA vaccination in SARS-CoV-2 recovered individuals primarily increased antibody responses in the short-term without significantly altering antibody decay rates or long-term B and T cell memory. Together, this study provides insights into the generation and evolution of vaccine-induced immunity to SARS-CoV-2, including variants of concern, and has implications for future booster strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/457229v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@16c64b1org.highwire.dtl.DTLVardef@146ca3aorg.highwire.dtl.DTLVardef@86b7edorg.highwire.dtl.DTLVardef@956879_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

An atlas of healthy and injured cell states and niches in the human kidney

Understanding kidney disease relies upon defining the complexity of cell types and states, their associated molecular profiles, and interactions within tissue neighborhoods. We have applied multiple single-cell or -nucleus assays (>400,000 nuclei/cells) and spatial imaging technologies to a broad spectrum of healthy reference (n = 42) and disease (n = 42) kidneys. This has provided a high resolution cellular atlas of 100 cell types that include rare and novel cell populations. The multi-omic approach provides detailed transcriptomic profiles, epigenomic regulatory factors, and spatial localizations for major cell types spanning the entire kidney. We further identify and define cellular states altered in kidney injury, encompassing cycling, adaptive or maladaptive repair, transitioning and degenerative states affecting several segments. Molecular signatures of these states permitted their localization within injury neighborhoods using spatial transcriptomics, and large-scale 3D imaging analysis of [~]1.2 million neighborhoods provided linkages to active immune responses. These analyses further defined biological pathways relevant to injury niches, including signatures underlying the transition from reference to predicted maladaptive states that were associated with a decline in kidney function during chronic kidney disease. This human kidney cell atlas, including injury cell states and neighborhoods, will be a valuable resource for future studies.

genomics↗

bioRxiv2020

In vivo visualization of pig vagus nerve 'vagotopy' using ultrasound

BackgroundPlacement of the clinical vagus nerve stimulating cuff is a standard surgical procedure based on anatomical landmarks, with limited patient specificity in terms of fascicular organization or vagal anatomy. As such, the therapeutic effects are generally limited by unwanted side effects of neck muscle contractions, demonstrated by previous studies to result from stimulation of 1) motor fibers near the cuff in the superior laryngeal and 2) motor fibers within the cuff projecting to the recurrent laryngeal. ObjectiveThe use of patient-specific visualization of vagus nerve fascicular organization could better inform clinical cuff placement and improve clinical outcomes. MethodsThe viability of ultrasound, with the transducer in the surgical pocket, to visualize vagus nerve fascicular organization (i.e. vagotopy) was characterized in a pig model. Ultrasound images were matched to post-mortem histology to confirm the utility of ultrasound in identifying fascicular organization. ResultsHigh-resolution ultrasound accurately depicted the vagotopy of the pig vagus nerve intra-operatively, as confirmed via histology. The stereotypical pseudo-unipolar cell body aggregation at the nodose ganglion was identifiable, and these sensory afferent fascicular bundles were traced down the length of the vagus nerve. Additionally, the superior and recurrent laryngeal nerves were identified via ultrasound. ConclusionsIntraoperative visualization of vagotopy and surrounding nerves using ultrasound is a novel approach to optimize stimulating cuff placement, avoid unwanted activation of motor nerve fibers implicated in off-target effects, and seed patient-specific models of vagal fiber activation to improve patient outcomes.

neuroscience↗

bioRxiv2020

The Histoplasma capsulatum DDR48 Gene Is Required For Survival Within Macrophages, Response To Oxidative Stress, And Resistance to Antifungal Drugs

Histoplasma capsulatum (Hc) is a systemic, dimorphic fungal pathogen that affects upwards of 500,000 individuals in the United States annually. Hc grows as a multicellular mold at environmental temperatures; whereas, upon inhalation into a human or other mammalian host, it transforms into a unicellular, pathogenic yeast. This manuscript is focused on characterizing the DNA damage-responsive gene HcDDR48. HcDDR48 was originally isolated via a subtractive DNA library enriched for transcripts enriched in the mold-phase of Hc growth. Upon further analysis we found that HcDDR48 is not just expressed in the mold morphotype, but both growth programs dependent upon the environment. We found that HcDDR48 is involved in oxidative stress response, antifungal drug resistance, and survival within resting and activated macrophages. Growth of ddr48{Delta} yeasts was severely decreased when exposed to the reactive oxygen species generator paraquat, as compared to wildtype controls. We also found that ddr48{Delta} yeasts were 2-times more sensitive to the antifungal drugs amphotericin b and ketoconazole. To test HcDDR48s involvement in vivo, we infected resting and activated RAW 264.7 murine macrophages with Hc yeasts and measured yeast survival 24-hours post-infection. We observed a significant decrease in yeast recovery in the ddr48{Delta} strain compared to wildtype Hc levels. Herein, we demonstrate the importance of maintaining a functional copy of HcDDR48 in order for Hc yeasts to sense and respond to numerous environmental and host-associated stressors. ImportanceHistoplasma capsulatum is an intracellular pathogen of phagocytes, where it subverts immune recognition and avoids killing by the innate immune system. Macrophages provide a permissive environment for Hc replication and killing only occurs upon the onset of the T-cell driven adaptive immune response. Hc has evolved numerous virulence factors that aid in its survival against host-derived ROS and RNS in vivo. While these virulence factors have been described in past years, only a few reports describing the regulation of these genes and how this intricate system leads to fungal survival. In this study, we characterized the stress response gene DDR48 and determined it to be indispensable for Hc survival within macrophages. HcDDR48 regulates transcript levels of superoxide dismutases and catalases responsible for detoxification of ROS and contributes to antifungal drug resistance. Our studies highlight DDR48 as a potential target to control Hc infection and decrease the severity of the disease process.

microbiology↗