Biology subjects

Cooper, K.

Publications and source records attributed to Cooper, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Zn2+ as a secondary messenger for exogenous redox potential sensed through Chemosensory Zinc-Binding (CZB) protein domains

Redox environments in nature are shaped by reactive oxygen species (ROS), oxygen availability, and metal ion chemistry, and exert profound effects on cell physiology and survival. While extensive work has characterized how cells resist oxidative damage, the mechanisms by which cells sense and navigate environmental redox conditions remain less well understood. Here, we identify a previously unrecognized and widespread mechanism of redox sensing in Salmonella enterica serovar Typhimurium mediated by the chemosensory zinc-binding (CZB) domain-containing receptor McpA. Using quantitative chemotaxis assays and live-cell imaging, we show that S. Typhimurium exhibits robust, concentration-dependent chemotaxis toward the neutrophil-derived oxidants HOCl and hydroperoxides, with attraction occurring at low, physiologically relevant concentrations below those that cause bactericidal effects, and this response requires McpA and its conserved zinc-binding cysteine. Whereas other Cys-Zn thiolate systems function through direct oxidation mechanisms, we find that the unique 3His,1Cys binding motif of CZBs responds to redox-dependent changes in Zn{superscript 2} speciation, whereby oxidizing conditions shift soluble, bioavailable Zn{superscript 2} into insoluble zinc precipitates. In this way, CZBs utilize the bioavailable Zn{superscript 2} pool as a secondary messenger of exogenous redox potential, and correspondingly, cells exhibit chemoattraction toward Zn{superscript 2}-depleted environments, including sources of ROS, but also toward oxygen-rich conditions that provide a metabolic growth advantage. The broad phylogenetic distribution of CZB domains is consistent with this Zn{superscript 2}-responsive mechanism being an ancient redox-sensing strategy, likely established early in bacterial evolution under changing planetary redox conditions and retained across diverse bacterial lineages. Significance StatementWe report a previously unknown mechanism of redox sensing that operates through changes in the bioavailability and speciation of Zn{superscript 2}, enabling bacteria to detect changes in exogenous redox potential with high sensitivity and navigate redox gradients. The broad phylogenetic distribution of chemosensory zinc-binding (CZB) domains suggests that this zinc-dependent sensory mechanism is an ancient and widespread strategy for detecting environmental redox gradients that arose early in bacterial evolution and may have been subsequently shaped or expanded in response to increasing atmospheric oxygen associated with the Great Oxygenation Event.

microbiology↗

bioRxiv2026

Assessment of the household antibiotic resistance gene, virulence factor genes, and pathogen profiles from three global cities

This study assessed the prevalence of antibiotic resistance genes (ARGs) and virulence factor genes (VFs), DNA viruses, and medically-relevant pathogens in three major cities around the globe - Mysuru (India), Dubai (United Arab Emirates), and Tucson (Arizona, United States of America). Ten households were sampled in each city, at ten sites in the bathroom, kitchen, and living spaces. The alpha diversity of ARGs significantly differed between household locations in each country (ANOVA, p<0.05) and beta diversity (dissimilarity) analysis showed a significant association between the ARGs and the geographic locations (PERMANOVA, p<0.01). A set of ARGs were found in every location across the dataset (the core ARG profile) included 25 different genes. The alpha diversity of virulence factors differed across the household locations within the three cities (ANOVA, p<0.01). The beta diversity of VFs was not well explained by geographic location or location within the household (PERMANOVA, p=0.129 (geographic), p=0.127 (household)). There were 341 unique VFs found in the study, but only 5 core VFs across the dataset. Bacterial pathogens detected across the household included Escherichia coli, Acinetobacter baumanii, Klebsiella pneumoniae, and more. The DNA (and bacteriophage) virome varied between countries and was more diverse in Tucson and Dubai (top viral families included Poxviridae and Orthoherpesviridae - two families which contain human pathogens - and Steitzviridae, a family of bacteriophages) compared to Mysuru, where nearly all viruses were a part of the Muvirus genus (a bacteriophage which contributes to horizontal gene transfer by phage transduction). ImportanceThe diversity of the built environment microbiome is not well characterized globally. Household occupants interact with the built microbiome on a daily basis, and the built microbiome may have the potential to influence human health. The presence of pathogens in the built environment and the key genes contributing to microorganism pathogenicity were investigated in this study, as information on this is lacking on an international scale. The diversity of ARG and VFs across the globe, as well as the presence of medically relevant pathogens within the house that were found in this study highlights the need to explore further the factors which influence the household microbiome, virome, and resistome. This may aid in identify how the build microbiome may be shaped by humans and influence human health. Impact StatementThis research contributes to the understanding of the built microbiome, specifically how it varies within the house, within cities, and across the globe. This can aid in our understanding of microbial dynamics in environments with heavy human influence and help develop and improve hygiene habits and products which are mindful of the existing microbiome.

microbiology↗

bioRxiv2026

Phenotypic Resistance of Ciprofloxacin and Azithromycin Resistant Campylobacter Isolates to an Extended Panel of Antibiotics.

In this study we explored phenotypic resistance using traditional Kirby-Bauer methods in human and animal derived Campylobacter isolates that were concurrently resistant to both azithromycin and ciprofloxacin to an expanded panel of antimicrobials, including clindamycin, fosfomycin, ampicillin sulbactam and tigecycline. Out of 236 Campylobacter isolates, over 85% of C. jejuni and C. coli were resistant to clindamycin, over 60% were resistant to ampicillin sulbactam and over 30% to fosfomycin. Less than 2% of isolates were resistant to tigecycline and there was no observed resistance to Imipenem.

microbiology↗

bioRxiv2025

An Evaluation of the Efficacy of Single-Echo and Multi-Echo fMRI Denoising Strategies

Resting-state functional magnetic resonance imaging (rsfMRI) is commonly used to study brain-wide patterns of inter-regional functional coupling (FC). However, the resulting signals are vulnerable to multiple sources of noise, such as those related to non-neuronal physiological fluctuations and head motion, which can alter FC estimates and influence their associations with behavioral outcomes. The best strategy for acquiring and processing rsfMRI data to mitigate noise remains an open question. In this study of 358 healthy individuals, we compared the denoising efficacy of 60 multi-echo (ME) and 30 single-echo (SE) rsfMRI preprocessing pipelines across six distinct measures of data quality. We also evaluated how each pipeline influences the effect sizes of FC-based predictive models of personality and cognitive measures estimated via cross-validated kernel ridge regression. We found that ME pipelines generally showed superior denoising efficacy to SE pipelines, but that no single pipeline was associated with both superior denoising efficacy and behavioural prediction. Using a heuristic scheme to rank pipelines across benchmark evaluations, we found that an ME acquisition combined with Automatic Removal of Motion Artifacts Independent Component Analysis (ICA-AROMA) and Regressor Interpolation at Progressive Time Delays (RIPTiDe) offered a reasonable compromise between denoising efficacy and brain-behavior predictions for both ME and SE data. In general, ME pipelines ranked more highly than SE pipelines. These findings support the use of ME acquisitions in future work but suggest that no single denoising pipeline should be considered optimal for all purposes.

neuroscience↗

bioRxiv2025

American black bear (Ursus americanus) as a potential host for Campylobacter jejuni

The Gram-negative bacterium Campylobacter jejuni is part of the commensal gut microbiota of numerous animal species and a leading cause of bacterial foodborne illness in humans. Most complete genomes of C. jejuni are from strains isolated from human clinical, poultry, and ruminant samples. Here, we characterized and compared the genomes of C. jejuni that were isolated from American black bears in three states in the southeastern United States. Despite the limited sample size (n=9), the isolates displayed substantial genotypic variability, including eight distinct sequence types (STs) and variable gene content encoding surface glycan structures such as capsular polysaccharides (CPS) and lipooligosaccharides (LOS). Phylogenetic analysis identified several C. jejuni host generalist strains among the isolates from bears that clustered with isolates from domestic poultry, cattle, and environmental sources. Three isolates (SKBC94, SKBC3, SKBC5) clustered with wildlife-associated strains, exhibiting mutations or deletions in loci associated with cytolethal distending toxin production and oxidative stress resistance, potentially influencing host-specific colonization. Additionally, strains SKBC3 and SKBC5 harbored distinct Entner-Doudoroff (E-D) loci, suggesting a potential evolutionary fitness advantage. This study provides the first evidence of C. jejuni colonization in American black bears, highlighting their potential role as reservoirs for diverse C. jejuni lineages from both anthropogenic and environmental sources. Further research is needed to determine the prevalence and host specificity of C. jejuni strains in black bears and their potential implications for public and wildlife health.

microbiology↗

bioRxiv2025

XPC loss-of-function triggers melanomagenesis through CDKN2A downregulation

We identified a novel XPC variant, c.2420+5G>A (XPCvar), in siblings with multiple melanomas, inherited alongside c.779+1G>T, which results in an absent or disrupted protein. However, they did not exhibit significantly higher nucleotide excision repair (NER) deficits compared to their unaffected parents, suggesting an NER-independent tumor suppressor function for XPC. XPC knockdown increased cell proliferation and tumorigenicity in vitro without affecting NER. Single-cell RNA sequencing revealed lower Cdkn2a in Xpcvar/var mouse melanocytes. Additionally, XPC knockdown downregulated CDKN2A in vitro. Furthermore, patient fibroblasts showed decreased p16INK4a, which was rescued by XPC overexpression. ChIP-PCR and luciferase assays confirmed XPC binding to the CDKN2A promoter, initiating transcription. Premature stop codon read-through, with gentamicin, restored XPC and p16INK4A in patient fibroblasts. These suggest that XPC regulates CDKN2A and XPC loss might promote melanomagenesis by downregulating CDKN2A, independent of its NER function. We provide preclinical evidence for potential preventive and/or therapeutic strategies for similarly affected individuals. STATEMENT OF SIGNIFICANCECharacterizing a novel XPC variant identified in patients with melanoma revealed a non-canonical role for XPC in regulating CDKN2A transcription. XPC binding to the CDKN2A promoter is crucial for CDKN2A expression and might suppress melanomagenesis. This expands our understanding of melanomagenesis and suggests potential therapies targeting XPC-mediated regulation of CDKN2A.

cancer biology↗

bioRxiv2024

Damage-induced IL-18 stimulates thymic NK Cells limiting endogenous tissue regeneration

Interleukin-18 is an acute phase pro-inflammatory molecule crucial for mediating viral clearance by activating Th1 CD4+, cytotoxic CD8+ T, and NK cells. Here, we show that mature IL-18 is generated in the thymus following numerous distinct forms of tissue damage, all of which cause caspase-1-mediated immunogenic cell death. We report that IL-18 stimulated cytotoxic NK cells limit endogenous thymic regeneration, a critical process that ensures restoration of immune competence after acute insults like stress, infection, chemotherapy, and radiation. NK cells suppressed thymus recovery by aberrantly targeting thymic epithelial cells (TECs), which act as the master regulators of organ function and regeneration. Together these studies reveal a novel pathway regulating tissue regeneration in the thymus and offer IL-18 as a potential therapeutic target to boost thymic function. Moreover, given the enthusiasm for IL-18 as a cancer immunotherapy for its capacity to elicit a type-1 immune response, these findings also offer insight into potential off-target effects.

immunology↗

bioRxiv2024

Immunocompetent murine model of Ewing sarcoma reveals role for TGFβ inhibition to enhance immune infiltrates in Ewing tumors during radiation

Ewing sarcoma (ES) is an aggressive cancer diagnosed in adolescents and young adults. The fusion oncoprotein (EWSR1::FLI1) that drives Ewing sarcoma is known to downregulate TGFBR2 expression (part of the TGF{beta} receptor). Because TGFBR2 is downregulated, it was thought that TGF{beta} likely plays an inconsequential role in Ewing biology. However, the expression of TGF{beta} in the Ewing tumor immune microenvironment (TIME) and functional impact of TGF{beta} in the TIME remains largely unknown given the historical lack of immunocompetent preclinical models. Here, we use single-cell RNAseq analysis of human Ewing tumors to show that immune cells, such as NK cells, are the largest source of TGF{beta} production in human Ewing tumors. We develop a humanized (immunocompetent) mouse model of ES and demonstrate distinct TME signatures and metastatic potential in these models as compared to tumors developed in immunodeficient mice. Using this humanized model, we study the effect of TGF{beta} inhibition on the Ewing TME during radiation therapy, a treatment that both enhances TGF{beta} activation and is used to treat aggressive ES. Utilizing a trivalent ligand TGF{beta} TRAP to inhibit TGF{beta}, we demonstrate that in combination with radiation, TGF{beta} inhibition both increases ES immune cell infiltration and decreases lung metastatic burden in vivo. The culmination of these data demonstrates the value of humanized models to address immunobiologic preclinical questions in Ewing sarcoma and suggests TGF{beta} inhibition as a promising intervention during radiation therapy to promote metastatic tumor control.

cancer biology↗

bioRxiv2023

Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of [≥] 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers health.

microbiology↗

bioRxiv2023

Polyploidy enhances desiccation tolerance in the grass Microchloa caffra

Desiccation tolerance evolved recurrently across diverse plant lineages to enable survival in water limited conditions. Many resurrection plants are polyploid and several groups have hypothesized that polyploidy enabled the evolution of desiccation tolerance. However, due to the vast evolutionary divergence between resurrection plant lineages, the rarity of desiccation tolerance, and the prevalence of polyploidy in plants, this hypothesis has been difficult to test. Here, we surveyed variation in morphological, reproductive, and desiccation tolerance traits across natural populations of a single species that has differing ploidies and tested for links between polyploidy and resilience. We sampled multiple populations of the resurrection grass Microchloa caffra across an environmental gradient ranging from mesic to xeric in South Africa. We describe two distinct ecotypes of M. caffra that occupy different ends of the environmental gradient and exhibit consistent differences in ploidy, morphological, reproductive, and desiccation tolerance traits in both field and common growth conditions. Interestingly, plants with more polyploid genomes were consistently more desiccation tolerant, less reproductive, and larger than plants with smaller genomes and lower ploidy. These data suggest that polyploidy enhances desiccation tolerance and that stronger selective pressures in increasingly xeric sites may play a role in maintaining and increasing desiccation tolerance.

plant biology↗

bioRxiv2023

Damage-induced pyroptosis drives endogenous thymic regeneration viainduction of Foxn1 by purinergic receptor activation

Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression of Foxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTP{gamma}S promotes rapid regeneration of the thymus in vivo following acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration. SUMMARYO_LIThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration. C_LIO_LIDamage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes. C_LIO_LIElevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage. C_LIO_LIExtracellular ATP release promotes Foxn1 expression in cTECs via activation of P2Y2 C_LIO_LITherapeutic targeting of the P2Y2 receptor promotes thymic regeneration. C_LI

immunology↗

bioRxiv2021

Activation of the Zinc-sensing receptor GPR39 promotes T cell reconstitution after hematopoietic stem cell transplant

Prolonged lymphopenia represents a major clinical problem after cytoreductive therapies such as chemotherapy and the conditioning required for hematopoietic stem cell transplant (HCT), contributing toward the risk of infections and malignant relapse. Restoration of T cell immunity is dependent on tissue regeneration in the thymus, the primary site of T cell development; although the capacity of the thymus to repair itself diminishes over lifespan. However, although boosting thymic function and T cell reconstitution is of considerable clinical importance, there are currently no approved therapies for treating lymphopenia. Here we found that Zinc (Zn), is critically important for both normal T cell development as well as repair after acute damage. Accumulated Zn in thymocytes during development was released into the extracellular milieu after HCT conditioning, where it triggered regeneration by stimulating endothelial cell-production of BMP4 via the cell surface receptor GPR39. Dietary supplementation of Zn was sufficient to promote thymic function in a mouse model of allogeneic HCT, including enhancing the number of recent thymic emigrants in circulation; although direct targeting of GPR39 with a small molecule agonist enhanced thymic function without the need for prior Zn accumulation in thymocytes. Together, these findings not only define an important pathway underlying tissue regeneration, but also offer an innovative preclinical approach to treat lymphopenia in HCT recipients. KEY POINTSO_LIThymocytes release zinc after HCT conditioning is sensed by GPR39 and promotes epithelial repair C_LIO_LIPharmacologic stimulation of GPR39 promotes T cell reconstitution after HCT C_LI

immunology↗

bioRxiv2021

Utilization efficiency of human milk oligosaccharides by human-associated Akkermansia is strain-dependent

Akkermansia muciniphila are mucin degrading bacteria found in the human gut and are often associated with positive human health. However, despite being detected as early as one month of age, little is known about the role of Akkermansia in the infant gut. Human milk oligosaccharides (HMOs) are abundant components of human milk and are structurally similar to the oligosaccharides that comprise mucin, the preferred growth substrate of human-associated Akkermansia. A limited subset of intestinal bacteria has been shown to grow well on HMOs and mucin. We therefore examined the ability of genomically diverse strains of Akkermansia to grow on HMOs. First, we screened 85 genomes representing the four known Akkermansia phylogroups to examine their metabolic potential to degrade HMOs. Furthermore, we examined the ability of representative isolates to grow on individual HMOs in a mucin background and analyzed the resulting metabolites. All Akkermansia genomes were equipped with an array of glycoside hydrolases associated with HMO-deconstruction. Representative strains were all able to grow on HMOs with varying efficiency and growth yield. Strain CSUN-19 belonging to the AmIV phylogroup, grew to the highest level in the presence of fucosylated and sialylated HMOs. This activity may be partially related to the increased copy numbers and/or the enzyme activities of the -fucosidases, -sialidases, and {beta}-galactosidases. Utilization of HMOs by strains of Akkermansia suggests that ingestion of HMOs by an infant may enrich for these potentially beneficial bacteria. Further studies are required to realize this opportunity and deliver long-lasting metabolic benefits to the human host. ImportanceHuman milk oligosaccharides (HMOs) are utilized by a limited subset of bacteria in the infant gut. Akkermansia are detected in infants as young as one month of age and are thought to contribute to the HMO deconstruction capacity of the infant. Here, using phylogenomics, we examined the genomic capacity of different Akkermansia phylogroups to potentially deconstruct HMOs. Furthermore, we experimentally showed that strains from all the currently known phylogroups of Akkermansia can deconstruct all the major types of HMOs, albeit with different utilization efficiencies. This study thus examines Akkermansia-HMO interactions that can potentially influence the gut microbial ecology during the first 1,000 days of life - a critical phase for the development of the gut microbiome and infant health. This study will be of interest to a wide range of scientists from microbiologists, glycochemists/glycobiologists, to functional food developers investigating Akkermansia as probiotics or functional foods containing milk oligosaccharides as prebiotics.

microbiology↗

bioRxiv2020

Attenuation of homeostatic signaling from apoptotic thymocytes triggers a global regenerative response in the thymus

The molecular triggers of organotypic tissue repair are unknown. The thymus, which is the primary site of T cell development, is a model of tissue damage and regeneration as it is particularly sensitive to insult, but also has a remarkable capacity for repair. However, acute and profound damage, such as that caused by common cytoreductive therapies or age-related decline, lead to involution of the thymus and prolonged T cell deficiency, precipitating life-threatening infections and malignant relapse. Consequently, there is an unmet need to boost thymic function and enhance T cell immunity. Here, we demonstrate an innate trigger of the reparative response in the thymus, centered on the attenuation of signaling directly downstream of apoptotic cell detection as thymocytes are depleted after acute damage. We found that the intracellular pattern recognition receptor NOD2, via induction of microRNA-29c, suppressed the induction of the regenerative factors IL-23 and BMP4, from thymic dendritic cells (DCs) and endothelial cells (ECs), respectively. During steady-state, when a high proportion of thymocytes are undergoing apoptosis (as a consequence of selection events during T cell development), this suppressive pathway is constitutively activated by the detection of exposed phosphatidylserine on apoptotic thymocytes by cell surface TAM receptors on DCs and ECs, with subsequent downstream activation of the Rho GTPase Rac1. However, after damage, when profound cell depletion occurs across the thymus, the TAM-Rac1-NOD2-miR29c pathway is abrogated, therefore triggering the increase in IL-23 and BMP4 levels. Importantly, this pathway could be modulated pharmacologically by inhibiting Rac1 GTPase activation with the small molecule inhibitor EHT1864, leading to increased thymic function and T cell recovery after acute damage. In conclusion, our work not only represents a novel regenerative strategy for restoring immune competence in patients whose thymic function has been compromised due to cytoreductive conditioning, infection, or age; but also, identifies a mechanism by which tissue regenerative responses are triggered.

immunology↗

bioRxiv2020

Snx4-mediated nucleophagy targets transcription factors controlling ATG gene expression

Autophagy is controlled in part by the repression and activation of Autophagy-related (ATG) gene transcription. Here, we demonstrate that the conserved Cdk8 Kinase Module (CKM) of the mediator complex represses transcription of several ATG genes. To relieve this repression following nitrogen starvation, Med13 is rapidly degraded via a novel selective autophagy mechanism. This pathway requires the core autophagy machinery but is independent of known nucleophagy systems. It requires the cytosolic filament nucleoporin Gle1, the sorting nexin Snx4-Atg20 heterodimer, and the scaffold protein Atg17. This suggests a model where Med13 traverses through the nuclear pore complex, passing from Gle1 to Snx4. Snx4 then transports Med13 to autophagosomes by binding to Atg17. This previously unidentified nucleophagy pathway also mediates the autophagic degradation of two transcriptional activators of ATG genes (Rim15, Msn2) suggesting that this mechanism targets transcription factors that regulate ATG expression. This system provides a new level of selectivity, permitting the cell to fine-tune the autophagic response by controlling the turnover of both positive and negative ATG transcription factors.

cell biology↗

bioRxiv2020

Dynamic aqueous multiphase reaction system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis

Recently, CRISPR-Cas technology has opened a new era of nucleic acid-based molecular diagnostics. However, current CRISPR-Cas-based nucleic acid biosensing has largely a lack of the quantitative detection ability and typically requires separate manual operations. Herein, we reported a dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis by taking advantage of density difference of sucrose concentration. In the DAMR system, recombinase polymerase amplification (RPA) and CRISPR-Cas12a derived fluorescent detection occurred in spatially separated but connected aqueous phases. Our DAMR system was utilized to quantitatively detect human papillomavirus (HPV) 16 and 18 DNAs with sensitivities of 10 and 100 copies within less than one hour. Multiplex detection of HPV16/18 in clinical human swab samples were successfully achieved in the DAMR system using 3D-printed microfluidic device. Furthermore, we demonstrated that target DNA in real human plasma samples can be directly amplified and detected in the DAMR system without complicated sample pre-treatment. As demonstrated, the DAMR system has shown great potential for development of next-generation point-of-care molecular diagnostics.

bioengineering↗

bioRxiv2019

Secondary ossification centers evolved to make endochondral bone growth possible under the weight-bearing demands of a terrestrial environment

Growth plate and articular cartilage constitute a single anatomical entity early in development, but later separate into two distinct structures by the secondary ossification center (SOC). The reason for such separation remains unknown. We found that evolutionarily SOC appears in animals conquering the land - amniotes. Analysis of ossification pattern in mammals with specialized extremities (whales, bats, jerboa) revealed that SOC development correlates with the extent of mechanical loads. Mathematical modelling revealed that SOC reduces mechanical stress within the growth plate. Functional experiments revealed high vulnerability of hypertrophic chondrocytes to mechanical stress and showed that SOC protects these cells from apoptosis caused by extensive loading. Atomic force microscopy showed that hypertrophic chondrocytes are the least mechanically stiff cells within the growth plate. Altogether, these findings suggest that SOC has evolved to protect the hypertrophic chondrocytes from the high mechanical stress encountered in the terrestrial environment.

evolutionary biology↗

bioRxiv2018

Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host-responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. In contrast to the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viremia was measurable 2 days after exposure and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of IFN-{gamma}, MCP-1, and IL-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of terminal livers and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host-response suggests that relatively small subsets of a hosts response to infection may be responsible for driving pathogenesis that results in a hemorrhagic fever. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host-response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.\n\nIMPORTANCEHost-response mechanisms involved in pathogenesis of VHFs remain poorly understood. An underlying challenge is separating beneficial, inconsequential, and detrimental host-responses during infection. The comparison of host-responses to infection with the same virus in biologically similar animals that have drastically different disease manifestations allows for the identification of pathogenic mechanisms. SHFV, a surrogate virus for human VHF-causing viruses likely causes subclinical infection in African monkeys such as patas monkeys but can cause severe disease in Asian macaque monkeys. Data from the accompanying article by Buechler et al. support that infection of macaques and baboons with non-SHFV simarteviruses can establish persistent or long-term subclinical infections. Baboons, macaques, and patas monkeys are relatively closely taxonomically related (Cercopithecidae: Cercopithecinae) and therefore offer a unique opportunity to dissect how host-response differences determine disease outcome in VHFs.

immunology↗