Biology subjects

Chen, A.

Publications and source records attributed to Chen, A..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Size Selection of Giant Unilamellar Vesicles (GUVs) via Modified cDICE Method

The production of giant unilamellar vesicles (GUVs) plays a pivotal role in various scientific disciplines, particularly in the development of synthetic cells. While numerous methods exist for GUV preparation, the modified continuous droplet interface crossing encapsulation (cDICE) method offers the advantages of simplicity and high encapsulation efficiency. However, a significant limitation of this technique is the generation of vesicles with a broad size distribution and the inability to control the desired size range. This raises a key question: Can the modified cDICE method be optimized to produce GUVs with controlled size distribution? In this study, we examined the effects of two experimental parameters--rotation time (tROT) and the angular frequency ({omega}) of the cDICE chamber--on the size distribution of GUVs. Our results show that reducing either the angular frequency or rotation time shifts the size distribution toward larger vesicles, enabling effective size selection. These findings are further supported by a physical model, which provides insights into the mechanisms underlying size selection. This work demonstrates that control over GUV size distribution can be achieved through straightforward adjustments of system parameters. The ability to fine-tune vesicle size offers researchers a powerful tool for developing customizable experimental systems for synthetic biology and related fields.

synthetic biology↗

bioRxiv2024

Monitoring lysosomal catabolism: a sensitive probe for assessing targeted lysosomal degradation of extracellular proteins

Extracellular targeted protein degradation (eTPD) is an emerging therapeutic field. The Lysosome targeting chimera (LYTAC) is a therapeutic modality that promotes degradation of extracellular drivers of disease in the lysosome. While widely available pH-sensitive probes may report on lysosome delivery, these probes do not necessarily report on the enzymatically active functional state of the lysosome. We report the development and application of a sensitive fluorescent probe, LysoLight Deep Red, to monitor catabolism of internalized proteins in the lysosome based on cleavage by cathepsin proteases. We demonstrate the application of Lysolight Deep Red to monitor the catabolic fate of therapeutic monoclonal antibodies, ASGPR-targeted LYTAC therapeutics and LYTAC targets in immortalized cell lines and in primary human hepatocytes.

cell biology↗

bioRxiv2024

Plasma Exosomes in Insulin Resistant Obesity Exacerbate Progression of Triple Negative Breast Cancer

Breast cancer, the most common cancer among women worldwide, continues to pose significant public health challenges. Among the subtypes of breast cancer, triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat due to the absence of receptors for estrogen, progesterone, or human epidermal growth factor receptor 2, rendering TNBC refractory to conventional targeted therapies. Emerging research underscores the exacerbating role of metabolic disorders, such as type 2 diabetes and obesity, on TNBC aggressiveness. Here, we investigate the critical cellular and molecular factors underlying this link. We explore the pivotal role of circulating plasma exosomes in modulating the tumor microenvironment and enhancing TNBC aggressiveness. We find that plasma exosomes from diet-induced obesity mice induce epithelial-mesenchymal transition features in TNBC cells, leading to increased migration in vitro and enhanced metastasis in vivo. We build on our previous reports demonstrating that plasma exosomes from obese, diabetic patients, and exosomes from insulin-resistant 3T3-L1 adipocytes, upregulate key transcriptional signatures of epithelial-mesenchymal transition in breast cancer. Bioinformatic analysis reveals that TNBC cells exhibit higher expression and activation of proteins related to the Rho-GTPase cascade, particularly the small Ras-related protein Rac1. Our approach suggests novel therapeutic targets and exosomal biomarkers, ultimately to improve prognosis for TNBC patients with co-morbid metabolic disorders.

cancer biology↗

bioRxiv2024

Plasma exosomes from individuals with type 2 diabetes drive breast cancer aggression in patient-derived organoids

Women with obesity-driven diabetes (T2D) are predisposed to more aggressive breast cancers, yet patient metabolic status does not fully inform current standards of care. We previously identified plasma exosomes as key mediators of intercellular communication and drivers of tumor progression; however, their effect on immune cells within the tumor microenvironment (TME) remains unclear. To model this exosomal signaling, we developed a novel method to generate patient-derived organoids (PDOs) from breast tumor resections, uniquely preserving native tumor-infiltrating lymphocytes (TILs) for the first time. This modifiable and reproducible system provides a robust platform for studying human tumor-immune interactions within the TME in vitro. After 3-day exosome treatment, we assessed the impact of T2D-derived exosomes on PDOs via single-cell RNA sequencing. Exosomes from T2D patient plasma triggered a 13.6-fold expansion of immunosuppressive TILs compared to non-diabetic exosome controls. This immune dysfunction may permit the survival of micrometastases and undermine immune checkpoint therapies, a known challenge for cancer patients with comorbid T2D. Tumor-intrinsic analysis revealed a 1.5-fold increase in intratumoral heterogeneity and approximately 2.3-fold upregulation of epithelial-to-mesenchymal transition, invasiveness, and cancer stemness, consistent with enhanced tumor aggressiveness and metastatic potential of these PDOs. These findings demonstrate how metabolic dysregulation in T2D disrupts tumor-immune crosstalk, profoundly impairing anti-tumor immunity and driving cancer progression through a previously underappreciated exosomal signaling pathway. These insights into the TME could inform personalized treatments for patients with this comorbidity.

cancer biology↗

bioRxiv2024

Acute Viral Infection Accelerates Neurodegeneration in a Mouse Model of ALS

While several viral infections have been associated with amyotrophic lateral sclerosis (ALS), the mechanism(s) through which they promote disease has remained almost entirely elusive. This study investigated the impact of common, acute viral infections prior to disease onset on ALS progression in the SOD1G93A mouse model. A single sublethal infection prior to onset of ALS clinical signs was associated with markedly accelerated ALS disease progression characterized by rapid loss of hindlimb function. Prior infection resulted in gliosis in the lumbar spine and upregulation of transcriptional pathways involved in inflammatory responses, metabolic dysregulation, and muscular dysfunction. Therapeutic suppression of gliosis with an anti-inflammatory small molecule, or administration of a direct-acting antiviral, was associated with significantly improved ALS clinical signs, akin to what was observed in uninfected animals. This study provides causal and mechanistic evidence that the immune response elicited by acute viral infections may be an important etiological factor that alters ALS disease trajectory, and provides insight into novel therapeutic and preventative strategies for ALS. One Sentence SummaryAcute viral infection with influenza A virus and SARS-CoV-2 accelerates the progression of ALS in SOD1G93A mice.

immunology↗

bioRxiv2024

Long read sequencing and assembly of wild diploid relatives and cultivars in support of banana breeding programs

Banana is an important fruit and staple crop, which is vital for food and income security in developing countries. A genomics initiative was undertaken as part of the Global Alliance against TR4 (Tropical Race 4 of Fusarium Wilt of Banana) Alliance, with Bayer Crop Science, International Institute of Tropical Agriculture, and University of Queensland to collectively accelerate the breeding efforts in Eastern Africa. The goal of this project is to develop genomics resources to improve banana breeding, productivity, and quality traits amid a changing climate and shifting disease pressures for breeding. Here, we present the PacBio HiFi sequencing, assembly, and comparative analysis of seven genomes of wild and edible diploid banana accessions belonging to subspecies considered important progenitors to cultivated banana varieties and part of breeding programs. This banana genome resource will help power genome editing, a pangenome effort, and conventional breeding programs for germplasm improvement.

genomics↗

bioRxiv2024

Brain Charts for the Rhesus Macaque Lifespan

Recent efforts to chart human brain growth across the lifespan using large-scale MRI data have provided reference standards for human brain development. However, similar models for nonhuman primate (NHP) growth are lacking. The rhesus macaque, a widely used NHP in translational neuroscience due to its similarities in brain anatomy, phylogenetics, cognitive, and social behaviors to humans, serves as an ideal NHP model. This study aimed to create normative growth charts for brain structure across the macaque lifespan, enhancing our understanding of neurodevelopment and aging, and facilitating cross-species translational research. Leveraging data from the PRIMatE Data Exchange (PRIME-DE) and other sources, we aggregated 1,522 MRI scans from 1,024 rhesus macaques. We mapped non-linear developmental trajectories for global and regional brain structural changes in volume, cortical thickness, and surface area over the lifespan. Our findings provided normative charts with centile scores for macaque brain structures and revealed key developmental milestones from prenatal stages to aging, highlighting both species-specific and comparable brain maturation patterns between macaques and humans. The charts offer a valuable resource for future NHP studies, particularly those with small sample sizes. Furthermore, the interactive open resource (https://interspeciesmap.childmind.org) supports cross-species comparisons to advance translational neuroscience research.

neuroscience↗

bioRxiv2024

Three-dimension transcriptomics maps of whole mouse embryo during organogenesis

Understanding mammalian development heavily relies on classical animal models like the house mouse (Mus musculus). Advanced spatial transcriptomics has enabled biologists to break new ground in studies of molecular dynamics and cellular patterning during embryonic development with spatiotemporal resolution. To construct a comprehensive developmental trajectory, current three-dimensional (3D) spatial transcriptomic profiling leverages mouse embryos from gastrulation (E5.5) to organogenesis (E13.5) in continuity. However, a crucial phase for early organogenesis between E9.5 and E11.5 was deficient. To unveil the mystery of this stage, we present the 3D transcriptomics of mouse embryos at E9.5 and E11.5, with a widely applicable reconstruction workflow that bypasses sophisticated bioinformatic calculations. As our 3D atlas is generated at single-cell resolution, we demonstrate how organogenetic processes can be interpreted at different levels of granularity, from local cellular interactions to whole embryonic regionalization. We release the open-access database MOSTA3D (Mouse Organogenesis Spatiotemporal Transcriptomic Atlas in Three-dimensional) and hope a broader community will contribute to extending this framework from conception to senility in the near future.

developmental biology↗

bioRxiv2024

Cell-type-specific striatal modulation of amygdalar acetylcholine in salience assignment

The salience assignment is pivotal for both natural and artificial intelligence. Pioneering studies established that basal forebrain cholinergic neurons process behaviorally relevant salient information. However, the neural circuit mechanism underlying salience assignment remains poorly understood. Here we show that the acetylcholine (ACh) level in the basolateral amygdala (BLA) dynamically represented behavioral salience. Distinct neuronal subpopulations in the nucleus accumbens (NAc), D1- and D2-expressing medium spiny neurons (MSNs), antagonistically and specifically promote and suppress ACh release in the BLA, but not the cortex and hippocampus. These striatal D1 and D2 MSNs regulate BLA ACh by disinhibiting and inhibiting cholinergic neurons in the basal forebrain subregion substantia innominata (SI), respectively. Optogenetic manipulations of the pathway from striatal D1 and D2 MSNs to the SI opposingly affect associative learning. Our findings uncover an unconventional role of striatal MSNs in salience assignment via regulating the salience-representing amygdalar ACh activity.

neuroscience↗

bioRxiv2024

Identification of targetable vulnerabilities of PLK1-overexpressing cancers by synthetic dosage lethality

Tumor heterogeneity poses a significant challenge in combating treatment resistance. Despite Polo-like kinase 1 (PLK1) being universally overexpressed in cancers and contributing to chromosomal instability (CIN), direct PLK1 inhibition hasnt yielded clinical progress. To address this, we utilized the synthetic dosage lethality (SDL) approach, targeting PLK1s genetic interactions for selective killing of overexpressed tumor cells while mitigating heterogeneity-associated challenges. Employing computational methods, we conducted a genome-wide shRNA screen, identifying 105 SDL candidates. Further in vivo CRISPR screening in a breast cancer xenograft model and in vitro CRISPR analysis validated these candidates. Employing Perturb-seq revealed IGF2BP2/IMP2 as a key SDL hit eliminating PLK1-overexpressing cells. Suppression of IGF2BP2, genetically or pharmacologically, downregulated PLK1 and limited tumor growth. Our findings strongly propose targeting PLK1s genetic interactions as a promising therapeutic approach, holding broad implications across multiple cancers where PLK1 is overexpressed.

cancer biology↗

bioRxiv2024

Reward history guides attentional selection in whisker somatosensory (S1) cortex

Prior reward is a potent cue for attentional capture, but the underlying neurobiology is largely unknown. In a novel whisker touch detection task, we show that mice flexibly shift attention between specific whiskers on a trial-by-trial timescale, guided by the recent history of stimulus-reward association. Two-photon calcium imaging and spike recordings revealed a robust neurobiological correlate of attention in the somatosensory cortex (S1), boosting sensory responses to the attended whisker in L2/3 and L5, but not L4. Attentional boosting in L2/3 pyramidal cells was topographically precise and whisker-specific, and shifted receptive fields toward the attended whisker. L2/3 VIP interneurons were broadly activated by whisker stimuli, motion, and arousal but did not carry a whisker-specific attentional signal, and thus did not mediate spatially focused tactile attention. Together, these findings establish a new model of focal attention in the mouse whisker tactile system, showing that the history of stimuli and rewards in the recent past can dynamically engage local modulation in cortical sensory maps to guide flexible shifts in ongoing behavior.

neuroscience↗

bioRxiv2024

Temporal dynamics analysis reveals that concurrent working memory load eliminates the Stroop effect through disrupting stimulus-response mapping

Concurrent verbal working memory task can eliminate the color-word Stroop effect. Previous research, based on specific and limited resources, suggested that the disappearance of the conflict effect was due to the memory information preempting the resources for distractors. However, it remains unclear which particular stage of Stroop conflict processing is influenced by working memory loads. In this study, electroencephalography (EEG) recordings with event-related potential (ERP) analyses, time-frequency analyses, multivariate pattern analyses (MVPA), and representational similarity analyses (RSA) were applied to provide an in-depth investigation of the aforementioned issue. Subjects were required to complete the single task (the classical manual color-word Stroop task) and the dual task (the Sternberg working memory task combined with the Stroop task), respectively. Behaviorally, the results indicated that the Stroop effect was eliminated in the dual-task condition. The EEG results showed that the concurrent working memory task did not modulate the P1, N450, and alpha bands. However, it modulated the sustained potential (SP), late theta (740-820 ms), and beta (920-1040 ms) power, showing no difference between congruent and incongruent trials in the dual-task condition but significant difference in the single-task condition. Importantly, the RSA results revealed that the neural activation pattern of the late theta was similar to the response interaction pattern. Together, these findings implied that concurrent working memory task eliminated the Stroop effect through disrupting stimulus-response mapping.

neuroscience↗

bioRxiv2024

Prkra is the double-stranded RNA sensor to mediate an embryonic integrated stress response

Double-stranded RNAs (dsRNAs), known as conserved pathogen-associated molecular patterns, are recognized by interferon-induced protein kinase R (PKR) to trigger an integrated stress response, characterized by the inhibition of global translation. However, the interferon system is inactive in pluripotent cells, thus the mechanism underlying dsRNA sensing and translational control is unclear. In this study, we utilized early zebrafish embryos as a model of pluripotent cells and discovered a PKR-independent blockage of translation initiation induced by dsRNA stimulation. Prkra dimer was identified as the genuine dsRNA sensor. Upon binding to dsRNAs, the dimerized dsRNA binding domain 3 becomes activated to sequester the eIF2 complexes from the translation machinery, leading to a hindrance in global protein synthesis. This distinctive embryonic stress response restricts RNA virus SVCV replication in zebrafish embryos but is also conserved in early mouse embryos and embryonic stem cells. Therefore, the Prkra-mediated dsRNA sensing and translation blocking mechanism potentially represents a common strategy for reestablishing physiological homeostasis in response to environmental stresses.

immunology↗

bioRxiv2024

Eco-evolutionary Guided Pathomic Analysis to Predict DCIS Upstaging

Cancers evolve in a dynamic ecosystem. Thus, characterizing cancers ecological dynamics is crucial to understanding cancer evolution and can lead to discovering novel biomarkers to predict disease progression. Ductal carcinoma in situ (DCIS) is an early-stage breast cancer characterized by abnormal epithelial cell growth confined within the milk ducts. In this study, we show that ecological analysis of hypoxia and acidosis biomarkers can significantly improve prediction of DCIS upstaging. First, we developed a novel eco-evolutionary designed approach to define habitats in the tumor intraductal microenvironment based on oxygen diffusion distance. Then, we identified cancer cells with metabolic phenotypes attributed to their habitats, including CA9 for hypoxia responding phenotype, and LAMP2b for acid adapted phenotype. Traditionally these markers have shown limited predictive capabilities for DCIS progression, if any. However, when analyzed from an ecological perspective, their power to differentiate between non-upstaged and upstaged DCIS increased significantly. Second, we discovered distinct niches with spatial patterns of these biomarkers and used the distribution of such niches to predict patient upstaging. The niches were characterized by pattern analysis of both cellular and spatial features. With a 5-fold validation on the biopsy cohort, we trained a random forest classifier to achieve the area under curve (AUC) of 0.74. Our results affirm the importance of tumor ecological features in eco-evolutionary-designed approaches for novel biomarkers discovery. SignificanceOur results affirm the importance of spatial and ecological features in eco-evolutionary-designed biomarkers discovery studies in the era of digital pathology.

cancer biology↗

bioRxiv2024

ComBatLS: A location- and scale-preserving method for multi-site image harmonization

Recent work has leveraged massive datasets and advanced harmonization methods to construct normative models of neuroanatomical features and benchmark individuals morphology. However, current harmonization tools do not preserve the effects of biological covariates including sex and age on features variances; this failure may induce error in normative scores, particularly when such factors are distributed unequally across sites. Here, we introduce a new extension of the popular ComBat harmonization method, ComBatLS, that preserves biological variance in features locations and scales. We use UK Biobank data to show that ComBatLS robustly replicates individuals normative scores better than other ComBat methods when subjects are assigned to sex-imbalanced synthetic "sites". Additionally, we demonstrate that ComBatLS significantly reduces sex biases in normative scores compared to traditional methods. Finally, we show that ComBatLS successfully harmonizes consortium data collected across over 50 studies. R implementation of ComBatLS is available at https://github.com/andy1764/ComBatFamily.

neuroscience↗

bioRxiv2024

A Coronaviral Pore-Replicase Complex Drives RNA Synthesis in Double Membrane Vesicles

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the non-structural viral membrane protein nsp4 is the key DMV pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, with broad spectrum anti-coronaviral activity.

biochemistry↗

bioRxiv2024

An Infusible Extracellular Matrix Biomaterial Improves Survival in a Model of Severe Systemic Inflammation

Excess systemic inflammation can often be lethal in septic and trauma patients due to onset of multiple organ dysfunction syndrome (MODS). As of right now, there are no effective immunomodulatory therapeutics that can promote survival within this patient population. Pro-regenerative extracellular matrix (ECM) biomaterials have shown success for treatment of local inflammation but have not been fully explored for treating systemic inflammation. Here, we demonstrate efficacy of an intravenously delivered infusible ECM (iECM) material, which promotes increased survival in a murine model of MODS by decreasing systemic mediators of inflammation. Lung and kidney failure are associated with higher mortality in MODS compared to other organ failures, and we demonstrate that iECM localizes primarily to kidney and lung tissues during systemic inflammation induced by endotoxin. iECM successfully lowered vascular permeability within lung tissue and lowered levels of inflammatory cytokine signaling such as IL-6, verified via ELISA and gene expression analyses. We also demonstrated that immune cell infiltration into lung tissue was modulated with iECM treatment, with an increase in neutrophil retention in the lung and decreases in pro-inflammatory macrophage presence. In summation, iECM improves survival from severe systemic inflammation by decreasing the local and systemic inflammatory signaling pathways that contribute to MODS. These results provide a strong rationale for translational studies of iECM treatment in systemic inflammatory syndromes, including sepsis and trauma.

bioengineering↗

bioRxiv2024

Investigating Subpopulation Dynamics in Clonal CHO-K1 Cells with Single-Cell RNA Sequencing

Chinese Hamster Ovary (CHO) cells are used to produce monoclonal antibodies and other biotherapeutics at industrial scale. Despite their ubiquitous nature in the biopharmaceutical industry, little is known about the behaviors of individual transfected clonal CHO cells. Most CHO cells are assessed on their ability to produce the protein of interest over time, known as their stability. But these CHO cells have primarily been studied in bulk, working under the assumption that these bulk samples are identical because of genetic clonality across the sample; however, this does not address other forms of cellular heterogeneity in these ostensibly clonal cells. It is possible these variable stability phenotypes reflect heterogeneity within the clonal samples. In this study, we performed single-cell RNA sequencing on two clonal CHO-K1 cell populations with different stability phenotypes over a 90 day culture period. Our data showed that the instability of the unstable clone was due in part to the emergence of a low-producing subpopulation in the aged samples. This low-producing subpopulation did not exhibit markers of cellular stress which were expressed in the higher-producing populations. Further multiomic investigation should be performed to better characterize this heterogeneity.

genomics↗