Biology subjects

Fang, R.

Publications and source records attributed to Fang, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

In vivo quantification of anterior and posterior chamber volumes in mice: implications for aqueous humor dynamics

PurposeAqueous humor inflow rate, a key parameter influencing aqueous humor dynamics, is typically measured by fluorophotometery. Analyzing fluorophotometric data depends, inter alia, on the volume of aqueous humor in the anterior, but not the posterior, chamber. Previous fluorophotometric studies of aqueous inflow rate in mice have assumed the ratio of anterior:posterior volumes in mice to be similar to those in humans. Our goal was to measure anterior and posterior chamber volumes in mice to facilitate better estimates of aqueous inflow rates. MethodsWe used standard near-infrared optical coherence tomography (OCT) and robotic visible-light OCT (vis-OCT) to visualize, reconstruct and quantify the volumes of the anterior and posterior chambers of the mouse eye in vivo. We used histology and micro-CT scans to validate relevant landmarks from ex vivo tissues to facilitate in vivo measurement. ResultsPosterior chamber volume is 1.1 times the anterior chamber volume in BALB/cAnNCrl mice, i.e. the anterior chamber constitutes about 47% of the total aqueous humor volume, which is very dissimilar to the situation in humans. Anterior chamber volumes in 2-month-old BALB/cAnNCrl and 7-month-old C57BL6/J mice were 1.55 {+/-} 0.36 {micro}L (n=10) and 2.41 {+/-} 0.29 {micro}L (n=8), respectively. This implies that previous studies likely over-estimated aqueous inflow rate by approximately two-fold. ConclusionsIt is necessary to reassess previously reported estimates of aqueous inflow rates, and thus aqueous humor dynamics in the mouse. For example, we now estimate that only 0-15% of aqueous humor drains via the pressure-independent (unconventional) route, similar to that seen in humans and monkeys.

physiology↗

bioRxiv2024

Post-flowering photoperiod sensitivity of soybean in pod-setting responses

The development of soybean (Glycine max) is regulated by photoperiod, with genes related to photoperiod sensitivity primarily focused on flowering time. However, their roles in post-flowering reproductive development and the mechanisms by which photoperiod affects them are not yet determined. In this study, we found that pod formation is sensitive to photoperiod. Long-day (LD) conditions tend to extend the time from flowering to pod formation (R1 to R3 stage), and the first wave of flowers tends to fall off. Additionally, photoperiod affects pistil morphology; under short-day (SD) conditions, the stigma has a curved hook-like structure that facilitates better interaction with the filaments when pollen is released, ultimately influencing the timing of pod formation. Photoperiod-insensitive mutants, lacking E1 family and Evening Complex genes, showed no difference in pod formation time under LD or SD conditions. Hormone content analysis and transcriptome data analysis indicated that various hormones, ROS signals, and the application of sucrose solution in vitro might influence floral organ abscission. HighlightPhotoperiod sensitivity after flowering affects the pod-setting time in soybean.

plant biology↗

bioRxiv2024

Freeform robotic optical coherence tomography beyond the optical field-of-view limit

Imaging complex, non-planar anatomies with optical coherence tomography (OCT) is limited by the optical field of view (FOV) in a single volumetric acquisition. Combining linear mechanical translation with OCT extends the FOV but suffers from inflexibility in imaging non-planar anatomies. We report the freeform robotic OCT to fill this gap. To address challenges in volumetric reconstruction associated with the robotic movement accuracy being two orders of magnitudes worse than OCT imaging resolution, we developed a volumetric registration algorithm based on simultaneous localization and mapping (SLAM) to overcome this limitation. We imaged the entire aqueous humor outflow pathway, whose imaging has the potential to customize glaucoma surgeries but is typically constrained by the FOV, circumferentially in mice as a test. We acquired volumetric OCT data at different robotic poses and reconstructed the entire anterior segment of the eye. The reconstructed volumes showed heterogeneous Schlemms canal (SC) morphology in the reconstructed anterior segment and revealed a segmental nature in the circumferential distribution of collector channels (CC) with spatial features as small as a few micrometers.

bioengineering↗

bioRxiv2023

Nucleosome wrapping state encodes principles of 3D genome organization

Nucleosome is the basic structural unit of the genome. During processes like DNA replication and gene transcription, the conformation of nucleosomes undergoes dynamic changes, including DNA unwrapping and rewrapping, as well as histone disassembly and assembly. However, the wrapping characteristics of nucleosomes across the entire genome, including region-specificity and their correlation with higher-order chromatin organization, remain to be studied. In this study, we investigated the wrapping length of DNA on nucleosomes across the whole genome using wrapping-seq. We discovered that the chromatin of mouse ES cells forms Nucleosome Wrapping Domains (NRDs), which is conserved in yeast and fly. We found that the degree of nucleosome wrapping decreases after DNA replication and is promoted by transcription. Furthermore, we observed that nucleosome wrapping states delineate 3D genome organization and DNA replication timing. In conclusion, we have revealed a novel principle of higher-order chromatin organization.

molecular biology↗

bioRxiv2023

Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer

Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping out fibroblasts heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in the definition of PDAC CAF phenotypes. We identify the myCAF transcriptional phenotype as uniquely dependent on proficient MAPK signalling. In addition, CAFs displaying elevated MAPK activity are specifically anchored to basal-like/squamous PDAC cells and define tumour subdomains with reduced frequency of CD8+ T cells. We characterize the single-cell transcriptome of mouse PDAC tumours in response to MAPK inhibition and identify gene expression signatures of MAPKhigh CAFs, which suggest immunoregulatory functions. Accordingly, a gene expression signature of MAPKhigh CAFs correlates with poor prognosis in several human cancer conditions, including PDAC, and with reduced response to immune checkpoint inhibition in immune-reactive solid tumours. Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a new strategy for targeting of myofibroblastic CAFs in vivo.

cancer biology↗

bioRxiv2023

ATR promotes mTORC1 activation via de novo cholesterol synthesis in p16-low cancer cells

DNA damage and cellular metabolism exhibit a complex interplay characterized by bidirectional feedback mechanisms. Key mediators of the DNA damage response and cellular metabolic regulation include Ataxia Telangiectasia and Rad3-related protein (ATR) and the mechanistic Target of Rapamycin Complex 1 (mTORC1), respectively. Previous studies have established ATR as a regulatory upstream factor of mTORC1 during replication stress; however, the precise mechanisms by which mTORC1 is activated in this context remain poorly defined. Additionally, the activity of this signaling axis in unperturbed cells has not been extensively investigated. Here, we demonstrate that ATR promotes mTORC1 activity across various cellular models under basal conditions. This effect is particularly enhanced in cells following the loss of p16, which we have previously associated with hyperactivation of mTORC1 signaling and here found have increased ATR activity. Mechanistically, we found that ATR promotes de novo cholesterol synthesis and mTORC1 activation through the upregulation of lanosterol synthase (LSS), independently of both CHK1 and the TSC complex. Furthermore, the attenuation of mTORC1 activity resulting from ATR inhibition was rescued by supplementation with lanosterol or cholesterol in multiple cellular contexts. This restoration corresponded with enhanced localization of mTOR to the lysosome. Collectively, our findings demonstrate a novel connection linking ATR and mTORC1 signaling through the modulation of cholesterol metabolism.

cancer biology↗

bioRxiv2023

Prothoracicotropic hormone controls female sexual receptivity through the function of ecdysone in pC1 neurons of Drosophila

Female sexual receptivity is essential for reproduction of a species. Neuropeptides play the main role in regulating female receptivity. However, whether neuropeptides regulate female sexual receptivity during the neurodevelopment is unknown. Here we found the peptide hormone prothoracicotropic hormone (PTTH), which belongs to the insect PG axis, negatively regulated virgin female receptivity through ecdysone during neurodevelopment in Drosophila melanogaster. We identified PTTH neurons as doublesex-positive neurons, they regulated virgin female receptivity before the metamorphosis during the 3rd-instar larval stage. PTTH deletion resulted in the increased EcR-A expression in the whole newly formed prepupae. Furthermore, the ecdysone receptor EcR-A in pC1 neurons positively regulated virgin female receptivity during metamorphosis. The decreased EcR-A in pC1 neurons induced abnormal morphological development of pC1 neurons without changing neural activity. Among all subtypes of pC1 neurons, the function of EcR-A in pC1b neurons was necessary for virgin female copulation rate. These suggested that the changes of synaptic connections between pC1b and other neurons decreased female copulation rate. Moreover, female receptivity significantly decreased when the expression of PTTH receptor Torso was reduced in pC1 neurons. This suggested that PTTH not only regulates female receptivity through ecdysone but also through affecting female receptivity associated neurons directly. The PG axis has similar functional strategy as the HPG axis in mammals to trigger the juvenile-adult transition. Our work suggests a general mechanism underlying which the neurodevelopment during maturation regulates female sexual receptivity.

neuroscience↗

bioRxiv2023

Three-dimensional single-cell transcriptome imaging of thick tissues

Multiplexed error-robust fluorescence in-situ hybridization (MERFISH) allows genome-scale imaging of RNAs in individual cells in intact tissues. To date, MERFISH has been applied to image thin tissue samples of [~]10-{micro}m thickness. Here, we present a thick-tissue three-dimensional (3D) MERFISH imaging method, which uses confocal microscopy for optical sectioning, deep learning for increasing imaging speed and quality, as well as sample preparation and imaging protocol optimized for image registration of thick samples. We demonstrated 3D MERFISH on mouse brain tissue sections of up to 200 {micro}m thickness with high detection efficiency and accuracy. We anticipate that 3D thick-tissue MERFISH imaging will broaden the scope of questions that can be addressed by spatial genomics.

neuroscience↗

bioRxiv2023

DeepDynaForecast: Phylogenetic-informed graph deep learning for epidemic transmission dynamic prediction

In the midst of an outbreak or sustained epidemic, reliable prediction of transmission risks and patterns of spread is critical to inform public health programs. Projections of growth or decline among specific risk groups can aid in optimizing interventions, particularly when resources are limited. Phylogenetic trees have been widely used in the detection of transmission chains and high-risk populations. Moreover, tree topology and the incorporation of population parameters (phylodynamics) can be useful to reconstruct the evolutionary dynamics of an epidemic across space and time among individuals. We now demonstrate the utility of phylodynamic trees for infection forecasting in addition to backtracking, developing a phylogeny-based deep learning system, called DeepDynaForecast. Our approach leverages a primal-dual graph learning structure with shortcut multi-layer aggregation, and it is suited for the early identification and prediction of transmission dynamics in emerging high-risk groups. We demonstrate the accuracy of DeepDynaForecast using simulated outbreak data and the utility of the learned model using empirical, large-scale data from the human immunodeficiency virus epidemic in Florida between 2012 and 2020. Our framework is available as open-source software (MIT license) at: https://github.com/lab-smile/DeepDynaForcast. Author SummaryDuring an outbreak or sustained epidemic, accurate prediction of patterns in transmission risk can reliably inform public health strategies. Projections indicating growth or decline of transmission for specific risk groups can significantly enhance the optimization of interventions, especially when resources are limited. To address this, we present DeepDynaForecast, a cutting-edge deep learning algorithm designed for forecasting pathogen transmission dynamics. Uniquely, DeepDynaForecast was trained on in-depth simulation data and used more information from the phylogenetic tree of pathogen sequence data than any other algorithm in the field to date, allowing classification of samples according to their dynamics (growth, static, or decline) with incredible accuracy. We evaluated the models performance using both simulated outbreak data and empirical, large-scale data from the HIV epidemic in Florida between 2012 and 2020. We conclude DeepDynaForecast represents a significant advancement in genomics-mediated pathogen transmission characterization and has the potential to catalyze new research directions within virology, molecular biology, and public health.

bioinformatics↗

bioRxiv2023

De novo purine metabolism is a metabolic vulnerability of cancers with low p16 expression

p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in [~]50% of all human cancers. In its canonical role, p16 inhibits the G1-S phase cell cycle progression through suppression of cyclin dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. Whether other nucleotide metabolic genes and pathways are affected by p16/CDKN2A loss and if these can be specifically targeted in p16/CDKN2A-low tumors has not been previously explored. Using CRISPR KO libraries in multiple isogenic human and mouse melanoma cell lines, we determined that many nucleotide metabolism genes are negatively enriched in p16/CDKN2A knockdown cells compared to controls. Indeed, many of the genes that are required for survival in the context of low p16/CDKN2A expression based on our CRISPR screens are upregulated in p16 knockdown melanoma cells and those with endogenously low CDKN2A expression. We determined that cells with low p16/Cdkn2a expression are sensitive to multiple inhibitors of de novo purine synthesis, including anti-folates. Tumors with p16 knockdown were more sensitive to the anti-folate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2A-low tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents.

cancer biology↗

bioRxiv2023

HUWE1 Amplifies Ubiquitin Modifications to Broadly Stimulate Clearance of Proteins and Aggregates

Selective breakdown of proteins and aggregates is crucial for maintaining normal cellular activities and is involved in the pathogenesis of diverse diseases. How the cell recognizes and tags these targets in different structural states for degradation by the proteasome and autophagy pathways has not been well understood. Here, we discovered that a HECT-family ubiquitin ligase HUWE1 is broadly required for the efficient degradation of soluble factors and for the clearance of protein aggregates/condensates. Underlying this capacity of HUWE1 is a novel Ubiquitin-Directed ubiquitin Ligase (UDL) activity which recognizes both soluble substrates and aggregates that carry a high density of ubiquitin chains and rapidly expand the ubiquitin modifications on these targets. Ubiquitin signal amplification by HUWE1 recruits the ubiquitin-dependent segregase p97/VCP to process these targets for subsequent degradation or clearance. HUWE1 controls the cytotoxicity of protein aggregates, mediates Targeted Protein Degradation and regulates cell-cycle transitions with its UDL activity.

molecular biology↗

bioRxiv2023

Multiscale imaging of corneal endothelium damage and effects of Rho Kinase inhibitor application in mouse models of acute ocular hypertension

We developed a multiscale optical imaging workflow, integrating and correlating visible-light optical coherence tomography, confocal laser scanning microscopy, and single-molecule localization microscopy to investigate the mouse cornea damages from the in-vivo tissue level to the nanoscopic single-molecule level. We used electron microscopy to validate the imaged nanoscopic structures. We imaged wild-type mice and mice with acute ocular hypertension and examined the effects of Rho Kinase inhibitor application. We defined four types of intercellular tight junction structures as healthy, compact, partially-distorted, and fully-distorted types by labeling the Zonula occludens-1 protein in the corneal endothelial cell layer. We correlated the statistics of the four types of tight junction structures with cornea thickness and intraocular pressure. We found that the population of fully-distorted tight junctions correlated well with the level of cornea edema, and applying Rho Kinase inhibitor reduced the population of fully-distorted tight junctions under acute ocular hypertension.

biophysics↗

bioRxiv2023

Quantify nanoscopic alterations associated with mitochondrial dysfunctions using three-dimensional single-molecule localization microscopy

The morphology of mitochondria provides insights into their functions. Three-dimensional (3D) single-molecule localization microscopy (SMLM) uniquely enables the analysis of mitochondrial morphological features individually, thanks to its high resolution. However, nearly all reported studies of mitochondrial morphology have been qualitative and without statistical analysis. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D SMLM images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to analyze mitochondrial morphological alterations associated with disease progression quantitatively.

biophysics↗

bioRxiv2023

Target-identification and mechanism-of-action studies of indole terpenoid mimics reveal that proper spindle microtubule assembly is essential for Cdh1-mediated proteolysis of CENP-A

Centromere protein A (CENP-A), a histone H3 variant specific to centromeres, is crucial for kinetochore positioning and chromosome segregation. However, its regulatory mechanism in human cells remains incompletely understood. We conducted a structure-activity relationship (SAR) study of the cell cycle-arresting indole terpenoid mimic JP18 and found two more potent analogues, (+)-6-Br-JP18 and (+)-6-Cl-JP18. Tubulin was identified as a potential cellular target of these halogenated analogues by using the drug affinity responsive target stability (DARTS) based method. X-ray crystallography analysis revealed that both molecules bind to the colchicine-binding site of {beta}-tubulin. Furthermore, we discovered that treatment of human cells with microtubule-targeting agents (MTAs), including these two compounds, led to CENP-A accumulation by destabilizing Cdh1, a co-activator of the APC/C E3 ubiquitin ligase. Our study establishes a link between microtubule dynamics and CENP-A accumulation using small-molecule tools and highlights the role of Cdh1 in CENP-A proteolysis.

cell biology↗

bioRxiv2023

Emergence of Emotion Selectivity in A Deep Neural Network Trained to Recognize Visual Objects

Recent neuroimaging studies have shown that the visual cortex plays an important role in representing the affective significance of visual input. The origin of these affect-specific visual representations is debated: they are intrinsic to the visual system versus they arise through reentry from frontal emotion processing structures such as the amygdala. We examined this problem by combining convolutional neural network (CNN) models of the human ventral visual cortex pre-trained on ImageNet with two datasets of affective images. Our results show that (1) in all layers of the CNN models, there were artificial neurons that responded consistently and selectively to neutral, pleasant, or unpleasant images and (2) lesioning these neurons by setting their output to 0 or enhancing these neurons by increasing their gain led to decreased or increased emotion recognition performance respectively. These results support the idea that the visual system may have the intrinsic ability to represent the affective significance of visual input and suggest that CNNs offer a fruitful platform for testing neuroscientific theories. Author SummaryThe present study shows that emotion selectivity can emerge in deep neural networks trained to recognize visual objects and the existence of the emotion-selective neurons underlies the ability of the network to recognize the emotional qualities in visual images. Obtained using two affective datasets (IAPS and NAPS) and replicated on two CNNs (VGG-16 and AlexNet), these results support the idea that the visual system may have an intrinsic ability to represent the motivational significance of sensory input and CNNs are a valuable platform for testing neuroscience ideas in a way that is not practical in empirical studies.

neuroscience↗

bioRxiv2023

Plant Arbovirus Mobilizes a Vector Salivary Protein to Initiate Plant Infection

Plant viruses typically rely on insect vectors for transmission between plants, with insect salivary proteins playing critical roles in this process. In this study, we demonstrate how Laodelphax striatellus salivary carbonic anhydrase (LssaCA) promotes rice stripe virus (RSV) infection in plants. We discovered that LssaCA directly binds to RSV nucleocapsid protein (NP) in insect salivary glands. This LssaCA-NP complex interacts with a rice thaumatin-like protein (OsTLP) that possess endo-{beta}-1,3-glucanase activity potentially degrading callose. Upon binding, the LssaCA-NP complex significantly enhances OsTLP enzymatic activity. We further clarify that both L. striatellus feeding and RSV infection induce callose deposition. The tripartite LssaCA-NP-OsTLP interaction enhances callose degradation, thereby facilitating RSV infection via its insect vector. This study provides new insights into complex virus-insect-plant tripartite interactions mediated by insect salivary proteins, with broad implications for numerous plant viruses transmitted by insect vectors.

plant biology↗

bioRxiv2022

Integrative analysis of the 3D genome and epigenome in mouse embryonic tissues

While a rich set of putative cis-regulatory sequences involved in mouse fetal development has been annotated recently based on chromatin accessibility and histone modification patterns, delineating their role in developmentally regulated gene expression continues to be challenging. To fill this gap, we mapped chromatin contacts between gene promoters and distal sequences genome-wide in seven mouse fetal tissues, and for one of them, across six developmental stages. We identified 248,620 long-range chromatin interactions centered at 14,138 protein-coding genes and characterized their tissue-to-tissue variations as well as developmental dynamics. Integrative analysis of the interactome with previous epigenome and transcriptome datasets from the same tissues revealed a strong correlation between the chromatin contacts and chromatin state at distal enhancers, as well as gene expression patterns at predicted target genes. We predicted target genes of 15,098 candidate enhancers, and used them to annotate target genes of homologous candidate enhancers in the human genome that harbor risk variants of human diseases. We present evidence that schizophrenia and other adult disease risk variants are frequently found in fetal enhancers, providing support for the hypothesis of fetal origins of adult diseases.

developmental biology↗

bioRxiv2022

Multiple forward scattering reduces the measured scattering coefficient of whole blood in visible-light optical coherence tomography

Optical properties of blood encode oxygen-dependent information. Noninvasive optical detection of these properties is increasingly desirable to extract biomarkers for tissue health. Recently, visible-light optical coherence tomography (vis-OCT) demonstrated retinal oxygen saturation (sO2) measurements using the depth-resolved spectrum of blood. Such measurements rely on differences between the absorption and scattering coefficients of oxygenated and deoxygenated blood. However, there is still broad disagreement, both theoretically and experimentally, on how vis-OCT measures bloods scattering coefficient. Incorrect assumptions of bloods optical properties can add additional uncertainties or biases into vis-OCTs sO2 model. Using Monte Carlo simulation of a retinal vessel, we determined that vis-OCT almost exclusively detects multiple-scattered photons in blood. Meanwhile, photons mostly forward scatter in blood within the visible spectral range, allowing photons to maintain ballistic paths and penetrate deeply, leading to a reduction in the measured scattering coefficient. We defined a scattering scaling factor (SSF) to account for such a reduction and found that SSF varied with measurement conditions, such as numerical aperture, depth resolution, and depth selection. We further experimentally validated SSF in ex vivo blood phantoms pre-set sO2 levels and in the human retina, both of which agreed well with our simulation.

bioengineering↗