Biology subjects

Gu, L.

Publications and source records attributed to Gu, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Viscoelastic Extracellular Matrix Enhances Epigenetic Remodeling and Cellular Plasticity

Extracellular matrices of living tissues exhibit viscoelastic properties, yet how these properties regulate chromatin and the epigenome remains unclear. Here, we show that viscoelastic substrates induce changes in nuclear architecture and epigenome, with more pronounced effects on softer surfaces. Fibroblasts on viscoelastic substrates display larger nuclei, lower chromatin compaction, and differential expression of distinct sets of genes related to the cytoskeleton and nuclear function compared to those on purely elastic surfaces. Slow-relaxing viscoelastic substrates reduce lamin A/C expression and enhance nuclear remodeling. These structural changes are accompanied by a global increase in euchromatin marks and local increase in chromatin accessibility at cis-regulatory elements associated with neuronal and pluripotent genes. Consequently, viscoelastic substrates improve the reprogramming efficiency from fibroblasts into neurons and induced pluripotent stem cells. Collectively, our findings unravel the roles of matrix viscoelasticity in epigenetic regulation and cell reprogramming, with implications for designing smart materials for cell fate engineering.

cell biology↗

bioRxiv2024

Rapid High-Throughput Isolation and purification of Chicken Myoblasts Based on Deterministic Lateral Displacement Microfluidic Chips

Myoblasts are defined as stem cells containing skeletal muscle cell precursors. However, there are some challenges associated with the purification of myoblast samples, including long culture times and ease of bacterial contamination. In this study, we propose a microfluidic myoblast cell enrichment and purification platform based on the principle of deterministic lateral displacement (DLD). To achieve this, we designed a DLD chip with three outlets and tested it on 11-day-old Wenchang chicken pectoral muscle tissue. A cell suspension was prepared using the collagenase method, pretreated, and then passed into the designed DLD chip for myoblast enrichment and purification. In this study, the number of myoblasts and the diameter of myoblasts increased slowly before E9, and the diameter of myofibers decreased and the number of myofibers increased rapidly after E9. The period when the number of muscle fibers is highest is E12, and the period when the diameter of muscle fibers increases again after the lowest point is also E12. After E12, the diameter of the muscle fibers increased and the number of muscle fibers decreased. At E12, myoblasts clustered and fused, and the proliferation of myoblasts was greatly reduced. E12 is both intact myoblasts and the most vigorous proliferation period, so the best time to determine isolation is E12. We attained a myoblast cell recovery rate of 80%, a target outlet collection purity of 99%, and a chip throughput of 50 m/min. This study provides a novel and effective method for the isolation and purification of skeletal muscle myoblasts.

cell biology↗

bioRxiv2024

A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions

Cortical myosin contraction and cell adhesion work together to promote tissue shape changes, but how they are modulated to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical accumulation of contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While levels of contractile myosin correlate with apical constriction, whether levels of adherens junctions modulate apical constriction is unknown. We identified a novel Drosophila gene moat that maintains low levels of Bazooka/Par3-dependent adherens junctions and thereby restricts apical constriction to ventral furrow cells with high-level contractile myosin. In moat mutants, abnormally high levels of Bazooka/Par3-dependent adherens junctions promote ectopic apical constriction in cells with low-level contractile myosin, insufficient for apical constriction in wild type. Such ectopic apical constriction expands infolding behavior from ventral furrow to ectodermal anterior midgut, which normally forms a later circular invagination. In moat mutant ventral furrow, a perturbed apical constriction gradient delays infolding. Our results indicate that levels of adherens junctions can modulate the outcome of apical constriction, providing an additional mechanism to define morphogenetic boundaries. Summary StatementCharacterization of a novel gene moat demonstrates ectopic expansion of apical constriction due to abnormally high levels of Bazooka/Par3-dependent adherens junctions without defects in early patterning gene expression.

developmental biology↗

bioRxiv2024

Substrate stiffness modulates the emergence and magnitude of senescence phenotypes

Cellular senescence is a major driver of aging and disease. Here we show that substrate stiffness modulates the emergence and magnitude of senescence phenotypes after exposure to senescence inducers. Using a primary dermal fibroblast model, we show that decreased substrate stiffness accelerates senescence-associated cell-cycle arrest and regulates the expression of conventional protein-based biomarkers of senescence. We found that the expression of these senescence biomarkers, namely p21WAF1/CIP1 and p16INK4a are mechanosensitive and are in-part regulated by myosin contractility through focal adhesion kinase (FAK)-ROCK signaling. Interestingly, at the protein level senescence-induced dermal fibroblasts on soft substrates (0.5 kPa) do not express p21WAF1/CIP1 and p16INK4a at comparable levels to induced cells on stiff substrates (4GPa). However, cells express CDKN1a, CDKN2a, and IL6 at the RNA level across both stiff and soft substrates. Moreover, when cells are transferred from soft to stiff substrates, senescent cells recover an elevated expression of p21WAF1/CIP1 and p16INK4a at levels comparable to senescence cells on stiff substrates, pointing to a mechanosensitive regulation of the senescence phenotype. Together, our results indicate that the emergent senescence phenotype depends critically on the local mechanical environments of cells and that senescent cells actively respond to changing mechanical cues.

molecular biology↗

bioRxiv2023

INHBA/Activin A promotes tumor growth and induces resistance to anti-PD-L1 therapy by suppressing IFN-γ signaling

Inhibin beta A (INHBA) and its homodimer activin A have pleiotropic effects on modulation of immune responses and tumor progression, respectively, but it remains uncertain whether tumors may release activin A to regulate anti-tumor immunity. As evidenced by our RNA-Seq and in vitro results, the interferon-{gamma} (IFN-{gamma}) signaling pathway was significantly down-regulated by tumor intrinsic activin A. Tumor INHBA deficiency led to lower expression of PD-L1 induced by IFN-{gamma}, resulting in poor responsiveness to anti-PD-L1 therapy. On the other hand, decreased secretion of IFN-{gamma}-stimulated chemokines, including C-X-C motif chemokine 9 (CXCL9) and 10 (CXCL10), impaired the infiltration of effector T cells into the tumor microenvironment. Furthermore, the activin A-specific antibody garetosmab improved anti-tumor immunity and its combination with the anti-PD-L1 antibody atezolizumab showed a superior therapeutic effect to monotherapy. Our findings reveal that INHBA/activin A is involved in anti-tumor immunity by inhibiting the IFN-{gamma} signaling pathway and considered to be a potential target to overcome anti-PD-L1 resistance in clinical cancer treatment.

immunology↗

bioRxiv2023

Multifrequency control of Faraday wave bioassembly for constructing multiscale hPSC-derived neuronal networks

Bioassembly is recently regarded as a critical alternative biofabrication technical route to bioprinting since it can directly manipulate millions of live cells to form multicellular structures with close intercellular proximity, improving contact-dependent cell communication and promoting the emergence of tissue-specific functions. However, acoustic bioassembly techniques are currently limited to generating cytoarchitecture with a single characteristic length which cannot faithfully mimic the multiscale cellular structures in native tissues. To overcome this challenge, herein we report a novel acoustic bioassembly technique that employs multifrequency control of Faraday waves to form multiscale cellular structures. By superimposing multiple sine wave signals with proper amplitude ratios, Faraday waves containing multiple wavelengths can be induced and enabled to generate multiscale structures in few seconds. Using this technique, we construct functional neuronal networks with multiscale connectivity that display spontaneous neuroelectrical activities. We anticipate this technique will find wide applications in tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2023

Development of attenuated coxsackievirus B3 vectored intranasal pre-emptive pan-coronavirus vaccine

SARS-CoV-2 has the ability to evade immunity, resulting in breakthrough infections even after vaccination. Similarly, zoonotic coronaviruses pose a risk of spillover to humans. There is an urgent need to develop a pre-emptive pan-coronavirus vaccine that can induce systemic and mucosal immunity. Here, we employed a combination of immune-informatics approaches to identify shared immunodominant linear B- and T-cell epitopes from SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs), as well as zoonotic coronaviruses. Epitope-guided vaccine were designed and the attenuated coxsackievirus B3 vectored intranasal vaccines rCVB3-EPI and rCVB3-RBD-trimer were constructed. The immunogenicity of these candidate vaccines was evaluated using Balb/c mice. The results demonstrated effective immune responses, including the production of SARS-CoV-2-specific IgG and sIgA antibodies, as well as T cell-mediated responses. However, further verification is required to assess cross-reactivity with various variants. Our intranasal pre-emptive pan-coronavirus vaccine design framework offers an appealing candidate for future vaccine development.

microbiology↗

bioRxiv2023

Calcium Sulfate Microparticle Size Modification for Improved Alginate Hydrogel Fabrication and Its Application in 3D Cell Culture

Calcium ion-crosslinked alginate hydrogels are widely used as a materials system for investigating cell behavior in 3D environments in vitro. Suspensions of calcium sulfate particles are often used as the source of Ca2+ to control the rate of gelation. However, the instability of calcium sulfate suspensions can increase chances of reduced homogeneity of the resulting gel and requires researchers proficiency. Here, we show that ball-milled calcium sulfate microparticles with smaller sizes can create more stable crosslinker suspensions than unprocessed or simply autoclaved calcium sulfate particles. In particular, 15 {micro}m ball-milled calcium sulfate microparticles result in gels that are more homogeneous with a balanced gelation rate, which facilitates fabrication of gels with consistent mechanical properties and reliable performance for 3D cell culture. Overall, these microparticles represent an improved method for alginate hydrogel fabrication that can increase experimental reliability and quality for 3D cell culture.

bioengineering↗

bioRxiv2023

Picomolar-Level Sensing of Cannabidiol by Metal Nanoparticles Functionalized with Chemically Induced Dimerization Binders

Simple and fast detection of small molecules is critical to health and environmental monitoring. Methods for chemical detection often use mass spectrometers or enzymes; the former relies on expensive equipment and the latter is limited to those that can act as enzyme substrates. Affinity reagents like antibodies can target a variety of small-molecule analytes, but the detection requires successful design of chemically conjugated targets or analogs for competitive binding assays. Here, we developed a generalizable method for highly sensitive and specific in-solution detection of small molecules, using cannabidiol (CBD) as an example. Our sensing platform uses gold nanoparticles (AuNPs) functionalized with a pair of chemically induced dimerization (CID) nanobody binders (nano-binders), where CID triggers AuNPs aggregation and sedimentation in the presence of CBD. Despite moderate binding affinities of the two nano-binders to CBD (KDs of [~]6 and [~]56 {micro}M), a scheme consisting of CBD-AuNP pre-analytical incubation, centrifugation, and electronic detection (ICED) was devised to demonstrate a high sensitivity (limit of detection of [~]100 picomolar) in urine and saliva, a relatively short assay time ([~]2 hours), a large dynamic range (5 logs), and a sufficiently high specificity to differentiate CBD from its analog, tetrahydrocannabinol. The high sensing performance was achieved with the multivalency of AuNP sensing, the ICED scheme that increases analyte concentrations in a small assay volume, and a portable electronic detector. This sensing system is readily coupled to other binders for wide molecular diagnostic applications.

bioengineering↗

bioRxiv2023

ccTCM: a quantitative component and compound platform for promoting the research of traditional Chinese medicine

Traditional Chinese medicine (TCM) databases play a vital role in bridging the gap between TCM and modern medicine, as well as in promoting the popularity of TCM. Elucidating the bioactive ingredients of Chinese medicinal materials is key to TCM modernization and new drug discovery. However, one drawback of current TCM databases is the lack of quantitative data on the constituents of Chinese medicinal materials. Herein, we present ccTCM, a web-based platform designed to provide a component and compound-content-based resource on TCM and analysis services for medical experts. In terms of design features, ccTCM combines resource distribution, similarity analysis, and molecular-mechanism analysis to accelerate the discovery of bioactive ingredients in TCM. ccTCM contains 273 Chinese medicinal materials commonly used in clinical settings, covering 29 functional classifications. By searching and comparing, we finally adopted 2043 studies, from which we collected the compounds contained in each TCM with content greater than 0.001%, and a total of 1 449 were extracted. Subsequently, we collected 40767 compound-target pairs by integrating multiple databases. Taken together, ccTCM is a versatile platform for that can be used by TCM scientists to perform scientific and clinical TCM studies based on quantified ingredients of Chinese medicinal materials. ccTCM is freely accessible at http://www.cctcm.org.cn.

bioinformatics↗

bioRxiv2023

Small extracellular vesicles promote stiffness-mediated metastasis

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiological matrix stiffness affects the quantity and protein cargo of small EVs produced by cancer cells, which in turn drive their metastasis. Primary patient breast tissue produces significantly more EVs from stiff tumor tissue than soft tumor adjacent tissue. EVs released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITG2{beta}1, ITG6{beta}4, ITG6{beta}1, CD44) compared to EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix (ECM) protein collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination through enhanced chemotaxis. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer associated fibroblast (CAF) phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/545937v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1be1dbdorg.highwire.dtl.DTLVardef@928710org.highwire.dtl.DTLVardef@1e14e9borg.highwire.dtl.DTLVardef@efdb7f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Toward single cell tattoos: Biotransfer printing of lithographic gold nanopatterns on live cells

Lithographic nanopatterning techniques like photolithography, electron-beam lithography, and nanoimprint lithography (NIL) have revolutionized modern-day electronics and optics. Yet, their application for creating nano-bio interfaces is limited by the cytotoxic and two-dimensional nature of conventional fabrication methods. Here, we present a biocompatible and cost-effective transfer process that leverages (a) NIL to define sub-300 nm gold (Au) nanopattern arrays, (b) amine functionalization of Au to transfer the NIL-arrays from a rigid substrate to a soft transfer layer, (c) alginate hydrogel as a flexible, degradable transfer layer, and (d) gelatin conjugation of the Au NIL-arrays to achieve conformal contact with live cells. We demonstrate biotransfer printing of the Au NIL-arrays on rat brains and live cells with high pattern fidelity and cell viability and observed differences in cell migration on the Au NIL-dot and NIL-wire printed hydrogels. We anticipate that this nanolithography-compatible biotransfer printing method could advance bionics, biosensing, and biohybrid tissue interfaces. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/542285v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@16f6e1corg.highwire.dtl.DTLVardef@a60bacorg.highwire.dtl.DTLVardef@1485d06org.highwire.dtl.DTLVardef@1d365c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Concurrent measurement of O2 and isoprene production during photosynthesis: pros, cons, and metabolic implications

During oxygenic photosynthesis, oxygen (O2) is generated from water photolysis, which provides reducing power to sustain CO2 assimilation. To date, traditional leaf gas-exchange experiments have been focused on net CO2 exchange (Anet), with limited observations of net oxygen production (NOP). Here, we present the first gas-exchange/fluorescence system, coupling CO2/H2O analysis (photosynthesis and transpiration) with NOP and isoprene emission measurements. This configuration allowed us to calculate the assimilatory quotient (AQ = Anet/NOP) and thus obtain a more complete picture of the photosynthetic redox budget via photosynthetic production of O2, electron transport rate (ETR), and isoprene biosynthesis. We used cottonwood leaves (Populus trichocarpa) and carried out response curves to light, CO2 and temperature along with 18O-labelling with 18O-enriched water. We found that Anet and NOP were linearly correlated across environmental variables with AQ of 1.27 +/- 0.12 regardless of light, CO2, and temperature. Anet and NOP had optimal temperatures (Topt) of 31{degrees}C, while ETR (35{degrees}C) and isoprene emissions (39{degrees}C) had distinctly higher Topt. Leaves labelled with H218O produced labeled (18O16O) oxygen with the same Topt as ETR (35{degrees}C). The results confirm a tight connection between water oxidation and ETR and are consistent with a suppression of Anet and NOP at high temperature driven by an acceleration of (photo)respiration. The findings support the view of isoprene biosynthesis primarily driven by excess photosynthetic ATP/NADPH not consumed by the Calvin cycle during photorespiratory conditions as an important thermotolerance mechanism linked with high rates of CO2 and O2 recycling. KeywordsPhotosynthesis, net oxygen production, gross oxygen production, H218O labeling One sentence summaryA leaf gas-exchange system is presented enabling a more complete picture of the photosynthetic redox budget and calculation of the assimilatory quotient.

plant biology↗

bioRxiv2023

Neural Mechanism Underlying NEergic Neurons-Modulated The Arousal Associated With Midazolam Anesthesia

The advent of midazolam holds profound implications for modern clinical practice. The hypnotic and sedative effects of midazolam afford it broad clinical applicability. However, the specific mechanisms underlying the modulation of altered consciousness by midazolam remain elusive. Herein, using pharmacology, optogenetics, chemogenetics, fiber photometry, and gene knockdown, we revealed the role of locus coeruleus (LC)-ventrolateral preoptic nucleus (VLPO) noradrenergic neural circuit in regulating midazolam-induced altered consciousness. This effect was mediated by 1 adrenergic receptors. Moreover, gamma-aminobutyric acid receptor type A (GABAA-R) represents a mechanistically crucial binding site in the LC for midazolam. Our findings will provide novel insights into the neural circuit mechanisms underlying the recovery of consciousness after midazolam administration and will help guide the timing of clinical dosing and propose effective intervention targets for timely recovery from midazolam-induced loss of consciousness.

neuroscience↗

bioRxiv2023

Angiotensin II receptor inhibition ameliorates liver fibrosis and enhances hepatocellular carcinoma infiltration by effector T cells

Although viral hepatocellular carcinoma (HCC) is declining, non-viral HCC, which often is the end-stage of non-alcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICI) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a small portion of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell mediated tumor regression and postulated that anti-fibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1 induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8+ T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with inhibition of TGF-{beta} receptor signaling, collagen deposition and depletion of immunosuppressive fibroblasts. SignificanceImmune checkpoint inhibitors are used in HCC treatment but overall response rates for single agent PD-1/PD-L1 blockers have remained stubbornly low. Using a mouse model of NASH-driven HCC, we show that co-treatment with the safe and inexpensive angiotensin II receptor inhibitor losartan substantially enhanced anti-PD-1 triggered HCC regression. Although losartan did not influence the reinvigoration of exhausted CD8+ T cells it considerably enhanced their intratumoral invasion, which we postulated to be compromised by peritumoral fibrosis. Indeed, the beneficial effect of losartan correlated with inhibition of TGF-{beta} signaling and collagen deposition, and depletion of immunosuppressive fibroblasts. Losartan should be evaluated for its adjuvant activity in HCC patients undergoing PD-1/PD-L1 blocking therapy.

cancer biology↗

bioRxiv2023

FBP1 is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness

Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is not clear. Fructose-1,6 bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia in affected individuals. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin-dependent. FBP1 prevents insulin hyperresponsiveness, independently of its catalytic activity, by interacting with AKT, PP2A-C and Aldolase-B (ALDOB) to specifically accelerate AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:AKT:PP2A-C:ALDOB complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, a complex disrupting peptide reverses diet-induced insulin resistance.

molecular biology↗

bioRxiv2023

Arginine shortage induces replication stress and confers genotoxic resistance by inhibiting histone H4 translation and promoting PCNA polyubiquitination

The unique arginine dependencies of cancer cell proliferation and survival creates metabolic vulnerability. Here, we investigate the impact of extracellular arginine availability on DNA replication and genotoxic resistance. Using DNA combing assays, we find that when extracellular arginine is limited, cancer cells are arrested at S-phase and DNA replication forks slow or stall instantly until arginine is re-supplied. The translation of new histone H4 is arginine-dependent and impacts DNA replication and the expression of newly synthesized histone H4 is reduced in the avascular nutrient-poor breast cancer xenograft tumor cores. Furthermore, we demonstrate that increased PCNA occupancy and HLTF-catalyzed PCNA K63-linked polyubiquitination protects arginine-starved cells from hydroxyurea-induced, DNA2-catalyzed nascent strand degradation. Finally, arginine-deprived cancer cells are tolerant to genotoxic insults in a PCNA K63-linked polyubiquitination-dependent manner. Together, these findings reveal that extracellular arginine is the "linchpin" for nutrient-regulated DNA replication. Such information could be leveraged to expand current modalities or design new drug targets against cancer.

cancer biology↗

bioRxiv2023

Ultrasensitive Proteomics Depicted an In-depth Landscape for Mouse Embryo

In recent years, single-cell or low-input multi-omics techniques have brought a revolution in the study of pre-implantation embryo development. However, single-cell or low-input proteome research in this field is relatively underdeveloped, due to the limited source of mammalian embryo samples, the objective reality of high abundance zona pellucida proteins, and the lack of hypersensitive proteome technology. Here, a comprehensive solution of ultrasensitive proteome technology was developed for single-cell or low-input mouse embryos. Both deep coverage route and high-throughput route could significantly reduce the starting material and enhance the proteomic depth without any customized instrument. Using the deep coverage route, an average of 2,665 or 4,585 protein groups can be identified from 1 or 20 mouse zygotes respectively. Using the high-throughput route, 300 single mouse zygotes can be analysis in 8 days with an average of 2,371 proteins identified. With its popularization, we believe researchers can choose deep coverage or high-throughput technology routes according to their own conditions.

systems biology↗