Biology subjects

Ouyang, L.

Publications and source records attributed to Ouyang, L..

10 recordsLinked to original sources

bioRxiv2026

Excluded Volume Effect-induced Shrinkage for Resolution Enhancement of Hydrogel Bioconstructs

Biofabrication of hydrogel materials is usually constrained by process-specific resolution limits. Here, we introduce excluded volume effect-induced shrinkage (EVER), a dehydration-enabled resolution enhancement strategy that achieves dimensional refinement via post-processing. In this approach, we treat biofabricated constructs with hydrophilic macromolecular crowding agent bath, with poloxamer as a representative. With EVER treatment, gelatin-based constructs can reach a dimension reduction of up to ~49% with an estimated volumetric reduction of ~87%. Shape fidelity was well retained after shrinkage across different constructs and could be stabilized by introducing secondary crosslinking. In extrusion bioprinted cell-laden constructs, myoblasts retained a considerable cell viability right after the shrinkage treatment and recovered significantly by day 1, with subsequent cell spreading and increasing proliferation over 7 days. Without the need of continued immersion in the shrinkage bath, EVER provides a hardware-independent route for refining hydrogel structures while retaining encapsulated cell compatibility.

bioengineering↗

bioRxiv2024

Microfiber-Templated Porogel Bioinks Enable Tubular Interfaces and Microvascularization Down to the Building Blocks for 3D Bioprinting

Vascularization is key to the biofabrication of large-scale tissues. Despite the progress, there remain some outstanding challenges, such as limited vessel density, difficulty in fabricating microvasculatures, and inhomogeneity of post-seeding cells. Here, we introduce a new form of bioink called microfiber-templated porogel (FTP) bioink to engineer vasculatures down to the filament building blocks of 3D bioprinted hydrogels. The cell-laden sacrificial microfibers (diameter ranges from 50-150 m) are embedded in the bioink to template tubular voids and deliver endothelial cells for in-situ endothelialization. The inclusion of softening hydrogel microfibers retains the desirable rheological properties of the bioink for extrusion-based bioprinting. Such bioinks can be printed into a well-defined 3D structure with tunable tubular porosities up to 55%. Compared to the conventional bulk bioink counterpart, the FTP bioink supports the significant growth and spread of endothelial cells either embedded in the matrix or sacrificial fibers, free of the post-cell seeding procedure. Furthermore, the bioprinted scaffolds based on FTP bioink are seen to significantly promote the in-growth of blood vessels and native tissues in vivo. The FTP bioink approach enables the engineering of tubular bio-interfaces within the building blocks and contributes to the in-situ endothelialization of microvasculatures, providing a versatile tool for the construction of customized vascularized tissue models.

bioengineering↗

bioRxiv2023

A multistage, dual voxel study of glutamate in the anterior cingulate cortex in schizophrenia supports a primary pyramidal dysfunction model of disorganization

BackgroundSchizophrenia is an illness where glutamatergic dysfunction in the anterior cingulate cortex (ACC) has been long suspected; Recent in vivo evidence (Adams et al. 2022) has implicated pyramidal dysfunction (reduced glutamate tone) as the primary pathophysiology contributing to subtle features, with a secondary disinhibition effect (higher glutamate tone) resulting in the later emergence of prominent clinical symptoms. We investigate if genetic high risk (GHR) for schizophrenia reduces glutamatergic tone in ACC when compared to the states of clinical high risk (CHR) and first episode schizophrenia (FES) where symptoms are already prominent. MethodsWe recruited 302 individuals across multiple stages of psychosis (CHR, n=63; GHR, n=76; FES, n=96) and healthy controls (n=67) and obtained proton magnetic resonance spectroscopy of glutamate from perigenual ACC (pACC) and dorsal ACC (dACC) using 3-Tesla scanner. ResultsGHR had lower Glu compared to CHR while CHR had higher Glu compared to FES and HC. Higher disorganization burden, but not any other symptom domain, was predicted by lower levels of Glu in the GHR group (dACC and pACC) and in the CHR group (pACC only). ConclusionsThe reduction in glutamatergic tone in GHR supports the case for a pyramidal dysfunction contributing to higher disorganization, indicating disorganization to be the core domain in the pathophysiology of schizophrenia. Higher glutamate (likely due to disinhibition) is apparent when psychotic symptoms are raising to be prominent (CHR), though at the full-blown stage of psychosis, the relationship between glutamate and symptoms ceases to be a simple linear one.

neuroscience↗

bioRxiv2022

Protein Language Model Predicts Mutation Pathogenicity and Clinical Prognosis

Accurately predicting the effects of mutations in cancer has the potential to improve existing treatments and identify novel therapeutic targets. In this paper, we evidence for the first time that the large-scale pre-trained protein language models (PPLMs) are zero-shot predictors for two clinically relevant tasks: identifying diseasecausing mutations and predicting patient survival rate. Then we benchmark a series of state-of-the-art (SOTA) PPLMs on 2279 protein variants across 20 cancer-related genes. Our empirical results show that the PPLMs outperform the SOTA baseline, EVE [1], trained on multiple sequence alignment (MSA) data. We also demonstrate that the evolutionary index score, generated from the PPLMs softmax layer, is good indicator for both mutation pathogenicity and patient survival rate. Our paper has taken a key step toward the clinical utility of large-scale PPLMs.

bioinformatics↗

bioRxiv2021

A new role of low barrier hydrogen bond in mediating protein stability by small molecules

Low barrier hydrogen bond (LBHB) is a special type of hydrogen bond which occurs where two heteroatoms with similar pKa values share a single proton resulting in an unusually strong and short hydrogen bond. LBHBs in protein play important roles in enzyme catalysis and maintaining protein structural integrity but its other biochemical roles are unknown. Here we report a novel function of LBHB in selectively inducing tubulin protein degradation. A tubulin inhibitor, 3-(3-Phenoxybenzyl) amino-{beta}-carboline (PAC), promotes selective degradation of {beta}-tubulin heterodimers by binding to the colchicine site of {beta}-tubulin. Biochemical studies have revealed that PAC specifically destabilizes tubulin, making it prone to aggregation that then predisposes it to ubiquitinylation and then degradation. Structural activity analyses have indicated that the destabilization is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and {beta}Glu198, which is identified as a LBHB. In contrast, another two tubulin inhibitors only forming normal hydrogen bonds with {beta}Glu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. Most importantly, we screened for compounds capable of forming LBHB with {beta}Glu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimers degradation in a PAC-like manner as expected. Our study has identified a novel approach for designing tubulin degraders, providing a unique example of LBHB function and suggests that designing small molecules to form LBHBs with protein residues resulting in the highly specific degradation of a target protein could be a new strategy for drug development.

biochemistry↗

bioRxiv2020

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin and provides insights into microtubule dynamic instability

Microtubule, composed of {beta}-tubulin heterodimers, remains as one of the most popular anticancer targets for decades. To date, anti-microtubule drugs are mainly functionally divided into microtubule-destabilizing and microtubule-stabilizing agents while microtubule- or tubulin-degradation agents are rarely reported. Six known binding sites on tubulin dimer are identified with five sites on {beta}-tubulin and only one site on -tubulin (pironetin site), hinting compounds binding to -tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our X-ray crystallography study reveals that, in addition binding to the vinblastine site, cevipabulin also binds to a novel site on -tubulin (named the seventh site) which located near the nonexchangeable GTP. Interestingly, we find the binding of cevipabulin to the seventh site induces tubulin degradation. As the non-exchangeable GTP has structural role and is important for the stability of tubulin dimers, we propose and confirm the tubulin degradation mechanism as: Cevipabulin at the seventh site puts the T5 loop outward to make the non-exchangeable GTP exchangeable, which reduces the stability of tubulin and results in its destabilization and degradation. Our results confirm a novel agent binding site on -tubulin and shed light on the development of tubulin degraders as a new generation of anti-microtubule drugs targeting this novel site.

cell biology↗

bioRxiv2020

Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p

Clinical use of antimicrobials faces great challenges from the emergence of multidrug resistant (MDR) pathogens. The overexpression of drug efflux pumps is one of the major contributors to MDR. It is considered as a promising approach to overcome MDR by reversing the function of drug efflux pumps. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to multidrug resistance. Here we report a counterintuitive case of reversing multidrug resistance in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria, and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. It results in the selective elimination of Mdr1p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time (MST) of mice with a blood-borne dissemination of Mdr1p overexpressed multidrug resistant candidiasis. This study provided a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.

microbiology↗

bioRxiv2020

Simple cloning of large natural product biosynthetic gene cluster by CRISPR/Cas12a-mediated fast direct capturing strategy

Directly cloning of biosynthetic gene clusters (BGCs) from microbial genomes has been revolutionizing the natural product-based drug discovery. However, it is still very challenging to efficiently clone, for example, large (> 80kb) and GC-rich (> 70%), streptomycete originating BGCs. In this study, we developed a simple, fast yet efficient and low-cost in vitro platform for direct cloning large BGCs from streptomycete genomic DNA, named as CAT-FISHING (CRISPR/Cas12a- and Agarose plug-based sysTem for Fast bIoSyntHetIc geNe cluster cloninG), by combining the advantages of CRISPR/Cas12a cleavage and bacterial artificial chromosome (BAC) library construction. CAT-FISHING was demonstrated by directly cloning large DNA fragments ranging from 47 to 139 kb with GC content of > 70% from the S. albus J1074 genome in a relatively efficient manner. Moreover, surugamides, encoded by a captured 87-kb BGC with GC content of 76%, was heterologously expressed in a Streptomyces chassis. These results indicate that CAT-FISHING is a powerful platform for BGCs batch cloning, which would be greatly beneficial to the natural products-based drug discovery. We believe that this system will lead a renaissance of interest in microorganisms as a source for drug development.

synthetic biology↗

bioRxiv2018

Microbial Cells Harboring a Mitochondrial Gene Are Capable of CO2 Capture

Global warming is escalating with increased temperatures reported worldwide. Given the enormous land mass on the planet, biological capture of CO2 remains a viable approach to mitigate the crisis as it is economical and easy to implement. In this study, a gene capable of CO2 capture was identified via selection in minimal media. This mitochondrial gene named as OG1 encodes the OK/SW-CL.16 protein and shares homology with cytochrome oxidase subunit III of various species and PII uridylyl-transferase from Loktanella vestfoldensis SKA53. CO2 capture experiments indicate that {delta}13C was substantially higher in the cells harboring the gene OG1 than the control in the nutrition-poor media. This study suggests that CO2 capture using engineered microorganisms in barren land can be exploited to address the soaring CO2 level in the atmosphere, opening up vast land resources to cope with global warming.\n\nIMPORTANCEGlobal warming crisis is deteriorating with increased CO2 levels in the atmosphere each year. Action must be taken before catastrophic consequences occur in the not-so-distant future. Biological capture of CO2 is a feasible approach to alleviate the current crisis. We have identified a mitochondrial gene which demonstrated CO2 utilization capability. Data presented in this study suggest that CO2 capture using engineered microorganisms can be harnessed to address the ever-rising CO2 level in the atmosphere.

bioengineering↗

bioRxiv2018

Metabolomics and proteomics analyses of grain yield reduction in rice under abrupt drought-flood alternation

HighlightAbrupt drought-flood alteration is a frequent meteorological disaster that occurs during summer in southern China and the Yangtze river basin, which often causes a large area reduction of rice yield. We previously reported abrupt drought-flood alteration effects on yield and its components, physiological characteristics, matter accumulation and translocation, rice quality of rice. However, the molecular mechanism of rice yield reduction caused by abrupt drought-flood alternation has not been reported.\n\nIn this study, four treatments were provided, no drought and no floods (control), drought without floods (duration of drought 10 d), no drought with floods (duration of floods 8 d), and abrupt drought-flood alteration (duration of drought 10 d and floods 8 d). The quantitative analysis of spike metabolites was proceeded by LC-MS (liquid chromatograph-mass spectrometry) firstly. Then the Heat-map, PCA, PLS-DA, OPLS-DA and response ranking test of OPLS-DA model methods were used to analysis the function of differential metabolites (DMs) during the rice panicle differentiation stage under abrupt drought-flood alteration. In addition, relative quantitative analysis of spike total proteins under the treatment was conducted iTRAQ (isobaric tags for relative and absolute quantification) and LC-MS. In this study, 5708 proteins were identified and 4803 proteins were quantified. The identification and analysis of DEPs function suggested that abrupt drought-flood alteration treatment can promote carbohydrate metabolic, stress response, oxidation-reduction, defense response, and energy reserve metabolic process, etc, during panicle differentiation stage. In this study relative quantitative proteomics, metabolomics and physiology data (soluble protein content, superoxide dismutase activity, hydrogen peroxidase activity, peroxidase activity, malondialdehyde content, free proline content, soluble sugar content and net photosynthetic rate) analysis were applied to explicit the response mechanism of rice panicle differentiation stage under abrupt drought-flood alteration and provides a theoretical basis for the disaster prevention and mitigation.\n\nAbstractAbrupt drought-flood alternation is a meteorological disaster that frequently occurs during summer in southern China and the Yangtze river basin, often causing a significant loss of rice production. In this study, a quantitative analysis of spike metabolites was conducted via liquid chromatograph-mass spectrometry (LC-MS), and Heat-map, PCA, PLS-DA, OPLS-DA, and a response ranking test of OPLS-DA model methods were used to analyze functions of differential metabolites (DMs) during the rice panicle differentiation stage under abrupt drought-flood alternation. The results showed that 102 DMs were identified from the rice spike between T1 (abrupt drought-flood alternation) and CK0 (control) treatment, 104 DMs were identified between T1 and CK1 (drought) treatment and 116 DMs were identified between T1 and CK2 (flood) treatment. In addition, a relative quantitative analysis of spike total proteins was conducted using isobaric tags for relative and absolute quantification (iTRAQ) and LC-MS. The identification and analysis of DEPs functions indicates that abrupt drought-flood alternation treatment can promote carbohydrate metabolic, stress response, oxidation-reduction, defense response, and energy reserve metabolic process during the panicle differentiation stage. In this study, relative quantitative metabolomics and proteomics analyses were applied to explore the response mechanism of rice panicle differentiation in response to abrupt drought-flood alternation.\n\nAbbreviations

plant biology↗