Biology subjects

Wang, J.

Publications and source records attributed to Wang, J..

At least 919 records · Page 51Linked to original sources

bioRxiv2024

Functional Connectivity Alterations in Cocaine Use Disorder: Insights from the Triple Network Model and the Addictions Neuroclinical Assessment Framework

Cocaine use disorder (CUD) disrupts functional connectivity within key brain networks, specifically the default mode network (DMN), salience network (SN), and central executive network (CEN). While the triple network model has been proposed to explain various psychiatric disorders, its applicability to CUD requires further exploration. In the present study, we built machine learning classifiers based on different combinations of DMN/SN/CEN to distinguish cocaine-use disorder (CUD) subjects from healthy control (HC) subjects. Among them, the combination of the SN and the CEN results in a remarkably high accuracy of 73.4% (sensitivity/specificity: 69.6%/78.6%, AUC: 0.78), outperforming the model based on the full triple network. This supports the hypothesis that during the binge/intoxication stage of addiction, the SN and the CEN play a more critical role than the DMN, consistent with the Addictions Neuroclinical Assessment (ANA) framework. Functional connectivity analysis revealed decreased connectivity within the DMN and the SN and increased connectivity within the CEN in CUD patients, suggesting that alterations in these networks could serve as biomarkers for addiction severity.

neuroscience↗

bioRxiv2024

Diverse quorum sensing systems regulate microbial communication and biogeochemical processes in deep-sea cold seeps

Quorum sensing is a fundamental chemical communication mechanism that enables microorganisms to coordinate behavior and adapt to environmental conditions. In deep-sea cold seep ecosystems, where diverse microbial communities thrive, quorum sensing is likely a key mechanism driving microbial interactions. However, the distribution, mechanisms, and ecological roles of quorum sensing in cold seeps remain poorly understood. To address this, we analyzed 173 metagenomes, 33 metatranscriptomes, and 18 metabolomes from 17 global cold seep sites. We identified 299,355 quorum sensing genes from the seep non-redundant gene catalog, representing 34 gene types across six quorum sensing systems, with distribution patterns influenced by sediment depth and seep type. These quorum sensing genes were present in 3,576 metagenome-assembled genomes from 12 archaeal and 108 bacterial phyla, revealing a complex network of intraspecies and interspecies communication. Microbial groups involved in key metabolic processes, such as sulfate-reducing bacteria, anaerobic methanotrophic archaea, diazotrophs and organohalide reducers, were extensively regulated by quorum sensing, influencing biogeochemical cycles of carbon, nitrogen, and sulfur. Phylogenetic analysis and protein domain identification highlighted the functional roles of key quorum sensing-related proteins (e.g., PDEs, CahR, RpfC/G and LuxR) in modulating microbial behaviors, such as motility and chemotaxis. Heterologous expression and metabolomic profiling further confirmed the activity of representative LuxI-R pairs and identified inhibitors of quorum sensing in cold seep sediments. Overall, these findings highlight complexity and significance of quorum sensing in microbial interactions, ecological adaptation, and biogeochemical cycling within cold seep ecosystems, advancing our understanding of microbial communication in the deep biosphere.

microbiology↗

bioRxiv2024

scCompass: An integrated cross-species scRNA-seq database for AI-ready

Emerging single-cell sequencing technology has generated large amounts of data, allowing analysis of cellular dynamics and gene regulation at the single-cell resolution. Advances in artificial intelligence enhance life sciences research by delivering critical insights and optimizing data analysis processes. However, inconsistent data processing quality and standards remain to be a major challenge. Here we propose scCompass, which provides a data quality solution to build a large-scale, cross-species and model-friendly single-cell data collection. By applying standardized data pre-processing, scCompass integrates and curates transcriptomic data from 13 species and nearly 105 million single cells. Using this extensive dataset, we are able to archieve stable expression genes (SEGs) and organ-specific expression genes (OSGs) in human and mouse. We provide different scalable datasets that can be easily adapted for AI model training and the pretrained checkpoints with state-of-the-art (SOTA) single-cell foundataion models. In summary, the AI-readiness of scCompass, which combined with user-friendly data sharing, visualization and online analysis, greatly simplifies data access and exploitation for researchers in single cell biology(http://www.bdbe.cn/kun).

bioinformatics↗

bioRxiv2024

A Multi-Property Optimizing Generative Adversarial Network for de novo Antimicrobial Peptide Design

Antimicrobial peptides (AMPs) play a crucial role in developing novel antiinfective drugs due to their broad-spectrum antimicrobial activity and lower likelihood of causing bacterial resistance. However, laboratory synthesis of AMPs is tedious and time-consuming. Existing computational methods have limited capability in optimizing multiple desired properties simultaneously. Here, we propose a Multi-Property Optimizing Generative Adversarial Network (MPOGAN), a feedback-loop framework that iteratively learns from data with multiple desired properties. This approach enables de novo design of AMPs with potent antimicrobial activity, reduced cytotoxicity, and diversity. Through extensive computational tests, MPOGAN exhibits superior performance in optimizing multiple desired properties of generated AMPs. Ten of the most promising candidates are chemically synthesized, with nine showing potent antimicrobial activity against three bacterial strains and low cytotoxicity against eukaryotic cells. MPOGAN offers a powerful computational approach for effective multi-property optimization of AMPs, thereby advancing the field of AI-aided drug discovery.

bioinformatics↗

bioRxiv2024

Characterization of FLOWERING LOCUS T related genes and their putative gene regulatory network in semi-winter Brassica napus cultivar Zhongshaung11

In many species, FLOWERING LOCUS T (FT)-like genes promote the floral transition by integrating environmental signals, in particular photoperiod, and internal cues. Here we show that Brassica napus contains 6 FT-like genes and 2 pseudogenes belonging to 3 orthogroups. All B. napus FT-like genes induce early flowering when expressed at the shoot apical meristems of Arabidopsis thaliana ft mutants, but BnaFT.C6 and non-orthologous FT-like genes do not encode fully functional mobile florigens. In the case of BnFT.C6, the functional change is associated with a T to C amino acid change that is restricted to semi-winter accessions. Expression of orthologs of FT is photoperiod-dependent and two distal enhancers are conserved; however, the homeologs BnaFT.A7 and BnaFT.C6 show rearrangements of DNA motifs binding NF-Y/CO and NF-Y transcriptional activator complexes between the promoter and downstream enhancers. Motif rearrangements correlate with differences in tissue-specific expression. Furthermore, homeologs with rearranged motifs could not be trans-activated by B. napus CO in transient assays although they genes show LD photoperiod-dependent expression. We propose that differential diurnal expression of NF-Y genes contributes to the photoperiod-dependent regulation of B. napus FT genes. Significance statementB. napus FT-like genes are a case study for gene retention and retraction after multiple rounds of polyploidisation. Retained B. napus FT-like genes show preferred expression in different developmental stages and amino-acid changes that affect florigen function.

plant biology↗

bioRxiv2024

Targeting IL-1/IRAK1/4 signaling in Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

Therapies for acute myeloid leukemia (AML) face formidable challenges due to relapse, often driven by leukemia stem cells (LSCs). Strategies targeting LSCs hold promise for enhancing outcomes, yet paired comparisons of functionally defined LSCs at diagnosis and relapse remain underexplored. We present transcriptome analyses of functionally defined LSC populations at diagnosis and relapse, revealing significant alterations in IL-1 signaling. Interleukin-1 receptor type I (IL1R1) and interleukin-1 receptor accessory protein (IL1RAP) were notably upregulated in leukemia stem and progenitor cells at both diagnosis and relapse. Knockdown of IL1R1 and IL1RAP reduced the clonogenicity and/or engraftment of primary human AML cells. In leukemic MLL-AF9 mice, Il1r1 knockout reduced LSC frequency and extended survival. To target IL-1 signaling at both diagnosis and relapse, we developed UR241-2, a novel interleukin-1 receptor-associated kinase 1 and 4 (IRAK1/4) inhibitor. UR241-2 robustly suppressed IL-1/IRAK1/4 signaling, including NF-{kappa}B activation and phosphorylation of p65 and p38, following IL-1 stimulation. UR241-2 selectively inhibited LSC clonogenicity in primary human AML cells at both diagnosis and relapse, while sparing normal hematopoietic stem and progenitor cells. It also reduced AML engraftment in leukemic mice. Our findings highlight the therapeutic potential of UR241-2 in targeting IL-1/IRAK1/4 signaling to eradicate LSCs and improve AML outcomes.

cancer biology↗

bioRxiv2024

DeFrND: detergent-free reconstitution into native nanodiscs with designer membrane scaffold peptides

Membrane scaffold proteins-based nanodiscs (NDs) have facilitated unprecedented structural and biophysical analysis of membrane proteins in a near-native lipid environment. However, successful reconstitution of membrane proteins in NDs requires prior solubilization and purification in detergents, which may impact their physiological structure and function. Furthermore, the detergent-mediated reconstitution of NDs is unlikely to recapitulate the precise composition or asymmetry of native membranes. To circumvent this fundamental limitation of traditional ND technology, we herein describe the development of membrane-solubilizing peptides to directly extract membrane proteins from native cell membranes into nanoscale discoids. By systematically protein engineering and screening, we created a new class of chemically modified Apolipoprotein-A1 mimetic peptides to enable the formation of detergent-free NDs (DeFrNDs) with high efficiency. NDs generated with these engineered membrane scaffold peptides are suitable for obtaining high-resolution structures using single-particle cryo-EM with native lipids. To further highlight the versatility of DeFrNDs, we directly extract a sampling of membrane signaling proteins with their surrounding native membranes for biochemical and biophysical interrogations.

biophysics↗

bioRxiv2024

Aramchol attenuates fibrosis in mouse models of biliary fibrosis and blocks the TGFβ-induced fibroinflammatory mediators in cholangiocytes

BackgroundFibroinflammatory cholangiopathies, such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), are characterized by inflammation and biliary fibrosis, driving disease-related complications. In biliary fibrosis, cholangiocytes activated by transforming growth factor-{beta} (TGF{beta}) release signals that recruit immune cells to drive inflammation and activate hepatic myofibroblasts to deposit the extracellular matrix (ECM). TGF{beta} regulates stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the synthesis of monounsaturated fatty acids, in stimulating fibroinflammatory lipid signaling. However, the role of SCD or its inhibitor, Aramchol, has not been investigated in biliary fibrosis or TGF{beta}-mediated cholangiocyte activation. Method10-16-week-old multi-drug resistance 2 knockout (Mdr2-/-) and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) diet-fed mice were orally gavaged daily with Aramchol at 12.5 mg/kg/day for 4 and 3 weeks, respectively. Liver and serum were harvested for the assessment of fibrosis and inflammation. Transformed human cholangiocyte cells (H69) and mouse large biliary epithelial cells (MLEs) were used to test the effects of the SCD inhibitor, Aramchol, at varying doses on TGF{beta}-mediated expression of fibroinflammatory signals and were confirmed in PSC-derived cholangiocytes (PSC-Cs) using ELISA, qPCR, and Western blot analyses. ResultsAramchol treatment of Mdr2-/- mice with established biliary fibrosis (treatment) and DDC diet-induced (prevention) models of cholestatic injury and fibrosis demonstrated significant reductions in both measures of ECM synthesis (mRNA expression of ECM components in the liver), collagen content of the liver (picrosirius red staining and hydroxyproline content) and myofibroblast activation (SMA staining). Il6 and Tnfa were also reduced with Aramchol in the liver. RNA-seq analysis of H69 cells showed that Aramchol co-treatment led to significant inhibition of TGF{beta}-induced hepatic fibrosis pathways while upregulating peroxisome proliferator-activated receptor (PPAR) signaling. SCD expression was significantly increased in TGF{beta}-treated H69 cells (2-fold, p<0.05). Aramchol in a dose-dependent manner significantly attenuated the increased expression of the fibrotic marker, plasminogen activator inhibitor-1 (PAI-1/SERPINE1), and hepatic stellate cell-activating genes (VEGFA and PDGFB) in TGF{beta}-activated H69 and MLEs. Aramchol also markedly reduced the expression of the inflammatory cytokine, interleukin 6 (IL6). SCD siRNA knockdown produced similar results in H69 cells. Furthermore, in PSC-Cs, the expressions of SCD, VEGFA and IL6 were significantly reduced with Aramchol. The expression of the anti-fibroinflammatory factors PPAR and -{gamma} were modestly increased in cholangiocyte cell lines with increased expression of PPAR-responsive genes and increased nuclear binding of DNA PPAR response elements with Aramchol co-treatment compared to TGF{beta} only. ConclusionAramchol, an SCD inhibitor, both attenuates and prevents biliary fibrosis in mouse models of cholestatic injury and fibrosis. This effect is partially due to Aramchol inhibiting TGF{beta}-induced fibroinflammatory mediators in cholangiocytes by upregulating PPAR and -{gamma} expression and activity. These findings, along with Aramchols excellent safety profile in clinical trials, provide the rationale for assessing Aramchol in further clinical studies in patients with biliary fibrosis, particularly PSC, where a treatment is desperately needed.

cell biology↗

bioRxiv2024

Intraflagellar transport protein IFT172 contains a C-terminal ubiquitin-binding U-box-like domain involved in ciliary signaling

Intraflagellar transport (IFT) is a fundamental process driving ciliogenesis in most eukaryotic organisms. IFT172, the largest protein of the IFT complex, plays a crucial role in cilium formation and several disease-causing IFT172 variants have been identified in ciliopathy patients. While IFT172 is tethered to the IFT-B complex via its N-terminal domains, the function of its C-terminal domains has remained elusive. Here, we reveal that the C-terminal part of IFT172 interacts with IFT-A complex subunits, providing a molecular basis for the role of IFT172 in bridging IFT-A and IFT-B complexes. We determine the crystal structure of the C-terminal part of IFT172, uncovering a conserved U-box-like domain often found in E3 ubiquitin ligases. This domain exhibits ubiquitin-binding properties and IFT172 undergoes ubiquitin conjugation in vitro, an activity which is reduced in the C1727R patient ciliopathy variant. We use CRISPR-engineered RPE-1 cells to demonstrate that the U-box-like domain is essential for IFT172 protein stability and proper cilium formation. Notably, RPE-1 cells with heterozygous deletion of the U-box domain show altered TGF-{beta} signaling responses, particularly in SMAD2 phosphorylation levels and AKT activation. Our findings suggest that IFT172, beyond its structural role in bridging IFT-A and IFT-B complexes within IFT trains, harbors a conserved U-box-like domain with potential involvement in ciliary ubiquitination processes and signaling, providing new insights into the molecular mechanisms underlying IFT172-related ciliopathies.

biochemistry↗

bioRxiv2024

Species-specific circular RNA circDS-1 enhances adaptive evolution in Talaromyces marneffei through regulation of dimorphic transition and virulence

Thermal adaptability is a crucial characteristic for mammalian pathogenic fungi that originally inhabit natural ecosystems. Thermally dimorphic fungi have evolved a unique ability to respond to host body temperature by shifting from mycelium to yeast. The high similarity of protein-coding genes between these fungi and their relatives suggests the indispensable but often overlooked roles of non-coding elements in fungal thermal adaptation. Here, we systematically delineated the landscape of full-length circRNAs in both mycelial and yeast conditions of Talaromyces marneffei, a typical thermally dimorphic fungus causing fatal Talaromycosis, by optimizing an integrative pipeline for circRNA detection utilizing next- and third-generation sequencing. We found T. marneffei circRNA demonstrated features such as shorter length, lower abundance, and circularization-biased splicing. We then identified and validated that circDS-1, independent of its parental gene, promotes the hyphae-to-yeast transition, maintains yeast morphology, and is involved in virulence regulation. Further analysis and experiments among Talaromyces confirmed that the generation of circDS-1 is driven by a T. marneffei-specific region in the flanking intron of circDS-1. Together, our findings not only provide fresh insights into the role of circRNA in fungal thermal adaptation but also reveal a novel molecular mechanism for the adaptive evolution of functional circRNAs derived from intronic mutations. Author SummaryIt is an essential characteristic for human pathogens to survive under human body temperature, which is obviously higher than environmental temperature. The morphology transformation between mycelia and yeasts of thermally dimorphic fungi are tightly associated with temperature. The limited divergences of protein-coding genes among their closest relatives are not able to explain these fungi thermal dimorphism. CircRNAs are non-coding RNAs that distribute ubiquitously across eukaryotes. However, lower eukaryotes have been relatively neglected in circRNA research, either from the biological function level or from the evolutionary level. Our study focused on the typical thermally dimorphic fungus, Talaromyces marneffei. We combined next-generation and third-generation sequencing technologies to map the full-length circRNA profile in both the saprophytic mycelial and pathogenic yeast forms of T. marneffei to identify functional circRNAs. Gene manipulation experiments were performed to evaluate the functional impact of the target circRNA circDS-1. We uncovered unique characteristics of T. marneffei circRNAs, such as shorter lengths, reduced abundance, and a preference for circularization-biased splicing events. CircDS-1, which functions independently from its host gene to promote the transition from hyphae to yeast form, maintain yeast morphology, and modulate virulence. Our study not only offers fresh perspectives on the role of circRNAs in fungal thermal adaptation, but also enhances our understanding of the adaptive evolution of pathogenic fungi as they transform from environmental to host environments.

microbiology↗

bioRxiv2024

DNA replication fork speed Acts as an Engine in Cortical Neurogenesis

DNA replication fork speed, which controls the rate of genome duplication, has emerged as a key regulator of cellular plasticity. However, its role in neurogenesis remains unexplored. Mini-chromosome maintenance complex (MCMs)-binding protein (MCMBP) functions as a chaperone for newly synthesized MCMs, increasing chromatin coverage to restrain fork speed. We demonstrate that selective deletion of MCMBP in neural progenitor radial glial cells (RGCs) accelerates fork speed, triggering widespread apoptosis, DNA damage, and micronuclei formation, ultimately activating p53 and causing microcephaly. Unexpectedly, concurrent deletion of Trp53 and Mcmbp further increased fork speed, leading to extensive RGC detachment from the ventricular zone and acquisition of outer-RGC characteristics. Mechanistically, we found that the MCM3 subunit coordinates DNA and centrosome duplication, thereby mediating RGC attachment. Behavioral analysis revealed that disruption of fork speed results in anxiety-like behavior in mice. These findings unveil a previously unrecognized role for replication fork speed in neurogenesis.

neuroscience↗

bioRxiv2024

Up sampled PSF enables high accuracy 3D super-resolution imaging with sparse sampling rate

Single-molecule localization microscopy (SMLM) provides nanoscale imaging, but pixel integration of acquired SMLM images limited the choice of sample rate which restricts the information content conveyed within each image. We propose an up-sampled point spread function (PSF) inverse modeling method for large-pixel single molecule localization, enabling precise 3D super-resolution imaging with sparse sampling rate. Our approach could reduce data volume or expand the field of view by nearly an order of magnitude, while maintaining high localization accuracy, greatly improving the imaging throughput with limited pixels offered by the existing cameras.

biophysics↗

bioRxiv2024

Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

bioengineering↗

bioRxiv2024

Nitrate Promoting Hair Growth Through Enhancing Wnt/β-catenin and Downregulating TGF-β/Samd/BMP signaling pathway in C57BL/6 Mice

Nitrate found widely distributed in vegetables and fruits. It has been proved to have many physiological functions through nitrate-nitrite-NO pathway, such as attenuate oxidative stress, enhance the skeletal muscle and tone skin vascular. But the function of nitrate in regulating hair growth is unclear. Here, we evaluated potential roles of nitrate on hair growth and its mechanisms by in vivo models. Mice treated with 10mM nitrate supplementation significantly promoting hair growth, along with an increase in the number of hair follicles and thickness of dermis. We further demonstrated that Wnt3a and {beta}-catenin were highly expressed in the nitrate-treated groups, while the expression of TGF-{beta}1 was attenuated. Moreover, nitrate also increased the expression levels of growth factors (Fgf, Igf-1, and Vegf). Our data indicated that nitrate supplementation could effectively accelerate hair regrowth through activating the Wnt/{beta}-catenin and inhibiting TGF-{beta}1/Smad/BMP signaling pathway. Therefore, these findings provide support for a potential therapeutic role of nitrate in preventing and treating alopecia.

biochemistry↗

bioRxiv2024

Neuron-astrocyte Coupling in Lateral Habenula Mediates Depressive-like Behaviors

The lateral habenular (LHb) neurons and astrocytes have been strongly implicated in depression etiology but it was not clear how the two dynamically interact during depression onset. Here, using multi-brain-region calcium photometry recording in freely-moving mice, we discover that stress induces a unique, bimodal neuronal response and a most rapid astrocytic response in the LHb. LHb astrocytic calcium requires the 1A-adrenergic receptor, and depends on a recurrent neural network between the LHb and locus coeruleus (LC). Through the gliotransmitter glutamate and ATP/Adenosine, LHb astrocytes mediate the second-wave activation of local LHb neurons as well as release of norepinephrine (NE). Activation or inhibition LHb astrocytic calcium signaling facilitates or prevents stress-induced depressive-like behaviors respectively. These results identify a stress-induced positive feedback loop in the LHb-LC axis, with astrocytes being a critical signaling relay. The identification of this prominent neuron-glia interaction may shed light on stress management and depression prevention.

neuroscience↗

bioRxiv2024

Targeted disruption of linkage-specific ubiquitylation reveals a key role of K29-linked ubiquitylation in epigenome integrity

Linkage-specific ubiquitin chains dictate the functional outcome of numerous critical ubiquitin-dependent signaling processes. However, the functions and targets of several poly-ubiquitin topologies remain poorly defined due to a paucity of tools for their specific detection and manipulation. To remedy this knowledge gap, we applied a cell-based ubiquitin replacement strategy enabling targeted conditional abrogation of each of the seven lysine-based ubiquitin chain types in human cells to profile system-wide impacts of disabling formation of individual chain types. Focusing on K29-linked ubiquitylation, we reveal a strong association of this linkage type with chromatin-associated proteins and show that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. We demonstrate that K29-linked ubiquitylation is essential for proteasomal degradation of SUV39H1 despite its extensive modification by K48-linked ubiquitylation, and that K29-linked ubiquitylation of SUV39H1 is catalyzed and reversed by TRIP12 and TRABID, respectively. Preventing K29-linked ubiquitylation-mediated control of SUV39H1 stability deregulates the H3K9me3 landscape, but not other histone marks. Collectively, our ubiquitin replacement cell line panel and datasets provide valuable resources for illuminating cellular functions of linkage-specific ubiquitin chains and establish a key role of K29-linked ubiquitylation in epigenome integrity.

cell biology↗

bioRxiv2024

Revolutionizing large-scale DNA synthesis with microchip-based massive in parallel synthesis system

DNA synthesis serves as the fundamental enabling technology of engineering biology, aiming to provide DNA molecules of designed composition, length, and complexity at scale and low cost. Current high-throughput DNA synthesis technologies rely on intricate chip manufacturing and microfluidic systems to provide large-scale synthetic oligonucleotides, at the expense of low concentration and limited compatibility in the processing of longer DNA constructs assembly. Here, we report a microchip-based massive in parallel synthesis (mMPS), pioneering an "identification-sorting-synthesis-recycling" iteration mechanism to microchips for high throughput DNA synthesis. In comparison to microarray-based methods, we demonstrate that our method can increase the DNA product concentration by 4 magnitudes (to picomole-scale per sequence) and greatly simplifies the downstream processes for large-scale gene synthesis construction. By the construction of 1.97 million-diversity variant libraries that cover 1,254 human protein domains, we demonstrated the uniformity of the constructed variant libraries using mMPS-derived oligos is greatly improved, with amino acid distribution highly consistent as designed. In addition, by synthesizing 285 1kb-to-3kb genes with varying degrees of sequence complexity from previously reported strains A501 and 3DAC, potential ancestor of early archaea and bacteria, our result shows that the overall gene assembly success rate using mMPS-derived oligos is increased by 10-fold in comparison to other methods. Our mMPS technology holds the potential to close the gap between the quality and cost of writing DNA in increasing demand across many sectors of research and industrial activities.

synthetic biology↗

bioRxiv2024

Microbial succession, community assembly and adaptation over five years in a newly discovered deep-sea cold seep

Deep-sea cold seeps, characterized by the release of hydrocarbon-rich fluids such as methane from the seafloor, create unique ecosystems with evolving biological communities. Microorganisms serve as pioneers, initially colonizing early-stage cold seeps and providing essential matter and energy for subsequent colonizers. However, microbial succession, community assembly, and adaptation in these environments remain largely unexplored due to challenges associated with locating early-stage cold seeps. Here, we collected sediment samples from a newly discovered early-stage cold seep site, QDN-W07, in the South China Sea at varying depths over five years. We also sampled two previously reported cold seep sites, QDN-S18 and HM-ROV01, for comparative analysis. Amplicon sequencing revealed a high relative abundance of ANME-3 (up to 99%) and Methylomonadaceae (up to 83%) in surface sediments, and indicated changes in microbial community structures over time. Dispersal limitation dominated community assembly, while the contribution of homogeneous selection decreased over time, accompanied by a declined in the intensity of microbial association. Metagenomic analysis of core microbiome, comprising 172 metagenomic-assembled genomes at QDN-W07, revealed tolerance to low temperatures and oxygen. Core microbiome genomes were enriched with genes related to aerobic methane oxidation, urea metabolism, and the oxidation of sulfur, ammonia and carbon monoxide. These results indicate that core microorganisms obtain energy from the oxidation of inorganic compounds, allowing them to thrive in cold, methane-rich and unstable conditions. Overall, this study enhances our understanding of deep biosphere life, biogeochemical cycling, and the ecological management of cold seep environments.

microbiology↗