Biology subjects

Shen, Z.

Publications and source records attributed to Shen, Z..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

CloneCoordinate: Open-source software for collaborative DNA construction

Custom DNA constructs have never been more common or important in the life sciences. Many researchers therefore devote substantial time and effort to molecular cloning, aided by abundant computer-aided design tools. However, support for managing and documenting the construction process, and for effectively handling and reducing the frequency of setbacks, is lacking. To address this need, we developed CloneCoordinate, a free, open-source electronic laboratory notebook specifically designed for cloning and fully implemented in Google Sheets. By maintaining a real-time, automatically prioritized task list, a uniform physical sample inventory, and standardized data structures, CloneCoordinate enables productive, collaborative cloning for individuals or teams. We demonstrate how the information captured by CloneCoordinate can be leveraged to troubleshoot assembly problems and provide data-driven insights into cloning efficiency. CloneCoordinate offers a new and uniquely accessible model for how to carry out, and iteratively improve on, real-world DNA assembly.

synthetic biology↗

bioRxiv2025

Single-cell spatial map of cis-regulatory elements for disease-related genes in the macaque cortex

Single-cell spatial transcriptomes have demonstrated molecular and cellular diversity in the brain1, 2, 3, 4, 5, 6, 7, 8, but gene regulatory mechanisms underlying transcriptomic profiles and disease pathogenesis remain largely unknown in primates. Here we performed single-nucleus Assay for Transposase-Accessible Chromatin followed by sequencing (snATAC-seq) for [~]1.6 million cells from 142 cortical regions of two male cynomolgus monkeys (Macaca fascicularis), and identified distinct chromatin accessibility profiles of cis-regulatory elements (CREs) for various cell types. By integrative analysis with large-scale spatial transcriptome data, we found that these CREs showed laminar and regional preferences, with their regional accessibility exhibiting striking dependence on the regions hierarchical level. Cross-species comparison of snATAC-seq data revealed human/macaque-enriched layer-4 glutamatergic neurons and LAMP5/LHX6-expressing GABAergic neurons as well as human/macaque-biased CREs for genes related to neurodevelopment and psychiatric diseases. Importantly, risk single-nucleotide polymorphisms for many brain disorders strongly associated with human/macaque-biased CREs in glutamatergic neuronal types and those for Alzhemers disease strongly associated with CREs exclusively in microglia. Our results provided the basis for understanding the spatial gene regulatory mechanisms underlying cellular diversity and disease pathogenesis in the primate cortex.

neuroscience↗

bioRxiv2025

Candida auris Metabolism and Growth Preferences in Physiologically Relevant Skin-like Conditions

Candida auris is an opportunistic, multidrug-resistant yeast with high capacity of skin colonization in healthcare settings, which can lead to subsequent infections with high mortality rates. Given the recent emergence of at least four distinct clades at the global scale, little remains known about how C. auris is so adept at growing on skin and the key genes and pathways it utilizes to metabolize the scarce nutrients available. Here, we identify the roles that conventional and alternative carbon metabolism genes and metabolic pathways have in facilitating C. auris growth through laboratory-based experiments and bioinformatics analyses. In artificial skin-like media, all four clades of C. auris were more capable of growing than C. albicans SC5314, a clinically relevant counterpart. By investigating the differential regulation of C. auris when growing in skin-like media as compared to rich fungal media, we uncovered hundreds of genes in multiple metabolic pathways. To further test the mechanisms of these metabolic pathways, we deleted several non-essential gene candidates including FOX2 (B9J08_002847), CAT2 (B9J08_000010), and ICL1 (B9J08_003374). The mutant strains all exhibited abrogated growth in skin-like media and demonstrated nutrient preferences that differed from the wild type. Thus, we propose a model of how C. auris has the capacity to metabolize nutrients that are naturally available on skin by changing its metabolic profile. Targeting these metabolic pathways to mitigate C. auris growth on skin is a potential avenue to explore in controlling the spread of this emerging human fungal pathogen. IMPORTANCECandida auris is an emerging fungal pathogen with skin as its primary site of colonization and subsequent transmission. Here, we show the importance of conventional and alternative carbon metabolism for C. auris ability to grow in artificial skin-like media. This knowledge provides a better understanding of C. auris metabolism and sheds light on genes and pathways that could be targeted to interfere with persistent skin colonization.

microbiology↗

bioRxiv2025

Comparison of Urinary Proteome Modifications Between Methamphetamine-Abstinent Rehabilitation Patients for Over Three Months and Healthy Individuals

Methamphetamine addiction is characterized by complex mechanisms and a high relapse rate, posing a serious public health threat. In this study, urinary proteome modifications of rehabilitation patients who had abstained from methamphetamine for over three months and those of healthy individuals were analyzed and compared. A total of 984 differentially modified peptides were identified, 362 of which showed changes from presence to absence or vice versa. Results from the randomized grouping test indicated that at least 74.85% of these differentially modified peptides were not randomly generated. Several proteins containing these peptides have been reported to be associated with methamphetamine, including prostaglandin-H2 D-isomerase, complement factor H, filamin-A, and plasminogen. Additionally, some significantly changed modified peptides and the proteins they are located in, which have not been previously reported to be associated with methamphetamine, may provide new insights into addiction rehabilitation. This study establishes a method for investigating drug addiction through urinary proteome modifications and observes differences between methamphetamine-abstinent rehabilitation patients and healthy individuals, which may help explain the drugs high relapse rate. It offers a new perspective on drug withdrawal research and holds potential for tracking and assessing the rehabilitation process of methamphetamine addiction.

biochemistry↗

bioRxiv2025

Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

Traumatic heterotopic ossification (tHO) is a musculoskeletal disorder characterized by ectopic bone formation in soft tissues following trauma. However, the etiology and mechanisms of tHO remain unknown, and no treatments are highly effective for tHO. Here, by performing single-cell RNA sequencing (scRNA-seq) on two tHO mouse models, we found that ciliary Hedgehog (Hh) and PI3K-Akt signaling in tendon stem/progenitor cells (TSPCs) is upregulated during tHO development, leading to the activation of GLI family zinc finger 2 (GLI2) transcription factor, which promotes mesoderm-specific transcript (MEST) gene expression. Untargeted lipidomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on detached cilia indicated that MEST could reduce ciliary sphingomyelin (SM) levels by inhibiting SM synthesis in TSPCs, creating a positive feedback loop that amplified ciliary Hh signaling, enhancing TSPCs osteochondrogenesis and driving tHO formation. Targeting ciliary genes intraflagellar transport 88 (IFT88) or ADP ribosylation factor like GTPase 3 (ARL3) restored normal TSPCs osteochondrogenesis in vitro and attenuated ectopic bone formation in vivo by suppressing Hh and PI3K-Akt signaling. AAV-mediated MEST inhibition or exogenous SM administration in vivo also alleviates tHO progression. These findings may provide novel insights into tHO pathogenesis and potential therapeutic strategies.

molecular biology↗

bioRxiv2025

Comparative molecular landscapes of immature neurons in the mammalian dentate gyrus across species reveal special features in humans

Immature dentate granule cells (imGCs) arising from adult hippocampal neurogenesis contribute to plasticity, learning and memory, but their evolutionary changes across species and specialized features in humans remain poorly understood. Here we performed machine learning-augmented analysis of published single-cell RNA-sequencing datasets and identified macaque imGCs with transcriptome-wide immature neuronal characteristics. Our cross-species comparisons among humans, monkeys, pigs, and mice showed few shared (such as DPYSL5), but mostly species-specific gene expression in imGCs that converged onto common biological processes regulating neuronal development. We further identified human-specific transcriptomic features of imGCs and demonstrated functional roles of human imGC-enriched expression of a family of proton-transporting vacuolar-type ATPase subtypes in development of imGCs derived from human pluripotent stem cells. Our study reveals divergent gene expression patterns but convergent biological processes in the molecular characteristics of imGCs across species, highlighting the importance of conducting independent molecular and functional analyses for adult neurogenesis in different species.

neuroscience↗

bioRxiv2025

Polo-like kinase 1 maintains transcription and chromosomal accessibility during mitosis

Transcription persists at low levels in mitotic cells and plays essential roles in mitotic fidelity and chromosomal dynamics. However, the detailed regulatory network of mitotic transcription remains largely unresolved. Here, we report the novel role of Polo-like kinase 1 (Plk1) in maintaining mitotic transcription. Using 5-ethynyl uridine (5-EU) labeling of nascent RNAs, we found that Plk1 inhibition leads to significant downregulation of nascent transcription in prometaphase cells. Chromatin-localized Plk1 activity is required for transcription regulation and mitotic fidelity. Plk1 sustains global chromosomal accessibility in mitosis, especially at promoter and transcription start site (promoter-TSS) regions, facilitating transcription factor binding and ensuring proper transcriptional activity. We identified SMC4, a common subunit of condensin I and II, as a potential Plk1 substrate. Plk1 activity is fundamental to these processes across non-transformed and transformed cell lines, underscoring its critical role in cell cycle regulation. This study elucidates a novel regulatory mechanism of global mitotic transcription, advancing our understanding of cell cycle control. Significance StatementO_LICells retain a low level of transcription during mitosis, while the regulatory network and specific contributions of mitotic transcription are not well understood. C_LIO_LIWe identify Polo-like kinase 1 (Plk1) as a novel regulator of mitotic transcription, crucial for chromosome condensation, genome accessibility, and maintaining mitotic fidelity. C_LIO_LIThis study enhances our understanding of Plk1s multifaceted role in mitotic progression, advancing cell cycle regulation knowledge, and informing new cancer therapies development. C_LI

cell biology↗

bioRxiv2024

Exploring the effects of metformin on the body by urine proteome

Metformin is the first-line medication for treating type 2 diabetes mellitus (T2DM), with over 200 million patients taking it daily. Its effects are extensive and play a positive role in multiple areas. Can we explore its effects and potential mechanisms by urine proteome? In this study, a total of 166 differential proteins were identified after rats were given a dose of 150 mg/(kg{middle dot}d) of metformin for 5 consecutive days, including complement component C6, pyruvate kinase, coagulation factor X, growth differentiation factor 15 (GDF15), carboxypeptidase A4, chymotrypsin-like elastase family member 1, and L-lactate dehydrogenase C chain (LDH-C). Several of these proteins have been reported to be directly affected by metformin or associated with the effects of metformin. Several biological pathways enriched by the differential proteins or proteins where the differentially modified peptides are located have been reported to be associated with metformin, including glutathione metabolic process, negative regulation of gluconeogenesis, and renin-angiotensin system. Additionally, some significantly enriched biological pathways that have not been reported to be related to the effects of metformin may provide clues for the study of metformins potential mechanisms. In conclusion, the application of urine proteome offers a comprehensive and systematic approach to exploring both the known and unknown effects of drugs, thus opening a new window to study the mechanisms of metformin.

biochemistry↗

bioRxiv2024

PERK Signaling Maintains Hematopoietic Stem Cell Pool Integrity under Endoplasmic Reticulum Stress by Promoting Proliferation

The integrity of the hematopoietic stem cell (HSC) pool relies on efficient long-term self-renewal and the timely removal of damaged or differentiation-prone HSCs. Previous studies have demonstrated the PERK branch of the unfolded protein response (UPR) drives specific programmed cell death programs to maintain HSC pool integrity in response to ER stress. However, the role of PERK in regulating HSC fate in vivo remains unclear. Here, we demonstrate that PERK is dispensable for normal hematopoiesis and HSC self-renewal under steady-state conditions. In contrast, PERK is activated to promote HSC proliferation and depletion in response to ER stress induced by the inactivation of ER-associated degradation (ERAD), via the knockout of key components of ERAD Sel1L or Hrd1. Inhibition of PERK, either through genetic knockout or knock-in of a point mutation that eliminates PERK kinase activity, significantly restores the HSC defects induced by Sel1L or Hrd1 knockout. Mechanistic studies reveal that ERAD deficiency does not lead to HSC death or ROS accumulation. Instead, PERK promotes the activation of mTOR signaling and drives abnormal proliferation of HSCs, impairing their self-renewal potential. This process removes stressed HSCs, thereby maintaining HSC pool integrity. Our study uncovers a PERK-centered strategy employed by HSCs to preserve their pool integrity independently of apoptosis. Key pointsO_LIPERK is not required for steady-state hematopoiesis but preserves hematopoietic stem cell pool integrity in response to increased ER stress. C_LIO_LIUnder ER stress induced by ERAD deficiency, PERK is activated to promote mTOR signaling and HSC hyper-proliferation, depleting damaged HSCs. C_LI

cell biology↗

bioRxiv2024

Perivascular macrophages convert physical wound signals into rapid vascular responses

Leukocytes detect distant wounds within seconds to minutes, which is essential for effective pathogen defense, tissue healing, and regeneration. Blood vessels must detect distant wounds just as rapidly to initiate local leukocyte extravasation, but the mechanism behind this immediate vascular response remains unclear. Using high-speed imaging of live zebrafish larvae, we investigated how blood vessels achieve rapid wound detection. We monitored two hallmark vascular responses: vessel dilation and serum exudation. Our experiments--including genetic, pharmacologic, and osmotic perturbations, along with chemogenetic leukocyte depletion--revealed that the cPla2 nuclear shape sensing pathway in perivascular macrophages converts a fast ([~]50 m/s) osmotic wound signal into a vessel-permeabilizing, 5-lipoxygenase (Alox5a) derived lipid within seconds of injury. These findings demonstrate that perivascular macrophages act as physicochemical relays, bridging osmotic wound signals and vascular responses. By uncovering this novel type of communication, we provide new insights into the coordination of immune and vascular responses to injury.

cell biology↗

bioRxiv2024

Deconstructing evolutionary histories of complex genomic rearrangements in lung malignancies

Somatic genomic rearrangements are hallmarks of cancer. Complex genomic rearrangements (CGRs) involving multiple intertwined structural alterations are often present in tumor genomes. CGRs frequently harbor oncogenic drivers, but their genomic architectures and etiologies are poorly understood. We used deep-coverage optical mapping technology to profile the genomic landscapes of normal lung tissues, benign pulmonary lesions, carcinoma in situ, and advanced carcinomas to examine the patterns of genome disorganization and instability in different stages of carcinogenesis in lung. Large rearrangements and CGRs were prevalent in the carcinomas. We developed omcplR to resolve the architecture of CGRs and predict their genesis from optical mapping data using genome-graph concept. We found that CGRs often arose from hierarchical combinations of multiple, localized simple structural variations, and harbored allelic heterogeneity at the affected loci. Rearrangement patterns and associated genomic signatures suggested that chromoanasynthesis was a likely prevalent mechanism driving complex genomic rearrangements. The early rearrangement junctions in intra-chromosomal CGRs were usually localized within the same chromatin domains, but the late junctions in advanced tumors had more heterogeneous contexts suggesting progressive organizational heterogeneity. The CGRs, especially the late events therein were under positive selection. A composite signature of genomic alterations including the CGRs captured the trajectory of progressive genomic disorganization and instability with carcinogenesis in lung and underscored the extent of genomic structural heterogeneity among the in-situ tumors.

genomics↗

bioRxiv2024

Topological rearrangements activate the HerA-DUF anti-phage defense system

Leveraging the rich structural information provided by AlphaFold, we used integrated experimental approaches to characterize the HerA-DUF4297 (DUF) anti-phage defense system, in which DUF is of unknown function. To infer the function of DUF, we performed structure-guided genomic analysis and found that DUF homologs are universally present in bacterial immune defense systems. One notable homolog of DUF is Cap4, a universal effector with nuclease activity in CBASS, the most prevalent anti-phage system in bacteria. To test the inferred nuclease function of DUF, we performed biochemical experiments and discovered that the DUF only exhibits activity against DNA substrates when it is bound by HerA. To understand how HerA activates DUF, we determined the structures of DUF and the HerA-DUF complex. DUF forms large oligomeric assemblies with or without HerA, suggesting that oligomerization per se is not sufficient for DUF activation. Instead, DUF activation requires dramatic topological rearrangements that propagate from HerA to the entire HerA-DUF complex, leading to reorganization of DUF for effective DNA cleavage. We further validated these structural insights by structure- guided mutagenesis. Together, these findings reveal dramatic topological rearrangements throughout the HerA-DUF complex, challenge the long-standing dogma that protein oligomerization alone activates immune signaling, and may inform the activation mechanism of CBASS.

biochemistry↗

bioRxiv2024

Astrocytic Ryk signaling coordinates scarring and wound healing after spinal cord injury

Wound healing after spinal cord injury involves highly coordinated interactions among multiple cell types, which is poorly understood. Astrocytes play a central role in creating a border against the non-neural lesion core. To do so, astrocytes undergo dramatic morphological changes by first thickening the processes and then elongating and overlap them. We show here show that the expression of a cell-surface receptor, Ryk, is induced in astrocytes after injury in both rodent and human spinal cord. Astrocyte-specific knockout of Ryk dramatically elongated the reactive astrocytes and accelerated the formation of the border and reduced the size of the scar. Astrocyte-specific knockout of Ryk also accelerated the injury responses of multiple cell types, including the resolution of neuroinflammation. Single cell transcriptomics analyses revealed a broad range of changes cell signaling among astrocytes, microglia, fibroblasts, endothelial cell, etc, after astrocyte-specific Ryk knockout, suggesting that Ryk not only regulates the injury response of astrocytes but may also regulate signals which coordinate the responses of multiple cell types. The elongation is mediated by NrCAM, a cell adhesion molecule induced by astrocyte-specific conditional knockout of Ryk after spinal cord injury. Our findings suggest a promising therapeutic target to accelerate wound healing and promote neuronal survival and enhance functional recovery.

neuroscience↗

bioRxiv2024

P450-Catalyzed Biaryl Macrocyclization of Leaderless Ribosomal Peptides

Macrocyclic peptides containing biaryl motifs, predominantly derived from natural products, are valuable scaffolds due to their structural rigidity and potent bioactivity. However, current synthetic methods remain constrained by the absence of broadly applicable strategies across chemocatalytic and biocatalytic platforms. Here, we discover a versatile P450 enzyme, GpeC, capable of facilitating oxidative C-C/O/N cross-coupling for peptide biaryl macrocyclization (PBC) with leader-independent activity. Crystal structure analysis of GpeC rationalizes leader-independent property and reveals adaptive recognition that enables access to diverse biaryl-linked macrocycles. GpeC exhibits exceptional substrate promiscuity, accommodating 19 of 20 canonical amino acids and diverse noncanonical analogs within a minimal tetrapeptide scaffold. The efficient semi-synthesis of the natural product Rubrin further demonstrated the versatility of GpeC. We further introduced lytic to tetraregion (LTT), a one-step, single enzyme synthesis for modular synthesis of biaryl-cyclized tetrapeptide. Overall, GpeCs robustness and programmability position it as a broadly applicable biocatalyst for the synthesis of biaryl macrocyclic peptides.

biochemistry↗

bioRxiv2024

Prevalence of Problematic Papers in Non-Coding RNA Research

This study examines the prevalence of problematic papers in the rapidly growing field of non-coding RNA (ncRNA) research. Analysis of 153,826 ncRNA papers during 2000-2023 reveals that around 1.79% have been retracted and an additional 5.68% have raised concerns on PubPeer. The number of problematic papers has steadily increased, peaking in 2019 when the concerning and retraction rates reached nearly 10.8% and 3.7%, respectively. These unreliable papers have been widely disseminated, accumulating hundreds of thousands of citations in academic literature, patents, clinical trials, and policy documents, posing a significant threat to research integrity and public health. The main issues identified include image manipulation, data falsification, fake peer reviews, and ethical lapses. The findings call for urgent, comprehensive scrutiny of ncRNA publications and broader reforms to address systemic problems driving the proliferation of problematic research.

molecular biology↗

bioRxiv2024

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgo) and the DNA Defense Module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

biophysics↗

bioRxiv2024

Dynamic Plk1 recruitment to the inner centromere

Mitosis is carefully orchestrated by reversible phosphorylation events. Polo-like kinase 1 (Plk1) regulates multiple functions across the kinetochore during mitotic progression. Recently, Bub1 (outer kinetochore) and CENP-U (inner kinetochore) were described as two major sites of Plk1 recruitment to the kinetochore. Here, we report an additional dynamic site of Plk1 recruitment to the inner centromere. Inner centromere docking occurs during late prometaphase and metaphase, exhibiting transient residency at multiple chromosomes. Chromosomes with inner centromere-localized Plk1 have end-on attached microtubules, diminished Spindle Assembly Checkpoint (SAC) components, and low Shugoshin 1 (Sgo1) levels at the inner centromere. Mechanistically, recruitment is driven by Cdk1 activity and requires Plk1s Polo-Box Domain (PBD). Moreover, inhibition of Bub1 or Protein Phosphatase 2A (PP2A) increases Plk1 recruitment and residency at the inner centromere. Collectively, our data identify a novel pathway for Plk1 recruitment to the inner centromere that is dynamically regulated by counteracting activities of Cdk1 and Bub1/PP2A.

cell biology↗

bioRxiv2024

The Mammalian KU70 C-terminus SAP Domain Is Required to Repair Exogenous DNA Damage

The mammalian non-homologous end joining (NHEJ) is required for V(D)J recombination as well as coping with exogenously induced DNA double strand breaks (DSBs). Initiated by the binding of KU70/KU80 (KU) dimer to DNA ends and the subsequent recruitment of the DNA- dependent protein kinase catalytic subunit (DNA-PKcs), NHEJ plays a key role in DNA repair. While there has been significant structural understandings of how KU70 participates in NHEJ, the specific function of its highly conserved C-terminal SAP domain remains elusive. In this study, we developed a novel mouse model by deleting the SAP domain but preserving the KU70 nuclear localization and its dimerization ability with KU80. We found that the KU70 SAP deletion did not affect the V(D)J recombination or animal development but significantly impaired the animals and cells in repairing exogenously induced DSBs. We further showed an inability of KU70-{Delta}SAP cells to retain the DNA Ligase IV (LIG4) and other NHEJ co-factors on chromatin, and a spreading pattern of DSB marker {gamma}H2AX in KU70-{Delta}SAP cells after DNA damage. Our findings suggest that a specific inhibition of the SAP function may offer an opportunity to modulate cell sensitivity to therapeutic DSB-inducing agents without interfering with the developmental function of KU70. KeyPointsO_LIGeneration of a novel transgenic mouse line lacking the C-terminal conserved KU70-SAP domain C_LIO_LIKU70-SAP defends against exogenous DSBs, but unessential for development and V(D)J recombination C_LIO_LIKU70-SAP aids in recruiting and retaining NHEJ components, such as LIG4, to DSB sites C_LI

cancer biology↗