Biology subjects

Kong, Y.

Publications and source records attributed to Kong, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Dysregulation of mitochondrial function by PLK1-mediated PDHA1 phosphorylation promotes Cr(VI)-associated lung cancer progression

Hexavalent chromium (Cr(VI)) is a class I environmental carcinogen known to induce lung epithelial cell transformation and promote lung cancer progression through alterations in the cell cycle and cellular energy metabolism. In this study, we investigated the role of polo-like kinase 1 (PLK1) in Cr(VI)-transformed (CrT) bronchial epithelial cells (BEAS-2B) and found that PLK1 expression was significantly upregulated in CrT cells, leading to impaired mitochondrial function and enhanced cell proliferation both in vitro and in vivo. High levels of PLK1 in CrT cells resulted in decreased mitochondrial activity due to defective modulation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), which is crucial for pyruvate/Acetyl-CoA conversion and carbon influx into the tricarboxylic acid (TCA) cycle. Mechanistically, we demonstrated that PLK1 directly phosphorylates PDHA1 at T57, leading to E1 collapse and PDHA1 degradation via activation of mitophagy. These defects resulted in the inhibition of oxidative phosphorylation and reduction of mitochondrial superoxide generation, ultimately leading to suppression of mitochondrial-mediated apoptotic response. Our findings highlight the role of PLK1 in metabolic reprogramming during Cr(VI)-associated cancer progression, providing new insights and a potential therapeutic target to inhibit Cr(VI)-induced cancer development. Moreover, PLK1 inhibitors may also have the potential to increase chemo-sensitivity of cancer cells by restoring normal mitochondrial function, thereby mitigating drug resistance caused by mitochondrial dysfunction and hyperpolarization.

cancer biology↗

bioRxiv2024

Cortico-spinal Mechanisms of Periphery Neuromodulation induced Analgesia

BackgroundTranscutaneous Electrical Nerve Stimulation (TENS) is widely used for pain management, yet how different parameters--Conventional (high frequency, low intensity) versus Acupuncture-Like (low frequency, high intensity)--modulate pain remains controversial. These modes produce variable analgesic effects, but their underlying brain-spinal mechanisms are unclear. Clarifying these pathways could refine TENS protocols, optimize clinical outcomes, and reduce opioid reliance, highlighting the need for more precise, non-pharmacological approaches to advanced pain care. MethodsThis single-blind, three-arm randomized trial recruited 95 healthy adults (18-30 years) and assigned them (1:1:1) to Conventional TENS, Acupuncture-Like TENS, or sham. Participants underwent a 30-minute TENS intervention on the left forearm (C5-C6 dermatome) with simultaneous brain-spinal fMRI before and after thermal nociceptive stimuli. The primary outcomes were changes in Numeric Rating Scale (NRS) pain scores and brain-spinal BOLD signals. Secondary outcomes included psycho-physiological interaction (PPI) and mediation analyses of periaqueductal gray (PAG) activity and brain- cord connectivity. FindingsBoth TENS modes significantly reduced pain but engaged distinct cortico-spinal pathways. Conventional TENS yielded local analgesia via dlPAG-driven spinal inhibition plus partial cortical involvement (PAG-vmPFC). By contrast, Acupuncture-Like TENS produced diffuse analgesia through vlPAG-linked top-down modulation reliant on spinal gating. Correlation and mediation analyses confirmed that Conventional TENS integrates spinal and cortical synergies, whereas Acupuncture-Like TENS is dominated by robust descending control. InterpretationDifferent TENS parameters yield distinct analgesic mechanisms. Conventional TENS couples direct spinal inhibition with partial cortical regulation, while Acupuncture-Like TENS relies heavily on top-down pathways passing through the spinal cord. Recognizing these unique descending networks can guide targeted TENS protocols for diverse pain conditions, optimizing clinical outcomes and reducing reliance on pharmacological approaches. FundingsThis work was supported by the National Key R&D Program of China (2022YFC3500603), the National Natural Science Foundation of China (32071061, 82072010, 82030121 and 32100861, 82330057), the Beijing Natural Science Foundation (IS23108, JQ22018).

neuroscience↗

bioRxiv2024

Increased regional P2X7R expression detected by GSK1482160 PET in a tauopathy mouse model

Neuroinflammation plays an important role in Alzheimers disease and primary tauopathies. The aim of the current study was to map [18F]GSK1482160 for imaging of purinergic P2X7R in Alzheimers disease and primary tauopathy mouse models. MicroPET was performed using [18F]GSK1482160 in widely used mouse models of Alzheimers disease (APP/PS1, 5xFAD and 3xTg), 4-repeat tauopathy (rTg4510) mice and age-matched wild-type mice. Increased uptake of [18F]GSK1482160 was observed in the cortex and basal forebrain of 7-month-old rTg4510 mice compared to age-matched wild-type mice and compared to 3-month-old rTg4510 mice. Nonparametric Spearmans rank analysis revealed a positive correlation between tau [18F]APN-1607 uptake and [18F]GSK1482160 in the hippocampus of rTg4510 mice. No significant differences in the uptake of [18F]GSK1482160 were observed between wild-type mice and APP/PS1 mice (5, 10 months), 5xFAD mice (3, 7 months) or 3xTg mice (10 months). Immunofluorescence staining further indicated the distribution of P2X7Rs in the brains of 7-month-old rTg4510 mice with accumulation of tau inclusion compared to wild-type mice. These findings provide in vivo imaging evidence for increased P2X7R in the brains of tauopathy model mice.

neuroscience↗

bioRxiv2023

Publication, Funding, and Experimental Data in Support of Human Reference Atlas Construction and Usage

Experts from 18 consortia are collaborating on the Human Reference Atlas (HRA) which aims to map the 37 trillion cells in the healthy human body. Information relevant for HRA construction and usage is held by experts (clinicians, pathologists, anatomists, single-cell experts), published in scholarly papers, and captured in experimental data. However, these data sources use different metadata schemes and cannot be cross-searched efficiently. This paper documents the compilation of a dataset, called HRAlit, that links the 136 HRA v1.4 digital objects (31 organs with 2,689 anatomical structures, 590 cell types, 1,770 biomarkers) to 583,117 experts; 7,103,180 publications; 896,680 funded projects, and 1,816 experimental datasets. The resulting HRAlit represents 23 tables with 21,704,001 records including 7 junction tables with 13,042,188 relationships. We demonstrate how HRAlit can be mined to identify leading experts, major papers, funding trends, or alignment with existing ontologies in support of systematic HRA construction and usage. Data and code are at https://github.com/cns-iu/hra-literature.

bioinformatics↗

bioRxiv2023

Reevaluating the Concept of Aging: Long-Term Stress Adaptation as a Key Factor in Yeast Aging

It has been demonstrated that short-term stress can enhance cellular responses and promote longevity, whereas long-term stress shortens lifespan. Understanding the relationship between short-term and long-term stress could offer new insights into comprehending and modulating age-related diseases. In this study, we investigate this relationship using transcriptomic and metabolomic analyses in the yeast model system (Saccharomyces cerevisiae). We employed three metabolic treatments: firstly, treating yeast cells with threshold levels of benzoic acid for 24 hours (Short-term [ST] Stressed Cells); secondly, treating yeast cells with threshold levels of benzoic acid for 500 hours, with sub-culturing every 24 hours (Long-term [LT] Stressed Cells); and thirdly, allowing the long-term stressed cells to grow for 16 hours without any benzoic acid (Recovered Cells). Here, we propose that aging is an evolutionarily conserved cellular adaptation mechanism in response to long-term stress exposure. Under short-term stressed conditions, prominent lifespan-extending metabolites such as trehalose and metabolites linked to tumor suppression in humans, such as 5-methylthioadenosine, were overexpressed. In contrast, LT Stressed Cells activated genes such as those responsible for epigenetic regulatory enzymes that govern the aging process, and secondary stress response genes, such as heat shock proteins (HSPs) which are associated with adaptation to cell damage but also often associated with aged cells. Chronological lifespan experiments showed that LT stressed cells lived a shorter lifespan compared to ST Stressed Cells. This suggests that the markers of aging (eg. HSPs, certain epigenetic regulators) are expressed in response to long-term stress to enable cell survival but have the long-term effect of reducing lifespan. In support of this hypothesis, we also show that genes exclusively activated in ST Stressed Cells are conserved solely in eukaryotes, while those significantly expressed in LT Stressed Cells (aging related) exhibit high conservation across all domains of life, with a majority having originated from bacteria hinting at the potential evolutionary benefit of aging.

cell biology↗

bioRxiv2023

Reduced SV2A and GABAA receptor levels in the brains of type 2 diabetic rats revealed by flumazenil PET

PurposeType 2 diabetes mellitus (T2DM) is associated with a greater risk of Alzheimers disease. Synaptic impairment and protein aggregates have been reported in the brains of T2DM models. Here, we assessed whether neurodegenerative changes in synaptic vesicle 2A (SV2A), {gamma};-aminobutyric acid type A (GABAA) receptor, amyloid-{beta}, tau and receptor for advanced glycosylation end product (RAGE) can be detected in vivo in T2DM rats. MethodsPositron emission tomography (PET) using [18F]SDM-8 (SV2A), [18F]flumazenil (GABAA receptor), [18F]florbetapir (amyloid-{beta}), [18F]PM-PBB3 (tau), and [18F]FPS-ZM1 (RAGE) was carried out in 12-month-old diabetic Zucker diabetic fatty (ZDF) and Sprague{square}Dawley (SD) rats. Proteomic profiling and pathway analysis of the hippocampus of ZDF and SD rats were performed. ResultsReduced cortical [18F]SDM-8 and cortical and hippocampal [18F]flumazenil uptake were observed in 12-month-old ZDF rats compared to SD rats. [18F]florbetapir and [18F]PM-PBB3 uptake were comparable in the brains of 12-month-old ZDF rats and SD rats. ConclusionThe findings provide in vivo evidence for regional reductions in SV2A and GABAA receptor levels in the brains of aged T2DM ZDF rats.

neuroscience↗

bioRxiv2023

Relationship between reactive astrocytes, by SMBT-1 imaging, with amyloid-beta, tau, glucose metabolism, and microgliosis in mouse models of Alzheimer's disease

PurposeReactive astrocytes play an important role in the development of Alzheimers disease (AD). Here, we aim to investigate the temporospatial relationship between reactive astrocytes, tau and amyloid-{beta}, glucose metabolism, and microgliosis by using multitracer imaging in AD transgenic mouse models. MethodsPositron emission tomography (PET) imaging with [18F]SMBT-1 (monoamine oxidase-B), [18F]florbetapir (A{beta}), [18F]PM-PBB3 (tau), [18F]FDG, and [18F]DPA-714 (translocator protein) was carried out in 5- and 10-month-old APP/PS1, 11-month-old 3xTg mice, and aged-matched wild-type mice. The brain regional referenced standard uptake value (SUVR) was computed with the cerebellum as the reference region. Immunofluorescence staining was performed in mouse brain tissue slices. Results[18F]SMBT-1 and [18F]florbetapir SUVRs were higher in the cortex and hippocampus of 10-month-old APP/PS1 mice than in 5-month-old APP/PS1 mice and wild-type mice. Reduced [18F]FDG SUVR was observed in the thalamus and midbrain of 5-month-old APP/PS1 mice compared to wild-type mice. No significant difference in brain regional [18F]DPA-714 SUVR was observed in 5- and 10-month-old APP/PS1 mice compared to wild- type mice. No significant difference in the SUVRs of any tracers was observed in 11-month-old 3xTg mice compared to age-matched wild-type mice. A positive correlation between the SUVRs of [18F]SMBT-1 and [18F]DPA-714 in the cortex was observed. Immunostaining validated the distribution of MAO-B and TSPO, amyloid and tau inclusions in brain tissue from 10-month-old APP/PS1 mice and limited changes in 11-month- old 3xTg mice. ConclusionThe findings provide in vivo evidence for reactive astrocytes along with amyloid plaque and tau deposition preceding microgliosis in animal models of AD pathologies.

neuroscience↗

bioRxiv2023

Single-cell analysis characterizes PLK1 as a promoter of immunosuppressive tumor microenvironment in LUAD

PLK1 (Polo-like kinase 1) plays a critical role in the progression of lung adenocarcinoma (LUAD). Recent studies have unveiled that targeting PLK1 improves the efficacy of immunotherapy, highlighting its important role in the regulation of tumor immunity. Nevertheless, our understanding of the intricate interplay between PLK1 and the tumor microenvironment (TME) remains incomplete. Here, using genetically engineered mouse model and single-cell RNA-seq analysis, we report that PLK1 promotes an immunosuppressive TME in LUAD, characterized with enhanced M2 polarization of tumor associated macrophages (TAM) and dampened antigen presentation process. Mechanistically, elevated PLK1 coincides with increased secretion of CXCL2 cytokine, which promotes M2 polarization of TAM and diminishes expression of class II major histocompatibility complex (MHC-II) in professional antigen-presenting cells. Furthermore, PLK1 negatively regulates MHC-II expression in cancer cells, which has been shown to be associated with compromised tumor immunity and unfavorable patient outcomes. Taken together, our results reveal PLK1 as a novel modulator of TME in LUAD and provide possible therapeutic interventions.

cancer biology↗

bioRxiv2023

Phosphorylation of AHR by PLK1 promotes metastasis of LUAD via DIO2-TH signaling

Metastasis of Lung adenocarcinoma (LUAD) is a major cause of death in patients. Aryl hydrocarbon receptor (AHR) is an important transcription factor involved in the initiation and progression of lung cancer. Polo-like kinase 1 (PLK1), a serine/threonine kinase, is an oncogene that promotes the malignancy of multiple cancer types. Nonetheless, the interaction between these two factors and significance in lung cancer remains to be determined. Here, we demonstrate that PLK1 phosphorylates AHR at S489 in LUAD, which leads to epithelial-mesenchymal transition (EMT) and metastatic events. RNA-seq analyses show that type 2 deiodinase (DIO2) is responsible for EMT and enhanced metastatic potential. DIO2 converts tetraiodothyronine (T4) to triiodothyronine (T3), which then activates thyroid hormone signaling. In vitro and in vivo experiments demonstrate that treatment with T3 or T4 promotes the metastasis of LUAD, whereas depletion of DIO2 or deiodinase inhibitor disrupts this property. Taken together, our results identify the phosphorylation of AHR by PLK1 as a mechanism leading to the progression of LUAD and provide possible therapeutic interventions for this event.

cancer biology↗

bioRxiv2023

Contact toxicity, antifeedant activity and oviposition preference of osthole against agricultural pests

Osthole, the dominant bioactive constituent in Cnidium monnieri, has been shown to exhibit acute insecticidal activities. However, its detailed toxicity, antifeedant and oviposition preference effects against agricultural pests has not been fully understood, which has greatly hindered its practical applications. This study is designed to investigate the contact toxicity, antifeedant activity and oviposition preference of osthole against three agricultural pests (T. urticae, M. persicae and B. dorsalis) to evaluate its potential agricultural applications. Our results showed that Cnidium monnieri (L.) Cusson (CMC) have a high osthole content of 11.4 mg/g. Osthole exhibited a comparable level of acute toxicity against T. urticae to four other coumarins found in CMC. Osthole demonstrated significant insecticidal activity against first instar nymphs and adults of T. urticae and M. persicae in a dose-dependent manner, but not against B. dorsalis adults. Osthole exposure reduced the fecundity and prolonged the developmental time of T. urticae and M. persicae. Leaf choice bioassays revealed potent antifeedant activity in T. urticae. Furthermore, female B. dorsalis showed a distinct preference for laying eggs in mango juice with 0.02 mg/mL osthole at 48 hours, a preference which persisted at 96 hours. These results provide valuable insights into the toxicity, repellent activity, and attractant activity of osthole, thereby contributing to its expanded use in pest control.

zoology↗

bioRxiv2023

Transformer with Convolution and Graph-Node co-embedding: An accurate and interpretable vision backbone for predicting gene expressions from local histopathological image

Inferring gene expressions from histopathological images has always been a fascinating but challenging task due to the huge differences between the two modal data. Previous works have used modified DenseNet121 to encode the local images and make gene expression predictions. And later works improved the prediction accuracy of gene expression by incorporating the coordinate information from images and using all spots in the tissue region as input. While these methods were limited in use due to model complexity, large demand on GPU memory, and insufficient encoding of local images, thus the results had low interpretability, relatively low accuracy, and over-smooth prediction of gene expression among neighbor spots. In this paper, we propose TCGN, (Transformer with Convolution and Graph-Node co-embedding method) for gene expression prediction from H&E stained pathological slide images. TCGN consists of convolutional layers, transformer encoders, and graph neural networks, and is the first to integrate these blocks in a general and interpretable computer vision backbone for histopathological image analysis. We trained TCGN and compared its performance with three existing methods on a publicly available spatial transcriptomic dataset. Even in the absence of the coordinates information and neighbor spots, TCGN still outperformed the existing methods by 5% and achieved 10 times higher prediction accuracy than the counterpart model. Besides its higher accuracy, our model is also small enough to be run on a personal computer and does not need complex building graph preprocessing compared to the existing methods. Moreover, TCGN is interpretable in recognizing special cell morphology and cell-cell interactions compared to models using all spots as input that are not interpretable. A more accurate omics information prediction from pathological images not only links genotypes to phenotypes so that we can predict more biomarkers that are expensive to test from histopathological images that are low-cost to obtain, but also provides a theoretical basis for future modeling of multi-modal data. Our results support that TCGN is a useful tool for inferring gene expressions from histopathological images and other potential histopathological image analysis studies. HighlightsO_LIFirst deep learning model to integrate CNN, GNN, and transformer for image analysis C_LIO_LIAn interpretable model that uses cell morphology and organizations to predict genes C_LIO_LIHigher gene expression prediction accuracy without global information C_LIO_LIAccurately predicted genes are related to immune escape and abnormal metabolism C_LIO_LIPredict important biomarkers for breast cancer accurately from cheaper images C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/542669v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1c6a4dforg.highwire.dtl.DTLVardef@7266d4org.highwire.dtl.DTLVardef@bcfd0aorg.highwire.dtl.DTLVardef@188d452_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

P-Selectin promotes SARS-CoV-2 interactions with platelets and the endothelium

COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood1,2. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium3, and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions. In vivo, authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.

immunology↗

bioRxiv2023

Thermal Stimulus Task fMRI in the Cervical Spinal Cord at 7 Tesla

PurposeAlthough functional MRI is widely applied in the brain, fMRI of the spinal cord is more technically demanding. Proximity to the vertebral column and lungs results in strong spatial inhomogeneity and temporal fluctuations in B0. Increasing field strength enables higher spatial resolution and improved sensitivity to BOLD signal, but amplifies the effects of B0 inhomogeneity. In this work, we present the first task fMRI in the spinal cord at 7 T. Further, we compare the performance of single-shot and multi-shot 2D EPI protocols, which differ in sensitivity to spatial and temporal B0 inhomogeneity. MethodsThe cervical spinal cords of 11 healthy volunteers were scanned at 7 T using single-shot 2D EPI at 0.75 mm in-plane resolution and multi-shot 2D EPI at 0.75 and 0.6 mm in-plane resolutions. All protocols used 3 mm slice thickness. For each protocol, the BOLD response to thirteen 10-second noxious thermal stimuli applied to the right thumb was acquired in a 10-minute fMRI run. Image quality, temporal SNR, and BOLD activation (percent signal change and z-stat) at both individual-and group-level were evaluated between the protocols. ResultsTemporal SNR was highest in single-shot and multi-shot 0.75 mm protocols. In group-level analyses, activation clusters appeared in all protocols in the ipsilateral dorsal quadrant at the expected C6 neurological level. In individual-level analyses, activation clusters at the expected level were detected in some, but not all subjects and protocols. Single-shot 0.75 mm generally produced the highest mean z-statistic, while multi-shot 0.60 mm produced the best-localized activation clusters and the least geometric distortion. Larger than expected within-subject segmental variation of BOLD activation along the cord was observed. ConclusionGroup-level sensory task fMRI of the cervical spinal cord is feasible at 7 T with single-shot or multi-shot EPI. The best choice of protocol will likely depend on the relative importance of sensitivity to activation versus spatial localization of activation for a given experiment. HighlightsFirst stimulus task fMRI results in the spinal cord at 7 T Single-shot 0.75 mm 2D EPI produced the highest mean z-statistic Multi-shot 0.60 mm 2D EPI provided the best-localized activation and least distortion

bioengineering↗

bioRxiv2023

A thin-film optogenetic visual prosthesis

Retinitis pigmentosa and macular degeneration lead to photoreceptor death and loss of visual perception. Despite recent progress, restorative technologies for photoreceptor degeneration remain largely unavailable. Here, we describe a novel optogenetic visual prosthesis (FlexLED) based on a combination of a thin-film retinal display and optogenetic activation of retinal ganglion cells (RGCs). The FlexLED implant is a 30 {micro}m thin, flexible, wireless {micro}LED display with 8,192 pixels, each with an emission area of 66 {micro}m2. The display is affixed to the retinal surface, and the electronics package is mounted under the conjunctiva in the form factor of a conventional glaucoma drainage implant. In a rabbit model of photoreceptor degeneration, optical stimulation of the retina using the FlexLED elicits activity in visual cortex. This technology is readily scalable to hundreds of thousands of pixels, providing a route towards an implantable optogenetic visual prosthesis capable of generating vision by stimulating RGCs at near-cellular resolution.

neuroscience↗

bioRxiv2023

Increased TCR signaling in regulatory T cells is disengaged from TCR affinity

Foxp3+ regulatory T cells (Tregs) are capable suppressors of aberrant self-reactivity. However, TCR affinity and specificities that support Treg function, and how these compare to autoimmune T cells remain unresolved. In this study, we used antigen agnostic and epitope-focused analyses to compare TCR repertoires of regulatory and effector T cells that spontaneously infiltrate pancreatic islets of non-obese diabetic mice. We show that effector and regulatory T cell-derived TCRs possess similar wide-ranging reactivity for self-antigen. Treg-derived TCRs varied in their capacity to confer optimal protective function, and Treg suppressive capacity was in part determined by effector TCR affinity. Interestingly, when expressing the same TCR, Tregs showed higher Nur77-GFP expression than Teffs, suggesting Treg-intrinsic ability to compete for antigen. Our findings provide a new insight into TCR-dependent and independent mechanisms that regulate Treg function and indicate a TCR-intrinsic insufficiency in tissue-specific Tregs that may contribute to the pathogenesis of type 1 diabetes.

immunology↗

bioRxiv2022

Comparative genomic analysis between Leonurus japonicus and Leonurus sibiricus

Leonurus japonicus Houtt. is an important medicinal plant in East Asia and is now widely recognized for its role in treating cerebral apoplexy and lowering blood lipids. Here, we report two sets of chromosome-level genome sequences for leonurine-producing Leonurus japonicus and for its closely related species leonurine-free Leonurus sibiricus, where 99.78% of 518.19 Mb of L. japonicus was assembled into ten pseudochromosomes with a contig N50 of 17.62 Mb and 99.33% of 472.29 Mb of L. sibiricus was assembled into nine pseudochromosomes with a contig N50 of 13.29 MB. The reference genomes of Leonurus will accelerate the decoding of novel bioactive molecules in medicinal plants, especially in the Lamiaceae family.

plant biology↗

bioRxiv2022

mEnrich-seq: Methylation-guided enrichment sequencing of bacterial taxa of interest from microbiome

Metagenomics has enabled the comprehensive study of microbiomes. However, many applications would benefit from a method that can sequence specific bacterial taxa of interest (pathogens, beneficial microbes, or low-abundance taxa), but not the vast background of other taxa in a microbiome sample. To address this need, we developed mEnrich-seq, a method that can enrich taxa of interest from metagenomic DNA before sequencing. The core idea is to exploit the self vs. non-self genome differentiation provided by natural bacterial DNA methylation and rationally choose methylation-sensitive restriction enzymes (REs), individually or in combination, to deplete host DNA and most background microbial DNA while enriching bacterial taxa of interest. This core idea is integrated with library preparation procedures in a way that only non-digested DNA libraries are sequenced. We performed in-depth evaluations of mEnrich-seq and demonstrated its use in several applications to enrich (up to 117-fold) genomic DNA of pathogenic or beneficial bacteria from human urine and fecal samples, including several species that are hard to culture or of low abundance. We also assessed the broad applicability of mEnrich-seq and found that 3130 (68.03%) of the 4601 strains with mapped methylomes to date can be targeted by at least one commercially available RE, representing 54.78% of the species examined in this analysis. mEnrich-seq provides microbiome researchers with a versatile and cost-effective approach for selective sequencing of diverse taxa of interest directly from the microbiome.

genomics↗

bioRxiv2022

Ultra-sensitive platelet proteome maps the O-glycosylation landscape and charts the response to thrombin dosage

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets tendency to pre-activate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1,300 proteins. Unbiased scanning for post-translational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterised sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal EMI domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new consensus sequence. Our data suggest that Protein O-fucosyltransferase 1 (POFUT1) is responsible for this modification. Secretion of MMRN1 was reduced in cells lacking POFUT1, supporting a key role of O-fucosylation in MMRN1 function. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3,800 proteins, which confirmed the platelet origin of releasate proteins by anti-correlation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The comprehensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis. Key PointsO_LIHigh-sensitivity proteome mapping of human platelets identifies O-glycosylation of platelet proteins at key functional sites C_LIO_LIPlatelet O-fucosyltransferase POFUT1 regulates the secretion of adhesive protein multimerin-1 (MMRN1) C_LI

biochemistry↗