Biology subjects

Xi, L.

Publications and source records attributed to Xi, L..

10 recordsLinked to original sources

bioRxiv2026

Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.

Genomic, transcriptomic and epigenomic characterization has accelerated the discovery of clinically-relevant alterations in cancer, predominantly using fresh frozen (FF) specimens. However, clinical molecular pathology laboratories prefer formalin-fixed paraffin-embedded (FFPE) methods, known to introduce artifacts at the nucleic acid level, over fresh frozen methods. Extending the multi-platform analysis to FFPE specimens for comprehensive clinical molecular diagnosis requires a thorough understanding of the consequence of formalin-fixation. We present a detailed multi-platform characterization of FFPE preservation using paired FF specimens as the 'gold standard'. DNA and RNA were obtained from 38 patients across 6 cancer types using a FFPE optimized co-isolation. The impact of FFPE on exome sequencing was dependent on filtering, where a minimum coverage or supporting read filter can mitigate FFPE-specific false positives. Copy number alterations, MSI assessment, mutational signatures, and DNA methylation were comparable between FFPE and FF. FFPE biases in RNA expression can be overcome when using biology-relevant genes and we describe a novel consequence of FFPE on miRNA species diversity. Collectively, this data provides a broad view of FFPE artifact and offers best practices for overcome these biases.

bioinformatics↗

bioRxiv2026

resolveS: An ultra-fast, memory-efficient and reference-free tool for RNA-seq Strandedness Detection

Accurate specification of RNA-seq library strandedness is essential for read counting, transcript assembly and antisense transcription analysis, yet this information is frequently missing from public metadata. We present resolveS, a fast and lightweight method that infers strandedness from alignments to a compact universal rRNA database, avoiding the need for organism-specific reference genomes and annotations. Across benchmark datasets, resolveS provides robust strandedness calls while maintaining very low runtime and memory usage, making it suitable for both routine quality control and large-scale reanalysis of public RNA-seq data.

bioinformatics↗

bioRxiv2024

Identification of nuclear pore proteins at plasmodesmata

Plasmodesmata (PD) mediate intercellular exchange of small molecules, RNAs and proteins between plant cells with an apparent exclusion limit for passive non-specific transport, and transport of specific cargo mediated by mediators. PD and nuclear pore complexes (NPC) are nanometer sized micropores with strikingly similar properties. Cargo translocation through NPC is mediated by phase separating FG-nucleoporins (FG-NUP). Here, bioinformatics, proteomics and fluorescence imaging identified FG-NUPs at PD. Transient expression of GFP fusions at low and intermediate expression levels supported dual localization of 12 NUPs to NPC and PD. Structured illumination microscopy detected the transmembrane anchor NUP CPR5 close to orifices of PD. cpr5 mutants showed reduced intercellular short-root (SHR) transport. However, transport defects cannot be excluded due to indirect effects in the mutants. Identification of FG-NUPs at PD is consistent with the recruitment of NUPs to form a PD pore gating complex consistent with phase separation domains as diffusion barriers at PD. Further analyses will be required to determine whether NUPs are bona fide PD components, or accumulate at PD in certain conditions, or may serve intermediate NPC storage.

cell biology↗

bioRxiv2022

A high confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution

Cells of multicellular organisms exchange nutrients, building blocks and information. In animals, this happens via gap junctions, in plants via plasmodesmata (PD). PD have striking properties, translocating a large range of molecules from ions, to metabolites, RNA and proteins up to 40 kDa. PD are hard to characterize due to being deeply embedded into cell walls and the presence of several membranes. While previous studies of protein composition of PD from angiosperms identified large lists of proteins, few were validated. Here, we developed a PD scoring approach in conjunction with systematic localization on a large scale to define a high-confidence PD proteome of Physcomitrium patens. This high confidence PD proteome comprises nearly 300 proteins, which together with the bona fide PD proteins from literature, are made available in the public PDDB database. Conservation of localization across plant species strengthens the reliability of plant PD proteomes and provides a basis for exploring the evolution of this important organelle. In particular, the P. patens PD proteome was highly enriched in cell wall modifying proteins. Callose-degrading glycolyl hydrolase family 17 (GHL17) proteins are presented as an abundant PD protein family with representatives across an evolutionary scale. Exclusively members of the alpha-clade of the GHL17 family are shown to be PD localized and their orthologs occur only in plant species which have developed PD. Members of the EXORDIUM-family and xyloglucan transglycosylases are additional cell-wall located proteins highly abundant in the P. patens PD proteome also showing evolutionary diversification of PD localized family members from other clade members.

plant biology↗

bioRxiv2022

PEP7 is a ligand for receptor kinase SIRK1 to regulate aquaporins and root growth

Plant receptor kinases constitute a large protein family that regulate various aspects of development and responses to external biotic and abiotic cues. Functional characterization of this protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified plasma membrane-intrinsic SUCROSE INDUCED RECEPTOR KINASE 1 (SIRK1) and QIAN SHOU KINASE 1 (QSK1) as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic conditions induced by sucrose. Here, we identified a member of the Elicitor Peptide (PEP) family, namely PEP7, as the specific ligand of receptor kinase SIRK1. PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44{+/-}0.79 {micro}M and is secreted to the apoplasm specifically in response to sucrose treatment. Stabilization of a signaling complex involving SIRK1, QSK1 and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application. Moreover, the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhance water influx into protoplasts. The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity, aquaporin phosphorylation and water influx activity. Our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module mediating sucrose-controlled water flux in plants.

plant biology↗

bioRxiv2020

Pharmacologic inhibition of Protein Kinase C α and Protein Kinase C β halts renal function decline indirectly, by blunting hyperphagia, and directly reduces adiposity in the ZSF1 rat model of type 2 diabetes

Type 2 diabetes (T2D) and its complications can have debilitating, sometimes fatal consequences. Despite advances that address some of the metabolic aspects of T2D, for many patients these approaches do not sufficiently control the disease. As a result, an emerging therapeutic strategy is to target the pathobiological mechanisms downstream of T2D metabolic derangement that can result in organ damage, morbidity, and mortality in afflicted individuals. One such proposed mechanism involves the Protein Kinase C (PKC) family members PKC and PKC{beta}, which have been linked to diabetes-induced tissue damage to organs including the kidneys. To evaluate the therapeutic potential of dual inhibition of PKC and PKC{beta} in the context of T2D, we have evaluated a potent and orally bioavailable inhibitor, herein referred to as Cmpd 1, in the ZSF1 rat model of leptin-receptor deficiency, obesity-driven T2D. Therapeutic dosing of Cmpd 1 virtually halted renal function decline but did so indirectly by blunting the hyperphagia response of these animals. Beyond this clear but indirect effect, Cmpd 1 had direct and prominent effects on body weight and in liver and inguinal white adipose tissue (iWAT) when administered to ZSF1 obese rats.

pharmacology and toxicology↗

bioRxiv2020

Atypical B cells are a normal component of immune responses to vaccination and infection in humans

The full diversity of the circulating human B cell compartment is unknown. Flow cytometry analysis suggests that in addition to naive and memory B cells, there exists a population of CD11c+, CD27- CD21- "atypical" B cells, that are associated with chronic or recurrent infection and autoimmunity. We used single cell RNA-seq approaches to examine the diversity of both antigen-specific B cells and total B cells in healthy subjects and individuals naturally-exposed to recurrent malaria infections. This analysis revealed two B cell lineages: a classical lineage of activated and resting memory B cells, and an atypical-like lineage. Surprisingly, the atypical lineage was common in both malaria exposed individuals and non-exposed healthy controls. Using barcoded antibodies in conjunction with our transcriptomic data, we found that atypical lineage cells in healthy individuals lack many atypical B markers and thus represent an undercounted cryptic population. We further determined using antigen specific probes that atypical cells can be induced by primary vaccination in humans and can be recalled upon boosting. Collectively these data suggest that atypical cells are not necessarily pathogenic but can be a normal component of B responses to antigen.

immunology↗

bioRxiv2019

Molecular profiling predicts meningioma recurrence and reveals loss of DREAM complex repression in aggressive tumors

Meningiomas account for one-third of all primary brain tumors. Although typically benign, about 20% of meningiomas are aggressive, and despite the rigor of the current histopathological classification system, there remains considerable uncertainty in predicting tumor behavior. Here, we analyzed 160 tumors from all three WHO grades (I-III) using clinical, gene expression and sequencing data. Unsupervised clustering analysis identified three molecular types (A, B, and C) that reliably predicted recurrence. These groups did not directly correlate with the WHO grading system, which would classify more than half of the tumors in the most aggressive molecular type as benign. Transcriptional and biochemical analyses revealed that aggressive meningiomas involve loss of the repressor function of the DREAM complex, which results in cell cycle activation; only tumors in this category tend to recur after full resection. These findings should improve our ability to predict recurrence and develop targeted treatments for these clinically challenging tumors.\n\nSignificance StatementMeningiomas are the most common primary brain tumors. Although most of these tumors are benign, one-fifth will recur despite apparently complete resection. Several studies have demonstrated that genomic approaches can yield important insights into the biology of these tumors. We performed RNA sequencing and whole-exome sequencing of 160 tumors from 140 patients, which identified three distinct groups of meningioma that correlate with recurrence better than the current WHO grading system. Our analysis also revealed that the most aggressive type was characterized by loss of the repressive DREAM complex. These findings should improve prognostication for patients and lead to viable therapeutic targets.

cancer biology↗

bioRxiv2019

De-risking drug discovery of intracellular targeting peptides: screening strategies to eliminate false-positive hits

Discovery of false-positive target binding, due to assay interference or aggregation, presents a significant problem for drug discovery programs. These issues may often be unrealized and could lead researchers astray if not subject to independent verification of reproducibility and/or on-target mechanism of action. Although well-documented for small molecules, this issue has not been widely explored for peptide modality. As a case study, we demonstrate that two purported KRas inhibitors, stapled peptide SAH-SOS1A and macrocyclic peptide cyclorasin 9A5, exemplify false-positive molecules - both in terms of their sub-micromolar KRas binding affinities and their on-target cellular activities. We observed that the apparent binding of fluorescein-labeled SAH-SOS1A given by a fluorescence polarization assay is sensitive to detergent. False-positive readouts can arise from peptide adsorption to the surface of microplates. Hence, we used surface plasmon resonance and isothermal titration calorimetry to unambiguously show that both SAH-SOS1A and cyclorasin 9A5 are non-binders for KRas. Thermal shift assay and hydrogen-deuterium exchange mass spectrometry further demonstrate that both peptides destabilize KRas and induce unfolding of the protein. Furthermore, both peptides caused significant release of intracellular lactate dehydrogenase, suggesting that membrane rupture rather than on-target activity is accountable for their reported cytotoxicity. Finally, both peptides exhibited off-target activities by inhibiting the proliferation of U-2 OS and A549 cells, despite their independency of the KRas signaling pathway. Our findings demonstrate the critical need to employ orthogonal binding assays and cellular counter-screens to de-risk false-positive molecules. More rigorous workflows should lead to improved data and help obviate inadvertent scientific conclusions.\n\nSignificance statementFalse positive molecule hits occur frequently in high-throughput screens and can contaminate the scientific literature. This has become an increasingly serious issue in small molecule drug discovery and chemical probe development and it is not surprising that peptides may be similarly prone to assay interference. Using KRas as a target and two known macrocyclic peptide inhibitors as a case study, we clearly show that reporter-free biophysical assays and cellular counter-screens offer the solution to detect and de-risk the potential of false-positive compounds. We further discuss the advantages, limitations and overall strategic importance of such methods.

biochemistry↗

bioRxiv2017

The ERA-related GTPase AtERG2 associated with mitochondria 18S RNA is essential for early embryo development in Arabidopsis

The ERA (E. coli RAS-like protein)-related GTPase (ERG) is a nuclear-encoded GTPase with two conserved domains: a GTPase domain and a K Homology domain. ERG plays a vital role in early seed development in Antirrhinum majus. However, the mechanism that regulates seed development remains unclear. Blasting the genome sequence revealed two homologies of ERG, AtERG1, and AtERG2 in Arabidopsis. In this study, we found that AtERG2 is localised in the mitochondria and binds mitochondrial 18S RNA. Promoter and transcript analyses indicated that AtERG2 was mainly expressed in the leaf vein, trichome, mature pollen, and ovule. The mutants of AtERG2 showed recessive lethal, gametophytic maternal effects, silique shortage, and early seed abortion, in which some seeds arrested in the zygotic stage at 1.5 days after pollination (DAP) and aborted at 2.0 DAP in aterg2-1 +/-. Reactive oxygen species (ROS) accumulated at 1.5 DAP in the arrested seeds, and the transcription of several ROS-responsible genes, WRKY40, ANAC017, and AOXla, was up-regulated in the aterg2-1 +/- seeds which were arrested 1.5 and 2.0 DAP but not in wild-type (WT) and aterg2-1 +/- seeds. The cell death-related gene BAG6 was also transcriptionally activated in aterg2-1 +/- seeds arrested at 2.0 DAP. Chloramphenicol treatment during pollination induced a similar phenotype and gene expression pattern but showed no transcriptional changes of ANAC017 in WT. These results suggested that AtERG2 promotes early seed development by affecting the maturation of the mitochondria ribosome small subunit and mitochondrial protein translation in Arabidopsis.

plant biology↗