Biology subjects

Bai, X.

Publications and source records attributed to Bai, X..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Vascular-water-exchange MRI (VEXI) enables the detection of subtle BBB breakdown in Alzheimer's disease without MRI contrast agent

Blood-brain barrier (BBB) impairment is an important pathophysiological process in Alzheimers disease (AD) and a potential biomarker for early diagnosis of AD. However, most current neuroimaging methods assessing BBB function need the injection of exogenous contrast agents (or tracers), which limits the application of these methods in a large population. In this study, we aim to explore the feasibility of vascular water exchange MRI (VEXI), a diffusion-MRI-based method to assess the BBB permeability to water molecules without using a contrast agent, in the detection of the BBB breakdown in AD. We tested VEXI on a 3T MRI scanner on three groups: AD patients (AD group), mild cognitive impairment (MCI) patients due to AD (MCI group), and the age-matched normal cognition subjects (NC group). Interestingly, we find that VEXI can detect the BBB permeability to water molecules increase in MCI and this BBB breakdown happens specifically in the hippocampus. This BBB breakdown gets worse and extends to more brain regions (orbital frontal cortex and thalamus) from MCI group to the AD group. Furthermore, we find that the BBB breakdown of these three regions detected by VEXI is correlated significantly with impairment of respective cognitive domains independent of age, sex and education. These results suggest VEXI is a promising method to assess the BBB breakdown in AD. HighlightsO_LIThe vascular water exchange MRI (VEXI) is a contrast-agent-free method to assess BBB permeability C_LIO_LIBBB breakdown happens specifically in the hippocampus, orbital frontal cortex, and thalamus in AD C_LIO_LIBBB breakdown detected by VEXI is significantly correlated with cognitive dysfunction C_LI

neuroscience↗

bioRxiv2022

Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

Obesity has always been considered a significant risk factor in OA progression, but the underlying mechanism of obesity-related inflammation in OA synovitis remains unclear. The present study found that synovial macrophages infiltrated and polarized in the obesity microenvironment and identified the essential role of M1 macrophages in impaired macrophage efferocytosis using pathology analysis of obesity-associated OA. The present study revealed that obese OA patients and ApoE-/- mice showed a more pronounced synovitis and enhanced macrophage infiltration in synovial tissue, accompanied by dominant M1 macrophage polarization. Obese OA mice had a more severe cartilage destruction and increased levels of synovial apoptotic cells than OA mice in the control group. Enhanced M1-polarized macrophages in obese synovium decreased growth arrest-specific 6 (GAS6) secretion, resulting in impaired macrophage efferocytosis in synovial apoptotic cells. Intracellular contents released by accumulated apoptotic cells further triggered an immune response and lead to a release of inflammatory factors, such as TNF-, IL-1{beta}, and IL-6, which induce chondrocyte homeostasis dysfunction in obese OA patients. Intra-articular injection of GAS6 restored the phagocytic capacity of macrophages, reduced the accumulation of local apoptotic cells, and decreased the levels of TUNEL- and caspase-3-positive cells, preserving cartilage thickness and preventing the progression of obesity-associated OA. Therefore, blocking M1 macrophage polarization or intra-articular injection of GAS6 is a potential therapeutic strategy for obesity-associated OA.

immunology↗

bioRxiv2022

Novel algorithms for improved detection and analysis of fluorescent signal fluctuations

Fluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra-as well as intercellular communication. Using advanced imaging technologies, fluorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and avnalysis of fluorescence signals requires to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation of fluorescent detection of messenger molecules and automated detection of fluorescence fluctuations. Author summaryAnalyzing molecular signalling is crucial in understanding intra- and intercellular communication. These signals are visualized using fluorescent dyes or genetically encoded fluorescence indicators. In the brain, Ca2+ signals of glial cells are essential in deciphering complex regulatory functions in health and disease. Due to signal heterogeneity, detection and analysis are highly challenging. They can be stationary, with low amplitude and localized in cell processes, occur as prominent somatic signals or propagate as waves across cellular networks. We have developed two algorithms to analyze fluorescence transients, each tackling a specific problem. PBasE performs automatic and adaptive background correction, removing basal fluorescence fluctuations. CoRoDe automatically extracts regions of interest, explicitly including temporal information to obtain a precise segmentation, which is essential for accurate transient extraction. Combined, these algorithms are able to detect regions exhibiting low amplitude transients with small spatial extent as well as large, high amplitude signals. Extracted transients are categorized based on their peak amplitude, allowing detailed analyses by comparing changes of specific properties. In order to make these algorithms accessible, an interactive application, called Msparkles, has been designed.

neuroscience↗

bioRxiv2022

Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV- 2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

microbiology↗

bioRxiv2022

Single cell multi-omic mapping of subclonal architecture and pathway phenotype in primary gastric and metastatic colon cancers

Single cell genomics provides a high-resolution profile of intratumoral heterogeneity and subclonal structure in primary and metastatic tumors. Notably, metastases and therapeutic resistant tumors often originate from distinct subclones. These distinct cellular populations are an important contributor to adaptation and resistance to ongoing therapy. Single cell DNA-sequencing (scDNA-seq) defines subclones but does not provide biological information about cell types. Single cell RNA-sequencing (scRNA-seq) provides biological information but is less useful for identifying different subclones. The integrated scDNA-seq and scRNA-seq data from the same tumor cell population provides both subclone structure and biology. To understand the cellular genomic variation of different subclones in primary and metastatic cancers, we developed an approach to integrate multi-omics data from both types of single cells. This joint data set represented thousands of normal and tumor cells derived from a set of primary gastric and metastatic colorectal cancers. The extensive cellular sampling provided robust characterization of the subclonal architecture of gastric and colorectal cancers. We reconstructed the subclonal architecture using the cells in G0/G1 phase. The scDNA-seq provided a ground truth for copy number-based subclones. From the scRNA-seq data, the epithelial cells in G0/G1 were identified and assigned to specific subclones by a correlation algorithm based on gene dosage. The inferred CNV profiles from the scRNA-seq epithelial cells were assigned subclones identified from the scDNA-seq. Afterward, we determined the biological pathway activities of specific clones. Overall, integrative multi-omics analysis of single-cell datasets is more informative than any individual genomic modality, provides deep insights into the intratumoral heterogeneity, and reveals subclonal biology.

genomics↗

bioRxiv2022

Single molecule methylation profiles of cell-free DNA in cancer with nanopore sequencing

Epigenetic characterization of cell-free DNA (cfDNA) is an emerging approach for characterizing diseases. We developed a strategy using nanopore-based single-molecule sequencing to measure cfDNA methylomes. This approach generated up to hundreds of millions of reads per cfDNA sample from cancer patients, an order of magnitude improvement over existing nanopore methods. Leveraging methylomes of matched tumors and immune cells, we characterized cfDNA methylomes of cancer patients for longitudinal monitoring during treatment.

genomics↗

bioRxiv2022

Brain injuries and complex motor learning suppress Olig2 in a subpopulation of oligodendrocyte precursor cells

Oligodendrocyte precursor cells (OPCs) are uniformly distributed in the mammalian brain, however their function is rather heterogeneous in respect to their origin, location, receptor/channel expression and age. The basic helix-loop-helix transcription factor Olig2 is expressed in all OPCs as a pivotal determinant of their differentiation. Here, we identified a subset (2-26%) of OPCs lacking Olig2 in various brain regions including cortex, corpus callosum, CA1 and dentate gyrus. These Olig2 negative (Olig2neg) OPCs were enriched in the juvenile brain and decreased subsequently with age, being rarely detectable in the adult brain. However, the loss of this population was not due to apoptosis or microglia-dependent phagocytosis. Unlike Olig2pos OPCs, these subset cells could not be labelled for the mitotic marker Ki67. And, accordingly, BrdU was incorporated only by a three-day long-term labeling but not by a two-hour short pulse, suggesting these cells do not proliferate any more but were derived from proliferating OPCs. The Olig2neg OPCs exhibited a less complex morphology than Olig2pos ones. Olig2neg OPCs preferentially remain in a precursor stage rather than differentiating into highly branched oligodendrocytes. Changing the adjacent brain environment, e.g. by acute injuries or by complex motor learning tasks stimulated the transition of Olig2pos OPCs to Olig2neg cells in the adult. Taken together, our results demonstrate that OPCs transiently suppress Olig2 upon changes of the brain activity. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/496631v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f8bc79org.highwire.dtl.DTLVardef@11dd807org.highwire.dtl.DTLVardef@51d2e9org.highwire.dtl.DTLVardef@1edf9a1_HPS_FORMAT_FIGEXP M_FIG C_FIG Main points: A subset of OPCs do not express Olig2, of which population peaks in the juvenile brain while wanes with age. Plastic changes of the brain by acute injuries or complex motor learning stop the expression of Olig2 in OPCs.

neuroscience↗

bioRxiv2022

Distinct cell states define the developmental trajectories of mucinous appendiceal neoplasms towards pseudomyxoma metastases

Appendiceal mucinous neoplasms (AMN) are rare tumors of the gastrointestinal tract. They metastasize with widespread abdominal dissemination leading to pseudomyxoma peritonei (PMP), a disease with poor prognosis. The cellular features of origin, differentiation and progression in AMN and PMP remain largely unknown. We characterized the distinct cell states of AMN and PMP using single-cell RNA-sequencing in 31 samples including AMNs, PMP metastases and matched normal tissues. We identified previously undescribed cellular features and heterogeneity in AMN and PMP. We discovered developmental cell states in tumor epithelial cells that ranged from progenitor phase to goblet cell differentiation, which was accompanied by elevated mucin gene expression. Metastatic PMP cells had a distinct cell state with increased mTOR and RAS signaling pathways and a series of upregulated cancer genes. We observed heterogeneity in a single PMP tumor as well as PMP metastases from the same patient. We validated our findings with immunohistochemistry, mass spectrometry on malignant ascites from PMP patients and gene expression data from an independent set of 63 PMPs.

cancer biology↗

bioRxiv2022

CRISPR screens reveal genetic determinants of PARP inhibitor sensitivity and resistance in prostate cancer

Prostate cancer (PCa) harboring BRCA1/2 mutations is often exquisitely sensitive to PARP inhibition. However, genomic alterations in other DNA damage response genes have not been consistently predictive of clinical response to PARP inhibitors (PARPis). Here, we perform genome-wide CRISPR-Cas9 knockout screens in BRCA1/2-proficient PCa cell lines and identify novel genes whose loss has a profound impact on PARPi sensitivity and resistance. Specifically, MMS22L deletion, frequently observed (up to 14%) in PCa, renders cells hypersensitive to PARPis by disrupting RAD51 loading required for homologous recombination repair, although this response is TP53-dependent. Unexpectedly, loss of CHEK2 confers resistance rather than sensitivity to PARPis in PCa cells through increased expression of BRCA2, a target of CHEK2-TP53-E2F7-mediated transcriptional repression. Combined PARP and ATR inhibition overcomes PARPi resistance caused by CHEK2 loss. Our findings may inform the use of PARPis beyond BRCA1/2-deficient tumors and support reevaluation of currently used biomarkers for PARPi treatment in PCa.

cancer biology↗

bioRxiv2022

Time-series monitoring of transgenic maize seedlings phenotyping exhibiting glyphosate tolerance

Developing herbicide resistant cultivars is one of the effective methods to solve the safety problem caused by the use of herbicide. In this study, hyperspectral image was used to develop more robust leaf chlorophyll content (LCC) prediction model based on different datasets to finally analyze the response of LCC to glyphosate-stress. Chlorophyll a fluorescence (ChlF) was used to dynamically monitor the photosynthetic physiological response of transgenic glyphosate-resistant and wild glyphosate-sensitive maize seedlings, and applying chemometrics methods to extract time-series features to screen resistant cultivars. Both the proposed two transfer strategies achieved the best prediction of LCC with a coefficient of determination value of 0.84, and relative root mean square error of 4.03 for the prediction set. Based on the predicted LCC and ChlF data, we found the antioxidant system of glyphosate-sensitive plants is too fragile to protect themselves from the damage, while glyphosate-resistant plants could overcome it by activating more powerful antioxidant system. {varphi}E0, VJ, {psi}E0, and M0 could be used to indicate damage caused by glyphosate to differentiate resistant cultivars. This study provided a new methodology to monitor LCC to finally analyze glyphosate tolerance in a time-series manner, and verified the feasibility of ChlF in crop breeding. HighlightThis study proposed a new methodology to monitor leaf chlorophyll content to finally analyze glyphosate tolerance in vivo, and verified the feasibility of chlorophyll a fluorescence in crop breeding.

plant biology↗

bioRxiv2022

Human endogenous retrovirus activation contributes to biliary atresia pathogenesis through re-education of resident macrophages

Biliary atresia (BA) is a life-threatening neonatal fibro-inflammatory disease characterized by hepatic fibrosis, cirrhosis, and end-stage liver failure. BA is also the most frequent indication of pediatric liver transplantation globally. Despite the devastating condition of BA, the pathogenesis mechanism is unknown. Viral infection has been suggested to be associated with BA, but definitive evidence to support this hypothesis is not available. To elucidate the virus-associated pathogenesis mechanism of BA and to understand the immune ecosystem, we performed single-cell transcriptomic and proteomic profiling of BA livers. We detected human endogenous virus (HERV) in infants with BA and their parents. HERV was mainly found in FOLR2+ resident macrophages, T cells, and NK cells. In addition, HERV activation re-educated the fetal-derived FOLR2+ resident macrophages, and reactive oxygen species scavenging neutrophil recruitment was impaired in patients with BA and HERV+, due to FOLR2+ resident macrophage re-education. Furthermore, we showed depletion of FOLR2+ resident macrophage and N-acetylcysteine treatment could rescue the liver damage in BA. Overall, our study revealed the HERV-associated immunopathology mechanism of BA. These results contribute to potential diagnosis and immunotherapy strategies for BA.

pathology↗

bioRxiv2022

Alpha-1-antitrypsin binds to the glucocorticoid receptor with biological significance in macrophages

Alpha-1-antitrypsin (AAT), a serine protease inhibitor produced mainly by the liver, is the third most abundant protein in plasma. While a canonical receptor for AAT has not been identified, AAT can be internalized into the cytoplasm and is known to affect gene regulation. Since AAT has significant anti-inflammatory properties affecting many cell types including macrophages, we examined whether AAT binds the cytoplasmic glucocorticoid receptor (GR) in macrophages. We report the novel finding that AAT binds to GR in macrophages using several approaches, including co-immunoprecipitation, mass spectrometry, microscale thermophoresis, and molecular modeling. The mass spectrometry data are available via ProteomeXchange with identifier PXD030989. We further demonstrate that AAT induction of angiopoietin-like 4 protein and AAT inhibition of lipopolysaccharide-induced nuclear factor-kappa B activation and interleukin-8 production are mediated, in part, through AAT-GR interaction. Furthermore, this interaction contributes to a host-protective role against mycobacteria in macrophages. The interaction of AAT and GR described in this study identifies a mechanism for the antiinflammatory and host-defense properties of AAT.

immunology↗

bioRxiv2022

SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex

Munc18-1 forms a template to recruit and organize assembly of the SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its N-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 N-terminal region can help nucleate interactions between the N-termini of the syntaxin-1 and synaptobrevin SNARE motifs while their C-termini bind to distal sites of Munc18-1. Mutagenesis, SNARE complex assembly assays and reconstitution experiments support a model whereby these interactions are critical to initiate SNARE complex assembly. One Sentence SummaryCryo-EM structures reveal key insights into the molecular mechanism of neuronal SNARE complex assembly templated by Munc18-1

biophysics↗

bioRxiv2022

The influence of leaf anatomical traits on photosynthetic in coffee leaves in Yunnan of China

Leaf photosynthesis is largely determined by anatomical features. This study aimed to reveal the quantitative effects of the anatomical structure of Coffea arabica leaves on photosynthesis. Pearsons correlation and path analysis were used to explore the correlation between leaf structure and photosynthesis. To calculate the comprehensive evaluation value of the correlation between leaf anatomical traits and photosynthetic parameters, the Criteria Importance Through Intercriteria Correlation (CRITIC) method was used to obtain the objective weight of each photosynthetic parameter. Most of the anatomical traits of the leaf significantly (P<0.05) to highly significantly (P<0.01) correlated with photosynthetic parameters, suggesting that anatomical traits greatly affected the photosynthetic capacity in Coffea arabica leaf. Likewise, path coefficient analysis revealed that the photosynthetic capacity of Arabic coffee leaves is strongly correlated, both directly and indirectly, with the stomatal characteristics of the leaves. The results of the comprehensive evaluation also indicated that leaf thickness (LT) and stomatal density (d) were the anatomical characteristics most closely related to photosynthesis. In these results, understanding the effects of the anatomical structure of coffee leaves on photosynthesis, may provide useful information for coffee breeding programs and the management of coffee plantations to increase photosynthesis.

biophysics↗

bioRxiv2022

Heparins enhance C1 esterase inhibitor activity: a promising remedy for acute hereditary angioedema

RationaleHereditary angioedema (HAE) is a potentially life-threatening illness most commonly due to deficiency or dysfunction of C1-esterase inhibitor (C1-INH). While specific treatments are available to thwart acute exacerbations, they are extremely costly and some can be associated with rare but serious side effects. The heparins are long known to augment C1-INH activity and case reports / series have documented their efficacy in treating HAE. Objectiveto determine if unfractionated heparin and two low-molecular weight heparins (enoxaparin and nadroparin) can augment C1-INH activity ex vivo in the sera of patients with HAE and in an in vitro biochemical assay. MethodsC1-INH activity in the absence or presence of the heparin formulations were analyzed by two different methods. To measure C1-INH activity ex vivo, a commercially available assay was utilized with patient sera, excess amounts of C1s, and a substrate of C1s which, upon cleavage by C1s, produces a chromogenic product. To determine biochemically the C1-INH activity in vitro, a pharmacologic grade C1-INH, recombinant C1s (C1s-CCP12SP), and a peptide substrate of C1s were employed. Microscale thermophoresis was used to determine whether C1-INH binds to heparin. Main resultsin patient sera, nadroparin was superior to enoxaparin and unfractionated heparin in augmenting C1-INH activity, followed by enoxaparin and then unfractionated heparin. In the in vitro biochemical assay, all three heparins augmented C1-INH-C1s binding linearly in a dose-dependent fashion. Microscale thermophoresis assay demonstrated that nadroparin binds to C1-INH, providing a mechanism by which heparin facilitates the interaction between C1-INH and the proteases known to produce bradykinin, the mediator of HAE. Conclusionlow-molecular weight heparin augments C1-INH activity and should be studied as a potential treatment for acute HAE.

pharmacology and toxicology↗

bioRxiv2022

Plasma proteomic biomarkers identify non-responders and reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses. Therefore, there is a critical need to identify early non-invasive biomarkers of response. We performed plasma proteomic analysis (>700 proteins) at three timepoints on 174 metastatic melanoma patients treated with ICB. We leverage independent training and testing cohorts to build a predictor of immunotherapy response that outperforms several tissue-based approaches. We found 217 differentially expressed proteins between ICB responders (R) and non-responders (NR), including a co-regulated module of proteins enriched in certain NR patients. By analyzing single-cell RNA-sequencing data of tumor biopsies from 32 patients, we dissected the relative contribution of cells in the tumor to proteins in circulation. The majority of proteins in the co-regulated NR module derived from tumor and myeloid cells. Amongst myeloid cells, we identified a subset of tumor-associated macrophages (TAMs) with a suppressive phenotype that expressed high levels of the co-regulated NR module, thus suggesting they are key drivers of non-response signatures. Together, our data demonstrates the utility of plasma proteomics in biomarker discovery and in understanding the biology of host response to tumors.

cancer biology↗

bioRxiv2022

Ex vivo and in vivo evidence that cigarette smoke-exposed T regulatory cells impair host immunity against Mycobacterium tuberculosis

A strong epidemiologic link exists between exposure to cigarette smoke (CS) and increased susceptibility to tuberculosis (TB). In vitro macrophage and in vivo murine studies showed that CS and nicotine impair host-protective immune cells against Mycobacterium tuberculosis (MTB) infection. However, little is known about how CS may affect immunosuppressive cells in the context of MTB infection. Thus, we investigated whether CS-exposed T regulatory cells (Tregs) could exacerbate MTB infection in co-culture with human macrophages and in the adoptive transfer of Tregs from air- and CS-exposed mice. We found that exposure of primary human Tregs to CS extract impaired the ability of human monocyte-derived macrophages to control an MTB infection by inhibiting phagosome-lysosome fusion and autophagosome formation. Neutralization of CTLA-4 on the CS extract-exposed Tregs abrogated the impaired control of MTB infection in macrophage and Treg co-cultures. In Foxp3+GFP+DTR+ (Thy1.2) mice depleted of endogenous Tregs, adoptive transfer of Tregs from donor CS-exposed B6.PL(Thy1.1) mice with subsequent MTB infection of the recipient Thy1.2 mice resulted in a greater burden of MTB in the lungs and spleens than those that received Tregs from airexposed mice. Mice that received Tregs from CS-exposed mice and then infected with MTB had modest but significantly reduced numbers of interleukin-12-positive dendritic cells and interferon-gamma-positive CD4+ T cells in the lungs and increased number of programmed cell death protein-1 positive CD4+ T cells in both the lungs and spleens.

immunology↗

bioRxiv2021

TK216 targets microtubules in Ewing sarcoma cells

Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11;22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for EWS patients. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding -tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.

cancer biology↗