Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 361 records · Page 20Linked to original sources

bioRxiv2024

Unraveling Axonal Transcriptional Landscapes: Insights from iPSC-Derived Cortical Neurons and Implications for Motor Neuron Degeneration

Neuronal function and pathology are deeply influenced by the distinct molecular profiles of the axon and soma. Traditional studies have often overlooked these differences due to the technical challenges of compartment specific analysis. In this study, we employ a robust RNA-sequencing (RNA-seq) approach, using microfluidic devices, to generate high-quality axonal transcriptomes from iPSC-derived cortical neurons (CNs). We achieve high specificity of axonal fractions, ensuring sample purity without contamination. Comparative analysis revealed a unique and specific transcriptional landscape in axonal compartments, characterized by diverse transcript types, including protein-coding mRNAs, RNAs encoding ribosomal proteins (RPs), mitochondrial-encoded RNAs, and long non-coding RNAs (lncRNAs). Previous works have reported the existence of transcription factors (TFs) in the axon. Here, we detect a set of TFs specific to the axon and indicative of their active participation in transcriptional regulation. To investigate transcripts and pathways essential for central motor neuron (MN) degeneration and maintenance we analyzed KIF1C-knockout (KO) CNs, modeling hereditary spastic paraplegia (HSP), a disorder associated with prominent length-dependent degeneration of central MN axons. We found that several key factors crucial for survival and health were absent in KIF1C-KO axons, highlighting a possible role of these also in other neurodegenerative diseases. Taken together, this study underscores the utility of microfluidic devices in studying compartment-specific transcriptomics in human neuronal models and reveals complex molecular dynamics of axonal biology. The impact of KIF1C on the axonal transcriptome not only deepens our understanding of MN diseases but also presents a promising avenue for exploration of compartment specific disease mechanisms.

neuroscience↗

bioRxiv2024

FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis

Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "Filament IDentification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of gently lysed cells. FilamentID identified the mitochondrial filaments as the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments being composed of acetyl-CoA synthetase Acs1ACSS2. The near-native high-resolution structures revealed the mechanism underlying polymerization and enabled us to perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores, and more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts. HIGHLIGHTSO_LIFilamentID: a multiscale imaging workflow to characterize cellular filaments of unknown composition C_LIO_LIThe conserved aldehyde dehydrogenase Ald4ALDH2 polymerizes into filament arrays within meiotic mitochondria C_LIO_LIThe conserved acetyl-CoA synthetase Acs1ACSS2 forms filament arrays in the nucleus and the cytoplasm C_LIO_LIMetabolites mediate Acs1 polymerization to store Acs1 in an inactive state in gametes and starved cells C_LIO_LIAcs1 filament formation is required for efficient return to growth from starvation stress C_LI

cell biology↗

bioRxiv2024

The Role of Morphological Adaptability in Vibrio cholerae's Motility and Pathogenicity

Vibrio cholerae, the etiological agent of cholera, exhibits remarkable adaptability to different environmental conditions by undergoing morphological changes that significantly contribute to its pathogenicity and impact the epidemiology of the disease globally. This study investigates the morphological adaptability of the clinically isolated V. cholerae O1 strain, specifically focusing on the motility and pathogenicity differences between the filamentous and original comma-shaped forms within diverse viscosity conditions. Utilizing the El Tor strain of V. cholerae O1, we induced the transformation into the filamentous form and performed a comparative analysis with the canonical comma-shaped morphology. Our approach involved assessing motility patterns, swimming speeds, rotation rates, kinematics, and reversal frequencies through dark-field microscopy and high-speed imaging techniques. The findings reveal that filamentous V. cholerae cell retains enhanced motility in viscous environments. This suggests an evolutionary adaptation enabling survival across a range of habitats, notably the human gastrointestinal tract. Filamentous forms demonstrated increased reversal behavior at mucin interfaces, hinting at an advantage in penetrating the mucus layer. Rabbit intestinal loop assays further showed that both morphological forms exhibit similar fluid accumulation ratios, thus indicating comparable pathogenic potentials. These results underscore the significance of V. choleraes morphological flexibility in adapting to environmental viscosity changes, shedding light on the bacteriums intricate survival and infection strategies. Our study provides critical insights into the dynamics of cholera, underlining the importance of considering bacterial morphology in developing effective cholera control strategies.

microbiology↗

bioRxiv2024

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

cell biology↗

bioRxiv2024

Decoding Continuous Character-based Language from Non-invasive Brain Recordings

Deciphering natural language from brain activity through non-invasive devices remains a formidable challenge. Previous non-invasive decoders either require multiple experiments with identical stimuli to pinpoint cortical regions and enhance signal-to-noise ratios in brain activity, or they are limited to discerning basic linguistic elements such as letters and words. We propose a novel approach to decoding continuous language from single-trial non-invasive fMRI recordings, in which a three-dimensional convolutional network augmented with information bottleneck is developed to automatically identify responsive voxels to stimuli, and a character-based decoder is designed for the semantic reconstruction of continuous language characterized by inherent character structures. The resulting decoder can produce intelligible textual sequences that faithfully capture the meaning of perceived speech both within and across subjects, while existing decoders exhibit significantly inferior performance in cross-subject contexts. The ability to decode continuous language from single trials across subjects demonstrates the promising applications of non-invasive language brain-computer interfaces in both healthcare and neuroscience.

neuroscience↗

bioRxiv2024

Curating models from BioModels: Developing a workflow for creating OMEX files

The reproducibility of computational biology models can be greatly facilitated by widely adopted standards and public repositories. We examined 50 models from the BioModels Database and attempted to validate the original curation and correct some of them if necessary. For each model, we reproduced these published results using Tellurium. Once reproduced we manually created a new set of files, with the model information stored by the Systems Biology Markup Language (SBML), and simulation instructions stored by the Simulation Experiment Description Markup Language (SED-ML), and everything included in an Open Modeling EXchange (OMEX) file, which could be used with a variety of simulators to reproduce the same results. On the one hand, the reproducibility procedure of 50 models developed a manual workflow that we would use to build an automatic platform to help users more easily curate and verify models in the future. On the other hand, these exercises allowed us to find the limitations and possible enhancement of the current curation and tooling to verify and curate models.

systems biology↗

bioRxiv2024

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

cell biology↗

bioRxiv2024

Cancer Stemness Online: A resource for investigating cancer stemness and associations with immune response

Cancer progression involves the gradual loss of a differentiated phenotype and acquisition of progenitor and stem-cell-like features, which are potential culprit in immunotherapy resistance. Although the state-of-art predictive computational methods have facilitated predicting the cancer stemness, currently there is no efficient resource that can meet various requirements of usage. Here, we presented the Cancer Stemness Online, an integrated resource for efficiently scoring cancer stemness potential at bulk and single-cell level. The resource integrates 8 robust predictive algorithms as well as 27 signature gene sets associated with cancer stemness for predicting the stemness scores. Downstream analyses were performed from five different aspects, including identifying the signature genes of cancer stemness, exploring the association with cancer hallmarks, cellular states, immune response and communication with immune cells, investigating the contributions for patient survival and the robustness analysis of cancer stemness among different methods. Moreover, the pre-calculated cancer stemness atlas for more than 40 cancer types can be accessed by users. Both the tables and diverse visualization for the analytical results are available for download. Together, Cancer Stemness Online is a powerful resource for scoring cancer stemness and going deeper and wider in the downstream functional interpretation, including immune response as well as cancer hallmark. Cancer Stemness Online is freely accessible at http://bio-bigdata.hrbmu.edu.cn/CancerStemnessOnline.

bioinformatics↗

bioRxiv2024

FTO-mediated m6A demethylation of ULK1 mRNA promotes autophagy and activation of hepatic stellate cells in liver fibrosis

The activation of hepatic stellate cells (HSCs) is the central link in the occurrence and development of liver fibrosis. Our previous studies showed that autophagy promotes HSCs activation and ultimately accelerates liver fibrosis. Unc-51-like autophagy activating kinase 1 (ULK1) is an autophagic initiator in mammals and N6-methyladenosine (m6A) modification is closely related to autophagy. In this study, we find that m6A demethylase fat mass and obesity-associated protein (FTO) is upregulated during HSCs activation and bile duct ligation (BDL)-induced hepatic fibrosis, which is the m6A methylase with the most significant difference in expression. Importantly, we identify that FTO overexpression aggravates HSCs activation and hepatic fibrosis via autophagy. Mechanistically, compared with other autophagy-related genes, ULK1 is the target of FTO due to FTO mainly mediates the m6A demethylation of ULK1 and upregulates its expression, thereby enhancing autophagy and activation of HSCs. Noteworthy, m6A reader YTH domain-containing protein 2 (YTHDC2) decreases ULK1 mRNA level via recognizing the m6A binding site and ultimately inhibits autophagy and activation of HSCs. Taken together, our findings highlight m6A-dependent ULK1 as an essential regulator of HSCs autophagy and reveal ULK1 as a novel potential therapeutic target for hepatic fibrosis treatment. Graphical Abstractm6A demethylases FTO promoted autophagy via recognizing the ULK1 m6A binding site, thus triggering HSCs activation, and eventually leading to liver fibrosis. In this process, YTHDC2 participated in the translation of ULK1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/584975v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@4fdd01org.highwire.dtl.DTLVardef@136791forg.highwire.dtl.DTLVardef@149f448org.highwire.dtl.DTLVardef@44ca9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Development of alcoholic liver disease model for drug evaluation from human induced pluripotent stem cell derived liver organoids

Alcoholic liver disease (ALD) poses a significant health challenge, demanding comprehensive research efforts to enhance our comprehension and treatment strategies. However, the development of effective treatments is hindered by the limitations of existing liver disease models. Liver organoids, characterized by their cellular complexity and three-dimensional (3D) tissue structure closely resembling the human liver, hold promise as ideal models for liver disease research. In this study, we employ a meticulously designed protocol involving the differentiation of human induced pluripotent stem cells (hiPSCs) into liver organoids. This process incorporates a precise combination of cytokines and small molecule compounds within a 3D culture system to guide the differentiation process. Subsequently, these differentiated liver organoids are subjected to ethanol treatment to induce ALD, thus establishing a disease model. Rigorous assessment through a series of experiments reveals that this model partially replicates key pathological features observed in clinical ALD, including cellular mitochondrial damage, elevated cellular reactive oxygen species (ROS) levels, fatty liver, and hepatocyte necrosis. In addition, this model offers potential utility in screening drugs for ALD treatment. Taken together, the liver organoids model of ALD, derived from hiPSCs differentiation, emerges as an invaluable platform for advancing our understanding and management of ALD in clinical settings.

cell biology↗

bioRxiv2024

A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma

Pediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Through in vitro pharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

cancer biology↗

bioRxiv2024

Feature-independent Encoding of Visual Salience inthe Mouse Superior Colliculus

Detecting conspicuous stimuli in a visual scene is crucial for animal survival, yet it remains debated how the brain encodes visual saliency. Here we investigate how visual saliency is represented in the superficial superior colliculus (sSC) of awake mice using two-photon calcium imaging. We report on a preference-independent saliency map in the sSC. Specifically, salient stimuli evoke stronger responses in both excitatory and inhibitory neurons compared to uniform stimuli, with similar encoding patterns observed in both neuron types. The largest response occurs when a salient stimulus is positioned at the receptive field center, with contextual effects extending [~]40{degrees} away from the center. The response amplitude correlates well with the saliency strength of stimuli and is not influenced by the orientation or motion direction preferences of neurons. However, saliency encoding does depend on specific visual features. Furthermore, neurons involved in saliency encoding exhibit weak orientation or direction selectivity, suggesting a complementary relationship between the saliency map and the feature map.

neuroscience↗

bioRxiv2024

A resource to enable chemical biology and drug discovery of WDR Proteins

Protein class-focused drug discovery has a long and successful history in pharmaceutical research, yet most members of druggable protein families remain unliganded, often for practical reasons. Here we combined experiment and computation to enable discovery of ligands for WD40 repeat (WDR) proteins, one of the largest human protein families. This resource includes expression clones, purification protocols, and a comprehensive assessment of the druggability for hundreds of WDR proteins. We solved 21 high resolution crystal structures, and have made available a suite of biophysical, biochemical, and cellular assays to facilitate the discovery and characterization of small molecule ligands. To this end, we use the resource in a hit-finding pilot involving DNA-encoded library (DEL) selection followed by machine learning (ML). This led to the discovery of first-in-class, drug-like ligands for 9 of 20 targets. This result demonstrates the broad ligandability of WDRs. This extensive resource of reagents and knowledge will enable further discovery of chemical tools and potential therapeutics for this important class of proteins.

biochemistry↗

bioRxiv2024

Diverse cell death signature based subtypes predict the prognosis and immune characteristics within glioma

BackgroundCell death plays an essential role in the pathogenesis, progression, drug resistance and recurrence of glioma. Although multiple cell death pathways are involved in glioma development, there is lack of a stratification and prognostic modelling for glioma based on the integration of diverse genes for cell deaths. MethodsIn this study, 1254 diverse cell death (DCD)-related genes were assessed using the ConsensusClusterPlus assessment to identify DCD patterns in glioma. CIBERSORT, ssGSEA, and ESTIMATE algorithms were applied to evaluate immune microenvironment differences between subtypes. LASSO Cox regression was used to screen prognosis-related DCD genes, and a risk score model was constructed. TMB, TIDE, immune infiltration, and immunotherapy response was analyzed to evaluate the immune characteristics. ResultsTwo DCD-related subgroups named Clusters 1 and 2, with distinct DCD levels, immune characteristics, and prognoses, were determined from glioma samples. A DCD-based risk score model was developed to assess DCD levels in glioma patients and divide patients into high- and low-risk groups. We found this risk model can be used as an independent prognostic factor for glioma patients. Notably, glioma patients with low risk scores exhibited subdued DCD activity, prolonged survival, and a favorable disposition towards benefiting from immune checkpoint blockade therapies. ConclusionsThis study established a novel signature classification and a risk model by comprehensively analyzing patterns of various DCDs to stratify glioma patients and to predict the prognosis and immune characteristics of glioma. We provided a theoretical basis for the clinical application of DCD-related genes in glioma prognosis and immunotherapy.

bioinformatics↗

bioRxiv2024

Impact of Glycan Depletion, Glycan Debranching and Increased Glycan Charge on HIV-1 Neutralization Sensitivity and Immunogenicity

Modifying HIV-1s envelope glycoprotein glycans can impact its neutralization sensitivity. The use of the knock out cell line GnT1-prevents the elaboration of complex-type glycans, and opens up the glycan shield, increasing bNAb vulnerability. Some bNAb precursors can bind to GnT1-trimers, supporting their use in vaccine priming. However, GnT1-trimers express poorly and exhibit very low infectious counts. Here, we describe two other potentially vaccine-relevant glycoengineered trimers, 1) To truncate complex glycans, we used of a cocktail of glycosidases, termed "NGAF3" (Neuraminidase, {beta}-Galactosidase, N-Acetylglucosaminidase and endoglycosidase F3). Like GnT1-trimers, NGAF3 reduced glycan clashes and increased bNAb potency while retaining a closed trimer conformation; 2) Modified by {beta}-1,4-galactosyltransferase 1 (B4GalT1) and {beta}-galactoside -2,6 sialyltransferase 1 (ST6Gal1) during Env biosynthesis. Glycan mass spectrometry revealed that NGAF3 removed glycan heads of 3 of 7 positions on the trimer that are largely occupied by complex glycans. It also revealed a novel B4GalT1 activity to favor glycan precursor conversion to hybrid glycans rather than complex glycans. A comparison to monomeric gp120 revealed that B4GalT1s new activity depends on tight glycan spacing. B4GalT1 affected more glycans than NGAF3 (6 out of 7 glycans), perhaps due to greater accessibility during Env folding rather than after folding. Surprisingly, the N611 glycan was unaffected by either modification. B4GalT1 and ST6Gal1 cooperatively increased the abundance of hybrid glycans and -2,6 hypersialylated termini. This occurred largely by amplifying the abundance of a limited number of hybrid glycan structures that are also present on unmodified trimers. In rabbit vaccinations, B4GalT1+ST6GalT1-modified virus-like particles reduced the frequency and titers of serum NAbs that showed a modest preference for modified glycans. Conversely, chronically HIV-1-infected donor plasma neutralizing antibody titers were 1.7- to 10.8- fold higher against B4GalT1+ST6GalT1-modified pseudovirus. Overall, our data provide tools for heterologous prime, boost and polishing vaccine regimens using modified glycans. AUTHOR SUMMARYAn HIV-1 vaccine remains one of the most significant biomedical challenges today. Vaccines often work by triggering virus fighting antibodies to stave off infection. For HIV-1, this has been exceedingly difficult because the target, called the HIV-1 envelope glycoprotein (Env) is extremely variable and carries a thick sugar coat, protecting it from all but a few rare broadly reactive antibodies (termed bNAbs) that sometimes develop during natural HIV-1 infection. Given these challenges, it is reasonable to propose that any successful vaccine will need to be rationally designed to trigger the rare bNAbs. To meet this goal might require more 3 different vaccine components. First, a vaccine "prime" to trigger rare bNAb proliferation, followed by heterologous "boosts" and "polishing" immunogens to select for bNAbs. In this study, we evaluated two potential immunogens. In one approach, we used cocktails of enzymes to strip Envs sugar coat, in the hope of reducing the barrier to stimulate bNAb precursors. In another, we used excess enzymes to build particular structures on Envs coat. We checked how well these two Env variants were recognized by antibodies. We also checked in fine detail how the sugars were changed at the molecular level. Finally, we immunized rabbits. Our data enrich the number of strategies available to further explore the concept of priming, boosting and polishing vaccine shots.

immunology↗

bioRxiv2024

Gustatory-neuron-supplied R-spondin-2 is required for taste bud replenishment

Taste buds undergo continuous cell turnover throughout life, and taste cell replenishment relies strictly on innervation, a phenomenon first described almost 150 years ago. Recently, we provided evidence that R-spondin 2 (Rspo2) may be the long-sought gustatory neuron-supplied factor that regulates taste stem cell activity, via its interaction with taste stem/progenitor cell-expressed receptor Rnf43/Znrf3. Yet, whether gustatory-neuron-supplied Rspo2 is strictly required for taste tissue maintenance has not been resolved. Here, we set out to determine the necessity of gustatory-neuron-supplied Rspo2 in taste tissue homeostasis using genetic approaches. We used a mouse line that harbors the neomycin-resistance gene (NeoR) in one of the intron regions of the Rspo2 gene, which results in reduced expression of Rspo2. The number of taste buds is significantly reduced in these mice, compared to wild-type mice, in both anterior and posterior tongue. This phenotypic change was completely reversed by removing NeoR from the Rspo2 gene, thus making it normal. We also combined adeno-associated virus (AAV)-based delivery of Cre recombinase with a mouse line amenable to Cre-based ablation of the Rspo2 exons encoding the receptor-binding domains. Such deletion of Rspo2 in the nodose-petrosal-jugular ganglion complex led to nearly complete loss of taste buds in the circumvallate papilla. Thus, we demonstrate that Rspo2 is the long-sought gustatory-neuron-supplied factor that acts on taste stem cells to maintain taste tissue homeostasis. SignificanceWe have known for 150 years that innervation is required to induce and maintain cell replacement in taste buds. Until recently, the identity of the inducing factor produced by neurons was unknown. We have shown that R-spondin alone is sufficient to substitute for neuronal input to induce taste bud regeneration. Using a genetic loss-of-function approach, we now demonstrate that gustatory-neuron-expressed Rspo2 is required to maintain taste tissue homeostasis. Altogether, our work reveals that Rspo2 is the long-sought neuron-supplied factor that regulates the activity of taste stem/progenitor cells.

neuroscience↗

bioRxiv2024

Jianpi Jiedu Recipe inhibits proliferation through reactive oxygen species-induced incomplete autophagy and reduces PD-L1 expression in colon cancer

BackgroundJianpi Jiedu Recipe has been used to treat digestive tract tumors in China since ancient times, and its reliability has been proven by clinical research. Currently, the specific biological mechanism of JPJDR in treating tumors is unclear. MethodologyCCK-8 assay was used to detect cell viability. Clone formation assay and EdU assay were used to detect cell proliferation potential. DCFH-DA probe and JC-1 probe were used to detect total intracellular reactive oxygen species and mitochondrial membrane potential, respectively. Western blotting and immunofluorescence were used to detect protein expression level and subcellular localization of cells. RFP-GFP-LC3B reporter system was used to observe the type of autophagy in cells. Xenograft tumor model was used to study the therapeutic effect of JPJDR in vivo. ResultsJPJDR has a good inhibitory effect on various colorectal cancer cells and effectively reduces the proliferation ability of HT29 cells. After treatment with JPJDR, the amount of reactive oxygen species in HT29 cells increased significantly and the mitochondrial membrane potential decreased. JPJDR induced the accumulation of autophagosomes in HT29 cells and was shown to be incomplete autophagy. At the same time, JPJDR reduced the expression of PD-L1.Meanwhile, JPJDR can exert a good therapeutic effect in xenograft tumor mice. ConclusionJPJDR is a low-toxic and effective anti-tumor agent that can effectively treat colon cancer in vitro and in vivo. Its mechanism may be through inducing mitochondrial dysfunction and incomplete autophagy injury to inhibit the proliferation of colon cancer cells.

pharmacology and toxicology↗

bioRxiv2024

Molecular basis of inhibition of the amino acid transporter B0AT1 (SLC6A19)

The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders. This discovery has resulted in a quest to identify high-affinity selective inhibitors of B0AT1 for the treatment of disorders of amino acid metabolism. Here we employed a medicinal chemistry approach to improve lead compounds that were identified in a previous high-throughput screen. This effort yielded a group of compounds that inhibited B0AT1 with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a substantial movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.

biochemistry↗