Biology subjects

Lin, C.

Publications and source records attributed to Lin, C..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Thiazolidinediones are partially effective bitter blockers

PurposeThe bad bitter taste of some medicines is a barrier to overcoming non-compliance with medication use, especially life-saving drugs given to children and the elderly. Here we evaluated a new class of bitter blockers (thiazolidinediones; TZDs). MethodsIn this study, two TZDs were tested, rosiglitazone (ROSI) and a simpler form of TZD, using a high-potency sweetener as a positive control (neohesperidin dihydrochalcone, NHDC). We tested bitter-blocking effects using the bitter drugs tenofovir alafenamide fumarate (TAF), a treatment for HIV and hepatitis B infection, and praziquantel (PRAZ), a treatment for schistosomiasis, by conducting taste testing with two separate taste panels: a general panel (N=97, 20-23 yrs, 82.5% female, all Eastern European) and a genetically informative panel (N=158, including 68 twin pairs, 18-82 yrs, 76% female, 87% European ancestry). Participants rated the bitterness intensity of the solutions on a 100-point generalized visual analog scale. FindingsParticipants in both taste panels rated the bitter drugs TAF and PRAZ as less bitter on average when mixed with NHDC than when sampled alone. ROSI partially suppressed the bitterness of TAF and PRAZ, but effectiveness differed between the two panels: bitterness was significantly reduced for PRAZ but not TAF in the general panel and for TAF but not PRAZ in the genetically informative panel. ROSI was a more effective blocker than the other TZD. ImplicationsThese results suggest that TZDs are partially effective bitter blockers, suggesting other TZDs should be designed and tested with more drugs and on diverse populations to define which ones work best with which drugs and for whom. The discovery of bitter receptor blockers can improve compliance with medication use.

physiology↗

bioRxiv2023

Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

Withdrawal StatementThe authors have withdrawn this manuscript owing to inaccuracies in the calculation of tuft cell numbers and errors in the selection of immunofluorescence images used to support our claims. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

immunology↗

bioRxiv2023

Activity-Induced MeCP2 Phosphorylation Regulates Retinogeniculate Synapse Refinement

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the brains capacity to respond to changes in neuronal activity and sensory experience. Here we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from that previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. SIGNIFICANCE STATEMENTRett syndrome (RTT) is an X-linked neurodevelopmental disorder that predominantly affects girls. RTT is caused by loss of function mutations in a single gene MeCP2. Girls with RTT develop normally during their first year of life, but then experience neurological abnormalities including breathing and movement difficulties, loss of speech, and seizures. This study investigates the function of the MeCP2 protein in the brain, and how MeCP2 activity is modulated by sensory experience in early life. Evidence is presented that sensory experience affects MeCP2 function, and that this is required for synaptic pruning in the brain. These findings provide insight into MeCP2 function, and clues as to what goes awry in the brain when the function of MeCP2 is disrupted.

neuroscience↗

bioRxiv2023

Predicting the impact of rare variants on RNA splicing in CAGI6

BackgroundVariants which disrupt splicing are a frequent cause of rare disease that have been under-ascertained clinically. Accurate and efficient methods to predict a variants impact on splicing are needed to interpret the growing number of variants of unknown significance (VUS) identified by exome and genome sequencing. Here we present the results of the CAGI6 Splicing VUS challenge, which invited predictions of the splicing impact of 56 variants ascertained clinically and functionally validated to determine splicing impact. ResultsThe performance of 12 prediction methods, along with SpliceAI and CADD, was compared on the 56 functionally validated variants. The maximum overall accuracy achieved was 82% from two different approaches, one weighting SpliceAI scores by minor allele frequency, and one applying the recently published Splicing Prediction Pipeline (SPiP). SPiP performed optimally in terms of sensitivity, while an ensemble method combining multiple prediction tools and information from databases exceeded all others for specificity. ConclusionsSeveral challenge methods equalled or exceeded the performance of SpliceAI, with ultimate choice of prediction method likely to depend on experimental or clinical aims. One quarter of the variants were incorrectly predicted by at least 50% of the methods, highlighting the need for further improvements to splicing prediction methods for successful clinical application.

bioinformatics↗

bioRxiv2023

High-resolution structure of mouse radial spoke and its in-situ structure in ependymal cilia revealed by cryo-EM and cryo-ET

Radial spokes (RS) transmit mechanochemical signals between the central pair (CP) and axonemal dynein arms to coordinate ciliary motility. However, the high-resolution structure of mammalian RS remains missing. Here, we present the high-resolution cryo-EM structure of mouse RS head-neck complex in both monomer and dimer forms and reveal the intrinsic dynamics of the dimer. We also map the genetic mutations related to primary ciliary dyskinesia and asthenospermia on the head-neck complex. Moreover, we present the unprecedented cryo-ET and sub-tomogram averaging map of mouse ependymal cilia, whose beating creates unidirectional cerebrospinal fluid flow, and build the models for RS1-3, IDAs, and N-DRC. Strikingly, our cryo-ET map reveals the lack of IDA-b/c/e and the absence of tektin filaments within the A-tubule of doublet microtubules in ependymal cilia compared with mammalian respiratory cilia and sperm flagella. This tissue-specific feature may represent an evolutionary choice driven by the functional requirements on ependymal cilia. Our findings reveal the RS head-neck assembly mechanism, shed light on the coordinated rigid and elastic RS-CP interaction modes beneficial for the regulation of asymmetric ciliary beating, and also facilitate understandings on the etiology of ciliary dyskinesia-related ciliopathies.

biophysics↗

bioRxiv2023

Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance

Among the signaling pathways that control the stem cell self-renewal and maintenance vs. acquisition of differentiated cell fates, those mediated by receptor tyrosine kinase (RTK) activation are well established as key players. CBL family ubiquitin ligases are negative regulators of RTKs but their physiological roles in regulating stem cell behaviors are unclear. While hematopoietic Cbl/Cblb knockout (KO) leads to a myeloproliferative disease due to expansion and reduced quiescence of hematopoietic stem cells, mammary epithelial KO led to stunted mammary gland development due to mammary stem cell depletion. Here, we examined the impact of inducible Cbl/Cblb double-KO (iDKO) selectively in the Lgr5-defined intestinal stem cell (ISC) compartment. Cbl/Cblb iDKO led to rapid loss of the Lgr5Hi ISC pool with a concomitant transient expansion of the Lgr5Lo transit amplifying population. LacZ reporter-based lineage tracing showed increased ISC commitment to differentiation, with propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single cell RNAseq analysis of organoids revealed Akt-mTOR pathway hyperactivation in iDKO ISCs and progeny cells, and pharmacological inhibition of the Akt-mTOR axis rescued the organoid maintenance and propagation defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

cell biology↗

bioRxiv2023

Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P. falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be utilized by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex-vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not impacted by lack of CD44. However, the rate of P. falciparum invasion was substantially reduced in CD44-null cRBCs relative to isogenic wild-type (WT) control cells, validating CD44 as an important host factor for this parasite. We identified two P. falciparum invasion ligands as binding partners for CD44, Erythrocyte Binding Antigen-175 (EBA-175) and EBA-140, and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors-glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model where P. falciparum exploits CD44 as a co-receptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

microbiology↗

bioRxiv2023

LIFR recruits HGF-producing neutrophils to promote liver injury repair and regeneration

The molecular links between tissue repair and tumorigenesis remain elusive. Here, we report that loss of the liver tumor suppressor Lifr in mouse hepatocytes impairs the recruitment and activity of reparative neutrophils, resulting in the inhibition of liver regeneration after partial hepatectomy or toxic injuries. On the other hand, overexpression of LIFR promotes liver repair and regeneration after injury. Interestingly, LIFR deficiency or overexpression does not affect hepatocyte proliferation ex vivo or in vitro. In response to physical or chemical damage to the liver, LIFR from hepatocytes promotes the secretion of the neutrophil chemoattractant CXCL1 (which binds CXCR2 to recruit neutrophils) and cholesterol in a STAT3-dependent manner. Cholesterol, in turn, acts on the recruited neutrophils to secrete hepatocyte growth factor (HGF) to accelerate hepatocyte proliferation and regeneration. Altogether, our findings reveal a LIFR-STAT3- CXCL1-CXCR2 axis and a LIFR-STAT3-cholesterol-HGF axis that mediate hepatic damage- induced crosstalk between hepatocytes and neutrophils to repair and regenerate the liver.

cell biology↗

bioRxiv2023

Reconstituting spore cortex peptidoglycan biosynthesis reveals a deacetylase that catalyzes transamidation

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-{delta}-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-{delta}-lactam synthesis in cells, but their combined ability to generate muramic-{delta}-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-{delta}-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-{delta}-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

biochemistry↗

bioRxiv2023

Inflammation induces bitter taste oversensitization via epigenetic changes in Tas2r gene clusters

T2R bitter receptors, encoded by Tas2r genes, are not only critical for bitter taste signal transduction but also important for defense against bacteria and parasites. However, little is known about whether and how Tas2r gene expression are regulated. Here we show that, in an inflammation model mimicking bacterial infection, the expression of many Tas2rs are significantly up-regulated and mice displayed markedly increased neural and behavioral responses to bitter compounds. Using single-cell assays for transposase-accessible chromatin with sequencing (scATAC-seq), we found that the chromatin accessibility of Tas2rs was highly cell type specific and inflammation increased the accessibility of many Tas2rs. scATAC-seq also revealed substantial chromatin remodeling in immune response genes in taste tissue stem cells, suggesting potential long-term effects. Together, our results suggest an epigenetic mechanism connecting inflammation, Tas2r gene regulation, and altered bitter taste, which may explain heightened bitter taste that can occur with infections and cancer treatments.

neuroscience↗

bioRxiv2023

Real-time spectral library matching for sample multiplexed quantitative proteomics.

Sample multiplexed quantitative proteomics has proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor co-isolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Towards this end, we sought to develop and implement a new real-time library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time as traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy in sample multiplexed analyses.

cell biology↗

bioRxiv2023

Simple Causal Relationships in Gene Expression Discovered through Deep Learned Collective Variables

Developments in high-content phenotypic screening with single-cell read-out hold the promise of revealing interactions and functional relationships between genes at the genomic scale scale. However, the high-dimensionality and noisiness of gene expression makes this endeavor highly challenging when treated as a conventional problem in causal machine learning, both because of the statistical power required and because of the limits on computational tractability. Here we take different tack, and propose a deep-learning approach that finds low-dimensional representations of gene expression in which the response to genetic perturbation is highly predictable. We demonstrate that the interactions between genes that are cooperative in these representations are highly consistent with known ground-truth in terms of causal ordering, functional relatedness, and synergistic impact on cell growth and death. Our novel, statistical physics-inspired approach provides a tractable means through which to examine the response the living cell to perturbation, employing coarse graining that reduces data requirements and focuses on identifying simple relationships between groups of genes. Author summaryUnderstanding the causal relationships between genes and the functions of a cells molecular components has long been a challenge in biology and biomedicine. With recent advancements in technologies that manipulate and measure the activity of thousands of genes at once at the single-cell level, scientists are now afforded with the opportunity to interrogate such relationships at scale. However, extracting useful information from the vast readouts of these technologies is non-trivial, in part due to their many-dimensional and noisy nature. Here we develop a machine learning model that allows for the interpretation of complex genetic perturbations in terms of a simple set of causal relations. By analyzing cooperative groups of genes identified by our model, we demonstrate the model can group genes accurately based on their biological function, their relative ordering up- or downstream in the flow of causation, and how their activities combine to affect cell growth and death. Our approach complements existing machine learning methods in providing a simple way to interpret causal mechanism governing genetic interactions and functional states of cells.

genomics↗

bioRxiv2023

Refined novel techniques for long term cryo-storage using vitrification and laser warming

Vitrification and ultra-rapid laser warming technique is an important approach for cryopreservation of animal embryos and oocytes, as well as other cells with medicinal, genetic, and agricultural value. We believe that the long term cryo-storage after vitrification and for the following laser warming has only been achieved to date using our customized device which was first attempted to be developed in our study. In the present study, we focused on developing alignment and bonding techniques for special cryo-jig which were assembling jig tool and jig holder in one piece. This newly produced customized cryo-jig was demonstrated to have significantly high laser striking accuracy of 95% and a successful rewarming rate of 62%. This study was experimentally demonstrate an refined novel device for improvement of laser striking accuracy after long term cryo-storage using vitrification and laser warming technique. In addition, the customized device described herein was successfully applied to a biological sample with over a thousand coral larvae in long term cryo-storage (The first cryo-repository for coral larvae; the results related to coral cryobanking and repositry described in this preprint will be published separately in details). We anticipate that our core findings will provide further examples of cryobanking applications that use vitrification and nano-laser warming to help a wide range of cells and tissues from diverse species.

bioengineering↗

bioRxiv2023

Combined genome-scale fitness and paralog synthetic lethality screens with just 44k clones: the IN4MER CRISPR/Cas12a multiplex knockout platform

Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call in4mer. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.

systems biology↗

bioRxiv2022

Ceramide synthase 6 (CerS6) promotes alcohol-induced fatty liver by promoting metabolic dysfunction and upregulating lipid droplet-associated proteins

ObjectiveAlcohol-associated liver disease (ALD) is the leading cause of liver-related mortality worldwide. Current strategies to manage ALD largely focus on advanced stage disease, however, metabolic changes such as glucose intolerance are apparent at the earliest stage of alcoholic steatosis and increase the risk of disease progression. Ceramides impair insulin signaling and accumulate in ALD, and metabolic pathways involving ceramide synthase 6 (CerS6) are perturbed in ALD during hepatic steatosis. In this study, we aimed to investigate the role of CerS6 in ALD development. MethodsC57BL/6 WT and CerS6 KO mice of both sexes were fed either a Lieber-DeCarli control (CON) or 15% ethanol (EtOH) diet for 6 weeks. In vivo metabolic tests including glucose and insulin tolerance tests (GTT and ITT) were performed. The mice were euthanized, and liver histology and lipid levels in serum and liver were measured. For in vitro studies, CerS6 was deleted in human hepatocytes and were incubated with EtOH and/or C16:0-ceramides. RNAseq analysis was performed in mice and in liver from patients with different stages of ALD and diseased controls. ResultsAfter six weeks on an EtOH diet, CerS6 KO mice had reduced body weight, food intake, and %fat mass compared to WT mice. Male (but not female) EtOH-fed KO mice showed significantly higher O2 consumption, CO2 production, respiratory exchange ratio, and energy expenditure (P<0.05 for all) during the dark period compared to EtOH-fed WT mice. In response to EtOH, WT mice developed mild hepatic steatosis, while steatosis was alleviated in KO mice as determined by H&E and ORO staining. KO mice showed significantly decreased long-chain ceramide species, especially C16:0 ceramides, in the serum and liver tissues compared to WT mice. CerS6 deletion decreased serum TG and NEFA only in male not female mice. CerS6 deletion improved glucose tolerance and insulin resistance in EtOH-fed mice of both sexes. RNAseq analysis revealed that 74 genes are significantly upregulated and 66 genes are downregulated by CerS6 deletion in EtOH-fed male mice, with key network pathways including TG biosynthetic process, positive regulation of lipid localization, and fat cell differentiation. Similar to RNAseq results, absence of CerS6 significantly decreased mRNA expression of lipid droplet associated proteins in EtOH-fed mice. In vitro, EtOH stimulation significantly increased PLIN2 protein expression in VL-17A cells while CerS6 deletion inhibited EtOH-mediated PLIN2 upregulation. C16:0-ceramide treatment significantly increased PLIN2 protein expression compared to CON. Importantly, progression of ALD in humans was associated with increased CerS6 hepatic expression. ConclusionsOur findings demonstrate that CerS6 deletion attenuates EtOH-induced weight gain and hepatic steatosis and improves glucose homeostasis in mice fed an EtOH diet. Notably, we unveil that CerS6 plays a major role as a regulator of lipid droplet biogenesis in alcoholic intra-hepatic lipid droplet formation. Together, our data suggest that CerS6 may be targeted for treatment for early stage ALD.

pathology↗

bioRxiv2022

SGC-CAMKK2-1: A chemical probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

cancer biology↗

bioRxiv2022

Improved in situ sequencing for high-resolution targeted spatial transcriptomic analysis in tissue sections

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, which is based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry, for highly multiplexed in situ gene expression profiling. Here, we present improved in situ sequencing (IISS) that exploits a new probing and barcoding approach, combined with advanced image analysis pipeline for high-resolution targeted spatial gene expression profiling. We developed an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation. The new encoding strategy resulted in higher signal intensity as well as improved specificity for in situ sequencing, while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics. We showed that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin embedded tissue sections for single cell level spatial gene expression analysis, based on which the developmental trajectory and cell-cell communication networks can also be constructed. In conclusion, our method is a versatile molecular tool for targeted spatial transcriptomic analysis.

molecular biology↗

bioRxiv2022

Locating cellular contents during cryoFIB milling by cellular secondary-electron imaging

Cryo-electron tomography (cryoET) is a powerful technique that enables the direct study of the molecular structure of tissues and cells. Cryo-focused ion beam (cryoFIB) milling plays an important role in preparation of high-quality thin lamellar samples for cryoET studies, promoting the rapid development of cryoET in recent years. However, locating the regions of interest in a large cell or tissue during cryoFIB milling remains a major challenge limiting cryoET applications on arbitrary biological samples. Here, we report an on-the-fly location method based on cellular secondary electron imaging (CSEI). CSEI is derived from a basic imaging function of the cryoFIB instruments and enables high-contrast imaging of the cellular contents of frozen hydrated biological samples, highlighted by that both fluorescent labels and additional devices are not required. The present work discusses the imaging principles and settings for optimizing CSEI. Tests on several commercially available cryoFIB instruments demonstrated that CSEI was feasible on mainstream instruments to observe all types of cellular contents and was reliable under different milling conditions. Assisted by CSEI, we established a simple milling-location workflow and tested it using the basal body of Chlamydomonas reinhardtii.

biophysics↗