Biology subjects

Cheng, C.

Publications and source records attributed to Cheng, C..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose non-fermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, a first-in-class, orally bioavailable proteolysis targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 (BRD4) and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise. Significance StatementThe mSWI/SNF complex is a promising therapeutic target for enhancer-driven cancers. PROTACs, which enable the targeting of "undruggable" proteins, often face the challenge of achieving oral bioavailability. Here, we present AU-24118, a first-in-class, orally bioavailable mSWI/SNF ATPase dual degrader with remarkable efficacy in in vitro and in vivo models. Additionally, our study describes two distinct mechanisms of resistance to PROTAC degraders, providing crucial insights into potential challenges facing their clinical application. These findings are critical for advancing PROTAC-based therapies to clinical settings as targeted therapies for cancers.

cancer biology↗

bioRxiv2024

MarmoPose: A Deep Learning-Based System for Real-time Multi-Marmoset 3D Pose Trackingp

The ability to track positions and poses (body parts) of multiple monkeys in a 3D space in real time is highly desired by non-human primate (NHP) researchers in behavioral and systems neuroscience because it allows both analyzing social behaviors among multiple NHPs and performing close-loop experiments (e.g., delivering sensory or optogenetics stimulation during a particular behavior). While a number of animal pose tracking systems have been reported, nearly all published work lacks the real-time analysis capacity. Existing methods for tracking freely moving animals have been developed primarily for rodents which typically move on a 2D space. In contrast, NHPs roam in a 3D space and move at a much faster speed than rodents. We have designed a real-time 3D pose tracking system (MarmoPose) based on deep learning to capture and quantify social behaviors in natural environment of a highly social NHP species, the common marmosets (Callithrix jacchus) which has risen to be an important NHP model in neuroscience research in recent years. This system has minimum hardware requirement and can accurately track the 3D poses (16 body locations) of multiple marmosets freely roaming in their homecage. It employs a marmoset skeleton model to optimize the 3D poses and estimate invisible body locations. Furthermore, it achieves high inference speed and provides an online processing module for real-time closed-loop experimental control based on the 3D poses of marmosets. While this system is optimized for marmosets, it can also be adapted for other large animal species in a typical housing environment with minimal modifications.

animal behavior and cognition↗

bioRxiv2024

Skeletal muscle effects of antisense oligonucleotides targeting glycogen synthase 1 in a mouse model of Pompe disease

Pompe disease (PD) is a progressive myopathy caused by the aberrant accumulation of glycogen in skeletal and cardiac muscle resulting from the deficiency of the enzyme acid alpha-glucosidase (GAA). Administration of recombinant human GAA as enzyme replacement therapy (ERT) works well in alleviating the cardiac manifestations of PD but loses sustained benefit in ameliorating the skeletal muscle pathology. The limited efficacy of ERT in skeletal muscle is partially attributable to its inability to curb the accumulation of new glycogen produced by the muscle enzyme glycogen synthase 1 (GYS1). Substrate reduction therapies aimed at knocking down GYS1 expression represent a promising avenue to improve Pompe myopathy. However, finding specific inhibitors for GYS1 is challenging given the presence of the highly homologous GYS2 in the liver. Antisense oligonucleotides (ASOs) are chemically modified oligomers that hybridize to their complementary target RNA to induce their degradation with exquisite specificity. In the present study, we show that ASO-mediated Gys1 knockdown in the Gaa-/- mouse model of PD led to a robust reduction in glycogen accumulation in skeletal and cardiac muscle. In addition, combining Gys1 ASO with ERT further reduced glycogen content in muscle, eliminated autophagic buildup and lysosomal dysfunction, and improved motor function in Gaa-/- mice. Our results provide a strong foundation for further validation of the use of Gys1 ASO, alone or in combination with ERT, as a therapy for PD. We propose that early administration of Gys1 ASO in combination with ERT may be the key to preventative treatment options in PD.

molecular biology↗

bioRxiv2023

Single cell dissection of developmental origins and transcriptional heterogeneity in B-cell acute lymphoblastic leukemia

Sequencing of bulk tumor populations has improved genetic classification and risk assessment of B-ALL, but does not directly examine intratumor heterogeneity or infer leukemia cellular origins. We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) and compared them to a reference map of normal human B-cell development established using both functional and molecular assays. Intra-sample heterogeneity was driven by cell cycle, metabolism, differentiation, and inflammation transcriptional programs. By inference of B lineage developmental state composition, nearly all samples possessed a high abundance of pro-B cells, with variation between samples mainly driven by sub-populations. However, ZNF384-r and DUX4- r B-ALL showed composition enrichment of hematopoietic stem cells, BCR::ABL1 and KMT2A-r ALL of Early Lymphoid progenitors, MEF2D-r and TCF3::PBX1 of Pre-B cells. Enrichment of Early Lymphoid progenitors correlated with high-risk clinical features. Understanding variation in transcriptional programs and developmental states of B-ALL by scRNA-seq refines existing clinical and genomic classifications and improves prediction of treatment outcome.

cancer biology↗

bioRxiv2023

pan-MHC and cross-Species Prediction of T Cell Receptor-Antigen Binding

Profiling the binding of T cell receptors (TCRs) of T cells to antigenic peptides presented by MHC proteins is one of the most important unsolved problems in modern immunology. Experimental methods to probe TCR-antigen interactions are slow, labor-intensive, costly, and yield moderate throughput. To address this problem, we developed pMTnet-omni, an Artificial Intelligence (AI) system based on hybrid protein sequence and structure information, to predict the pairing of TCRs of {beta} T cells with peptide-MHC complexes (pMHCs). pMTnet-omni is capable of handling peptides presented by both class I and II pMHCs, and capable of handling both human and mouse TCR-pMHC pairs, through information sharing enabled this hybrid design. pMTnet-omni achieves a high overall Area Under the Curve of Receiver Operator Characteristics (AUROC) of 0.888, which surpasses competing tools by a large margin. We showed that pMTnet-omni can distinguish binding affinity of TCRs with similar sequences. Across a range of datasets from various biological contexts, pMTnet-omni characterized the longitudinal evolution and spatial heterogeneity of TCR-pMHC interactions and their functional impact. We successfully developed a biomarker based on pMTnet-omni for predicting immune-related adverse events of immune checkpoint inhibitor (ICI) treatment in a cohort of 57 ICI-treated patients. pMTnet-omni represents a major advance towards developing a clinically usable AI system for TCR-pMHC pairing prediction that can aid the design and implementation of TCR-based immunotherapeutics.

immunology↗

bioRxiv2023

Increased FAN1 expression by mRNA-LNP attenuates CAG repeat expansion in Huntington patients' iPSC-derived astrocytes

Expansion of repeat sequences within the human genome can lead to disease pathogenesis, such as Huntingtons Disease, primarily affecting the nervous system. Genome-wide association studies (GWAS) of age-at-onset in Huntingtons disease (HD) patients demonstrated DNA mismatch repair (MMR) genes are modifiers of somatic expansion and may be potential therapeutic targets for repeat expansion (RE) disorders. FAN1, a Fanconi anemia-associated nuclease, has been reported as an influencer of repeat expansion in the RE mouse models. Here, we show the first demonstration that FAN1 knock-out in HD patient-derived fibroblasts and results in increased CAG repeat length. We also develop a robust novel cell-based platform using stem cell technology to produce the HD patients iPSC-derived astrocytes (iAstro). This platform is a disease-relevant system and has a significantly wider assay window, making it more suitable to assess the effect of gene modulation on CAG repeats. A substantial and exponential increase in repeat instability was exhibited in this HD patients iPSC-derived astrocytes platform. Over-expression of FAN1 protein via FAN1 plasmid transfection in this platform reduced CAG repeat instability, suggesting that upregulation of FAN1 protein may have a potential protective effect in CAG repeat expansion for a therapeutic setting. We leveraged the mRNA-LNP modality to enhance FAN1 protein expression and revealed that codon-optimized FAN1 mRNA-LNP robustly prevented increased CAG repeat in HD patients iPSC-derived astrocytes platform. The data from these cell-based platforms highlight that FAN1 plays a protective role in attenuating expanded somatic HTT CAG repeats and shed light on new therapeutic directions against repeat expansion disorders.

neuroscience↗

bioRxiv2023

Structure of scavenger receptor SCARF1 and its interaction with lipoproteins

SCARF1 (Scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDL) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.

biophysics↗

bioRxiv2023

Somatic mutation detection workflow validity distinctly influences clinical decision.

Evaluating robustness of somatic mutation detections is essential when utilizing whole exome sequencing (WES) for treatment decision-making. A comprehensive evaluation was conducted using tumor WES from the FDA-led Sequencing Quality Control Phase 2 (SEQC2) project, in which multiple library kits sequenced identical DNA materials across three labs to benchmark analytical validity. These workflows included various read aligner (BWA, Bowtie2, DRAGEN-Aligner, DRAGMAP, and HISAT2) and mutation caller (Mutect2, TNscope, DRAGEN-Caller, and DeepVariant) combinations. The results revealed that DRAGEN exhibited superior performance, achieving mean F1-scores of 0.966 and 0.791 for SNV and INDEL detection, respectively. Among open-source software, BWA Mutect2 and HISAT2 Mutect2 combinations showed the highest mean F1-scores for SNV (0.949) and IN-DEL (0.722), respectively. The analyses indicated that high-quality data can be analyzed as having worse results, and vice versa. Evaluations of COSMIC reported mutations unveiled discrepancies across enrichment kits. IDT enrichment kits showed a higher false negative rate, while Agilent WES kits tended to miss mutations in CBL and IDH1, and Roche library kits tended to miss the mutations in PIK3CB. For drug-related biomarkers, Sentieon TNscope tended to underestimate tumor mutation burden and overlook crucial drug-resistance mutations such as FLT3 (c.G1879A: p.A627T) for cytarabine resistance in leukemia and MAP2K1 (c.G199A:p.D67N) for BRAF inhibitors in melanoma. The findings highlight the importance of robust bioinformatic analysis in identifying tumor mutations and guiding clinical decision-making. HighlightsO_LIMutation callers had a significantly higher effect on overall sensitivity than aligners. C_LIO_LIBenchmarking analyses demonstrated that high-quality sequencing reads can be analyzed as having worse results, and vice versa. C_LIO_LIDRAGEN exhibited the best performance among other aligner-caller combinations. C_LIO_LIThe combination of BWA with Mutect2 and HISAT2 with Mutect2 yielded the highest mean F1 scores for detecting SNVs and INDELs by open-source software, respectively. C_LIO_LISentieon TNscope tended to underestimate the tumor mutation burden and missed several drug-resistant mutations. C_LI

bioinformatics↗

bioRxiv2023

Integrating population-level and cell-based signatures for drug repositioning

Drug repositioning presents a streamlined and cost-efficient way to expand the range of therapeutic possibilities. Drugs with human genetic evidence are more likely to advance successfully through clinical trials towards FDA approval. Single gene-based drug repositioning methods have been implemented, but approaches leveraging a broad spectrum of molecular signatures remain underexplored. We propose a framework called "TReD" (Transcriptome-informed Reversal Distance) that integrates population-level disease signatures robust to reverse causality and cell-based, drug-induced transcriptome response profiles. TReD embeds the disease signature and drug response profiles in a high-dimensional normed space, quantifying the reversal potential of candidate drugs in a disease-related cell-based screening. Here, we implemented this framework to identify potential therapeutics relevant to COVID-19 and type 2 diabetes (T2D). For COVID-19, we identified 36 drugs showing potential reversal roles. Notably, nearly 70% (25/36) of the drugs have been linked to COVID-19 from other studies, with seven drugs supported by ongoing/completed clinical trials. For T2D, we observed reversal signals for 16 compounds on multiple disease signatures. Five drugs are supported by published literature, affirming potential therapeutic value. In summary, we propose a comprehensive genetics-anchored framework integrating population-level signatures and cell-based screening that has the potential to accelerate the search for new therapeutic strategies.

bioinformatics↗

bioRxiv2023

Zinc is Essential for the Copper Reductase Activity of Yeast Nucleosomes

The histone H3-H4 tetramer is a copper reductase enzyme, facilitating the production of cuprous (Cu1+) ions for distribution to copper-dependent enzymes. It was, however, unknown if this enzymatic activity occurred within nucleosomes. To investigate this, we obtained native nucleosomes from Saccharomyces cerevisiae using micrococcal nuclease digestion of chromatin in isolated nuclei and ion-exchange chromatographic purification. The purified nucleosomal fragments robustly reduced Cu2+ to Cu1+ ions, with the optimal activity dependent on the presence of zinc ions. Mutation of the histone H3 histidine 113 (H3H113) residue at the active site substantially reduced the enzymatic activity of nucleosomes, underscoring the catalytic role of histone H3. Consistently, limiting zinc ions reduced intracellular Cu1+ levels and compromised growth, phenotypes that were mitigated by genetically enhancing the copper reductase activity of histone H3. These results indicate that yeast nucleosomes possess copper reductase activity, suggesting that the fundamental unit of eukaryotic chromatin is an enzyme complex.

biochemistry↗

bioRxiv2023

The Effect of Mechanical Stimuli on the Phenotypic Plasticity of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells in a 3D Hydrogel

IntroductionVascular smooth muscle cells (VSMCs) play a pivotal role in vascular homeostasis, with dysregulation leading to vascular complications. Human induced pluripotent stem cell (hiPSC)-derived VSMCs offer prospects for personalized disease modeling and regenerative strategies. Current research lacks comparative studies on the impact of 3D substrate properties under cyclic strain on phenotype adaptation in hiPSC-derived VSMCs. Here we investigated the potential of human mural cells derived from hiPSC-derived organoids (ODMCs) to undergo phenotypical adaptation under various biological and 3D mechanical stimuli. Methods and resultsODMCs were cultured in 2D conditions with synthetic or contractile differentiation medium, or 3D Gelatin Methacryloyl (GelMa) substrates with varying degrees of functionalization and percentages to modulate material stiffness, elasticity, and crosslink density. Cells in 3D substrates were exposed to cyclic unidirectional strain. Phenotype characterization was conducted using specific markers through immunofluorescence and gene expression analysis. Under static 2D culture, ODMCs derived from hiPSCs exhibited a VSMC phenotype, expressing key mural markers, and demonstrated a level of phenotypic plasticity like primary human vSMCs. In static 3D culture, higher substrate stiffness, lower elasticity and higher crosslink density promoted a contractile phenotype in ODMCs and vSMCs. Dynamic stimulation in 3D substrate promoted a switch towards a contractile phenotype in both cell types. ConclusionOur study demonstrates a phenotypic plasticity of human ODMCs in response to 2D biological and 3D mechanical stimuli that equals that of primary human vSMCs. These findings may contribute to the advancement of tailored approaches for vascular disease modelling and regenerative strategies

cell biology↗

bioRxiv2023

CDK19 and CDK8 Mediator kinases drive androgen-independent in vivo growth of castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) remains incurable due to its high plasticity. We found that Mediator kinases CDK8 and CDK19, pleiotropic regulators of transcriptional reprogramming, are differentially affected by androgen, which downregulates CDK8 and upregulates CDK19. Accordingly, expression of CDK8 decreases while CDK19 increases during prostate carcinogenesis, but both CDK19 and CDK8 are upregulated in metastatic CRPC. Genetic inactivation of CDK8 and CDK19 suppresses CRPC tumor growth in castrated male mice and renders CRPC responsive to androgen deprivation. Restoration of active CDK19 or CDK8 kinases reverses this phenotype, indicating that CRPC becomes dependent on Mediator kinase activity for in vivo growth under the conditions of androgen deprivation. Selective CDK8/19 inhibitors suppress androgen-independent growth of cell line-based and patient-derived CRPC xenografts, whereas prolonged inhibitor treatment induces tumor regression and even leads to cures. Mediator kinase activity was found to affect tumor and stromal gene expression preferentially in castrated mice, orchestrating castration-induced transcriptional reprogramming. These results warrant the exploration of Mediator kinase inhibitors for CRPC therapy.

cancer biology↗

bioRxiv2023

Circulating metabolites of Borneolum syntheticum (Bingpian) inhibit foam-cell formation in macrophages induced by oxidized low-density lipoprotein

Coronary heart disease is caused by the accumulation of atherosclerotic plaques which narrow the arteries over time. The plaques are formed by cholesterol deposits in the arterial intima and lead to the symptom of angina pectoris. Borneolum syntheticum (Bingpian) has been extensively used as a component in Chinese herbal medicines for cardiovascular diseases. This investigation aimed to examine Bingpian metabolism and its effects on anti-atherosclerotic activities. Major circulating Bingpian compounds were detected in human subjects who received a Bingpian-containing medicine. In vitro and rat studies were also conducted to facilitate the understanding of disposition factors that govern the systemic exposure to Bingpian compounds. Although Bingpian constituents, borneol (1) and isoborneol (2), are efficiently absorbed in the intestine, extensive hepatic first-pass glucuronidation, which is mediated predominantly by UGT2B7, coupled with MRP3 and MRP4-mediated efflux of the glucuronides into the blood, and oxidation, which is mediated by CYP2A6, CYP2B6, and CYP3A, result in the formation of metabolites borneol-2-O-glucuronide (M1G), isoborneol-2-O-glucuronide (M2G), and camphor (3) as the major circulating Bingpian compounds instead of the unchanged 1 and 2. Glucuronides are predominantly eliminated through renal excretion, which involves both glomerular filtration and OAT3- and OAT4-mediated tubular secretion. Furthermore, M1G, M2G, and 3, as well as 1 and 2, displayed inhibitory effects on oxidized low-density lipoprotein-induced foam-cell formation in macrophages. The findings emphasized that the metabolites must be given priority in pharmacodynamic studies of Bingpian. Comprehensive integration of pharmacokinetic and pharmacodynamic studies facilitates understanding how Bingpian functions in the body to provide therapeutic benefits.

pharmacology and toxicology↗

bioRxiv2023

Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

Non-small cell lung cancers (NSCLCs) treated with tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) almost invariably relapse in the long term, due to the emergence of subpopulations of resistant cells. Here we show that the lack of sensitivity of these cells to EGFR-TKIs constitutes a vulnerability that can be potentially targeted. Through a DNA barcoding approach, we demonstrate that the clinically approved drug sorafenib specifically abolishes the selective advantage of EGFR-TKI-resistant cells, while preserving the response of EGFR-TKI-sensitive cells, thus resulting in overall inhibition of clonal evolution within the tumor cell mass population. Sorafenib is active against multiple mechanisms of resistance/tolerance to EGFR-TKIs and its effects depend on early inhibition of MAPK interacting kinase (MNK) activity and signal transducer and activator of transcription 3 (STAT3) phosphorylation, and later down-regulation of MCL1 and EGFR. Using several xenograft and allograft models to recapitulate different mechanisms and kinetics of acquired resistance, we show that the sorafenib-EGFR-TKI combination can substantially delay tumor growth and promote the recruitment of inflammatory cells. Together, our findings indicate that sorafenib can substantially prolong the response to EGFR-TKIs by targeting NSCLC capacity to adapt to treatment through the emergence of resistant cells.

cancer biology↗

bioRxiv2023

Identification of differentially recognized T cell epitopes in the spectrum of Mtb infection

Tuberculosis caused by Mycobacterium tuberculosis is one of the leading causes of death from a single infectious agent. Identifying dominant epitopes and comparing their reactivity in different tuberculosis (TB) infection states can help design diagnostics and vaccines. We performed a proteome-wide screen of 20,610 Mtb derived peptides in 21 Active TB (ATB) patients 3-4 months post-diagnosis of pulmonary TB (mid-treatment) using an IFN{gamma} and IL-17 Fluorospot assay. Responses were mediated exclusively by IFN{gamma} and identified a total of 137 unique epitopes, with each patient recognizing, on average, 8 individual epitopes and 22 epitopes (16%) recognized by 2 or more participants. Responses were predominantly directed against antigens part of the cell wall and cell processes category. Testing 517 peptides spanning TB vaccine candidates and ESAT- 6 and CFP10 antigens also revealed differential recognition between ATB participants mid-treatment and healthy IGRA+ participants of several vaccine antigens. An ATB-specific peptide pool consisting of epitopes exclusively recognized by participants mid-treatment, allowed distinguishing participants with active pulmonary TB from healthy interferon-gamma release assay (IGRA)+/- participants from diverse geographical locations. Analysis of longitudinal samples indicated decreased reactivity during treatment for pulmonary TB. Together, these results show that a proteome-wide screen of T cell reactivity identifies epitopes and antigens that are differentially recognized depending on the Mtb infection stage. These have potential use in developing diagnostics and vaccine candidates and measuring correlates of protection.

immunology↗

bioRxiv2023

Targeting ABCB4 using mRNA-LNP for the treatment of rare liver diseases

Mutations in the ABCB4 gene lead to a wide-spectrum of rare liver diseases including progressive familial intrahepatic cholestasis type 3 (PFIC3) and low-phospholipid associated cholelithiasis (LPAC) syndrome. PFIC3 patients develop symptoms during late infancy, including severe itching, jaundice, and failure to thrive. The condition may progress to liver failure during childhood or adulthood. This is a highly unmet medical condition where liver transplantation is the only option to correct this disease. Recently, exciting data suggested that restoration of the ABCB4 function via gene replacement could rescue liver phenotypes associated with ABCB4 dysfunction in a preclinical PFIC3 mouse model. Here, we used mRNA LNP platform to determine expression and durability of ABCB4 in the liver of wildtype mice. In addition, we generated Abcb4-/- mice to study the efficacy of systemic delivery of ABCB4 mRNA LNP. We observed a robust and durable expression of hABCB4 up to 72 hours post systemic dosing in the liver of wild-type mice. Systemic administration of hABCB4 mRNA achieved a remarkable restoration of phosphatidylcholine levels in bile, a significant decrease in liver stiffness as measured by shear wave elastography, and amelioration of liver histopathology including fibrosis and ductular reaction. We conclude that administration of hABCB4 mRNA LNPs was sufficient to ameliorate fibrosis markers in the PFIC3 mouse model. Our data suggests that gene replacement using mRNA LNP modality could provide an excellent opportunity for patients with biliary diseases.

molecular biology↗

bioRxiv2023

Microbially-derived indole-3-acetate alleviates diet induced steatosis and inflammation in mice

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. There is growing evidence that dysbiosis of the intestinal microbiota and disruption of microbiota-host interactions contribute to the pathology of NAFLD. We previously demonstrated that gut microbiota derived tryptophan metabolite indole-3-acetate (I3A) was decreased in both cecum and liver of high-fat diet-fed mice and attenuated the expression of inflammatory cytokines in macrophages and TNF-a and fatty acid induced inflammatory responses in an aryl-hydrocarbon receptor (AhR) dependent manner in hepatocytes. In this study, we investigated the effect of orally administered I3A in a mouse model of diet induced NAFLD. Western diet (WD)-fed mice given sugar water (SW) with I3A showed dramatically decreased serum ALT, hepatic TG, liver steatosis, hepatocyte ballooning, lobular inflammation, and hepatic production of inflammatory cytokines, compared to WD-fed mice given only SW. Metagenomic analysis show that I3A administration did not significantly modify the intestinal microbiome, suggesting that I3As beneficial effects likely reflect the metabolites direct actions on the liver. Administration of I3A partially reversed WD induced alterations of liver metabolome and proteome, notably, decreasing expression of several enzymes in hepatic lipogenesis and {beta}- oxidation. Mechanistically, we also show that AMP-activated protein kinase (AMPK) mediates the anti-inflammatory effects of I3A in macrophages. The potency of I3A in alleviating liver steatosis and inflammation clearly demonstrates its potential as a therapeutic modality for preventing the progression of steatosis to NASH.

cell biology↗

bioRxiv2023

Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D

The polyextremophilic Cyanidiales are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal {beta}-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/530181v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@dd010dorg.highwire.dtl.DTLVardef@1700e5corg.highwire.dtl.DTLVardef@1bee8eaorg.highwire.dtl.DTLVardef@ad67da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗