Biology subjects

Yan, S.

Publications and source records attributed to Yan, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Epicardial extracellular vesicles modulate gene expression following ischemia-reperfusion injury in heart-on-a-chip

The epicardium is an essential regulator of cardiac development, homeostasis, and injury, yet the composition and impacts of epicardial cell-secreted extracellular vesicles (EVs) remain incompletely understood. Here, we harness an epicardial Biowire platform integrating human stem cell derived epicardial cells with functional myocardium and apply transcriptomics to reveal enriched EV transport in tissues containing epicardial cells. Profiling epicardial-EVs identified key miRNAs and their conservation through stimulated epithelial-to-mesenchymal transition. Supplementation of epicardial-EVs to tissues undergoing ischemia-reperfusion injury influenced gene expression associated with reduction of extracellular matrix remodeling, fibroblast activation, and suppression of cell-ECM interactions. Correlation of EV-miRNAs with mRNA targets highlighted the role of miR-30d-5p, miR-9-5p, miR-16-5p, and the let-7 family in moderating deleterious fibrotic activation and matrix remodelling during myocardial injury in vitro. TeaserCells from the hearts outer surface secrete tiny packages of biomolecules that influence how the heart responds to injury

bioengineering↗

bioRxiv2025

PARP1 promotes replication-independent DNA double-strand break formation after acute DNA-methylation damage

Poly-ADP-Ribose Polymerase 1 (PARP1) is a potent regulator of DNA damage response signaling through the recruitment of DNA damage repair proteins to damage sites, and its catalytic function of converting Nicotinamide adenine dinucleotide (NAD+) into poly-ADP-ribose (PAR) which covalently modifies hundreds of protein substrates in a process known as PARylation. However, PARP1s role in the recognition, processing, and intracellular signaling downstream of DNA damage in cells remains incompletely understood, especially in a replication-independent context. Here, we show that cells exposed to high doses of the methylating agent Methyl Methanesulfonate (MMS) generate DNA double-strand breaks (DSBs) in a base excision repair (BER)-dependent and DNA replication-independent manner. The capacity of cells to generate DSBs after MMS exposure relies heavily on intracellular NAD+ availability and PARP1s catalytic production of PAR. In our experimental system, we show that acute MMS exposure causes NAD+ exhaustion in a PARP1-dependent manner, which results in a temporal-dependent loss of downstream PARP1 activity. This functional loss of PARP1 signaling in later timepoints leads to the loss of BER-dependent single-strand break (SSB)-to-DSB conversion, as well as silencing of ATR-Chk1 signaling in both cycling and non-cycling cells, demonstrating a novel PARP1-dependent regulatory mechanism for both ATR-Chk1 signaling and BER-associated processes following methylation challenge. Additionally, we provide experimental evidence supporting the role of PARP1 and NAD+ in promoting the exonuclease-mediated SSB-to-DSB conversion. These findings support a previously uncharacterized mechanism of PARP1-mediated replication-independent DSB generation and provide insight into checkpoint signaling by integrating DDR with PARP1s consumption of NAD+ and production of PAR.

molecular biology↗

bioRxiv2025

Excretory-secretory products of the fish-borne parasite Anisakis simplex L3 larvae possess allergens and unusual glycan modifications

Anisakis simplex is a parasitic aquatic nematode, which may cause mild-to-severe gastrointestinal allergic reactions (Anisakiasis) with clinical symptoms, such as rhinitis and urticaria in humans who accidentally consume raw or undercooked marine products contaminated with infective L3 Anisakis larvae. Several Anisakis excretory/secretory (E/S) products and somatic proteins are known to be involved in IgE-mediated allergic reactions. In comparison to vertebrates, nematodes have a distinct machinery to glycosylate their proteins and unusual glycan structures have been reported previously, many of which play immunogenic and immunomodulatory roles in host-parasite interactions. While an early study indicated that O-glycans participate the cross-reactivity of antibodies in allergy patients to A. simplex somatic antigens, the N-glycosylation pattern of Anisakis and the potential role of N-glycans in allergic reactions remained unknown. The aim of this study was to characterise N-glycans and the associated glycoproteins from Anisakis E/S products using mass spectrometry. We collected E/S products from larvae culture and released N-glycans from trypsinised proteins using PNGase Ar. Native glycans were pyridylaminated prior to HPLC separation and MALDI-TOF-MS/MS analysis. In addition, hydrofluoric acid and glycosidase digestions were performed to aid structural characterisation. MS data of 5h and 24h E/S products indicated the presence of pauci-mannose and core fucosylated N-glycans as major species; tri-fucosylated and methylated glycans as well as complex-type and phosphorylcholine-substituted glycans were also detected. In addition, E/S products were subject to proteomics analysis which revealed a set of proteins with conserved domains associated with allergens. Our study provides the first insight into the N-glycosylation machinery of Anisakis and highlights the needs for investigating whether and which N-glycans are indubitably involved in the modulation of allergic responses.

biochemistry↗

bioRxiv2025

Astrocytic CREB regulates transcriptional, neuronal, and behavioral responses to cocaine

Drug addiction is characterized by neuronal adaptations that support a shift from goal-directed behaviors to habitual, compulsive drug-seeking with persistent effects on cognition and decision-making. Emerging evidence increasingly indicates that astrocytes are also involved in nervous system disorders, including addiction, but the cocaine-induced astrocyte-specific transcriptome has not yet been investigated. We utilized whole cell sorting of astrocytes, RNA-sequencing, and bioinformatic approaches to characterize the astrocyte transcriptome in the nucleus accumbens (NAc), a key brain region involved in reward-processing, following cocaine self-administration, prolonged abstinence, and "relapse" in male mice. We found that astrocytes exhibit robust and contextually-specific transcriptional signatures that converge strongly with human cocaine use disorder. Bioinformatic analysis revealed CREB as a highly ranked predicted upstream regulator of cocaine-induced transcriptional regulation in NAc astrocytes, and CUT&RUN-sequencing mapped increased CREB binding across the astrocyte genome in response to cocaine. Viral-mediated manipulation of CREB activity selectively in NAc astrocytes, in combination with several measures of addiction-related behaviors including conditioned place preference and self-administration, revealed that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. This effect is sex-specific, with no change in astrocytic CREB activity or CPP found in females. Subsequent experiments identify potential molecular mechanisms of astrocytic CREBs influence through modulating astrocytic Ca2+ signaling in response to cocaine. Finally, we show that astrocytic CREB selectively modulates D1-type medium spiny neurons in NAc to control cocaine-related behaviors. Together, these data demonstrate that the astrocyte transcriptome responds robustly to cocaine and that CREB mediates cocaines effects on gene expression in astrocytes, with consequent effects on neuronal activity and rewarding responses to the drug.

neuroscience↗

bioRxiv2025

Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

ALK and ROS1 fusions are emerging as key drivers of infant-type hemispheric gliomas (IHG). With diverse gene partners, the impact of ALK and ROS1 oncoprotein heterogeneity on glioma biology remains unknown. We developed an integrative phospho-proteomic and transcriptomic approach to discover biological functions regulated by five IHG-associated fusions: CCDC88A::ALK, PPP1CB::ALK, GOPC::ROS1, CLIP1::ROS1 and KIF21A::ROS1. Here, we report fusion-specific oncogenic functions conferred by the 5 gene partner, including increased cell motility driven by microtubule-interacting fusions CCDC88A::ALK and CLIP1::ROS1. All studied fusions converged on a non-canonical STAT3 activation. Using affinity purification mass spectrometry, we identify SHP2 in direct interaction with all three ROS1 oncoproteins, but with none of the ALK oncoproteins, which in turn interact with SHC1/SHC3. ROS1 fusions phosphorylated SHP2 to a larger extent than ALK fusions, and analyses of downstream pathways suggested MAPK-independent, non-canonical SHP2-driven functions. Our findings reveal both common and fusion-specific dependencies, offering opportunities to optimize therapeutic strategies for pediatric gliomas.

cancer biology↗

bioRxiv2025

Safety and Efficacy of a Novel Glycoengineered Recombinant Vaccine Candidate against Haemonchus contortus in Sheep

Haemonchus contortus is considered the most pathogenic nematode in small ruminants and South American camelids, causing significant production losses and threatening animal welfare worldwide. Compared to the use of anthelmintic drugs, to which resistance is increasing, vaccination is considered a more sustainable control strategy. The safety and efficacy of a novel glycoengineered vaccine produced in Hi5 insect cells was evaluated in a randomized, controlled vaccination-and-challenge trial. For this, 35 male Jura x Lacaune sheep were assigned to five groups (n=7). Three experimental groups were vaccinated three times subcutaneously with 1 ml of either the commercial Barbervax(R) vaccine, the novel glycoengineered vaccine, consisting of a cocktail of five antigens (H11, H11-1, H11-2, H11-4 and GA1), or its non-glycoengineered counterpart before challenge with 5,000 H. contortus third-stage larvae. Clinical assessments, differential blood counts, serum antibody responses and fecal egg counts were evaluated at least once per week within the 16-week trial period. The abomasal worm burden was counted 40 days after challenge. The results of the mixed model showed a lower degree of anemia (mean packed cell volume (PCV) of 26.77%; mean hemoglobin (Hb) of 9.03 g/dl), in sheep vaccinated with the glycoengineered antigen cocktail compared to the unvaccinated group (PCV of 24.34%; mean Hb of 7.84 g/dl) and to the sheep vaccinated with the non-glycoengineered antigens (PCV of 26.11%; mean Hb of 8.53 g/dl). However, the degree of anemia was lower in sheep vaccinated with native antigens (Barbervax(R)) (PCV of 29.10%; mean Hb of 9.84 g/dl). Serum IgG and IgE antibodies were elevated in all vaccinated groups. Sheep vaccinated with the glycoengineered antigens showed a reduction in abomasal worm burden of 25.36% and a reduction of fecal egg shedding of 81.09%, the group vaccinated with non-glycoengineered antigen showed no worm reduction and a fecal egg count reduction of 32.72%. While there was no significant difference in egg shedding between sheep vaccinated with the glycoengineered antigens and sheep vaccinated with Barbervax(R) (98.32%; p = 0.33), the Barbervax(R) group showed a greater reduction in worm burden (86.40%). The results emphasize that glycoengineering of vaccine candidates is essential to achieve protective immunity against H. contortus in sheep and that the novel glycoengineered vaccine produced in insect cells clearly has the potential to reduce fecal egg shedding and worm burden, paving the way for the development of highly effective recombinant vaccines against this and other parasitic worms in the future.

molecular biology↗

bioRxiv2025

Glycoengineering of nematode antigens using insect cells: a promising approach for producing bioactive vaccine antigens of the barber's pole worm Haemonchus contortus

The H11 antigens, located on the intestinal microvilli of Haemonchus contortus, comprise a group of homologous aminopeptidases essential for the parasites digestion of blood meals. Native H11 proteins are promising vaccine antigens, capable of eliciting robust protective immunity against H. contortus in sheep and goats. However, recombinant forms of H11, produced either in conventional expression systems or in transgenic Caenorhabditis elegans, failed to replicate the protective efficacy of the native form, most likely due to two critical factors: improper glycosylation and protein misfolding. To address these limitations, we developed a novel strategy to produce recombinant Haemonchus antigens in glycoengineered insect cells. By introducing three C. elegans genes that alter the native N-glycosylation pathways of Hi5 insect cells we successfully expressed soluble H11 and GA1 antigens featuring nematode-specific glycan epitopes, including tri-fucosylated structures and the Gal{beta}1,4Fuc motif. The glycoengineered H11 proteins retained aminopeptidase activity and stimulated cytokine secretion from ovine peripheral blood mononuclear cells in vitro. These findings establish a platform for producing bioactive vaccine antigens against the parasitic nematode H. contortus.

bioengineering↗

bioRxiv2025

Effect of Shufeng Xingbi Therapy on Th1/Th2 immune balance and intestinal flora in rats with allergic rhinitis

ObjectiveTo study the effect of Shufeng Xingbi Therapy (SFXBT) on Th1/Th2 immune balance and intestinal flora in allergic rhinitis (AR) rats. Methods32 SD rats were randomly divided into control group, OVA group, antibiotic + SFXBT group and acetic acid + SFXBT group. Evaluate the AR behavioral score, observe the pathological changes of nasal mucosa with H&E, detect the composition and content of colonic contents with 16S rDNA, the levels of serum IgE, IL-4 and short-chain fatty acid (SCFAs) with ELISA, the mRNA expressions of STAT5, STAT6 and GATA3 in nasal mucosa with RT-qPCR, and the protein expressions of IL-4, STAT5, STAT6 and GATA3 in nasal mucosa with Western Blot. ResultsCompared with the OVA group, the AR behavioral score in the antibiotic + SFXBT group and acetic acid + SFXBT group decreased (P < 0.01), and the pathological changes of nasal mucosa were alleviated. At the door level, the relative abundance of Firmicutes in feces increased significantly, while the relative abundance of Bacteroidetes decreased significantly. At the genus level, The relative abundance of fecal Lactobacillus, Romboutsia, Allobaculum and Dubosiella increased significantly, the levels of serum IgE and IL-4 decreased (P < 0.05), the content of SCFAs increased significantly (P < 0.05), and the expression levels of STAT5, STAT6 and GATA3 mRNA and protein in nasal mucosa decreased significantly (P < 0.05). ConclusionShufeng Xingbi Therapy can significantly improve the inflammatory symptoms of nasal mucosa in AR rats, and its mechanism may be closely related to regulating Th1/Th2 immune balance and intestinal flora.

immunology↗

bioRxiv2024

Probing mechanical selection in diverse eukaryotic genomes through accurate prediction of 3D DNA mechanics

Connections between the mechanical properties of DNA and biological functions have been speculative due to the lack of methods to measure or predict DNA mechanics at scale. Recently, a proxy for DNA mechanics, cyclizability, was measured by loop-seq and enabled genome-scale investigation of DNA mechanics. Here, we use this dataset to build a computational model predicting bias-corrected intrinsic cyclizability, with near-perfect accuracy, solely based on DNA sequence. Further, the model predicts intrinsic bending direction in 3D space. Using this tool, we aimed to probe mechanical selection - that is, the evolutionary selection of DNA sequence based on its mechanical properties - in diverse circumstances. First, we found that the intrinsic bend direction of DNA sequences correlated with the observed bending in known protein-DNA complex structures, suggesting that many proteins co-evolved with their DNA partners to capture DNA in its intrinsically preferred bent conformation. We then applied our model to large-scale yeast population genetics data and showed that centromere DNA element II, whose consensus sequence is unknown, leaving its sequence-specific role unclear, is under mechanical selection to increase the stability of inner-kinetochore structure and to facilitate centromeric histone recruitment. Finally, in silico evolution under strong mechanical selection discovered hallucinated sequences with cyclizability values so extreme that they required experimental validation, yet, found in nature in the densely packed mitochondrial(mt) DNA of Namystynia karyoxenos, an ocean-dwelling protist with extreme mitochondrial gene fragmentation. The need to transmit an extraordinarily large amount of mtDNA, estimated to be > 600 Mb, in combination with the absence of mtDNA compaction proteins may have pushed mechanical selection to the extreme. Similarly extreme DNA mechanics are observed in bird microchromosomes, although the functional consequence is not yet clear. The discovery of eccentric DNA mechanics in unrelated unicellular and multicellular eukaryotes suggests that we can predict extreme natural biology which can arise through strong selection. Our methods offer a way to study the biological functions of DNA mechanics in any genome and to engineer DNA sequences with desired mechanical properties.

biophysics↗

bioRxiv2024

Discovery of a Potent and Selective Inhibitor of Human NLRP3 with a Novel Binding Modality and Mechanism of Action

The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1{beta} and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA encoded library screen to identify novel NLRP3 binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950 binding pocket. Using humanized NLRP3 mice we show that a derivative of BAL-0028 inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that BAL-0028 inhibits select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than does MCC950. BAL-0028 thus represents a new modality for NLRP3 inhibition in inflammatory diseases. SUMMARYNLRP3 is a target for anti-inflammatory therapies and can be inhibited by the tool compound MCC950. We describe the characterization of a new small molecule inhibitor of NLRP3 BAL-0028 that has a distinct mechanism of action and binding site.

immunology↗

bioRxiv2024

Bird Name-a-thon: Categorizing English bird names using crowdsourcing

Common names of species are important for communicating with the general public. In principle, these names should provide an accessible way to engage with and identify species. The common names of species have historically been labile without standard guidelines, even within a language. Currently, there is no systematic assessment of how often common names communicate identifiable and biologically relevant characteristics about species. This is a salient issue in ornithology, where common names are used more often than scientific names for species of birds in written and spoken English, even by professional researchers. To gain a better understanding of the types of terminology used in the English-language common names of bird species, a group of 85 professional ornithologists and non-professional contributors classified unique descriptors in the common names of all recognized species of birds. In the AvianLexiconAtlas database produced by this work, each species common name is assigned to one of ten categories associated with aspects of avian biology, ecology, or human culture. Across 10,906 species of birds, 89% have names describing the biology of the species, while the remaining 11% of species have names derived from human cultural references, human names, or local non-English languages. Species with common names based on features of avian biology are more likely to be related to each other or be from the same geographic region. The crowdsourced data collection also revealed that many common names contain specialized or historic terminology unknown to many of the data collectors, and we include these terms in a glossary and gazetteer alongside the dataset. The AvianLexiconAtlas can be used as a quantitative resource to assess the state of terminology in English-language common names of birds. Future research using the database can shed light on historical approaches to nomenclature and how people engage with species through their names.

zoology↗

bioRxiv2024

Core Tri-fucosylation of Nematode N-glycans Requires Golgi α-mannosidase III Activity that Impacts Animal Growth and Behaviours

Many nematodes possess N-glycans with complex core chitobiose modifications, which is a feature observed in various free-living and parasitic nematodes but is absent in mammals. Using Caenorhabditis elegans as a model to study N-glycan biosynthesis, we demonstrated that the core N-acetylglucosamine (GlcNAc) residues can be modified by three fucosyltransferases in the Golgi, namely FUT-1, FUT-6 and FUT-8. While the asparagine-linked GlcNAc is modified with a 1,3- and 1,6-linked fucose by FUT-1 and FUT-8 respectively, the distal GlcNAc residue is 1,3-fucosylated solely by FUT-6. Interestingly, FUT-6 can only fucosylate N-glycan structures lacking the 1,6-mannose upper arm, indicating that a specific -mannosidase is required to generate substrates for subsequent FUT-6 activity. By analysing the N-glycomes of aman-3 mutants (tm5400 and a CRISPR/Cas9 knockout, hex-2;hex-3;aman-3) using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of the aman-3 gene abolishes 1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. To further investigate it, we recombinantly expressed AMAN-3 in insect cells and characterised its enzymatic activity in vitro. In contrast to the classical Golgi -mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent 1,6-mannosidase activity towards N-glycans. Using AMAN-3 and other recombinant C. elegans glycoenzymes, we remodelled a fluorescein conjugated-Man5GlcNAc2 structure; we were able to mimic N-glycan biosynthesis in the Golgi and generate a tri-fucosylated glycan in vitro. We performed confocal microscopy studies using a knock-in strain (aman-3::eGFP) and could show the Golgi localisation of AMAN-3. In addition, using a high-content computer-assisted C. elegans analysis platform, we observed that AMAN-3 deficient worms display significant developmental delays, morphological and behavioural alterations in comparison to the wild type. Therefore, our data suggested that AMAN-3 participates in nematode N-glycan biosynthesis in the Golgi and generates substrates for FUT-6; thereby, this enzyme is essential for the formation of the unusual tri-fucosylated chitobiose cores of nematode N-glycans, which may play important roles in nematode development and behaviour. BackgroundTri-fucosylation of N-glycan core is a conserved feature seen in the N-glycomes of several nematode species. However, beyond the three core fucosyltransferases, we know very little about the biosynthesis and biological function of these core modifications. ResultsComparative glycomics data revealed that aman-3 mutants possess underfucosylated N-glycomes. Biochemical characterisation of AMAN-3 clarified its optimal reaction conditions and substrate specificity and, we demonstrated a Golgi localisation. Thereafter in vitro reconstruction of biosynthesis of a core tri-fucosylated N-glycan was achieved using 8 recombinant C. elegans glycoenzymes. Notably, aman-3 deficient worms exhibited significant developmental and behavioural changes. ConclusionAMAN-3 is a Golgi -mannosidase required for core fucosylation of the distal N-acetylglucosamine of N-glycoproteins. SignificanceThis study elucidates the key role of a novel Golgi -mannosidase in the biosynthesis of the unusual N-glycans of C. elegans and related nematodes, thereby setting the stage for new approaches to study the roles of glycan in the biology and immunology of nematode glycoproteins.

biochemistry↗

bioRxiv2024

An intricate balancing act: Upstream and downstream frameshift co-regulatory elements

Targeting ribosomal frameshifting has emerged as a potential therapeutic intervention strategy against Covid-19. During ribosomal translation, a fraction of elongating ribosomes slips by one base in the 5' direction and enters a new reading frame for viral protein synthesis. Any interference with this process profoundly affects viral replication and propagation. For Covid-19, two RNA sites associated with ribosomal frameshifting for SARS-CoV-2 are positioned on the 5' and 3' of the frameshifting residues. Although much attention has been on the 3' frameshift element (FSE), the 5' stem-loop (attenuator hairpin, AH) can play a role. The formation of AH has been suggested to occur as refolding of the 3' RNA structure is triggered by ribosomal unwinding. However, the attenuation activity and the relationship between the two regions are unknown. To gain more insight into these two related viral RNAs and to further enrich our understanding of ribosomal frameshifting for SARS-CoV-2, we explore the RNA folding of both 5' and 3' regions associated with frameshifting. Using our graph-theory-based modeling tools to represent RNA secondary structures, "RAG" (RNA-As-Graphs), and conformational landscapes to analyze length-dependent conformational distributions, we show that AH coexists with the 3-stem pseudoknot of the 3' FSE (graph 3_6 in our dual graph notation) and alternative pseudoknot (graph 3_3) but less likely with other 3' FSE alternative folds (such as 3-way junction 3_5). This is because an alternative length-dependent Stem 1 (AS1) can disrupt the FSE pseudoknots and trigger other folds. In addition, we design four mutants for long lengths that stabilize or disrupt AH, AS1 or FSE pseudoknot to illustrate the deduced AH/AS1 roles and favor the 3_5, 3_6 or stem-loop. These mutants further show how a strengthened pseudoknot can result from a weakened AS1, while a dominant stem-loop occurs with a strengthened AS1. These structural and mutational insights into both ends of the FSE in SARS-CoV-2 advance our understanding of the SARS-CoV-2 frameshifting mechanism by suggesting a sequence of length-dependent folds, which in turn define potential therapeutic intervention techniques involving both elements. Our work also highlights the complexity of viral landscapes with length-dependent folds, and challenges in analyzing these multiple conformations.

biochemistry↗

bioRxiv2024

Synthetic genomes unveil the effects of synonymous recoding

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1-4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement--recoding--is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organisms genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

synthetic biology↗

bioRxiv2024

Proteogenomic Reprogramming to a Functional Human Totipotent Stem Cell State via a PARP-DUX4 Regulatory Axis

PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome and proteome during zygotic genome activation (ZGA), totipotent, and pluripotent embryonic stages. Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naive reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells (TIRN-SC) concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.g., TPRXL, HOX clusters), eight-cell (8C)-specific (e.g., DUXA, GSC, GATA6), primitive endoderm-specific (e.g., GATA4, SOX17), trophectoderm-specific (e.g., CDX2, TFAP2C), and naive epiblast-specific (e.g., DNMT3L, NANOG, POU5F1(OCT4)) factors; all in a hybrid, combinatorial single-cell manner. Mapping of proteomic and single-cell expressions of TIRN-SC against human preimplantation embryo references identified them as relatively homogenous 4C-8C stage populations. Injection of TIRN cells into murine 8C-16C-staged embryos resulted in efficient totipotent-like single cell contributions of human cells to both extra-embryonic (trophectoderm, placenta) and embryonic (neural, fetal liver, hematopoietic) lineages in human-murine blastocyst and fetal chimeras. Pairing of proteome with ubiquitinome analyses of TIRN-SC revealed a global shutdown of ADP-ribosylation, and a perturbed TNKS/PARP1 equilibrium which not only impacted the protein levels of hundreds of TNKS/PARP1 substrates via a rewiring of the ubiquitin-proteosome system (UPS), but also de-repressed expression of hundreds of developmental genes associated with PARP1 suppression. ChIP-Seq analysis of core NANOG-SOX2-OCT4 (NSO) pluripotency factors in TIRN-SC identified reprogrammed DUX4-accessible distal and cis-regulatory enhancer regions that were co-bound by PARP1 (NSOP). These NSOP enhancer regions possessed co-binding motifs for hundreds of the same ZGA-associated, embryonic, and extraembryonic lineage-specifying pioneer factors (e.g., HOX, FOX, GATA, SOX, TBX, CDX families) that were concurrently co-expressed in TIRN-SC; suggesting that PARP1 and DUX4 cooperate with NSO pluripotency core factors to regulate the epigenetic plasticity of a human totipotency program. These findings provide the first demonstration that global, proteome-wide perturbations of post-translational modifications (i.e., ADP-ribosylation, ubiquitination) can regulate epigenetic reprogramming during human embryogenesis. Totipotent TIRN-SC will provide a valuable cell culture model for studying the proteogenomic regulation of lineage specification from human blastomere stages and may facilitate the efficient generation of human organs in interspecies chimeras.

developmental biology↗

bioRxiv2024

Large scale loss-of-function mutations during chicken evolution and domestication

Evolution and domestication are often driven by genetic innovations, yet the role of gene loss remains debated. Here, we present comparative genomic analyses of four indigenous chicken breeds from Yunnan Province, China and a reference red jungle fowl genome (GRCg6a). We identify extensive gene presence-absence variation and large numbers of pseudogenes, revealing highly dynamic gene repertoires among closely related chickens. By reconstructing ancestral gene content, we estimate that the common ancestor harbored at least 21,972 genes, of which 7,993 are dispensable. Each lineage has independently lost thousands of genes through both complete gene loss and pseudogenization. These loss-of-function events are non-random: pseudogenization mutations are biased toward gene termini, frequently fixed in populations, and enriched in specific biological pathways. Notably, patterns of gene loss recapitulate phylogenetic relationships, suggesting that loss-of-function mutations are shaped by selection rather than neutral drift. Analysis of four other chicken genomes assembled using PacBio HiFi reads draws the same conclusion. Thus, our results support a model in which large-scale loss-of-function mutations are a major driver of chicken evolution and domestication, consistent with the "less-is-more" hypothesis of adaptive evolution.

bioinformatics↗

bioRxiv2024

AHR activation accelerates the resolution of TGF-β1 induced fibroblast activation and promotes alveolar type 1 cell regeneration in alveolar organoids

Regeneration of the alveolar epithelium is necessary to restore tissue architecture and gas exchange capabilities in chronic pulmonary diseases such as fibrosing interstitial lung disease. While it is known alveolar type 2 (AT2) cells give rise to alveolar type 1 (AT1) cells to repair the alveolar epithelium after injury, methods to promote this process under pathological settings are poorly understood. Here, using a complex 3D organoid culture with TGF-{beta}1 dependent impaired AT1 spheroid formation, we performed a high-throughput screen (HTS) with [~]16,800 compounds to identify small molecules that increase number of AT1 spheroids. Longitudinal single cell RNA sequencing (scRNA-seq) revealed that DB-11-BE87 increased AT1 regeneration by reducing TGF-{beta}1 induced fibroblast activation, concurrently with AHR activation in those cells. These studies highlight a novel HTS system to identify factors that can promote AT1 differentiation and suggest AHR activation as a method to counteract pathological TGF-{beta}1 signaling in pulmonary disease.

cell biology↗

bioRxiv2024

Abolished frameshifting for predicted structure-stabilizing SARS-CoV-2 mutants: Implications to alternative conformations and their statistical structural analyses

The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.

molecular biology↗