Biology subjects

Fan, E.

Publications and source records attributed to Fan, E..

11 recordsLinked to original sources

bioRxiv2026

Hunting for microsatellite instability in long-read data with Owl

Microsatellite instability (MSI) is a key biomarker of mismatch repair deficiency and response to immunotherapy, yet most existing genomic detection methods are optimized for short-read sequencing and rely on small panels of homopolymer markers, limiting the ability to characterize genome-wide and motif-specific patterns of instability. Here we present Owl, a bioinformatic tool for quantifying MSI from long-read (PacBio) genomic data. Owl leverages a genome-wide marker set of more than 140,000 microsatellite repeats ranging from 1-6 bp in length to measure MSI across a phased genome. Using a wrap-around alignment algorithm, Owl constructs repeat-length distributions at each marker site and flags somatic instability using the coefficient of variation. We applied Owl to screen for markers with stable coverage, phasing, and baseline variation across 131 diverse genomes from the Human Pangenome Reference Consortium, where Owl scores ranged from 1.4% to 5.4% of markers exceeding the instability threshold. When applied to 19 cancer cell lines and one diffuse astrocytoma tumor-normal pair, Owl identified five MSI-high genomes with 15-18% unstable markers and showed close concordance with an Illumina DRAGEN MSI assay for the astrocytoma sample. Motif-level analyses revealed shared enrichment of short homopolymer and dinucleotide (A- and AT-rich) repeats across MSI-high cancers, and additionally uncovered a distinct pattern of elevated GGAA microsatellite instability in Ewing sarcoma cell lines, consistent with the known role of the EWS::FLI1 fusion protein at GGAA-rich regulatory elements. Owl is implemented in Rust and integrated into the PacBio HiFi Somatic workflow, providing a scalable framework for MSI analysis from long-read sequencing focused on repeat instability specifically in tumor samples.

bioinformatics↗

bioRxiv2026

Spore-Based Biocomposite Thermoplastic Polyesters with Enhanced Toughness and Programmable Disintegration

Thermoplastic polyesters are widely used in commodity and high-performance applications due to their tunable and exceptional properties, versatile performance, and increasing relevance in sustainable materials. Integrating biological functionality into these polymers offers a promising route to enhance performance and end-of-life behavior beyond what conventional additives can achieve. Here, we report the generalization of an embedded spore-based engineered living material concept to three representative thermoplastic polyesters; polycaprolactone (PCL), polylactic acid (PLA), and poly(butylene adipate-co-terephthalate) (PBAT). Heat-shock-tolerized Bacillus subtilis spores were compounded with each polyester as a living biofiller via hot melt extrusion. The resulting biocomposite polyesters retained high spore viability (>90%) after extrusion and exhibited improved mechanical performance (up to 41% toughness improvement compared to neat polymers). End-of-life behavior was evaluated in a microbially-limited composting environment, where spore-containing PCL exhibited nearly complete disintegration within five months, corresponding to a [~]7-fold increase in degradation kinetics relative to neat PCL. Finally, 3D printing of biocomposite PCL was demonstrated through fused deposition modeling and direct ink writing methodologies. Together, this work demonstrated the successful extension of spore-based engineered living materials from thermoplastic polyurethane to multiple thermoplastic polyesters. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/707801v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@4eaafaorg.highwire.dtl.DTLVardef@bb17c2org.highwire.dtl.DTLVardef@114e4ceorg.highwire.dtl.DTLVardef@b9bd0c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Orthosteric Molecular Glue Inhibits COP9 Signalosome with Substrate-Dependent Potency

Orthosteric inhibitors block enzyme active sites and prevent substrates from binding. Enhancing their specificity through substrate dependence seems inherently unlikely, as their mechanism hinges on direct competition rather than selective recognition. Here, we show that a molecular glue mechanism unexpectedly imparts substrate-dependent potency to CSN5i-3, an orthosteric inhibitor of the COP9 signalosome (CSN). We first confirm that CSN5i-3 inhibits CSN, which catalyzes NEDD8 deconjugation from the cullin-RING ubiquitin ligases (CRLs), by occupying the active site of its catalytic subunit, CSN5, and directly competing with the iso-peptide bond substrate. Curiously, the orthosteric inhibitor binds free CSN with only micromolar affinity, yet achieves nanomolar potency in blocking its deneddylase activity. Cryo-EM structures of the enzyme-substrate-inhibitor complex reveal that active site-engaged CSN5i-3 occludes the substrate iso-peptide linkage while simultaneously extending an NEDD8-binding exosite of CSN5, acting as a molecular glue to cement the NEDD8-CSN5 interaction. The cooperativity of this tri-molecular CSN5i-3-NEDD8-CSN5 assembly, in turn, sequesters CSN5i-3 at its binding site, conferring high potency to the orthosteric inhibitor despite its low affinity for the free enzyme. Together, our findings highlight the modest affinity requirements of molecule glues for individual target proteins and establish "orthosteric molecular glue inhibitors" as a new class of substrate-dependent enzyme antagonists.

molecular biology↗

bioRxiv2025

Quantifying HLA transcripts by genotype in chimeric mixtures at single-cell resolution

Gene products from the highly variable major histocompatibility locus, including HLA, are essential for self-recognition and immune surveillance of malignancy. Following allogeneic hematopoietic cell transplantation (alloHCT), genetic and epigenetic alterations in HLA can drive disease recurrence, making precise HLA assessment critical for determining future therapy. However, current methods lack the sensitivity to quantify HLA transcripts at the single-cell level, limiting their clinical utility. We introduce scrHLA-typing, a novel technique that accurately identifies and quantifies HLA transcripts in single cells using long-read sequencing. When applied to samples from patients with post-transplant relapse, scrHLA-typing successfully detected HLA allele-specific expression, across a range of levels of donor-recipient chimerism, at clinically actionable levels. By characterizing allele expression in residual leukemia cells, our assay identified differences in expression patterns among patients. This capability highlights scrHLA-typings potential to improve risk stratification and guide the selection of appropriate salvage therapies, enhancing personalized treatment strategies after relapse.

molecular biology↗

bioRxiv2025

Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1+Tcf1+ stem-like reservoir and overcomes resistance to PD-1 axis blockade

PD-1+Tcf1+ stem-like cells are critical mediators of endogenous T cell responses to PD-1/PD-L1 blockade and are maintained by MHC-dependent interactions with professional antigen-presenting cells (APCs). Unlike conventional T cells, CAR-T cells are activated by intact antigen expressed on tumors, not by peptide/MHC expressed on APCs, restricting their activation to the hostile tumor microenvironment (TME) that may impair preservation of this critical stem-like subset. Indeed, in an autochthonous model of ROR1+ lung cancer that we developed, CAR-T cells targeting the tumor-associated antigen ROR1 were uniformly Tcf1+ prior to infusion but rapidly downregulated Tcf1 in vivo and became terminally exhausted, similar to observations in patients, resulting in faster attrition and no enhancement in response to PD-L1 blockade. We hypothesized that overexpression of AP-1 family transcription factors, which can regulate T cell exhaustion, could enable CAR-Ts to maintain this critical PD-1+Tcf1+ subset within tumors independently of APCs and sensitize them to PD-1/PD-L1 blockade. Overexpression of the AP-1 TF c-Jun, but not BATF, improved preservation of PD-1+Tcf1+ CAR-T cells within tumors in a cell-intrinsic manner that correlated with increased persistence deeper within tumors. Notably, c-Jun overexpression alone was insufficient to prevent CAR-T exhaustion in the lung TME, in contrast to prior work in xenograft models, with progressive CAR-T dysfunction correlated with PD-1-dependent downregulation of c-Jun. However, c-Jun overexpression dramatically sensitized CAR-Ts to PD-L1 blockade, which restored c-Jun levels in CAR-Ts, drove log-fold expansion of CAR-Ts within tumors, and induced nearly complete eradication of ROR1+ tumor in highly aggressive models of lung cancer. Altogether, our data show that combination with PD-L1 blockade is necessary to unleash the full potential of c-Jun-overexpressing CAR-T cells in aggressive solid tumors like lung cancer and suggest that strategies to enhance formation of intratumoral PD-1+Tcf1+ reservoirs can overcome CAR-T resistance to PD-1 blockade.

immunology↗

bioRxiv2024

Individual G-quadruplex targeting with ligand-functionalized CRISPR-Cas9 uncovers transcriptional-dependent functional responses

The development of selective ligands to target DNA G-quadruplexes (G4s) and i-motifs (iMs) has revealed their relevance in transcriptional regulation. However, most of these ligands are unable to target individual G4s or iMs in the genome, severely limiting their scope. Herein, we describe a new Approach to Target Exact Nucleic Acid alternative structures (ATENA) that relies on the chemical conjugation of established G4 and iM ligands to a catalytically inactive Cas9 protein (dCas9), enabling their individual targeting in living cells. ATENA demonstrated that the selective targeting of the G4 present in the oncogene c-MYC leads to the suppression of transcripts regulated exclusively by one of its promoters (P1). Conversely, targeting the c-MYC iMs on the opposite strand leads to the selective increase of P1-driven transcripts. ATENA revealed that G4-mediated transcriptional responses are highly ligand-specific, with different ligands eliciting markedly different effects at the same G4-site. We further demonstrated that the basal expression levels of the gene targeted can be used to predict the transcriptional impact associated with G4-stabilization. Our study provides an innovative platform to investigate G4- and iM-biology with high precision and unveils the therapeutic relevance of individual DNA structures with unprecedented selectivity.

cell biology↗

bioRxiv2022

Bacterial growth-promoting properties of pooled urine and individual urines from post-menopausal women with or without a urinary tract infection can vary substantially

Urinary tract infections (UTIs), mostly caused by uropathogenic E. coli (UPEC), are common bacterial infections that affect a majority of women and often recur. Genomic and transcriptomic analyses have not identified a common set of virulence genes which has suggested conserved virulence functions instead of virulence genes, multiple virulence mechanisms, and complex host-pathogen interactions. One aspect of the host-pathogen interaction is rapid UPEC growth in urine in vivo. When bacteria are grown in pooled urine, an averaged urine is assumed to diminish individual variation. We grew a non-pathogenic and pathogenic E. coli strains in urine from individuals who never had a UTI, had a UTI history but no current infection, and had a UTI history with a current infection. Bacterial growth showed large variations in individual urines and pooled urine supported significantly more growth than predicted for never and history groups but not the current group. UPEC strains, but not the non-pathogenic strain, were resistant to urinary inhibitory factors e.g., antimicrobial peptides based on an indirect inoculation-density effect assay. Total nutrient content tended to be higher in current group urine than never and history group urine. We propose that pooling optimizes a nutrient mixture in never and history group urines, which are often studied, whereas urine from current group individuals appear to have a more optimal nutrient mixture. We conclude that pooled urine is not "an average urine", and that the best comparisons of results between labs using pooled urine would also include results with a standardized synthetic urine.

microbiology↗

bioRxiv2021

Spontaneous selection of Cryptosporidium drug resistance in a calf model of infection

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last [~]10 years with numerous lead compounds identified including several tRNA synthetase inhibitors. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC50S of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence. ImportanceCryptosporidium is a leading protozoan cause of diarrhea in young children with no reliable treatment. We report results of a dairy calf drug efficacy trial and the spontaneous emergence of drug resistance. Cryptosporidium parvum infected calves initially improved with drug treatment, but infection relapsed in two animals. Parasites shed by each recrudescent calf had mutations in the gene encoding the drug target that altered its amino acid sequence. Recapitulation of the drug target mutations by CRISPR/Cas9 genome editing resulted in highly drug-resistant parasites, and recombinant mutant enzymes were resistant to inhibition. This is the first report of naturally emerging Cryptosporidium drug resistance. There is a currently a great opportunity to impact public health with new drugs to treat cryptosporidiosis, and this report highlights the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence in order to realize their full potential. One-sentence summaryDrug-target point mutations mediating anticryptosporidial resistance spontaneously arose in the dairy calf C. parvum infection model.

microbiology↗

bioRxiv2020

Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice, were assessed in pregnant mice. Drugs were emulsified in corn oil and applied by gavage for 5 days. Five BKIs did not affect pregnancy, 5 BKIs exhibited 15-35% of neonatal mortality, and 5 compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilized eggs to 0.2-50M of BKIs and microscopical monitoring of embryo development in a blinded manner during 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows to calculate an impact score (Si) that indicates at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for 9 compounds no clear correlation between Si and pregnancy outcome was visible. However, those 3 BKIs affecting zebrafish embryos only at high concentrations (40M or higher) did not impair mouse pregnancy at all, and those 3 compounds that inhibited zebrafish embryo development already at 0.2M showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has a limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude, that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

pathology↗

bioRxiv2019

Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model

ObjectivesMethionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. To properly analyze the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites.\n\nMethodsSystematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and 1717 was tested for efficacy in the mouse infection model.\n\nResultsCell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with noninvasive whole-animal imaging and fecal analysis. Compound 1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which is supported by data from enzymatic and phenotypic cell assays.\n\nConclusionsThe new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by 1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

microbiology↗

bioRxiv2019

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

\"Bairihua\", a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development. Here, a SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in Bairihua and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in \"Bairihua\". Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time. Taken together, our results implied a novel pathway that ChuSPL9 regulated flowering development, but not flowering transition, with the participation of ChuHMGA. Further investments need to be done to verify the details of this pathway.

plant biology↗