Biology subjects

Tan, E.

Publications and source records attributed to Tan, E..

12 recordsLinked to original sources

bioRxiv2026

Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

Recent advances in AI-based structural biology have made de novo protein binder design increasingly effective, yet performance remains highly target dependent. For instance, the cancer surface antigen Nectin-4, an immunoglobulin-like cell adhesion protein, proved particularly challenging for RFdiffusion-based minibinder generation, yielding substantially fewer high-quality candidates than related targets. To address this bottleneck, we integrated an evolutionary genetic algorithm (GA) with AI-driven design. GA selection with tunable stringency was coupled with diversification via partial diffusion or direct sequence editing, enabling efficient exploration of sequence-structure space and rapid enrichment of promising candidates. This AI-GA pipeline quickly produced large and diverse minibinder panels with very good in silico quality metrics and is compatible with inputs from multiple design algorithms. Pooled, large-scale experimental screening identified highly stable Nectin-4 minibinders with single-digit nanomolar down to subnanomolar affinities. Lead binders were further engineered into Nectin-4-specific flow cytometry detection reagents and potent bispecific T cell engagers, demonstrating functional activity beyond binding. Together, these results show that evolutionary refinement can unlock challenging targets and accelerate de novo protein design for next-generation cancer biologics.

bioengineering↗

bioRxiv2026

Mitochondrial dsRNA: A Hidden Source of Immunogenic RNA in ADAR1 Deficiency

Double-stranded RNA (dsRNA) triggers immune responses during viral infections, but self-derived dsRNA can activate similar pathways. To prevent this, the body relies on mechanisms like ADAR1, an RNA-editing enzyme essential for immune regulation. Dysfunction of ADAR1 is linked to various diseases, yet the nature and role of dsRNAs accumulating in its absence remain unclear. Here, we identify mitochondrial dsRNA (mt-dsRNA), transcribed from the mitochondrial genome, as a major contributor to the endogenous dsRNA pool in ADAR1-deficient human and murine cells. We propose a "Draw-and-Release" model, where ADAR1 loss increases mitochondrial reactive oxygen species (mtROS), causing mt-dsRNA accumulation in the mitochondrial matrix ("Draw" phase) and its immune-activating release into the cytosol upon mitochondrial protein dysfunction ("Release" phase). This study highlights the importance of mitochondrial integrity in mitigating ADAR1-related pathologies.

cell biology↗

bioRxiv2025

Development of AI-designed protein binders for detection and targeting of cancer cell surface proteins

Artificial intelligence (AI)-based protein design opens new avenues for the rapid generation of new research tools and therapeutics, but experimental validation lags behind the computational design throughput. Here, we present a scalable workflow for the discovery and validation of AI-designed protein binders. Leveraging the RFdiffusion protein design pipeline with a custom filter for stable alpha-helical bundle folds, we construct libraries of thousands of AI-binders against cancer-associated surface proteins. Mammalian cell-surface and phage display screening yield multiple high-affinity PD-L1 binders but fewer hits for CD276 (B7-H3) and VTCN1 (B7-H4), reflecting the target-dependent efficiency of RFdiffusion in generating high-quality designs. Using our experimentally validated AI-designed binder libraries, we benchmark freely available structure prediction models. We find that interface predicted template modelling (ipTM) scores by Chai-1 with ESM embedding correlate well with experimental success and even predict deleterious effects of binding interface mutations. To demonstrate the versatility of AI-binders as research tools, we deploy them in CAR-T cells and also assemble them with fluorophore-labeled streptavidin into tetravalent quattrobinders, which achieve antibody-comparable staining of endogenous PD-L1 by flow cytometry. With high production yields and accessible structural models, AI-designed quattrobinders are versatile and cost-effective research tools amenable to community-driven validation and optimization.

bioengineering↗

bioRxiv2025

NKG7 is a stable marker of cytotoxicity across immune contexts and within the tumor microenvironment

Cytotoxicity is a cornerstone of immune defense, critical for combating tumors and infections. This process relies on the coordinated action of granzymes and pore-forming proteins, with Granzyme B (GZMB) and Perforin (PRF1) being key markers and the most widely studied molecules pertaining to cytotoxicity. However, other human granzymes and cytotoxic components remain underexplored, despite growing evidence of their distinct, context-dependent roles. Natural Killer Cell Granule Protein 7 (NKG7) has recently emerged as a crucial cytotoxicity regulator, yet its expression patterns and function are poorly understood. Using large publicly available single-cell RNA sequencing atlases, we performed a comprehensive profiling of cytotoxicity across immune subsets and tissues. Our analysis highlights NKG7 expression as a strong marker of cytotoxicity, exhibiting a strong correlation with overall cytotoxic activity (r = 0.97) and surpassing traditional markers such as Granzyme B and Perforin in reliability. Furthermore, NKG7 expression is notably consistent across diverse immune subsets and tissues, reinforcing its versatility and robustness as a cytotoxicity marker. These findings position NKG7 as an invaluable tool for evaluating immune responses and a reliable indicator of cytotoxic functionality across biological and clinical contexts.

immunology↗

bioRxiv2024

Diurnal.plant.tools in 2024: expanding to Marchantia polymorpha and four angiosperms

Diurnal gene expression is a pervasive phenomenon occurring across all kingdoms of life, orchestrating adaptive responses to daily environmental fluctuations and thus enhancing organismal fitness. Our understanding of the plant circadian clock is primarily derived from studies in Arabidopsis and direct comparisons are difficult due to differences in gene family sizes. To this end, the identification of functional orthologs based on diurnal and tissue expression is necessary. The diurnal.plant.tools database constitutes a repository of gene expression profiles from 17 members of the Archaeplastida lineage, with built-in tools facilitating cross-species comparisons. In this database update, we expand the dataset with diurnal gene expression from 4 agriculturally significant crop species and Marchantia, a plant of evolutionary significance. Notably, the inclusion of diurnal gene expression data for Marchantia enables researchers to glean insights into the evolutionary trajectories of the circadian clock and other biological processes spanning from algae to angiosperms. Moreover, by integrating data from tissue, and perturbation gene expression datasets for Marchantia from other databases, the diurnal dataset complements it to offer a comprehensive overview of gene functions across varied biological contexts. This expanded database serves as a valuable resource for elucidating the intricacies of diurnal gene regulation and its evolutionary underpinnings in plant biology.

plant biology↗

bioRxiv2023

Temporal Insights into Molecular and Cellular Responses during rAAV Production in HEK293T Cells.

The gene therapy field is actively pursuing cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for therapeutic applications, which demand high dosages. Enhanced yield is essential but presents technical and cost challenges. Strategies like suspension cell culture, transfection optimization, and cultivation conditions have shown moderate success but fall short for large-scale applications. In this study, we explore the host cell proteome of HEK293T cell lines used for AAV2 production under different transfection conditions (standard, sub-optimal, optimal) using SWATH-MS. To understand molecular and cellular mechanisms, we created a tailored spectral library for HEK293T-AAV interactions. Our gene ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. These conditions increased rAAV titre by 50% compared to standard protocols. Furthermore, we identified alterations in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts in optimal transfection conditions. This study provides insights into cellular mechanisms during rAAV production in HEK293T cells and offers potential advancements in scalability and cost-efficiency for gene therapy vector production. SignificanceGenerating AAV is challenging and the amount produced is limited, despite the success of gene therapy. Thus, it is necessary to enhance the productivity of host cells through various engineering techniques. The replication and transduction of viruses cause numerous modifications to the host cells proteome. Viruses exploit the processes of the host cell, influencing the availability, post-translational adjustments, associations, or placement of its proteins during the replication/growth cycle. Comprehending the fluctuations in biological processes during AAV replication is essential to grasping how virus-host relations shape the result of infection and viral protein expression.

molecular biology↗

bioRxiv2023

Background matching can reduce responsiveness of jumping spiders to stimuli in motion

Motion and camouflage were previously considered to be mutually exclusive, as sudden movements can be easily detected. Background matching, for instance, is a well-known, effective camouflage strategy where the color and pattern of a stationary animal match its surrounding background. However, background matching may lose its efficacy when the animal moves, as the boundaries of the animal become more defined against its background. Recent evidence shows otherwise, as camouflaged objects can be less detectable than uncamouflaged objects even while in motion. Here, we explored if the detectability of computer-generated stimuli varies with the speed of motion, background (matching and unmatching) and size of stimuli in six species of jumping spiders (Araneae: Salticidae). Our results showed that in general, the responsiveness of all six salticid species tested decreased with increasing stimulus speed regardless of whether the stimuli were conspicuousness or camouflaged. Importantly, salticid responses to camouflaged stimuli were significantly lower compared to conspicuous stimuli. There were significant differences in motion detectability across species when the stimuli were conspicuous, suggesting differences in visual acuity in closely related species of jumping spiders. Furthermore, small stimuli elicited significantly lower responses than large stimuli across species and speeds. Our results thus suggest that background matching is effective even when stimuli are in motion, reducing the detectability of moving stimuli. Summary StatementContrary to belief, stimuli in motion can remain camouflaged against their backgrounds. Using computer-generated stimuli, we show that smaller and faster stimuli against camouflaged background elicit lower responses from jumping spiders.

animal behavior and cognition↗

bioRxiv2021

PEDOT:PSS-Modified Cotton Conductive Thread for Mass Manufacturing of Textile-Based Electrical Wearable Sensors by Computerized Embroidery

The textile industry has advanced processes that allow computerized manufacturing of garments at large volumes with precise visual patterns. The industry, however, is not able to mass fabricate clothes with seamlessly integrated wearable sensors, using its precise methods of fabrication (such as computerized embroidery). This is due to the lack of conductive threads compatible with standard manufacturing methods used in industry. In this work, we report a low-cost poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS)-modified cotton conductive thread (PECOTEX) that is compatible with computerized embroidery. The PECOTEX was produced using a crosslinking reaction between PEDOT:PSS and cotton thread using divinyl sulfone as the crosslinker. We extensively characterized and optimized our formulations to create a mechanically robust conductive thread that can be produced in large quantities in a roll-to-roll fashion. Using PECOTEX and a domestic computerized embroidery machine, we produced a series of wearable electrical sensors including a facemask for monitoring breathing, a t-shirt for monitoring heart activity and textile-based gas sensors for monitoring ammonia as technology demonstrators. PECOTEX has the potential to enable mass manufacturing of new classes of low-cost wearable sensors integrated into everyday clothes.

bioengineering↗

bioRxiv2021

Protist.guru: a comparative transcriptomics database for the protist kingdom

SummaryDuring the last few decades, the study of microbial ecology has been enabled by molecular and genomic data. DNA sequencing has revealed the surprising extent of microbial diversity and how microbial processes run global ecosystems. However, significant gaps in our understanding of the microbial world remain, and one example is that microbial eukaryotes, or protists, are still neglected. To address this gap, we used gene expression data from 15 distinct protist species to create protist.guru: an online database equipped with tools for identifying functional co-expression networks, gene families, and enriched gene clusters. Here, we show how our database can be used to reveal genes involved in essential pathways, such as the synthesis of secondary carotenoids in Haematococcus lacustris. We expect protist.guru to serve as a valuable resource for protistologists, as well as a catalyst for discoveries and new insights into the biological processes of microbial eukaryotes. AvailabilityThe database and co-expression networks are freely available from http://protist.guru/. The expression matrices and sample annotations are found in the supplementary data.

evolutionary biology↗

bioRxiv2021

Developmental plasticity in male courtship in Bicyclus anynana butterflies is driven by hormone regulation of the yellow gene

The organizational role for hormones in the regulation of sexual behavior is currently poorly explored. Previous work showed that seasonal variation in levels of the steroid hormone 20-hydroxyecdysone (20E) during pupal development regulates plasticity in male courtship behavior in Bicyclus anynana butterflies. Wet season (WS) males, reared at high temperature, have high levels of 20-hydroxyecdysone (20E) during pupation and become active courters. Dry season (DS) males, reared at low temperatures, have lower levels of 20E and lower courtship rates. Rescue of WS courtship rates can be achieved via injection of 20E into DS male pupae, but it is still unknown whether 20E alters gene expression in the pupal brain, and if so, the identity of those targets. Using transcriptomics, qPCR, and behavioral assays with a transgenic knockout, we show that higher expression levels of the yellow gene in DS male pupal brains, relative to WS brains, represses courtship in DS males. Furthermore, injecting DS males with 20E downregulates yellow to WS levels 4 hours post-injection, revealing a hormone sensitive window that determines courtship behavior. These findings are in striking contrast to Drosophila, where yellow is required for active male courtship behavior. We conclude that 20E plays an organizational role during pupal brain development by regulating the expression of yellow, which is a repressor of the neural circuity for male courtship behavior in B. anynana. This work shows that similar to vertebrates, hormones can also play an organizational role in insect brains, leading to permanent changes in adult sexual behavior. Significance StatementBehavioral plasticity in adult insects is known to be regulated by hormones, which activate neural circuits in response to environmental cues. Here, we show that hormones can also regulate adult behavioral plasticity by altering gene expression during brain development, adjusting the insects behavior to predictable seasonal environmental variation. We show that seasonal changes in the hormone 20E alters expression of the yellow gene in the developing pupal brain of Bicyclus anynana butterflies, which leads to differences in male courtship behavior between the dry and wet seasonal forms. This work provides one of the first examples of the organizational role of hormones in altering gene expression and adult sexual behavior in the developing insect brain.

neuroscience↗

bioRxiv2020

The Irish potato famine pathogen subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How and why adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phythophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway, while antagonizing antimicrobial autophagy. Here we show that PexRD54 induces autophagosome formation by bridging small GTPase Rab8a-decorated vesicles with autophagic compartments labelled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing that specific trafficking pathways underpin selective autophagy. We discovered that Rab8a contributes to basal immunity against P. infestans, but PexRD54 diverts a sub-population of Rab8a vesicles to lipid droplets that associate with autophagosomes. These are then diverted towards pathogen feeding structures that are accommodated within the host cells. We propose that PexRD54 mimics starvation-induced autophagy by channeling host endomembrane trafficking towards the pathogen interface possibly to acquire nutrients. This work reveals that effectors can interconnect independent host compartments to stimulate complex cellular processes that benefit the pathogen. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/000117v1_ufig1.gif" ALT="Figure 1"> View larger version (83K): org.highwire.dtl.DTLVardef@bfc9fforg.highwire.dtl.DTLVardef@166b5f8org.highwire.dtl.DTLVardef@34d581org.highwire.dtl.DTLVardef@ad397f_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2017

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp Marsupenaeus japonicus reared at different salinity

Shrimps inhabiting around the coastal area can survive in a wide range of salinity. However, the molecular mechanisms involved in their adaptation to different environmental salinity have remained largely unknown. In the present study, we reared kuruma shrimp Marsupenaeus japonicus at 1.7 %, 3.4 % and 4.0 % salinity. After rearing for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA-seq analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after rearing for 24 h. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. Notably, the increased glutamine content at high salinity appeared to be relevant to the increase of the expression level of the gene encoding glutamate-ammonia ligase which functions in the glutamine metabolism. Furthermore, the alanine content increased at high salinity was likely to be associated with the decrease in the expression levels of the alanine-glyoxylate transaminase gene. Thus, the changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are considered to be regulated by the changes in the expression levels of genes related to amino acid metabolism.\n\nSummary statesmentKuruma shrimp Marsupenaeus japonicus changes free amino acid contents and associated gene expression levels in their muscle to adjust effectively to different salinity.

zoology↗