Biology subjects

Huang, C.-Y.

Publications and source records attributed to Huang, C.-Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Antibody evasion and receptor binding of SARS-CoV-2 RW.1.1

The continued evolution of SARS-CoV-2 is shaped by changes in antibody evasion and receptor engagement that influence viral fitness. RW.1.1, an emerging descendant of XFJ carrying five additional spike substitutions, has recently increased in frequency in North America. Here, we characterized the serum antibody evasion, monoclonal antibody sensitivity, and ACE2 receptor engagement of RW.1.1. Pseudovirus neutralization assays using sera from adults showed that RW.1.1 was not more resistant to serum neutralization than currently circulating variants, with neutralizing titers comparable to XFG, BA.3.2.2, and NB.1.8.1 in the tested cohort. Neutralization of RW.1.1 also did not differ significantly among adults, children, and infants and toddlers. Despite the absence of increased overall serum antibody resistance, monoclonal antibody neutralization assays revealed substantial resistance to several RBD class 1 antibodies and increased resistance to a subset of class 1/4 antibodies. Moreover, RW.1.1 exhibited reduced ACE2 receptor engagement compared with XFG, which itself has reduced receptor engagement relative to earlier JN.1 subvariants. Thus, RW.1.1 has continued to expand despite a further reduction in receptor engagement and without a substantial increase in overall serum antibody evasion. These findings suggest that the fitness of emerging SARS-CoV-2 variants may depend not only on the magnitude of antibody evasion but also on the specific components of the polyclonal antibody response that are evaded, highlighting the increasingly complex interplay between population immunity and receptor engagement in shaping SARS-CoV-2 evolution.

microbiology↗

bioRxiv2026

A dynein-driven nucleokinesis program enables neural crest migration through confined tissues in vivo

Cell migration through confined tissue environments requires precise coordination between the nucleus and the cytoskeleton. Translocation of the nucleus through small pores is a limiting step in confined cell migration. Work in immune and cancer cells has illuminated a key role for actin polymerization and Myosin-II contractility in facilitating nucleus translocation through confinement. However, the microtubule cytoskeleton can also adaptively stabilise under confinement. Microtubule-dependent nucleokinesis constitutes an evolutionarily conserved cellular program ensuring directional nucleus translocation by deployment of cytoplasmic dynein motors. However, whether microtubule-driven nucleokinesis can operate during cell migration through confinement remained so far unexplored. The zebrafish neural crest (NC) provides a unique in vivo system to address this question. NC cells are a highly migratory, multipotent precursors of the vertebrate peripheral nervous system, which traverse diverse environments along the anterior-posterior axis of the embryo. In the head, cranial neural crest (cNC) cells migrate through loosely organised tissues, whereas trunk neural crest (tNC) cells navigate narrowly confined tissue spaces. Here, we show that tNC cells engage a microtubule motor-driven program of nuclear deformation and translocation during confined migration in vivo. Under confinement, tNC cells reorganize their microtubules from a perinuclear meshwork into a polarized, centrosome-associated bundle positioned ahead of the nucleus. Laser ablation reveals that this microtubule array actively pulls and deforms the nucleus. Using pharmacological and genetic approaches, we discover that dynein-dependent pulling forces enable nucleus translocation through confinement. Strikingly, this process occurs independently of Rho/ROCK/myosin II-mediated contractility, revealing a novel neuronal-like mode of nucleokinesis operating in confined tissue environments. Together, our findings uncover a conserved nuclear translocation program deployed during neural crest migration and suggest that microtubule-based nucleokinesis represents a fundamental strategy for navigating tissue confinement in vivo during both central and peripheral nervous system development.

cell biology↗

bioRxiv2026

A Monoclonal Antibody Panel to Track Ongoing Antigenic Evolution of SARS-CoV-2 Variants

SARS-CoV-2 continues to evolve, generating successive variants with enhanced resistance to antibody-mediated neutralization. However, many existing monoclonal antibody (mAb) repertoires were derived from exposures to ancestral or early Omicron strains and thus incompletely capture the antigenic landscape of newly emerging strains. Here, we generated a mAb panel by immunizing naive, unimprinted VelocImmune humanized mice with spike from the KP.3.1.1 variant, and isolated and characterized 11 neutralizing antibodies targeting diverse epitopes of recently evolved antigenic variants. We then integrated these antibodies with previously characterized mAbs to establish an expanded toolbox for profiling antigenic evolution among emerging variants. Application of this panel to currently emerging SARS-CoV-2 variants provided mechanistic insight into recent antigenic evolution, highlighting the potential role of altered receptor-binding domain conformation. Our data also revealed clear differences in antibody-evasion profiles between JN.1-derived lineages and BA.3.2 lineages. Together, our findings establish an updated mAb panel for monitoring SARS-CoV-2 antigenic evolution.

microbiology↗

bioRxiv2026

Effects of periodic group housing opportunities on reproductive performance and welfare in sows

ObjectiveStall housing of pregnant sows raises welfare concerns, whereas conventional group housing systems often reduce space efficiency. This study evaluated the effects of periodic group housing (PG) on reproductive performance and welfare compared with continuous stall housing (CS). MethodsSows in the CS group (n = 15) were continuously housed in stalls. In the PG group (n = 15), sows were housed in groups of three and allocated 1 day of group housing and 6 days of stall housing per week over 10 weeks. During group housing sessions, the sows had access to a group housing area containing sawdust. Behavioral observations and salivary cortisol measurements were conducted on the first day of the stall housing session in weeks 1, 6, and 10. Behavioral indices were expressed as proportions based on 90 sampling points recorded at 1-min intervals. ResultsThe number of stillbirths was significantly lower in the PG group than in the CS group (0.63 vs. 1.49 piglets per litter). whereas other reproductive outcomes, including total litter size and average birth weight, did not differ. In older parity sows, the PG treatment markedly increased the proportion of time spent lying, suggesting reduced discomfort associated with restricted movement. Furthermore, the proportion of exploratory behavior decreased markedly, and drinking behavior showed a decreasing trend across parity levels in the PG group, suggesting partial satisfaction of motivations for environmental exploration and oral manipulation. The proportion of oral abnormal behavior showed a pronounced interaction between housing treatment and experimental week, increasing from week 1 to week 6 in the PG group. Salivary cortisol concentrations did not differ between the groups. ConclusionPG may improve reproductive performance and partially satisfy the behavioral motivations restricted under continuous stall housing. This system may represent a practical alternative for improving animal welfare while minimizing economic losses.

animal behavior and cognition↗

bioRxiv2026

Protein Response to ACL Injury in Humans Show Early Cartilage Remodeling and Differences by Sex

ObjectiveAnterior cruciate ligament (ACL) tears increase the risk for developing posttraumatic osteoarthritis (PTOA). Females have greater risk for both. However, studies defining sex-specific protein responses in human cartilage after ACL injury are lacking. We hypothesize that articular cartilages response to an injurious environment differs depending on sex. DesignWe compared the proteomic profiles of normal cartilage with injured cartilage harvested from the intercondylar area during ACL surgery. Sex-specific injury effects were estimated through contrasts between Injured Male and Normal Male and between Injured Female and Normal Female. Pathway enrichment analysis was done using gene ontology (GO) and compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the Matrisome AnalyzeR. ResultsFrom the 2,188 proteins identified, males and females shared 1,121 upregulated and 23 downregulated proteins in injured compared to normal cartilage. Analysis of ECM proteins and enriched pathways revealed mostly similar male and female responses to an injurious environment, with evidence of early cartilage remodeling in both sexes. Nevertheless, more than 240 proteins were affected specifically by sex, and significant sex differences were found in inflammation, ECM-related, and metabolic pathways. Males were enriched mostly in "ECM-receptor interaction", while females were enriched in "Citrate cycle (TCA cycle)", "Fatty acid degradation", and "Fatty acid metabolism" pathways. ConclusionArticular cartilage shows signs of remodeling soon after ACL injury, even when only exposed to an injurious environment rather than being physically impacted. Sex differences were observed in inflammation, metabolic pathways, and ECM synthesis.

biochemistry↗

bioRxiv2026

Robust volumetric multiplex staining of centimeter-scale FFPE tissues guided by neural network-based optimization

Archived formalin-fixed, paraffin-embedded (FFPE) tissues, including brain-bank specimens, are valuable resources for disease research, but rapid cyclic immunolabeling of intact specimens remains challenging. Here, we present FIDELITY, an epoxy-free workflow that balances optical transparency, immunostaining performance, and tissue integrity for multi-cycle protein profiling in centimeter-thick FFPE specimens. Neural network-guided optimization identified an SDS-glycine formulation that integrates delipidation and epitope retrieval while preserving tissue stiffness, replacing prolonged epoxy reinforcement and subsequent high-temperature antigen retrieval with a single aqueous pretreatment. Application to human Alzheimer's disease specimens enabled volumetric imaging and quantitative analysis while retaining compatibility with conventional histopathology. Its compatibility with active electrophoretic staining further supported rapid labeling and five rounds of immunolabeling in a whole FFPE mouse brain. Two-round labeling of a human glioma specimen further demonstrated the applicability of the cyclic workflow to human pathological tissue. Together, FIDELITY provides a practical platform for large-volume, multiplexed protein profiling of intact FFPE specimens.

neuroscience↗

bioRxiv2026

Time perception follows Weber's law in Drosophila

The ability to perceive time is essential for adaptive behavior, enabling organisms to respond to change, coordinate actions, and predict future events in dynamic environments and during social interactions. However, its evolutionary origins and underlying neural mechanisms remain poorly understood. Here, we demonstrate that the fruit fly, Drosophila melanogaster, can perceive time intervals ranging from sub-second to a few seconds and use them to predict the location of potential food sources. Using a behavioral paradigm in which flies learn to associate temporal patterns of sound with food rewards, we show that their ability to discriminate between two time intervals depends on the ratio of their durations rather than their absolute difference. This proportional relationship follows Webers law, a fundamental principle of sensory discrimination. Moreover, flies can generalize learned temporal rules to novel stimuli and across sensory modalities, suggesting they form an abstract representation of time. Finally, we identify the mushroom body as a critical neural circuit for temporal learning. These findings reveal unexpected timing capabilities in Drosophila, providing new insights into the evolutionary origin of temporal cognition and establishing Drosophila as a genetically tractable model for investigating the neural basis of time perception.

neuroscience↗

bioRxiv2025

Structural basis for non-AUG translation regulation by 5MPs

The cellular proteome is regulated by translation initiation on AUG or non-canonical (non-AUG) start codons1-3. Non-AUG initiation remodels proteome during stress and is implicated in cancer and other diseases4-6. The eIF5-mimic proteins (5MPs) restrict non-AUG start codon usage and thereby reprogram proteoform expression from mRNAs with alternative start sites, such as the oncogenic c-Myc7-10. The mechanism by which 5MPs induce such translational reprogramming remains unknown. Here, using in extracto cryo-electron microscopy (cryo-EM) and biochemical assays, we report that translational repression by 5MP strongly depends on the sequence context near the AUG or non-AUG codons. Cryo-EM structures of 5MP-bound 48S pre-initiation complexes (PICs) from native cell extracts reveal that 5MP binds at the A site of the small ribosomal subunit, stabilizing an expanded open-head conformation of the PIC scanning along mRNA. The N-terminal region of 5MP blocks the A site, whereas the C-terminal domain docks at eIF2{beta} and the initiator tRNAMet outside the P site (i.e., Pout). These findings indicate that 5MP protein directly biases the initiating 48S complexes toward the open conformation, promoting mRNA scanning and inhibiting initiation at suboptimal start codons.

biophysics↗

bioRxiv2025

An Atlas of Extrachromosomal DNA Structures Illuminates Its Evolution and Biogenesis in Cancer

Extrachromosomal circular DNA (ecDNA) is a prevalent driver of oncogene amplification across diverse types of cancers. Leveraging single-molecule long-read sequencing and a de novo circular genome assembler, ecLego3, we charted a comprehensive atlas of ecDNA structures from longitudinal pairs of primary and recurrent glioblastoma (GBM). Our approach resolved 23 ecDNA genomes representing major oncogenes associated with GBM pathogenesis and revealed a dynamic configuration characterized by pronounced structural variation and multi-species co-existence within individual tumors. Evolutionary trajectories were shaped by hierarchical accrual of structural variants, predominated by deletions and inversions, giving rise to novel oncogene isoforms and enhancer repositioning. Such structural plasticity promotes intratumor heterogeneity and tumor fitness. Multiomic analyses at single cell resolution linked ecDNA abundance and structure to transcriptional output, uncovering non-linear scaling and synergistic co-selection among multiple ecDNA species. Comparative profiling revealed locus-dependent configuration, a contraction of ecDNA diversity upon recurrence, and convergent structural architectures across patients, implicating repeat-mediated genomic rearrangements and replication stress in ecDNA biogenesis. These findings elucidate ecDNA-driven mechanisms of tumor adaptation and highlight new molecular vulnerabilities, informing potential biomarkers and therapeutic strategies for high-grade gliomas.

cancer biology↗

bioRxiv2025

Phenotypes and Cellular Mechanics of Primary Human Aorta- and Pluripotent Stem Cell-derived Vascular Smooth Muscle Cells

AimsMature, contractile vascular smooth muscle cells (vSMC) in the medial layer of muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Our aim was to define the mechanical properties of well-characterized in vitro-differentiated vSMC compared to vSMC derived from adult human aorta (AoSMC). Methods and ResultsWe generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines, and we obtained proliferating AoSMC. Immature, synthetic hPSC-vSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMCs are non-proliferative, have elevated levels of contractile proteins, and can undergo phenotype switching in vitro into a proliferative form. Immunostaining of contractile proteins present in vSMCs (CNN1, MYH11, TAGLN, ACTA2) display similar cell-to-cell heterogeneities between AoSMC and hPSC-vSMCs. The fluorescent signals are relatively uniform at sub-confluency, but highly variable at confluency. Human PSC-vSMC maintain doubling rates more readily and are more easily enriched using lactate medium than AoSMC. The single cell mechanics of AoSMC and hPSC-vSMC, assessed using micropost array detectors (mPADs), are highly comparable and have an average force production per micropost in the nN range. When cultivated to form humanized smooth muscle cell microtissues (SMTs), developed forces normalized to cell numbers are similar to that seen using microarray post detectors. Maximum developed forces in these tissues could be measured in response to endothelin-1. ConclusionsThese results demonstrate that synthetic hPSC-vSMCs and proliferating AoSMCs are highly comparable, and mechanically similar, but results with hPSC-vSMC are more reproducible. This study establishes the hPSC-vSMCs as a reproducible system to study how changes in cellular mechanics may contribute to development and disease. Translational PerspectiveThe mechanical properties and responses of lineage-specific human vSMC undergoing phenotype switching during perinatal development and vascular pathologies remain poorly understood. We have employed paraxial-mesoderm derived hPSC-vSMC, which recapitulate phenotypic properties of AoSMC, to define the mechanical properties of synthetic and contractile vSMCs. We quantitatively measured the forces of individual vSMC and of multicellular vSMC constructs. By defining the mechanical changes of normal vSMCs to phenotype switching, this validated, adaptable system advance studies of vSMC mechanics that may contribute to vascular pathologies in patients.

cell biology↗

bioRxiv2025

Prolonged oscillating kisspeptin neuron activity underlies the preovulatory luteinizing hormone surge in mice

The population of kisspeptin neurons located in the rostral periventricular area of the third ventricle (RP3V) is thought to have a key role in generating the GnRH surge that triggers ovulation. Using a modified GCaMP fibre photometry procedure, we have been able to record the in vivo population activity of RP3VKISS neurons across the estrous cycle of female mice. A marked increase in GCaMP activity was detected beginning on the afternoon of proestrus that lasted in total for 13{+/-}1 hours. This was comprised of slow baseline oscillations with a period of 91{+/-}4 min and associated with high frequency rapid transients. Very little oscillating baseline or transient activity was detected at other stages of the estrous cycle. Concurrent blood sampling showed that the peak of the LH surge occurred 3.5{+/-}1.1 h after the first baseline RP3VKISS neuron baseline oscillation on the afternoon of proestrus. The time of onset of RP3VKISS neuron oscillations varied between mice and across subsequent proestrous stages in the same mice. To assess the impact of estradiol on RP3VKISS neuron activity, mice were ovariectomized and given an incremental estradiol replacement regimen. Minimal patterned GCaMP activity was found in OVX mice, and this was not changed acutely by any of the estradiol treatments. However, on the afternoon of the expected LH surge, the same oscillating baseline activity with associated transients occurred for 7.1{+/-}0.5 h. These observations reveal an unexpected prolonged oscillatory pattern of RP3VKISS neuron activity that is dependent on estrogen and underlies the preovulatory LH surge as well as potentially other facets of reproductive behavior.

neuroscience↗

bioRxiv2025

Single-cell-resolved calcium and organelle dynamics in resistosome-mediated cell death

Plant nucleotide-binding domain leucine-rich repeat-containing (NLR) proteins act as intracellular immune receptors that assemble into resistosomes to execute immune responses. However, the subcellular processes during cell death following resistosome activation remain unclear. Here, we visualized the changes in calcium signaling and organelle behavior after activation of the NRC4 (NLR-required for cell death 4) resistosome. We found that NRC4 membrane enrichment coincided with calcium influx. This is followed by sequential mitochondria and plastid disruption, endoplasmic reticulum fragmentation and cytoskeleton depolymerization. Subsequent loss of plasma membrane integrity, nuclear shrinkage, and vacuolar collapse mark the terminal stage of cell death. Our findings reveal a spatiotemporally-resolved cascade of subcellular events downstream of resistosome activation, providing new mechanistic insight into the execution phase of plant immune cell death.

plant biology↗

bioRxiv2024

Rhizobium rhizogenes A4-derived strains mediate hyper-efficient transient gene expression in Nicotiana benthamiana and other solanaceous plants

Agroinfiltration, a method utilizing agrobacteria to transfer DNA into plant cells, is widely used for transient gene expression in plants. Besides the commonly used Agrobacterium strains, Rhizobium rhizogenes can also introduce foreign DNA into host plants for gene expression. While many R. rhizogenes strains have been known for inducing hairy root symptoms, their use for transient expression has not been fully explored. Here, we showed that R. rhizogenes A4 outperformed all other tested agrobacterial strains in agroinfiltration experiments on leaves of Nicotiana benthamiana and other solanaceous plants. By conducting an agroinfiltration screening in N. benthamiana leaves using various agrobacterial strains carrying the RUBY reporter gene cassette, we discovered that A4 mediates the strongest and fastest transient expression. Utilizing the genomic information, we developed a collection of disarmed and modified strains derived from A4. By performing vacuum infiltration assays, we demonstrated that these A4-derived strains efficiently transiently transform 6-week-old N. benthamiana leaves, showing less sensitivity to the age of plants compared to the laboratory strain GV3101. Furthermore, we performed agroinfiltration using AS109, an A4-derived disarmed strain, on the leaves of tomato, pepper, and eggplant. Remarkably, AS109 mediated transient gene expression on tested solanaceous plants more effectively than all the tested commonly used agrobacterial strains. This discovery paves the way for establishing R. rhizogenes A4-derived strains as a new option for enhancing transient expression in N. benthamiana and facilitating the functional study of plant genes in other solanaceous species.

plant biology↗

bioRxiv2024

The Taiwan Precision Medicine Initiative: A Cohort for Large-Scale Studies

The Taiwan Precision Medicine Initiative (TPMI), a project initiated by the Academia Sinica in collaboration with 16 major medical centers around Taiwan, has recruited 565,390 participants who consented to provide DNA samples for genetic profiling and grant access to their electronic medical records (EMR) for studies to develop precision medicine. Access to the EMR is both retrospective and prospective, allowing researchers to conduct prospective studies over time. Genetic profiling is done with population-optimized SNP arrays for the Han Chinese populations that enable genetic analyses such as genome-wide association, phenome-wide association, and polygenic risk score studies to evaluate common disease risk and pharmacogenetic response. Furthermore, the TPMI participants agree to be contacted for future research opportunities related to their genetic risks and receive personalized genetic risk profiles with health management recommendations. TPMI has established the TPMI Data Access Platform (TDAP), a central database and analysis platform that both safeguards the security of the data and facilitates academic research. The TPMI is the largest non-European cohort that merges genetic profiles with EMR in the world. With a cohort that can be followed over time, it can be utilized to validate genetic risk prediction models, conduct clinical trials to show the efficacy of risk-based health management, and optimize health policies based on genetic risks. In this report, we describe the TPMI study design, the population and genetic characteristics of the TPMI cohort, and the power it provides to conduct crucial studies in developing precision medicine on a population and personal level. As Han Chinese represent almost 20% of the worlds population, the results of TPMI studies will benefit >1.4 billion people around the world and serve as a model for developing population-based precision medicine.

genetics↗

bioRxiv2024

Challenging the Conventional Treatment Initiation Paradigm: Early Detection of Irreversible Cellular Damage in Cardiac Biopsies of Fabry Disease Before the Formation of Gb3 Inclusion Bodies

BackgroundFabry disease (FD) is a lysosomal storage disorder impacting multiple organs, including the heart. We investigated whether early-stage globotriaosylceramide (Gb3) accumulation, before occurrence of inclusion bodies, could cause significant stress and irreversible damages of the cardiomyocytes in FD patients. To assess the cellular stress and irreversible damage of cardiomyocytes in FD during early-stage Gb3 accumulation before the occurrence of typical pathology. MethodsImmunofluorescent (IF) staining or Western blotting were performed on fibroblasts from FD patients and myocardial biopsies from G3Stg/GLAko mice and FD patients. Notably, all biopsies exhibited detectable Gb3 accumulation under IF but lacked typical FD (Gb3 inclusion body) pathology. Staining targeted nuclear factor-{kappa}B (NF-{kappa}B), interleukin-18 (IL-18), phospho-p42/44 mitogen-activated protein kinase (MAPK), and inducible nitric oxide synthase (iNOS) as inflammatory and oxidative stress markers. Alpha-smooth muscle actin (-SMA) IF staining was conducted to detect myofibroblasts. ResultsFibroblasts from FD patients, in conjunction with cardiomyocytes from both G3Stg/GLAko mice and FD patients, exhibited significant accumulation of inflammatory markers such as NF-{kappa}B IL-18 and phospho-p42/44 MAPK, as well as the oxidative stress marker iNOS. Despite the absence of typical FD pathology, the presence of fibrosis was confirmed in myocardial biopsies from these patients through strong positive staining of -SMA. ConclusionsSignificant cellular stress and even irreversible damage may occur before the onset of typical pathological changes in cardiomyocytes of FD. Based on our findings, treatment should be initiated much earlier than we currently thought to prevent irreversible damage and improve the prognosis of FD patients.

pathology↗

bioRxiv2024

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating suicide exon and Cre recombinase

Chemical-inducible gene expression systems have been frequently used to regulate gene expression for functional genomics in various plant species. However, a convenient chemical-inducible system that can tightly regulate transgene expression in Nicotiana benthamiana is still missing. In this study, we developed a tightly regulated copper-inducible system that can be used to regulate transgene expression and perform cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system showed the least concerns of leakiness issues. Using the MoClo-based synthetic biology approach, we optimized the design of the copper-inducible system and incorporated the use of the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested NLRs and their matching AVRs, and it can also be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings provide new approaches for both fundamental and translational studies in plant functional genomics.

plant biology↗

bioRxiv2023

Functional divergence shaped the network architecture of plant immune receptors

In solanaceous plants, several sensor NLRs and their helper NLRs, known as NRC, form a complex network to confer immunity against pathogens. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, the evolution and molecular basis of the complex network structure remained elusive. Here we demonstrated that functional divergence of NRC3 variants has shaped the genetic architecture of the NLR network. Natural NRC3 variants form three allelic groups displaying distinct compatibilities with sensor NLRs. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility, with two residues critical for subfunctionalization. Co-functioning Rpi-blb2 and NRC3 variants showed stronger transient interactions upon effector detection, with NRC3 membrane-associated complexes forming subsequently. Our findings reveal how mutations in helper NLRs, particularly NRC3, have driven the evolution of their transient interactions with sensor NLRs, leading to subfunctionalization and contributing significantly to the complexity of the NRC network in plant immunity. TeaserHelper NLR subfunctionalization alters transient interactions with sensor NLRs, enhancing plant immune system complexity.

plant biology↗

bioRxiv2023

Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate binding pocket. Of 631 fragments soaked, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (2 hits) or at crystal packing interfaces (3 hits). Notably, two fragments with a pose sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. Our structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. SynopsisAn X-ray crystallographic screen on SARS-CoV-2 3CL protease resulted in 29 fragment hits, including two isatin-based reversible covalent binders, and revealed a strong influence of the crystal form used for fragment soaking on the bound conformation of three additional reference fragments.

biophysics↗