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Despite the recent advancement of proteolysis-targeting chimera (PROTAC) development, they remain predominantly dependent on two E3 ligases, CRBN and VHL, which are ubiquitously expressed in all types of cells. Recently, efforts to discover tissue-specific E3 ligase ligands get attention as a promising strategy to enable tissue-specific protein degradation and avoid off-target tissue effects. Advancing this line of research, we discover a ligand of KLHL41, a muscle-specific E3 ligase, through virtual screening. Building on the KLHL41 ligand, we develop KBD-1, a muscle-specific BRD4-targeting PROTAC with micromolar activity. To enhance degradation efficiency, we employ a two-body kinetic strategy, resulting in the covalent PROTAC cKBD-1, which achieves sub-nanomolar activity. cKBD-1 demonstrates muscle-specific BRD4 degradation through KLHL41 recruitment both in vitro and in vivo. Moreover, the KLHL41 ligand enables AR-targeting PROTAC development, demonstrating its broad applicability. These findings highlight the potential of KLHL41 as a platform for tissue-specific protein degradation and its applicability in therapeutic development.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the aggregation of Lewy bodies, composed of the protein α-synuclein, and the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta. The management of PD seeks to mitigate motor symptoms by substituting diminished endogenous dopamine; nevertheless, it does not halt disease progression. Various animal models have been employed to elucidate the etiology of PD and to discover disease-modifying treatments. Zebrafish serve as a PD model owing to their capacity for high-throughput screening. This review presents updates on the currently available zebrafish models of PD, encompassing both chemically induced and genetically based models, and discusses their advantages and limitations. This review also delineates numerous investigative strategies that utilize the zebrafish PD model and summarizes the findings of previous studies. Taken together, further studies, including the investigation of the regeneration mechanism of DA neurons, neurobehavioral testing of adult zebrafish reflecting PD-associated neurocognitive impairment, and a reliable gene-based model providing precise gene knockout and reproducibility, may assist in elucidating the critical pathways that trigger PD and its progression, alongside potential targets to hinder this progression.

The overexpression of the transcriptional enhanced associate domain (TEAD), which regulates gene transcription linked to cell growth, drives the proliferation in cases of hepatocellular carcinoma (HCC). In order to discover novel TEAD inhibitors that are more effective and have better efficacy and pharmacokinetic properties for treating HCC, this study employed a cyclization strategy to generate a novel indole-based scaffold of TEAD inhibitors. A comprehensive and systematic structure-activity relationship (SAR) analysis identified the most promising compound: LC-TD-05, a non-covalent, partial TEAD inhibitor with selective activity against TEAD1, TEAD2 and TEAD4, but reduced potency against TEAD3. LC-TD-05 exhibits good potency against TEAD1/2/4 (TEAD1 IC50 = 116.6 ± 21.7 nM, TEAD2 IC50 = 168.7 ± 17.1 nM, TEAD4 IC50 = 68.3 ± 18.2 nM), demonstrates favorable oral bioavailability (F = 53.7%), and exhibits significant anti-tumor activity in HCC LM3 models in vitro (LM3 cell IC50 = 248 ± 27.9 nM) and in vivo (TGI = 75%). Overall, this study provides a novel scaffold for TEAD inhibitors, enabling more effective interventions against HCC.

Allergic rhinitis (AR), asthma, and their combined allergic rhinitis and asthma syndrome (CARAS) frequently coexist. However, the underlying pathophysiological and metabolic mechanisms, as well as reliable diagnostic differentiation, remain challenging. Carboxyl-containing metabolites (CCMs) have been implicated in the pathogenesis of these conditions; therefore, this study aimed to comprehensively profile serum CCMs in pediatric patients. Sera from 63 children with AR, 41 with asthma, and 90 with CARAS, sensitized to Dermatophagoides farina and/or Dermatophagoides pteronyssinus, were analyzed using ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry coupled with 5-(diisopropylamino) amylamine derivatization. The results identified 100 differentially expressed metabolites common across them, alongside disease-specific alterations of 23, 17, and 31 unique to AR, asthma, and CARAS, respectively. These findings reveal that allergic airway diseases share a core metabolic disturbances that may reflect immune dysregulation and potentially involve gut dysbiosis, and yet exhibit distinct profiles: AR shows localized amino acid perturbations, asthma displays systemic lipid remodeling, and CARAS manifests as a distinct comorbid phenotype combining both features.

Fossil evidence indicates that millipedes were the first animals adapted to life on land about 425 million years ago, becoming the very first land animals and beating vertebrates by a staggering 50 million years.1,2 These multi-legged arthropods provide a vital ecological role in forests by decomposing coarse organic matter and contributing to the formation of nutrient-rich soils.3,4 To date, 14,232 species have been described, with at least as many still awaiting discovery.5 Despite their ecological significance and ancient origins in the Ordovician, the evolutionary relationships among millipedes have remained unresolved, and a synthesis of the 16 orders that comprise the class Diplopoda had never been attempted. In this study, we analyzed the last two remaining unsampled orders, Siphonocryptida and Siphoniulida, two rare paleoendemics whose placement had been unresolved until now. Our results show that all extant diplopod orders except one were present by the end of the Jurassic, that millipedes evolved potent terpenoid alkaloid chemical defenses 261 million years ago, and that Siphonocryptida is a derived lineage of Polyzoniida. Early millipede lineages possessed sophisticated sensory structures, including compound eyes and Tömösváry organs, which were repeatedly lost over 459 million years of diversification. These findings provide a robust framework for understanding the evolution of the earliest fully terrestrial animals and support ongoing efforts to discover and describe thousands of new millipede species.

Bacteriocins are antimicrobial peptides/proteins that are widely distributed among bacteria and are gathering traction as natural alternatives to antibiotics, modulators of the microbiota, and interbacterial signaling peptides. The Hungate1000 is a culture collection of isolated prokaryotic microorganisms and their genomes from ruminant animals that aims to expand the knowledge base of rumen ecology. In this study, 410 rumen-isolated prokaryotes within the collection were mined to expand upon the bacteriocin-producing potential of the rumen. A total of 408 novel bacteriocin gene clusters were identified across 308 genomes. Bacteriocins in novel species within the Hungate1000 were identified, such as Pseudobutyrivibrio sp. UC1225, which has two novel natural nisin variants, Clostridium sp. DSM 8431 with a novel peptide 81% identity to amylocyclicin and Lachnobacterium C7 encoding a novel circular bacteriocin with 55% identity to the circular bacteriocin NKR-5-3B. A novel class II lanthipeptide gene cluster was also identified containing eight distinct core peptides encoded within the genome of a novel Butyrivibrio species. Bacteriocin biosynthetic potential was noted within species unknown to produce bacteriocins, such as Lachnobacterium bovis DSM 14045, Lachnospira multipara D15d, Eubacterium callanderi NLAE-zl-G225, Eisenbergiella tayi NLAE-zl-G231, and Muricomes contorta NLAE-zl-C134. The frequency of putative bacteriocin production within ruminal strains was 30%, doubling the frequency previously suggested in the mammalian gastrointestinal tract. This number increases to ~70% when encompassing groups of peptides with limited knowledge of antibacterial activity, such as ranthipeptides and auto-inducing peptides. We also show that the bacteriocin core peptides mined from the Hungate1000 culture collection are found in the microbiomes of other ruminant animals and the human gut microbiome. These findings highlight the Hungate1000 as a rich biosynthetic reservoir of cultured strains that can be experimentally explored for functional antimicrobial activity. The presence of diverse bacteriocin-producing lineages in rumen-associated microbes provides a foundation for future strategies aimed at targeted microbiome modulation, including approaches to improve rumen function and potentially mitigate enteric methane emissions using bacterial strains or their natural products.IMPORTANCEBacteriocins are gathering traction as a possible alternative to antibiotics in some instances. Therefore, it is crucial to discover novel bacteriocins to expand the bacteriocin knowledge base if these peptides are to be translated to the clinic for use in humans or developed as veterinary interventions to modulate rumen function. Here, we use in silico methods to identify the biosynthetic potential of the Hungate1000 culture collection of rumen bacterial strains. We discover 408 novel bacteriocin gene clusters across 308 genomes and identify that the frequency of bacteriocin gene clusters is over 30%, which is double the incidence rate from previous studies of the mammalian gastrointestinal tract. This number increases to approximately 70% when including bacteriocin classes such as ranthipeptides and cyclic-lactone-autoinducer peptides. Together, these findings position the rumen microbiome as a rich and underexplored reservoir of antimicrobial diversity, with potential for the development of targeted microbiome-modulating therapeutics, livestock interventions aimed at improving rumen function, and strategies aligned with One Health goals, including antimicrobial stewardship and methane mitigation.

Sex and gender are often used interchangeably, though they refer to distinct concepts -the former relating to biological differences grounded in chromosomal and hormonal features and the latter to socially constructed norms, roles, and identity. In preclinical research with non-human animals, the prevailing view is that only sex differences are measurable because it is commonly accepted that rodents do not express a gender identity. However, we argue that rodents express context-specific, sex-specific behaviors which fulfill the defining requirement of gender-like norms. To explore whether sex-specific behavioral patterns in non-human animals provide evidence of gender-like norms, we focus on rodents, the most common non-human animal model. We cite examples of how their sex-specific behavior is shaped by their environment. Importantly we acknowledge that gender norms are malleable depending on the physical and social contexts - a dynamic mix of spatial environment, material composition, and social structure that jointly shapes how behavior unfolds and its meaning. Thus, our analysis examines these gender-like norms across three levels. First, we compare natural and vivarium environments to reveal the malleability of gender-like norms. We then explore how the material composition in the physical context of the vivarium cage (e.g., cage type, enrichment) can shift these norms. Finally, we show the social context within the cage (e.g., sex composition, rank, familiarity) modifies individual interactions which in turn, affects the expression of gender-like norms. Throughout we provide experimental design recommendations that advocate for the explicit consideration of both sex and gendered behavior in rodent models. Incorporating these gender considerations will yield more effective and translatable animal models, significantly improving our ability to discover and test treatments for human toxicology, physiology, psychopathology, and neurological diseases.

Perceptual systems in humans and many other animals are able to segment scenes into regions that are likely to be physically meaningful. This ability depends on having low-level mechanisms that can accurately categorize whether local image patches are samples from the same or different kinds of texture. We find that using spatial proximity as a proxy for same-different ground truth makes it possible to train accurate decision variables and bounds directly from arbitrary natural images with no feedback. We also find that performance can be further improved by using proximity as a ground truth for adjusting the final decision variables and bounds for the current image/scene. These surprising findings result from the simple fact that under a wide range of conditions proximity discrimination (near vs. far) and texture discrimination (same vs. different) have mathematically identical decision bounds if the same image features are used for both tasks. We used the decision variables and bounds trained on natural images as the initial steps in a hierarchical Bayesian observer (HBO) model of texture discrimination [9]. Given the relative simplicity of this HBO model, it did an excellent job of segmenting images having randomly shaped regions containing arbitrary natural textures. We suggest that the proximity proxy is something that natural selection could discover and exploit for any same-different task where the task-relevant stimulus features also vary systematically with distance in space and/or time. For example, natural selection could have created developmental learning/plasticity mechanisms that exploit the proximity proxy.

STING is an important component in the host innate immune system where its activation by cyclic dinucleotides culminates in the production of interferons and pro-inflammatory cytokines that mediate host defence against infection. While the mechanisms that govern STING-induced interferon production have been comprehensively characterised, how pro-inflammatory cytokines are produced downstream of STING remains less understood. Here we discover that IRF3 is critical for effective STING-mediated inflammatory cytokine production from macrophages as those lacking IRF3 display significant defects. Interestingly, the loss of IRF3 does not impact the activation of the prominent pro-inflammatory transcription factor, NF-κB, but rather affects the AP-1 transcriptional complex. We further discover the role of IRF3 in STING inflammatory responses is independent of its phosphorylation and distinct from its role as a transcription factor for induction of type I interferons. This additional activity of IRF3 is dependent on its recruitment to the previously defined IRF3 binding motif within the C-terminal tail of STING. Hence, our findings reveal an unexpected noncanonical function of IRF3 that is critical for mediating STING-induced pro-inflammatory cytokines from macrophages.

CRISPR technologies are transforming cardiovascular therapy development by creating an increasingly seamless pipeline from potential target discovery to clinical translation. What began as a genome-editing tool has evolved into a versatile platform that enables researchers to precisely interrogate and modulate cardiac biology with tools such as base- and prime-editors, and CRISPR inhibition and activation. In this review, we follow the use of CRISPR across the stages of biomedical research through to bench-to-bedside application. This review begins by addressing how genome-wide and focused CRISPR screens discover developmental regulators, disease drivers, and drug-response pathways, making the first steps in identifying therapeutic targets. We then explore how CRISPR engineering creates progressively more relevant disease model systems to validate mechanisms of disease and test interventions, helping bridge the translational gaps between the lab and the clinic. Finally, we consider how CRISPR technologies are beginning to enter cardiovascular clinical trials, while highlighting the key challenges that still limit this translation. By linking the latest advances of modern CRISPR platforms to the stages of therapeutic development, this review highlights how CRISPR technology is reshaping the pipeline from molecular insight to clinical innovation in cardiac disease.

Ubiquitin-fold modifier 1 (UFM1) covalently modifies protein substrates (UFMylation) and alters their biological functions. Genetic screening disclosed that enzymes in the UFMylation system play critical roles in regulating autophagy. However, it is still elusive which protein is UFMylated and how this modification modulates autophagy. Here, our quantitative proteomics and biochemical experiments identify SQSTM1/p62 as a UFMylation substrate and discover its two major UFMylation sites, K420 and K435. Mutating them to Arg (p622KR) completely abolishes the effect of p62 on autophagic activity. Fusion of UFM1ΔC4 to p622KR (p622KR-UFM1ΔC4) restores the p62-mediated pathogenic autophagic degradation in primary cortical neurons and Huntington's disease mouse striatum. Mechanistically, p62 UFMylation enhances its interaction with LC3, augments autophagic flux, and eliminates pathogenic mutant huntingtin. Collectively, this work discovers a new post-translational modification, UFMylation, on p62 and establishes this modification as a key regulator of autophagy that promotes the clearance of mutant huntingtin, offering a potential target for therapeutic intervention.

The manufacture of platelets from induced pluripotent stem cells (iPSCs) in a cost-effective, controlled and reproducible manner is a critical challenge for transfusion and regenerative medicine applications. Here we employ a high throughput, combinatorial screening method (CombiCult®) to discover new efficient protocols for generation of mature megakaryocytes (MKs) and platelets from human iPSC (hiPSC) lines. The CombiCult® platform was adapted to encapsulate hiPSCs cells in alginate by electro-spraying and cell differentiation was initiated in alginate beads, circumventing the need for stromal inducer cells, extracellular matrix materials and the process of embryoid body (EB) formation. Both feeder-dependent and feeder-free iPSC lines were multiplexed in one screen, allowing discovery of multiple serum-free feeder-free protocols that direct MK differentiation from diverse cell lines. These protocols provide high yield, high purity and low-cost methods for MK production compatible with large-scale suspension format bioreactor systems. Platelets produced by these methods (iPLT) phenotypically and functionally resemble human donor platelets following in vitro activation by platelet agonists. Furthermore, following transfusion in NOD-SCID mice, resting iPLT showed no evidence of pulmonary thrombo-embolism or heightened tissue accumulation up to 72 h post infusion and exhibited similar responses to donor platelets in a murine model of pulmonary platelet aggregation.

Azole-resistant Aspergillus infections are a source of increasing concern with limited alternative therapeutic options. However, as most infections are still caused by azole-susceptible Aspergillus strains, there is a need to better understand fungal responses to azole antifungals. To this end, we discover that a long non-coding RNA, afu-182, is a major regulator of cyp51-independent sub-MIC azole response. We observe that loss of afu-182 leads to increased surface attached growth and poor treated disease outcomes in a murine model of invasive pulmonary aspergillosis upon azole treatment. In contrast, overexpression of afu-182 significantly reduces fungal burden in animals treated with the azole drug, posaconazole. Importantly, afu-182 levels decrease upon azole exposure and in an azole adaptation experiment, continuous exposure to low dose azole led to MIC increase in an afu-182 dependent manner. Whole transcriptome analyses revealed that azole drug treatment leads to an increase in transcripts of genes encoding 7-transmembrane domain proteins of the RTA1 family, and these proteins are negatively regulated by afu-182. Two RTA1 family genes have individual and combined effects and are sufficient to increase fungal susceptibility to azole drugs in the WT strain. Taken together, our data show a role of the long non-coding RNA afu-182 in regulating Aspergillus fumigatus response to azole drugs both in vitro and in vivo.

The insect chitin metabolism system is an ideal target for species-selective pesticide development, in which OfChi-h and OfChtI jointly mediate chitin degradation. However, existing Chitinase inhibitors face limitations in efficacy and specificity, necessitating innovative computer-aided driven lead discovery strategies. Herein, a dual-target virtual screening was performed to discover triazinobenzoxazepine derivatives (TBZs) as novel Chitinase inhibitors. Compound TBZ-15 emerged as a potent dual inhibitor with Ki values of 0.64 μM and 0.43 μM against OfChi-h and OfChtI, respectively. TBZ-4 and TBZ-15 displayed stronger insecticidal activity against Ostrinia furnacalis (O. furnacalis) and Plutella xylostella (P. xylostella) than hexaflumuron, and TBZ-4 achieved better control efficacy than chlorbenzuron. Additionally, scanning electron microscopy observation and DFT/ESP calculations clarified their action mechanism. Toxicity prediction confirmed their safety to nontarget organisms. This work demonstrates TBZs as eco-friendly insecticides and highlights the value of computer-aided strategy for sustainable agrochemical development.

While T-cell receptors (TCRs) hold immense therapeutic potential, their clinical translation is bottlenecked by the scarcity of high-affinity, specific clones and the instability of recombinant formats. Existing display technologies fall short: phage display often fails to produce properly folded TCRs, and mammalian display is low-throughput. This review is the first to comprehensively synthesize how yeast surface display (YSD) is uniquely positioned to overcome these hurdles and build a new pipeline for TCR based drug discovery. We articulate how YSD's eukaryotic machinery enables the robust expression and engineering of complex TCRs and peptide-MHC (pMHC) libraries. We then critically catalog and evaluate pioneering strategies where YSD has been used to evolve TCRs with enhanced affinity and stability while minimizing cross-reactivity and discover novel antigenic targets through unbiased library-to-library screening. By mapping this largely pre-clinical landscape, our work establishes a foundational framework that bridges fundamental protein engineering with future therapeutic applications. We conclude by outlining a translational roadmap, arguing that YSD derived molecules are poised to become the cornerstone of safer, more effective, and personalized T-cell therapies.

Human adults can extract regularities through implicit learning, resulting in non-conscious knowledge, or through explicit learning, leading to conscious and reportable knowledge. Experiments aiming to disentangle implicit from explicit learning are limited by their heavy reliance on verbal instructions. This prevents the creation of a fully implicit situation and restricts the populations studied, effectively excluding non-verbal individuals. To address these limitations, the current study validates a non-verbal version of a standard test to assess implicit and explicit sequence knowledge: the Process Dissociation Procedure (PDP). In the PDP, after a learning phase where participants are exposed to spatial regularities, their control over sequence reproduction-a hallmark of explicit learning-is probed by contrasting two conditions: an inclusion condition, where participants must reproduce the sequences they have learned, and an exclusion condition, where they must avoid reproducing these. Departing from conventional design, inclusion/exclusion instructions were removed, and participants had to discover the task rules through trial and error. At the end of the experiment, they were asked to describe their understanding of the task. Data from 32 human adults showed that ceiling-level performance in inclusion and exclusion conditions occurred only in the subgroup of participants who verbally reported both the PDP rules and the presence of sequences. In contrast, others performed around the chance level. These results confirm that awareness is necessary for controlling sequence reproduction. This first non-verbal adaptation of the PDP provides a promising method to disentangle explicit from implicit learning in children and non-human animals.

Clonorchis sinensis (C. sinensis) infection causes serious pathological changes of hepatobiliary system such as hyperplasia of the biliary mucosa, inflammation and periductal fibrosis. The excretory-secretory products of C. sinensis (CsESPs) play critical roles in triggering inflammation and subsequent activation of hepatic stellate cells (HSCs). Yet, how CsESPs induce hepatic fibrosis through breaking the barrier of biliary epithelium remains unclear. Previous studies have confirmed that interleukin-17A (IL-17A) promoted fibrosis in some liver diseases. In the present study, the IL-17A levels in the serum of C. sinensis infected patients and healthy people were compared. C. sinensis infected mouse model was applied to discover the expression of IL-17A, especially its localization in the biliary epithelium. Cells and bile duct organoid models were established to evaluate the effect of CsESPs on the production of IL-17A by biliary epithelium and subsequent activation of HSCs. The results indicated that the levels of IL-17A were higher in the serum of patients and mice infected with C. sinensis than in the healthy people and control mice respectively. Infected mouse liver showed increased collagen deposition and marked hyperplasia of the intrahepatic bile duct with significant expression of IL-17A. CsESPs-stimulated human cholangiocarcinoma cells (RBE) displayed elevated proliferation ability and produced higher level of IL-17A. Supernatant of CsESPs-RBE cells activated human hepatic stellate cells (LX-2) with upregulated production of α-SMA and collagen I. Knocking down of IL-17A in RBE cells by lentivirus attenuated the expression of α-SMA and collagen I in LX-2 cells incubated with supernatants of CsESPs-stimulated RBE cells. Under stimulation of CsESPs, the bile duct organoids became swelled with thickened and deformable walls and prominent IL-17A signals. These findings suggest that CsESPs may activate HSCs through a new pathway of stimulating biliary epithelium to produce IL-17A.

The aim of this study is to conduct a thorough examination of the most recent derivatives that have been studied for their potential in treating Parkinson's disease and to establish the connection between the chemical structure of these derivatives and their effectiveness as therapeutic agents. A thorough examination of the available literature was carried out to gather data about the identification of pertinent research on derivatives aimed at addressing Parkinson's disease. The literature was analyzed to determine derivative chemical structures, modes of action, and preclinical and clinical results. To discover novel derivatives with therapeutic potential for Parkinson's disease, our investigation uncovered a variety of such chemical compounds. These derivatives cover a wide range of chemical classes and demonstrate different ways of working, such as impacting neurotransmitter systems, acting as antioxidants, and providing neuroprotective effects. In addition, an examination of the SAR (Structure-activity relationship) uncovered important structural characteristics linked to improved therapeutic effectiveness. This manuscript offers a thorough examination of the most recent derivatives aimed at addressing Parkinson's disease and their potential for therapeutic use. The understanding of SAR and pharmacological attributes provides insights into the development and refining of novel medications for Parkinson's disease treatment, promoting advancement in the field of neurotherapeutics.

Genetic introgression from Neanderthals and Denisovans shaped modern human genomes; however, introgressed structural variants (SVs ≥ 50 base pairs) remain challenging to discover. We integrated high-quality phased assemblies from four new Papua New Guinea (PNG) haploid genomes with 94 published assemblies of diverse ancestry to infer an introgressed SV map. Introgressed SVs are enriched in genes (47%), including critical genomic disorder regions, and are most abundant in PNG genomes. We identified 11 centromeres likely derived from archaic hominins, adding unexplored diversity to centromere genomics. Pangenome genotyping of these 98 assemblies across 1363 samples revealed 16 adaptive SVs, many associated with immune-related genes and expression, in the PNG genomes. We hypothesize that archaic SVs contributed to reproductive success, underscoring introgression as a major force in human adaptive evolution.