bioRxiv @biorxivpreprint@qoto.org

Background: Anxiety is a common symptom of several mental health disorders and adversely affects motivated behaviors. Anxiety can emerge from associating risk of future harm while engaged in goal-guided actions. Using a recently developed behavioral paradigm to model this aspect of anxiety, we investigated the role of two cortical subregions, the prelimbic medial frontal cortex (PL) and lateral orbitofrontal cortex (lOFC), which have been implicated in anxiety and outcome expectation, in flexible representation of actions associated with harm risk. Methods: A seek-take reward-guided instrumental task design was used to train animals to associate the seek action with a variable risk of punishment. After learning, animals underwent extinction training for this association. Fiber photometry was used to measure and compare neuronal activity in PL and lOFC during learning and extinction. Results: Animals increased action suppression in response to punishment contingencies. This increase dissipated after extinction training. These behavioral changes were associated with region specific changes in neuronal activity. PL neuronal activity preferentially adapted to threat of punishment whereas lOFC activity adapted to safe aspects of the task. Moreover, correlated activity between these regions was suppressed during actions associated with harm risk suggesting that these regions may guide behavior independently under anxiety. Conclusions: These findings suggest the PL and lOFC serve distinct but complementary roles in the representation of learned anxiety. This dissociation may provide a mechanism for how overlapping cortical systems are implicated in reward-guided action execution during anxiety. ### Competing Interest Statement The authors have declared no competing interest.

The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo where it promotes immune escape of HIV-infected cells and viral persistence. While these features identify Nef as an attractive antiretroviral drug target, Nef lacks enzymatic activity and an active site, complicating development of occupancy-based drugs. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on a previously reported hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the Cereblon E3 ubiquitin ligase, resulting in ubiquitylation of Nef and proteolytic degradation. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation of Nef is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo. ### Competing Interest Statement The authors have declared no competing interest.

The MYCN oncoprotein broadly binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here we show that MYCN forms stable high molecular weight complexes with the exosome and multiple RNA-binding proteins. In cells, MYCN binds to thousands of intronic RNAs; recombinant MYCN directly binds RNA via a short, highly conserved sequence termed MYCBoxI. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. At promoters, MYCN is then replaced by the MNT(MXD6) repressor protein, which inhibits MYCN-dependent transcription. MYCN promotes the degradation of its bound introns via the nuclear exosome targeting (NEXT) complex. Our data demonstrate that MYCN is an RNA-binding protein that regulates nascent transcript turnover and show that competition between its RNA- and DNA-bound states links the dynamics of the MYCN/MAX/MXD network to mRNA processing. ### Competing Interest Statement M.E. is a founder and shareholder of Tucana Biosciences.

Understanding the utilization of tunnels and water transport within enzymes is crucial for the catalytic function of enzymes, as water molecules can stabilize bound substrates and help with unbinding processes of products and inhibitors. Since the choice of water models for molecular dynamics simulations was shown to determine the accuracy of various calculated properties of the bulk solvent and solvated proteins, we have investigated if and to what extent the water transport through the enzyme tunnels depends on the selection of the water model. Here, we have focused on simulating enzymes with various well-defined tunnel geometries. In a systematic investigation using haloalkane dehalogenase as a model system, we focused on the well-established TIP3P, OPC, and TIP4P-Ew water models to explore their impact on using tunnels for water molecules transport. The TIP3P water model showed significantly faster migration, resulting in the transport of approximately 2.5 times more water molecules in comparison to OPC and 2.0 times greater than the TIP4P-Ew. The increase in migration of TIP3P water molecules was mainly due to faster transit times, and in the case of narrower tunnels, greater concurrent transport was evident as well. We have observed similar behavior in two different enzymes with buried active sites and different tunnel network topologies, indicating that our findings are likely not restricted to a particular enzyme family. Our study emphasizes the critical importance of water models in comprehending the use of enzyme tunnels for small molecule transport. Given the significant role of water availability in various stages of the catalytic cycle and solvation of substrates, products, and drugs, choosing an appropriate water model might be crucial for accurate simulations of complex enzymatic reactions, rational enzyme design, and predicting drug residence times. ### Competing Interest Statement The authors have declared no competing interest.

Background: The natural barrier function of the epidermal skin layer poses a significant challenge to nanoparticle-mediated topical delivery. A key factor in this barrier function is the thickness of the stratum corneum (SC) layer within the epidermis, which varies across different anatomical sites. The epidermis from the palms and soles, for instance, have thicker SC compared to those from other areas. Previous studies have attempted to bypass the SC layer for nanoparticle penetration by using physical disruption; however, these studies have mostly focused on non-thick skin. Objective: In this study, we investigate the role of mechano-physical strategies on SC of thick skin for transdermal nanoparticle penetration. Methods: We characterize and compare two mechano-physical strategies, namely tape-stripping and microneedle abrasion, for epidermal disruption in both thick and thin skin. Furthermore, we examine the impact of SC disruption in thick and thin skin on the penetration of topically applied 100 nm sized polystyrene nanoparticles using an ex-vivo model. Results: Our findings show that tape-stripping reduced the overall thickness of SC in thick skin by 87%, from 67.4 ± 17.3 μm to 8.2 ± 8.5 μm, whereas it reduced thin skin SC by only 38%, from 9.9 ± 0.6 μm to 6.2 ± 3.2 μm. Compared to non-thick skin, SC disruption in thick skin resulted in higher nanoparticle diffusion. Conclusion: Tape-stripping effectively reduces SC thickness of thick skin and can be potentially utilized for enhanced penetration of topically applied nanoparticles in skin conditions that affect thick skin. ### Competing Interest Statement The authors have declared no competing interest.

Microglia, the resident immune cells of the central nervous system, play a pivotal role in maintaining brain homeostasis and responding to various pathological conditions1,2. Neuroinflammation, characterized by the activation of microglia and subsequent release of pro-inflammatory cytokines, has been implicated in the pathogenesis of numerous neurodegenerative diseases. Among these cytokines, chemokine (C-C motif) ligand 2 (CCL2) has gained significant attention due to its role in attracting immune cells and its potential contribution to neuronal inflammation. This review aims to comprehensively elucidate the involvement of microglial CCL2 in neuronal inflammation and its relevance to neurodegenerative diseases. We discuss the molecular mechanisms underlying CCL2 production, its receptors on neurons, and the downstream signaling pathways that initiate and perpetuate neuroinflammation. Furthermore, we explore the bidirectional communication between microglia and neurons, highlighting how neuronal dysfunction can trigger microglial CCL2 release and subsequent immune responses3-5. Additionally, we examine the implications of CCL2-mediated neuroinflammation in neurodegenerative disorders such as Alzheimer's disease6,7, Parkinson's disease8, and amyotrophic lateral sclerosis. Lastly, we discuss potential therapeutic strategies targeting the microglial CCL2 axis to modulate neuroinflammation and ameliorate neurodegenerative processes. ### Competing Interest Statement The authors have declared no competing interest.

The biodiversity crisis is set to prune the Tree of Life in a way that threatens billions of years of evolutionary history. To secure this evolutionary heritage along with the benefits it provides to humanity, there is a need to understand where in space the greatest losses are predicted to occur. We therefore present threatened evolutionary history mapped for all tetrapod groups, globally and within Biodiversity Hotspots, and identify priority regions of Evolutionarily Distinct and Globally Endangered (EDGE) species at both a grid cell and national level. We find that threatened evolutionary history peaks in Cameroon, whilst EDGE species richness peaks in Madagascar. We refined and advanced the 2013 EDGE Zone concept for spatially prioritising phylogenetic diversity using a novel complementarity procedure with uncertainty incorporated for 33,628 tetrapod species. This involved using extinction risk, phylogenetic, and spatial data to iteratively select areas with the highest accumulated threatened evolutionary history driven by unique species compositions. We identify 25 priority EDGE Zones, which are insufficiently protected and disproportionately exposed to high levels of human pressure. Together, the 25 EDGE Zones occupy 0.723% of the world's surface but harbour one-third of the world's threatened evolutionary history, half of which is endemic to these grid cells. They also contain part of the distribution of 918 EDGE tetrapod species, representing near one-third of all EDGE species, with 480 being endemic. Our tetrapod EDGE Zones highlight areas of immediate concern for researchers, practitioners, policymakers, and communicators looking to safeguard the Tree of Life. ### Competing Interest Statement The authors have declared no competing interest.

The Claudin-15 (CLDN15) channel is important for nutrient, electrolyte, and water transport in the gastrointestinal tract. We used cell culture studies and molecular dynamics simulations to elucidate its structure and permeability mechanisms. We provide a model that underscores the crucial role of the D55 residue in the CLDN15 selectivity filter, which interacts with permeating cations. Our studies demonstrated the mechanisms whereby the size and charge of the D55 residue influence paracellular permeability. By altering D55 to larger, negatively charged glutamic acid (E) or similarly sized neutral asparagine (N), we observed changes in pore size and selectivity, respectively. D55E mutation decreased pore size, favoring small ion permeability without affecting charge selectivity, while D55N mutation led to reduced charge selectivity without markedly altering size selectivity. These findings shed light on the complex interplay of size and charge selectivity of CLDN15 channels. This knowledge can inform the development of strategies to modulate the function of CLDN15 and similar channels, which has implications for tight junction modulation in health and disease. ### Competing Interest Statement The authors have declared no competing interest.

The physical structure and dynamics of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them into specific functional architectures. Mechanical force has been shown to modulate the interactions between several ABPs and individual actin filaments, but it is unclear how this phenomenon contributes to the emergent force-responsive functional dynamics of actin networks. Here, we engineer filament linker complexes and combine them with photo-micropatterning of myosin motor proteins to produce an in vitro reconstitution platform for examining how force impacts the behavior of ABPs within multi-filament assemblies. Our system enables the monitoring of dozens of actin networks with varying architectures simultaneously using total internal reflection fluorescence microscopy, facilitating detailed dissection of the interplay between force-modulated ABP binding and network geometry. We apply our system to study a dimeric form of the critical cell-cell adhesion protein α-catenin, a model force-sensitive ABP. We find that myosin forces increase α-catenin's engagement of small filament bundles embedded within networks. This activity is absent in a force-sensing deficient mutant, whose binding scales linearly with bundle size in both the presence and absence of force. These data are consistent with filaments in smaller bundles bearing greater per-filament loads that enhance α-catenin binding, a mechanism that could equalize α-catenin's distribution across actin-myosin networks of varying sizes in cells to regularize their stability and composition. ### Competing Interest Statement The authors have declared no competing interest.

The Human Immunodeficiency Virus Type 1 (HIV-1) continues to be a major global health issue, with infection leading to Acquired Immunodeficiency Syndrome (AIDS). Despite advances in antiviral therapy, the need for new and more effective treatments remains critical. In this study, we determined the structure of the disulfide stabilised HIV-1 capsid protein hexamer in complex with the novel antiviral capsid inhibitor compound 11l. The structure revealed the presence of six 11l molecules bound to the capsid hexamer in a conserved site shared by nuclear import factors, and other capsid inhibitor compounds such as PF74 and GS-6207. The 11l compound exhibits disorder in the benzo-sulfonamide groups and disruption in the loop between helix 8 and 9 for the capsid C-terminal domain, an important 2-fold symmetry axis for hexamer formation. Our findings provide insights into the mechanism of action of 11l as a capsid inhibitor and antiviral. These results contribute to the ongoing efforts to develop more effective antiviral treatments for the global burden of HIV and AIDS. ### Competing Interest Statement The authors have declared no competing interest.

The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition and also they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion, thus understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan biosynthesis. Using carbohydrate gel electrophoresis we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are more porous, suggesting the xylan substitutions directed by both BdXAX1A andGUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials. ### Competing Interest Statement The authors have declared no competing interest.

Native mass spectrometry (native MS) is a powerful technique that provides information on stoichiometry, interactions, homogeneity and shape of protein complexes. However, the extent of deviation between protein structures in the mass spectrometer and in solution remains a matter of debate. Here, we uncover the gas-phase structure of β-galactosidase using single particle electron cryomicroscopy (cryo-EM) down to 2.6 A resolution, enabled by soft-landing of mass-selected protein complexes onto cold TEM grids and in-situ ice coating. We find that large parts of the secondary and tertiary structure are retained from solution, with dehydration-driven subunit reorientation leading to consistent compaction in the gas phase. Our work enables visualizing the structure of gas-phase protein complexes from numerous experimental scenarios at side-chain resolution and demonstrates the possibility of more controlled cryo-EM sample preparation. ### Competing Interest Statement T.K.E., A.M., A.B., and A.K are employees of Thermo Fisher Scientific, manufacturer of Q Exactive UHMR, Aquilos, and Krios instruments used in this research.

Sterile insect techniques can be an efficient solution for reducing or eliminating certain insect pest populations. It is widely and efficiently used in agriculture against fruit flies, including the Mediterranean fly, Ceratitis capitata. The re-mating tendency of medfly females and the fact that sterile males are often released with slight residual fertility can at first sight appear to be detrimental for the successful implementation of SIT. Obtaining the right balance between sterility level and male quality is the key to a cost-efficient program. Since field experimental approaches can be impacted by many environmental variables, it is difficult to get a clear understanding on how some specific parameters can affect, alone or in combination, the efficacy of sterile males release. The use of models not only helps to gather knowledge, but it also helps simulating different situations scenarii and can be easily adapted to local values from the field population and sterile male production. In this study, we consider how the minimal release ratio is affected by female re-mating rate (with an equal or higher tendency to re-mating after an insemination by a sterile male) and male residual fertility. Different scenarios were explored: continuous versus periodic releases, with and without ginger aromatherapy, which is known to enhance sterile male competitiveness. Parameters values were chosen from the literature on peach host fruit when available to reflect what could be expected in the Corsican context, for which SIT against the medfly is under consideration. Our results clearly show that low residual fertility is not necessarily detrimental to SIT performance. In contrast, re-mating of wild females provides contrasting results: when re-mating occurs without a change in the refractory period, it is beneficial because it requires the releases of less sterile males. On the other hand, if females mated with a sterile male have a faster re-mating, it is detrimental for the SIT program as the amount of sterile males to release can become too large. We also confirmed that the use of ginger aromatherapy is more than an essential step for SIT success against medfly. Our model can be easily adapted to different context and species, in order to help understanding release strategies and guiding decision making. ### Competing Interest Statement The authors have declared no competing interest.

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse substrates remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways which act on substrates based on their topology. NAC is required for efficient targeting of polytopic proteins whereas signal-anchored proteins require TTC1, a novel cytosolic chaperone which physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems. ### Competing Interest Statement .S.W. declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics, and Third Rock Ventures. R.M.V. is a consultant and equity holder in Gate Bioscience.

Herpes zoster remains an important global health issue and mainly occurs in aged and immunocompromised individuals with an early exposure history to Varicella Zoster Virus (VZV). Although the licensed vaccine Shingrix has a remarkably high efficacy, undesired reactogenicity and increasing global demand causing vaccine shortage urged the development of improved or novel VZV vaccines. In this study, we developed a novel VZV mRNA vaccine candidate (named as ZOSAL) containing sequence-optimized mRNAs encoding full-length glycoprotein E encapsulated in an ionizable lipid nanoparticle. In mice and rhesus macaques, ZOSAL demonstrated superior immunogenicity and safety in multiple aspects over Shingrix, especially in the induction of strong T cell immunity. Transcriptomic analysis revealed that both ZOSAL and Shingrix could robustly activate innate immune compartments, especially Type-I IFN signaling and antigen processing/presentation. Multivariate correlation analysis further identified several early factors of innate compartments that can predict the magnitude of T cell responses, which further increased our understanding of the mode of action of two different VZV vaccine modalities. Collectively, our data demonstrated the superiority of VZV mRNA vaccine over licensed subunit vaccine. The mRNA platform therefore holds prospects for further investigations in next-generation VZV vaccine development. ### Competing Interest Statement J.H. is an employee of Firestone Biotechnologies, which focuses on the development of novel ionizable lipids and LNP formulation. Firestone has filed a patent on the novel ionizable lipid (YX-02) used in this study. All other authors declare no conflict of interest.

Proteasomes are essential for protein homeostasis in mammalian cells and in protozoan parasites such as Trichomonas vaginalis (Tv). Tv and other protozoan 20S proteasomes have been validated as druggable targets. However, in the case of Tv 20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant Tv20S by expressing all seven alpha and seven beta subunits together with the Ump-1 chaperone in insect cells. We isolated recombinant proteasome and showed that it was biochemically indistinguishable from the native enzyme. We confirmed that the recombinant Tv20S is inhibited by the natural product marizomib (MZB) and the recently developed peptide inhibitor carmaphycin-17 (CP-17). Specifically, MZB binds to the beta1, beta2 and beta5 subunits, while CP-17 binds the beta2 and beta5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8A resolution. The structures revealed the overall fold of the Tv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards Tv20S as compared to human proteasome and provides the platform for the development of Tv20S inhibitors for treatment of trichomoniasis. ### Competing Interest Statement The authors have declared no competing interest.

The functional properties of RNA-binding proteins (RBPs) require allosteric regulation through inter-domain communication. Despite the foundational importance of allostery to biological regulation, almost no studies have been conducted to describe the biophysical nature by which inter-domain communication manifests in RBPs. Here, we show through high-pressure studies with hnRNP A1 that inter-domain communication is vital for the unique stability of its N-terminal domain containing a tandem of RNA Recognition Motifs (RRMs). Despite high sequence similarity and nearly identical tertiary structures, the two RRMs exhibit drastically different stability under pressure. RRM2 unfolds completely under high-pressure as an individual domain, but when appended to RRM1, it remains stable. Variants in which inter-domain communication is disrupted between the tandem RRMs show a large decrease in stability under pressure. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered C-terminus to engage in protein-protein interactions and more importantly, they also influenced the RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions. ### Competing Interest Statement The authors have declared no competing interest.

Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin. ### Competing Interest Statement The authors have declared no competing interest.

As a major source of genetic and regulatory variation in their host genome, transposable elements (TEs) have gained a growing interest in research on humans and model organisms. In this species, integrative analysis of multi-omics data has shed light on the regulatory functions of TEs. However, there remains a notable gap in our understanding of TEs in domesticated animals. we annotated TEs in the genomes of pigs, cattle, and chickens, respectively, and systematically compared the genome distributions and amplification patterns of TEs across these three species. Furthermore, by integrating multi-tissue RNA-seq, ATAC-seq, and histone modification ChIP-seq data, we explored the expression atlas of TEs and their contribution to cis-regulatory elements (CREs) in different tissues of the three species. Most importantly, we developed a novel computational framework that revealed TE-mediated gene regulatory networks (TE-GRNs) underlying tissue-related biological processes. To demonstrate the power of this approach, we applied our framework to analyze liver tissues across the three different species. Overall, our research provides novel insights into the regulatory functions of TEs in livestock animals and highlights a computational framework to uncover TE-GRNs in various biological contexts. ### Competing Interest Statement The authors have declared no competing interest.

Objective: Inflammatory bowel disease (IBD) is a persistent inflammatory condition affecting the gastrointestinal tract, presenting significant challenges in its management and treatment. Despite the knowledge that within-host bacterial evolution occurs in the intestine, the disease has so far rarely been studied from an evolutionary perspective. In this study, we aimed to investigate resident bacterial evolution during intestinal inflammation, and whether- and how disease-related bacterial genetic changes may present trade-offs with potential therapeutic importance. Design: Here, we perform an in vivo evolution experiment of E. coli in a gnotobiotic mouse model of IBD, followed by multiomic analyses to identify disease-specific genetic and phenotypic changes in bacteria evolved in an inflamed versus non-inflamed control environment. Results: Our results demonstrate distinct evolutionary changes in E. coli specific to inflammation, including a single nucleotide variant that independently reached high frequency in all inflamed mice. Using ex vivo fitness assays, we find that these changes are associated with a higher fitness in an inflamed environment compared to isolates derived from non-inflamed mice. Further, using large-scale phenotypic assays, we show that bacterial adaptation to inflammation results in clinically relevant phenotypes, which intriguingly include collateral sensitivity towards antibiotics. Conclusions: Bacterial evolution in an inflamed gut yields specific genetic and phenotypic signatures. These results may serve as a basis for developing novel, evolution-informed treatment approaches for patients with intestinal inflammation. ### Competing Interest Statement The authors have declared no competing interest.