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Biology subjects

Brown, N.

Publications and source records attributed to Brown, N..

At least 19 recordsLinked to original sources

From Flocks to Swarms: Rewriting the Rules of Collective Cell Migration

From flocking birds to swarming insects, collective motion enables groups to accomplish tasks beyond the capabilities of individuals. These behaviors emerge from local interactions among group members, but the interaction rules that organize collectives at the cellular scale remain poorly understood, in part because they are difficult to manipulate experimentally. Here we develop a cell-machine interface that enables us to rewrite the rules of collective migration. Cell-cell interaction rules are specified in software and implemented in living cells through real-time computer vision and closed-loop optogenetic control. Using a single engineered cell line, we program cells to self-organize into flocks, to propagate regenerative signal relays that recapitulate the interaction logic of immune-cell swarms, and to collectively navigate guidance cues that individual cells cannot. Our work shows that altering simple rules of cell-cell interaction is sufficient to generate new emergent collective capabilities. This approach provides an experimental framework for discovering the organizational principles of multicellular coordination and designing synthetic collectives with capabilities beyond those of their individual members.

synthetic biology↗

PAMalytics: a no-code application for structured validation of bioacoustic detections

1. Passive acoustic monitoring (PAM) is increasingly used for ecological research, biodiversity monitoring, assessment, and reporting. Automated species classifiers make it feasible to process large audio datasets but generate numerous detections that often need validation before use in downstream analyses or formal outputs. 2. Method development in PAM has focused on classifier building and downstream models that account for imperfect detection, yet the practical step between these - post-classification validation - remains weakly supported and is often implemented through ad hoc workflows. This increases manual handling, creates scope for transcription or consolidation errors, limits transparency and makes it difficult to document what was reviewed. 3. We introduce PAMalytics, an open-source, no-code, local browser-based application to support post-classification validation as a standardised workflow stage. PAMalytics ingests detections from any classifier, allows users to define how detections are sampled for review, and presents selected detections alongside their spectrograms with audio playback in one unified interface. Sampling strategy and review decisions are tracked alongside reviewer identity improving traceability and reproducibility across the validation workflow. 4. Case studies with Conservation International Cambodia and Imperial College London demonstrate PAMalytics in two validation settings. In Cambodia, gibbon predictions from a large, uneven dataset were sampled within sites, with likely classifier errors prioritised for validation. At Imperial, Amazon bird detections were sampled across each species classifier-confidence range before biodiversity metrics were derived. In both cases, PAMalytics reduced manual handling and validation time. By turning an ad hoc step into an accessible, structured workflow for conservation practitioners, PAMalytics fills a practical gap in the PAM bioacoustics pipeline and strengthens the link between automated detections and evidence used in biodiversity monitoring and reporting.

ecology↗

Performance of pathogen identification and resistance gene expression tests using ASTar(R) remnant bacterial suspension in Gram-negative contrived positive blood cultures

IntroductionRapid pathogen identification, resistance detection, and susceptibility profiling improve antimicrobial prescribing and associated outcomes, but fragmented workflows lead to inefficiencies and are costly. We evaluated a research-use-only (RUO) approach using ASTar(R) remnant bacterial suspension from routine AST for MALDI-TOF MS pathogen identification and Lateral Flow Assay (LFA)-based detection of targeted resistance mechanisms. MethodsGram-negative (GN) bacterial strains from reference and curated resistance collections (CDC1, ARLG2, ATCC3) [n=119] were contrived into blood culture bottles and processed in the ASTar System using the ASTar BC G-Kit (Q-linea AB, Sweden). Under RUO conditions, remnant bacterial suspensions were collected ~1-2 h after ASTar run initiation and analyzed using NG Test CTX-M Multi, NG Test CARBA-5, NG Test Acineto-5 RUO, and MALDI-TOF MS. ResultsMean ({+/-} SD) remnant suspension volume was 2722 L ({+/-} 300 L). All samples yielded high-confidence MALDI-TOF MS scores (>2.0), with five initially scoring <2.0 and resolving on repeat testing. LFA results showed full agreement with reference isolates for blaCTX-M positive/negative (30/30) and with 60 or 61 target carbapenemase-positive/negative isolates. Testing of a subset of samples to mimic reflex workflows with ASTar phenotypic results did not affect LFA performance (n=26; 23 Enterobacterales, 3 P. aeruginosa and 9 A. baumannii). Cost savings can be realised versus commercial multiplex PCR. ConclusionThis integrated approach of ~6 h rapid phenotypic AST with same-run identification and resistance detection (1-2 h from instrument start) or reflex testing upon availability of ASTar results may support earlier susceptibility results and offer cost savings to current workflows.

microbiology↗

Once yearly cell-based therapy for sustained and dose tunable delivery of monoclonal antibodies

Over 200 monoclonal antibodies (mAbs) are approved for clinical use, yet their therapeutic potential is constrained by dependence on repeated injections or infusions that drive non-adherence, limit access in low-resource settings, and generate peak-trough pharmacokinetics linked to adverse effects and reduced efficacy. Here, we developed an immunomodulatory encapsulated cell-based biologics factory that overcomes mAb instability, immunogenicity, and the fibrotic foreign body response that have limited previous approaches, enabling continuous in situ production of therapeutic antibodies from a single administration. Screening chemically modified alginate biomaterials in immunocompetent mice identified a lead immunomodulatory alginate formulation that sustains stable serum titers of the HIV-neutralizing mAb 3BNC117 for one year. Single-cell RNA sequencing revealed that this formulation promotes a local anti-inflammatory, pro-resolving immune niche that attenuates fibrosis. The platforms versatility was demonstrated by production of thirteen diverse mAbs from an allogeneic cell chassis, with sustained in vivo delivery of a subset including ipilimumab, pembrolizumab, adalimumab, and PGT121. Integration into a retrievable macrodevice enabled on-demand therapeutic termination and re-implantation for dose-proportional tuning. In a non-human primates, subcutaneous implantation maintained stable ipilimumab titers for over six months with no detectable toxicity, anti-drug antibodies, or adverse events, and dose-dependent exposure was confirmed across a three-dose escalation. These results demonstrate a clinically translatable platform offering a practical strategy to replace frequent injections with single-administration therapy.

bioengineering↗

Large scale antibiotic-phage synergy studies reveal key combinations for urinary tract infection and urosepsis treatments

The growing problem of AMR infections in healthcare has prompted the search for alternative treatments, with increasing interest in bacteriophages. However, most bacteriophage-antibiotic interactions are incompletely understood, and the benefits of combining them remains context dependent. In this study, we screened thousands of phage-antibiotic combinations to assess interaction outcomes in clinical E. coli and K. pneumoniae isolates. By integrating bacteriophages into an existing, scalable clinical MIC determination platform, we identified shifts in antibiotic MIC and susceptibility, revealing patterns of additivity and antagonism. Overall, interactions showed a species-specific profile; additive interactions predominated, particularly for E. coli. Hierarchical clustering highlighted frequent positive interactions between {beta}-lactams and Tequatroviruses. Notably, closely related phages sometimes displayed divergent phenotypes, indicating that interaction outcomes cannot be inferred solely from taxonomic relatedness or genomic similarity. Taken together, these results establish a foundation for rational, evidence-based development of phage-antibiotic therapies to restore and broaden treatment options against multidrug-resistant infections.

microbiology↗

Genomic diversity of British native oaks: species differentiation, hybridisation and triploidy

The morphological continuum between Britains two native oak species helped Darwin formulate his "one long argument" in The Origin of Species. Here we comprehensively analyse the genome-wide differentiation of 418 individuals from 60 British oak populations. Using 7 million biallelic SNPs, we find evidence for two species with islands of variation containing 8,230 SNPs of which 2,005 are within gene annotations. We find extensive hybridisation and back-crossing between the species, and this is biased in favour of introgression from Quercus robur into Q. petraea. Chloroplasts are shared between the species, mainly of haplotypes derived from Iberian glacial refugia. Genomic allocation of species and hybrids allows analysis of niche differentiation, showing that a larger Q. robur component is disproportionately found in warmer and more thermally variable environments with alkaline soils, while a larger Q. petraea component is found in more topographically complex terrain with high rainfall. This leads us to emphasise the balance between the opposing forces of hybridisation and niche differentiation in explaining the continuum between the two species. Using long-term field data, we show that stem growth over three decades was higher in Q. robur than in Q. petraea or hybrids, but this difference could be explained by environmental factors. In contrast, the five triploid trees in our sample grew significantly faster than diploids even after accounting for environmental effects, suggesting that triploids could be a valuable economic resource.

evolutionary biology↗

Genomic basis for resistance to acute oak decline and mildew infection in English oak

English oak (Q. robur) in Britain is at risk from a young age due to powdery mildew and at maturity due to acute oak decline (AOD). The contribution of oak genetic diversity to these conditions is a critical question that until recently would have required extensive clonal or family trials of juvenile and mature oak trees. Here we overcome this practical burden with genomic approaches inspired by medicine. We sequence 1868 oak trees from 78 sites and identify 1491 trees exclusively of the species Q. robur, which we analyse. Using single nucleotide polymorphism (SNP) based estimates of heritability ([Formula]), we estimate 20.9% of variation in AOD presence is genetically determined. For mildew symptoms, [Formula] is 27.8%. Using genome-wide association studies, no SNP markers show a significant association with AOD, but 183 are significantly associated with mildew. Genomic prediction models for AOD and its symptoms, using thousands of loci, give low to moderate accuracies, ranging from 0.187 to 0.617. These results suggest a highly polygenic heritable component of susceptibility to AOD, which perhaps reflects the number of biotic and abiotic factors known to contribute to it. In contrast, powdery mildew, caused by a single fungus, has an oligogenic heritable component controlled by fewer loci of large effect. These results could be used to inform breeding programs to develop trees more resistant to AOD and mildew.

evolutionary biology↗

Latent Regulatory Programs Generate Synthetic T Cell States with Enhanced Therapeutic Potential

Transcription factors (TFs) govern cell fate through coordinated gene-regulatory networks, yet the full potential of these networks to generate non-native, therapeutically advantageous cell states in vivo remains largely unexplored. We hypothesized that systematic gain-of-function (GOF) overexpression of TFs in CD8 T cells, central mediators of immune protection, could reveal latent, or "hidden," regulatory programs capable of generating synthetic T cell states with therapeutic utility. To test this, we developed single-cell GOF sequencing (scGOF-seq), a multiplexed platform for unbiased, in vivo mapping of GOF effects on T cell fate in immunocompetent mouse models of infection and cancer. scGOF-seq uncovered unexpected regulators of T cell differentiation and accumulation, including SOX2, OCT4, and GATA2, which are normally silenced during T cell differentiation. Notably, outside its native regulatory context, supraphysiologic cMyc GOF reprogrammed CD8 T cells into a synthetic stem-effector hybrid state, enabling >5,000-fold antigen-dependent expansion and antitumor activity, contrasting sharply with its native function in driving terminal differentiation. scGOF-seq further identified TF modules that cooperate with cMyc GOF to promote robust CD8 T cell responses in solid tumors. Together, these findings establish GOF perturbation as a powerful strategy for revealing latent immune regulatory programs and engineering synthetic immune states with therapeutic potential. One-Sentence SummaryIn vivo single-cell gain-of-function screening reveals latent transcriptional programs that can reprogram T cells into highly functional synthetic states.

immunology↗

A single-cell transcriptomic atlas of peripheral blood immune cells spanning progressive canine leishmaniosis

Dogs play a major role in sustaining transmission of Leishmania infantum to people, thus prevention and treatment of canine leishmaniosis (CanL) to reduce transmission represents an unmet public health need. Although advances have been made in understanding how immunopathology correlates to infectiousness as CanL progresses, the immune mechanisms underlying transition between disease stages and terminal decline remain ill-defined. To address this knowledge gap, we generated a comprehensive atlas of peripheral immune cells from control dogs and naturally-infected dogs using single-cell RNA sequencing. The LeishDog Atlas captures the cellular and transcriptional complexity underlying CanL, tracing shifts in immune composition and gene expression across progressive, well-defined disease stages. Notably, we identified distinct myeloid, CD4+ and CD8+ T cell phenotypes and transcriptional states associated with disease progression. This resource provides a valuable framework for understanding systemic immune dysregulation in CanL, including determinants of T cell exhaustion and myeloid cell activation, and establishes a foundation for comparative, translational, and mechanistic studies of CanL immunopathology and transmissibility.

immunology↗

Environment and disease have tissue-specific effects on the tree microbiome

Trees are essential for ecosystem function, but due to their long lifespan, are disproportionately impacted by climate change and disease. Tree-associated microbiota are critical for tree health and resilience, but the composition and function of tree microbiomes across different tissue types, and how environmental factors and disease impact tree microbiomes, is poorly understood. Oak trees are major constituents of forests of the Northern hemisphere, but are increasingly impacted by climate and disease. Here, we studied the oak microbiome across Britain, combining 16S rRNA gene and ITS microbial community profiling and shotgun metagenomics of leaf, stem and root/rhizosphere samples, and developed a three-level occupancy model to describe microbiota distribution across the landscape. We show that oak leaf, stem and root/rhizosphere tissues harbour taxonomically and functionally distinct microbiota and identified differences in tissue-specific effects of environmental variables (e.g. temperature, rainfall, ion deposition) on microbiome composition and function. We generated 1657 bacterial, archaeal and fungal metagenome-assembled genomes representing key members of the oak microbiome. Furthermore, the stem microbiome of oak trees with symptoms of Acute Oak Decline, a complex decline disease driven by abiotic and biotic stressors, exhibited reduced bacterial and fungal richness and altered microbiome function. This work represents the most comprehensive microbiome study of a tree species to date. Understanding how tree-associated microbiota respond to environmental change and disease across different tissues is crucial to predict future climate and disease impacts on tree microbiome function, and inform translational approaches to modulate tree microbiomes for plant health.

microbiology↗

Risk assessment for condylar stress fracture in elite racing Thoroughbreds using standing computed tomography-based virtual mechanical testing

BackgroundCondylar stress fracture of the third metacarpal bone is a common catastrophic musculoskeletal injury in Thoroughbred racehorses. Condylar stress fracture is associated with parasagittal groove (PSG) subchondral osteolysis. Standing computed tomography (sCT) imaging allows for sensitive identification of this fatigue-induced structural change, an early form of subchondral bone injury (SBI), but there is not yet an objective method for identifying racehorses at heightened risk of condylar stress fracture. ObjectivesTo estimate PSG first principal strain in elite Thoroughbred racehorses that have undergone subjective risk assessment using sCT fetlock screening. Study designRetrospective clinical study. MethodsWe used fetlock sCT images from 9 thoracic limbs from 7 Thoroughbred racehorses. A tuned, validated 3D finite element (FE) analysis approach was used as a virtual mechanical test to estimate PSG first principal strain in the distal third metacarpal bone (MC3) from these joints. Virtual mechanical testing results were compared with a subjective clinical risk assessment using an established screening approach by Racing Victoria. ResultsMC3 condyles with PSG SBI consistently and significantly displayed increased levels of first principal strain throughout the PSG. We found focal strain concentrations associated with the anatomical location of SBI compared to condyles with no evidence of SBI in the PSG. Clinical diagnosis of SBI with focal osteolysis, FE-predicted strain elevation, and clinical risk assessment were concordant with recall and precision of 0.86. Main limitationsThe sample size was small, and our virtual mechanical testing protocol does not account for whole-joint physiology. ConclusionsRisk assessment through screening with sCT is an established approach to injury prevention in racing Thoroughbreds. Concordance of a current clinical risk assessment approach by Racing Victoria with objective FE analysis of principal strain in sites of SBI in the present study suggests 3D FE analysis using a validated pipeline is an important new approach for routine assessment of risk of MC3 condylar stress fracture in Thoroughbred racehorses.

bioengineering↗

Hi-C for genome-wide detection of enhancer-hijacking rearrangements in routine lymphoid cancer biopsies

Standard techniques for detecting genomic rearrangements in formalin-fixed paraffin-embedded (FFPE) biopsies have important limitations. We performed FFPE-compatible Hi-C on 44 clinical biopsies comprising large B-cell lymphomas (n=18), plasma cell neoplasms (n=14), and other diverse lymphoid cancers, identifying consistent topological differences between malignant B cell and plasma cell states. Hi-C detected expected oncogene rearrangements at high concordance with fluorescence in-situ hybridization (FISH) and supported enhancer-hijacking in recurrent rearrangements of BCL2, CCND1, and MYC, plus unanticipated variants involving homologous loci. Hi-C identified unanticipated non-coding rearrangements involving PD-1 ligand genes and other loci of potential therapeutic relevance, distinguished between functionally divergent classes of BCL6 rearrangements, and provided topological information supporting the interpretation of atypical MYC rearrangements. In biopsies lacking MYC-activating rearrangements, Hi-C revealed differential interactions with functionally-validated disease-specific native MYC locus enhancers. FFPE-compatible Hi-C detects oncogene rearrangements and their topological consequences at genome-wide scale, finding clinically-relevant drivers that are missed by standard approaches.

genomics↗

Unlocking plant health survey data: an approach to quantify the sensitivity and specificity of visual inspections

Invasive plant pests and pathogens cause significant environmental and economic damage. Visual inspection remains a central tenet of plant health surveys, but its sensitivity (probability of correctly identifying the presence of a pest) and specificity (probability of correctly identifying the absence of a pest) is usually ignored. These parameters facilitate calculation of surveillance metrics which are critical for effective contingency planning and outbreak management. To address this, twenty-three citizen scientist surveyors assessed up to 175 oak trees for three symptoms of acute oak decline. The same trees were also assessed by an expert who has monitored these trees annually for over a decade. The sensitivity and specificity of surveyors was calculated using the expert data as the gold-standard. The utility of a workflow utilising Bayesian modelling was then examined using simulated data to estimate these parameters in the absence of a rarely available gold-standard dataset. There was large variation in sensitivity and specificity between surveyors and symptoms, although the sensitivity was positively related to the number of symptoms on a tree. By leveraging surveyor observations of two symptoms from a minimum of 80 trees on two sites, with knowledge of whether a site has higher ([~]0.6) or lower ([~]0.3) true disease prevalence we show that sensitivity and specificity can be estimated without gold-standard data. We highlight that sensitivity and specificity will depend on the symptoms of a pest or disease, the individual surveyor, and the survey protocol. This has consequences for how surveys are designed to detect and monitor outbreaks, as well as the interpretation of survey data that is used to inform outbreak management. Author summaryThe increasing occurrence of emerging plant pests and diseases is affecting both agricultural and natural ecosystems. Effective management and control of such pests and diseases is much easier when they are detected early. Currently, visual surveys underpin plant health surveillance, but basic metrics of the reliability of visual detection such as the sensitivity (probability of correctly identifying a positive) and specificity (probability of correctly identifying a negative) are not routinely quantified. In this study, we first quantify the sensitivity and specificity of 23 trained citizen scientist surveyors at detecting three symptoms of acute oak decline, by comparing their symptom classifications against a dataset from an expert who has conducted long-term monitoring of these trees. We demonstrate how individuals vary greatly in their ability to detect symptoms, and how different symptoms are associated with different detection error. Secondly, based on this dataset we outline a workflow developed for scenarios realistic in plant health which utilises Bayesian modelling to estimate these parameters in the absence of a rarely available gold-standard expert dataset. In summary, our results highlight variation in the reliability of visual detection, and we provide a workflow to calculate this and facilitate optimisation of risk-based surveillance strategies in plant health.

ecology↗

Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering

Protein quality control is crucial for cellular homeostasis, involving the heat shock response, the ubiquitin-proteasome system, and the autophagy-lysosome pathway. Central to these systems are the chaperone homologs heat shock protein 70 (HSP70) and heat shock cognate 70 (HSC70), which manage protein folding and degradation. This study investigated the impact of the C-terminal phosphorylation of HSP70 on its interaction with the co-chaperone CHIP (C-terminus of HSC70 interacting protein), an E3 ligase that ubiquitinates protein substrates for degradation. Using both cell-free and cell-based approaches, including X-ray crystallography, biolayer interferometry, and live cell biocomplementation assays, we demonstrate that phosphorylation at HSP70 T636 reduces CHIPs binding affinity, shifting the preference toward other co-chaperones like HOP. Structural analysis reveals that phosphorylation disrupts key hydrogen bonds, altering binding dynamics. We engineered a CHIP variant (CHIP-G132N) to restore binding affinity to phosphorylated HSP70. While CHIP-G132N effectively restored binding without additional functional domains, its effectiveness was diminished in full-length phosphomimetic constructs in cell-free and in-cell assays, suggesting that additional interactions may influence binding. Functional assays indicate that phosphorylation of HSP70 affects its stability and degradation, with implications for diseases such as cancer and neurodegeneration. Our findings highlight the complexity of chaperone-co-chaperone interactions and underscore the importance of post-translational modifications in regulating protein quality control mechanisms. By elucidating the molecular details of HSP70 and CHIP interactions, our study provides a foundation for developing therapeutic interventions for diseases characterized by proteostasis imbalance.

biochemistry↗

ENaC contributes to macrophage dysfunction in cystic fibrosis

BackgroundCystic fibrosis (CF) is a chronic systemic disease caused by dysfunctional or absent cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in human immune cells and plays a role in regulating innate immunity both directly and indirectly. Besides CFTR, research indicates that the epithelial sodium channel (ENaC) also contributes to dysfunction in CF airway epithelial cells. However, the impact of non-CFTR ion channel dysfunction on CF immune responses is not yet fully understood. A precise understanding of how CF immune function is regulated by ion channels may allow antibiotic-and mutation-agnostic treatment approaches to chronic bacterial infection and inflammation. Therefore, we hypothesized that ENaC is aberrantly expressed in CF macrophages and directly contributes to impaired phagocytic and inflammatory functions. MethodsENaC expression was characterized in human immune cells isolated from CF and non-CF blood donors. Monocyte-derived macrophage (MDM) function and bacterial killing was tested in the setting of ENaC modulation. ResultsBaseline expression of ENaC in human CF MDMs, lymphocytes, and granulocytes was increased at both the transcript and protein level relative to non-CF controls and persisted after exposure to bacteria. Inhibition of CFTR in non-CF MDMs resulted in ENaC overexpression. CFTR modulator treatment reduced but did not eliminate ENaC overexpression in CF MDMs. Interestingly, ENaC inhibition with Amiloride increased CFTR expression. Amiloride-treated CF MDMs also showed normalized ROS production, improved autophagy, and decreased pro-inflammatory cytokine production. Finally, results from an ion channel microarray indicated that sodium channel expression in CF MDMs normalized after Amiloride treatment with minimal effect on other ion channels. DiscussionENaC is overexpressed in CF immune cells and is associated with abnormal macrophage function. ENaC modulation in immune cells is a novel potential therapeutic target for infection control in CF, either in combination with CFTR modulators, or as a sole agent for patients not currently eligible for CFTR modulators.

immunology↗

USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

The CMG helicase (CDC45-MCM2-7-GINS) unwinds DNA as a component of eukaryotic replisomes. Replisome (dis)assembly is tightly coordinated with cell cycle progression to ensure genome stability. However, factors that prevent premature CMG unloading and replisome disassembly are poorly described. Since disassembly is catalyzed by ubiquitination, deubiquitinases (DUBs) represent attractive candidates for safeguarding against untimely and deleterious CMG unloading. We combined a targeted loss-of-function screen with quantitative, single-cell analysis to identify human USP37 as a key DUB preventing replisome disassembly. We demonstrate that USP37 maintains active replisomes on S-phase chromatin and promotes normal cell cycle progression. Proteomics and enzyme assays revealed USP37 interacts with the CMG complex to deubiquitinate MCM7, thus antagonizing replisome disassembly. Significantly, USP37 protects normal epithelial cells from oncoprotein-induced replication stress. Our findings reveal USP37 to be critical to the maintenance of replisomes in S-phase and suggest USP37-targeting as a potential strategy for treating malignancies with defective DNA replication control.

cell biology↗

Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain

Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory and excitatory neurons exhibited substantial changes in translation after peripheral nerve injury. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity. Conversely, reducing translation in spinal PV neurons prevented the nerve injury-induced decrease in excitability but did not alleviate mechanical hypersensitivity. Together, these findings advance our understanding of translational control mechanisms in the spinal cord during neuropathic pain and highlight their cell-type- and phase-specific contributions to gene expression and pain hypersensitivity.

neuroscience↗

Mechanisms of USP18 deISGylation revealed by comparative analysis with its human paralog USP41

The ubiquitin-like protein ISG15 (interferon-stimulated gene 15) regulates the host response to bacterial and viral infections through its conjugation to proteins (ISGylation) following interferon production. ISGylation is antagonized by the highly specific cysteine protease USP18, which is the major deISGylating enzyme. However, mechanisms underlying USP18s extraordinary specificity towards ISG15 remains elusive. Here, we show that USP18 interacts with its paralog USP41, whose catalytic domain shares 97% identity with USP18. However, USP41 does not act as a deISGylase, which led us to perform a comparative analysis to decipher the basis for this difference, revealing molecular determinants of USP18s specificity towards ISG15. We found that USP18 C-terminus, as well as a conserved Leucine at position 198, are essential for its enzymatic activity and likely act as functional surfaces based on AlphaFold predictions. Finally, we propose that USP41 antagonizes conjugation of the understudied ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) from substrates in a catalytic-independent manner. Altogether, our results offer new insights into USP18s specificity towards ISG15, while identifying USP41 as a negative regulator of FAT10 conjugation.

biochemistry↗