Biology subjects

Ferguson, L.

Publications and source records attributed to Ferguson, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Nanoparticle Albumin-Bound Paclitaxel Targets Pulmonary Neutrophils

Nanoparticle albumin-bound paclitaxel (Abraxane) was among the first clinically approved nanomedicines and is a first-line chemotherapeutic. It enhances therapeutic efficacy and reduces side effects of paclitaxel. Despite its extensive clinical use, mechanisms of Abraxane's therapeutic effects are the subject of ongoing study. This work posits a new mechanism for Abraxane's effects in lung cancers and metastasis. Tracing the albumin component of Abraxane with radioisotopes or fluorophores, we find that Abraxane homes to the lungs. Abraxane's lung tropism is elevated by inflammation and is eliminated in complement protein C3 knockout mice, showing that complement pathway drives the uptake. Flow cytometry and histology show that neutrophils are the dominant cell type taking up Abraxane in the lungs. We demonstrate Abraxane's tropism to pulmonary neutrophils in mice and in human donor lungs. We note previously undocumented side effects associated with Abraxane's lung tropism, including pro-thrombotic and pro-inflammatory responses in acute lung inflammation. We show that Abraxane's efficacy against lung metastases is affected when lung uptake is eliminated by C3 knockout. Our results provide insight into why Abraxane is effective in lung cancer and metastases. These findings may be used to improve outcomes in patients receiving Abraxane and help design next-generation cancer nanomedicines.

bioengineering↗

bioRxiv2026

Evidence from three taxonomically distinct species for a non-AhR mechanism of developmental neurotoxicity of an environmentally derived mixture of polycyclic aromatic hydrocarbons

Typical environmental exposures to the toxic class of chemicals known as polycyclic aromatic hydrocarbons (PAHs) involve complex mixtures; however, relatively few mechanistic toxicity studies have evaluated them as environmental mixtures, instead focusing on individual compounds or simple mixtures. In this study, we first derived Republic Sediment Extract (REPSE), a complex PAH mixture extracted from sediment at the Republic Creosoting site of the Elizabeth River in Norfolk, Virginia. After characterizing the PAH contents of REPSE, we evaluated its mechanisms of developmental neurotoxicity in three evolutionarily distinct taxa, leveraging the unique strengths of Atlantic killifish, zebrafish, and Caenorhabditis elegans as model species, with a focus on the Aryl hydrocarbon Receptor (AhR) pathway. Embryonic REPSE exposure caused induction of CYP1A in both fish species at sub-teratogenic concentrations, consistent with activation of the canonical AhR pathway. These sub-teratogenic exposures nevertheless induced neurotoxicity across both fish species, altering neurobehavioral phenotypes in fish, and induced dopaminergic neuronal damage in worms, again at non-teratogenic concentrations. To determine whether these effects were linked to canonical AhR response pathways, we examined killifish offspring from the pollution-adapted Republic Creosoting population, which exhibited characteristic recalcitrance to CYP1A induction, but remained susceptible to the neurobehavioral effects of REPSE. The induction of neuronal damage in worms provides orthogonal evidence for a non-AhR mechanism, because C. elegans AhR is not transcriptionally activated by PAHs as in vertebrates. Further probing of potential mechanisms underlying REPSE-induced neurotoxicity in worms revealed altered neuronal redox status (roGFP) and energy availability (ATP:ADP ratio). Collectively, our multispecies approach reveals conserved mechanisms of PAH mixture neurotoxicity, including effects that extend beyond canonical AhR signaling.

pharmacology and toxicology↗

bioRxiv2026

OTTR-CLASH: improved biochemical and bioinformatic identification of Argonaute 2-mediated microRNA-target RNA interactions

Various methods have detected miRNA-target interactions via immunoprecipitation of UV-crosslinked Argonaute ribonucleoprotein complexes, followed by intermolecular ligation of bound miRNAs to target strands, forming chimeric RNAs. To date, these methods have relied on conventional viral reverse transcriptases (RTs) to generate cDNAs for sequencing. However, crosslinked RNAs often retain adducts after purification, which can make them poor templates for viral RTs. Here, we adapted OTTR (Ordered Two-Template Relay) techniques to generate cDNAs from Ago2-bound RNAs. OTTR makes use of a modified retroelement-encoded RT, which is strongly processive even on templates with modifications or adducts. We show that this "OTTR-CLASH" method increases the frequency of generating chimeric RNAs compared to previous methods. We also developed an improved bioinformatic pipeline for analysis of these data, and we use this to catalog miRNA-target interactions not previously described in the literature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/738487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@16d62f8org.highwire.dtl.DTLVardef@7c8da4org.highwire.dtl.DTLVardef@13727fborg.highwire.dtl.DTLVardef@21f443_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

The Blood RNA Stability Atlas: defining temporal structure and trait-state programs in the human whole-blood transcriptome

Whole-blood RNA biomarkers are widely used for diagnosis and disease monitoring, but their utility depends not only on abundance but also on temporal stability, a property that is not routinely incorporated into biomarker design. We analyzed 968 longitudinal whole-blood transcriptomes from 165 healthy individuals across eight independent studies spanning diverse platforms, time scales (50 minutes to 16 weeks), and common environmental exposures. Using a cross-study analytical framework integrating variance partitioning, repeatability, and time-associated differential expression, we quantified temporal stability for 6,064 RNAs and classified transcripts into "trait" (stable) and "state" (dynamic) categories representing the extremes of longitudinal changes in transcript abundance. We identified 1,118 trait RNAs exhibiting stable within-individual levels of abundance but substantial inter-individual variability, enriched for whole-blood eGenes (P = 6.0 x 10-{superscript 2}), supporting a genetic basis for stability. In contrast, 1,504 state RNAs showed context-dependent temporal variation and were enriched for translation and RNA-binding pathways. Integration with genetic datasets revealed that 4,395 (72%) blood transcripts were linked to at least one whole-blood eQTL, collectively associated with 18,358 GWAS trait relationships, providing disease-relevant context for transcript stability. We developed the Blood RNA Stability Atlas to integrate these features and demonstrate both top-down (disease-to-gene) and bottom-up (gene-to-context) applications for biomarker prioritization and interpretation. These findings establish temporal stability as a defining property of the blood transcriptome and provide a practical, publicly accessible framework for distinguishing stable baseline abundance levels from context-dependent transcriptional responses, informing biomarker selection, study design, and hypothesis generation.

genomics↗

bioRxiv2026

Preconception Chronic Intermittent Ethanol Exposure Impacts Offspring Transcriptomes with Sex and Tissue Specific Effects

Alcohol use disorder demonstrates [~]50% heritability, much of which remains unexplained by genetic sequence alone. Chronic alcohol exposure before conception changes offspring phenotypes through epigenetic mechanisms that are still being elucidated. Preconception ethanol exposure studies have focused on paternal exposure, neglecting maternal and biparental exposure. To address this, we exposed adult male and female mice to five cycles of chronic intermittent ethanol vapor interleaved with two bottle choice ethanol drinking and mated them to produce male and female F1 offspring with paternal, maternal, or biparental preconception ethanol exposure or controls. Whole blood and medial prefrontal cortex from adult, ethanol-naive offspring underwent RNA-sequencing. We also analyzed previously unpublished RNA-sequencing data from male and female preimplantation embryos derived from preconception ethanol-exposed sires. Here, we report transcriptomic patterns of preconception ethanol exposure that depend on the exposed parent, offspring sex, and tissue which suggest metabolic and immune dysfunction in offspring.

neuroscience↗

bioRxiv2026

Antidepressant mechanism and treatment response define distinct hippocampal-amygdala circuit biomarkers during emotional memory in humans

Antidepressant efficacy varies widely, yet the circuit-level mechanisms that distinguish treatment responders from non-responders remain poorly understood in humans. Here, we used high-resolution neuroimaging of hippocampal-amygdala networks during an emotional mnemonic discrimination task that taxes hippocampal pattern separation to examine how antidepressant mechanism of action and perceived treatment response shape emotional memory circuitry (N = 117). Participants included individuals taking single-action antidepressants (selective serotonin reuptake inhibitors), multi-action antidepressants (serotonin-norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, or polypharmacy), and unmedicated controls matched on current depression severity. Antidepressant mechanism and treatment response were associated with distinct patterns of activity in hippocampal subfields (dentate gyrus (DG)/CA3 and CA1) and amygdala subnuclei, including the basolateral amygdala (BLA) and central amygdala (CEA), during emotional mnemonic discrimination. Among non-responders, the relative balance of hippocampal activity differed by antidepressant mechanism: those taking single-action antidepressants showed greater DG/CA3 than CA1 activity, whereas those taking multi-action antidepressants showed the opposite pattern. This suggests mechanistically specific differences in hippocampal computations associated with ineffective treatment. These effects were localized to the anterior hippocampus, with no significant effects observed in posterior regions. In contrast, responders exhibited stronger DG/CA3-BLA coactivation during negative mnemonic discrimination, a pattern absent in non-responders and unmedicated controls. Antidepressant-associated differences in amygdala subnuclei activity persisted beyond current symptom severity, suggesting medication-related modulation of emotional memory circuits rather than effects driven solely by depression severity. These findings provide evidence in humans that antidepressant use is associated with altered hippocampal-amygdala circuitry in a manner that depends on both pharmacological mechanism and treatment efficacy. Identifying circuit-level signatures of treatment response may inform mechanistically guided approaches to antidepressant selection and monitoring.

neuroscience↗

bioRxiv2026

Temporal cortex astrocytic Gi-GPCR signaling regulates learned threat responses

Astrocytes are increasingly recognized as dynamic modulators of brain circuit function, memory processing, and behavior. Emerging evidence suggests that astrocytic G-protein-coupled receptors (GPCRs) are key regulators of these processes through their influence on intracellular signaling and neuron-glia interactions. Here, we show that the repertoire of functionally expressed GPCRs in cortical astrocytes is broader than previously appreciated. Yet, how distinct GPCR pathways contribute to behavioral regulation remains unknown for most brain areas and behavioral contexts. We therefore investigated the role of astrocytic GPCR signaling in the temporal cortex, a region that integrates multimodal sensory information and learned fear associations. Using chemogenetic tools to selectively activate distinct GPCR pathways in astrocytes, we demonstrate that Gi-coupled GPCR signaling, but not Gs- or Gq-coupled signaling, enhances fear memory retrieval. In vivo fiber photometry revealed that temporal cortex astrocytes exhibit robust Ca2+ transients to neutral, conditioned auditory and aversive sensory stimuli. Notably, astrocytic Gi-GPCR activation attenuated cue-evoked Ca2+ transients during memory retrieval. Together, these findings identify astrocytic GPCR signaling as a pathway-specific regulator of fear memory retrieval and suggest that astrocytic Gi-GPCR signaling modulates the processing of sensory cues to drive defensive behavior.

neuroscience↗

bioRxiv2025

Translational Downregulation of 5' TOP mRNAs During T Cell Exhaustion

T cell exhaustion is a dysfunctional state that arises during chronic infections and cancer, characterized by impaired effector functions and sustained expression of inhibitory receptors. While transcriptional, epigenetic, and metabolic rewiring have been well documented in exhausted T cells, a comprehensive understanding of how translation is regulated in this state remains incomplete. To address this gap, we performed ribosome profiling and RNA sequencing on exhausted human CD8+ T cells to globally assess translational control. Our analyses reveal a marked repression of 5 terminal oligopyrimidine (TOP) mRNAs during exhaustion. Unexpectedly, we demonstrate that this translational repression occurs despite evidence of elevated mTOR activity in exhausted T cells. These findings uncover a previously unknown layer of translational control in exhausted T cells.

molecular biology↗

bioRxiv2025

Key molecular and cellular events of table olive fruit abscission zone formation during natural maturation and after ethephon treatment

Abscission zones are specialized cell layers that enable organ detachment, yet their morphology and regulation vary among species. Olive (Olea europaea), a non-climacteric fruit, requires high concentrations of ethylene-releasing compounds to reduce fruit removal force (FRF) for mechanical harvest. To elucidate mechanisms of fruit abscission zone (FAZ) development in olive, we integrated physiological, transcriptomic, and cellular analyses during natural maturation and after ethephon treatment. Olive fruits emitted low ethylene at color transition, coinciding with declining FRF, and application of 1500 ppm ethephon reduced FRF after one week. Transcriptome analyses of FAZ tissues, using a mesocarp-subtraction strategy to isolate FAZ-specific responses, identified 733 FAZ-specific genes shared between natural maturation and ethephon treatments, including genes of {beta}-1,3-glucanases, pectate lyases, and the phenylpropanoid pathway. Microscopy revealed lignification and alkalization of FAZ, accompanied by reduced low-methylesterified homogalacturonan and non-fucosylated xyloglucan. Increased glucanase activity and reduction in plasmodesmata callose likely facilitate cellular communication during abscission. Further, this study provides the first evidence of FAZ alkalization and implicates transporter upregulation in pH regulation preceding abscission. Our findings advance understanding of abscission biology in non-climacteric fruits and delineate conserved features of FAZ development. Summary StatementIntegrated physiological, transcriptomic, and cellular analyses reveal that fruit abscission zone development in olive involves lignification, alkalization, and coordinated cell wall remodeling, identifying {beta}-1,3-glucanases, pH-regulating transporters, and pectate lyases that facilitate cell separation in a non-climacteric fruit.

plant biology↗

bioRxiv2025

The macroecology of immunity: predominant influence of climate on invertebrate immune response

The immune system is the primary defense against parasites. With the ever-increasing rate of disease, epidemiologic models considering geographic variation in immune responses could prove useful. Despite increasing interest in the macroecology of parasitism and infectious diseases, we know little about the macroecology of immune responses. Host characteristics, parasite exposure, and environmental factors can all affect immunity, but how these factors interact to shape spatial variation in the strength of immune responses remains unexplored. We captured odonates (dragonflies and damselflies) and their conspicuous ectoparasitic mites across a geographic area spanning the temperate and boreal forest biomes in eastern Canada. We then conducted immune response bioassays on 1,237 individuals from 63 odonate species. We used linear regressions and structural equation models to relate immune responses to host body size, parasite load, pH, temperature, and precipitation while accounting for evolutionary relationships among host species. We found significant differences in the strength of immune response among host individuals, and this variation was best explained by climatic conditions, specifically decreasing with precipitation and, to a lesser degree, temperature. While host species significantly differed in immune response strength, we found no effect of host body size, evolutionary relationships among hosts, or parasitism on immune response. Our study investigating the drivers of immune response across dozens of species spread across two biomes is the most comprehensive to date. Climatic conditions have a strong influence on host immune response, regardless of host characteristics or parasitism rates. In this specific case, strong immune responses were associated with low levels of annual precipitation, which could relate to the role of cuticular melanin content in desiccation resistance, and the melanin-based encapsulation response being a byproduct of this adaptation. A spatially-explicit understanding of the biological processes affecting immunity could improve epidemiological models of disease risk that inform disease management globally.

immunology↗

bioRxiv2025

miRNA-Mediated Regulation of Gene Expression During Early Activation in Jurkat Cells

BackgroundT cell activation induces substantial changes in gene expression by rapidly increasing transcription and translation. Additionally, microRNAs play a crucial role in regulating protein expression in T cell physiology, adding a layer of complexity by fine-tuning protein levels. While various miRNAs have been implicated in T cell function, a systematic analysis of differentially expressed miRNAs during early T cell activation and identification of their mRNA targets remains mostly unknown. ResultsWe investigated dynamic changes in global gene expression during early T cell activation using a multi-omics approach combining small RNA-seq, mRNA-seq and ribosome profiling. Our results show that most differential expression changes occur by 5 hours post-activation, with translational upregulation predominating over downregulation. From 5 to 12 hours, we observed modest transcriptional and translational reprogramming. We identified 9 miRNAs that are differentially expressed (DE) during early activation, with most changes occurring as early as 5 hours. We calculated translation efficiency (TE) and classified genes based on changes in both mRNA abundance and ribosome-protected fragments (RPFs). By integrating TE and miRNA expression data, we examined the relationship between TE group-specific regulation patterns and the number of miRNA binding sites. Interestingly, rather than observing a uniform downregulation of targets with 4 or more predicted DE miRNA binding sites, we found distinct regulatory patterns that varied with both activation time point and TE category. ConclusionsOur data provide new insights into how genes associated with key events in T cell activation such as translation, cell proliferation, and immune signaling are regulated at both the transcriptional and translational levels. The observation that most regulatory changes occur within the first 5 hours post-activation highlights the rapid and coordinated nature of T cell responses. The differential patterns of target regulation, based on translation efficiency groups and miRNA binding site density, suggest a context-dependent role for miRNAs in shaping protein output. Future experiments will be required to functionally validate specific miRNA-target interactions and to explore their relevance in primary T cells in vivo. This study also lays the groundwork for identifying miRNA-based regulatory circuits for therapeutic modulation of T cell activity.

genomics↗

bioRxiv2025

Endosome transcriptomics reveal trafficking of Cajal bodies into multivesicular bodies

All eukaryotic cells secrete exosomes, a type of extracellular vesicles (EVs) derived from the endocytic compartments known as multivesicular bodies (MVBs), or late endosomes (LEs). Exosomes contain a diverse range of cargo such as nucleic acids, proteins, lipids and small molecules but whether these contents have a biological function remains an area of intense investigation. Over the last decade, numerous studies have described the transcriptome of exosomes but very little is known about the RNA content of the MVBs, the source compartment for exosome biogenesis. Here we determine the small-RNA transcriptome of highly purified MVBs and report that various classes of nuclear small regulatory RNAs such as small-Cajal body associated RNAs (scaRNAs), small-nucleolar RNAs (snoRNAs) and small-nuclear RNAs (snRNAs) traffic to MVBs. We show that this RNA-trafficking requires the function of ESCRT machinery but is independent of canonical LC3 lipidation mediated selective autophagy. Furthermore, blocking the activity of a PI3K Class 3 enzyme, VPS34, required for recruitment of the ESCRT machinery to the endosome, prevents the turnover of these nuclear RNAs in MVBs. Our results provide a mechanism for targeting nuclear ribonucleoprotein complexes (RNPs), such as Cajal bodies, for degradation and turnover by the cytoplasmic endo-lysosomal pathway. Significance StatementEndosomes are cytoplasmic, membrane-bound subcellular organelles that are sites for biogenesis of exosomes, a class of extracellular vesicles, thought to mediate intercellular communication via their packaged cargo such as RNA. Previous studies have focused on the transcriptome of exosomes however very little is known about the identity of RNAs and mechanisms by which they are sorted into endosomes. Here we report a comprehensive endosome transcriptome and provide evidence that several nuclear RNA-protein complexes (RNPs) sort into endosomes, a previously unappreciated phenomenon. We show that this process requires the activity of endosomal sorting complexes and phospholipids characteristic of cellular endocytic compartments. Our study provides a mechanism for recycling and disposal of unwanted nuclear RNPs by the cytoplasmic endolysosomal pathway.

biochemistry↗

bioRxiv2025

Reference Point-Dependent Reinforcement Learning in Humans and Rats

Previous studies indicate that rewards and punishments in reinforcement learning are encoded in a relative manner. Reference point-dependence, a valuation bias shared by eminent adaptation level and prospect theories, is often cited as the underlying computational mechanism. However, whether these behavioural and computational mechanisms are preserved across species is unknown. To fill this gap, we designed a reinforcement learning task that was adapted for both humans (homo sapiens sapiens) and rats (rattus norvegicus). Behavioural analyses indicated robust relative value encoding in both species. At the computational level, reference point-dependence provided a reliable account of reinforcement learning behaviour in humans and rats. Despite these major similarities between species, some distinct differences in behavioural and computational modelling parameters were also observed. Overall, our study demonstrates that relative value encoding is a robust feature of reinforcement learning that is conserved across human and rat species.

molecular biology↗

bioRxiv2025

DeorphaNN: Virtual screening of GPCR peptide agonists using AlphaFold-predicted active state complexes and deep learning embeddings

Peptide-activated G protein-coupled receptors (GPCRs) regulate critical physiological processes such as metabolism, neural signalling, and endocrine function through their interaction with neuropeptides and peptide hormones. Despite their importance, identifying endogenous peptide agonists for GPCRs remains challenging, particularly for orphan receptors without known ligands. Recent advances in deep learning-based protein structure prediction, exemplified by AlphaFold (AF), have shown application beyond structural modelling, including protein-protein interaction prediction. Given that GPCR-peptide agonist interactions represent a specialized form of protein-protein interaction, we leveraged a dataset of experimentally validated agonist and non-agonist GPCR-peptide interactions from Caenorhabditis elegans to evaluate AF-Multimers ability to distinguish agonist-bound complexes. When modelling GPCR-peptide complexes, AF-Multimer confidence metrics partially discriminate agonist from non-agonist interactions, with improved discrimination achieved by utilizing AF-Multistate-derived active-state templates. We further investigated whether embeddings from the hidden layer of AF-Multimers neural network could distinguish agonist from non-agonist complexes. Feature performance analysis reveals that AF- Multimers pair representations outperform single representations, with distinct subregions of the pair representation providing complementary predictive signals. Building on these insights, we developed DeorphaNN, a graph neural network integrating active-state GPCR-peptide structural predictions, interatomic interactions, and deep learning embeddings to prioritize GPCR-peptide agonist interactions. DeorphaNN generalizes across datasets derived from different species, including annelids and humans, and successfully uncovered peptide agonists for two orphan GPCRs. DeorphaNN offers a novel computational resource to accelerate deorphanization by prioritizing GPCR-peptide agonist candidates for AI-guided experimental validation.

bioinformatics↗

bioRxiv2025

Context-dependent translation inhibition as a novel oncology therapeutic modality

Inhibitors of protein synthesis, including anisomycin, homoharringtonine, and other natural products bind in the peptidyl-transferase center (PTC) of the eukaryotic ribosome to inhibit translation. Recent work has demonstrated that some PTC-binding antibiotics act in a sequence-selective manner, inhibiting translation elongation at specific amino acids while the polypeptide is engaged in the PTC. However, this phenomenon has yet to be documented for compounds that inhibit translation by the human ribosome. Here we use structure-based design to guide synthesis of molecules called interdictors that bind to the human ribosome PTC and act in a context-selective manner to inhibit translation elongation. Using ribosome profiling, in combination with in vitro biochemistry and cryo-electron microscopy, we characterize the context selectivity of unique analogues and observe their preferred interactions with nascent chain residues with complementary properties. Furthermore, we present a structure for an interdictor bound to a portion of the MYC protein at [~] 1.9 [A] resolution and identify resulting structural rearrangements in both the nascent chain and ribosomal RNA. In cells, we document how these compounds differentially impact the ribotoxic stress response pathway which monitors ribosome collisions and can trigger apoptosis. Finally, we confirm their tumor growth inhibition activity after oral dosing in cell line derived xenografts in mice using the MDA-MB-231 model for triple-negative breast cancer. Together, our data establish sequence-selective inhibition of translation as a novel small-molecule therapeutic modality for historically difficult to address cancers by targeting translation of oncogenic dependency factors in the human ribosome PTC.

cancer biology↗

bioRxiv2024

Improved precision, sensitivity, and adaptability of Ordered Two-Template Relay cDNA library preparation for RNA sequencing

Sequencing RNAs that are biologically processed or degraded to less than [~]100 nucleotides typically involves multi-step, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced Ordered Two-Template Relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced non-productive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 picograms (less than 1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or post-transcriptional modifications.

biochemistry↗

bioRxiv2024

Non-immunogenic and reversible transgene expression using self-amplifying RNA

Self-amplifying RNA (saRNA) has the potential to provide durable, non-integrating transgene expression for transient gene therapy. However, its auto-replicative nature mimics viral infection, triggering innate immune responses that shut down cap-dependent translation, degrade cellular mRNA, induce cell death, and release cytokines. In non-immunotherapy applications, this immune activation is undesirable as it limits transgene expression, induces unintended changes in host gene expression, depletes transfected cells, and promotes inflammation--ultimately undermining therapeutic outcomes. Moreover, the use of exogenous immune suppressants to mitigate these effects often increases treatment complexity and the risk of unintended systemic side effects. To address these challenges, we developed a strategy to encode broad-spectrum innate immune suppression directly within saRNA. This approach leverages cap-independent translation to bypass saRNA-triggered translation shutdown, enabling the expression of multiple inhibitors targeting diverse double-stranded RNA-sensing and inflammatory signalling pathways. In mouse primary fibroblast-like synoviocytes--key cells in joint pathologies--this strategy eliminates the need for external immune inhibitors, reduces cytotoxicity and antiviral cytokine secretion, and enables sustained transgene expression that can be controlled with a small-molecule antiviral. Together, these findings support the development of reversible immune-evasive saRNA constructs for transient gene therapy applications that avoid persistent immune activation and eliminate the need for external immune suppressants. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/614636v6_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@18c1f36org.highwire.dtl.DTLVardef@554f8borg.highwire.dtl.DTLVardef@d9370dorg.highwire.dtl.DTLVardef@ad5ff2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

In a nutshell: pistachio genome and kernel development

Pistachio is a sustainable nut crop with exceptional climate resilience and nutritional value. To advance pistachios as a future food source and a model system for hard-shelled fruits, we generated a chromosome-scale reference genome of the most widely grown pistachio cultivar (Pistacia vera Kerman) and a spatiotemporal developmental study of the hull, shell, and kernel. Our study defined four distinct stages of pistachio growth and maturation by integrating tissue-level physiological and molecular data from thousands of nuts across twenty-four time points over three growing seasons. Transcriptional and metabolic changes in the kernel elucidate molecular pathways governing nutritional quality, such as the accumulation of unsaturated fatty acids, which are vital for shelf-life and dietary value. This work yields new knowledge and resources that will inform other woody crops and facilitate further improvement of pistachio as a globally significant, sustainable, and nutritious crop.

plant biology↗