Biology subjects

Johnson, R.

Publications and source records attributed to Johnson, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Indole-3-propionic acid nano-prodrugs sustain increased lymphoid exposure and alleviate experimental autoimmune arthritis

Chronic inflammatory diseases, including rheumatoid arthritis (RA), currently require sustained control of inflammation. Although long-acting therapies can reduce dosing frequency, those that rely on systemic immunosuppression may still compromise protective immunity. Microbiota-derived metabolites offer a complementary starting point for immune modulation through naturally occurring host-microbe signals. Indole-3-propionic acid (IPA), a gut microbial tryptophan metabolite naturally present in human circulation, has newly recognized immunoregulatory activity, yet its effects across disease contexts and its potential for therapeutic use remain incompletely defined. Here, we developed polymeric IPA nano-prodrugs (pIPANP) by conjugating IPA to a methacrylamide polymer through ester linkages and formulating the polymer into injectable nanoparticles. Following subcutaneous administration of fluorescently-labeled pIPANP, injection-site fluorescence remained detectable for at least 35 days, with labeled material detected in multiple lymph nodes. LC-MS/MS confirmed higher lymph-node IPA concentrations in mice treated with the pIPANPs compared to free IPA control at every sampled time point over 14 days. In collagen antibody-induced arthritis (CAIA), pIPANPs reduced clinical disease severity and paw swelling under both prophylaxis and therapeutic administration. pIPANP treatment increased the relative representation of CD11cCD103CD11b- cells with a conventional dendritic cell (cDC)1-associated phenotype and reduced costimulatory-marker frequencies in both CD11cCD103 and CD11cCD11b populations, most prominently in hock-draining lymph nodes. These changes were accompanied by increased PD-1 and IFN-{gamma} frequencies among CD4 and CD8 T cells. Fluorescent nanoparticle tracking showed preferential association with several myeloid populations, while single-cell profiling revealed divergent interferon-response signatures across splenic populations. These findings reveal arthritis-associated immunomodulatory activity of polymeric IPA and support its further development as a microbiome-inspired, long-acting approach to inflammatory arthritis.

bioengineering↗

bioRxiv2026

Polarized F-actin establishes cell interactions required for the formation of a stem cell niche

Lifelong stem cells are maintained by a cellular microenvironment called the niche, which enables tissue homeostasis. Proper niche construction is essential for persistent function, but studying niche formation is challenged by the inaccessibility of most niches to in vivo visualization during development. Innovations imaging the Drosophila testis are now allowing investigation of niche inception. F-actin polarizes to precise cell interfaces during testis niche assembly. Yet it is unknown whether polarization directs niche cell motility, or reflects adhesive sorting in response to formation of niche cell contacts. By adapting a method to optogenetically manipulate cortical F-actin via disruption of Rho1, we interrogate the role for cytoskeletal polarization during niche formation with tissue and temporal specificity. Rho1-mediated disruption of F-actin polarization caused defects in niche anterior assembly and architecture. Also, fewer cells adopted bona fide niche identity, given diminished Fas3, N-Cadherin, and Islet. These disrupted niches fail in signaling to germ cells to establish stem cell identity. We reveal that polarized F-actin is crucial for establishing cell contacts to form a functional niche, and to maintain cell identity in the developing tissue. Summary StatementOptogenetic cortical localization of cytoskeletal disruptors reveals tissue and temporal specific requirements for F-actin polarization in establishing a functional stem cell niche.

developmental biology↗

bioRxiv2026

Organisational principles of long non-coding RNAs revealed by exon deletion

Long non-coding RNAs (lncRNAs) regulate cell phenotypes in health and disease, yet how function is encoded in their sequence remains poorly understood. Current models propose a modular architecture composed of discrete functional elements, but this is based on a limited set of paradigmatic examples and methods for mapping function to sequence are limited in scope and resolution. Here, we establish a high-throughput CRISPR-Cas9 strategy for dissecting lncRNA functional architecture at exon resolution. Using cell fitness as a phenotypic readout, we screened 358 exons from 107 lncRNAs across four human cell lines. We report that (1) a large proportion of exons have no detectable function, (2) a minority of exons are functional in any given cell line (19-111 exons), equivalent to one-fifth of total transcript nucleotides on average, and (3) functionality is enriched towards the 5 end of the transcript. We developed a database of putative lncRNA functional elements, ElementaLdb, and demonstrated through statistical and experimental analyses that lncRNA function depends on transposable elements, microRNA response elements and RNA binding protein sites. These sub-genic functional maps expand the catalogue of experimentally defined lncRNA functional elements by an order of magnitude, illuminate molecular mechanisms and broadly support a modular organisation for lncRNAs.

genomics↗

bioRxiv2026

Pan-cancer discovery of driver mutations in long noncoding RNAs reveals widespread functional rewiring of RNA regulatory elements

Most somatic mutations in cancer occur outside protein-coding genes, yet the functional impact of these mutations remains largely unknown. Long noncoding RNAs (lncRNAs) represent a major class of cancer-promoting genes whose molecular mechanisms are poorly understood. While individual driver mutations in lncRNAs have been identified, detecting such driver lncRNAs at scale requires large tumour genome cohorts. We analyse 12,631 cancer genomes from the 100,000 Genomes Project (100kGP) and identify 121 lncRNAs under positive selection across 19 cancer types. These driver lncRNAs are independently supported by functional genomic screens, germline predisposing variants, mutual exclusivity with protein-coding drivers, and independent oncogenic lncRNA catalogues. Overall, approximately two-thirds of analysed tumours harbour at least one lncRNA driver mutation. Leveraging the depth of this dataset, we demonstrate that somatic mutations preferentially target and remodel RNA-binding protein (RBP) interaction sites to potentiate oncogenic lncRNAs, including MALAT1, SNHG14 and NEAT1. From these data, we derive a model in which somatic mutations liberate oncogenic lncRNAs from repressive RNA:protein interactions. This work expands the number and nature of cancer driver genes, identifies targets for RNA-directed therapies, and demonstrates that with large tumour mutation catalogues we can dissect the molecular mechanisms of noncoding genes.

Cancer Biology↗

bioRxiv2026

Aquifer microbial communities differentially display metabolisms capable of secondary effects on uranium speciation across a former metal processing site

Groundwater contamination presents challenges across world, yet remediation solutions in variably oxidized regions are limited and many co-interactions between contaminant metals and microbial reactions occur. Here we present a genomic and metabolic study into the biogeochemistry of a uranium-contaminated surficial aquifer site in Riverton, WY. We identified unique communities that varied based on geochemistry, geography, and compartment, matching microbial subsurface studies. Cross-site metabolism tests showed communities had functional capabilities of nitrogen respiration, manganese reduction, iron reduction, and sulfide oxidization. No sites showed evidence of microbial U-bioreduction nor ammonium oxidation. Only former tailings area groundwater and ditch surface water sites nearest a retention pond, and a downgradient oxbow lake exhibited sulfate reduction metabolisms. This was contrary to our hypothesis of near-river downgradient groundwater sites having U and S reduction capability. Most communities which showed S reduction capacity exhibited Fe oxidation capacity. Modeling demonstrated U as calcium uranyl carbonates. Based on our metabolism tests and known mineral and microbial metabolism reduction potentials, this suggests U reduction could only be achieved via abiotic reaction with biogenic sulfide. Of eleven sites tested, it is possible in four. This has impact on future site-specific remediation plans and understanding of microbial reactions in variably reduced zones. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/729369v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1926923org.highwire.dtl.DTLVardef@13486f9org.highwire.dtl.DTLVardef@1895a91org.highwire.dtl.DTLVardef@990213_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract TextWe performed microbial membership and metabolism measurements across a uranium-contaminated sites surface and ground waters, then performed analyses relating these metrics to geochemistry at the site. Findings showed variations in the membership, yet mainly similar functional capabilities. Metabolic differences were explained in relationship to uranium cycling and remediation implications.

microbiology↗

bioRxiv2026

Linking Infection, Immunity, and Symptoms for Age-Dependent Influenza Severity

Influenza infection results from tightly coupled interactions between viral replication, host immune responses, and the emergence of clinical symptoms. While mathematical models have extensively characterized viral and immune dynamics, the mechanistic link between immune activity and disease severity remains poorly understood. Here, we develop an integrative, within-host modeling framework that explicitly connects infection dynamics, immune responses, and symptom manifestation through a unified dynamical system. Using murine influenza data, we incorporate key immune components alongside a mechanistic representation of symptom progression, quantified via host weight loss.Our analysis identifies inflammatory signaling, particularly TNF- mediated pathways, as a central driver linking immune activity to symptom severity. Importantly, we demonstrate that age-dependent alterations in immune regulation reshape this coupling: aged hosts exhibit prolonged inflammatory responses that amplify and sustain symptom burden despite comparable viral kinetics. These results highlight that disease severity cannot be inferred from viral load alone, but instead emerges from the dynamical interplay between immune regulation and host physiology. This framework provides a quantitative basis for understanding age-specific morbidity and offers a foundation for designing interventions that target immune-mediated pathology rather than viral replication alone.

systems biology↗

bioRxiv2026

Deer tick virus genotypes are perpetuated by different modes of transmission

Deer tick virus (DTV), or lineage II Powassan virus, is an emergent tick-borne encephalitis virus in North America. Survivors frequently sustain neurologic sequelae. Nationally reported cases have been increasing. DTV is thought to be maintained in nature by multiple modes including horizontal transmission (from viremic host to tick), cofeeding transmission (between ticks feeding nearby) and by transovarial transmission (female to progeny). Analysis of the relative importance of each mode has been hindered by low enzootic transmission. In 2021, Marthas Vineyard, Massachusetts experienced an epizootic that allowed us to probe the modes of transmission on the island. We detected virus in 7.8% of questing deer tick nymphs (161 of 2063) and in 0.3% of lone star nymphs (2 of 678). Infected ticks had a highly focal distribution; 56% of infected ticks derived from only 4 of 71 collection sites. Tick mitochondrial genome sequencing demonstrated that infected ticks were not more likely to be siblings than negative ticks and, therefore, were unlikely to have inherited the infection. Whole viral genome sequencing revealed the presence of 3 genotypes, 58% were type1, 0.6% type2, and 13.7% type3. Tick host bloodmeal identification analyses determined that nymphs infected with type1 were significantly associated with having fed on shrews (50 of 94 type1 ticks, odds ratio=2.3, p<0.001). This is consistent with shrews serving as a reservoir. Ticks infected with type3, however, had no host associations, consistent with infection acquired by cofeeding. It may be that local DTV genetic variation is shaped by transmission modes or host associations. ImportanceDeer tick virus (DTV; Powassan lineage II) is a tick-borne encephalitis virus that causes a rare zoonosis in North America. Cases have been increasingly reported within the last decade. Is the recent risk trend due to increased transmission? How this virus is perpetuated in nature is not well understood. We took advantage of a natural epizootic on Marthas Vineyard to probe how the ticks there had become infected. Using a combination of viral whole genome sequencing and bloodmeal remnant identification in ticks, we find that the mode of transmission varied by viral genotype. One genotype is associated with ticks that had fed on shrews, and another did not depend on a specific reservoir host. Host associations may drive genetic diversity of deer tick virus and thus local host population dynamics may influence zoonotic risk.

ecology↗

bioRxiv2025

Direct measurement of sub-kilobase chromatin structure reveals that linker histone H1 broadly compacts chromatin, with differential impact amongst epigenetic states

Chromatin compaction by linker histone H1 family proteins is a long-standing model for transcriptional repression. However, the biophysical and conformational details of such compaction in situ, at the kilobase- and sub-kilobase length scale relevant to the activity of transcriptional regulatory elements, remain under debate. Rather than inferring such compaction from indirect measurements of features like DNA accessibility, we sought to directly probe sub-kilobase contacts between nearby nucleosomes. We developed an improved version of radiation-induced correlated cleavage with sequencing (RICC-seq), which we term RICC-seq 2.0, and used it in parallel with Micro-C to cross-validate our measurements of chromatin structure in both diverse cell types with different levels of linker histone and different levels of chromatin compaction, as well as a CRISPRi system for pan-H1 depletion. Using this system, we find that chromatin fiber de-compaction upon H1 depletion is global across the genome, reducing the contrast in inter-nucleosome contacts between acetylated chromatin and the rest of the genome. Surprisingly, this does not dramatically change higher-order chromatin organization such as nuclear compartments. Nevertheless, we observe a broad increase in accessibility at tens of thousands of sites and an increase in expression of over a thousand genes, which are enriched in polycomb repressive complex targets. Investigating the local chromatin compaction at upregulated genes as opposed to genes that do not change transcription, we observe that upregulated genes are not specifically de-compacted. Rather, our data support a model in which linker histone globally induces local compaction of nucleosome contacts and an increase in linker lengths, and repression by PRC1/2 is particularly dependent on these local features of chromatin architecture.

genomics↗

bioRxiv2025

CRISPR-mediated transcriptional activation as a mutation-independent therapeutic strategy for SYNGAP1-related intellectual disability

Synaptic Ras GTPase-activating protein (SynGAP) regulates synaptic strength and neuronal signaling, with essential roles in cortical development and synaptic plasticity. Heterozygous loss-of-function variants in SYNGAP1 cause SYNGAP1-related intellectual disability (SRID), a severe neurodevelopmental disorder characterized by epilepsy, developmental delay, and autism. SYNGAP1 mutations often result in haploinsufficiency, providing a strong rationale for gene-targeted therapies. However, no treatment currently addresses the underlying genetic cause of SRID. Here, we developed a CRISPR-mediated transcriptional activation (CRISPRa) approach to upregulate the functional Syngap1 allele in a SRID mouse model. CRISPRa activated Syngap1, normalized SynGAP protein expression and downstream signaling, and rescued working memory deficits. We validated the translational potential of this strategy in human induced pluripotent stem cell (hiPSC)-derived excitatory cortical neurons. CRISPRa rescued SYNGAP1 in two distinct loss-of-function variant lines. Together, these findings demonstrate the feasibility of mutation-independent transcriptional activation as a therapeutic approach for SRID and its broader applicability to haploinsufficiency disorders.

neuroscience↗

bioRxiv2025

Robust CRISPR Screens Identify TPL1 as a Novel Long Noncoding RNA Driving Triple-Negative Breast Cancer Hallmarks

Despite the growing catalog of long noncoding RNAs (lncRNAs), the functional roles of their vast majority in cancer remain poorly defined. To systematically explore lncRNA dependencies in triple-negative breast cancer (TNBC), we compiled a comprehensive annotation by merging GENCODE, BIGTranscriptome, and MiTranscriptome databases and performed a CRISPR-Cas9 deletion screen targeting 1,029 TNBC-enriched lncRNAs. The screen revealed several essential lncRNAs and those modulating doxorubicin sensitivity, with TPL1 emerging among top hits. TPL1 silencing significantly impaired TNBC cell proliferation in both 2D and 3D cultures and reduced invasive capacity in an organ-on-chip model. Transcriptomic and proteomic profiling following TPL1 knockdown revealed downregulation of genes involved in ECM-receptor interaction, focal adhesion, cell migration, and PI3K-Akt signaling. Mechanistically, TPL1 directly interacted with key proteins including EIF4B, MDM2, TARBP2, TLE5, and GTPase RAN, suggesting TPL1 could regulate RNA processing, transcriptional repression, and translation, as well as modulate GTPase signaling pathways. Additionally, TPL1 functioned as a competing endogenous RNA (ceRNA), sequestering miR-10396b-5p, miR-486-3p, and miR-450a-2-3p, among others, thereby modulating expression of pro-tumorigenic targets. Clinically, TPL1 was significantly overexpressed in TNBC tissues, particularly in the BLIS subtype. Collectively, our findings highlight TPL1 as a key regulator of TNBC molecular networks and a promising therapeutic target.

cancer biology↗

bioRxiv2025

Generation and maintenance of apical rib-like actin fibers in epithelial support cells of the Drosophila eye

Heterogeneity and complexity of cytoskeletal structures, and how these are regulated, is poorly understood. Here we use cells of the Drosophila pupal eye as models to explore diversity in the actin cytoskeleton. We found that different F-actin structures emerge in primary (1{degrees}), secondary (2{degrees}), and tertiary (3{degrees}) pigment cells as they mature. 1{degrees} cells became characterized by dense accumulations of apical F-actin that we termed Apical Ribs of Actin Fibers (ARAFs). The formins Diaphanous and Dishevelled Associated Activator of Morphogenesis are essential for generation of ARAFs, which are connected into a network by -Actinin, the villin Quail, and Spectrins, and linked to the apical membrane by Quail and Spectrins. ARAFs are contractile, stress-fiber-like, and connect to adherens junctions. Impairing ARAFs indicated that this network maintains cortical tension and is crucial for 1{degrees}s to achieve their characteristic shapes. Our evaluation of the three-dimensional shape of 1{degrees}s reveals that ARAFs are essential for the rounding and elevation of the apical membrane. Hence, a toolkit of conserved actin regulatory proteins builds and maintains a network of apical stress fibers that governs the morphology of the cell. Summary StatementThis study describes a novel F-actin network that shapes the apical region of cells of the Drosophila eye and uncovers a set of proteins that build and maintain the network.

developmental biology↗

bioRxiv2025

Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Library screening and selection methods can determine the binding activities of individual members of large protein libraries given a physical link between protein and nucleotide sequence, which enables identification of functional molecules by DNA sequencing. However, the solution properties of individual protein molecules cannot be probed using such approaches because they are completely altered by DNA attachment. Mass spectrometry enables parallel evaluation of protein properties amenable to physical fractionation such as solubility and oligomeric state, but current approaches are limited to libraries of 1,000 or fewer proteins. Here, we improved mass spectrometry barcoding by co-synthesizing proteins with barcodes optimized to be highly multiplexable and minimally perturbative, scaling to libraries of >5,000 proteins. We use these barcodes together with mass spectrometry to assay the solution behavior of libraries of de novo-designed monomeric scaffolds, oligomers, binding proteins and nanocages, rapidly identifying design failure modes and successes.

biochemistry↗

bioRxiv2024

Enhancer lncRNA LOC730338 limits the innate immune response against lymphoma cells, inhibiting ADAR2-dependent alternative transcription.

Chronic antigenic stimulation is a central factor in the development of marginal zone lymphoma (MZL). While the pharmacological inhibition of the B-cell receptor (BCR) signaling by Brutons tyrosine kinase (BTK) inhibitors is initially effective, the development of resistance remains a challenge in treating MZL and other B-cell malignancies. Enhancer activation remodeling is a key epigenetic mechanism that enables tumor adaptation during therapy. The most active regulatory regions cluster in super-enhancers and produce enhancer RNAs (eRNAs), a class of unstable noncoding transcripts that primarily serve as scaffolds for chromatin looping. However, when stabilized, these eRNAs can evolve into long noncoding RNA (lncRNAs) with distinct functions. To investigate enhancer-associated long non-coding RNAs (elncRNAs) involved in shaping BCR pathway dependence, we conducted a CRISPR interference (CRISPRi) screen in MZL cells. We identified LOC730338, an elncRNA linked to A-to-I RNA editing, which we renamed ADARreg. ADARreg renders tumor cells refractory to BCR pathway inhibition by modulating ADAR2 nuclear translocation and altering RNA modification patterns in key regulatory isoforms through coordinated control of RNA stability and localization, as revealed through subcellular direct RNA sequencing (DRS). In addition, ADARreg induces an immune-suppressive transcriptional program, increasing the production of inhibitory cytokines and receptors that diminish NK cell-mediated cytotoxicity. Together, these findings uncover a novel role for elncRNAs in orchestrating immune evasion and provide a potential therapeutic strategy to overcome resistance in lymphoma and other immune-related diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/627805v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1c3fa92org.highwire.dtl.DTLVardef@177b234org.highwire.dtl.DTLVardef@154a8fdorg.highwire.dtl.DTLVardef@122acfd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe enhancer-associated long non-coding RNA ADARreg modulates RNA editing and alters the stability and localization of key immune-related transcripts, thereby enabling tumor immune evasion. C_LIO_LIADARreg has prognostic and therapeutic relevance, linking resistance mechanisms and immune modulation, which supports the development of RNA-based therapeutic strategies in cancer. C_LI

cancer biology↗

bioRxiv2024

UCE phylogenomics inform the systematics and geographic range evolution of the harvester ant genus Pogonomyrmex

The harvester ant genus Pogonomyrmex is common in arid regions of North America, South America, and Hispaniola. Here, we use ultraconserved elements (UCEs) to infer the most comprehensive phylogeny of the genus to date. Our inferred topology largely supports previously hypothesized Pogonomyrmex evolutionary relationships and species groups. Our divergence dating analysis substantially pushes back the age of both Pogonomyrmex and the Pogonomyrmecini tribe, suggesting an early Eocene origin for Pogonomyrmex. We infer that Pogonomyrmex likely originated within the Pacific dominion ecoregion of Northern South America. Pogonomyrmex (excluding the Pogonomyrmex mayri clade) likely adapted to cooler and drier climates in the middle Eocene. The genus spread to North America in the late Eocene, likely aided by the ephemeral volcanic islands of the Panama Arc. A smaller group of Pogonomyrmex dispersed to Mesoamerica and North America in the Oligocene and to Hispaniola in the Miocene, most likely out of Mesoamerica. Our results suggest that adaptations to arid environments allowed Pogonomyrmex to spread and diversify in regions of the Americas that were actively becoming cooler and drier due to orogeny and other geologic processes.

zoology↗

bioRxiv2024

Cxcl10 is required for survival during SARS-CoV-2 infection in mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, remains endemic worldwide [~]5 years since the first documented case. Severe COVID-19 is widely considered to be caused by a dysregulated immune response to SARS-CoV-2 within the respiratory tract. Circulating levels of the chemokine CXCL10 are strongly positively associated with poor outcome; however, its precise role in pathogenesis and its suitability as a therapeutic target have remained undefined. Here, we challenged 4-6 month old C57BL/6 mice genetically deficient in Cxcl10 with a mouse-adapted strain of SARS-CoV-2. Infected male, but not female, Cxcl10-/- mice displayed increased mortality compared to wild type controls. Histopathological damage, inflammatory gene induction and virus load in the lungs of male mice 4 days post infection and before death were not broadly influenced by Cxcl10 deficiency. However, accumulation of B cells and both CD4+ and CD8+ T cells in the lung parenchyma of infected mice was reduced in the absence of Cxcl10. Thus, during acute SARS-CoV-2 infection, Cxcl10 regulates lymphocyte infiltration in the lung and confers protection against mortality. Our preclinical model results do not support targeting CXCL10 therapeutically in severe COVID-19.

immunology↗

bioRxiv2024

The dual Ras Association (RA) Domains of Drosophila Canoe have differential roles in linking cell junctions to the cytoskeleton during morphogenesis

During embryonic development and adult homeostasis epithelial cells must change shape and move without disrupting the tissues dynamic architecture. This requires robust linkage of cell-cell adherens junctions to the force-generating actomyosin cytoskeleton. Drosophila Canoe and mammalian Afadin play key roles in this linkage. One central task for the field is defining how upstream inputs from Ras-family GTPases regulate Canoe and Afadin. They are unusual in that they share two tandem Ras-association (RA) domains, which, when deleted, virtually eliminate Canoe function. Previous work in vitro suggested RA1 and RA2 differ in their ability to bind GTPases, but their individual functions in vivo remain unknown. Combining bioinformatic and biochemical approaches, we find that both RA1 and RA2 bind to active Rap1 with similar affinities, and that conserved N-terminal extensions play a role in binding. We created Drosophila canoe mutants to test RA1 and RA2 function in vivo. Despite their similar affinities for Rap1, RA1 and RA2 play strikingly different roles. Deleting RA1 virtually eliminates Canoe function in morphogenesis, while mutants lacking RA2 are viable and fertile but have defects in junctional reinforcement in embryos and during pupal eye development. These data significantly expand our understanding of how adherens junction:cytoskeletal linkage is regulated.

cell biology↗

bioRxiv2024

Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas1,2. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation5,6,7,8. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization6,10. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

neuroscience↗

bioRxiv2024

A transformer model for de novo sequencing of data-independent acquisition mass spectrometry data

A core computational challenge in the analysis of mass spectrometry data is the de novo sequencing problem, in which the generating amino acid sequence is inferred directly from an observed fragmentation spectrum without the use of a sequence database. Recently, deep learning models have made significant advances in de novo sequencing by learning from massive datasets of high-confidence labeled mass spectra. However, these methods are primarily designed for data-dependent acquisition (DDA) experiments. Over the past decade, the field of mass spectrometry has been moving toward using data-independent acquisition (DIA) protocols for the analysis of complex proteomic samples due to their superior specificity and reproducibility. Hence, we present a new de novo sequencing model called Cascadia, which uses a transformer architecture to handle the more complex data generated by DIA protocols. In comparisons with existing approaches for de novo sequencing of DIA data, Cascadia achieves state-of-the-art performance across a range of instruments and experimental protocols. Additionally, we demonstrate Cascadias ability to accurately discover de novo coding variants and peptides from the variable region of antibodies.

bioinformatics↗