Biology subjects

McCormick, C.

Publications and source records attributed to McCormick, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Skiaviridae: a large DNA virus family that persistently infects thraustochytrid protists

Genomics and metagenomics have transformed our understanding of the diversity of large DNA viruses infecting eukaryotic microorganisms. Using long-read sequencing, we discovered ubiquitous, co-culturing nucleocytoviruses in several thraustochytrid protists. These viruses are closely related to SmDNAV, an 'orphan' virus of the thraustochytrid Sicyoidochytrium minutum. Transcriptomics, proteomics and electron microscopy demonstrate viral gene expression and particle formation in these cultures without obvious defects in culture growth. We also found SmDNAV-type viruses associated with previously sequenced thraustochytrids. Phylogenetic analysis reveals that the SmDNAV and SmDNAV-like genomes belong to a hitherto unrecognized order of Nucleocytoviricota, here named "Skiavirales". All fifteen identified skiavirus genomes lack numerous viral hallmark genes including DNA-dependent RNA polymerase; all but three also lack DNA polymerase family B (PolB). PolB phylogeny suggests a specific relationship between Skiavirales and the recently discovered mirusviruses, which belong to a different viral realm. Gene exchange between skiaviruses, co-occurring mirusviruses, and host nuclear genomes shows that persistent infection by diverse large DNA viruses provides an opportunity for virus-virus and virus-host co-evolution in thraustochytrids and, perhaps, other microbial eukaryotes.

evolutionary biology↗

bioRxiv2026

Blindness reshapes mental time travel: From perceptual scenes to conceptual scaffolds

How can humans remember and imagine without vision? Mental time travel, the ability to re-experience past events and envision future ones, is widely assumed to rely on visual imagery and the construction of mental scenes. Blindness provides a critical test of this assumption. Across behavioral interviews, language analyses, and multimodal neuroimaging in congenitally blind, late-blind, and sighted individuals, we show that blind individuals, even those blind from birth, mentally time travel as vividly as sighted people, but construct their inner worlds differently. Sighted participants relied on perceptual detail and activated classic scene-processing regions, whereas blind participants emphasized thoughts and emotions and recruited reorganized occipital cortex. Connectivity analyses revealed strengthened coupling between occipital and medial temporal regions, indicating adaptive reconfiguration of the episodic system. The brain does not require images to imagine: it flexibly builds internal experiences using the representational resources available. Summary ParagraphHow humans reconstruct events that are no longer available to the senses is a fundamental but unresolved question. Remembering the past and imagining the future, known as mental time travel, is a defining feature of human cognition, shaping identity and guiding decisions{superscript 1},{superscript 2}. Prevailing theories assume that such constructions depend on visual imagery, with the minds eye reconstructing events on a visuospatial stage3,. Blindness provides a critical test of this assumption. Across extensive behavioral interviews and multimodal neuroimaging, we find that mental time travel remains phenomenologically intact in blindness, but its scaffolding changes fundamentally. Sighted individuals rely on perceptual detail and activate regions specialized for visual scenes, whereas blind individuals, whether blind from birth or later in life, emphasize thoughts and emotions and reorganize occipital cortex for conceptual strategies,. These contrasting strategies map onto distinct neural signatures, revealing a dissociation between perceptual and conceptual routes to episodic simulation. Together, these findings reveal that the brains capacity to construct internal experience rests on conceptual scaffolding, not perceptual re-creation.

neuroscience↗

bioRxiv2026

Chd8 haploinsufficiency leads to molecular layer heterotopias and age-dependent cortical expansion

Mutations in the chromatin remodeler CHD8 are associated with autism and macrocephaly. While mouse models of Chd8 haploinsufficiency recapitulate brain overgrowth, the specific cellular mechanisms and developmental timing that lead to these anatomical abnormalities remain poorly understood. Here, we conducted 3D imaging of Chd8V986*/+ mouse brains using magnetic resonance imaging followed by tissue clearing and cellular resolution light-sheet microscopy across embryonic and postnatal developmental stages. We found that brain overgrowth occurs postnatally, driven by an increase in non-neuronal cells prior to volumetric expansion. Unexpectedly, we identified prevalent molecular layer heterotopias (MLH) within the frontal cortex of Chd8V986*/+ mice composed of neurons breaking through the pial surface during embryonic development and persisting throughout life. Increased incidence of MLH, previously identified in individuals with idiopathic autism, was replicated across three independent Chd8+/- mouse models and present across multiple genomic backgrounds, establishing aberrant neuronal migration as a core feature of Chd8 haploinsufficiency.

neuroscience↗

bioRxiv2026

Genome-wide single-cell perturbation screens with VIPerturb-seq

CRISPR-based screening combined with single-cell sequencing (i.e., Perturb-seq) enables systematic mapping of genetic perturbations to molecular phenotypes. While Perturb-seq is well-suited to profile targeted subsets of regulators, scaling to genome-wide screens presents substantial cost and throughput challenges. Here we introduce VIPerturb-seq, a platform to facilitate routine genome-wide Perturb-seq experiments using probe-based detection workflows. We describe a split probe strategy for detection of genome-wide CRISPR libraries in fixed cells that enables (i) support for phenotypic enrichment of Very Important Perturbations (VIPs) prior to single-cell profiling, and (ii) compatibility with combinatorial indexing workflows to further improve Perturb-seq throughput by 50-fold. Using a genome-wide CRISPRi library (GuEST-List), we demonstrate VIPerturb-seq on three genome-wide screens representing both unbiased and phenotypically enriched workflows. Our results demonstrate how the sensitivity, scalability, and efficiency of VIPerturb-seq can enable both individual labs with targeted research questions and large data generation platforms aiming to construct virtual cells.

genomics↗

bioRxiv2025

Stimulation of cholesterol efflux inhibits herpesvirus nuclear egress

Cholesterol in herpesvirus envelopes and host membranes supports membrane fusion during virus entry. However, little is known about how cholesterol affects other aspects of the viral replication cycle. Here, using an infection model that begins with reactivation from latency, bypassing the entry step, we demonstrate that depleting intracellular cholesterol inhibits the production of infectious Kaposis sarcoma-associated herpesvirus (KSHV). Treatment of latently infected cells with the liver X receptor (LXR) agonist 22(R)-hydroxycholesterol (22OH) increased expression of sterol response genes, including the cholesterol efflux pump ATP binding cassette subfamily A member 1 (ABCA1), and reduced intracellular cholesterol. 22OH treatment initially enhanced lytic reactivation, but diminished viral protein accumulation during late replication, while altering accumulation of host proteins that regulate the cell cycle and innate immunity. Cholesterol efflux led to an increase in the proportion of capsids lacking viral genomes in the nucleus and a reduction in nucleocapsids accessing the cytoplasm. Next, we used a herpes simplex virus (HSV) infection model to explore if 22OH affected the conserved herpesvirus nuclear egress complex (NEC), comprised of two viral proteins that accumulate at the inner nuclear membrane and facilitate primary envelopment. We found that 22OH inhibited HSV-1 and HSV-2 replication and spread in plaque reduction assays and impaired localization of NEC proteins pUL31 and pUL34 to the nuclear periphery. These findings indicate that cholesterol efflux inhibits herpesvirus replication by interfering with the formation of nucleocapsids competent to complete nuclear egress. IMPORTANCECholesterol plays key roles in regulating the fluidity of cellular membranes and the trafficking and lateral diffusion of transmembrane proteins. Herpesviruses encode a variety of transmembrane proteins that traffic to different cellular compartments, while herpesvirus capsids and tegument proteins interact with nuclear and trans-Golgi network membranes throughout the primary and secondary envelopment process, respectively. This produces viral particles with cholesterol as an envelope component which is required to support membrane fusion during entry. Our discovery that cholesterol efflux inhibits KSHV nuclear egress suggests that cholesterol plays important supportive roles at this stage of lytic replication. Moreover, we observed that cholesterol efflux mediated by oxysterol treatment has widespread effects on host cell protein expression, resulting in a non-classical antiviral state. Cholesterol likely supports the proper trafficking of viral proteins to nuclear sites of virion assembly or is influencing the fluidity of nuclear membranes used for viral primary envelopment and de-envelopment during nuclear egress.

microbiology↗

bioRxiv2025

Lower Airway Dysbiosis in NTM+ Bronchiectasis is Associated with NET-Predominant Severe Phenotypes

RationaleThe discoveries of neutrophilic inflammation and Pseudomonas-dominant pulmonary dysbiosis have helped pave the way for host-directed therapy in bronchiectasis. Substantial knowledge gaps remain about the interplay between neutrophilic signatures and microbes in non-tuberculous mycobacterial lung disease (NTM-LD), a phenotypically diverse lung infection that is increasingly prevalent in the United States and other parts of the world. ObjectivesEvaluate the lower airway microbiota and neutrophilic traits in NTM- and NTM+ bronchiectasis. Methods16S rRNA gene sequencing, cell counts and neutrophil extracellular trap (NET) immunoassays were performed on bronchoscopic lower airway samples in 200 bronchiectasis subjects (108 NTM-, 92 NTM+). A preclinical model of oral commensal micro-aspiration and NTM infection was used to profile the murine lower airways with flow cytometry and a NET assay. Measurements and Main ResultsLower airways of NTM+ bronchiectasis patients were enriched with Mycobacterium and oral commensals (e.g., Veillonella, Prevotella). NET levels were higher in NTM+ BAL. Mycobacterium and oral commensals co-occurred with NET and neutrophils in network studies. Distinct oral commensal taxa associated with severe disease phenotypes such as cavitary disease and exacerbators. In a murine micro-aspiration model, the combination of oral commensals and Mycobacterium led to a sustained pro-inflammatory immune response marked by an increase in Th17, {gamma}8T cells, PD-1+ T lymphocytes as well as higher NET levels. ConclusionsOur analyses showed that distinct microbiome features beyond the primary pathogen can contribute to neutrophilic inflammation and severe disease phenotypes in bronchiectasis/ NTM-LD.

immunology↗

bioRxiv2025

Coronavirus M proteins disperse the trans-Golgi network and inhibit anterograde protein trafficking in the secretory pathway

Coronaviruses (CoVs) encode Spike, Membrane (M), and Envelope (E) transmembrane proteins that are translated and processed at the endoplasmic reticulum (ER) and traverse the secretory pathway to converge at sites of virus assembly. Three transmembrane ER resident proteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK), sense the accumulation of unfolded proteins in the ER and initiate the unfolded protein response (UPR) to increase ER protein folding capacity. We observed UPR modulation by numerous CoV proteins, including Spike, which broadly activated all three arms of the UPR. By contrast, M selectively inhibited the ATF6 arm of the UPR, either when it was activated by CoV proteins like Spike, or when it was activated by chemical stimuli of ER stress; however, M was unable to inhibit Spike-mediated activation of IRE1 or PERK. ATF6 inhibition was conserved amongst all human CoV M proteins. Amongst the UPR sensors, ATF6 has a unique activation mechanism whereby ER stress triggers translocation to the Golgi where ATF6 is processed by resident proteases to release the ATF6-N bZIP transcription factor. Because M had no effect on the function of the ATF6-N transcription factor, we reasoned that it may act upstream by inhibiting ATF6 trafficking in the secretory pathway. Indeed, we observed that ectopically expressed M inhibited several processes that require ER-to-Golgi transport, including sterol regulatory element binding protein-2 (SREBP2)-mediated activation of sterol responses and stimulator of interferon response cGAMP interactor 1 (STING1)-mediated activation of interferon responses. M also inhibited the secretion of a soluble Gaussia luciferase reporter protein. Using a Retention Using Selective Hooks (RUSH) cargo sorting assay, we observed that M accumulated in the cis-Golgi and inhibited further anterograde transport of a transmembrane reporter protein beyond this compartment, while dispersing the trans-Golgi network (TGN). We also observed a conserved TGN dispersal phenotype in cells infected with SARS-CoV-2, hCoV-OC43, or hCoV-229E. We determined that M is present in both detergent resistant and detergent soluble membranes and that M increased cholesterol abundance at the cis-Golgi. Together, these observations suggest that CoV M proteins disrupt the TGN and impede normal anterograde traffic in the canonical secretory pathway, potentially by increasing cholesterol levels at the cis-Golgi . Because CoV egress does not require the TGN, this mechanism could allow the virus to selectively interfere with host responses to infection without impeding egress of nascent virions. IMPORTANCEMany viruses encode proteins that limit host antiviral responses. Coronaviruses encode a remarkably diverse array of proteins that antagonize host responses at discrete steps including the detection of viral products, signal transduction, host mRNA processing and nuclear export, and protein synthesis. Here, we describe a new form of viral antagonism of host antiviral responses by remodelling the secretory pathway, dispersing a distal portion of the Golgi network and causing accumulation of proteins early in the Golgi. This is achieved by an abundant viral structural transmembrane glycoprotein M, which is best known for its role as the central player in the assembly of new viruses. This newly discovered function of M allows it to limit host antiviral responses that depend on trafficking in the late secretory pathway, while maintaining its role in virus assembly in the early secretory pathway.

microbiology↗

bioRxiv2025

Seeing more than schemas: the vmPFC represents imagery- rich mental scenarios

Mental imagery varies dramatically across individuals, from vivid scene construction to the complete absence of visual experience, as seen in aphantasia. While the ventromedial prefrontal cortex (vmPFC) is traditionally associated with abstract, schematic representations, emerging theories suggest it may also contribute to constructing vivid, visual mental content. To test this, we developed a novel 7T fMRI experiment varying imagery demands across conditions: Prior to scanning, participants memorized richly detailed scenarios, more constrained, stationary objects, and finally semantic definitions for each of three abstract German words (e.g., hope). During fMRI and eye-tracking, the same word was presented across trials, but participants vividly re-engaged with one of three distinct representations (i.e., scenarios, objects, and definitions), allowing for comparison across richly different cognitive modes triggered by identical visual input. Univariate analyses confirmed previous findings; highlighting the roles of the vmPFC, hippocampus, parahippocampal cortex, and visual-perceptual cortex in imagery-rich scenario construction. We further performed multivoxel pattern analysis (MVPA) to examine distributed neural representations, which can reveal the informational content of mental imagery beyond activation magnitude. Critically, the vmPFC was the only brain region where MVPA classifier accuracy was higher for scenario construction than for object and abstract conditions, directly supporting our hypothesis that the vmPFC encodes imagery-rich details rather than solely abstract, schematic information. Eye movement variability also distinguished between conditions. These findings advance our understanding of vmPFC function, emphasising its role in representing vivid mental content. HighlightsO_LI{middle dot} vmPFC distinctly represents imagery-rich scenarios in MVPA analysis C_LIO_LIUnivariate fMRI reveals increased HC and PHC activity during scenario construction C_LIO_LIEye movements vary by imagery type, distinguishing scenarios from objects and definitions C_LI

neuroscience↗

bioRxiv2025

Elucidating directed neural dynamics of scene construction across memory and imagination

Autobiographical memory (AM) and imagination both rely on the brains ability to construct vivid, coherent mental representations of past or imagined experiences. A central cognitive process underlying these functions is scene construction--the mental generation of spatially organized, imagery-rich representations of environments. Using ultra-high field 7T fMRI combined with Dynamic Causal Modeling (DCM), we examined directed effective connectivity among key nodes of the default mode network: the ventromedial prefrontal cortex (vmPFC), hippocampus, and precuneus during object imagery, single-scene construction, and AM retrieval. Our results revealed distinct patterns of network dynamics depending on task demands. During AM retrieval, effective connectivity was characterized by vmPFC-driven top-down modulation primarily targeting the precuneus, supporting the temporal and self-relevant structuring of episodic memory. In contrast, extended scenario imagination engaged hippocampal bidirectional influences with both vmPFC and precuneus, reflecting the dynamic simulation and integration of unfolding events. Single Scene construction shifted network leadership to the precuneus, which exerted modulatory effects on both vmPFC and hippocampus, consistent with its pivotal role in spatial integration and the generation of coherent mental scenes. Object imagery showed minimal stable connectivity within this network, suggesting limited engagement of the hippocampal-default mode circuit during simpler visual representations. Together, our findings highlight a flexible, task-dependent reorganization of effective connectivity within the vmPFC- hippocampus-precuneus network during the construction of rich mental experiences. Scene construction emerges as a unifying mechanism linking memory and imagination, with the direction and strength of neural interactions adapting according to whether temporal or spatial demands predominate. HighlightsO_LIAutobiographical memory is reflected by vmPFC-driven effective connectivity C_LIO_LIEffective connectivity varies flexibly amongst vmPFC, hippocampus and precuneus C_LIO_LIScene construction emerges as a core process across memory and imagination C_LI

neuroscience↗

bioRxiv2025

From single scenes to extended scenarios: the role of the ventromedial prefrontal cortex in the construction of imagery-rich events

Mental events are fundamental to daily cognition, including the recollection of past experiences, the anticipation of future scenarios, and engagement in imaginative, fictitious thought. Typically, these temporally extended mental events unfold within coherent spatial contexts, rich in naturalistic scenes and objects. However, there remains a significant gap in understanding how these events are represented in the brain. This study aimed to investigate the neural patterns involved in the construction of temporally extended mental events. Using ultra-high field functional magnetic resonance imaging, we examined brain regions previously implicated in this cognitive process, including the ventromedial prefrontal cortex (vmPFC), hippocampus, and posterior neocortex. We employed a novel experimental paradigm in which participants engaged in three forms of mental imagery: single objects (e.g., "a black espresso"), single scenes (e.g., "a busy cafe"), and extended scenarios (e.g., "meeting a friend for coffee"). We identified a shared neural network - comprising the vmPFC, hippocampus, and posterior neocortex - engaged across all forms of mental imagery. However, we observed a hierarchical organization in their contributions: the posterior neocortex supported the construction of objects, scenes, and scenarios, while the hippocampus primarily contributed to scenes and scenarios. The vmPFC exhibited a stepwise increase in activation, peaking during scenario construction. These findings suggest that the construction of mental events emerges from the close interaction of perceptual details provided by the posterior neocortex, spatial coherence from the hippocampus, and the integration of those elements into a coherent, temporally extended mental event by the vmPFC - the "movies" of the mind.

neuroscience↗

bioRxiv2025

Assay for Rapid Quantification of Capped and Tailed Intact mRNA

The rapid development of mRNA vaccines since the COVID-19 pandemic has highlighted the potential of mRNA technology for vaccine and therapeutic applications. However, evaluating mRNA quality, including its integrity, 5' capping efficiency, and 3' poly(A) tailing, remains complex and challenging. Current analytical methods provide detailed insights, but are often limited by long analysis times, complex sample preparation, and the need for specialized expertise. The 5 CapQ assay addresses these challenges and provides a novel, rapid, and straightforward method that offers a single, unique measurement of intact mRNA with both a 5'cap and 3'poly(A) tail. This 2-hour benchtop-based assay provides quantitative or relative analysis, with high accuracy and precision without extensive sample processing. The 5 CapQ assay offers a significant advancement in mRNA analytics to alleviate bottlenecks in applications including in vitro transcription (IVT) reaction optimization, post-transcriptional capping optimization, and assessment of batch-to-batch IVT bioprocess consistency.

molecular biology↗

bioRxiv2025

Primary envelopment of Kaposi's sarcoma-associated herpesvirus at the nucleoplasmic reticulum

Herpesvirus egress begins with primary envelopment of newly assembled capsids at the inner nuclear membrane (INM) facilitated by a viral nuclear egress complex (NEC). Primary envelopment has been observed at the peripheral INM as well as at structures known as nuclear infoldings. Nuclear infoldings resulting from invaginations of the INM are known as Type-I nucleoplasmic reticulum (NR), whereas infoldings of both INM and outer nuclear membrane (ONM) are known as Type-II NR. Here, we report that Kaposis sarcoma-associated herpesvirus (KSHV) reactivation from latency and lytic cycle progression correlates with increases in both types of NR invaginations, but primary envelopment was restricted to Type-I NR and the peripheral INM. Over a time-course of infection, DNA-containing KSHV C-capsids were frequently observed budding into nuclear infoldings contiguous with the INM and sparsely decorated with nuclear lamina, characteristic of Type-I NR. These Type-I NR structures co-localized with puncta containing CTP:phosphocholine cytidylyltransferase (CCT), the enzyme that catalyzes the rate-limiting step in phosphatidylcholine (PtdCho) synthesis, and drives de novo membrane biogenesis and membrane curvature required for NR expansion; this may provide sufficient Type-I NR to match requirements for KSHV nuclear egress. The selective utilization of the Type-I NR for primary envelopment despite the concurrent expansion of the Type-II NR suggests a mechanism to target C-capsids into the Type-I NR. We also observed accumulation of de-enveloped KSHV C-capsids in 2nd order nuclear infoldings, suggesting that primary envelopment and de-envelopment can occur not only at the nuclear periphery, but also at the Type-I NR. ImportanceHerpesvirus capsids assemble in the cell nucleus but are too large to exit via nuclear pores. Instead, they invade the inner nuclear membrane and acquire a provisional lipid envelope that is quickly shed through fusion with the outer nuclear membrane, a neat trick that delivers the capsid to the cytoplasm for subsequent steps in assembly and egress. Dynamic rearrangements of the nuclear membrane include invaginations that plumb the depths of the nucleus to facilitate cargo trafficking. Here, we demonstrate that during Kaposis sarcoma-associated herpesvirus (KSHV) replication, nuclear membrane infolding and expansion events increase, coinciding with recruitment of a host enzyme required for phosphatidyl choline synthesis to these sites. We observed an abundance of KSHV capsids at infoldings of the inner nuclear membrane, as well as free capsids in the interior of second order infoldings suggestive of de-envelopment events occurring prior to infold retraction. This complements a more well-established mechanism of KSHV egress at the nuclear periphery, and indeed, we observed concomitant egress of KSHV capsids via both mechanisms from the same nucleus. Similar observations for other herpesviruses by other groups suggests that this versatility in nuclear egress mechanisms is conserved.

microbiology↗

bioRxiv2024

A yeast-based reverse genetics system to generate HCoV-OC43 reporter viruses encoding an eighth sgRNA

Coronaviruses have large, positive-sense single-stranded RNA genomes that challenge conventional strategies for mutagenesis. Here, we report the development of a new reverse genetics system for the endemic human coronavirus (HCoV) OC43 that utilizes transformation-associated recombination (TAR) to assemble complete viral genomes from dsDNA genome fragments via homologous recombination in Saccharomyces cerevisiae. Following cDNA synthesis from HCoV-OC43 viral RNA, we used TAR to capture fragments of the HCoV-OC43 genome to store as sequence-validated dsDNA parts. We performed combinatorial assembly in yeast to obtain an intact dsDNA copy of the HCoV-OC43 genome sufficient to launch viral replication upon introduction into human cells, yielding the yeast assembled OC43YA virus. We also expanded the OC43YA genome by inserting an eighth body transcription regulatory sequence (B-TRS) and an mClover3-H2B reporter gene between the M and N genes, designed to allow the reporter protein to be translated from its own subgenomic mRNA. We thoroughly evaluated OC43YA and the OC43-mCloYA reporter virus, and demonstrated comparable viral gene expression, fitness in cell culture, and susceptibility to antivirals, compared to their natural progenitor. In summary, this new HCoV-OC43 reverse genetics system provides a modular platform for mutagenesis and combinatorial assembly of HCoV-OC43 genomes, and demonstrates the feasibility of expanding the genome while avoiding disruption of native coding sequences. IMPORTANCEHuman coronavirus OC43 (HCoV-OC43) is an endemic human coronavirus that typically causes relatively mild respiratory illnesses and displays seasonal patterns of infection. We developed a new system to assemble DNA copies of HCoV-OC43 genomes and generate recombinant viruses for research purposes. This system uses yeast, first to capture segments of DNA encompassing the entire RNA-based viral genome, and then to stitch them together into complete DNA genome copies that can be amplified in bacteria and introduced into human cells to initiate an infectious cycle, ultimately yielding recombinant viruses with comparable properties to their natural progenitors. We also devised a strategy to expand the viral genome, adding a gene for a reporter protein encoded by an additional eighth subgenomic mRNA. This yeast-based genome assembly system provides a modular platform for rapid mutagenesis and combinatorial assembly of HCoV-OC43 genomes and demonstrates the feasibility of expanding the genome.

synthetic biology↗

bioRxiv2023

Dual inhibition of coronavirus Mpro and PLpro enzymes by phenothiazines and their antiviral activity

Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 Mpro and PLpro protease activity in vitro was identified. In silico docking studies also predicted binding of the phenothiazine to the active sites of Mpro and PLpro from distantly related alphacoronavirus, HCoV-229E (229E) and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cell culture infection models. It was further demonstrated that the compound inhibited 229E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/557219v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@fa2c92org.highwire.dtl.DTLVardef@844484org.highwire.dtl.DTLVardef@1ae5d5aorg.highwire.dtl.DTLVardef@730133_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICoronavirus cysteine proteases Mpro and PLpro are targets for novel antiviral agents C_LIO_LIPhenothiazine ureas inhibit SARS-CoV-2 Mpro and PLpro protease activity C_LIO_LISome phenothiazine ureas inhibit replication of diverse coronaviruses with minimal cytotoxicity C_LIO_LIPhenothiazine ureas inhibit early stages of coronavirus replication consistent with failure of viral polyprotein cleavage C_LI

microbiology↗

bioRxiv2023

Hippocampal-occipital connectivity reflects autobiographical memory deficits in aphantasia

Aphantasia refers to reduced or absent visual imagery. While most of us can readily recall decade-old personal experiences (autobiographical memories, AM) with vivid mental images, there is a dearth of information about whether the loss of visual imagery in aphantasics affects their AM retrieval. The hippocampus is thought to be a crucial hub in a brain-wide network underlying AM. One important question is whether this network, especially the connectivity of the hippocampus, is altered in aphantasia. In the current study, we tested 14 congenital aphantasics and 16 demographically matched controls in an AM fMRI task to investigate how key brain regions (i.e., hippocampus and visual-perceptual cortices) interact with each other during AM re-experiencing. All participants were interviewed regarding their autobiographical memory to examine their episodic and semantic recall of specific events. Aphantasics reported more difficulties in recalling AM, were less confident about their memories, and described less internal and emotional details than controls. Neurally, aphantasics displayed decreased hippocampal and increased visual-perceptual cortex activation during AM retrieval compared to controls. In addition, controls showed strong negative functional connectivity between the hippocampus and the visual cortex during AM and resting-state functional connectivity between these two brain structures predicted better visualization skills. Our results indicate that visual mental imagery plays an important role in detail-rich vivid AM, and that this type of cognitive function is supported by the functional connection between the hippocampus and the visual-perceptual cortex.

neuroscience↗

bioRxiv2023

RNAi-mediated regulation of alg-3 and alg-4 coordinates the spermatogenesis developmental program in C. elegans

Coordination of gene regulatory networks is necessary for proper execution of cellular programs throughout development. In C. elegans, spermatogenesis and oogenesis occur during different life stages (L4 and adult, respectively) within the hermaphroditic germline tissue. Yet, it remains unclear how these developmental programs are robustly executed, particularly during stressful conditions. Here we show evolutionarily conserved RNA interference (RNAi) pathways act to restrict expression of spermatogenesis genes to the L4 stage. Our findings indicate a novel RNAi-mediated regulatory cascade is essential for properly coordinating the spermatogenesis developmental program, particularly during heat stress. This is achieved through RNAi-mediated genetic switches that regulate the expression of the Argonautes, ALG-3 and ALG-4, to control ALG-3/4 pathway function. Moreover, this work provides key insights into the different molecular mechanisms RNAi pathways employ to maintain both maternal and paternal germ cells reproductive potential, and further highlights the complexities and importance of RNAi-mediated gene regulation in development.

genetics↗

bioRxiv2022

30-Minute Highly Multiplexed VaxArray Immunoassay for Pneumococcal Vaccine Antigen Characterization

Pneumonia accounts for over 20% of deaths worldwide in children aged 1 to 5 years, disproportionately affecting lower- and middle-income countries. Effective, highly multivalent pneumococcal vaccines are available to decrease disease burden, with numerous new vaccines currently under development to serve a variety of worldwide markets. However, pneumococcal conjugate vaccines are among the hardest biologics to manufacture and characterize due to their complexity and heterogeneity. Current characterization methods are often inherently singleplex, requiring separate tests for each serotype present. In addition, identity and quantity are often determined with separate methods. We have developed the VaxArray Pneumococcal Assay for applications in identity, quantity, and stability testing of pneumococcal polysaccharide and pneumococcal conjugate vaccines. The VaxArray Pneumococcal Assay has a time to result of less than 30 minutes and is an off-the-shelf multiplexed, microarray-based immunoassay kit that can identify and simultaneously quantify 23 pneumococcal polysaccharide serotypes common to many on-market and in-development vaccines. Here, we highlight the potential of the assay for identity testing by showing high reactivity and serotype-specificity to a wide variety of native polysaccharides, CRM197-conjugated polysaccharides, and drug product. The assay also has vaccine-relevant lower limits of quantification in the low to mid ng/mL range and can be used for accurate quantification even in adjuvanted vaccines. Excellent correlation to the anthrone assay is demonstrated, with VaxArray resulting in significantly improved precision over this antiquated chemical method.

immunology↗

bioRxiv2022

Synthesis of Long RNA with a Site-Specific Modification by Enzymatic Splint Ligation

Synthesis of RNA molecules that contain an internal site-specific modification is important for RNA research and therapeutics. While solid-state synthesis is attainable for such RNA in the range of 100 nucleotides (nts), it is currently impossible with kilobase (kb)-long RNA. Instead, long RNA with an internal modification is usually assembled in an enzymatic 3-part splint ligation to join a short RNA oligonucleotide, containing the site-specific modification, with both a left-arm and a right-arm long RNA that are synthesized by in vitro transcription. However, long RNAs have structural heterogeneity and those synthesized by in vitro transcription have 3-end sequence heterogeneity, which together substantially reduce the yield of 3-part splint ligation. Here we describe a method of 3-part splint ligation with an enhanced efficiency utilizing a ribozyme cleavage reaction to address the 3-end sequence heterogeneity and involving DNA disruptors proximal to the ligation sites to address the structural heterogeneity. The yields of the synthesized kb-long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing.

biochemistry↗