Biology subjects

Johnston, C. M.

Publications and source records attributed to Johnston, C. M..

9 recordsLinked to original sources

bioRxiv2026

Striatal M4 Receptors Mediate M4 PAM Antipsychotic-like Efficacy Independently of Normalizing Striatal Dopamine Release

Muscarinic acetylcholine receptor (mAChR) drugs have emerged as a promising new class of antipsychotics, highlighted by the FDA approval of Cobenfy, the first treatment for schizophrenia that does not directly target dopamine receptors. The M4 receptor subtype is thought to be a key mediator of antipsychotic-like efficacy. However, the in vivo circuit mechanisms underlying the antipsychotic-like efficacy observed with M4-selective positive allosteric modulators (PAMs) remain incompletely understood. Here, we used fiber photometry in awake, behaving mice to assess how the M4 PAM VU0467154 (VU'154) affects striatal dopamine (DA) transmission under basal and amphetamine-stimulated conditions. We also generated and validated dorsal striatum-specific M4 knockout mice (DS-M4-KO) to test the role of striatal M4 receptors in regulating DA release and antipsychotic-like effects. VU'154 dose-dependently reduced the amplitude of striatal DA release events under basal conditions without altering event frequency. However, VU'154 had no significant effects on tonic DA levels under basal conditions, as assessed using the D1 PAM DETQ. Despite robustly attenuating AHL, VU'154 produced little to no effect on amphetamine-induced DA levels, indicating that antipsychotic-like efficacy was dissociated from changes in striatal DA levels. Notably, VU'154 efficacy in AHL was absent in DS-M4-KO mice, demonstrating that M4 receptors in the dorsal striatum are required for antipsychotic-like effects. Combined with previous studies implicating a key role for M4 receptors on D1-expressing neurons, these findings suggest that M4 PAMs produce antipsychotic-like efficacy primarily through postsynaptic mechanisms in the dorsal striatum, rather than through broad normalization of amphetamine-induced DA release.

pharmacology and toxicology↗

bioRxiv2026

Detection of merbecoviruses in Danish bats using a novel broad-range RT-qPCR assay

Bat-associated merbecoviruses comprise a genetically diverse group of coronaviruses with variable receptor usage and host range, highlighting the need for efficient tools for their surveillance. Here, we developed and optimized a broad-range reverse transcription quantitative PCR (RT-qPCR) assay for the detection of bat-associated merbecoviruses, including the lineage previously identified in brown long-eared bats (Plecotus auritus) in Denmark. In silico analysis predicted detection of 898 of 941 (95.4%) merbecovirus sequences, and the assay detected all bat-associated merbecoviruses included in the experimental panel. The assay was applied to 33 bats sampled through the Danish national bat surveillance program in 2025. Merbecovirus RNA was detected in two samples, one from a P. auritus and one from a Nathusius' pipistrelle (Pipistrellus nathusii), representing the first detection of a merbecovirus in P. nathusii in Denmark. Metagenomic next-generation sequencing recovered a complete merbecovirus genome from the P. nathusii sample. Phylogenetic analysis assigned the virus to the MOW clade and showed that it was closely related to the Russian MOW15-22 virus, sharing 98.67% nucleotide identity across the complete genome and 99.85% amino acid identity in the spike protein. Limited sequence data obtained from the P. auritus sample was consistent with the merbecovirus lineage previously identified in this species in Denmark. Together, these findings demonstrate the utility of broad-range RT-qPCR combined with metagenomic sequencing for surveillance and genomic characterization of bat-associated merbecoviruses.

microbiology↗

bioRxiv2026

Efficient primer design for broad-range virus detection: A semi-automated workflow using sequence clustering and varVAMP

Broad-range RT-qPCR assays are essential for detecting highly diverse viruses within virus families, but designing suitable primers remains challenging because of extensive sequence diversity and biases in public sequence databases. Here, we present a semi-automated workflow that combines sequence clustering and varVAMP to streamline pan-RT-qPCR primer design. The workflow reduces sequence redundancy, improves the representation of viral diversity, and facilitates the identification of broadly reactive primer candidates. We demonstrate the workflow using the arboviral genera Orthoflavivirus and Alphavirus as proof-of-concept datasets, showing its applicability to large and genetically diverse viral sequence collections. The workflow provides a practical, scalable, and reproducible framework for developing broad-range primer candidates and may support surveillance, diagnostics, and preparedness for emerging or divergent viruses.

microbiology↗

bioRxiv2026

Targeted metagenomic recovery of coronaviruses from wildlife samples

Coronaviruses (CoVs) are diverse RNA viruses infecting a wide range of hosts, with significant implications for public health, animal production and welfare. Bats are key reservoirs of mammalian CoVs and have contributed to the emergence and circulation of several zoonotic viruses in a wildlife context, while mustelids represent important hosts at the human-animal interface, as highlighted by SARS-CoV-2 outbreaks in farmed mink. Using metagenomic next-generation sequencing, we screened bats and wild mustelids and recovered and characterized full-length CoV genomes. Building on previous findings in Danish wildlife, including alpha-CoVs and a MERS-related beta-CoV in bats, this study expands current knowledge of coronavirus diversity and evolution in wildlife, emphasizing their relevance for zoonotic emergence.

microbiology↗

bioRxiv2024

Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

Herpes simplex virus (HSV) infection has worldwide public health concerns and lifelong medical impacts. The standard therapy, acyclovir, has limited efficacy in preventing HSV subclinical virus shedding, and drug resistance occurs in immunocompromised patients, highlighting the need for novel therapeutics. HSV manifests in the skin and mucosal epithelium. Here, we found acyclovir significantly less effective in skin-derived keratinocytes than donor-matched fibroblasts. To recapitulate in vivo tissue architecture, we 3D bioprinted human skin equivalents (HSE) in a 96-well plate format amenable for antiviral screening and preclinical testing. We screened a library of 738 compounds with broad targets and mechanisms of action and identified potent antivirals, including 23 known or experimental HSV treatments, validating the translational relevance of our assay. Unlike acyclovir, antivirals against HSV helicase/primase or host replication pathways displayed similar potency across cell types and donor sources in 2D and 3D models. Our 3D bioprinted platform allowed for integrating patient-derived cells and incorporating genetic variability early in drug development. The reduced potency in keratinocytes helps explain the limited benefit acyclovir and its congeners play in reducing sexual transmission. These data indicate that the 3D bioprinted HSE assay platform provides a more physiologically relevant approach to identifying potential antivirals for HSV. One Sentence SummaryHigh-throughput screen using 3D bioprinted human skin equivalents to identify antivirals against HSV and evaluate cell-type specific effects.

bioengineering↗

bioRxiv2024

Improving antibody-mediated protection against HSV infection by eliminating interactions with the viral Fc receptor gE/gI

Herpes simplex virus (HSV) encodes surface glycoproteins that are host defense evasion molecules, allowing the virus to escape immune clearance. In addition to their role in neuropathogenesis and cell-cell spread, glycoproteins E and I (gE/gI) form a viral Fc receptor (vFcR) for most subclasses and allotypes of human IgG and promote evasion of humoral immune responses. While monoclonal antibodies (mAbs) protect mice from neonatal HSV (nHSV) infections, the impact of the vFcR on mAb-mediated protection by binding to IgG is unknown. Using HSV-1 with intact and ablated gE-mediated IgG Fc binding, and Fc-engineered antibodies with modified ability to interact with gE/gI, we investigated the role of the vFcR in viral pathogenesis and mAb-mediated protection from nHSV. The gD-specific human mAb HSV8 modified to lack binding to gE exhibited enhanced neutralization and in vivo protection compared to its native IgG1 form. This improved protection by the engineered mAbs was dependent on the presence of the vFcR. Human IgG3 allotypes lacking vFcR binding also exhibited enhanced antiviral activity in vivo, suggesting that vaccines that robustly induce IgG3 responses could show enhanced protection. suggesting the value of vaccination strategies that robustly induce this subclass. Lastly, analysis of longitudinal responses to acute primary genital infection in humans raised the possibility that unlike most viruses, HSV may exhibited slow induction of IgG3. In summary, this study demonstrates that mAbs lacking the ability to interact with the vFcR can exhibit improved protection from HSV--offering new prospects for antibody-based interventions. One Sentence Summary: The herpes simplex virus neutralizing antibody HSV8 demonstrates improved activity in vitro and in vivo when its IgG Fc domain lacks the ability to bind the viral Fc receptor glycoprotein E/I complex through either Fc engineering or natural human IgG3 allotypes.

immunology↗

bioRxiv2023

Release of fragmented host, cell-free, genomic DNA into the circulation of pigs during infection by virulent African swine fever virus

African swine fever virus (ASFV) causes a severe hemorrhagic disease in domestic pigs resulting in high case fatality rates. The virus replicates in circulating cells of the monocyte-macrophage lineage and within lymphoid tissues, e.g. tonsils, spleen and lymph nodes. The infection results in high fever and a variety of clinical signs from about 3 days post infection. In this study, it was observed that one of many changes resulting from ASFV- infection within pigs was a large (>1000-fold) increase in the level of circulating cell-free DNA (cfDNA), including the beta-actin gene, derived from the nuclei of host cells, in the serum. This change occurred in parallel with the increase in circulating ASFV DNA. In addition, elevated levels (about 30-fold higher) of host mitochondrial DNA (mtDNA) were detected in serum from ASFV-infected pigs, but with a much higher baseline level of mtDNA in sera from uninfected pigs. The host derived cfDNA is derived from dead cells which may, or may not, have been infected. For comparison, the release of the cellular enzyme, lactate dehydrogenase (LDH), a commonly used marker of cellular damage, was also found to be elevated during the infection. The cfDNA is readily detected in serum and is a more sensitive host marker of ASFV infection than the release of mtDNA or LDH. In addition, sera from pigs infected by classical swine fever virus (CSFV), which causes a clinically similar disease as ASFV, were also tested but this infection did not result in the release of cfDNA, mtDNA or LDH. Author summaryAfrican swine fever virus causes a severe hemorrhagic disease in domestic pigs and wild boar, which often leads to death within a week. The infection results in a spectrum of different clinical signs and other changes within infected animals. In this study, we have shown, for the first time, that one consequence of infection by a highly virulent strain of this virus is the release into the blood of host genomic DNA, in a highly fragmented form. We found an increase of >1000-fold in the level of this cell-free DNA within the serum of infected animals. Furthermore, we also showed that the level of the small circular DNA from the cell mitochondria is also elevated in serum from infected animals as is the cellular enzyme lactate dehydrogenase but these changes were less marked and occurred later. The increase in the level of the cell-free host DNA is coincident with the increase in level of the viral DNA within blood and may act as a marker for infection by a highly virulent form of the virus. Remarkably, pigs infected by classical swine fever virus, which produces similar clinical signs, did not have elevated levels of these markers in their serum.

microbiology↗

bioRxiv2020

Verification of CRISPR editing by XdropTM Indirect Sequence Capture followed by short- and long- read sequencing

Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the gene editing site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop, a new microfluidic technology that allows targeted enrichment of long regions (~ 100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene edited region in 4 cell lines on long- and short -read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kb large insertions in 3 of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.

genomics↗

bioRxiv2019

In vitro evolution of herpes simplex virus 1 (HSV-1) reveals selection for syncytia and other minor variants in Vero cell culture

The large dsDNA virus HSV-1 is often considered to be genetically stable, however it is known to rapidly evolve in response to strong selective pressures such as antiviral drug treatment. Deep sequencing analysis has revealed that clinical and laboratory isolates of this virus exist as populations that contain a mixture of minor alleles or variants, similar to many RNA viruses. Classical virology methods often used plaque-purified virus populations to demonstrate consistent genetic inheritance of viral traits. Plaque purification represents a severe genetic bottleneck which may or may not be representative of natural transmission of HSV-1. Since HSV-1 has a low error rate polymerase but exhibits substantial genetic diversity, the virus likely uses other mechanisms to generate genetic diversity, including recombination, contraction and expansion of tandem repeats, and imprecise DNA repair mechanisms. We sought to study the evolution of HSV-1 in vitro, to examine the impact of this genetic diversity in evolution, in the setting of standard laboratory conditions for viral cell culture, and in the absence of strong selective pressures. We found that a mixed population of HSV-1 was more able to evolve and adapt in culture than a plaque-purified population, though this adaptation generally occurred in a minority of the viral population. We found that certain genetic variants appeared to be positively selected for rapid growth and spread in Vero cell culture, a phenotype which was also observed in clinical samples during their first passages in culture. In the case of a minor variant that induces a visually observable syncytial phenotype, we found that changes in minor variant frequency can have a large effect on the overall phenotype of a viral population.\n\nAuthor SummaryHerpes simplex virus type 1 (HSV-1) is a common virus, affecting over half of the adult human population, although it presents variable levels of disease burden and frequency of symptomatic recurrence. Antiviral treatments for HSV-1 infections are available, but thus far attempts at vaccine development have been foiled by insufficient immunity and/or viral escape. As a virus with a double-stranded DNA genome, HSV-1 is generally considered to be genetically stable and to have limited evolutionary potential. As these two statements are in conflict, we examined the ability of HSV-1 to evolve in a standardized cell culture setting. We utilized two HSV-1 isolates in this experiment, one with multiple viral genotypes present, which is similar to the viral populations seen in clinical settings, and one with a highly clonal viral population, which is similar to those often used in laboratory settings. After multiple rounds of replication, we analyzed the sequences of each passaged population. We found that the mixed viral population changed substantially over passage, and we were able to track specific genetic variants to phenotypic traits. By comparison, evolution in the clonal virus population was more limited. These data indicate that HSV-1 is capable of evolving rapidly, and that this evolution is facilitated by diversity in the viral population.

microbiology↗