The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although ...genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability)
, studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans
, but have little effect on the brains of mice
; this probably reflects evolutionarily divergent functions of ASPM
. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity
than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients
, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates
. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.
Gyrencephalic species develop folds in the cerebral cortex in a stereotypic manner, but the genetic mechanisms underlying this patterning process are unknown. We present a large‐scale transcriptomic ...analysis of individual germinal layers in the developing cortex of the gyrencephalic ferret, comparing between regions prospective of fold and fissure. We find unique transcriptional signatures in each germinal compartment, where thousands of genes are differentially expressed between regions, including ~80% of genes mutated in human cortical malformations. These regional differences emerge from the existence of discrete domains of gene expression, which occur at multiple locations across the developing cortex of ferret and human, but not the lissencephalic mouse. Complex expression patterns emerge late during development and map the eventual location of folds or fissures. Protomaps of gene expression within germinal layers may contribute to define cortical folds or functional areas, but our findings demonstrate that they distinguish the development of gyrencephalic cortices.
Synopsis
Complex patterns of gene expression emerge in germinal layers during early cortical development of gyrencephalic animals. These modular expression patterns map the eventual location of folds and fissures.
Microarray analysis of developing ferret cerebral cortex reveals transcriptomic differences between prospective folds and fissures.
Differential gene expression delineates mosaic patterns along proliferative zones prior to the emergence of folds.
Some mosaics of gene expression correlate with the prospective location of folds versus fissures.
Differentially expressed genes in our microarray analysis include 80% of those mutated in human cortical malformations.
Complex patterns of gene expression emerge in germinal layers during early cortical development of gyrencephalic animals. These modular expression patterns map the eventual location of folds and fissures.
Since the initial report in 1911, the domestic ferret has become an invaluable biomedical research model. While widely recognized for its utility in influenza virus research, ferrets are used for a ...variety of infectious and noninfectious disease models due to the anatomical, metabolic, and physiological features they share with humans and their susceptibility to many human pathogens. However, there are limitations to the model that must be overcome for maximal utility for the scientific community. Here, we describe important recent advances that will accelerate biomedical research with this animal model.
Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation ...and immunization
. Here we show that computationally designed, two-component nanoparticle immunogens
induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines
.
Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process ...constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We found severe acute respiratory syndrome coronavirus 2 RNA in 6 (8.4%) of 71 ferrets in central Spain and isolated and sequenced virus from 1 oral and 1 rectal swab specimen. Natural infection ...occurs in kept ferrets when virus circulation among humans is high. However, small ferret collections probably cannot maintain virus circulation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ...ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret
, -
, and -
are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections.
IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.
Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus ...(IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid--α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.
Folds in the cerebral cortex in mammals are believed to be key structures for accommodating increased cortical neurons in the cranial cavity. However, the mechanisms underlying cortical folding ...remain largely unknown, mainly because genetic manipulations for the gyrencephalic brain have been unavailable. By combining in utero electroporation and the CRISPR/Cas9 system, we succeeded in efficient gene knockout of Cdk5, which is mutated in some patients with classical lissencephaly, in the gyrencephalic brains of ferrets. We show that Cdk5 knockout in the ferret cerebral cortex markedly impaired cortical folding. Furthermore, the results obtained from the introduction of dominant-negative Cdk5 into specific cortical layers suggest that Cdk5 function in upper-layer neurons is more important for cortical folding than that in lower-layer neurons. Cdk5 inhibition induced severe migration defects in cortical neurons. Taken together, our findings suggest that the appropriate positioning of upper-layer neurons is critical for cortical folding.
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•Efficient gene knockout in the ferret cerebral cortex was achieved using CRISPR/Cas9•Cdk5 knockout in the ferret cerebral cortex markedly impairs cortical folding•Appropriate positioning of upper-layer neurons is critical for cortical folding
Shinmyo et al. describe a highly efficient gene knockout method for the folded cerebral cortex of ferrets using the CRISPR/Cas9 system. Loss-of-function studies of the Cdk5 gene suggest that appropriate positioning of upper-layer neurons is crucial for cortical folding.
Mutations in the cystic fibrosis transmembrane conductance regulator (
) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs, including the lung. We developed a CF ferret ...model of a scarless G551→D substitution in
(
), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated nonfluorescent Y66S-EGFP (enhanced green fluorescent protein) mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. Recombinant adeno-associated virus (rAAV) vectors were used to deliver two DNA templates and sgRNAs for dual-gene editing at the
and
genes, followed by fluorescence-activated cell sorting of
-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and
-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl
transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the
locus by HDR and nonhomologous end joining (NHEJ) were assessed by targeted gene next-generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the
-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR (clustered regularly interspaced short palindromic repeats) cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously coedit at two genomic loci using rAAV may have utility as a model system for optimizing gene-editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.
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