PHF6, while inherited variants identified in males might be hypomorph or have weaker effects on protein stability. This might contribute to the different phenotypes in male versus female individuals with BFLS. Furthermore, we used CRISPR/Cas9 to induce knockout of PHF6 in SK-N-BE (2) cells which were then differentiated to neuron-like cells in order to model nervous system related consequences of PHF6 loss. Transcriptome analysis revealed a broad deregulation of genes involved in chromatin and transcriptional regulation as well as in axon and neuron development. Subsequently, we could demonstrate that PHF6 is indeed required for proper neuron proliferation, neurite outgrowth and migration. Impairment of these processes might therefore contribute to the neurodevelopmental and cognitive dysfunction in BFLS.Pathogenic variants in PHD finger protein 6 (PHF6) cause Borjeson–Forssman–Lehmann syndrome (BFLS), a rare X-linked neurodevelopmental disorder, which manifests variably in both males and females. To investigate the mechanisms behind overlapping but distinct clinical aspects between genders, we assessed the consequences of individual variants with structural modelling and molecular techniques. We found evidence that de novo variants occurring in females are more severe and result in loss of  PHF6 [MIM: 300414]) have been identified to cause Borjeson–Forssman–Lehmann syndrome (BFLS [MIM: #301900]), an X-linked syndromic neurodevelopmental disorder (NDD)2, affecting both male and female individuals. In males, it is characterized by mild to severe intellectual disability (ID), epilepsy, a distinct facial gestalt, obesity and hypogonadism4. In these families, female carriers are usually unaffected or present with mild symptoms, only3. Recently, de novo variants in PHF6 were identified in affected females with NDDs, partly overlapping with BFLS in males but additionally with very distinct phenotypic aspects7. They presented with mild to severe ID, a characteristic facial gestalt, finger and toe anomalies, irregularly shaped teeth and oligodontia5.Variants in the gene encoding PHD finger protein 6  and in limb buds8. The expression pattern is therefore in line with the neurodevelopmental phenotype, facial dysmorphism and finger/toe anomalies in affected human individuals8.PHF6 encodes a protein with two extended atypical PHD-like zinc finger domains (ePHD), two nuclear and one nucleolar localization sequences2. Due to the DNA-binding domains9 and its co-localization with transcriptionally active euchromatin in the nucleus8, a role as transcriptional regulator and/or chromatin remodeler has been suggested (reviewed in10). Furthermore, PHF6 functions as a histone reader recognizing H2BK12 acetylation via the extended part of its C-terminal PHD, and additionally as a histone writer/E3 ubiquitin ligase by triggering H2BK120 ubiquitination through the extended part of N-terminal ePHD during trophectoderm development11.Several protein-interaction partners of PHF6 have been identified, most of them indeed involved in transcriptional and chromatin regulation. Interaction of PHF6 with the PAF1 transcription elongation complex13 might play a role in driving neuronal migration12, and by interaction with upstream binding factor UBTF14 (MIM: 600673), it might be involved in ribosomal DNA transcription15. In addition, PHF6 was demonstrated to interact with histone deacetylase 1 (HDAC1 [MIM: 601241])16 and the ATP-dependent chromatin remodelling NuRD complex16. More recently it was shown that PHF6 is required to maintain a precise chromatin landscape in leukemia cells17. It is also regulating transcriptional networks by its chromatin-binding capacities, dependent on the nutritional state18.19.Furthermore, a role of PHF6 deficiency in impaired cell proliferation, cell cycle arrest and increased DNA damage at the rDNA locus has been controversially discussedPHF6 revealed a broad deregulation of genes enriched for chromatin organization, transcription, neuron generation and nervous system development. Analysis of these PHF6 knockout cells showed altered neurite outgrowth, proliferation and migration. Our observations therefore implicate these processes in the pathomechanism of BFLS.We could now show that both truncating and missense variants in females result in loss of PHF6 while structural modelling indicated that variants identified in males might be less severe. Furthermore, RNA sequencing on human neuron-like cells upon CRISPR/Cas9 mediated knockout of PHF6 have been reported as pathogenic29, though missense variants seem to be significantly more frequent in males  and truncating variants (7 independent families versus 11 cases/families) more frequent in females .In male and female individuals with BFLS both truncating and missense variants in PHF6 identified in males24 and one missense variant identified in a female with BFLS4. Apart from two variants located close to the N-terminal ePHD, all missense variants were residing in either the N-terminal or the C-terminal ePHD ; c.940A>G, p.)23] and the only missense variant reported in females )4 in the C-terminal ePHD. Additionally, we analyzed one C-terminal truncating variant reported in males )2 and two truncating variants reported in females in the C-terminal ePHD ; c.955C>T, p.(Arg319*))4  23] G, p.Ile34Val)23] G, p.Ile34Val)23] CL) Fig. . In contG, p.Ile34Val)23] G, p.Ile34Val)23] G, p.Ile34Val)23] CL) Fig. .PHF6 carrying variants identified in females led to altered subcellular localization of PHF6 with formation of aggregates and markedly decreased protein levels, while PHF6 containing variants identified in males behaved more similar to wildtype PHF6. Though tested in a rather “artificial” situation by overexpression, variants identified in females or males behaved differently with more severe consequences for “female” variants.Taken together, overexpression of PHF6 4. Fibroblasts of male individuals with BFLS were not available for this study. As the X-inactivation in fibroblasts had been random in the investigated females (in contrast to skewed X-inactivation in blood)4, we were able to assess both the mutant and wildtype PHF6 allele. Utilizing immunofluorescence, western blot and Sanger sequencing, we found that only wildtype gene and protein were expressed in fibroblasts of the three individuals  mediated decay32. Indeed, variant c.914G>T was predicted to create two novel miRNA binding sites at this locus , indicating a common loss-of-function mechanism in females with BFLS.In accordance with mRNA expression levels, western blot analysis on fibroblasts showed a reduction of protein levels to 50–70% in all three affected individuals, respectively. Western blots were normalized to tubulin levels Fig. . In contPHF6 a more suitable model for the female form of BFLS in a neuronal context than overexpression of the mutant protein. Utilizing CRISPR/Cas9 we generated PHF6 KO in neuroblastoma SK-N-BE (2) cells. KO was confirmed by sequencing and western blot analysis in three different cell lines  of ing Fig. .Figure 3PHF6 KO with genes deregulated after dosage alteration of a known chromatin remodeler or transcription factor. We retrieved published datasets39 containing gene expression changes caused by KO or knockdown (KD) of several genes with a clinical or possibly functional overlap to PHF6. KDM5C (MIM: 314690)  is a demethylase which acts as a transcriptional repressor through the REST complex40. Variants in KDM5C are implicated in an X-linked NDD with mild to severe ID, epilepsy, short statue, mild facial dysmorphism including large ears, and hypogonadism (MRXSJ [MIM #300534]), partly mirroring the phenotype observed in males with BFLS44. Additionally, KDM5C also contains two PHD zinc finger domains42. The BAF complex  is an ATP dependent chromatin remodeling complex45. Variants in several subunits of the BAF complex are causative for Coffin–Siris syndrome , a NDD with multiple anomalies47, phenotypically overlapping with BFLS due to de novo variants in PHF6 in young female individuals5. BMI1 (MIM: 164831)  represents a major component of the polycomb group complex 1 (PcG 1), which acts as an epigenetic repressor49. As our set of deregulated genes after PHF6 KO contained a large number of homeobox (Hox) genes  (TCF4 KD SH-SY5Y neuroblastoma cells)39, a transcription factor with a major role in nervous system development52 and implicated in severe NDD Pitt-Hopkins syndrome (MIM: #610954). It promotes differentiation of neurons, thus being a good fit for differentiation of SK-N-BE (2) cells into neuron-like cells53. In addition, we used a so far unpublished list of deregulated genes in individuals with pathogenic variants in TCF4 (patient blood cells) (Supplementary Table This prompted us to compare deregulated genes (either up- or downregulated) upon PHF6 KO were significantly overlapping with deregulated gene sets upon KO of either a Baf complex subunit or Bmi1  . Overlapping deregulated genes between TCF4 KD and PHF6 KO were enriched for genes involved in regulation of nervous system development, neurogenesis and cell migration . . Fig. . This woPHF6 KO were enriched for GO terms such as neuron development, synaptic signaling, axon development and generation of neurons  enriched for known NDD associated genes. Of 1,336 confirmed ID genes , 1,250 were expressed in differentiated SK-N-BE (2) cells, and 92 were contained in the deregulated gene set. This is significantly higher than the expected number of 72 genes by chance   and MAP1B (MIM: 157129), both important for neurite cytoskeleton55.Furthermore, also downregulated genes after ons Fig. e, sugges05) Fig. . Overlap56 or agouti-related peptide (AgRP) neurons18 upon PHF6 KO. We found that 18 genes were overlappingly deregulated in murine cerebral cortex and our neuron-like cells, and that 38 genes were overlappingly deregulated in murine AgRP neurons and our neuron-like cells. The overlap in both cases was significantly higher than by chance   cells for neurite outgrowth by measuring the total length of their protrusions and length and number of primary and secondary neurites. Cells with PHF6 KO had significantly  increased total neurite length compared to control cells  cells under normal culture conditions  , indicating that only a minor fraction of the cells were fully differentiated  Fig. c. Firstl10) Fig. a, S9. NePHF6 KO cells migrated significantly slower and filled a significantly smaller area of the wound after 24 h than control cells   cells under normal culture conditions using a wound healing assay. These observations support a possible role of PHF6 in proper neurite outgrowth and neuron development, which might be altered in BFLS.PHF6 belongs to the growing list of X-chromosomal genes, in which variants not only cause X-chromosomal recessive NDDs in males but also severe NDDs in females when occurring de novo62. Factors contributing to different manifestation in males versus females might include X-inactivation, functional mosaicism due to X-inactivation, localisation and nature of variants, different functional consequences of sex specific variants, and dosage related expression effects62. Although the mutational spectrum for PHF6 includes truncating and missense variants for both genders, there are no overlapping variants reported yet occurring in both X-chromosomal recessive BFLS families and de novo in females. Additionally, variants identified in females seem to represent the severe end of the mutational spectrum. There are more truncating variants than in males, and the only missense variant is located within the C-terminal ePHD, while variant localisation in males is more variable4. It might therefore be speculated that variants found in females might have a more severe effect than variants found in males and might be lethal when occurring hemizygously. This is supported by the fact that complete knockout of PHF6 is lethal in mice63. Such a severe effect would be in line with our observation of complete loss of mutant PHF6 on both mRNA and/or protein level in fibroblasts of three females, independent from the variant . In a previous study on variants identified in male individuals with BFLS, a hypomorphic effect was discussed56. This is supported by several findings. A mouse model with CRISPR/Cas9 mediated knock-in of the specific c.296G>T (p.(Cys99Phe)) variant showed unaltered mRNA expression and only reduced protein levels of mutant PHF656. Furthermore, while the only missense variant identified in females is residing in the C-terminal ePHD, missense variants identified in males are located in either the N-terminal or the C-terminal ePHD. The N-terminal ePHD mediates the interaction of PHF6 with UBTF14, and the C-terminal ePHD interacts with double stranded DNA (dsDNA) through its positively charged region9. NMR spectra analysis of the four missense variants of male origin in the C-terminal ePHD did not reveal a structural impairment, while dsDNA binding abilities were still almost completely abolished9. Whereas structural modelling in our study indicated a severe effect on protein structure for variants in the N-terminal ePHD, it is predicted to be less severe for male variants in the C-terminal ePHD. In contrast, the female missense variant in the C-terminal ePHD appears to have a severe effect on protein structure. This suggests that in males a more severe structural effect on the N-terminal ePHD is required to result in a comparable impairment of PHF6 function as from variants in the C-terminal ePHD. As a logic implication, a severe alteration in the important C-terminal ePHD might be lethal in males and result in a severe NDD in heterozygous females. The association of inherited variants in X-linked recessive BFLS families with no or only mild phenotypes in carrier females might therefore be due to a less severe effect on protein function. However, we cannot exclude that variants in PHF6 occurring in male individuals also result in complete loss of protein, especially those with high destabilizing effects on the N-terminal ePHD.PHF6 is a suitable model for BFLS, it is interesting to see that there are also differences in the subcellular localisation and protein levels depending on the variant occurrence in males or females.Though it is debatable—in light of the observations from fibroblasts—if overexpression of mutant PHF6 variants in females might contribute to their severe and distinct phenotype. This suggests a possible genotype–phenotype correlation regarding gender-based localisation and severity of PHF6 variants.Therefore, in addition to X-inactivation related functional mosaicism, also more severe functional consequences of pathogenic 9, its nuclear localisation8 and co-localisation with euchromatin8, and its interaction with several chromatin remodelling or transcription regulating proteins and complexes64. Two recent studies used ChIP (chromatin immunoprecipitation) sequencing to show that PHF6 binds chromatin similar to typical chromatin remodelers18. Furthermore, transcriptional deregulation as a consequence of Phf6 knockdown or knockout was demonstrated in the nervous system of two independent mouse models56 and one rat model12. Using a human, neuron-like cell line in our study, we also found a broad deregulation of genes upon KO of PHF6. Deregulated genes significantly overlapped with deregulated genes after dosage alterations of known chromatin remodelers or transcription factors such as KDM5C, the BAF complex, BMI1 and TCF4, suggesting that PHF6 might act in a similar way. Interestingly, all overlapping genes were enriched for genes involved in either neuron generation and differentiation, or even more specific in axon development and guidance and synaptic signalling, proposing a specific role for PHF6 in these processes. In detail, the highest overlap was found with deregulated genes after either Kdm5c KO in the frontal cortex or Bmi1 KO. Pathogenic variants in KDM5C are associated with a NDD, partly mirroring the phenotype observed in males with BFLS44. Therefore, commonly deregulated genes, most prominently involved in synaptic signalling, might contribute to these similarities. BMI1 is a major component of the polycomb group complex 1 (PRC1), which is an essential epigenetic repressor of multiple regulatory genes involved in embryonic development65. This is also in line with a recent study identifying PHF6 as a transcriptional repressor of activity-dependent immediate-early genes18 and other studies discussing repressing effects17. However, repressing function might only apply for specific pathways, as deregulated genes both in our as well as other transcriptome studies56 indicated both upregulating and downregulating capacities. Furthermore, Bmi1 is required for the repression of Hox genes, preserving the undifferentiated state of stem cells and preventing inappropriate differentiation50. We also observed an upregulation of Hox genes after PHF6 KO, suggesting a potential similar mechanism. Interestingly, deregulated genes upon PHF6 KO also significantly overlapped with deregulated genes after KO of BAF (SWI/SNF) complex subunits in mouse . Variants in multiple subunits of the BAF are causative for Coffin–Siris syndrome, a NDD with multiple anomalies47. As phenotypic overlap between BFLS in females and CSS has been acknowledged before67, commonly deregulated genes, especially enriched for genes involved in neuron generation, development and differentiation, might contribute to the phenotypic similarities between these disorders.There are several lines of evidence pointing to PHF6 acting as a chromatin/transcription regulator, among them its domain structureKiaa2022 (KIAA2022 [MIM: 300524]), which is implicated in an X-chromosomal NDD in both males and females (MRX98 [MIM: #300912]), in mice resulted in longer or shortened apical neurites dependent on the developmental timepoint68. Also shRNA mediated knockdown of Ankrd11 (ANKRD11 [MIM: 611192]), of which haploinsufficiency in humans is associated with neurodevelopmental KBG syndrome (MIM: #148050), resulted in an increased number of shortened neurites in a mouse model69. Apart from neurite development also aberrant migration and differentiation of neurons and precursor cells can be observed in NDDs73. During radial migration, pyramidal neurons undergo a transition from multipolar to bipolar morphology in the intermediate zone (reviewed in 74). Impairment of this transition process in cortical pyramidal neurons and subsequently radial migration defects were described for the Ankrd11 mouse model69. In accordance, we observed significantly slower migration of SK-N-BE (2) cells after PHF6 KO compared to controls. In combination with the observation of an increased number of neurites this might be in line with a disturbed transition from multi- to bipolar neurons. Impaired migration has been discussed in the context with BFLS before12. An RNAi based knockdown of Phf6 in the developing mouse cerebral cortex at E14 showed impaired migration of cortical neurons as well as an increased number of multipolar neurons and a concomitantly reduced number of bipolar neurons12. However, in another mouse model carrying a patient specific Phf6 variant (p.Cys99Phe), no migration defects were reported56. Of note, there is also a report on two adult female individuals with the identical de novo duplication of exons 4 and 5 in PHF6 and presenting with seizures, in whom MRI anomalies were described that would be compatible with malformations of cortical development based on abnormal neuronal migration75. Furthermore, PHF6 is directly targeted and suppressed by miR-128 in the developing cortex. Their interaction is critical for proper migration and dendritic outgrowth of upper layer neurons76.Deregulated genes in our study were mainly enriched for genes involved in broad processes such as transcriptional and chromatin regulation, but also for more specific processes such as axon and neuron development. Following-up this observation in neuron-like cells, we indeed observed a significantly increased total length of protrusions, an increased number of primary neurites and increased axon length. Altered neurite development has been demonstrated in several other NDD models before. For example, shRNA mediated knockdown of 73. Differentiating SK-N-BE (2) cells after PHF6 KO proliferated significantly slower than control cells, while SK-N-BE (2) cells under normal cell culture conditions did not exhibit changes Previous studies showed that stable knockdown of PHF6 in HeLa cells under normal cell culture conditions resulted in decreased cell proliferation starting on day five14, while CRISPR/Cas9 mediated Phf6 KO in B-ALL cells did not show any proliferation changes17, and Phf6 KO in hematopoietic stem cells resulted in better growth77. This might indicate that the effect of PHF6 loss on proliferation might be cell type specific. For differentiating SK-N-BE (2) cells, we investigated if underlying mechanisms might be apoptosis, cell death, accelerated differentiation or cell cycle changes. A role of PHF6 in cell cycle regulation has been controversially discussed19. We could not detect changes in cell cycle regulation or any of these other processes, leaving the reason for the proliferation defect unclear.Apart from altered neurite outgrowth and migration we also observed altered proliferation in our cell model, another anomaly often observed in NDDsTaken together, our observations suggest that PHF6 might act as a context-specific epigenetic activator/repressor and is required for correct migration, proliferation and development of neurons. Also dependent on the severity of variants , impairment of these processes might contribute to the neurodevelopmental and cognitive dysfunction in BFLS.9). This structure also served as a template to model the N-terminal ePHD, which exhibits 47% sequence identity to the C-terminal ePHD. Modelling was performed with HHpred78 and Modeller79. The structural effect of the variants was assessed using VIPUR30, which is designed to distinguish between neutral and deleterious protein variants by modelling their effect on the three-dimensional protein structure.The structural effects of variants located in the C-terminal ePHD were assessed based on the crystal structure of this domain  or legal guardians (affected adult participants). The work described here was conducted in accordance with relevant guidelines and regulations.4 with de novo variants in PHF6. HEK-293, SK-N-BE (2) cells and fibroblasts were cultured at 37 °C and 5% CO2 under sterile conditions. For differentiation of neuroblastoma SK-N-BE (2) cells into neuron-like cells, they were seeded into 6-well plates and differentiated for 10 days using 10 µM all-trans retinoic acid and 25 µM caffeic acid. Medium was changed every 2–3 days (protocol was adapted from80).Skin fibroblasts were obtained from three previously published female individualsPHF6 was amplified from cDNA derived from whole blood and cloned into a pCR 2.1-TOPO vector (Thermo Fisher Scientific). After site-directed mutagenesis using a modified version of the QuikChange Site-Directed Mutagenesis protocol (Stratagene), wildtype and mutant PHF6 cDNAs were transferred into an N-terminal HA-tagged CMV expression vector. C-terminal myc-tagged UBE3A in a pCMV3 expression vector was used as transfection control.Human For immunofluorescence experiments, cells were grown on poly-lysine coated coverslips and transiently transfected with the plasmids using jetPrime (Polyplus-transfection) following manufacturer’s instructions. Cells were fixated with 4% paraformaldehyde in PBS for 10 min at room temperature (RT) or with MetOH + EGTA for 10 min at − 20 °C 24 or 48 h post transfection. Cells were stained with rabbit polyclonal anti-PHF6 antibody , or rabbit anti-HA  and mouse monoclonal anti-Nucleolin antibody , and with Alexa Fluor 488 goat anti-mouse , and/or Alexa Fluor 546 donkey anti-rabbit , and DAPI  for nuclei counterstaining. Cells were analyzed with a Zeiss Axio Imager Z2 Apotome microscope with a 63 × objective. For analysis of patient fibroblasts, at least 63 cells per cell line were analyzed, and the number of PHF6 expressing cells was counted. For analysis of HA-tagged mutant and wildtype PHF6, at least 40 cells per construct were analyzed.RNA was extracted from cells using the RNeasy Mini Kit (QIAGEN) following manufacturer’s instructions. DNase digestion was performed on-column with the RNase-free DNase kit (QIAGEN). Reverse transcription of RNA into cDNA was performed using the SuperScript II reverse transcriptase (Thermo Fisher Scientific).Genomic DNA (gDNA) was extracted from cells using the DNeasy Blood & Tissue Kit (QIAGEN) following manufacturer’s instructions.PHF6 on RNA and gDNA level, Exon 9 was amplified  and sequenced.For analysis of patient variants in For protein analysis, cells were lysed in Lysis Puffer  and frozen for 1 h at − 80 °C.For western immunoblotting, the Mini PROTEAN TGX stain free electrophorese system  in combination with the Trans-Blot Turbo Transfer System  was used. After blocking, membrane was incubated with either rabbit polyclonal anti-PHF6 , mouse monoclonal anti-PHF6 , mouse anti-tubulin , mouse anti-myc  or rabbit anti-HA  antibody for 1.5 h at RT or overnight at 4 °C. Whole protein or alpha-tubulin were used as loading controls. C-terminal myc-tagged UBE3A was used as transfection control. Secondary, HRP-conjugated antibody  was applied for 1 h. Blots were stained with SuperSignal West Femto Maximum Sensitivity Substrate, scanned using the ChemiDoc Imaging System , and analyzed using the Image Lab software version 6.0.0 (Bio-Rad). Mutant HA-tagged PHF6 was normalized using transfection control UBE3A (myc-tagged).PHF6 specific crRNA sequences were created using the CRISPR Targets Track from UCSC Genome Browser on Human Feb. 2009 (GRCh37/hg19) Assembly. Two crRNAs were designed, targeting exon 2 or 9, respectively. The guide sequence, efficiency as well as potential off targets can be found in Supplementary Table SK-N-BE (2) cells were subjected to CRISPR/Cas9 mediated KO using the GeneArt CRISPR Nuclease Vector Kit (Life Technologies). 81; then reads were mapped against the human reference genome  using the STAR aligner v. 2.5.4a82, and a STAR genome directory created by supplying the Ensembl gtf annotation file (release 87). Read counts per gene were obtained using featureCounts program v. 1.6.183 and the Ensembl gtf annotation file.RNA sequencing was executed in-house at the NGS core unit of the Medical Faculty of the FAU Erlangen. The TruSeq Stranded mRNA LT Sample Prep Kit  was used for library preparation. Libraries were subjected to single-end sequencing (101 bp) on a HiSeq-2500 platform . The obtained reads were converted to .fastq format and demultiplexed using bcl2fastq v2.17.1.14. Quality filtering was performed using cutadapt v. 1.1584. In particular, differential expression analysis and principal component analysis (PCA) was performed with the DESeq2 package v.1.20.085. Heatmap was created using the pheatmap package v.1.0.12 applying the standard settings 86.Following analyses were performed using R version 3.5.039 and one in house so far unpublished list of deregulated genes after TCF4 dosage alterations from RNA sequencing and transcriptome analysis of RNA from peripheral blood, collected and extracted with the PaxGene system  from three individuals with variants in TCF4  and performed RNA sequencing36. The BAF-complex deregulated gene dataset was retrieved from a conditional dKO Baf155/Baf170 mouse model where both genes where knocked out in late cortical neurogenesis. RNA was extracted from the pallium of E17.5 embryos and RNA sequencing was performed37. The BMI1 deregulated gene dataset was retrieved from adult brain tissue from a Bmi1 KO mouse model38. The TCF4 deregulated gene dataset was retrieved from TCF4 KD SH-SY5Y neuroblastoma cells39. Firstly, deregulated genes in mice were converted to human gene IDs using BioMart87. A common background was generated by using all genes with a base mean > 2 (if applicable) that were present in both data sets. Next, deregulated genes present in shared background were computed. Lastly, overlap of these deregulated genes was computed. Lists can be found in Supplementary Table Enrichment analysis was performed using four already published data sets54, June 2020), the number of confirmed ID genes present in SK-N-BE (2) transcriptome data was analyzed and the overlap with deregulated genes was computed. Statistical testing was performed using hypergeometric testing. GO term analysis was performed on overlapping genes.For comparison with confirmed ID genes  cells were transfected with 1 µg of mCD8-GFP using jetPRIME (Polyplus-transfection) following manufacturer’s instructions on day eight of differentiation. 24 h post transfection medium was changed. Cells were subjected to immunofluorescence analysis at differentiation day 10 as described above using only DAPI as counterstaining.88 in Fiji89 and respective number was counted. Three PHF6 KO and three control cell lines were evaluated, for each line at least 30 cells were measured. The experiment was performed in biological triplicates. Data was tested for normality using the Kolmogorov–Smirnov test. Testing for statistical significance was performed using a two-tailed t test.Cells were analyzed with a Zeiss Axio Imager Z2 Apotome microscope. Primary and secondary neurite length was measured using the NeuronJ pluginPHF6 KO and three control cell lines were seeded in 96-well plates and either kept in normal medium for 4 days or differentiated for 10 days. Every day, four wells per cell line were treated with the XTT reagent following manufacturer’s instructions and incubated for 4 h. The color change was measured using a plate reader at wave length 660 nm and 450 nm. The specific absorbance of each well was determined using following formula: Specific Absorbance = [Abs450 nm(Test) −Abs450 nm(Blank)] −Abs660 nm(Test). The experiment was performed in biological duplicates and technical quadruplicates. Data was tested for normality using the Kolmogorov–Smirnov test. Testing for statistical significance was performed using a two-tailed t test.Proliferation of undifferentiated and differentiating cells was assessed using the CyQUANT XTT Cell Viability Assay (invitrogen). Three PHF6 KO and control cells was assessed using four well culture inserts (ibidi) following manufacturer’s instructions. SK-N-BE (2) cells were seeded into each well of the culture inserts, which were removed after 24 h. Resulting cell gaps were photographed every 4 h up to 24 h. The growth area was measured using the Wound healing tool90 on Fiji89. The experiment was performed in biological triplicates using four control cell lines and eight PHF6 KO cell lines. Data was tested for normality using the Kolmogorov–Smirnov test. Testing for statistical significance was performed using a two-tailed t test.Migration of Supplementary Figures.Supplementary Tables.


SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 ± 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN. With more than 5 million cases confirmed and a death toll exceeding several hundred thousand individuals, a search for antiviral therapies as well as vaccine candidates is of utmost urgency. Non-infectious virus like particles (VLPs) displaying essential viral proteins can be used to study the structural properties of the SARS-CoV-2 virions and due to their maximum immunogenicity are also vaccine candidates3. VLPs are released from cells with similar mechanisms as fully infectious virions and resemble the shape and composition of fully infectious virions4. Most Coronaviruses are pathogens of zoonotic nature with different viruses infecting avian (IBV), bovine (BCoV), porcine (TGEV), feline (FIPV) and murine(MHV) species4. There is evidence that Bat-SARS-CoV is the origin of the SARS-CoV virus which first appeared in human hosts in 20035. The genome of SARS- CoV-2 has ~ 90% similarity to genome of coronaviruses previously identified in bat populations in China6.COVID-19 is a pandemic disease caused by infection of SARS-CoV-2 virus7. Similar results are reported for IBV8. Similarly, expression of M, E and S proteins are shown to result in release of morphologically identical particles to wild type SARS-CoV virus10. It is known that TGEV VLPs can induce an IFNα response in their hosts7. More recently human MERS-CoV VLPs were also shown to elicit an immune response and serve as vaccine candidates11.VLPs with similar sizes to wild type TGEV are released by expression of E and M proteins of TGEV12, we set out to create SARS-CoV-2 VLPs by expressing S, M and E proteins of SARS-CoV-2 in mammalian cells. Since SARS-CoV-2 virions have been reported to survive on solid surfaces in dry conditions for many hours13, we tested the structural integrity of the SARS- CoV-2 VLPs attached to dry glass using Atomic Force Microscopy (AFM).Given the similarities between SARS-CoV-2 and SARS-CoV virusesPlasmid preparation and VLP harvest. SARS-CoV-2 M, S and E protein genes were identified from the full genome sequence of the virus1, these genes were then humanized and inserted in CMV driven mammalian expression vectors (see supplement for complete plasmid sequences). 24 h after transfecting a monolayer of 293 T cells with a cocktail of S, M and E plasmids, VLPs were harvested from the supernatant (see supplement for further details). The supernatant was filtered using 0.2um filter and VLPs were captured in a 40–10% sucrose step gradient and concentrated using AMICON spin filters . Total protein yield for purified VLP stock varied somewhat but was typically above 5 mg/mL. The purified VLPs were stable for at least a week when stored at ~ 4 °C.Immunogold electron microscopy. VLPs were incubated with 1:1000 dilution of anti-S antibody  and 1:5 dilution of goat anti-mouse IgG nanogold conjugates . Negative stain electron microscopy was performed on VLPs by applying 3.5 μL of VLPs to a glow-discharged formvar-carbon coated EM grid  followed by two de-ionized water washes and staining with 1% uranyl acetate. Imaging was performed in a JOEL JEM1400-Plus microscope with an accelerating voltage of 120 keV.Western Blot analysis. After triple washing the cells with PBS, 293 T cells were suspended in 100ul of RIPA buffer . 10 μl of VLPs or cell extracts were then denatured by Laemmli sample buffer  with 5% BME and boiled at 95C for 10 min. The proteins were separated by SDS-PAGE and then transferred to a PVDF membrane . Membranes were stained with 1:1000 dilution of Anti-SARS-CoV SΔ10 antibody  along with 1:1000 dilution of Anti-Membrane Protein (2019-nCoV) Polyclonal Antibody  and then immunoprobed with appropriate infrared secondary antibody. Both antibodies were confirmed to be specific to intended antigens  in PeakForce QNM in air mode at room temperature. Glass coverslips were functionalized with anti-S antibody as follows. Glass was first cleaned and functionalized with biotin-PEG-silane as previously described15. The surfaces were then incubated with neutravidin  followed by a brief dd-H20 wash to get rid of excess neutravidin and incubation with biotinylated anti-S antibody . Surfaces thus prepared were generally devoid of debris but sometimes had step-edge character suggesting that antibody coating was less than a full monolayer . Our measured VLPs are also consistent with limited electron microscopy data available for the fully infectious SARS-CoV-2 virions observed in patient samples14.We further characterized the purified VLPs via electron microscopy. Figure 16 although most prior work has been done in liquid. Our observations represent an upper estimate of the rupture force for SARS-CoV-2. This demonstrates that SARS-CoV-2 VLPs can be disrupted by direct application of moderate mechanical perturbations and open the door for future studies of VLP mechanics and integrity.The structural integrity of VLPs can inform their practical applications as well as serve as an estimate of the stability of fully infectious SARS-CoV-2 virions. We therefore further characterized the VLPs on functionalized surfaces with atomic force microscopy (AFM) Fig.  & S6. VL17.Expression of just M, S, and E proteins Fig.  was suffSupplementary Information 1.


We thus evaluated (1) synaptic dynamics by combining electrocorticography and multiunit activity recording in the time and time-frequency domain, (2) non-synaptic dynamics by recording modifications in light scattering induced by changes in the membrane configuration related to cell activity using the fast optical signal, and (3) vascular dynamics using functional near-infrared spectroscopy and, independently but simultaneously to the electrocorticography, the changes in cerebral blood flow using diffuse correlation spectroscopy. The first observed alterations in the measured signals occurred in the hemodynamic compartments a few seconds before the peak of the IIS. These hemodynamic changes were followed by changes in coherence and then synchronization between the deep and superficial neural networks in the 1 s preceding the IIS peaks. Finally, changes in light scattering before the epileptic spikes suggest a change in membrane configuration before the IIS. Our multimodal, multiscale approach highlights the complexity of (1) interactions between the various neuronal, vascular, and extracellular compartments, (2) neural interactions between various layers, (3) the synaptic mechanisms (coherence and synchronization), and (4) non-synaptic mechanisms that take place in the neuronal network around the time of the IISs in a very specific cerebral hemodynamic environment.Interictal spikes (IISs) may result from a disturbance of the intimate functional balance between various neuronal (synaptic and non-synaptic), vascular, and metabolic compartments. To better characterize the complex interactions within these compartments at different scales we developed a simultaneous multimodal-multiscale approach and measure their activity around the time of the IIS. We performed such measurements in an epileptic rat model ( A multimodal multiscale analysis  was performed on penicillin-induced interictal epileptic spikes in rats.Hemodynamic activities are modified well before (5 s) the interictal spike (IIS).Between the cortical layers, interactions are modified ~1 s before the IIS.The configuration of the cell membranes is modified ~0.5 s before the IIS.Our results highlight the complex interactions that occur before the IIS at different scales and in different compartments .Epilepsy is a common disorder of the central nervous system that affects ~0.6% of the global population –3, makinThe ictal phase is characterized by clinical or subclinical seizures, the diagnosis of which is electro-clinical, often combining EEG analysis and a clinical report of the semiology of the seizures.+], ephaptic conduction, cellular environment, etc.) mechanisms that propel the neural network to transient hypersynchronization , poly-spikes, and spike waves, associated or not with disorganization of the background brain activity. The occurrence of IIS within epileptic networks is generally related to changes in the excitability threshold by the interaction of synaptic (excitatory and/or inhibitory) and non-synaptic , vascular, and metabolic compartments.Synaptic compartment: Epileptic spikes are underpinned by complex neuronal inhibitory and excitatory interactions between deep and superficial cortical layers ((1) l layers . These il layers , 8. The l layers . The neul layers  at the pl layers . They arl layers , 12.Non-synaptic compartment: Non-synaptic disturbances are likely to contribute to IISs. Among various non-synaptic events (including communication across gap junctions) that facilitate neuronal activation during the IIS, neural activation causes cellular and glial swelling that can reduce the extracellular space ((2) ar space , 14 and ar space , 16 thatar space . This enar space . The appar space .Vascular compartment: The neurovascular coupling induced by IISs has been widely studied and has shown that the duration and frequency of IISs modulate hemodynamic responses that can be measured in animals by intrinsic optical signals or in humans by fMRI (2) and deoxyhemoglobin (HbR)] prior to epileptic spikes (5–6 s) in animals using fNIRS ((3)  by fMRI –22. The  by fMRI . Howeverng fNIRS , 21 and ng fNIRS  and fMRIng fNIRS , 26, sugHere, we developed a multimodal-multiscale approach to address several issues.(1) The mechanisms  that are likely to contribute to the emergence of epileptic spikes in a longer time frame than that of the early activation of inhibitory interneurons.(2) The dynamics of the various compartments around the IIS.(3) Whether such multimodal multiscale analysis can provide new information on the mechanisms that propel neurons to the hyperactivation and synchronization that results in an epileptic spike.(4) The type of information time-frequency analysis, combined with wavelet coherence analysis, of the interaction between the different layers before the IIS can provide.(5) Whether the complexity of the neuronal activation/inhibition described in human studies  can be o2 and HbR around the time of the IIS are associated with changes in CBF.(6) Whether the previously described hemodynamic changes in HbO(7) Whether individual neuronal activity modifies their rate of discharge simultaneously with the hemodynamic changes that occur prior to the epileptic spikes recorded at the surface.(8) The neuronal and hemodynamic environment in which changes in cellular configuration occur.(9) Whether such simultaneous changes in cell configuration have a neuronal or hemodynamic counterpart.We developed a multimodal approach to study the various compartments in epileptic rats to address these issues using techniques that offer good time resolution. This multimodal multi-scale approach allows a better understanding of the emergence of interictal epileptic spikes as signatures/biomarkers of epileptic disorders.(1) We evaluated the synaptic dynamics  by combining ECoG and multi-unit activity recordings of different cortical layers. We further developed a time-frequency analysis (TFR) of the local field potential (LFP), consisting of an analysis of the current source density (CSD), and wavelet coherence analysis of the neuronal interaction between different layers, together with an analysis of the rate of discharge of unitary recordings.(2) We evaluated the non-synaptic dynamics, specifically changes in the extracellular space, by recording modifications in light scattering induced by changes in the membrane configuration related to cell activity using the FOS technique.2 and HbR using fNIRS and, independently but simultaneously to ECoG, the changes in CBF using diffuse correlation spectroscopy (DCS) recordings.(3) We evaluated the vascular activity (hemodynamic changes around the IIS) by simultaneously recording the changes in HbOOur main objective was to characterize the concomitance of the neuronal, hemodynamic, and/or cellular configuration changes that occur around the IIS using a multimodal multi-scale approach. Our multimodal approach follows clinical practice by building a body of evidence from all clinical, structural, hemodynamic, and metabolic data using paraclinical examinations such as EEG, MEG, SEEG with MUA, MRI, fMRI, PET, and SPECT and sometimes by combining these analyses  with semiological information.ad libitum. The protocol was approved by the Ethics Committee of the French Ministry of Research (ref: APAFIS#1464-2015081710033478). Every effort was made to limit the number of rats and their suffering.Recordings were made on 43 male adult Sprague-Dawley  rats (260 and 550 g) using a multimodal-multiscale approach . The ratEach rat was weighed and anesthetized using urethane (1.25–1.5 g/kg) with a single intraperitoneal injection. The body temperature and respiratory and heart rates were monitored during the experimental procedure to follow the vital signs of the rats and the level of anesthesia. Tracheotomy was not required since respiratory rate was not altered during the recording period. Approximately 1 h after the injection, the surgical protocol was started. Part of the scalp was removed and the animal was placed in a stereotaxic frame to expose the skull. A 10 × 15-mm field of bone was exposed, the periosteum removed, and eight craniotomy holes (diameter of 1.5–2 mm) drilled at determined coordinates . A smallMLE electrodes were placed at half the distance between the emitters and detectors to probe the trough of the banana shape made by the photon pathway. The position of the MLE was the same as the penicillin G injection site (U1). First, to minimize errors related to probing different neuronal populations, the ECoG electrodes, which were always present, were located as close as possible to the MLE site as a control measurement. The distance between the MLE and ECoG holes was only a few millimeters. The position of EcoG electrodes are therefore not completely symmetric. For injection and MLE electrode placement, the EcoG has to be slightly moved laterally. Although, EcoG were only used as control for the LFP recorded on the most superficial MLE channel that was used as an “ECoG” like electrode for further analysis and for the zero signal alignment of the different multimodal measurements (peak of the IIS). Second, the injected penicillin G probably spread to adjacent areas. Third, it is well-known that an IIS recorded at the surface (EEG and to a lesser extent ECoG) are supported by a relatively wide area of synchronized neurons. It is therefore very likely that the region involved in the epileptic discharge recruited neurons located in a relatively wide area encompassing the structures probed by the various modalities. Because ECoG and the most superficial electrodes provide the same information (see the following figure), the most superficial channel of the MLE was used as the reference electrode instead of the ECoG electrode. This allows using the same sampling rate and facilitates the further analysis.Three electrocardiogram (ECG) electrodes were positioned (two in the skin of the front paws and one in the skin of the left-rear paw) to monitor the vital signs of the animals. A piezoelectric device was placed beneath the ventral side of the rat (close to diaphragm) to monitor respiration. A body temperature of 36–38°C was maintained by inserting a rectal thermometer connected to a proportional integral derivative (PID) to control a heating pad. At the end of the experiment, the rats were killed by urethane overdose following the guidelines for animal euthanasia of the animal facility.A receptors (®) and allowed to flow over the cortical surface. The doses of penicillin were determined based on values obtained from the literature , ECoG, functional fNIRS, FOS, and DCS. An external TTL trigger was used between the electrophysiological  and hemodynamic measurement systems  to synchronize the recording devices. All recordings were performed in a dark room and monitored in an adjacent room to not interfere with the acquired signal.2) spaced 150 μm apart. Introduction of the device was carefully performed to avoid any neuronal damage caused by excessive insertion speed and damage to the MLE by any bone sliver from the trepanation. The condition of the electrodes was verified before each experiment and they were cleaned as required with an enzymatic detergent. The reference electrode was inserted through the right part of the occipital bone, through a stainless-steel screw. The MLE probe was mounted on a probe holder attached to a manual micro-manipulator  grounded to a Faraday-cage. After MLE probe insertion, the ECoG monopolar gold-plated electrodes were inserted into the appropriate holes almost perpendicular to the cortical surface, with a reference needle electrode inserted in the neck muscle. The MLE and ECoG signals were then amplified by a multi-channel system setup and acquired by a CED 1401 (Cambridge Electronic Design Ltd.) acquisition-board interface. The MLE and ECoG signal sampling rates were set to 20 kHz and 1,024 Hz per channel, respectively. The raw signal of the deeper MLE channel was filtered online  to monitor the multi-unit activity during each recording.The 16-site MLE  was inserted 2.5–3 mm at an angle of 90° to the cortical surface to record the entire columnar cortex (6 layers) . Each ML2] and [HbR]) were performed by fNIRS , whereas changes in light scattering (changes in membrane properties) were performed by FOS . The near-infrared (NIR) intensity fluctuations were measured by DCS .The optical measurement for monitoring changes in light absorption  and light scattering (FOS). The cerebral hemodynamic activities were measured by fNIRS by placing one pair of optical fiber light emitters (each pair with 690 and 830 nm wavelengths) caudally from Bregma AP −2.5 mm to ML 2.5 mm over both hemispheres. Their respective optical fiber light detectors were placed rostrally from Bregma AP +2.5 mm to ML 2.5 mm over both hemispheres. The optodes were inserted into the holes and secured perpendicularly to the cortical surface. A distance of 5 mm was set between each emitter and detector pair to probe a banana-shaped photon trajectory corresponding to the desired deepness of 1.7 mm [one-third of the emitter-detector distance ].A DCS device was used to invasively quantify changes in CBF. The DCS measures blood flow by optical modality using intensity fluctuations of NIR light . The ligSpike2, Cambridge Electronic Design, and CED). IISs were detected semi-automatically and clustered using a template-matching technique (Spike2). All IISs with <80% of the points inside the template were rejected.We wished to evaluate the changes in electrical, hemodynamic, and cellular configuration around the IIS. Thus, the first step was to define T0. The IIS peaks were identified using the signal of the MLE channel closest to the surface of the cortex. As commonly observed in penicillin rat models , 37, the/x)·100%, where σ is the standard deviation of the IIS firing rate and x the mean of the IIS firing rate. The impact of the time interval between two spikes (ISI) on the proportion of the neuronal population involved in the generation of the IIS (amplitude) was evaluated by contrasting the ISI against the amplitude of the IIS for each rat. Then, the individual amplitude was normalized to develop a heat map to highlight the spot density of the relationship between the normalized amplitude and the ISI values to group all the rats in a single analysis. Kolmogorov–Smirnov tests were used on the histograms to clarify the tendency of the normalized peak voltage and the ISI from 0 to 5 s.We selected the population of IISs that fulfilled the objective of the analysis of the 500-ms pre-spike period (baseline between −800 to −600 ms) by characterizing the firing rate, inter-spike interval (ISI), and spike amplitude. First, the dispersion of the firing rate of the IIS was evaluated by calculating the coefficients of rhythmicity of the firing rate for each rat. CV =  were considered for each IIS set to avoid, or at least minimize, the effect of the previous IIS.CSDplotter MATLAB toolbox  , 39 anal toolbox  on the L toolbox .Power changes of the neuronal activity related to the IISs were quantified by averaging the root mean square (RMS) of the LFPs ± 1.5 s around T0 for each rat independently. Finally, all data were averaged and the difference between the superficial and deep channels evaluated.t-test was applied to the LFP (baseline −800 to −600 ms before the IIS peak) to evaluate the point at which the changes in LFP signal became significant.A Bonferroni corrected Spike2) on the filtered data  An optimal threshold (>3 standard deviations) was applied to isolate the events correctly from the background noise. Moreover, the refractory period (2 ms) was respected and principal component analysis (PCA) with the k-means algorithm applied.Single unit activity was carefully sorted using an amplitude threshold (t-test was performed with a baseline selected between −1.5 and −1 s (5–9 Hz).A wavelet coherency was applied to each possible pair of MLE recording probe sites to investigate the spectral perturbations and correlation in the time-frequency domain. The coherence values were highlighted and their phase amplitude plotted with arrows. An average analysis, based on the extraction of 200 windows (T0 ±1.5 s) for each rat, was then performed and averaged for each rat. Then, z-score normalization was applied and the grand-average for all the results calculated. To extract significant changes in the cohenrence between deep and supeficial channels, a time-frequency window was selected between  with lower coherence values and a ®. The time-frequency representation was calculated for each IIS epoch for all 16 contacts of the MLE. Frequencies were sampled (Gaussian filter) in 2-Hz steps and windows were sampled in 25-ms steps, corresponding to a time-frequency resolution of ±2.83 Hz and ±3 9.4 ms for each time-frequency bin . In addition, TFRs were expressed as the relative power change from baseline activity for a time-frequency bin compared to the mean power over the baseline epoch for that frequency, TFR = {P−Pbaseline(f)}/Pbaseline(f) × 100 where P = power at time t and frequency f and Pbaseline(f) = mean activity at frequency f over the baseline epoch. This procedure yielded TFRs containing phase-locked as well as non-phase-locked responses.Time-frequency analysis was performed for frequencies between 4 and 50 Hz to characterize phase-locked and non-phase-locked changes in neuronal activity that occurred around the IISs during each selected epoch. The same non-overlapping IIS epochs lasting 2 s were considered for each IIS. The TFRs of ECoG activity were compared to the baseline segment, lasting 400 ms (−800 to −600 ms before T0 of each IIS). Time-frequency analysis was performed on the window between −800 and + 800 ms around T0. TFR was performed according to the procedures described by Hoechstetter et al.  and impl2 and HbR were calculated using the modified Beer-Lambert law (https://homer-fnirs.org/). Hemoglobin (HbT) concentrations were calculated by summing the HbO2 and HbR concentrations.The concentrations of HbObert law , based o2 and HbR concentrations were band-pass filtered  to reject very slow drift of the baseline and cardiac artifacts. Hemodynamic changes around IIS activity in the ipsilateral and contralateral hemispheres were studied by deconvolution within a 40-s window of hemoglobin concentration data (±20 s around T0). The changes in HbO2 and HbR occurring around the IIS were compared to the baseline segment (lasting −20 to −10 s before T0).The HbOp < 0.05) by applying t-tests.Compared to other physiological signals, FOS is so small that it cannot be observed directly. These signals need to be separated from the noise, for which the signal may be several orders of magnitude larger. Cardiac artifacts are the most common and powerful physiological artifacts that affect optical signals. Respiratory artifacts were eliminated without shifting the signal delay using sixth-order low-pass and third-order high-pass zero-phase Butterworth filters to filter the signal between 2 and 20 Hz. Baseline correction from −800 to −600 ms was then performed. Cardiac artifacts were eliminated by applying an independent component analysis (ICA) algorithm plus one classification method to all filtered signals, almost eliminating such noise. The epochs (−800/+800 ms) around the peak of the T0 spikes were selected and then averaged. Finally, time bins were designated with a significant difference of responses from baseline .First, all recorded IISs on ECoG data were detected automatically. The negative peak (T0) of each IIS was detected as the zero time-point for the analysis of the regional CBF responses (rCBF) associated with the IISs. ISIs were defined as the time-course of IIS sequences. The hemodynamic response function (HRF) is a model that indirectly reflects neuronal activity in response to an event. In general, a measured hemodynamic signal can be modeled as the linear convolution between the explicit timing of events and a specific shape of the HRF, with additive noise  and calculated a grand-average based on each rat coherency  . The wav −370 ms . After TThere was a nested increase in coherencies at 12–20 Hz between the deep and superficial channels, with a maximum at around 100 ms. The decrease in coherency in the post-IIS period was more pronounced than that in the pre-IIS period.We recorded various types of firing activity for the motor cortex. We generally observed the best unit-activity signal for electrodes sampling layer VI. Each of the 19 rats selected for unit-activity firing analysis developed similar IISs on the ECoG . The IIS was characterized by a biphasic wave, with a first peak of ~70 ms in duration, followed by a slow wave that peaked at ~200 ms, lasting 400–500 ms . Based oWe classified different types of units based on this segmentation . The mosIn summary, focusing on the temporal dynamics of unit activity that were modulated during the pre-spike periods, unit-activity increased during the spike (Periods 3 and 4), regardless of the pattern of discharge during Periods 1 and 2. The discharge of unit activity during the post-spike periods was more complex and consisted of either an increase, decrease, or no change, with no specific relationship with the discharge patterns during the pre-spike periods.We analyzed the hemodynamic activity of 18 rats , using a total of 17,995 IIS events . The pre2 (increase) and HbR (decrease) were recorded between −10 and −2 s  signal. Then, HbO2 and CBF simultaneously significantly increased, with a peak occurring at ~3 s after T0, whereas HbR significantly decreased, with a trough at 7 s after T0. This corresponds to a positive BOLD signal. Then, HbO2 and CBF simultaneously significantly decreased until 8 s after T0, whereas HbR once again increased, with a peak at 10 s. This corresponds to a second negative BOLD signal. Considering the baseline to be between 0 and −2 s  (p < 0.05) from baseline (−800 to −600 ms) in six rats. Subsequently, the detected light intensity reached its peak and then decreased until reaching the zero line at −100 ms. From −100 to T0, the intensity of the detected light continued to significantly drop. Then, a sharp increase in detected light intensity occurred from T0, crossing the zero line at 70 ms and reaching its maximum at 160 ms. Finally, the signal progressively decreased up to 360 ms.FOS results were broken down into several periods according to the fluctuations of the intensity of detected scattered photons that occurred around the IISs  . The perIn the present study, we focused on simultaneous changes in neural and vascular activity and the membrane configuration of neural cells around the IIS, especially those preceding the IIS by a few hundred milliseconds. These changes were preceded and followed by complex alterations within the spike onset zone (injection zone) which corresponds to the region where epileptic spikes start and from where they propagate . The mec2, HbR, and CBF) a few seconds before the peak of the IIS. These hemodynamic changes were followed by changes in coherence and then synchronization between the deep and superficial neural networks in the 1 s preceding the IIS peaks. Finally, changes in light scattering that occurred before the epileptic spikes support a change in membrane configuration before the IIS.The first alteration in the measured signals that we observed was in the hemodynamic compartments  by using the deconvolution method, which can efficiently estimate the hemodynamic response of a randomly-spaced event , 48. Alta priori assumption about the direction of neurovascular coupling. A baseline that covered all the data  but rather enhanced the measured changes that occurred before the IISs and the dynamics of the various slopes.We chose a baseline distant from the IIS (−20 to −10 s) rather than one next to it (−2 to −0 s) to evaluate changes in the dynamics of the various domains that occur before the IIS. A baseline close to the IIS makes an the data  would haa priori assumption was made that the IIS itself would induce the hemodynamic response. Linear and non-linear modulations of the CBF dynamics were thus demonstrated from the IIS using laser Doppler flow , in agreement with previous studies performed by fNIRS on rats , 21 and ler flow .a priori assumptions about the direction of the relationship between the neuronal and vascular system and using a baseline distant from the IIS, the very initial changes corresponded to low positive neurovascular coupling with (1) an increase in HbO2, (2) a decrease in HbR, and (3) a slight increase in CBF, which was not significant but for which the variability increased. This is consistent with the positive BOLD signal described in a fMRI study  spike discharge of certain neurons evaluated by LFP and MUA, (2) synchronization and coherencies between different cortical layers, and (3) neural membrane properties evaluated by FOS (see below).From 5 s before the IIS, CBF decreased concomitantly with a decrease in HbOeriments . When theriments . We obseeriments , 61, 64.2 and CBF (approximately −500 ms before the IIS peak), CBF increased concomitantly with the increase in HbO2 and decrease in HbR. This corresponds to the classical positive BOLD signal in response to an IIS (2 and CBF) become linearly and non-linearly modulated by neuronal activity , as in the present study  are modulated for 15 s. Such long-lasting modulation has not been described by laser Doppler flow, in which the data were smoothed with a 1.5-Hz Fourier filter  argue for coupling with neuronal activity. Glial cells should also be considered; their membrane potential is known to be mainly affected by the extracellular potassium ion concentration. In penicillin-induced epileptic rats, they show strong transient depolarization concomitant with spikes in the EEG. These depolarizations reach their maximum value within 100 ms and repolarize over a period lasting between 1.5 and 7.0 s  when using a method with poor temporal resolution (on the order of 1 s), such as fMRI . However, we observed more-or-less concomitant neuronal and cellular changes during phase III, resulting from hypoxic stress. The first dynamics to be modified were the changes in spectral coherence between the deep and superficial layers, which occurred symmetrically around the IIS (−1 to +1 s), simultaneously with the rising slope of CBF and HbOWe estimated the cross-correlation between oscillations in different cortical layers  by applying time-frequency domain wavelet analysis. Wavelet-coherence analysis allows quantification of the similarity of the time-frequency (correlation) between the LFP and MUA activities of different channels , 75. OurNeuronal activity changes start from the increase in CBF. We identified three classes of changes in unit activity . This is comparable to what has been previously described in epileptic patients . WaveletAnalyzing the FOS provides information on non-synaptic mechanisms involved in the emergence of the IIS. Changes in the optical properties of the neuronal tissue consisted of an increase in light detection, corresponding to a decrease in scattering, suggesting the shrinking of neurons or astrocytes, resulting in an increase in the extracellular space  and thus2 levels, the firing rate of the neurons increase, although a decrease has not been observed , concomitant or not with a source in layers V and VI at the peak of the IIS, followed by a second source in layers V–VI (post-IIS), in complete accordance with the results of Castro-Alamancos  and UlbeDuring the spike period, a decrease in light detection/increase in light scattering is consistent with the swelling of activated neuronal cells , resulting in a reduction of the extracellular space and thus an increase in the bioavailability of extracellular molecules that is specific to the site of injection  .2. Based on ECoG, this period can be divided into two periods: Period 5-SW1, corresponding to the ascending part of the slow-wave [0 to +300 ms], and Period 6-SW2, corresponding to the descending slope of the slow-wave, between +300 to +500 ms. Concomitant with the ascending portion of the slow-wave (SW1), the neuronal activity either increased, decreased, or did not change relative to baseline, again suggesting complex interactions involving different types of neurons (excitatory or inhibitory) that participate in generating the slow-wave  the neurogenic hypothesis: changes in the neuronal dynamics were not visible because they involved non-neuronal activity or that of astrocytes, which do not develop an electrical signature , 21; (2)Alternatively, slight changes in spontaneous vascular (and/or hemodynamic) oscillations in an atmosphere of interictal hypermetabolism in the epileptic focus, as observed in focal penicillin-induced epilepsy in rats , may be 2, which may alter the oxygen partial pressures within the neuronal tissue and thus the dynamics of oxygen exchange. Such non-synaptic events occurred between −300 and +300 ms around the IIS, with a swing between shrinking-swelling-shrinking of the cellular compartments, including neurons and/or astrocytes. We previously have discussed the relationship between the changes in optical properties and structural changes in neural tissues [see  the difficulty in maintaining a correct level of anesthesia, (2) technical issues (noisy data), and (3) changes in IIS morphology during the recording. We paid particular attention to probe similar brain structures and placed the electrical and optical probes as close as possible to each other for the recordings of the various modalities, but it was impossible to place them in exactly the same position. Our main objective in this first study was to characterize complex interactions between the different neuronal and hemodynamic and extracellular compartments. Further study are required to investigate the specificity according to the different IIS patterns and to the type of epilepsy in different species. Despite the use of several multidimensional methods, which have still their own limitations, the biological process behind the interictal spikes were not completely monitored and require further studies, notably at the cellular level.in vitro analysis at the cellular level. This multimodal approach highlights the complexity of spatio-temporal interactions between different cortical layers. It also shed new light on the specific hemodynamic and non-synaptic environment dynamics which initialize the neuronal changes that propels neurons to hypersynchronization of the interictal epileptic spike.This multimodal (ECoG-MLE-fNIRS-FOS-DCS) study also describes the neuronal, hemodynamic and membrane configuration changes observed in epileptic rats in response to endogenous hypersynchronization related to IIS. The intimate mechanisms supporting the concomitant changes during epileptic spikes deserve further The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by Ethical Committee of the French Ministry of Research (ref: APAFIS#1464-2015081710033478). Ethical Committee of the French Ministry of Research (ref: APAFIS#1464-2015081710033478).FW and MMah contributed to conception and design of the study. CA-R and MMah designed the MLE setup. MN and MMah designed the DCS setup. MMah designed the fNIRS/FOS setup. CA-R and MN participates in data acquisition. CA-R and MMah analyzed LFP/MUA data. MN analyzed DCS data. CA-R and MMah analyzed fNIRS data. MMah and MMan analyzed FOS data. CA-R, MN, and MMah performed the statistical analysis. CA-R, MMah, MN, and FW wrote the first draft of the manuscript. FW and MMah contributed to project administration. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4 Escherichia coli, leading to higher mortality. IBV infection also permanently destroys the oviducts of immature females and pullets, resulting in limited egg production for a long time and birds that fail to come into production  [Infectious bronchitis (IB) is an acute respiratory viral disease of chickens and caused by infectious bronchitis virus (IBV), a member of the coronavirus family. Chickens of all ages and breeds are susceptible to IBV . IBV inf layers) . In addi layers) . TherefoIBV contains four structural proteins that are the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins . The S pVaccination is still the main effective means for the preventing and controlling of the disease. However, traditional inactivated and attenuated vaccines cannot provide complete and effective protection due to the lack of serotype matching between the vaccine and field variant strains ,12,13. LDespite the widespread application of IBV vaccines, IB has been a continuing problem in the poultry industry. Many IBV variant strains have been isolated and identified. Studies have shown that the genotypes and serotypes of the majority of IBV isolates are different from those of traditional vaccine strains in China ,23,24. IAmong new vaccines, the VLP vaccine is considered one of the most promising due to its high safety and good immunogenicity . VLPs arIt is unclear how many combinations of structural proteins can assemble into IBV VLPs. To date, there have been few studies on IBV VLPs. A previous study showed that IBV VLPs, containing the M and S proteins of the H120 strain, could elicit humoral immunity comparable to that achieved with the H120 inactivated vaccine and a cellular immune response higher than that induced by the H120 inactivated vaccine . IBV VLPAt present, there have been no reports on comparisons of the immunogenicity and protection efficiency among SME-VLPs, SM-VLPs and ME-VLPs, carrying S, M and E proteins; S and M proteins; and M and E proteins, respectively. In this study, three VLPs, SME-VLPs, SM-VLPs and ME-VLPs, from the dominant serotype representative strain GX-YL5 in southern China were constructed. It is worth mentioning that southern China is the region with the largest production of local breeds of chickens, about 5 billion annually, in the country. In order to screen the VLP vaccine candidates for IB, the immunogenicity and protection efficacy of the three VLP vaccines were evaluated and compared in specific-pathogen-free (SPF) chickens. This is the first report on the construction and immunogenicity comparison of three VLPs carrying different combinations of structural proteins of IBV. This study provides a basis for the exploration of VLP vaccine candidates for IB.Sf9 insect cells were cultured in serum-free Sf-900TM II SFM  and SIM SF Sf9  at 27 °C. The virulent GX-YL5 strain  of IBV was the dominant serotype representative isolate and had a heterologous serotype compared to the H120 vaccine strain .TM/HBM-TOPO vector  to obtain the recombinant transposon vectors pFastBacTM/HBM-TOPO-S, pFastBacTM/HBM-TOPO-M and pFastBacTM/HBM-TOPO-E, respectively. The three recombinant transposon vectors were chemically transformed into competent Escherichia coli DH10Bac cells to obtain the recombinant bacmids rHBM-S, rHBM-M and rHBM-E by blue/white selection and sequencing using M13 primers. The purified recombinant bacmids were transfected into sf9 cells with Cellfectin reagent following the manufacturer’s instructions (Invitrogen) to obtain recombinant baculovirus rHBM-S, rHBM-M and rHBM-E. The three recombinant S, M and E proteins were identified by immunofluorescence staining analysis and Western blotting. For immunofluorescence staining analysis, the expression of protein was detected using an anti-His mouse monoclonal antibody  at a dilution of 1:200 and FITC-conjugated goat anti-mouse IgG secondary antibody . Cell nuclei were stained with DAPI . Chicken polyclonal antiserum (1:2000 dilution) was used as the primary antibody, and an HRP-conjugated goat anti-chicken IgG , as the secondary antibody for the verification of the three recombinant proteins by Western blotting. The Image J software was applied to analyze the quantification of the intensity of each protein band, and the results are presented as the ratios of the proteins of interest to an internal reference protein.The S, M and E genes of the GX-YL5 strain were amplified by reverse transcription polymerase chain reaction (RT-PCR) and subcloned into the pFastBacg for 2 h at 4°C. After the sediments were resuspended in PBS, the solutions were loaded on a discontinuous sucrose gradient of 20%, 40% and 60% sucrose and ultracentrifuged at 80,000× g for 3 h at 4°C. The VLPs at the interface between the 40% and 60% sucrose were collected and pelleted by ultracentrifugation at 80,000× g for 2 h at 4 °C.Three IBV VLPs, SME-VLPs, SM-VLPs and ME-VLPs, were prepared by co-infection with three different combinations of recombinant baculovirus  in suspension-cultured Sf9 cells. The cell culture supernatants were collected and ultracentrifuged at 80,000× Three VLPs were identified by Western blotting as previously described . An anti6 median infective dose of the trachea organ ring (TOC-ID50) of the IBV GX-YL5 strain in 0.2 mL by the nasal–ocular route. The animal experiments were approved of by the Animal Care & Welfare Committee of Guangxi University  and were performed in accordance with animal ethics guidelines and approved protocols.A total of 120 10-day-old specific-pathogen-free (SPF) chickens were randomly divided into 6 groups, with 20 birds per group. The chickens were immunized 2 times (at weeks 0 and 2) with IBV VLPs or other vaccines at two-week intervals. The inoculation dose for the SME-VLP and SM-VLP groups contained 2 µg of S proteins. The ME-VLP group was vaccinated with VLPs containing 2 µg of M proteins. The birds in the GX-YL5 group were immunized with the OEV of GX-YL5 containing 2 µg of S proteins. The birds in the H120 group were immunized with the commercial attenuated H120 vaccine according to the manufacturer’s protocols. A total of 20 birds were immunized with PBS as a negative control. Besides the birds in the H120 group being immunized by the nasal–ocular route, the birds in the other groups were intramuscularly (i.m.) immunized. At 14 days post-vaccination (dpv), booster vaccinations were performed with the same program and doses as the prime vaccinations. At 14 days post-booster (dpb), all the birds from each group were challenged with a 10Serum samples were collected from the inoculated chickens at 0, 7, 14, 21 and 28 dpv. A commercial enzyme-linked immunosorbent assay (ELISA) IBV antibody test kit  was used to evaluate the IBV-specific antibodies in the sera following the manufacturer’s instructions.At 0, 14 and 28 dpv, the IBV-neutralizing antibody titers in the sera from the vaccinated chickens were determined by a trachea organ ring (TOC) neutralization test following our previous description . TOCs we+, CD4+ and CD8+ T lymphocytes using a BD AccuriTMC6 Flow Cytometer . All the staining reactions were conducted according to the manufacturer’s protocols.Peripheral blood samples were collected from the wing veins of 10 birds in each group at 0, 7, 14, 21 and 28 dpv. Peripheral blood lymphocytes were isolated from the peripheral blood samples. The samples were diluted with equal volumes of sterile PBS, overlaid onto chicken peripheral blood lymphocyte isolation liquid  and centrifuged at 500 g for 20 min at room temperature. The peripheral blood lymphocytes at the interface were collected and washed with sterile PBS. A final volume of 0.1 mL was stained with 3 µL of Mouse Anti-Chicken CD3-PE, 2 µL of Mouse Anti-Chicken CD4-PE/CY7 and 2 µL of Mouse Anti-Chicken CD8α-FITC antibodies , respectively. The concentrations of the Mouse Anti-Chicken CD3-PE, CD4-PE/CY7 and CD8α-FITC antibodies were adjusted to 0.3, 0.1, and 1 µg/0.1 mL, respectively. All the samples were assessed for the percentages of CD3Serum samples were collected from the birds at 0, 14 and 28 dpv. The concentrations of IL-4 and IFN-γ in the sera were determined using ELISA Kits  according to the manufacturer’s protocols, respectively.Tear and oral swabs were collected at 0, 7, 14, 21 and 28 dpv. The sIgA concentrations were evaluated using an ELISA Kit . The sIgA antibody detection procedures were completed in accordance with the instructions.TM multisource total RNA miniprep kit . The viral RNA levels were assessed by real-time quantitative PCR (qPCR) targeting the nucleocapsid (N) gene, as previously described [At 5 days post-challenge (dpc), 10 birds in each group were sacrificed to collect the trachea and kidney tissue samples. The remaining birds were observed until 14 dpc. The tear and oral swabs were collected at 2, 4, 6, 8, 10, 12 and 14 dpc. The RNA in the tracheas, kidneys, and tear and oral swabs was extracted using a commercial AxyPrepescribed .Ten birds from each group were euthanized at 5 dpc. Ten tracheal rings from each trachea approximately 1–2 mm thick were cut. The ciliostasis test was conducted, and the protection scores for each trachea was calculated as previously described .p < 0.05) were considered significant, and those less than 0.01 were considered highly significant.All the data are presented as geometric means with 95% CIs. Statistical analysis was performed by a one-way analysis of variance (ANOVA) using the SPSS 24.0 software. P values less than 0.05  were detected in the three VLPs . Circulap < 0.05 or p < 0.01). The highest antibodies in each immunized group were observed at 28 dpv. At 14, 21 and 28 dpv, the birds in the GX-YL5 and H120 groups had higher antibody titers than those of the VLP-vaccinated groups (p < 0.05), while no significant difference was noted between the GX-YL5 and H120 groups. The antibody levels of the SME-VLP and SM-VLP groups were all significantly higher compared to those of the ME-VLP group (p < 0.05) at 14 dpv, but there was no significant difference in the antibody levels among the three VLP groups at 21 and 28 dpv.At 0, 7, 14, 21 and 28 dpv, the levels of IBV-specific antibodies were analyzed by ELISA. The antibody levels in all the vaccinated groups continued to rise from 7 to 28 dpv . The antp < 0.05) (p < 0.05). The highest IBV-neutralizing antibody titers in all the immunized groups were observed at 28 dpv. At 28 dpv, the SME-VLP and GX-YL5 groups had the highest and same IBV-neutralizing antibody titers. There were no significant differences in antibody titers among the SM-VLP and ME-VLP groups at 28 dpv.The IBV-neutralizing antibody titers in the sera were evaluated by TOC neutralization tests at 0, 14 and 28 dpv. The results demonstrate that all the vaccinated groups had higher neutralizing antibody levels than the negative control group at 14 and 28 dpv ( < 0.05) . At 14 d+, CD4+ and CD8+ T lymphocytes in all the vaccinated groups were rising at 7 dpv (p < 0.01) from 14 to 28 dpv. At 21 and 28 dpv, the H120 group presented the highest percentages of CD3+ T lymphocytes, which were significantly higher than those of the VLP and GX-YL5 groups (p < 0.05). At 21 and 28 dpv, the percentages of CD4+ and CD8+ T lymphocytes in the SME-VLP and H120 groups were significantly higher than those of the other vaccinated groups (p < 0.05), but there were no significant differences between the SME-VLP and H120 groups. No significant differences were noted among the SM-VLP, ME-VLP and GX-YL5 groups at 28 dpv.The results demonstrate that the percentages of CD3at 7 dpv . All thep < 0.01). At 28 dpv, the concentrations of IL-4 of all the vaccinated groups were significantly higher than those of the nonvaccinated group (p < 0.01). In addition, the IL-4 levels of the SME-VLP and H120 groups were significantly higher than those of the SM-VLP, ME-VLP and GX-YL5 groups (p < 0.05), and there were no significant differences between the H120 and SME-VLP groups. Higher IFN-γ levels than in the negative control group were successfully induced in all the vaccinated groups on the 14 and 28 dpv. At 14 dpv, the IFN-γ levels in the SME-VLP, SM-VLP and ME-VLP groups were comparable to those of the OEV of GX-YL5 and H120 groups, and they showed no significant differences among the vaccinated groups. At 28 dpv, the IFN-γ levels in the SME-VLP and H120 groups were significantly higher than those of the other immunized groups (p < 0.05), but no significant differences were observed between the SME-VLP and H120 groups. There was no significant difference in the IFN-γ levels among the SM-VLP, ME-VLP and GX-YL5 groups at 28 dpv.The concentrations of IL-4 and IFN-γ in the sera from all the immunized groups were analyzed at 0, 14 and 28 dpv . At 14 dp < 0.01) compared to the negative control group from 14 to 28 dpv. At 14 dpv, the H120 group had higher sIgA levels in the oral swabs than the other immunized groups (p < 0.05). At 21 and 28 dpv, the SME-VLP and H120 groups had higher levels of sIgA in the tear and oral swabs than the SM-VLP, ME-VLP and GX-YL5 groups (p < 0.05), but no significant differences between the SME-VLP and H120 groups were apparent. Furthermore, at 21 and 28 dpv, no significant differences were observed among the SM-VLP, ME-VLP and GX-YL5 groups.The sIgA levels were detected in tear and oral collections at 0, 7, 14, 21 and 28 dpv . The sIgp < 0.05). According to the viral RNA levels in the tracheas, the immunized groups ordered from lowest to highest were the SME-VLP group, ME-VLP group, SM-VLP group, GX-YL5 group and H120 group, but no significant differences were observed among the three VLP groups. The viral RNA levels in the kidneys of the SME-VLP, ME-VLP, GX-YL5 and H120 groups were significantly lower than those in the SM-VLP group (p < 0.05), but no significant differences were noted among the SME-VLP, ME-VLP, GX-YL5 and H120 groups. Under the IBV strain GX-YL5 challenge, no apparent symptoms were observed in the vaccinated birds. The birds in the negative control group showed depression, gasp, tracheal rales and watery eyes in the early days of the challenge. At 5 dpc, all the vaccinated birds had significantly lower viral RNA levels in the tracheas and kidneys compared to the negative control . The virp < 0.05 or p < 0.01) (p < 0.05). At 4 and 6 dpc, the viral RNA levels in the tear swabs of the SME-VLP and GX-YL5 groups were significantly lower than those of the three other immunized groups (p < 0.05), but no significant differences were noted between the SME-VLP and GX-YL5 groups. At 2, 4 and 6 dpc, the viral RNA levels in the oral swabs of the SME-VLP and GX-YL5 groups were significantly lower than those in the SM-VLP, ME-VLP and H120 groups (p < 0.05), and no significant differences were observed between the SME-VLP and GX-YL5 groups. The virus shedding in the tear and oral swabs was monitored after the IBV challenge. The results show that the vaccinated birds had significantly lower viral loads compared to the negative controls from 2 to 6 dpc ( < 0.01) . The virAt 5 dpc, the ciliostasis of each group was measured, and the protection scores were determined . The higIB is a highly contagious disease and brings serious economic losses to the poultry industry. Today, live-attenuated and inactivated vaccines are frequently used to control and prevent this disease in commercial chickens. However, the current commercial vaccines have defects in safety or immunogenicity . Live-at+ and CD8+ T lymphocytes, and IL-4 and IFN-γ concentrations were higher than those observed with the GX-YL5 inactivated vaccine; the sIgA levels of the tear and oral fluids were equivalent to those seen with the attenuated H120 vaccine and higher than those with the inactivated GX-YL5 vaccine. In the challenge study, the SME-VLPs induced significantly lower viral RNA levels in the trachea and higher protection scores than those of the GX-YL5 and H120 groups, viral RNA levels in the kidneys and tear and oral swabs comparable to those in the GX-YL5 group, and significantly lower virus shedding in the tear and oral swabs compared to the H120 and negative control groups. Hence, our results suggest that among the three VLPs, the SME-VLPs could efficiently stimulate the vaccinated birds to generate the strongest humoral, cellular and mucosal immune responses, and showed good protective efficacy against virulent IBV challenges, indicating that it would be useful as an appealing vaccine candidate for the development of IB vaccines in the future.In this study, SME-VLPs, SM-VLPs and ME-VLPs, carrying the S, M and E proteins, the S and M proteins, and the M and E proteins, respectively, of the dominant serotype representative strain GX-YL5 in southern China were constructed. All the results of Western blotting, TEM and IEM proved that the SME-VLPs, SM-VLPs and ME-VLPs were successfully assembled. As far as we know, IBV VLPs were observed by IEM for the first time in our study. In addition, we firstly systematically investigated the humoral, cellular and mucosal immune responses to different combinations of IBV VLP vaccines. Among the three VLPs, it was found that the SME-VLPs could induce neutralizing antibodies equivalent to those induced by the inactivated GX-YL5 vaccine and higher than those with the attenuated H120 vaccine; the percentages of CD4The VLPs carrying the M and S proteins of the H120 strain induced comparable humoral immune responses and a higher cellular immune response than the inactivated H120 vaccine . Another+ and CD8+ T lymphocytes than the inactivated GX-YL5 viruses, comparable to those of the H120 group. A previous study showed that rHBM-S1-N and rHBM-N generated higher CD4+ and CD8+ T lymphocyte percentages compared to those induced by a H120 vaccine [Additionally, our study showed that SME-VLPs could induce significantly higher percentages of CD4 vaccine . In anot vaccine . IFN-γ a vaccine . MERS-Co vaccine . However vaccine . A recen vaccine . Our stu vaccine ,41.IBV is a respiratory pathogen and firstly enters the body through the respiratory tract. For vaccines for respiratory disease, it is very critical to stimulate the host to produce mucosal immune responses in addition to the systemic immune responses. Past findings showed that sIgA was detected in mucosal secretions and tissues in mice immunized intranasally but not in mice immunized intraperitoneally with SARS-CoV VLPs formed from the S, E and M proteins . To dateIn the challenge study, the vaccinated birds had significantly lower viral RNA levels in the tracheas and kidneys, and significantly lower virus shedding in the tear and oral swabs compared to the nonvaccinated birds, and the VLP-vaccinated birds had higher protection scores, indicating that the three VLPs could provide protection against the IBV challenge. At 5 dpc, the SME-VLP group had the lowest viral RNA levels in the tracheas, which were significantly lower than those with the GX-YL5 and H120 vaccines; the viral RNA levels in the kidneys of the SME-VLP and ME-VLP groups were significantly lower than those of the SM-VLP group. From 2 to 6 dpc, the SME-VLP and GX-YL5 groups demonstrated significantly lower virus shedding in the tear and oral swabs compared to the SM-VLP and ME-VLP groups, and no significant differences were observed between the SME-VLP and GX-YL5 groups. The SME-VLP group had the highest protection scores compared to the SM-VLP and ME-VLP groups. Therefore, collectively, the SME-VLPs provided better protection against the IBV challenge than the ME-VLPs and SM-VLPs, especially in reducing the shedding of the virus in the tear and oral swabs in early infection, which was consistent with the systemic and mucosal immune responses induced by the SME-VLPs. In addition, our results show that the SME-VLPs provided better protection in the trachea compared to the inactivated GX-YL5 vaccine, which may be due to the higher cellular immune response and mucosal immune responses compared with those elicited by the GX-YL5 inactivated vaccine, which agrees with our previous study . In the Many factors affect the immune response, such as the route of administration of an immunogen, frequency, dose, time interval and animal health. A previous report showed that higher sIgA was detected in mucosal secretions and tissues in mice immunized intranasally than those immunized intraperitoneally with SARS-CoV VLPs . In our In summary, SME-VLPs, SM-VLPs and ME-VLPs, which carried the S, M and E proteins; the S and M proteins; and the M and E proteins, respectively, were firstly constructed and compared for immunogenicity in the present study. Among the three VLPs, the SME-VLPs could stimulate the strongest humoral, cellular and mucosal immune responses, and provide efficient protection against a virulent IBV challenge. To our knowledge, our study showed, for the first time, that IBV VLPs were identified by IEM. In addition, the humoral, cellular and local mucosal immune responses to and immunogenicity of different combinations of IBV VLP vaccines were systematically investigated for the first time. This work suggests that the SME-VLPs could serve as an appealing vaccine candidate for the IB vaccine.


This editorial aims to summarize the eight scientific papers published in the Special Issue “Genetics and Molecular Pathogenesis of Non-ischemic Cardiomyopathies”.Cardiomyopathies are primary diseases of the myocardium, and are leading causes of sudden cardiac death (SCD), particularly in athletes and the young. Diagnosis of affected individuals remains challenging, and a resolutive therapy is still lacking, as current treatments can only attenuate the symptoms but not prevent or rescue the phenotype (1). Genetics and genomics along with studies in cellular and animal models are fundamental for delineating the genetic bases of cardiomyopathies and to elucidate the pathogenesis of these diseases. This could greatly improve diagnosis and prognostic evaluation of the affected individuals as well as their family members and lead to the identification of novel targets for more effective treatments.The papers published in this Special Issue discuss several aspects of genetics, genomics and mechanistic studies in the field of primary cardiomyopathies.The role of genetic screening by next generation sequencing has been discussed by several papers in the Special Issue ,2,3,4,5.A comprehensive review by Patel et al.  describeACM incomplete penetrance and phenotypic variability suggest that the causal gene is not the only determinant of the phenotype and that other factors may modulate the disease manifestations. Among them, microRNAs (miRNAs)—small noncoding RNAs with a primary role as regulators of gene expression and cell–cell communication—have been proposed as potential players by several authors. The paper by Bueno Marinas et al.  publisheThe effects of lifestyle and in particular the effects of sexual interactions on the cardiac function, clinical presentation and survival have been studied in a paper published in this Special Issue in an experimental mouse model of dilated cardiomyopathy (DCM) . It has The role of the extracellular matrix (ECM) and of the cytoskeleton on the pathogenesis of cardiomyopathies has never been discussed in detail. In the Special Issue, two papers published by two research groups discuss these novel topics. HF is the final stage of several cardiovascular diseases, and HF with a preserved ejection fraction (HFpEF) represents half of the cases. However, no treatment has yet been shown to reduce morbidity and mortality in patients with HFpEF. The opinion letter from Hochman-Mendez et al.  discusseACTN2), Filamin C (FLNC) and Dystrophin (DMD) have been described as causal genes in several cardiomyopathies. These genes encode for cytoskeleton proteins that not only have structural function but also regulate cell behavior through mechano-transduction. In cardiac myocytes, ACTN2 and FLNC localize to the Z-disc of the sarcomeres and interact with the intercalated disc (ID) components, while DMD localizes to the inner face of the plasma membrane. Z-disks, IDs and plasma membrane are important signaling hubs for cardiac myocytes. As ACTN2, FLNC and DMD, functioning as cross-linkers, interact with all these structures, mutations in these proteins are expected to have deleterious effects on cardiac myocytes structure and function, including abnormal activation of molecular pathways. Further mechanistic studies are needed to reveal the effects of mutations in these cytoskeleton elements on signaling pathways implicated in the onset and development of inherited cardiomyopathies. The findings could facilitate the development of novel therapeutic drugs to treat inherited cardiomyopathies.The paper of Pecorari et al.  describeThe review by Gao et al.  focuses In summary, the papers published in the present Special Issue describe the causal and the contributing factors of the phenotypic expression of non-ischemic cardiomyopathies, including genetic background, circulating microRNAs, paracrine factors, extracellular matrix modifications, cellular and molecular interactions and external factors such as lifestyle. Moreover, the published papers highlight the complexity of the pathogenesis of these diseases and the necessity of further studies in order to improve the diagnosis and allow the development of novel personalized treatments to prevent and/or attenuate the phenotype of patients with cardiomyopathies.