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Journal of Virology Sep 2023Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the...
Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-S strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-S belongs to the virulent strains. Then, the recombination PEDV (rPEDV-E)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-S strain (rPEDV-E) as the backbone. Compared with the rPEDV-E strain, the release and infectivity of the rPEDV-E strain were significantly reduced , but stronger interferon (IFN) responses were triggered both and . The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-E caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-E. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-E group. In conclusion, the deletion of a 7-aa fragment in the E protein (E) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying E mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that E mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.
Topics: Animals; Coronavirus Infections; Interferons; Porcine epidemic diarrhea virus; RNA; Swine; Swine Diseases; Vaccines, Attenuated; Sequence Deletion; Viral Envelope Proteins
PubMed: 37681956
DOI: 10.1128/jvi.00847-23 -
Science Advances Dec 2023Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular...
Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.
Topics: Humans; Synthetic Lethal Mutations; Liver Neoplasms; Chromosome Deletion; Chromosome Aberrations; Chromosomes; CRISPR-Cas Systems
PubMed: 38134284
DOI: 10.1126/sciadv.adh1442 -
Nature Communications Mar 2024Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. To identify additional genetic factors, we analyzed exome sequences in a large cohort of...
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. To identify additional genetic factors, we analyzed exome sequences in a large cohort of Chinese ALS patients and found a homozygous variant (p.L700P) in PCDHA9 in three unrelated patients. We generated Pcdhα9 mutant mice harboring either orthologous point mutation or deletion mutation. These mice develop progressive spinal motor loss, muscle atrophy, and structural/functional abnormalities of the neuromuscular junction, leading to paralysis and early lethality. TDP-43 pathology is detected in the spinal motor neurons of aged mutant mice. Mechanistically, we demonstrate that Pcdha9 mutation causes aberrant activation of FAK and PYK2 in aging spinal cord, and dramatically reduced NKA-α1 expression in motor neurons. Our single nucleus multi-omics analysis reveals disturbed signaling involved in cell adhesion, ion transport, synapse organization, and neuronal survival in aged mutant mice. Together, our results present PCDHA9 as a potential ALS gene and provide insights into its pathogenesis.
Topics: Humans; Mice; Animals; Aged; Amyotrophic Lateral Sclerosis; Neurodegenerative Diseases; Mice, Transgenic; Motor Neurons; Spinal Cord
PubMed: 38467605
DOI: 10.1038/s41467-024-46333-5 -
Translational Pediatrics Aug 2023Primary ciliary dyskinesia (PCD) is a clinically heterogeneous group of autosomal or, less frequently, X-chromosomal recessive inheritance syndrome of motile cilia...
BACKGROUND
Primary ciliary dyskinesia (PCD) is a clinically heterogeneous group of autosomal or, less frequently, X-chromosomal recessive inheritance syndrome of motile cilia dysfunction characterized by neonatal respiratory distress, oto-sino-pulmonary disease, infertility and situs inversus. Recently, type 43 PCD (CILD43, OMIM#618699) was established by autosomal-dominant loss-of-function mutations identified in Forkhead box J1 (). However, the functional validation of mutations in humans and mice has not been fully performed. Here we studied a three-generation family with heterotaxy and proband with complex congenital heart disease (CHD).
METHODS
We performed whole-exome sequencing to investigate the causative variant of this family and generated gene knock-in mice carrying the human equivalent mutation by homologous recombination. Then, microscopy analysis was used to characterize the phenotype and ciliary ultrastructure of the model. Effects of the variant on heart anomaly were preliminarily explored through transcriptome sequencing.
RESULTS
A novel heterozygous deletion variant (c.1129delC/p.Leu377Trpfs*76) of was discovered that exerts a dominant-negative effect (DNE) . Notably, both homozygous () and heterozygous () mice developed situs inversus, hydrocephalus and showed a disruption of trachea cilia structure, whereas these abnormalities were only observed in previously reported , not mice. Thus, a more severe phenotype and higher expressivity of our mouse model further indicated the DNE of this mutation. Meanwhile, several cardiomyopathy-related genes were differentially expressed in the homozygous knock-in mouse hearts, pointing to a probable function in cardiac pathology.
CONCLUSIONS
Overall, our study results showed that c.1129delC mutation in was regarded as the cause of situs inversus in this family and this mutant showed a capacity of DNE over wild-type FOXJ1, causing more serious consequences than the allelic deletion of .
PubMed: 37692537
DOI: 10.21037/tp-23-27 -
Frontiers in Endocrinology 2023Bi-allelic variants in cause the ultrarare bone fragility disorder "spinal muscular atrophy with congenital bone fractures-2" (SMABF2). However, the mechanism by which...
Bi-allelic variants in cause the ultrarare bone fragility disorder "spinal muscular atrophy with congenital bone fractures-2" (SMABF2). However, the mechanism by which ASCC1 dysfunction leads to this musculoskeletal condition and the nature of the associated bone defect are poorly understood. By exome sequencing, we identified a novel homozygous deletion in in a female infant. She was born with severe muscular hypotonia, inability to breathe and swallow, and virtual absence of spontaneous movements; showed progressive brain atrophy, gracile long bones, very slender ribs, and a femur fracture; and died from respiratory failure aged 3 months. A transiliac bone sample taken postmortem revealed a distinct microstructural bone phenotype with low trabecular bone volume, low bone remodeling, disordered collagen organization, and an abnormally high bone marrow adiposity. Proteomics, RNA sequencing, and qPCR in patient-derived skin fibroblasts confirmed that ASCC1 was hardly expressed on protein and RNA levels compared with healthy controls. Furthermore, we demonstrate that mutated is associated with a downregulation of , the master regulator of osteoblastogenesis, and , which is involved in osteoblast and adipocyte differentiation. It also exerts an inhibitory effect on TGF-β/SMAD signaling, which is important for bone development. Additionally, knockdown of ASCC1 in human mesenchymal stromal cells (hMSCs) suppressed their differentiation capacity into osteoblasts while increasing their differentiation into adipocytes. This resulted in reduced mineralization and elevated formation of lipid droplets. These findings shed light onto the pathophysiologic mechanisms underlying SMABF2 and assign a new biological role to ASCC1 acting as an important pro-osteoblastogenic and anti-adipogenic regulator.
Topics: Infant; Humans; Female; Homozygote; Sequence Deletion; Cell Differentiation; Adipogenesis; Proteins; Carrier Proteins
PubMed: 37455927
DOI: 10.3389/fendo.2023.1137573 -
F1000Research 2022Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the gene. The full mutation spectrum of the gene in...
BACKGROUND
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the gene. The full mutation spectrum of the gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the gene in Indonesia to guide future development and application of feasible therapeutic strategies.
METHODS
This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the gene.
RESULTS
Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy.
CONCLUSION
This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of cases in the country.
Topics: Humans; Male; Cross-Sectional Studies; Dystrophin; Gene Deletion; Indonesia; Muscular Dystrophy, Duchenne; Mutation
PubMed: 38009102
DOI: 10.12688/f1000research.73476.3 -
IScience Oct 2023The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding mammalian oocytes. Recently, numerous mutations in genes encoding ZP proteins have been shown...
The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding mammalian oocytes. Recently, numerous mutations in genes encoding ZP proteins have been shown to be possibly related to oocyte abnormality and female infertility; few reports have confirmed the functions of these mutations in living animal models. Here, we identified a novel heterozygous missense mutation (NM_001376231.1:c.1616C>T, p.Thr539Met) in from a primary infertile female. We showed that the mutation reduced ZP2 expression and impeded ZP2 secretion in cell lines. Furthermore, we constructed the mouse model with the mutation () using CRISPR-Cas9. female mice had normal fertility though generated oocytes with the thin ZP, whereas female mice were completely infertile due to degeneration of oocytes without ZP. Additionally, ZP deletion impaired folliculogenesis and caused female infertility in mice. Our study not only expands the spectrum of mutation sites but also, more importantly, increases the understanding of pathogenic mechanisms of mutations.
PubMed: 37736051
DOI: 10.1016/j.isci.2023.107828 -
Nature Communications Sep 2023Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate...
Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.
Topics: Mice; Animals; CRISPR-Cas Systems; Gene Editing; Gene Deletion; RNA, Guide, CRISPR-Cas Systems; Mice, Knockout; Muscle Fibers, Skeletal
PubMed: 37777530
DOI: 10.1038/s41467-023-41769-7 -
Science Advances Aug 2023The 1.6-megabase deletion at chromosome 3q29 (3q29Del) is the strongest identified genetic risk factor for schizophrenia, but the effects of this variant on...
The 1.6-megabase deletion at chromosome 3q29 (3q29Del) is the strongest identified genetic risk factor for schizophrenia, but the effects of this variant on neurodevelopment are not well understood. We interrogated the developing neural transcriptome in two experimental model systems with complementary advantages: isogenic human cortical organoids and isocortex from the 3q29Del mouse model. We profiled transcriptomes from isogenic cortical organoids that were aged for 2 and 12 months, as well as perinatal mouse isocortex, all at single-cell resolution. Systematic pathway analysis implicated dysregulation of mitochondrial function and energy metabolism. These molecular signatures were supported by analysis of oxidative phosphorylation protein complex expression in mouse brain and assays of mitochondrial function in engineered cell lines, which revealed a lack of metabolic flexibility and a contribution of the 3q29 gene Together, these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species.
Topics: Child; Humans; Animals; Mice; Aged; Schizophrenia; Chromosome Deletion; Developmental Disabilities; Neocortex; Intellectual Disability
PubMed: 37585521
DOI: 10.1126/sciadv.adh0558 -
Scientific Reports Nov 2023Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation is brain metastasis (BM)-prone. We determined the impact of this hallmark, along...
Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation is brain metastasis (BM)-prone. We determined the impact of this hallmark, along with EGFR subtype and generation of tyrosine kinase inhibitor (TKI) treatment, on patients' outcome. 553 metastatic EGFR-mutant NSCLC patients received front-line EGFR-TKI treatment. Progression-free survival (PFS), overall survival (OS) and secondary T790M rate were analysed. BM was observed in 211 (38.2%) patients. BM (HR 1.20 [95% CI 0.99-1.48]; p = 0.053), ECOG PS 0-1 (HR 0.71 [95% CI 0.54-0.93]; p = 0.014) and afatinib treatment (HR 0.81 [95% CI 0.66-0.99]; p = 0.045) were associated with PFS. Afatinib-treated patients without BM demonstrated a significantly longer PFS (16.3 months) compared to afatinib-treated patients with BM (13.7 months) and to gefitinib/erlotinib-treated patients with (11.1 months) or without BM (14.2 months; p < 0.001). CNS-only progression trended higher in afatinib-treated patients. ECOG PS 0-1 (HR 0.41 [95% CI 0.31-0.56]; p < 0.001) and EGFR L858R mutation (HR 1.46 [95% CI 1.13-1.88]; p = 0.003), but not BM, were the predictors for OS. BM (OR 2.02 [95% CI 1.02-4.08]; p = 0.040), afatinib treatment (OR 0.26 [95% CI 0.12-0.50]; p < 0.001) and EGFR L858R mutation (OR 0.55 [95% CI 0.28-1.05]; p = 0.070) were associated with secondary T790M rate. In BM patients, gefitinib/erlotinib-treated ones with 19 deletion mutation and afatinib-treated ones with L858R mutation had the highest and the lowest T790M rate (94.4% vs. 27.3%, p < 0.001), respectively. BM and generation of EGFR-TKI jointly impact PFS and secondary T790M rate in patients with EGFR-mutant NSCLC, whereas OS was mainly associated with EGFR subtype.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Afatinib; Erlotinib Hydrochloride; Gefitinib; ErbB Receptors; Lung Neoplasms; Protein Kinase Inhibitors; Mutation; Treatment Outcome; Brain Neoplasms
PubMed: 37989860
DOI: 10.1038/s41598-023-45815-8