Recovery of Recombinant Canine Distemper Virus That Expresses CPV-2a VP2: Uncovering the Mutation Profile of Recombinant Undergoing 50 Serial Passages In Vitro

Canine distemper and canine parvoviral enteritis are infections caused by the canine distemper virus (CDV) and canine parvovirus type 2 (CPV-2), respectively. They are two common infectious diseases that cause high morbidity and mortality in affected dogs. Combination vaccines have been broadly used to protect dogs from infections of CDV, CPV-2, and other viruses. VP2 is the most abundant protein of the CPV-2 capsid. It elicits potent immunity in animals and, therefore, is widely used for designing subunit antigen-based vaccines. In this study, we rescued a recombinant CDV (QN vaccine strain) using reverse genetics. The recombinant CDV (rCDV-VP2) was demonstrated to express stably the VP2 in cells for at least 33 serial passages in vitro. Unfortunately, a nonsense mutation was initially identified in the VP2 open reading frame (ORF) at passage-34 (P34) and gradually became predominant in rCDV-VP2 quasispecies with passaging. Neither test strip detection nor indirect immunofluorescence assay demonstrated the expression of the VP2 at P50. The P50 rCDV-VP2 was subjected to next-generation sequencing, which totally identified 17 single-nucleotide variations (SNVs), consisting of 11 transitions and 6 transversions. Out of the 17 SNVs, 1 and 9 were identified as nonsense and missense mutations, respectively. Since the nonsense mutation arose in the VP2 ORF as early as P34, an earlier rCDV-VP2 progeny should be selected for the vaccination of animals in future experiments.

Expressing the pro-apoptotic Reaper protein via insertion into the structural open reading frame of Sindbis virus reduces the ability to infect Aedes aegypti mosquitoes

Arboviruses continue to threaten a significant portion of the human population, and a better understanding is needed of the determinants of successful arbovirus infection of arthropod vectors. Avoiding apoptosis has been shown to be one such determinant. Previous work showed that a Sindbis virus (SINV) construct called MRE/rpr that expresses the pro-apoptotic protein Reaper via a duplicated subgenomic promoter had a reduced ability to orally infect Aedes aegypti mosquitoes at 3 days post-blood meal (PBM), but this difference diminished over time as virus variants containing deletions in the inserted reaper gene rapidly predominated. The goal of this study was to generate a SINV construct that more stably expressed Reaper, in order to further clarify the effect of midgut apoptosis on disseminated infection in Ae. aegypti. We did this by inserting reaper as an in-frame fusion into the structural open reading frame (ORF) of SINV. This construct, MRE/rprORF, successfully expressed Reaper, replicated similarly to MRE/rpr in cell lines, and induced apoptosis in cultured cells and in mosquito midgut tissue. Mosquitoes that fed on blood containing MRE/rprORF developed less midgut and disseminated infection when compared to MRE/rpr or a control virus up to at least 7 days PBM, when less than 50% of mosquitoes that ingested MRE/rprORF had detectable disseminated infection, compared with around 80% or more of mosquitoes fed with MRE/rpr or control virus. However, virus titer in mosquitoes infected with MRE/rprORF was not significantly different from control virus, suggesting that induction of apoptosis by expression of Reaper by this method can reduce infection prevalence, but if infection is established then apoptosis induced by this method has limited ability to continue to suppress replication.

Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

Variants of the HNF4A and HNF1A genes in patients with impaired glucose metabolism and dyslipidemia

Maturity onset diabetes of the young is a dominantly inherited form of monogenic diabetes, diagnosed mainly before the age of 35 years. Mutations in the HNF1A and HNF4A genes are associated with diabetes mellitus of the HNF1A-MODY and HNF4A-MODY subtypes, respectively. These two forms of MODY are characterized by dyslipidemia in addition to impaired glucose metabolism due to the altered function HNF1A and HNF4A proteins. The aim of this study was a genetic analysis of young patients with the MODY phenotype and dyslipidemia with a burdened family history. Material and methods. The probands underwent targeted DNA sequencing using the Illumina MiSeq NGS System. The target panel included the coding regions and splicing sites of MODY-associated genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, KCNJ11, ABCC8, and APPL1. Results. A heterozygous single nucleotide deletion NM_000457.4: c.153del (3’rule) was found in proband P1 in the HNF4A gene. In proband P2, single nucleotide deletion NM_000545.8: c.335del (3 ‘rule) in the HNF1A gene was detected in a heterozygous state. Both variants are located in the coding parts of the genes, led to a shift in the reading frame and have not been described in the literature and databases earlier. Conclusions. Taking into account the phenotypic features of probands, we assume that the variants NM_000545.8: c.335del (rule 3) in the HNF1A gene and NM_000457.4: c.153del (rule 3) of the HNF4A gene are associated with different MODY subtypes in these individuals. After verification of MODY-HNF1A and MODY-HNF4A diagnosis, it is necessary to monitor the lipid profile parameters (total cholesterol, low and high density lipoprotein cholesterol, triglycerides) and prescribe appropriate drug therapy.

HDAC6 Interacts With Poly (GA) and Modulates its Accumulation in c9FTD/ALS

The aberrant translation of a repeat expansion in chromosome 9 open reading frame 72 (C9orf72), the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), results in the accumulation of toxic dipeptide repeat (DPR) proteins in the central nervous system We have found that, among the sense DPR proteins, HDAC6 specifically interacts with the poly (GA) and co-localizes with inclusions in both patient tissue and a mouse model of this disease (c9FTD/ALS). Overexpression of HDAC6 increased poly (GA) levels in cultured cells independently of HDAC6 deacetylase activity, suggesting that HDAC6 can modulate poly (GA) pathology through a mechanism that depends upon their physical interaction. Moreover, decreasing HDAC6 expression by stereotaxic injection of antisense oligonucleotides significantly reduced the number of poly (GA) inclusions in c9FTD/ALS mice. These findings suggest that pharmacologically reducing HDAC6 levels could be of therapeutic value in c9FTD/ALS.

Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

Molecular Characterization of a Novel Cathepsin L in Macrobrachium nipponense and Its Function in Ovary Maturation

Cathepsin L genes, which belonged to cysteine proteases, were a series of multifunctional protease and played important roles in a lot of pathological and physiological processes. In this study, we analyzed the characteristics a cathepsin L (named Mn-CL2) in the female oriental river prawn, Macrobrachium nipponense which was involved in ovary maturation. The Mn-CL2 was1,582 bp in length, including a 978 bp open reading frame that encoded 326 amino acids. The Mn-CL2 was classified into the cathepsin L group by phylogenetic analysis. Real-time PCR (qPCR) analysis indicated that Mn-CL2 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different ovarian stages, Mn-CL2 expression in the hepatopancreas and ovaries peaked before ovarian maturation. In situ hybridization studies revealed that Mn-CL2 was localized in the oocyte of the ovary. Injection of Mn-CL2 dsRNA significantly reduced the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CL2 dsRNA on ovary maturation. These results suggest that Mn-CL2 has a key role in promoting ovary maturation.

Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

Characterization of a C-Type Lectin Domain-Containing Protein with Antibacterial Activity from Pacific Abalone (Haliotis discus hannai)

Genes that influence the growth of Pacific abalone (Haliotis discus hannai) may improve the productivity of the aquaculture industry. Previous research demonstrated that the differential expression of a gene encoding a C-type lectin domain-containing protein (CTLD) was associated with a faster growth in Pacific abalone. We analyzed this gene and identified an open reading frame that consisted of 145 amino acids. The sequence showed a significant homology to other genes that encode CTLDs in the genus Haliotis. Expression profiling analysis at different developmental stages and from various tissues showed that the gene was first expressed at approximately 50 days after fertilization (shell length of 2.47 ± 0.13 mm). In adult Pacific abalone, the gene was strongly expressed in the epipodium, gill, and mantle. Recombinant Pacific abalone CTLD purified from Escherichia coli exhibited antimicrobial activity against several Gram-positive bacteria (Bacillus subtilis, Streptococcus iniae, and Lactococcus garvieae) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). We also performed bacterial agglutination assays in the presence of Ca2+, as well as bacterial binding assays in the presence of the detergent dodecyl maltoside. Incubation with E. coli and B. subtilis cells suggested that the CTLD stimulated Ca2+-dependent bacterial agglutination. Our results suggest that this novel Pacific abalone CTLD is important for the pathogen recognition in the gastropod host defense mechanism.

Loss of p19Arf Promotes Fibroblast Survival During Leucine Deprivation

Fibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here we show that prolonged leucine deprivation resulted in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation.