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.

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.

Neurotoxic Amyloidogenic Peptides in the Proteome of SARS-COV2: Potential Implications for Neurological Symptoms in COVID-19

COVID-19 is primarily known as a respiratory disease caused by the virus SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, cognitive and psychiatric issues, severe headaches, and even stroke are reported in as many as 30% of cases and can persist even after the infection is over (so-called ‘long COVID’). These neurological symptoms are thought to be caused by brain inflammation, caused by the virus infecting the central nervous system of COVID-19 patients, however we still don’t understand the molecular mechanisms that trigger these symptoms. The neurological effects of COVID-19 share many similarities to neurodegenerative diseases such as Alzheimer’s and Parkinson’s in which the presence of cytotoxic protein-based amyloid aggregates is a common etiological feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we performed a bioinformatic scan of the SARS-CoV-2 proteome, detecting peptide fragments that were predicted to be highly amyloidogenic. We selected two of these peptides from the open reading frame 6 (ORF6) and open reading frame 10 (ORF10) proteins. The amyloidogenic virus-derived proteins studied in this work did not include spike (S) protein or any other proteins that have been modified to function as antigens in any current vaccines. We discovered that these ORF protein fragments rapidly self-assemble into amyloid aggregates. Furthermore, these amyloid assemblies were shown to be highly toxic to a neuronal cell line. We introduce and support the idea that cytotoxic amyloid aggregates of SARS-CoV-2 proteins are causing some of the neurological symptoms commonly found in COVID-19 and contributing to long COVID.

Differential expression of chromosome 20 open reading frame 24 in triple negative breast cancer.

Women diagnosed with triple negative breast cancer can benefit neither from endocrine therapy nor from HER2-targeted therapies (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding chromosome 20 open reading frame 24, C20orf24, when comparing the tumor cells of patients with triple negative breast cancer to normal mammary ductal cells (2). C20orf24 was also differentially expressed in bulk tumor in human breast cancer (3). C20orf24 mRNA was present at significantly increased quantities in TNBC tumor cells relative to normal mammary ductal cells. Analysis of human survival data revealed that expression of C20orf24 in primary tumors of the breast was correlated with overall survival in patients with luminal A type cancer, while within triple negative breast cancer, primary tumor expression of C20orf24 was correlated with overall survival in patients with basal-like 1 and immunomodulatory subtype disease. C20orf24 may be of relevance to initiation, maintenance or progression of triple negative breast cancers.

Unveiling a Ghost Proteome in the Glioblastoma Non-Coding RNAs

Glioblastoma is the most common brain cancer in adults. Nevertheless, the median survival time is 15 months, if treated with at least a near total resection and followed by radiotherapy in association with temozolomide. In glioblastoma (GBM), variations of non-coding ribonucleic acid (ncRNA) expression have been demonstrated in tumor processes, especially in the regulation of major signaling pathways. Moreover, many ncRNAs present in their sequences an Open Reading Frame (ORF) allowing their translations into proteins, so-called alternative proteins (AltProt) and constituting the “ghost proteome.” This neglected world in GBM has been shown to be implicated in protein–protein interaction (PPI) with reference proteins (RefProt) reflecting involvement in signaling pathways linked to cellular mobility and transfer RNA regulation. More recently, clinical studies have revealed that AltProt is also involved in the patient’s survival and bad prognosis. We thus propose to review the ncRNAs involved in GBM and highlight their function in the disease.

STUDI BIOINFORMASI URUTAN ASAM AMINO DAN STRUKTUR 3D PROTEIN ALDOLASE KELAS II (AldII) DARI Uncultured Acidilobus sp.

Pada penelitian sebelumnya fragmen gen 1,9 kb telah berhasil diisolasi dari Kawah Domas, Jawa Barat melalui pendekatan metagenom. Fragmen tersebut diketahui mengandung daerah Open Reading Frame (ORF) utuh dari gen pengkode aldolase kelas II dari uncultured Acidilobus sp. yang kemudian disebut sebagai aldII. Fragmen gen aldII tersebut berhasil diekspresikan menjadi protein termostabil aldolase kelas II yang kemudian disebut sebagai AldII. Penelitian ini bertujuan untuk melakukan studi bioinformasi terhadap protein AldII tersebut. Protein AldII kemudian diketahui memiliki massa molekul ~21,2 kDa dengan rumus molekul C940H1539N261O281S8. Total residu bermuatan negatif (Asp + Glu) sebanyak 22 residu, sedangkan total residu bermuatan positif (Arg + Lys) adalah 18 residu. Nilai pI teoritis AldII sebesar 5,86. Hasil perhitungan indeks kestabilan protein ini adalah 36,61 dan diklasifikasikan sebagai protein yang stabil. Lewat penjajaran dengan homologi terdekat, ditemukan daerah lestari yang dapat menunjukan residu yang mungkin berperan dalam pengikatan logam dan sisi aktif. Prediksi struktur 3D dilakukan secara ab initio, menunjukan adanya 6 struktur β-sheet dan 6 struktur α-heliks. Dengan demikian dapat disimpulkan bahwa protein AldII dari uncultured Acidilobus sp. diduga memiliki aktivitas enzimatik.

Mitonuclear Genetic Interactions in the Basidiomycete Heterobasidion parviporum Involve a Non-conserved Mitochondrial Open Reading Frame

The mitochondrial and nuclear genomes of Eukaryotes are inherited separately and consequently follow distinct evolutionary paths. Nevertheless, the encoding of many mitochondrial proteins by the nuclear genome shows the high level of integration they have reached, which makes mitonuclear genetic interactions all the more conceivable. For each species, natural selection has fostered the evolution of coadapted alleles in both genomes, but a population-wise divergence of such alleles could lead to important phenotypic variation, and, ultimately, to speciation. In this study in the Basidiomycete Heterobasidion parviporum, we have investigated the genetic basis of phenotypic variation among laboratory-designed heterokaryons carrying the same pair of haploid nuclei, but a different mitochondrial genome. Radial growth rate data of thirteen unrelated homokaryotic parents and of their heterokaryotic offspring were combined with SNP data extracted from parental genome sequences to identify nuclear and mitochondrial loci involved in mitonuclear interactions. Two nuclear loci encoding mitochondrial proteins appeared as best candidates to engage in a genetic interaction affecting radial growth rate with a non-conserved mitochondrial open reading frame of unknown function and not reported apart from the Russulales order of Basidiomycete fungi. We believe our approach could be useful to investigate several important traits of fungal biology where mitonuclear interactions play a role, including virulence of fungal pathogens.

Translation rescue by targeting Ppp1r15a upstream open reading frame in vivo

The eIF2 initiation complex is central to maintaining a functional translation machinery. Extreme stress such as life-threatening sepsis exposes vulnerabilities in this tightly regulated system, resulting in an imbalance between the opposing actions of kinases and phosphatases on the main regulatory subunit eIF2α. Here, we report that translation shutdown is a hallmark of established sepsis-induced kidney injury brought about by excessive eIF2α phosphorylation and sustained by blunted expression of the counterregulatory phosphatase subunit Ppp1r15a. We determined that the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic approaches enabled the derepression of Ppp1r15a, salvaged translation and improved kidney function in an endotoxemia model. We also found that the loss of this uORF has broad effects on the composition and phosphorylation status of the immunopeptidome that extended beyond the eIF2α axis. Collectively, our findings define the breath and potency of the highly conserved Ppp1r15a uORF and provide a paradigm for the design of uORF-based translation rheostat strategies. The ability to accurately control the dynamics of translation during sepsis will open new paths for the development of therapies at codon level precision.

SARS-CoV-2 Genome Sequences, Including One with an ORF7a Deletion, Obtained from Patients in Bangladesh

Genomic sequences from a complete SARS-CoV-2 open reading frame (ORF) were obtained from 24 patients diagnosed in May 2020 in Dhaka, Bangladesh. All sequences belonged to clade 20A or 20B, and none were variants of concern. Interestingly, one sequence showed a 161-nucleotide deletion in ORF7a.