Search Results (96)

Background: The mammalian mitochondrial genome has long been considered to encode only 13 proteins. However, a recent study identified a nested alternative open reading frame (nAltORF) within the primate mitochondrial cytb gene, which we designate ncytb, that is reportedly translated in the cytosol using the standard genetic code. This discovery challenges conventional understanding and raises questions about the prevalence, conservation, and translational adaptation of such ORFs. Methods: This study conducted a comprehensive bioinformatic analysis of nested ncytb genes in 289 primate and 380 rodent mitochondrial cytb sequences. Results: Nested ncytb genes meeting the criteria (>150 codons, standard genetic code) were identified in only 10.73% of primate and 20.53% of rodent species, suggesting a patchy phylogenetic distribution. While their encoded proteins showed homology to the previously reported protein encoded by the Homo sapiens nested ncytb gene, overall amino acid conservation was low, and characteristic protein domains or signal peptides were generally not predicted. Crucially, the Kozak consensus sequences surrounding the putative start codons of these ncytb genes were exclusively “weak” or “adequate”, with none classified as “strong” or “optimal”. Codon Adaptation Index (CAI) and Relative Codon Deoptimization Index (RCDI) analyses of the nested ncytb genes revealed neither significant adaptation nor deoptimization to the codon usage of nuclear and mitochondrial genes. Furthermore, cosine similarity analysis indicated that ncytb genes exhibit significantly lower codon usage similarity to both nuclear and mitochondrial gene sets compared to their host cytb genes. Conclusions: These findings collectively suggest that while ncytb genes exist in some mammals, their inconsistent presence, weak translational initiation signals, and lack of adaptation to cytosolic codon usage characterize them as dispensable genetic elements rather than core functional genes. Full article

Parkinson’s disease (PD) is the second most common neurodegenerative disease. It primarily affects the motor system but is also associated with a range of cognitive impairments that can manifest early in disease progression, indicating its multifaceted nature. In this paper, we performed a meta-analysis of transcriptomics and proteomics data using MultiOmicsIntegrator to gain insights into the post-transcriptional modifications and deregulated pathways associated with this disease. Our results reveal differential isoform usage between control and PD patient brain samples that result in enriched alternative splicing events, including an extended UTR length, domain loss, and the upregulation of non-coding isoforms. We found that Inositol-Requiring Enzyme 1 (IRE1) is active in PD samples and examined the role of its downstream signaling through X-box binding mRNA 1 (XBP1) and regulated IRE1-dependent decay (RIDD). We identified several RIDD candidates and showed that the enriched alternative splicing events observed are associated with RIDD. Moreover, in vitro mRNA cleavage assays demonstrated that OSBPL3, C16orf74, and SLC6A1 mRNAs are targets of IRE1 RNAse activity. Finally, a pathway enrichment analysis of both XBP1s and RIDD targets in the PD samples uncovered associations with processes such as immune response, oxidative stress, signal transduction, and cell–cell communication that have previously been linked to PD. These findings highlight a potential regulatory role of IRE in PD. Full article

The high pathogenicity rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA) has resulted in substantial economic losses for humans and the breeding industry. Consequently, there is an urgent need to develop new alternatives to mitigate antibiotic use. Phage therapy has demonstrated promising results in numerous studies. In this study, lytic phages targeting CRPA were isolated from feces and river water samples in Shandong, China. A total of 94 phage strains with CRPA as hosts were obtained, exhibiting lysis rates that ranged from 29% to 76% for P. aeruginosa derived from humans and different types of animals (n = 246). We further examined five representative phages, the host bacteria of which were CRPA from clinical patients and poultry, and these phages included two myoviruses and three podoviruses. Their optimal multiplicities of infection (MOIs) ranged from 10⁻³ to 10⁻⁵, with latent periods of less than 5 to 15 min and burst durations of 140 to 175 min, resulting in burst sizes of 133 to 352 PFU/cell. All five phages exhibited the ability to survive at temperatures up to 60 °C and within pH levels of 3 to 11. Whole-genome sequencing revealed that these five phages were all double-stranded DNA phages and did not possess resistance genes or virulence factors. The two myoviruses, sharing similar sequences, were classified into the genus Pakpunavirus, with a size of 92,509 bp and 92,293 bp, 149 to 152 ORFs and 20 to 22 tRNAs. In contrast, the three similar podoviruses belong to the genus Phikmvvirus and all contained a perforin–lyase system, with a size of 43.35 kb, a GC content of 62%, 49 to 50 ORFs and 16 to 20 tRNAs. A spray disinfection experiment demonstrated that the phage cocktail exhibited a high sterilization effect after spraying and showed good efficacy against cement and metal surfaces. This study provides foundational information for further research into the elimination of CRPA in the environment. Full article

Long non-coding RNA (lncRNA) is a non-coding RNA longer than 200 nucleotides, crucial for functions like cell cycle regulation and gene transcription. Accurate localization prediction from sequence information is vital for understanding lncRNA’s biological roles. Computational methods offer an effective alternative to traditional experimental methods for annotating lncRNA subcellular positions. Existing machine learning-based methods are limited and often overlook regions with coding potential that affect the function of lncRNA. Therefore, we propose a new model called LncSL. For feature encoding, both lncRNA sequences and amino acid sequences from open reading frames (ORFs) are employed. And we selected the most suitable features by CatBoost and integrated them into a new feature set. Additionally, a voting process with seven feature selection algorithms identified the higher contributive features for training our final stacked model. Additionally, an automatic model selection strategy is constructed to find a better performance meta-model for assembling LncSL. This study specifically focuses on predicting the subcellular localization of lncRNA in the nucleus and cytoplasm. On two benchmark datasets called S1 and S2 datasets, LncSL outperformed existing methods by 6.3% to 12.3% in the Matthew’s correlation coefficient on a balanced test dataset. On an unbalanced independent test dataset sourced from S1, LncSL improved by 4.7% to 18.6% in the Matthew’s correlation coefficient, which further demonstrates that LncSL is superior to other compared methods. In all, this study presents an effective method for predicting lncRNA subcellular localization through enhancing sequence information, which is always overlooked by traditional methods, and addressing contributive meta-model selection problems, which can offer new insights for other bioinformatics problems. Full article

Background/Objectives: Antimicrobial resistance (AMR) is a major public health threat, which is exacerbated by the lack of new antibiotics and the emergence of multidrug-resistant (MDR) superbugs. Comprehensive efforts and alternative strategies to combat AMR are urgently needed to prevent social, medical, and economic consequences. Pseudomonas aeruginosa is a pathogen responsible for a wide range of infections, from soft tissue infections to life-threatening conditions such as bacteremia and pneumonia. Bacteriophages have been considered as a potential therapeutic option to treat bacterial infections. Our aim was to isolate phages able to infect MDR P. aeruginosa strains. Methods: We isolated two lytic phages, using the conventional double layer agar technique (DLA), from samples obtained from the influent of a wastewater treatment plant in Concepción, Chile. The phages, designated as PaCCP1 and PaCCP2, were observed by electron microscopy and their host range was determined against multiple P. aeruginosa strains using DLA. Moreover, their genomes were sequenced and analyzed. Results: Phage PaCCP1 is a member of the Septimatrevirus genus and phage PaCCP2 is a member of the Pbunavirus genus. Both phages are tailed and contain dsDNA. The genome of PaCCP1 is 43,176 bp in length with a GC content of 54.4%, encoding 59 ORFs, one of them being a tRNA gene. The genome of PaCCP2 is 66,333 bp in length with a GC content of 55.6%, encoding 102 non-tRNA ORFs. PaCCP1 is capable of infecting five strains of P. aeruginosa, whereas phage PaCCP2 is capable of infecting three strains of P. aeruginosa. Both phages do not contain bacterial virulence or AMR genes and contain three and six putative Anti-CRISPR proteins. Conclusions: Phages PaCCP1 and PaCCP2 show promise as effective treatments for MDR P. aeruginosa strains, offering a potential strategy for controlling this clinically important pathogen through phage therapy. Full article

Hairy vetch (Vicia villosa Roth) and smooth vetch (V. villosa Roth var. glabrescens) are important cover crops and legume forage with great economic and ecological values. Due to the large and highly heterozygous genome, full-length transcriptome reconstruction is a cost-effective route to mining their genetic resources. In this study, a hybrid sequencing approach combining SMRT and NGS technologies was applied. The results showed that 28,747 and 40,600 high-quality non-redundant transcripts with an average length of 1808 bp and 1768 bp were generated from hairy vetch and smooth vetch, including 24,864 and 35,035 open reading frames (ORFs), respectively. More than 96% of transcripts were annotated to the public databases, and around 25% of isoforms underwent alternative splicing (AS) events. In addition, 987 and 1587 high-confidence lncRNAs were identified in two vetches. Interestingly, smooth vetch contains more specific transcripts and orthologous clusters than hairy vetch, revealing intraspecific transcript diversity. The phylogeny revealed that they were clustered together and closely related to the genus Pisum. Furthermore, the estimation of Ka/Ks ratios showed that purifying selection was the predominant force. A putative 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHD/SDH) gene underwent strong positive selection and might regulate phenotypic differences between hairy vetch and smooth vetch. Overall, our study provides a vital characterization of two full-length transcriptomes in Vicia villosa, which will be valuable for their molecular research and breeding. Full article

The rise of antibiotic-resistant bacterial infections necessitates alternative therapeutic strategies, such as phage therapy. This study investigates the potential of phage vB_PmuM_CFP3 (CFP3) as a therapeutic agent against avian cholera caused by Pasteurella multocida (P. multocida). Phage CFP3 was isolated from the feces and wastewater of a laying hen farm and underwent comprehensive biological characterization, including host range, lytic activity, and environmental stability. Transmission electron microscopy revealed CFP3′s typical myovirus morphology, with a head diameter of approximately 60 nm and a tail length of about 120 nm. CFP3 demonstrated high stability across a pH range of 4–10 and temperatures of 30–40 °C, making it suitable for oral administration in poultry. The phage exhibited a latent period of about 90 min and an optimal multiplicity of infection (MOI) of 1. Despite its narrow host range, with a lysis rate of 28.2% against avian-derived type A P. multocida, CFP3′s specificity minimizes impact on non-target bacteria. Whole-genome sequencing revealed a 32,696 bp linear double-stranded DNA genome with 46 predicted open reading frames (ORFs) and no tRNA or antibiotic resistance genes, enhancing its safety profile. Phylogenetic analysis indicated a close evolutionary relationship with Haemophilus phages HP1, HP2, and Pasteurella phage F108. While CFP3 shows promise as a precision therapeutic tool, further in vivo studies are required to evaluate its efficacy and safety. Future research should focus on expanding the phage library, optimizing phage mixtures, and exploring synergistic effects with other antimicrobial strategies. This study provides foundational data supporting the development of CFP3 as a viable alternative to antibiotics for controlling avian cholera. Full article

Pseudomonas aeruginosa is a bacteria responsible for many hospital-acquired infections. Phages are promising alternatives for treating P. aeruginosa infections, which are often intrinsically resistant. The combination of phage and antibiotics in clearing bacterial infection holds promise due to increasing reports of enhanced effectiveness when both are used together. The aim of the study is to isolate and characterize a novel P. aeruginosa phage and determine its effectiveness in in vitro combination with antibiotics in controlling P. aeruginosa. In this study, a novel jumbo myophage HPP-Temi infecting P. aeruginosa Pa9 (PP334386) was isolated from household sewage. Electron micrographs of the phage were obtained to determine the morphological features of HPP-Temi virions. Complete genome analysis and a combination of Pseudomonas phage HPP-Temi with antibiotics were examined. The phage HPP-Temi was able to productively infect P. aeruginosa ATCC 9027 but was unable to infect a closely related genus. The phage was stable at 4–37 °C, 0.5% NaCl, and pH 8 for at least one hour. The HPP-Temi genome is a 302,719-bp-long dsDNA molecule with a GC content of 46.46%. The genome was predicted to have 436 ORFs and 7 tRNA genes. No virulence factor-related genes, antimicrobial resistance, or temperate lifestyle-associated genes were found in the phage HPP-Temi genome. Phage HPP-Temi is most closely related to the known or tentative representatives of the Pawinskivirus genus and can be proposed as a representative for the creation of a novel phage species in that genus. The phage and antibiotics (Ciprofloxacin) combination at varying phage titers (10³, 10⁶, 10⁹) were used against P. aeruginosa Pa9 (PP334386) at 3.0 × 10⁸ CFU/mL, which was carried out in triplicate. The result showed that combining antibiotics with phage significantly reduced the bacteria count at 10³ and 10⁶ titers, while no growth was observed at 10⁹ PFU/mL. This suggests that the effect of phage HPP-Temi in combination with antibiotics is a potential and promising agent for the control of P. aeruginosa infections. Full article

The 18-exon human TKFC gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as tkfc type by their intron-17 splice), databases contain evidence for alternative TKFC transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no. DQ344550). It results from an alternative 3′ splice-site in intron 17. The cloned cDNA contains an ORF also spanning from exon 2 to exon 18 of the TKFC gene but with a 56-nt insertion between exons 17 and 18 (classified as tkfc_ins56 type). This insertion introduces an in-frame stop, and the resulting ORF codes for a shorter TKFC variant, which, after expression, is enzymatically inactive. TKFC intron-17 splicing was found to be differentially expressed in human tissues. In a multiple-tissue northern blot using oligonucleotide probes, the liver showed a strong expression of the tkfc-like splice of intron 17, and the heart preferentially expressed the tkfc_ins56-like splice. Through a comparison to global expression data from massive-expression studies of human tissues, it was inferred that the intestine preferentially expresses TKFC transcripts that contain neither of those splices. An analysis of transcript levels quantified by RNA-Seq in the GTEX database revealed an exception to this picture due to the occurrence of a non-coding short transcript with a tkfc-like splice. Altogether, the results support the occurrence of potentially relevant transcript variants of the TKFC gene, differentially expressed in human tissues. (This work is dedicated in memoriam to Professor Antonio Sillero, 1938–2024, for his lifelong mentoring and his pioneering work on triokinase). Full article

Baculoviral vectors (BVs) derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability. Full article

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) has emerged with the rise of the aquaculture of Atlantic salmon (Salmo salar). The lack of cell culture cultivation has hampered the study of this infection. In this study, samples from naturally PMCV-infected Atlantic salmon from different commercial farms were collected and used. In situ hybridization (ISH) revealed intense staining of PMCV RNA in myocardial cells in the spongiform layer of the heart ventricle but almost no staining in the compact layer. In the kidneys, only sporadic staining was seen. Viral RNA was present in all organs, with the highest loads in the heart, kidney, and spleen. The high viral PMCV RNA loads in the heart were due to extensive viral mRNA transcription. The high ratio of viral mRNA to viral genomic dsRNA indicated active transcription but limited production of new viral particles. This suggests that the histopathological changes in the heart are caused by viral mRNA and corresponding viral proteins and not by virus particle formation. The production of full-length transcripts is regulated, with a reduction in the relative number of ORF3-containing transcripts at high transcription rates. Efforts to identify alternative hosts, such as fungi, were inconclusive, as fungal sequences were found inconsistently in the salmon tissue samples. The results of this study reinforce the need for further research to fully understand PMCV’s life cycle and potential alternative hosts and its whereabouts when it is not infecting the hearts of the Atlantic salmon. Full article

Feline calicivirus (FCV) is a highly contagious virus that causes upper respiratory tract disease, commonly known as cat flu. It is widely distributed worldwide and poses a major threat to feline health. Therefore, it is essential to find an efficient and rapid method for detecting FCV. In this study, the colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, using neutral red as an indicator, was developed and validated to target the ORF2 gene of FCV for the first time. Additionally, the study compared the diagnostic abilities of polymerase chain reaction (PCR), nested PCR, and RT-LAMP assays for detecting FCV in clinical samples. The optimized RT-LAMP amplification was carried out at 56.3 °C. The technique visually detected FCV within 70 min, with a limit of detection of 14.3 × 10¹ copies/µL, and showed no cross-reactivity with other feline pathogens. Out of 54 oropharyngeal swab samples, 17 tested positive for FCV using both nested PCR and RT-LAMP, while only one tested positive using conventional PCR. The positivity rate was higher with nested PCR and RT-LAMP (31.48%) compared to conventional PCR (1.85%). Consequently, these results demonstrated the effectiveness of the colorimetric RT-LAMP assay developed in this study as an alternative for diagnosing FCV in cats. Full article

The intrinsically disordered polyglutamine-binding protein 1 (PQBP1) has been linked to various cellular processes including transcription, alternative splicing, translation and innate immunity. Mutations in PQBP1 are causative for neurodevelopmental conditions collectively termed as the Renpenning syndrome spectrum. Intriguingly, cells of Renpenning syndrome patients exhibit a reduced innate immune response against human immunodeficiency virus 1 (HIV-1). PQBP1 is responsible for the initiation of a two-step recognition process of HIV-1 reverse-transcribed DNA products, ensuring a type 1 interferon response. Recent investigations revealed that PQBP1 also binds to the p17 protein of avian reovirus (ARV) and is affected by the ORF52 of Kaposi’s sarcoma-associated herpesvirus (KSHV), possibly also playing a role in the innate immune response towards these RNA- and DNA-viruses. Moreover, PQBP1-mediated microglia activation in the context of tauopathies has been reported, highlighting the role of PQBP1 in sensing exogenous pathogenic species and innate immune response in the central nervous system. Its unstructured nature, the promiscuous binding of various proteins and its presence in various tissues indicate the versatile roles of PQBP1 in cellular regulation. Here, we systematically review the available data on the structure of PQBP1 and its cellular functions and interactome, as well as possible implications for innate immune responses and neurodegenerative disorders. Full article

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome. Full article

The B169L protein (pB169L) of African swine fever virus (ASFV) is a structural protein with an unidentified function during the virus replication. The sequences of the B169L gene and the downstream B438L gene are separated by short intergenic regions. However, the regulatory mode of the gene transcription remains unknown. Here, we identified two distinct promoter regions and two transcription start sites (TSSs) located upstream of the open reading frame (ORF) of B438L. Using the promoter reporter system, we demonstrated that the cis activity of the ORF proximal promoter exhibited significantly higher levels compared with that of the distal promoter located in the B169L gene. Furthermore, transfection with the plasmids with two different promoters for B438L could initiate the transcription and expression of the B438L gene in HEK293T cells, and the cis activity of the ORF proximal promoter also displayed higher activities compared with the distal promoter. Interestingly, the B438L distal promoter also initiated the transcription of the alternatively spliced B169L mRNA (B169L mRNA2) encoding a truncated pB169L (tpB169L) (amino acids 92–169), and the gene transcription efficiency was increased upon mutation of the initiation codon located upstream of the alternatively spliced B169L gene. Taken together, we demonstrated that the distal promoter of B438L gene initiates the transcription of both the B438L mRNA and B169L mRNA2. Comprehensive analysis of the transcriptional regulatory mode of the B438L gene is beneficial for the understanding of the association of B438L protein and pB169L and the construction of the gene-deleted ASFV. Full article