Search Results (9)

Echinostomatidae is a taxonomically complex group with substantial species diversity and richness. The vast majority of species in this family parasitize birds and mammals, including humans, causing significant economic losses and medical costs. In this study, Echinostoma miyagawai (Digenea, Echinostomatidae) and Patagifer bilobus (Digenea, Echinostomatidae) were isolated from domestic duck and Grus japonensis, respectively. The nearly complete ribosomal transcription unit (rTU) sequences of two echinostomes were obtained, with the rTU for P. bilobus being obtained for the first time. The nearly complete rTU sequence of P. bilobus (6790 bp) and E. miyagawai (6893 bp) encompass the small-subunit (18S) ribosomal DNA (rDNA), internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and large-subunit (28S) rDNA. The complete lengths of 18S, ITS1, 5.8S, ITS2, and 28S sequences for E. miyagawai are 1989 bp, 444 bp, 162 bp, 431 bp, and 3858 bp, respectively. For P. bilobus, complete or nearly complete lengths of these sequences are 1929 bp (nearly complete), 419 bp, 162 bp, 432 bp, and 3848 bp (nearly complete), respectively. The 18S, ITS, and 28S sequences of E. miyagawai show the highest sequence similarity with other E. miyagawai. The ITS and 28S sequences of P. bilobus show the highest sequence similarity with other P. bilobus, while 18S sequence shows the highest similarity with E. miyagawai. This is likely due to the unavailability of the 18S sequence of P. bilobus in GenBank. Repeat sequences were identified in 18S, ITS1, ITS2, and 28S sequences, with the 28S sequence containing the most repeats and the 5.8S sequence having none. The results of phylogenetic reconstruction indicated that E. miyagawai clusters with other Echinostoma spp., while P. bilobus clusters with other Patagifer spp., forming sister taxa. This study not only provides the first rTU sequence for P. bilobus but also reinforces the sister group status of Patagifer to Echinostoma through phylogenetic evidence. Finally, this study represents the first record of the G. japonensis as a new host for P. bilobus and the first report of a bird from the crane family (Gruidae) as a host for any echinostome species. These findings are significant as they expand our understanding of the host range and ecological interactions of Echinostomatidae. The data obtained provide a valuable resource of molecular markers for studying the taxonomy, population genetics, and systematics of the family Echinostomatoidea. This research contributes to a more comprehensive understanding of the evolutionary relationships and biodiversity within this complex group of parasites, which is crucial for developing effective strategies to mitigate their impact on both wildlife and human health. Full article

Argonaute proteins, guided by small RNAs, play crucial roles in gene regulation and genome protection through RNA interference (RNAi)-related mechanisms. Ribosomal RNAs (rRNAs), encoded by repeated rDNA units, constitute the core of the ribosome being the most abundant cellular transcripts. rDNA clusters also serve as sources of small RNAs, which are loaded into Argonaute proteins and are able to regulate rDNA itself or affect other gene targets. In this review, we consider the impact of small RNA pathways, specifically siRNAs and piRNAs, on rRNA gene regulation. Data from diverse eukaryotic organisms suggest the potential involvement of small RNAs in various molecular processes related to the rDNA transcription and rRNA fate. Endogenous siRNAs are integral to the chromatin-based silencing of rDNA loci in plants and have been shown to repress rDNA transcription in animals. Small RNAs also play a role in maintaining the integrity of rDNA clusters and may function in the cellular response to rDNA damage. Studies on the impact of RNAi and small RNAs on rRNA provide vast opportunities for future exploration. Full article

The keystone of ribosome biogenesis is the transcription of 45S rDNA. The Arabidopsis thaliana genome contains hundreds of 45S rDNA units; however, they are not all transcribed. Notably, 45S rDNA units contain insertions/deletions revealing the existence of heterogeneous rRNA genes and, likely, heterogeneous ribosomes for rRNAs. In order to obtain an overall picture of 45S rDNA diversity sustaining the synthesis of rRNAs and, subsequently, of ribosomes in natura, we took advantage of 320 new occurrences of Arabidopsis thaliana as a metapopulation named At66, sampled from 0 to 1900 m of altitude in the eastern Pyrenees in France. We found that the 45S rDNA copy number is very dynamic in natura and identified new genotypes for both 5′ and 3′ External Transcribed Spacers (ETS). Interestingly, the highest 5′ETS genotype diversity is found in altitude while the highest 3′ETS genotype diversity is found at sea level. Structural analysis of 45S rDNA also shows conservation in natura of specific 5′ETS and 3′ETS sequences/features required to control rDNA expression and the processing of rRNAs. In conclusion, At66 is a worthwhile natural laboratory, and unraveled 45S rDNA diversity represents an interesting starting material to select subsets for rDNA transcription and alter the rRNA composition of ribosomes both intra- and inter-site. Full article

Transcriptome studies have reported the dysregulation of cell cycle-related genes and the global inhibition of host mRNA translation in COVID-19 cases. However, the key genes and cellular mechanisms that are most affected by the severe outcome of this disease remain unclear. For this work, the RNA-seq approach was used to study the differential expression in buffy coat cells of two groups of people infected with SARS-CoV-2: (a) Mild, with mild symptoms; and (b) SARS (Severe Acute Respiratory Syndrome), who were admitted to the intensive care unit with the severe COVID-19 outcome. Transcriptomic analysis revealed 1009 up-regulated and 501 down-regulated genes in the SARS group, with 10% of both being composed of long non-coding RNA. Ribosome and cell cycle pathways were enriched among down-regulated genes. The most connected proteins among the differentially expressed genes involved transport dysregulation, proteasome degradation, interferon response, cytokinesis failure, and host translation inhibition. Furthermore, interactome analysis showed Fibrillarin to be one of the key genes affected by SARS-CoV-2. This protein interacts directly with the N protein and long non-coding RNAs affecting transcription, translation, and ribosomal processes. This work reveals a group of dysregulated processes, including translation and cell cycle, as key pathways altered in severe COVID-19 outcomes. Full article

In this study, we invented and construct novel candidate HIV-1 vaccines. Through genetic and protein engineering, we unknowingly constructed an HIV-1-derived transgene with a homopolymeric run of 11 cytidines, which was inserted into an adenovirus vaccine vector. Here, we describe the virus rescue, three rounds of clonal purification and preparation of good manufacturing practise (GMP) starting material assessed for genetic stability in five additional virus passages. Throughout these steps, quality control assays indicated the presence of the transgene in the virus genome, expression of the correct transgene product and immunogenicity in mice. However, DNA sequencing of the transgene revealed additional cytidines inserted into the original 11-cytidine region, and the GMP manufacture had to be aborted. Subsequent analyses indicated that as little as 1/25th of the virus dose used for confirmation of protein expression (10⁶ cells at a multiplicity of infection of 10) and murine immunogenicity (10⁸ infectious units per animal) met the quality acceptance criteria. Similar frameshifts in the expressed proteins were reproduced in a one-reaction in vitro transcription/translation employing phage T7 polymerase and E. coli ribosomes. Thus, the most likely mechanism for addition of extra cytidines into the ChAdOx1.tHIVconsv6 genome is that the adenovirus DNA polymerase lost its fidelity on a stretch of 11 cytidines, which informs future adenovirus vaccine designs. Full article

In human cells, each rDNA unit consists of the ~13 kb long ribosomal part and ~30 kb long intergenic spacer (IGS). The ribosomal part, transcribed by RNA polymerase I (pol I), includes genes coding for 18S, 5.8S, and 28S RNAs of the ribosomal particles, as well as their four transcribed spacers. Being highly repetitive, intensively transcribed, and abundantly methylated, rDNA is a very fragile site of the genome, with high risk of instability leading to cancer. Multiple small mutations, considerable expansion or contraction of the rDNA locus, and abnormally enhanced pol I transcription are usual symptoms of transformation. Recently it was found that both IGS and the ribosomal part of the locus contain many functional/potentially functional regions producing non-coding RNAs, which participate in the pol I activity regulation, stress reactions, and development of the malignant phenotype. Thus, there are solid reasons to believe that rDNA locus plays crucial role in carcinogenesis. In this review we discuss the data concerning the human rDNA and its closely associated factors as both targets and drivers of the pathways essential for carcinogenesis. We also examine whether variability in the structure of the locus may be blamed for the malignant transformation. Additionally, we consider the prospects of therapy focused on the activity of rDNA. Full article

The bean (Phaseolus vulgaris) pathogen Pseudomonas syringae pv. phaseolicola NPS3121 synthesizes phaseolotoxin in a thermoregulated way, with optimum production at 18 °C. Gene PSPPH_4550 was previously shown to be thermoregulated and required for phaseolotoxin biosynthesis. Here, we established that PSPPH_4550 is part of a cluster of 16 genes, the Pbo cluster, included in a genomic island with a limited distribution in P. syringae and unrelated to the possession of the phaseolotoxin biosynthesis cluster. We identified typical non-ribosomal peptide synthetase, and polyketide synthetase domains in several of the pbo deduced products. RT-PCR and the analysis of polar mutants showed that the Pbo cluster is organized in four transcriptional units, including one monocistronic and three polycistronic. Operons pboA and pboO are both essential for phaseolotoxin biosynthesis, while pboK and pboJ only influence the amount of toxin produced. The three polycistronic units were transcribed at high levels at 18 °C but not at 28 °C, whereas gene pboJ was constitutively expressed. Together, our data suggest that the Pbo cluster synthesizes secondary metabolite(s), which could participate in the regulation of phaseolotoxin biosynthesis. Full article

Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid–host interactions. Full article

Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and gene therapy purposes. A majority of these NDV vectors express only a single FG or two FGs from suboptimal insertion sites in the NDV genome, obtaining various levels of FG expression. To improve the FG expression, we generated NDV LaSota vaccine strain-based recombinant viruses to express two FGs, green fluorescent protein (GFP) and red fluorescent protein (RFP) genes, from the identified optimal insertion sites, through a combination of the independent transcription unit (ITU) and the internal ribosomal entry site (IRES) dependent expression approaches. Biological assessments showed that these recombinants expressing two FGs were slightly attenuated with approximately one order of magnitude lower in virus titers than those containing a single FG. The FG expression efficiencies from two-FG viruses were also lower than those from the single-FG viruses. However, the expression of two FGs from the optimal insertion sites was significantly (p < 0.05) higher than those from the suboptimal insertion sites. The expression of FGs through the ITU approach was approximately 4-fold more efficient than that through the IRES-dependent approach. These results suggest that the NDV LaSota vector could efficiently express two FGs from the identified optimal insertions sites. The ITU strategy could be used for the expression of a higher amount of FG products, whereas the IRES tactic might be useful when a lower amount of FG products are needed. Full article