Coding Sequence Research Articles

Comparative Genome Analysis of Piscine Vibrio vulnificus: Virulence-Associated Metabolic Pathways

Vibriosis caused by Vibrio vulnificus is a major problem in aquatic animals, particularly brown marble groupers (Epinephelus fuscoguttatus). V. vulnificus biotype I has recently been isolated and classified into subgroups SUKU_G1, SUKU_G2, and SUKU_G3 according to the different types of virulence genes. In a previous study, we have shown that biotype I V. vulnificus strains were classified into three subgroups according to the different types of virulence genes, which exhibited different phenotypes in terms of growth rate and virulence. To gain insight into the different genetic features revealed by the potential virulence mechanisms of V. vulnificus in relation to a spectrum of pathogenesis, comparative genomic analyses of three biotype I V. vulnificus strains belonging to different subgroups (SUKU_G1, SUKU_G2, and SUKU_G3) were performed. The V. vulnificus genome is composed of two circular chromosomes with average sizes of 3 Mbp and 1.7 Mbp that are evolutionarily related based on the analysis of orthologous genes. A comparative genome analysis of V. vulnificus revealed 5200 coding sequences, of which 3887 represented the core genome and the remaining 1313 constituted the dispensable genome. The most virulent isolate (SUKU_G1) carries unique enzymes that are important for lipopolysaccharide (LPS) and capsular polysaccharide (CPS) synthesis, as well as flagellar glycosylation, and harbors another type of repeat in toxin (RTX) and bacterial defense mechanisms. The less virulent isolate (SUKU_G2) shares enzymes related to CPS biosynthesis or flagellar glycosylation, while the avirulent isolate (SUKU_G3) and a less virulent isolate (SUKU_G2) share enzymes related to the production of rare sugars. Interestingly, the isolates from the three subgroups containing specific CMP-N-acetylneuraminate-producing enzymes that are correlated with their growth abilities. Collectively, these observations provide an understanding of the molecular mechanisms underlying disease pathogenesis and support the development of strategies for bacterial disease prevention and control.

Exon disruptive variants in Populus trichocarpa associated with wood properties exhibit distinct gene expression patterns.

Forest trees may harbor naturally occurring exon disruptive variants (DVs) in their gene sequences, which potentially impact important ecological and economic phenotypic traits. However, the abundance and molecular regulation of these variants remain largely unexplored. Here, 24,420 DVs were identified by screening 1014 Populus trichocarpa full genomes. The identified DVs were predominantly heterozygous with allelic frequencies below 5% (only 26% of DVs had frequencies greater than 5%). Using common garden-grown trees, DVs were assessed for gene expression variation in the developing xylem, revealing that their gene expression can be significantly altered, particularly for homozygous DVs (in the range of 27%-38% of cases depending on the studied common garden). DVs were further investigated for their correlations with 13 wood quality traits, revealing that, among the 148 discovered DV associations, 15 correlated with more than one wood property and six genes had more than one DV in their coding sequences associated with wood traits. Approximately one-third of DVs correlated with wood property variation also showed significant gene expression variation, confirming their non-spurious impact. These findings offer potential avenues for targeted introduction of homozygous mutations using tree biotechnology, and while the exact mechanisms by which DVs may directly influence wood formation remain to be unraveled, this study lays the groundwork for further investigation.

High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes.

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both mRNA and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using HCR Flow-FISH for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to characterize full-length mRNA isoforms and observe remarkable uniformity of mRNA isoforms from the transgenic units. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for comparing native and synthetic gene regulation and for building more robust transgenic systems.

Cell-free supernatant of Lactobacillus gasseri 1A-TV shows a promising activity to eradicate carbapenem-resistant Klebsiella pneumoniae colonization.

The use of beneficial bacteria like Lactobacillus spp. is a potential innovative approach to fight antibiotic-resistant pathogens. Klebsiella pneumoniae is one of the most concerning multi drug-resistant (MDR) pathogens, and its ability to colonize the human gut is considered to be the main reason for recurrent infections in critically ill patients. In this study, Lactobacillus gasseri 1A-TV, already described for its probiotic activity, was characterized at the genomic level. Moreover, its cell-free supernatant (CFS) was tested for antimicrobial activity against extended-spectrum β-lactamase (ESBL)- and carbapenemase (KPC)-producing K. pneumoniae clinical isolates. Whole-genome sequencing showed that the L. gasseri 1A-TV genome was of 2,018,898 bp in size with 34.9% GC content, containing 1,937 putative protein coding sequences, 55 tRNA, and 4 rRNA detected by RAST and classified in 20 functional groups by Cluster of Orthologous Genes (COG). BAGEL4 (BActeriocin GEnome minimal tooL) and the antiSMASH 7.0 pipeline identified two bacteriocin biosynthetic gene clusters (BBGCs), namely, BBGC1 that comprises two class IIc bacteriocins including gassericin A-like bacteriocin, and BBGC2 carrying the class III bacteriocin helveticin J. Strikingly, 1A-TV CFS inhibited the growth of all K. pneumoniae isolates only after 8 h of incubation, showing a bactericidal effect at 24 h and interfering, even at lower concentrations, with the biofilm production of biofilm-producer strains independently of a bactericidal effect. NMR analysis of CFS identified and quantified several metabolites involved in carbohydrate metabolism and amino acid metabolism, and organic acids like ethanol, lactate, acetate, and succinate. Finally, in vitro assays of 1A-TV showed significant co-aggregation effects against carbapenem-resistant K. pneumoniae, namely, strains 1, 2, 3, and 7. Our findings highlight the antimicrobial activity of 1A-TV as a probiotic candidate or its CFS as a natural bioproduct active against MDR K. pneumoniae strains, underlining the importance of novel therapeutic strategies for prevention and control of ESBL- and carbapenemase-producing K. pneumoniae colonization.

Characterization and Biocontrol Efficacy of Bacillus velezensis GYUN-1190 against Apple Bitter Rot.

The application of synthetic fungicides has resulted in environmental pollution and adverse effects on non-target species. To reduce the use of agrochemicals, crop disease management requires microbial biological control agents. Bacillus-related genera produce secondary metabolites to control fungal pathogens. Bacillus velezensis GYUN-1190, isolated from soil, showed antagonistic activity against Colletotrichum fructicola, the apple anthracnose pathogen. Volatile organic compounds and culture filtrate (CF) from GYUN-1190 inhibited C. fructicola growth in vitro, by 80.9% and 30.25%, respectively. The CF of GYUN-1190 inhibited pathogen spore germination more than cell suspensions at 10 8 cfu/ml. Furthermore, GYUN-1190 CF is effective in inhibiting C. fructicola mycelial growth in vitro, and it suppresses apple fruit bitter rot more effectively than GYUN-1190 cell suspensions and pyraclostrobin in planta. The mycelial growth of C. fructicola was completely inhibited 48 h after immersion into the CF, in compared with positive controls and GYUN-1190 cell suspensions. The genetic mechanism underlying the biocontrol features of GYUN-1190 was defined using its whole-genome sequence, which was closely compared to similar strains. It consisted of 4,240,653 bp with 45.9% GC content, with 4,142 coding sequences, 87 tRNA, and 28 rRNA genes. The genomic investigation found 14 putative secondary metabolite biosynthetic gene clusters. The investigation suggests that B. velezensis GYUN-1190 might be more effective than chemical fungicides and could address its potential as a biological control agent.

Phylogenetic conflict between species tree and mitochondrial gene trees in Murininae (Mammalia, Chiroptera) and validation of the genus Harpiola

AbstractThe subfamily Murininae is renowned for its inherent taxonomic challenges associated with sampling difficulties and morphological similarities. At present, three genera are acknowledged within the subfamily; however, their phylogenetic interrelations and systematic classification remain debated. In this study, the separation of Harpiola at the genus level was robustly supported by our phylogenomic analyses based on the mitochondrial genome, coding sequences (CDSs) and ultraconserved elements (UCEs) from 12 individuals covering all three genera of Murininae. Notably, a distinctive mito‐nuclear discordance emerged, with the nuclear genealogy ((Harpiocephalus, Harpiola), Murina) contrasting with the mitochondrial genealogy (Harpiocephalus, (Harpiola, Murina)). The integration of these findings with inferences of demographic history reveals that dramatic environmental changes during the Pleistocene glacial and inter‐glacial cycles have shaped the current distribution of Murininae. Moreover, the detection of extensive gene flow between ancient lineages of Harpiola and Murina suggests that an ancestral ‘ghost’ Murina lineage may have contributed its mitochondrial DNA, along with a limited portion of nuclear DNA, to Harpiola in a bygone hybridization zone. In addition to molecular analyses, we employed traditional and geometric morphometric analyses of skulls to differentiate the three genera. Harpiocephalus is readily distinguishable, but Harpiola and certain species of Murina exhibit overlapping characteristics both morphometrically and geometrically which may be the outcome of ancient introgression events. This finding highlights the importance of fine‐scale morphological distinctions within the latter genera, which may be the outcome of ancient introgression events.

Genome-wide identification of SSR markers from coding regions for endangered Argania spinosa L. skeels and construction of SSR database: AsSSRdb.

Microsatellites [simple sequence repeats (SSRs)] are one of the most widely used sources of genetic markers, particularly prevalent in plants. Despite their importance in various applications, a comprehensive genome-wide identification of coding sequence (CDS)-associated SSR markers in the Argania spinosa L. genome has yet to be conducted. In this study, 66 280 CDSs containing 5351 SSRs within 4535 A. spinosa L. CDSs were identified. Among these, tri-nucleotide motifs (58.96%) were the most common, followed by hexa-nucleotide (15.71%) and di-nucleotide motifs (13.32%). The predominant SSR motif in the tri-nucleotide category was AAG (24.4%), while AG (94.1%) was the most abundant among di-nucleotide repeats. Furthermore, the extracted CDSs containing SSRs were subjected to functional annotation; 3396 CDSs (74.88%) exhibited homology with known proteins, 3341 CDSs (73.7%) were assigned Gene Ontology terms, 1004 CDSs were annotated with Enzyme Commission numbers, and 832 (18.3%) were annotated with KEGG pathways. A total of 3475 primer pairs were designed, out of which 3264 were successfully validated in silico against the A. spinosa L. genome, with 99.6% representing high-resolution markers yielding no more than three products. Additionally, the SSR markers demonstrated a low rate of transferability through in-silico verification in two species within the Sapotaceae family. Furthermore, we developed an online database, the "Argania spinosa L. SSR database: https://as-fmmdb.shinyapps.io/asssrdb/" (AsSSRdb) to provide access to the CDS-associated SSRs identified in this study. Overall, this research provides valuable marker resources for DNA fingerprinting, genetic studies, and molecular breeding in argan and related species. Database URL: https://as-fmmdb.shinyapps.io/asssrdb/.

Genomic Characterization of Achromobacter genogroup 20 and identification of a potential species-specific marker

ObjectiveTo describe the genome of Achromobacter genogroup 20 and explore the presence of resistance determinants. MethodsIsolate 413638 was identified through nrdA and MLST analysis. Antimicrobial susceptibility testing was performed according CLSI 2024. NGS was conducted using Illumina MiSeq, on the NextSeq500 platform with 150 bp paired-end reads, and de novo assembly was assessed using Unicycler (Galaxy). Coding sequences were predicted and confirmed with RAST and BLAST, and resistance determinants were evaluated using Resfinder and manual curation. All Achromobacter spp. genomes were obtained from NCBI, and the presence of blaOXA was investigated. Average nucleotide identity (ANI) and tetranucleotide analysis (TETRA) were calculated. Phylogenetic analysis of the new OXA variant was conducted against other species-specific markers in Achromobacter. ResultsIsolate 413638 was identified as Achromobacter genogroup 20 ST365, showing resistance to third- and fourth-generation cephalosporins, aminoglycosides, and fluoroquinolones. The genome included coding sequences for three putative β-lactamases (one new OXA type-class D β -lactamase and two new class C), 32 efflux pump proteins, two aminoglycoside-modifying enzymes and a dihydropteroate synthase; also, substitutions in parC and parE were detected. The OXA enzyme, designated OXA-1238 (PP446293), differs from OXA-114a by 85 amino acids, with 69% identity. In silico analysis found OXA-1238 variants in three additional Achromobacter spp. genomes, with 97% identity. Based on ANI and tetra analysis, the three genomes corresponded to Achromobacter genogroup 20. ConclusionSeveral resistance markers were found, probably contributing to the resistance profile observed in Achromobacter genogroup 20 ST365. The new OXA variant identified, OXA-1238, could constitute a useful molecular marker for species identification.

Pulsed Orthogonal Time Frequency Space: A Fast Acquisition and High-Precision Measurement Signal for Low Earth Orbit Position, Navigation, and Timing

The recent rapid development of low Earth orbit (LEO) constellation-based navigation techniques has enhanced the ability of position, navigation, and timing (PNT) services in deep attenuation and interference environments. However, existing navigation modulations face the challenges of high acquisition complexity and do not improve measurement precision at the same signal strength. We propose a pulsed orthogonal time frequency space (Pulse-OTFS) signal, which naturally converts continuous signals into pulses through a special delay-Doppler domain pseudorandom noise (PRN) code sequence arrangement. The performance evaluation indicates that the proposed signal reduces at least 89.4% of the acquisition complexity. The delay measurement accuracy is about 8 dB better than that of the traditional binary phase shift keying (BPSK) signals with the same bandwidth. It also provides superior compatibility and anti-multipath performance. The advantages of fast acquisition and high-precision measurement are verified by processing the real signal in the developed software receiver. As Pulse-OTFS occupies only one time slot of a signal period, it can be easily integrated with OTFS-modulated communication signals and used as a navigation signal from broadband LEO satellites as an effective complement to the global navigation satellite system (GNSS).

Molecular Genetic Characteristics of the Mutant Strain B-162/2 of the Bacteria <i>Pseudomonas chlororaphis</i> subsp. <i>aurantiaca</i>

The production of microorganisms that produce biologically active compounds for agriculture, the chemical, veterinary and pharmaceutical industries as well as for environmental protection continues to be an important direction of microbial biotechnology. One of the most effective approaches to the production of producers is chemical mutagenesis, which, in combination with the right breeding strategy, makes it possible to obtain highly productive strains. A significant disadvantage of chemical mutagenesis is the large number of induced mutations in the genomes of mutant strains, which makes it difficult to identify genes and, accordingly, biosynthetic pathways involved in the production of a given compound. The solution to this problem is modern technologies of genome sequencing and analysis, which make it possible to identify new genes and unknown biochemical pathways involved in the formation of biologically active compounds. The aim of the work was to analyse the genome of the mutant strain B-162/2 of the bacterium Pseudomonas chlororaphis subsp. aurantiaca, which is capable of increased production of biologically active compounds of the phenazine series and is resistant to hydrogen peroxide. When analysing the genome of strain B-162/2 in full size, 6482 coding sequences and 64 coding RNA sequences were identified. Comparison of the genome of the B-162/2 strain with the genome of the wild type B-162 allowed the identification of 39 mutations, 5 of which are localised in intergenic regions, and 34 affected coding sequences. Of the mutations detected, 14 led to a radical amino acid substitution in the proteins and 2 led to the formation of premature stop codons (methyl group sensor and MFS-type transporter). Several substitutions with high values of the Grantham coefficient were found, which could possibly lead to a change in the activity of the proteins concerned. The presence of three regions with phage genes in the genome of the B-162/2 strain was detected.

Codon optimality modulates cellular stress and innate immune responses triggered by exogenous RNAs.

The COVID-19 mRNA vaccines demonstrated the power of mRNA medicines. Despite advancements in sequence design, evidence regarding the preferential use of synonymous codons on cellular stress and innate immune responses is lacking. To this end, we developed a proprietary codon optimality matrix to re-engineer the coding sequences of three luciferase reporters. We demonstrate that optimal mRNAs elicited dramatic increases in luciferase activities compared to non-optimal sequences. Notably, transfecting an optimal RNA affects the translation of other RNAs in the cell including control transcripts in dual luciferase assays. This held true in multiple cell lines and for an unrelated reporter. Further, non-optimal mRNAs preferentially activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2α, a central event of the integrated stress response. Using nucleoside-modified or circular RNAs partially or fully abrogated these responses. Finally, we show that circularizing RNAs enhances both RNA lifespan and durability of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. However, we also show that RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Hence, RNA sequence engineering, chemical, and topological modifications must all be combined to elicit favorable therapeutic outcomes.

Mobile Element Insertion in the APOB Exon 3 Coding Sequence: A New Challenge in Hypobetalipoproteinemia Diagnosis.

Mobile elements (ME) can transpose by copy-and-paste mechanisms. A heterozygous insertion in APOB exon 3 coding sequence was suspected in a patient with hypobetalipoproteinemia (HBL), by gel electrophoresis of the PCR products. An insertion of a 85 bp fragment flanked by a polyA stretch and a target replication site duplication was identified as a ME insertion (MEI) from the AluYa5 subfamily, NM_000384.3(APOB):c.135_136ins(160). Then, the DNA was reanalyzed using our NGS custom panel. Routine analysis did not reveal any causative variant, but manual inspection of the alignments and MELT enabled us to detect this MEI from NGS data. A functional study revealed that this MEI introduces a stop codon p.(Phe46Alafs*2) and additionally leads to p.(Lys41Serfs*2) due to an exon skipping. This is the first report of a MEI into APOB, as a cause of HBL. Furthermore, our study highlights the value of including MEI-callers in routine pipelines to improve primary dyslipidemia diagnosis.

Mutational Landscape of KIT Proto-Oncogene Coding Sequence in 62 Canine Cutaneous and Subcutaneous Mast Cell Tumors

Canine mast cell tumors (MCTs) are common skin neoplasms with varying biological behaviors. The KIT proto-oncogene plays a key role in the development of these tumors, and internal tandem duplications on exon 11 are usually associated with more aggressive behavior, increased local recurrence, and decreased survival time. However, apart from exons 8–11 and 17, there is limited understanding of the overall KIT mutational landscape in canine MCTs. This work aims to analyze the entire KIT coding sequence (21 exons) in a cohort of 62 MCTs, which included 38 cutaneous and 24 subcutaneous tumors, and potentially identify new variants. In addition to confirming previously reported activating KIT mutations in exons 8, 9, and 11, we identified new variants in exons 2, 3, 5, 16, and the 3′ untranslated region (UTR). Notably, these last variants include an amino acid change (Asp/His) in exon 16. Additionally, we confirmed a differential prevalence of KIT variants in cutaneous and subcutaneous MCTs. These findings enhance our understanding of the KIT proto-oncogene coding sequence and provide valuable information for future confirmatory studies.

The Intra-Articular Delivery of a Low-Dose Adeno-Associated Virus-IL-1 Receptor Antagonist Vector Alleviates the Progress of Arthritis in an Osteoarthritis Rat Model

Background/Objectives: Interleukin-1 (IL-1) is a pivotal mediator in the pathological progression of osteoarthritis (OA), playing a central role in disease progression. However, the rapid clearance of IL-1 receptor antagonist (IL-1Ra) from the joints may hinder the efficacy of intra-articular IL-1Ra injections in reducing OA-associated pain or cartilage degradation. Methods: Sustaining sufficient levels of IL-1Ra within the joints via adeno-associated virus (AAV)-mediated gene therapy presents a promising therapeutic strategy for OA. In this study, we constructed an IL-1Ra expression cassette employing intron insertion in the coding sequence (CDS) region to enhance protein expression levels. Furthermore, we incorporated precisely targeted liver-specific microRNA (miRNA) sequences to specifically downregulate transgene expression within hepatic tissues, thereby ensuring more targeted and controlled regulation of gene expression. Results: A rat model of OA was employed to compare the efficacy of AAV5 and AAV9 for IL-1Ra delivery at both high and low doses. It was observed that low-dose, but not high-dose, AAV9-IL-1Ra resulted in a significant reduction in joint swelling, accompanied by a decrease in the diameter of the affected area and the preservation of biomarkers associated with trabecular bone integrity. Conclusions: These results highlight the great potential of AAV9-IL-1Ra in osteoarthritis therapy, with the promise of achieving long-term improvement through a single intra-articular injection.

Genome sequences of five Klebsiella bacteriophages that belong to the genus Jiaodavirus.

We describe the genomes of five lytic Klebsiella pneumoniae myophages, therapeutic candidates, that belong to the family Straboviridae and genus Jiaodavirus. The genomes ranged from 165,574 to 169,768 bp, with ca. 40% GC content, contained 289-300 coding sequences, had 15-16 tRNA genes, and no terminal repeats.

Expression of Viral DNA Polymerase in Synthetic Recombinant Adeno-Associated Virus Producer Cell Line Enhances Full Particle Productivity.

Recombinant adeno-associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high-quality products is required. We have developed a synthetic biology strategy to generate HEK293-based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction. One such cell line, GX6B, produced up to 106 capsids per cell, but only a much lower level of rAAV genomes. The low AAV genome titer limited its rAAV productivity and increased empty viral particle content. To boost AAV genome amplification, the coding sequence of the DNA polymerase complex (UL30/UL42) from helper Herpes Simplex Virus type 1 (HSV-1) was placed under an inducible promoter control and integrated into GX6B genome at a relatively low level. The resulting clones produced significantly higher titer of viral genomes, while their capsid level was unaffected. As a result, the encapsidated rAAV2 titer and the full particle content were significantly increased. We further demonstrated that this strategy of expressing HSV-1 DNA polymerase to increase full particle productivity could be implemented in a synthetic cell line producing another serotype rAAV8.

Genomic tools for post-elimination measles molecular epidemiology using Canadian surveillance data from 2018-2020.

Measles is caused by the highly infectious measles virus, MeV, for which there is an effective vaccine. Monitoring of progress of measles elimination requires enhanced surveillance and tracking of MeV strains, including documenting the absence of an endemically circulating strain. Due to a reduction in the number of circulating genotypes, additional sequence information, beyond the standardized 450 nucleotide window of the nucleoprotein (N450), is required to corroborate the information from epidemiological investigations and, ideally, fill in gaps in the surveillance data. This study applies MeV sequencing tools, namely the N450, the non-coding region between the matrix and fusion genes (MF-NCR), and the complete coding sequence of the genome (WGS-t), to clinical specimens obtained from cases occurring over a three-year time period in Canada. This data was systematically analyzed, including with Bayesian evolutionary analysis by sampling trees (BEAST) of the WGS-t. Of the 143 reported cases, N450, MF-NCR, and WGS-t sequences were obtained from 101, 81, and 75 cases, respectively. The BEAST analysis confirmed that the two most frequently detected lineages (B3 named strain MVi/Marikina City.PHL/10.18 and D8 named strain MVs/Gir Somnath.IND/42.16) were the result of repeated importations. Of the 16 outbreaks occurring during the study period, the analysis conclusively corroborated the epidemiological information for 13. BEAST analysis of the WGS-t convincingly demonstrated the expansion of two outbreaks by the inclusion of additional contemporary cases for which the epidemiological investigation had been unable to identify links. Furthermore, the analysis revealed the existence of three additional unrecognized outbreaks among the cases categorized as unknown source. One outbreak was without WGS-t and could not be resolved. Measles WGS-t data corroborated and expanded upon the outbreak analysis from traditional epidemiological investigations of measles outbreaks. However, both are needed for fulsome investigations in elimination settings.

WSSV induces Rubicon expression to regulate innate immune response in Penaeus vannamei

Rubicon, the RUN domain Beclin-1-interacting and cysteine-rich domain-containing protein plays an important role in facilitating viral replication. In this study, an involvement of P. vannamei Rubicon or PvRUBCN during white spot syndrome virus (WSSV) infection and its roles in regulation of apoptosis and innate immune response were investigated. The full-length coding sequence of PvRUBCN was 3708 bp encoding the protein of 1235 amino acids. PvRUBCN contained three conserved domains including the RUN, the PI3K-binding, and the Rubicon homology domains. PvRUBCN was grouped with the closely related RUBCN from the Penaeid species with more than 95 % identity. It was highly expressed in nerve followed by intestine, and gill. Its expression was induced upon WSSV challenge at 12 and 48 hpi. Suppression of PvRUBCN by dsRNA upon WSSV challenge resulted in more than 80 % reduction in PvRUBCN mRNA expression. Knockdown of PvRUBCN mRNA significantly decreased the WSSV copy number and prolonged the survival rate of WSSV-infected shrimp. In addition, the caspase3, a key regulator of the apoptosis pathway was significantly down-regulated. The interferon like genes including Vago4 was dramatically decreased at 72 hpi whereas Vago5 demonstrated slight reduction at 24 hpi and increase at 72 and 120 hpi, respectively. On the other hand, the expressions of the genes involved in the prophenoloxidase pathway including PPO1 and PPO2 were not changed. Taken together, PvRUBCN played important roles in the antiviral immunity in response to WSSV infection.

Aurora kinase as a putative target to tick control.

Aurora kinases (AURK) play a central role in controlling cell cycle in a wide range of organisms. They belong to the family of serine-threonine kinase proteins. Their role in the cell cycle includes, among others, the entry into mitosis, maturation of the centrosome and formation of the mitotic spindle. In mammals, 3 isoforms have been described: A, B and C, which are distinguished mainly by their function throughout the cell cycle. Two aurora kinase coding sequences have been identified in the transcriptome of the cattle tick Rhipicephalus microplus (Rm-AURKA and Rm-AURKB) containing the aurora kinase-specific domain. For both isoforms, the highest number of AURK coding transcripts is found in ovaries. Based on deduced amino acid sequences, it was possible to identify non-conserved threonine residues which are essential to AURK functions in vertebrates and which are not present in R. microplus sequences. A pan AURK inhibitor (CCT137690) caused cell viability decline in the BME26 tick embryonic cell line. In silico docking assay showed an interaction between Aurora kinase and CCT137690 with exclusive interaction sites in Rm-AURKA. The characterization of exclusive regions of the enzyme will enable new studies aimed at promoting species-specific enzymatic inhibition in ectoparasites.

Genomic characterisation of an extended-spectrum β-Lactamase-producing Klebsiella pneumoniae isolate assigned to a novel sequence type (6914)

BackgroundCow milk, which is sometimes consumed raw, hosts a plethora of microorganisms, some of which are beneficial, while others raise food safety concerns. In this study, the draft genome of an extended-spectrum β-lactamase-producing Klebsiella pneumoniae subsp. pneumoniae strain Cow102, isolated from raw cow milk used to produce traditional foods in Nigeria, is reported.ResultThe genome has a total length of 5,359,907 bp, with 70 contigs and a GC content of 57.35%. A total of 5,244 protein coding sequences were detected with 31% mapped to a subsystem, and genes coding for amino acids and derivatives being the most prevalent. Multilocus sequence typing revealed that the strain had new allelic profile assigned to the novel 6914 sequence type possessing capsular and lipopolysaccharide antigen K locus 122 with an unknown K type (KL122) and O locus O1/O2v2 with type O2afg, respectively. A total of 28 resistance-related genes, 98 virulence-related genes, two plasmids and five phages were identified in the genome. The resistance genes oqxA, oqxB and an IS3 belonging to cluster 204 were traced to bacteriophage Escher 500,465. Comparative analysis predicted one strain specific orthologous group comprising three genes.ConclusionThis report of a novel sequence type (ST6914) in K. pneumoniae presents a new allelic profile, indicating ongoing evolution and diversification within the species. Its uniqueness suggests it may represent a locally evolved lineage, although further sampling would be necessary to confirm this hypothesis. The strain’s multidrug resistance, virulence gene repertoire, and isolation from animal milk render it a potentially significant public health concern, underscoring the importance of genomic surveillance in non-clinical settings to detect emerging strains. Further research is required to fully characterise the capsular K type of ST6914.