Search Results (73)

H. pylori detection via the stool antigen test (SAT) requires 100 times more cells than nested PCR (NPCR) for a 454 bp amplicon, but is significantly more sensitive in identifying positive stool samples. To understand this contradiction, we developed an NPCR assay to amplify a shorter 148 bp segment of the 16S rRNA gene. The assay was extremely sensitive and reliable when adhering to particular rules commonly used in forensic laboratories. The SAT and NPCR for long and short amplicons were compared using stool samples from 208 gastroenterological patients, of which 27.9% were identified as positive according to the SAT and only 6.25% according to the 454 bp NPCR amplicon, but 51.0% in the short 148 bp NPCR. Among 100 asymptomatic volunteers, the prevalence was 35% in the SAT assay and 22% in the long NPCR, but as much as 66.6% of positives were determined in the short 148 bp NPCR. The specificity of the PCR product was determined via DNA sequencing, which confirmed H. pylori’s origin in all NPCR-positive samples. Apparently, the stool contains mostly short fragments of H. pylori DNA, and the most plausible explanation for the SAT/NPCR paradox is the degradation of H. pylori DNA in the digestive system. Full article

E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G₁-S phase transition and DNA synthesis with its heterodimeric partners (DP1 or DP2). The dimerization domain is the E2Fs and DP1 protein interaction interface and is believed to function in protein dimerization. In this study, eight E2F transcription factors (PcE2F1–8) of large yellow croaker Pseudosciaena crocea and one dimerization partner (PcDP1) are identified in the genome of large yellow croakers. The prediction of E2Fs conserved domains revealed that PcE2F1–6 has one DNA-binding domain (DBD) and one dimerization-binding domain (DD), while PcE2F7–8 only possess two duplicate DBDs but not DD, indicating that E2F7–8 cannot form the E2F/DP1 heterodimer. To explore whether PcDP1 is a partner of PcE2F1–6, the ORF of PcE2F1–6 was cloned. Subsequently, its sequence characteristics, the expression pattern in healthy fish, and subcellular co-localization were analyzed, and an interaction between PcDP1 and PcE2F1–6 were detected directly by yeast two-hybrid and BiFC. The PcE2F1, PcE2F2, PcE2F3, PcE2F4, PcE2F5, and PcE2F6 genes encode a protein of 454, 448, 444, 392, 362, and 396 amino acids, respectively, with accession numbers QFZ93593.1, QFZ93594.1, QFZ93595.1, QFZ93596.1, QFZ93597.1, and QFZ93598.1, respectively. Sequence characteristics analysis found that PcE2F1–5 but not PcE2F6 proteins share the pocket protein-binding domain sequestering in dimerization domains and transactivation domains. The PcE2F1,2,4 proteins possess one nuclear localization signal (NLS), and PcE2F3 protein possess two NLSs, but there is no NLS in PcE2F5 and 6 protein. Moreover, PcE2F4 also contains one NES. However, PcE2F1–6 proteins were all located in nucleus by using Euk-mPloc 2.0 programs and were confirmed by performing the Cherry and EGFP reporter assay. Regarding co-expression of DP1, only E2F4 can transfer DP1’s subcellular location from cytoplasm to the nucleus. RT-qPCR analysis indicated that PcE2F1–6 are constitutively and tissue specifically expressed in all of the tissues tested of a healthy large yellow croaker. The PcE2F1–6, except for PcE2F3, mRNA levels were all detected higher in the liver. PcE2F1–4 were also highly specifically expressed in the kidney, PcE2F4,6 in the brain, and PcE2F5 in the spleen of a healthy large yellow croaker, respectively. Using a yeast two-hybrid system, PcE2F4 interacting with PcDP1 was identified. The interaction between PcE2F4 and PcDP1 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Collectively, these results indicate that an interaction between PcE2F4 and PcDP1 was detected, which may form heterodimer E2F4/DP1 to regulate cell cycles and immune-related pathways in large yellow croakers. Full article

Fusarium species are among the most significant pathogens causing root rot in Panax notoginseng. In this study, a strain of Trichoderma hamatum was isolated from the rhizosphere soil of P. notoginseng and subjected to whole-genome sequencing. Plate confrontation experiments were conducted to investigate the antagonistic effects of T. hamatum against Fusarium oxysporum, Fusarium solani, and Fusarium acutatum, the primary Fusarium species causing root rot. Whole-genome sequencing revealed 10,774 predicted genes in T. hamatum, of which 454 were associated with carbohydrate-active enzymes (CAZymes) involved in fungal cell wall degradation. Additionally, 11 biosynthetic gene clusters (BGCs) associated with antimicrobial production were identified, highlighting the biocontrol potential of T. hamatum. In plate confrontation experiments, T. hamatum showed substantial inhibition rates of 68.07%, 70.63%, and 66.12% against F. oxysporum, F. solani, and F. acutatum, respectively. Scanning electron microscopy suggested the hyperparasitism of T. hamatum against F. solani, which was characterized by spore production that adhered to the pathogen, thereby inhibiting its growth. These findings provide a theoretical foundation to enhance understanding of the biological control mechanisms of T. hamatum, supporting its potential applications in sustainable agriculture. Full article

The use of lactic acid bacteria for developing functional foods is increasing for their ability to synthesize beneficial metabolites such as vitamin B (riboflavin, RF) and postbiotic compounds. Here, the spontaneous mutant FS54 B2 was isolated by treatment of the dextran-producing Weissella confusa FS54 strain with roseoflavin. FS54 B2 overproduced RF (4.9 mg/L) in synthetic medium. The FMN riboswitch is responsible for the regulation of RF biosynthesis, and sequencing of the coding DNA revealed that FS54 B2 carries the G131U mutation. FS54 B2 retained the capacity of FS54 to synthesize high levels of dextran (3.8 g/L) in synthetic medium. The fermentation capacities of the two Weissella strains was tested in commercial oat-, soy- and rice-based beverages. The best substrate for FS54 B2 was the oat-based drink, in which, after fermentation, the following were detected: RF (2.4 mg/L), dextran (5.3 mg/L), potential prebiotics (oligosaccharides (panose (5.1 g/L), isomaltose (753 mg/L) and isomaltotriose (454 mg/L)) and the antioxidant mannitol (16.3 g/L). pH-lowering ability and cell viability after one month of storage period were confirmed. As far as we know, this is the first time that an RF-overproducing W. confusa strain has been isolated, characterized and tested for its potential use in the development of functional beverages. Full article

The increasing number of antibiotic-resistant bacterial pathogens is a serious problem in medicine. Endolysins are bacteriolytic enzymes of bacteriophages, and a promising group of enzymes with antibacterial properties. Endolysins of bacteriophages infecting Gram-positive bacteria have a modular domain organization. This feature can be used to design enzymes with new or improved properties by modifying or shuffling individual domains. This work is a detailed analysis 1of the diversity of endolysin domains found in bacteriophages infecting bacilli. During the course of the work, a database of endolysins of such bacteriophages was created, and their domain structures were analyzed using the NCBI database, RASTtk, BLASTp, HHpred, and InterPro programs. A phylogenetic analysis of endolysins was performed using MEGA X. In 438 phage genomes, 454 genes of endolysins were found. In the endolysin sequences found, eight different types of catalytic domains and seven types of cell wall binding domains were identified. The analysis showed that many types of endolysin domains have not yet been characterized experimentally. Studies of the properties of such domains will help to reveal the potential of endolysins for the creation of new antibacterial agents. Full article

Background/Objectives: The giant red shrimp, Aristaeomorpha foliacea, is a valuable marine fishing resource. The conservation of species, especially exploited ones, depends on a good knowledge of their biology, as well as the development of appropriate management plans based on the identification of genetically differentiated units or genetic stocks. Microsatellites are widely used molecular markers to detect genetic stocks in penaeoid shrimps and prawns. This study aimed to develop and characterize new microsatellites for A. foliacea. Methods: Next-generation sequencing based on 454 pyrosequencing revealed 58 candidate microsatellite loci for A. foliacea. These were tested on a panel of 8 individuals representative of its worldwide geographical distribution, and 19 polymorphic loci were identified and subsequently validated and characterized in 30 individuals from a single population in the Mediterranean Sea. Results: As a result, 10 polymorphic loci were identified, which did not present linkage disequilibrium and showed a range of alleles per locus and an observed and expected heterozygosity of 2–10, 0.0667–0.5567, and 0.0661–0.8511, respectively. Nine out of these loci were under Hardy–Weinberg equilibrium and showed a combined exclusion probability of 0.9202 and 0.9968 in parentage and identity analysis, respectively. Conclusions: This set of loci will provide a strong set of tools to (i) perform parentage studies and (ii) examine connectivity patterns (horizontal and vertical), including examining the population structure of this species at a variety of geographical scales and, particularly, between exploited populations in shallow waters and deeper unexploited populations. Full article

The systematic analysis of measurement data allows a large amount of information to be obtained from existing measurements in a short period of time. Especially in vehicle development, many measurements are performed, and large amounts of data are collected in the process of emission calibration. With the introduction of Real Driving Emissions Tests, the need for targeted analysis for efficient and robust calibration of a vehicle has further increased. With countless possible test scenarios, test-by-test analysis is no longer possible with the current state-of-the-art in calibration, as it takes too much time and can disregard relevant data when analyzed manually. In this article, therefore, a methodology is presented that automatically analyzes exhaust measurement data in the context of emission calibration and identifies emission-related critical sequences. For this purpose, moving analyzing windows are used, which evaluate the exhaust emissions in each sample of the measurement. The detected events are stored in tabular form and are particularly suitable for condensing the collected measurement data to a required amount for optimization purposes. It is shown how different window settings influence the amount and duration of detected events. With the example used, a total amount of 454 events can be identified from 60 measurements, reducing 184,623 s of measurements to a relevant amount of 12,823 s. Full article

Fungi play key roles in forest soils and provide benefits to trees via mycorrhizal symbioses. After severe disturbance, forest regrowth can be impeded because of changes in fungal communities. In 2013–2014, soil fungi in forest floor and mineral soil were examined by Roche 454 pyrosequencing in undisturbed, harvested, and burned jack pine stands in a forested area near Fort Chipewyan, Alberta. These fungal communities were compared with jack pine, white spruce, and larch stands in Gateway Hill, a nearby certified reclaimed area. In 2014, a more detailed sampling of forestry and reclamation jack pine sites examined fungi in soil fractions using two high-throughput sequencing platforms and a sporocarp survey. The significances of compositional and functional differences in fungal communities between the forested and reclamation sites were assessed using permutation tests of partially constrained ordinations, accounting for confounding factors by variance partitioning. Taxa associated with the forestry area were primarily ectomycorrhizal. Fungal richness and diversity were greater in soils from the reclamation sites and included significantly more pathogenic taxa and taxa with unknown functional properties. Fungal community dissimilarities may have been artefacts of historical legacies or, alternatively, may have resulted from contrasting niche differentiation between forestry and reclamation sites. Full article

Background: Fibrolamellar carcinoma (FLC) is a rare type of liver cancer that primarily affects adolescents and young adults without prior liver disease or viral infections. Patients with FLC generally have non-specific symptoms, are often diagnosed at a later stage, and experience a higher frequency of metastases compared to patients with other liver cancers. A fusion transcript of DNAJB1 and PRKACA, which can lead to increased activity of PKA and cellular proliferation, has been identified in all FLC patients, but the exact mechanism through which FLC develops remains unclear. In this study, we investigated common lncRNA profiles in various FLC samples using bioinformatics analyses. Methods: We analyzed differentially expressed (DE) lncRNAs from three RNA sequencing datasets. Using lncRNAs and DE mRNAs, we predicted potential lncRNA target genes and performed Gene Ontology (GO) and KEGG analyses with the DE lncRNA target genes. Moreover, we screened for small-molecule compounds that could act as therapeutic targets for FLC. Results: We identified 308 DE lncRNAs from the RNA sequencing datasets. In addition, we performed a trans-target prediction analysis and identified 454 co-expressed pairs in FLC. The GO analysis showed that the lncRNA-related up-regulated mRNAs were enriched in the regulation of protein kinase C signaling and cAMP catabolic processes, while lncRNA-related down-regulated mRNAs were enriched in steroid, retinol, cholesterol, and xenobiotic metabolic processes. The analysis of small-molecule compounds for FLC treatment identified vitexin, chlorthalidone, triamterene, and amiloride, among other compounds. Conclusions: We identified potential therapeutic targets for FLC, including lncRNA target genes as well as small-molecule compounds that could potentially be used as treatments. Our findings could contribute to furthering our understanding of FLC and providing potential avenues for diagnosis and treatment. Full article

Whole-Exome Sequencing (WES) has proven valuable in the characterization of underlying genetic defects in most rare diseases (RDs). Copy Number Variants (CNVs) were initially thought to escape detection. Recent technological advances enabled CNV calling from WES data with the use of accurate and highly sensitive bioinformatic tools. Amongst 920 patients referred for WES, 454 unresolved cases were further analysed using the ExomeDepth algorithm. CNVs were called, evaluated and categorized according to ACMG/ClinGen recommendations. Causative CNVs were identified in 40 patients, increasing the diagnostic yield of WES from 50.7% (466/920) to 55% (506/920). Twenty-two CNVs were available for validation and were all confirmed; of these, five were novel. Implementation of the ExomeDepth tool promoted effective identification of phenotype-relevant and/or novel CNVs. Among the advantages of calling CNVs from WES data, characterization of complex genotypes comprising both CNVs and SNVs minimizes cost and time to final diagnosis, while allowing differentiation between true or false homozygosity, as well as compound heterozygosity of variants in AR genes. The use of a specific algorithm for calling CNVs from WES data enables ancillary detection of different types of causative genetic variants, making WES a critical first-tier diagnostic test for patients with RDs. Full article

As a valuable Chinese traditional medicinal species, Chaenomeles speciosa (Sweet) Nakai (C. speciosa) is a natural resource with significant economic and ornamental value. However, its genetic information is not well understood. In this study, the complete mitochondrial genome of C. speciosa was assembled and characterized to explore the repeat sequences, recombination events, rearrangements, and IGT, to predict RNA editing sites, and to clarify the phylogenetic and evolutionary relationship. The C. speciosa mitochondrial genome was found to have two circular chromosomes as its major conformation, with a total length of 436,464 bp and 45.2% GC content. The mitochondrial genome contained 54 genes, including 33 unique protein-coding genes, 18 tRNAs, and 3 rRNA genes. Seven pairs of repeat sequences involving recombination events were analyzed. Both the repeat pairs, R1 and R2, played significant roles in mediating the major and minor conformations. In total, 18 MTPTs were identified, 6 of which were complete tRNA genes. There were 454 RNA editing sites in the 33 protein-coding sequences predicted by the PREPACT3 program. A phylogenetic analysis based on 22 species of mitochondrial genomes was constructed and indicated highly conserved PCG sequences. Synteny analyses showed extensive genomic rearrangements in the mitochondrial genome of C. speciosa and closely related species. This work is the first to report the C. speciosa mitochondrial genome, which is of great significance for conducting additional genetic studies on this organism. Full article

The aim of this study was to investigate and clarify the ambiguous taxonomy of Actinomyces naeslundii and its closely related species using state-of-the-art high-throughput sequencing techniques, and, furthermore, to determine whether sub-clusters identified within Actinomyces oris and Actinomyces naeslundii in a previous study by multi locus sequence typing (MLST) using concatenation of seven housekeeping genes should either be classified as subspecies or distinct species. The strains in this study were broadly classified under Actinomyces naeslundii group as A. naeslundii genospecies I and genospecies II. Based on MLST data analysis, these were further classified as A. oris and A. naeslundii. The whole genome sequencing of selected strains of A. oris (n = 17) and A. naeslundii (n = 19) was carried out using Illumina Genome Analyzer IIxe and Roche 454 allowing paired-end and single-reads sequencing, respectively. The sequences obtained were aligned using CLC Genomic workbench version 5.1 and annotated using RAST (Rapid Annotation using Subsystem Technology) release version 59 accessible online. Additionally, genomes of seven publicly available strains of Actinomyces (k20, MG1, c505, OT175, OT171, OT170, and A. johnsonii) were also included. Comparative genomic analysis (CGA) using Mauve, Progressive Mauve, gene-by-gene, Core, and Pan Genome, and finally Digital DNA-DNA homology (DDH) analysis was carried out. DDH values were obtained using in silico genome–genome comparison. Evolutionary analysis using ClonalFrame was also undertaken. The mutation and recombination events were compared using chi-square test among A. oris and A. naeslundii isolates (analysis methods are not included in the study). CGA results were consistent with previous traditional classification using MLST. It was found that strains of Actinomyces k20, MG1, c505, and OT175 clustered in A. oris group of isolates, while OT171, OT170, and A. johnsonii appeared as separate branches. Similar clustering to MLST was observed for other isolates. The mutation and recombination events were significantly higher in A. oris than A. naeslundii, highlighting the diversity of A. oris strains in the oral cavity. These findings suggest that A. oris forms six distinct groups, whereas A. naeslundii forms three. The correct designation of isolates will help in the identification of clinical Actinomyces isolates found in dental plaque. Easily accessible online genomic sequence data will also accelerate the investigation of the biochemical characterisation and pathogenesis of this important group of micro-organisms. Full article

Many lncRNAs have been shown to play a vital role in aging processes. However, how lncRNAs regulate seed aging remains unknown. In this study, we performed whole transcriptome strand-specific RNA sequencing of samples from rice embryos, analyzed the differences in expression of rice seed lncRNAs before and after artificial aging treatment (AAT), and systematically screened 6002 rice lncRNAs. During the AAT period, the expression levels of most lncRNAs (454) were downregulated and only four were upregulated among the 458 differentially expressed lncRNAs (DELs). Cis- or trans-regulated target genes of the four upregulated lncRNAs were mainly related to base repair, while 454 downregulated lncRNAs were related to plant–pathogen interaction, plant hormones, energy metabolism, and secondary metabolism. The pathways of DEL target genes were similar with those of differentially expressed mRNAs (DEGs). A competing endogenous RNA (ceRNA) network composed of 34 lncRNAs, 24 microRNAs (miRNA), and 161 mRNAs was obtained. The cDNA sequence of lncRNA LNC_037529 was obtained by rapid amplification of cDNA ends (RACE) cloning with a total length of 1325 bp, a conserved 5′ end, and a non-conserved 3′ end. Together, our findings indicate that genome-wide selection for lncRNA downregulation was an important mechanism for rice seed aging. LncRNAs can be used as markers of seed aging in rice. These findings provide a future path to decipher the underlying mechanism associated with lncRNAs in seed aging. Full article

Background: We examined associations between NFκB1 polymorphisms and influenza A (H1N1) clinical outcomes in Canadian. Methods: A total of thirty-six Caucasian patients admitted to the intensive care unit (ICU) in hospitals in Canada were recruited during the 2009 H1N1 pandemic. Genomic DNA was extracted from the whole blood samples. The NFkB1 gene was targeted for genotyping using next-generation sequencing technology—Roche 454. Results: A total of 136 single nucleotide polymorphisms (SNPs) were discovered within the NFκB1 gene. Among them, 63 SNPs were significantly enriched in patients admitted in the ICU (p < 0.05) compared with the British Caucasian population in the 1000 Genomes study. These enriched SNPs are mainly intron variants, and only two are exon SNPs from the non-transcribing portion of the NFκB1 gene. Conclusions: Genetic variations in the NFκB1 gene could influence clinical outcomes of pandemic H1N1 infections. Our findings showed that sequence variations of the NFκB1 gene might influence patient response to influenza infection. Full article

Tinospora cordifolia, commonly known as “Giloe” in India, is a shrub belonging to the family Menispermaceae. It is an important medicinal plant known for its antipyretic, anti-inflammatory, antispasmodic, and antidiabetic properties and is used in the treatment of jaundice, gout, and rheumatism. Despite its economic importance, the limited information related to its genomic resources prohibits its judicious exploitation through molecular breeding or biotechnological approaches. In this study, we generated a meta-transcriptome assembly of 43,090 non-redundant transcripts by merging the RNASeq data obtained from Roche 454 GS-FLX, and Illumina platforms, and report the first transcriptome-based database for simple sequence repeats and transcription factors (“TinoTranscriptDB” (Tinospora cordifolia Transcriptome Database)). We annotated 26,716 (62%) of the total transcripts successfully from National Center for Biotechnology Information non-redundant protein (NCBI-NR), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and Pfam databases. This database contains information of 2620 perfect simple sequence repeats (P-SSRs) with a relative abundance of 340.12 (loci/Mb), and relative density of 6309.29 (bp/Mb). Excluding mono-nucleotides, the most abundant SSR motifs were tri-nucleotides (54.31%), followed by di-nucleotides (37.51%), tetra-nucleotides (4.54%), penta-nucleotides (3.16%) and hexa-nucleotides (0.45%). Additionally, we also identified 4,311 transcription factors (TFs) and categorized them into 55 sub-families. This database is expected to fill the gap in genomic resource availability in T. cordifolia and thus accelerate molecular breeding and related functional and other applied studies aimed towards genetic improvements of T. cordifolia and related species. Full article