Search Results (84)

The deep terrestrial subsurface is the largest reservoir of Earth’s freshwater resources as well as the largest habitat for prokaryotic life. However, the deep-subsurface microbiome, especially its spatial distribution across countries/continents, is still poorly understood. In this study, we compiled and compared 30 16S rRNA gene amplicon libraries from three deep fractured aquifers in different parts of the world (depth range of tens of meters to 2.4 km below surface) to understand the spatial distribution and functions of deep-subsurface microbial community, and to test for the presence of core taxa. The results revealed spatially heterogenous microbial community composition at both the local and the global scales, even at the phylum level. Environmental filtering was identified as an important driver of the microbial community structure of deep groundwaters. Despite the spatial heterogeneity, the three aquifers share a core microbiome at the genus level. Only one family, Comamonadaceae, was present in all the 30 samples analyzed. Several other families were also prevalent, including Hydrogenophilaceae, Omnitrophaceae, BSV26 (Candidatus Kryptonia), and an unclassified Thermodesulfovibrionia. FAPROTAX functional prediction indicated that chemoheterotrophic functions predominate, and the core microbial genera, together with the dominant genera, collectively govern the functional characteristics. Taken together, our findings provide new insights into the spatial heterogeneity and functional potential of deep-subsurface ecosystems across the globe. Full article

Background/Objectives: Lung cancer remains among the most frequently diagnosed malignancies in Romania, with a high mortality rate. Beyond EGFR mutations, clinically relevant genetic alterations in non-small cell lung cancer (NSCLC) include fusions involving ALK, ROS1, RET, and NTRK1/2/3. This study aimed to determine the prevalence of these mutations in a Romanian cohort and evaluate their associations with clinicopathological features. Methods: DNA and RNA were simultaneously extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections using the Genexus Purification System (ThermoFisher Scientific). Concentrations were quantified fluorometrically, and gene fusions were analyzed with Ion Torrent NGS (Ion GeneStudio S5) with the Oncomine Focus Assay (ThermoFisher Scientific). Library preparation was automated with the Ion Chef System, and data interpretation was conducted using Ion Reporter. Results: Among 721 newly diagnosed NSCLC patients, 28 (3.88%) harbored gene fusions. Adenocarcinoma prevailed among fusion-positive cases (85.7%). The subgroup included 15 males and 13 females, with a mean age of 63.25 years (range 43–83). ALK fusions were most frequent (1.66% of the cohort; 42.86% of positives), predominantly EML4::ALK. ROS1 fusions were detected in five patients (0.7%), most frequently CD74::ROS1. RET fusions occurred in 1.11%. Rare fusions included one ETV6::NTRK3, one PTPRZ1::MET, and one FGFR3::TACC3 co-occurring with EGFR L858R. Conclusions: Gene fusions were present in a minority of NSCLC cases, with ALK, ROS1, and RET being the most clinically relevant. These alterations were mutually exclusive with common drivers such as EGFR or KRAS. Detection of rare fusions highlights the therapeutic potential of comprehensive NGS profiling in Romanian NSCLC patients. Full article

Dragonflies and damselflies are important indicator species for quality and health of (semi-)aquatic habitats. Hitherto, 78 species of Odonata have been reported for Austria. Ecological data, Red List assessments, and a dragonfly association index exist, but population- and species-level genetic data are largely lacking. In this study, we establish a comprehensive reference DNA barcode library for Austrian dragonflies and damselflies based on the standard barcoding marker COI. Because of the increasing significance of environmental DNA (eDNA) analyses, we also sequenced a segment of the mitochondrial 16S rRNA gene, a marker often used in eDNA metabarcoding approaches. In total, we provide 786 new COI barcode sequences and 867 new 16S sequences for future applications. Sequencing success was >90 percent for both markers. Identification success was similar for both markers and exceeded 90 percent. Difficulties were only encountered in the genera Anax Leach, 1815, Chalcolestes Kennedy, 1920, Coenagrion Kirby, 1890 and Somatochlora Selys, 1871, with low interspecific genetic distances and, consequently, BIN (barcode index number) sharing. In Anax, however, individual sequences clustered together in species-specific groups in the COI tree. Irrespective of these challenges, the results suggest that both markers perform well within most odonate families in terms of sequencing success and species identification and can be used for reliably delimiting Austrian species, monitoring, and eDNA approaches. Full article

Background/Objectives: Postoperative pneumonia and other infectious complications after robotic-assisted minimally invasive esophagectomy still contribute to morbidity and mortality. Selective oral decontamination of the esophagus prior to surgery might reduce the rate of infectious complications. However, its impact on the esophageal microbiota is unknown. Therefore, this study aimed to analyze whether selective oral decontamination of the esophagus prior to surgery reduces postoperative pneumonia rates and alters the esophageal microbiome. Methods: We conducted a proof-of-principle study including 22 patients who underwent robotic-assisted minimally invasive esophagectomy. Thirteen patients were treated with 50 mg amphotericin B, 8 mg tobramycin, and 10 mg colistin orally 7 days prior to surgery, intraoperatively, and 5 days postoperatively. The remaining nine patients received standard-of-care treatment (no oral decontamination). The esophageal microbiome was assessed using 16S rRNA gene amplicon libraries which were annotated using the Ribosomal Data Project. The incidence of postoperative (at discharge from hospital or 30 days, whichever was later) infectious complications was assessed. Results: Selective oral decontamination was associated with reduced overall rates of infectious complications (7.7% vs. 55.5%, p = 0.008) and postoperative pneumonia (0% vs. 33.3%, p = 0.007). Alterations in the esophageal microbiome depending on selective oral decontamination were detectable. The microbiomes of patients with infectious complications showed higher abundances of Neisseria and lower abundances of Streptococcus than samples without infectious complications. Conclusions: Selective oral decontamination reduced the rate of postoperative complications, postoperative pneumonia in particular, after robot-assisted esophagectomy. Alterations in the microbiome were also evident following decontamination. Further studies with larger sample sizes are necessary to confirm these data. Full article

While the gut microbiota of obese children in Mexico has been studied, its relationship with depressive and anxiety symptoms in obese adults remains unexplored. The aim of this study was to describe the gut microbiota profile of Mexican adults with obesity and its association with depression and anxiety. We sequenced the V3-V4 region of the 16S rRNA gene from stool samples of obese adults categorized into four groups: control (OCG), with depressive symptoms (OD), with anxiety symptoms (OAx), or with both (ODAx). Alpha diversity was assessed using t-tests, beta diversity was assessed with PERMANOVA, and taxonomic differences was assessed with LEfSe. Associations between bacterial genera and clinical variables were analyzed using the Maaslin2 library. Bacteroidota was the most prevalent phylum, and Prevotella was the dominant enterotype across all groups. Although overall diversity did not differ significantly, 30 distinct taxonomic biomarkers were identified among groups as follows: 4 in OCG (Firmicutes), 5 in OD (Firmicutes, Bacteroidota), 13 in OAx (Firmicutes, Bacteroidetes, Fusobacteroidota, Proteobacteria), and 8 in ODAx (Firmicutes). This is the first study to identify distinct gut microbiota profiles in obese Mexican adults with depressive and anxiety symptoms. These findings suggest important microbial biomarkers for improving the diagnosis and treatment of mental health conditions in obesity. Full article

Small RNAs (sRNAs) are pivotal in regulating gene expression and are involved in a diverse array of biological processes. Among these, microRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) have been extensively investigated over the past decades. We conducted an in-depth analysis of deep sequencing data from the gymnosperm Ginkgo biloba, encompassing sRNA, transcriptome, and degradome libraries. Our analysis identified a total of 746 miRNAs and 654 phasiRNA precursor (PHAS) loci, with 526 (80%) of the PHAS loci predicted to be triggered by 515 miRNAs (69%). Several miRNA-PHAS modules, particularly the miR159/miR319-PHAS module, were found to potentially regulate reproductive development by targeting GAMYB genes and triggering phasiRNA biogenesis. The miR390-PHAS module appears to be involved in flavonoid biosynthesis by targeting key enzyme genes such as chalcone synthase (CHS) and anthocyanin synthase (ANS). Through target gene identification and coexpression analysis, we uncovered two distinct models of complex regulatory networks: growth-related factors like ARF and GRF seem to be regulated exclusively by miRNAs (Model 1), while certain disease resistance-related genes are predicted to be regulated by both miRNAs and phasiRNAs (Model 2), indicating diverse regulatory mechanisms across different biological processes. Overall, our study provides a comprehensive annotation of miRNA and PHAS loci in G. biloba and elucidates a post-transcriptional regulatory network, offering novel insights into sRNA research in gymnosperms. Full article

Background/Objectives: Massively parallel sequencing technologies have advanced chronic lymphocytic leukemia (CLL) diagnostics and precision oncology. Illumina platforms, while offering robust performance, require substantial infrastructure investment and a large number of samples for cost-efficiency. Conversely, third-generation long-read nanopore sequencing from Oxford Nanopore Technologies (ONT) can significantly reduce sequencing costs, making it a valuable tool in resource-limited settings. However, nanopore sequencing faces challenges with lower accuracy and throughput than Illumina platforms, necessitating additional computational strategies. In this paper, we demonstrate that integrating publicly available short-read data with in-house generated ONT data, along with the application of machine learning approaches, enables the characterization of the CLL transcriptome landscape, the identification of clinically relevant molecular subtypes, and the assignment of these subtypes to nanopore-sequenced samples. Methods: Public Illumina RNA sequencing data for 608 CLL samples were obtained from the CLL-Map Portal. CLL transcriptome analysis, gene module identification, and transcriptomic subtype classification were performed using the oposSOM R package for high-dimensional data visualization with self-organizing maps. Eight CLL patients were recruited from the Hematology Center After Prof. R. Yeolyan (Yerevan, Armenia). Sequencing libraries were prepared from blood total RNA using the PCR-cDNA sequencing-barcoding kit (SQK-PCB109) following the manufacturer’s protocol and sequenced on an R9.4.1 flow cell for 24–48 h. Raw reads were converted to TPM values. These data were projected into the SOMs space using the supervised SOMs portrayal (supSOM) approach to predict the SOMs portrait of new samples using support vector machine regression. Results: The CLL transcriptomic landscape reveals disruptions in gene modules (spots) associated with T cell cytotoxicity, B and T cell activation, inflammation, cell cycle, DNA repair, proliferation, and splicing. A specific gene module contained genes associated with poor prognosis in CLL. Accordingly, CLL samples were classified into T-cell cytotoxic, immune, proliferative, splicing, and three mixed types: proliferative–immune, proliferative–splicing, and proliferative–immune–splicing. These transcriptomic subtypes were associated with survival orthogonal to gender and mutation status. Using supervised machine learning approaches, transcriptomic subtypes were assigned to patient samples sequenced with nanopore sequencing. Conclusions: This study demonstrates that the CLL transcriptome landscape can be parsed into functional modules, revealing distinct molecular subtypes based on proliferative and immune activity, with important implications for prognosis and treatment that are orthogonal to other molecular classifications. Additionally, the integration of nanopore sequencing with public datasets and machine learning offers a cost-effective approach to molecular subtyping and prognostic prediction, facilitating more accessible and personalized CLL care. Full article

Understanding the interactions between the cervico-vaginal microbiome, immune responses, and sexually transmitted infections (STIs) is crucial for developing targeted diagnostic and therapeutic strategies. Although microbiome analyses are not yet standard practice, integrating them into routine diagnostics could enhance personalized medicine and therapies. We investigated the extent to which partial 16S short-read amplicon microbiome analyses could inform on the presence of six commonly encountered STI-causing pathogens in a patient cohort referred for colposcopy, and whether relevant taxonomic or diagnostic discrepancies occur when using vaginal rather than cervical swabs. The study cohort included cervical and vaginal samples collected from women referred for colposcopy at the University Hospital Bonn between November 2021 and February 2022, due to an abnormal PAP smear or positive hrHPV results. 16S rRNA gene sequencing libraries were prepared targeting the V1–V2 and V4 regions of the 16S RNA gene and sequenced on the Illumina MiSeq. PCR diagnostics for common STI-causing pathogens were conducted using the Allplex STI Essential Assay Kit (Seegene, Seoul, Republic of Korea). Concerning the bacterial microbiome, no significant differences were found between vaginal and cervical samples in terms of prevalence of taxa present or diversity. A total of 95 patients and 171 samples tested positive for at least one among Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydophila trachomatis or Neisseria gonorrhoeae. Sequencing the V1–V2 region enabled detection of one-third to half of the PCR-positive samples, with the detection likelihood increasing at lower cycle threshold (Ct) values. In contrast, sequencing the V4 region was less effective overall, yielding fewer species-level identifications and a higher proportion of undetermined taxa. We demonstrate that the vaginal microbiome closely mirrors the cervical microbiome, a relationship that has not been explored previously, but which broadens the possibilities for microbiome analysis and pathogen detection and establishes vaginal swabs as a reliable method for detecting the investigated pathogens, with sensitivities comparable with or superior to endocervical swabs. On the other hand, the sensitivity of partial 16S amplicon sequencing appears insufficient for effective STI diagnostics, as it fails to reliably identify or even detect pathogens at higher Ct values. Full article

Schistosomiasis, a parasitic disease caused by worms of the genus Schistosoma, affects >250 million people worldwide. With no available vaccine, treatment relies solely on one drug—praziquantel—underscoring the urgent need for new therapies. We identified a tegumental, non-neuronal acetylcholinesterase (AChE) from Schistosoma mansoni—SmTAChE—as a promising drug target. RNA interference confirmed its essential role in parasite survival, as gene suppression significantly reduced parasite recovery from infected animals. Here, we produced functionally active recombinant SmTAChE by using a mammalian expression system. Biochemical characterization confirmed its identity as a true acetylcholinesterase, with the highest turnover rate (K_cat = 373 ± 39 s⁻¹) and catalytic efficiency (K_cat/K_m = 1.17 × 10⁶ M⁻¹·S⁻¹) for acetylthiocholine. Additionally, rSmTAChE was inhibited by classical AChE-specific inhibitors but not by a butyrylcholinesterase-specific inhibitor. To identify novel SmTAChE inhibitors, we developed a high-throughput chemical screening protocol (Z′ factor > 0.9) and screened a 1894-compound validation library. Twelve compounds reproducibly inhibited rSmTAChE by >30% at 7.5 µM, including known AChE inhibitors like physostigmine and new selective inhibitors. Notably, compound #2 preferentially inhibited rSmTAChE (IC₅₀ = 0.74 µM) over human AChE (IC₅₀ = 151 µM), thus providing a foundation for developing parasite-specific therapies targeting SmTAChE and potentially leading to new treatments for schistosomiasis. Full article

Inflammation can positively and negatively affect tumorigenesis based on the duration, scope, and sequence of related events through the regulation of signaling pathways. A transcriptomic analysis of five pulmonary arterial hypertension, twelve Crohn’s disease, and twelve ulcerative colitis high throughput sequencing datasets using R language specialized libraries and gene enrichment analyses identified a regulatory network in each inflammatory disease. IRF9 and LINC01089 in pulmonary arterial hypertension are related to the regulation of signaling pathways like MAPK, NOTCH, human papillomavirus, and hepatitis c infection. ZNF91 and TP53TG1 in Crohn’s disease are related to the regulation of PPAR, MAPK, and metabolic signaling pathways. ZNF91, VDR, DLEU1, SATB2-AS1, and TP53TG1 in ulcerative colitis are related to the regulation of PPAR, AMPK, and metabolic signaling pathways. The activation of the transcriptomic network and signaling pathways might be related to the interaction of the characteristic microbiota of the inflammatory disease, with the lung and gut cell receptors present in membrane rafts and complexes. The transcriptomic analysis highlights the impact of several coding and non-coding RNAs, suggesting their relationship with the unlocking of cell phenotypic plasticity for the acquisition of the hallmarks of cancer during lung and gut cell adaptation to inflammatory phenotypes. Full article

Background: The satellite DNA (satDNA) PcP190 has been identified in multiple frog species from seven phylogenetically distant families within Hyloidea, indicating its broad distribution. This satDNA consists of repeats of approximately 190 bp and exhibits a highly conserved region (CR) of 120 bp, which is similar to the transcribed region of 5S ribosomal DNA (rDNA), and a hypervariable region (HR) that varies in size and nucleotide composition among and within species. Here, to improve our understanding of PcP190 satDNA, we searched for evidence of its transcription in the available transcriptomes of Rhinella marina (Bufonidae) and Engystomops pustulosus (Leptodactylidae), two phylogenetically distantly related species. Methods: We first characterized the 5S rDNA and PcP190 sequences in these species by searching for them in available genome assemblies. Next, we used the PcP190 (CR and HR) and 5S rDNA sequences of each species as queries to search for these sequences in RNA-seq libraries. Results: We identified two types of 5S rDNA in each analyzed species, with a new type found in E. pustulosus. Our results also revealed a novel type of PcP190 sequence in R. marina and a new subtype of PcP-1 in E. pustulosus. Transcriptome analyses confirmed the expected transcription of the 5S rRNA gene and showed transcription of both the CR and HR of the PcP190 satDNA in both species and in different tissues. Conclusions: As the entire repeat of this satDNA is susceptible to transcription, the high variability observed in the HR cannot be attributed to transcriptional activity confined to the CR. Full article

To date, only a few microbial community studies of cold seeps at the South China Sea (SCS) have been reported. The cold seep dominated by tubeworms was discovered at South Yungan East Ridge (SYER) offshore southwestern Taiwan by miniROV. The tubeworms were identified and proposed as Paraescarpia formosa sp. nov. through morphological and phylogenetic analyses. The endosymbionts in the trunk of P. formosa analyzed by a 16S rRNA gene clone library represented only one phylotype, which belonged to the family Sedimenticolaceae in Gammaproteobacteria. In addition, the archaeal and bacterial communities in the habitat of tubeworm P. formosa were investigated by using high-phylogenetic-resolution full-length 16S rRNA gene amplicon sequencing. The results showed that anerobic methane-oxidizing archaea (ANME)-1b was most abundant and ANME-2ab was minor in a consortia of the anerobic oxidation of methane (AOM). The known sulfate-reducing bacteria (SRB) partners in AOM consortia, such as SEEP-SRB1, -SRB2, and -SRB4, Desulfococcus and Desulfobulbus, occurred in a small population (0–5.7%) at the SYER cold seep, and it was suggested that ANME-1b and ANME-2ab might be coupled with multiple SRB in AOM consortia. Besides AOM consortia, various methanogenic archaea, including Bathyarchaeota (Subgroup-8), Methanocellales, Methanomicrobiales, Methanosarcinales, Methanofastidiosales and Methanomassiliicoccales, were identified, and sulfur-oxidizing bacteria Sulfurovum and Sulfurimonas in phylum Epsilonbacteraeota were dominant. This study revealed the first investigation of microbiota in and around tubeworm P. formosa discovered at the SYER cold seep offshore southwestern Taiwan. We could gain insights into the chemosynthetic communities in the deep sea, especially regarding the cold seep ecosystems at the SCS. Full article

Planktonic unicellular cyanobacteria are the dominant biomass producers and carbon fixers in the global ocean ecosystem, but they are not abundant in polar seawater. The interseasonal dynamics of picocyanobacterial (PC) abundance, picophytoplankton primary production, and phylogenetic diversity of PC Synechococcus were studied in the sub-Arctic White Sea. The PC abundance varied from 0.2–0.3 × 10⁶ cells/L in February to 5.2–16.7 × 10⁶ cells/L in July. Picophytoplankton primary production ranged from 0.22 mg C/m³ per day in winter to 11.32 mg C/m³ per day in summer. Synechococcus abundance positively correlated with water temperature and river discharge that increased in recent years in the White Sea. Phylogenetic analysis of the 16S rRNA gene and ITS region clone libraries from the White Sea and Barents Sea eDNA revealed picocyanobacterial sequences related to marine Synechococcus subclusters 5.1-I, 5.I-IV, 5.2, and 5.3. All Synechococcus S5.1-I were common in the White and Barents seas and were consistently present in the picophytoplankton composition throughout the year. Synechococcus S5.2 and S5.3 appear in the PC community in summer, suggesting their river origin, and Synechococcus S5.1-IV inhabits only the Barents Sea and was not detected in the White Sea. A unique Synechococcus phylotype was revealed. It is expected that the increase in the abundance of PC and their increasing role in ecosystem functioning, as well as the enrichment of the species composition with new phylotypes in the semi-enclosed sub-Arctic White Sea, which is vulnerable to the effects of climate change, will be characteristic of all Arctic seas in general. Full article

Grafting is important for increasing the resistance of grapevines to environmental stress, improving fruit quality, and shortening the reproductive period. In this study, ‘Cabernet Sauvignon’ (CS) grafted on the resistant rootstock 140R (CS/140R), self-grafted grapevines of the resistant rootstock 140R (140R/140R), and self-grafted grapevines of CS (CS/CS) were subjected to high-throughput sequencing; small RNA (sRNA) libraries were constructed, and miRNAs responsive to the grafting process were identified. A total of 177 known miRNAs and 267 novel miRNAs were identified. Many miRNAs responsive to the grafting process were significantly down-regulated in CS/140R leaves relative to CS/CS leaves, such as vvi-miR171c, vvi-miR171e, et al., suggesting that the expression of these miRNAs might be affected by grafting. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the differentially expressed miRNAs regulated the expression of genes in the phenylpropanoid synthesis pathway. Grapevine leaves transiently overexpressing vvi-miR171c were assayed, and the expression of the target gene, VvMYB154, and the resveratrol content were decreased, indicating that vvi-miR171c negatively regulates the expression of VvMYB154. In sum, 140R increased the resveratrol content of the scion by grafting, down-regulating the expression of vvi-miR171c. These results provide new information that will aid future analyses of the effects of grafting on the content of secondary metabolites. Full article

The objective of this study was to evaluate the diversity and prevalence of tick-borne protists in the Republic of Korea via DNA barcoding using 18S rRNA gene fragments and PCR. Between 2021 and 2022, questing ticks were collected using the flagging method, with a total of 13,375 ticks collected and pooled into 1003 samples. Of these, 50 tick pools were selected for DNA barcoding targeting the V4 and V9 regions of 18S rRNA using the MiSeq platform. A taxonomic analysis of the amplicon sequence variants identified three genera of protozoa, namely Hepatozoon canis, Theileria luwenshuni, and Gregarine sp. However, the number and abundance of protists detected were different depending on the primer sets, and T. gondii was not identified in DNA barcoding. Furthermore, conventional PCR confirmed the presence of H. canis, Toxoplasma gondii, T. luwenshuni, and Theileria sp. in the collected ticks. This study identified H. canis and T. gondii in Ixodes nipponensis for the first time. It demonstrated that the results of DNA barcoding using 18S rRNA gene fragments can vary depending on the primer sets and further optimization is required for library construction to identify tick-borne protists in ticks. Despite these limitations, the findings highlight the potential of DNA barcoding using 18S rRNA gene fragments for screening the diversity of tick-borne protists in ticks. Full article