Summary
Porcine circovirus type 3 (PCV3) is a novel circovirus species recently discovered in USA and China in cases of porcine dermatitis and nephropathy syndrome, reproductive failure, respiratory ...disease and multisystemic inflammation. This study reports on the first identification of PCV3 in Europe, in serum from pigs from Polish farms. A total of 1,050 serum samples were collected between 2014 and 2017 from sows and 3–20 weeks old pigs from 14 commercial farms representing different regions of Poland, different size and health status. The samples were pooled by 4–6 and tested with real‐time PCR for PCV3. PCV3 DNA was detected in 12 of 14 farms (85.7%). On the PCV3‐positive farms, the virus was detected in 5.9% to 65% serum pools. PCV3 was most common among weaned pigs and finishers (26.1% and 28.0% of serum pools, respectively). Sequence analysis of 359 nucleotide fragment of ORF2 showed highest identity of 99.7% to PCV3‐US/SD2016 from USA. Our results indicate that PCV3 is a common virus among Polish pigs but no links to unexplained disease conditions were established.
Environmental DNA (eDNA) analysis has successfully detected organisms in various aquatic environments. However, there is little basic information on eDNA, including the eDNA shedding and degradation ...processes. This study focused on water temperature and fish biomass and showed that eDNA shedding, degradation, and size distribution varied depending on water temperature and fish biomass. The tank experiments consisted of four temperature levels and three fish biomass levels. The total eDNA and size‐fractioned eDNA from Japanese Jack Mackerels (Trachurus japonicus) were quantified before and after removing the fish. The results showed that the eDNA shedding rate increased at higher water temperature and larger fish biomass, and the eDNA decay rate also increased at higher temperature and fish biomass. In addition, the small‐sized eDNA fractions were proportionally larger at higher temperatures, and these proportions varied among fish biomass. After removing the fish from the tanks, the percentage of eDNA temporally decreased when the eDNA size fraction was >10 µm, while the smaller size fractions increased. These results have the potential to make the use of eDNA analysis more widespread in the future.
This study showed that the Japanese Jack Mackerel eDNA shedding rate increased at higher water temperatures and larger fish biomass, and the most supported model for the eDNA decay curves included both temperature and fish density as explanatory variables. In addition, eDNA size distribution varied depending on temperature, fish biomass, and time passage.
Biodiversity monitoring via environmental DNA, particularly metabarcoding, is evolving into a powerful assessment tool for riverine systems. However, for metabarcoding to be fully integrated into ...standardized monitoring programmes, some current challenges concerning sampling design, laboratory workflow, and data analysis need to be overcome. Here, we review some of these major challenges and potential solutions. We further illustrate three potential pitfalls, namely the choice of suitable metabarcoding primers, the necessity of complete reference databases, and varying assay sensitivities, by a reappraisal of our‐own recently carried out metabarcoding study in the Volga headwaters. TaqMan qPCRs had detected catfish (Silurus glanis) and European eel (Anguilla anguilla), whereas metabarcoding had not, in the same samples. Furthermore, after extending the genetic reference database by 12 additional species and re‐analysing the metabarcoding data, we additionally detected the Siberian spiny loach (Cobitis sibirica) and Ukrainian brook lamprey (Eudontomyzon mariae) and reassigned the operational taxonomic units previously assigned to Misgurnus fossilis to Cobitis sibirica. In silico analysis of metabarcoding primer efficiencies revealed considerable variability among primer pairs and among target species, which could lead to strong primer bias and potential false‐negatives in metabarcoding studies if not properly compensated for. These results highlight some of the pitfalls of eDNA‐metabarcoding as a means of monitoring fish biodiversity in large rivers, which need to be considered in order to fully unleash the full potential of these approaches for freshwater biodiversity monitoring.
A novel labeled nucleotide exerts very low near‐IR fluorescence in water but lights‐up (up to a 348‐fold increase) after polymerase incorporation to DNA. It can be used to directly visualize ...enzymatic DNA synthesis in vitro and the labeled DNA probes are also responsive to secondary structure changes or interactions with small molecules or proteins. Its application was demonstrated on real‐time PCR detection of RNA from SARS‐CoV‐2 virus. More information can be found in the Communication by M. Hocek et al. on page 11950.
Arbuscular mycorrhizal fungi (AMF) are ubiquitous soil fungi, forming mutualistic symbiosis with a majority of terrestrial plant species. They are abundant in nearly all soils, less diverse than soil ...prokaryotes and other intensively studied soil organisms and thus are promising candidates for universal indicators of land management legacies and soil quality degradation. However, insufficient data on how the composition of indigenous AMF varies along soil and landscape gradients have hampered the definition of baselines and effect thresholds to date. Here, indigenous AMF communities in 154 agricultural soils collected across Switzerland were profiled by quantitative real‐time PCR with taxon‐specific markers for six widespread AMF species. To identify the key determinants of AMF community composition, the profiles were related to soil properties, land management and site geography. Our results indicate a number of well‐supported dependencies between abundances of certain AMF taxa and soil properties such as pH, soil fertility and texture, and a surprising lack of effect of available soil phosphorus on the AMF community profiles. Site geography, especially the altitude and large geographical distance, strongly affected AMF communities. Unexpected was the apparent lack of a strong land management effect on the AMF communities as compared to the other predictors, which could be due to the rarity of highly intensive and unsustainable land management in Swiss agriculture. In spite of the extensive coverage of large geographical and soil gradients, we did not identify any taxon suitable as an indicator of land use among the six taxa we studied.
Acute myeloid leukemia (AML) involves abnormal differentiation and clonal proliferation of myeloid progenitor cells in the bone marrow. The Wilms tumor 1 (WT-1) gene regulates cell growth, apoptosis, ...and differentiation, but its biological role in AML is poorly understood.
Evaluation of RNA expression and molecular functions of WT-1 gene in the context of other genetic alterations in AML.
Prospective clinical research
Tertiary cancer care referral center
A total of 112 diagnosed cases of AML, having blast percentage of ≥20% blasts in peripheral blood or bone marrow on Day-0 and treated with induction therapy for 28 days were enrolled.
WT-1 gene expression was assessed using RNA extracted from blood/bone marrow samples. The molecular functions of WT-1 were analyzed by performing gene set enrichment analysis (GSEA), and the relationship of WT-1 expression with immune checkpoints was analyzed using the Sangerbox 3.0 database. Kaplan–Meier survival analysis was performed to estimate the prognostic significance of the WT-1 gene in AML.
Out of 112 patients, 73 were males, and 39 were females. A total of 97 (86.60%) cases showed increased expression of the WT-1 gene at Day-0 as compared to cases in complete remission (P=<0.001). WT-1 expression was inversely correlated with normal hematopoiesis and positively correlated with age, high marrow blast counts, M4 subtype, and inferior outcomes compared to patients with low WT-1 expression levels. In GSEA, the WT-1 gene displayed an important role in misregulating DNA-binding transcription factor activity, RNA and protein binding, as well as negative regulation of cell growth and cell population proliferation. In immune checkpoint analysis, WT-1 gene expression was positively correlated with CD28, CD40, CD44, CD48, CD80, CD70, CD27, CD86. In survival analysis, poorer overall survival was seen in patients having higher WT-1 gene expression.
Increased expression of the WT-1 gene positively correlates with the leukemic burden in most cases of AML. The gene can be considered a promising molecular marker for early diagnosis, MRD detection, and a target for developing novel therapeutic approaches against AML.
SERB, DST, Government of India
The ten-eleven translocation 1 gene (TET1) is a member of the TET methyl cytosine dioxygenase family of enzymes (TET1, TET2, and TET3). TET1 has a role in various physiological and pathological ...processes. It is an epigenetic regulator of the DNA and RNA demethylation processes. Therefore, it regulates gene expression at both the transcriptional and post-transcriptional levels.
We studied the expression levels of the TET1 gene in patients with acute myeloid leukemia (AML) and its correlation with their clinical and pathological criteria.
This study was done in 37 patients who were newly diagnosed with AML. Bone marrow (BM) samples were analyzed using real-time PCR 7500s. Twenty subjects, apparently healthy, were included as a control group and matched by age and sex.
There was a significant correlation between the expression levels of the TET1 gene in patients with AML and their clinical and pathological criteria. It has been found that expression levels of the TET1 gene were higher in patients with AML than in the control group. Our study reported a significant TET1 gene upregulation in 62.2% of the patients with AML. Significantly higher TET1 levels were found in 66.7% of patients with AML not otherwise specified (NOS), 66.7% of patients with AML without maturation, 83.3% of patients with AML with maturation, and 61.9% of patients with AML with monocytic differentiation. Regarding AML with recurrent cytogenetic abnormalities, 100% of patients with AML carrying t(8;21) showed TET1 downregulation, but there was no difference in TET1 expression levels among AML patients with t(15;17). This study showed a significant relationship between TET1 gene levels and the percentage of blast cells in peripheral blood (PB). In BM, the higher percentage of blast cells was associated with the upregulated TET1 gene.
TET1 exerts an oncogenic role in AML and is upregulated in patients with the disease. A relationship was found between TET1 expression levels and blast percentages in the PB and BM of patients with AML. The higher percentage of blast cells was associated with the upregulated TET1 gene.
The fate of antibiotic resistance genes (ARGs) in environmental microbial communities is of primary concern as prodromal of a potential transfer to pathogenic bacteria. Although of diverse origin, ...the persistence of ARGs in aquatic environments is highly influenced by anthropic activities, allowing potential control actions in well‐studied environments. However, knowledge of abundance and space–time distribution of ARGs in ecosystems is still scarce. Using quantitative real‐time PCR, we investigated the presence and the abundance of twelve ARGs (against tetracyclines, β‐lactams, aminoglycosides, quinolones and sulphonamides) at different sampling sites, depths and seasons, in Lake Maggiore, a large subalpine lake, and in the area of its watershed. We then evaluated the correlation between each ARG and a number of ecological parameters in the water column in the deepest part of the lake. Our results suggest the constitutive presence of at least four ARGs within the bacterial community with a high proportion of bacteria potentially resistant to tetracyclines and sulphonamides. The presence of these ARGs was independent of the total bacterial density and temperature. The dynamics of tet(A) and sulII genes were, however, positively correlated with dissolved oxygen and negatively to chlorophyll a, suggesting that the resistant microbes inhabit specific niches. These observations indicate that the lake is a reservoir of antibiotic resistances, highlighting the need of a deeper understanding of the sources of ARGs and the factors allowing their persistence in waters.
Effective detection of pathogens from complex substrates is a challenging task. Molecular approaches such as real‐time PCR can detect pathogens present even in low quantities. However, weak real‐time ...PCR signals, as represented by high cycle threshold (Ct) values, may be questionable. Therefore, setting a reliable Ct threshold to declare a positive reaction is important for specific detection. In this study, five methods were assessed for their performance in determining a Ct cut‐off value. These methods were based on the widely used probability of detection (POD) or receiver‐operating characteristic (ROC) approaches. Two important forest pathogens, Hymenoscyphus fraxineus and Fusarium circinatum, were used to set up three experimental frameworks that combined two types of substrates (seed lots and spore traps) and different PCR machines. The ROC‐based method emerged as the most complete and flexible method under various experimental conditions. It was demonstrated that the ROC method leads to a cut‐off value below which late Ct results can reliably be considered indicative of positive test results. This cut‐off value must be determined for each experimental approach used. The method based on the distribution of a previously determined set of Ct values corresponding to false‐positives appeared to be better adapted to detecting false‐negative results, and thus useful for testing potentially invasive pathogens.
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