Search Results (1,278)

This study developed a rapid and reliable SYBR-Green semiplex PCR assay for simulta-neous detection of major carbapenem resistance genes in Klebsiella pneumoniae and Acinetobacter baumannii. Two primer sets were used: one to detect blaKPC, blaNDM-1, and blaOXA-48 genes in K. pneumoniae and blaOXA-23 in A. baumannii, and another to amplify conserved 16S rRNA gene regions as internal controls. The intra- and inter-assay coeffi-cient of variation ranged from 0.03% to 3.8%. Standard curves exhibited excellent linearity across six logarithmic scales (10¹–10⁶ DNA copies/µL), with detection limits of 10–10² DNA copies/mL. Melting temperatures (Tm) were: 88.85 °C (KPIC), 90.65 °C (NDM-1), 89.45 °C (KPC), 84.23 °C (OXA-48), 87.81 °C (OXA-23), and 80.67 °C (ABIC). The SYBR-Green Semiplex PCR assay offers a rapid (65 min turnaround), cost-effective, and sensitive method for early detection of carbapenem-resistant pathogens, enabling timely targeted therapy and improved infection control by potentially reducing empirical antibiotic use before culture confirmation. Full article

Potassium (K) is present in soils mainly in minerals, including feldspar. However, most of it is unavailable to plants. In the in-dyked alluvial soils of the Mekong Delta, available K is typically low despite the abundance of K-bearing feldspar, leading to nutrient imbalances and yield constraints. This study aimed to (i) select potential feldspar-potassium-solubilizing purple nonsulfur bacteria (K-PNSB), (ii) determine their ability to enhance hybrid maize seed vigor (Zea mays L.), and (iii) evaluate their effects on the growth of maize seedlings. Fifty-eight K-PNSB strains were isolated from maize-cultivated in-dyked alluvial soils, with soluble K concentrations ranging from 0.108 to 15.0 mg L⁻¹. Among these, strain M-Sl-03 released the highest K concentration under microaerobic light conditions, whereas strains M-Sl-01 and M-Sl-06 produced best under aerobic dark conditions. In addition, two more strains, M-Sl-02 and M-Wa-06, were also selected for their K solubilization ability. The selected strains were identified as Cereibacter sphaeroides strains M-Sl-01 and M-Sl-02, Rhodopseudomonas palustris strain M-Sl-03, and Rhodoplanes pokkaliisoli strains M-Sl-03 and M-Wa-06, according to their 16S rDNA region. None of them exhibited toxicity to germinating maize seeds. Both individual strains and the five-strain mixture significantly improved seed vigor. At a 1:1000 dilution, individual and mixed inoculants increased the vigor index of maize seeds by 47.5–68.8%. In addition, the selected PNSB strains contributed to improving the growth of maize seedlings, particularly plant height and root dry biomass. These promising strains have potential for application as biofertilizers to support hybrid maize cultivation. Full article

Environmental DNA (eDNA) has become a promising tool for phytoplankton surveys. However, the accuracy of eDNA-based detection is related to primer selection across diverse environments, and optimal primer pairs selection on phytoplankton community in human impacted ecosystems is still lacking. The aim of this study is to evaluate how primer selection shapes phytoplankton community profiles by eDNA biomonitoring diverse anthropogenically disturbed aquatic systems (rivers, reservoirs, and seas). Four primer pairs targeting the 18S rRNA (V9-1 and V9-2), chloroplast rbcL, and ITS regions, were explored and our results revealed that primer choice critically governed the accuracy of phytoplankton profiling. Significant variations in annotated phytoplankton eDNA sequences in different groups of primer pairs were observed, where the primers 18SV9-1 and rbcL demonstrated superior specificity, amplifying >90% of phytoplankton OTUs. 18S-targeted primers detected the highest species richness, while the ITS primer showed the lowest. Alpha diversity was highest and most consistent for 18S primers. Beta diversity ordination (nMDS/Bray–Curtis) further highlighted primer-dependent community structuring in which 18S primers effectively clustered reservoir and marine samples separately, whereas primer rbcL discriminated habitat-specific signatures across three ecosystems. The primer ITS failed to distinguish among different habitats. Overall, our data demonstrated the critical role of primer optimization in eDNA-based phytoplankton studies, and could provide methodological guidelines for the design of effective monitoring protocols in rapidly urbanizing aquatic systems. Full article

The Red-legged Partridge (Alectoris rufa, Phasianidae) is a non-migrant gamebird endemic to southwestern Europe that was introduced into Mediterranean and Atlantic islands in historical times. This is the case for Madeira, Portugal, where a population morphologically assigned to A. r. hispanica has been present since the XV century. We assessed its genetic identity using 2248 (Cytochrome-b, Cyt-b + Control Region, CR) and 297 bp-long (CR) mitochondrial DNA sequences obtained from modern and archival (1900–1964, including Caccabis rufa maderensis syntypes) partridges, respectively. These sequences were compared against an already published dataset covering the entire Iberian A. rufa range. We found that all the haplotypes of modern birds from Madeira were private to this island. The putative subspecies was confirmed, and northern Portugal with northwestern Spain turned out to host the closest mainland populations. This result was in line with the origin of the first human settlers of Madeira from, among other historical provinces, Douro Litoral and Minho, the latter neighboring Galicia. Despite relatively recent A. rufa importations from continental Europe, we did not find any significant change over time in the haplotypic pattern of Madeiran partridges as well as any evidence for maternal introgression from species such as the congeneric Chukar Partridge (A. chukar). Studies relying on genome-wide markers and including the only captive-bred population of Madeira are needed to gain more comprehensive information for the management of the local A. rufa. Full article

Both absolute and presumably active rDNA (with a hypomethylated promoter region) copy number (CN) in the haploid human sperm genome are highly variable among individuals. Using a combination of droplet digital PCR and deep bisulfite sequencing, we have quantified absolute and presumably active rDNA CN in sperm samples (N = 190) with normal (NSPs) vs. abnormal semen parameters (ASPs), as well as in samples leading or not leading to a clinical pregnancy. ASP samples had a significantly lower presumably active CN (104 ± 31) than normozoospermic samples (115 ± 31). The loss of presumably active rDNA copies is explained by an increased promoter methylation (13.9% in ASP vs. 12.1% in NSP). When correcting for confounding factors, most importantly semen quality, samples not leading to a clinical pregnancy after IVF/ICSI displayed a significantly lower absolute (225 ± 51) and presumably active CN (103 ± 30) than samples with pregnancy (249 ± 62 and 115 ± 31, respectively). This between-group difference was most noticeable in normozoospermic males: absolute CN 220 ± 54; presumably active CN 107 ± 32 in samples without pregnancy and absolute CN 246 ± 63; presumably active CN 120 ± 28 in samples with pregnancy. We propose that absolute/active rDNA CN in sperm is a modulating factor contributing to idiopathic male infertility. In NSP samples, presumably active CN increases with absolute CN, which may have a positive impact on fertility and ART outcome. Our results suggest that approximately 60 active sperm rDNA copies are sufficient to establish a pregnancy. Full article

The number of known mitochondrial genomes in Buprestidae is limited, especially in Chrysochroinae, which seriously hinders the phylogenetic study of this family. The mitogenomes of Capnodis miliaris, Lamprodila cupreosplendens, Sphenoptera insidiosa and Philocteanus rubroaureus were sequenced, assembled and annotated in this study. The mitogenomes of these four species are typical circular double-stranded DNA molecules, containing 13 protein-coding genes (PCGS), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a control region (CR). The total lengths of these four mitogenomes are moderate, ranging from 15,778 bp to 16,230 bp. Additionally, their A + T content ranges from 68.76% to 73.47%, showing positive AT-skew values ranging from 0.098 to 0.181. Relative Synonymous Codon Usage (RSCU) analysis indicated that TTT (Phe), ATT (Ile), TCT (Ser2), and TTA (Leu2) are the most frequently used codons. The gene arrangement of four mitogenomes is consistent with the previously reported Buprestidae mitogenomes. Most of the PCGs use ATN as the start codon, with TAA as the stop codon or an incomplete stop codon T-. Phylogenetic trees were constructed based on the PCGs and rRNAs using both maximum-likelihood and Bayesian inference methods. The phylogenetic results showed that Julodinae, Polycestinae, Buprestinae and Agrilinae are monophyletic groups, and Chrysochroinae is a paraphyletic group. As the number of Buprestidae mitogenomes used for polyogenetic analysis increases, the topology of phylogenetic tree shows differences compared to previous studies. Full article

Sapindus mukorrosi (Sm) seeds have been used in Chinese medicine for treating gingival disease, suggesting that Sm may modulate oral bacteria and alleviate gingival inflammation. However, the hydrophobicity of seed oil limits its use in the aqueous oral environment. Therefore, the artificial saliva-infused Sm seed aqueous extract (SMa) was developed and applied to our ex vivo model to test its anti-bacterial effect. Unstimulated whole saliva from seven patients with Stage III/IV, Grade C periodontitis was cultured for 8 h with or without SMa. The bacterial count was measured based on the optical density and bacterial DNA concentration. The salivary microbiome was sequenced via next-generation sequencing over the 16S rRNA gene V3-V4 hypervariable regions. The bacterial DNA concentration in the SMa group was significantly lower than the Without-SMa group after 6 to 8 h of culture. No significant difference in alpha and beta diversity was observed between the two groups. The relative abundance of Porphyromonas was reduced, while that of Veillonella was elevated in the SMa group compared to the Without-SMa group. The findings indicated that the antibacterial effects of SMa are manifested primarily through bacterial growth inhibition, with the minor modulation of specific taxa. Full article

Dinoflagellate resting cysts are critical to dinoflagellate ecology, acting as a key seed source for initiating harmful algal blooms (HABs) through their germination. However, the in situ germination dynamics of these cysts remain poorly understood due to technical challenges. To overcome this, we utilized the Germlings Harvester (GEHA), an in situ germination device we designed, to collect water samples containing dinoflagellate cysts germinated from marine sediments in Jiaozhou Bay, China, after 5 and 20 days of incubation. By combining the GEHA with metabarcoding analysis targeting 28S rDNA-specific primers for dinoflagellates, we identified 44 dinoflagellate species spanning 31 genera, 18 families, and 7 orders. Of these, 12 species were linked to HABs or recognized as toxic, including Azadinium poporum, Alexandrium leei, Alexandrium pacificum, Akashiwo sanguinea, Karlodinium veneficum, Stoeckeria algicida, and Luciella masanensis. Additionally, five species were newly identified as cyst producers, and one symbiotic dinoflagellate, Effrenium voratum, was detected. Our results also found that germinated dinoflagellate species increased from 23 to 34 with extended incubation, and the ratio of mixotrophic to heterotrophic species was approximately 2:1 in the samples of in situ sediments and seawater outside GEHA, as well as across germination durations (Sg-5 d vs. Sg-20 d). These findings provide essential field evidence for the role of resting cysts in driving HAB formation in this region and highlight the efficacy of the GEHA-based approach for studying in situ cyst germination dynamics, offering a robust tool for monitoring, early warning, prevention, and forecasting of HABs. Full article

This study evaluated the genetic diversity of 33 Coffea canephora genotypes through morphophysiological and molecular analyses, aiming to identify promising genotypes for pre-breeding purposes in the southern region of Espírito Santo, Brazil. Cutting-propagated seedlings were evaluated 120 days after planting, considering height, stem and crown diameter, number of leaves, fresh and dry shoot and root weight, chlorophyll content, and root characteristics. Molecular analysis was performed on 32 genotypes; one was excluded due to absent DNA, and 18 ISSR markers were used. Morphological data were analyzed by ANOVA, Scott–Knott’s mean test, principal component analysis, and cluster analysis. The results revealed significant diversity among genotypes. The first two principal components explained 75.5% of the total variability. Genotypes 2, 3, 4, 5, 6, 10, 32, and 33 stood out as those that produced the most vigorous seedlings. Molecular analysis also revealed genetic diversity among genotypes, with the formation of 16 groups, while the morphophysiological analysis revealed four groups. The Mantel test demonstrated a small but significant positive difference (r = 0.228; p = 0.018) between the genetic and morphophysiological distances of the genotypes. This diversity indicates that the genotypes evaluated are promising for use in C. canephora breeding programs. Full article

Between 1% and 2% of the world’s population is sensitised to mites. Aetiological diagnosis is key to the management of allergic patients. However, methods based solely on morphological criteria are ambiguous in many cases. Polymerase chain reaction of ribosomal genes represents a valuable complementary approach. The 5 most representative species (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Tyrophagus putrescentiae, Blomia tropicalis and Lepidoglyphus destructor) were selected as sources of allergens. They were first identified morphologically and the 18S rRNA gene sequences were obtained from the GenBank database. Alignment of the nucleotide sequences of the 18S rRNA ribosomal gene enabled the identification of the conserved and divergent regions in all of them. The alignment allowed the design of a pair of oligonucleotides in conserved regions of the gene, to amplify the sequence of interest in each of the species. We performed genomic DNA extraction, quantification and purity. PCR, using oligonucleotides designed to amplify the 18S sequence fragment of interest, showed the exact size for each species. Amplification, efficiency curves and melting points resulting from the amplification of the 18S amplicon of the five species were obtained. The oligonucleotides designed for real-time PCR studies, allow species identification by amplifying the specific fragment of each species using real-time PCR. Full article

The water deer, although an internationally endangered species, is designated as a nuisance wild animal in South Korea and occupies a unique ecological niche. Studying the gut microbiome of this species is crucial for understanding its ecology. We amplified 16S rRNA DNA and compared the gut microbiomes of wild water deer from three regions with those of captive water deer from one region. Our results showed that the gut microbiome diversity of water deer did not differ significantly across regions in the wild but decreased significantly when raised in captivity. The similar microbiomes of water deer living in different regions are believed to be due to dietary diversity rather than dietary homogeneity. Furthermore, the monotony of the food supply appears to lead to significant variation in captive environments. From a conservation biology and biorestoration perspective, we suggest the importance of conserving the gut environments of animals conserved and restored outside their native habitats. Full article

Azaleas (Ericaceae) are among the most diverse ornamental plants, celebrated for their cultural and economic significance. R. simsii has been extensively utilized in horticulture as a parent species for both “pot azalea” cultivars and various cultivars grown in the warmer regions of China. From 2021 to 2023, approximately 15% of R. simsii in nurseries situated in the Xuanwu District, Nanjing, exhibited symptoms of wilting and chlorosis. Investigations revealed that these symptoms were caused by a pathogen responsible for crown and root rot. Strains were isolated from the roots of affected plants. The morphology of the colonies was predominantly radial to stellate, characterized by intercalary and terminal hyphal swelling. The sporangia appeared spherical, pyriform, or ovoid with a single papillae. For accurate identification, the 28S rDNA gene (Large subunit, LSU), cytochrome oxidase subunit I (COXI), and cytochrome oxidase subunit II (COXII) genes were amplified through PCR and then sequenced. The species was identified as P. vexans after completing the phylogenetic analysis. Healthy R. simsii plants were infected with zoospores and developed symptoms similar to those of natural infection. Furthermore, the morphological characteristics of the isolates from the experimentally infected plants were similar to those of the original inoculated strains. This study identified P. vexans as the pathogen causing root rot in R. simsii. During the sampling process, several strains were isolated from the rhizosphere soil of healthy rhododendron plants. Based on this, research was immediately initiated to explore whether there are specific bacterial species in the soil that have the potential to inhibit the occurrence of root rot. Additionally, an endophytic bacterial strain BL1 was isolated from rhizosphere soil and subjected to Whole-Genome Shotgun (WGS) sequencing, thus constructing a bacterial genome framework for this isolate. The strain BL1 was identified as Bacillus licheniformis. To our knowledge, this is the first report of the occurrence of P. vexans causing crown and root rot of R. simsii in China. In this study, we also focused on exploring the potential of biological control agents against P. vexans. The isolation and identification of the endophytic bacterial strain BL1 (Bacillus licheniformis) from the rhizosphere soil of healthy soil show strong in vitro antagonism, identifying it as a promising candidate for future biological control studies of root rot in R. simsii. The genomic component analysis and coding gene annotation of BL1 provide insights into its genetic makeup and potential mechanisms of action against pathogens. However, these findings are based on in vitro assays. Therefore, further research, including in planta experiments, is essential to confirm the efficacy of BL1 in controlling P. vexans infections in R. simsii and to evaluate its potential for practical application. Full article

The Ponto-Caspian invader, the gravel snail Lithoglyphus naticoides (C. Pfeiffer, 1828), is infected with a diverse community of digenetic trematodes in its colonized range and most often serves as first intermediate host. We have performed the parasitological examination of L. naticoides snails sampled in Kaunas water reservoir (Lithuania) and found yet unknown strigeid metacercariae of the tetracotyle type using these snails as second intermediate host. In this study, we report data on morphology and molecular analysis based on two markers, the partial 28S rDNA gene and the ITS2 region of these metacercariae. Based on the comparative molecular and phylogenetic analysis, the metacercaria detected in L. naticoides was identified as Cotylurus cornutus (Rudolphi 1809) Szidat, 1928. Differences in metacercariae infection between snail sexes were assessed. The prevalence of infection in L. naticoides was significantly higher in males than in females. Additional molecular markers of tetracotyle of C. cornutus from Stagnicola palustris (O. F. Müller, 1774) and furcocercaria of Cotylurus strigeoides Dubois, 1958 from Physa fontinalis (Linnaeus, 1758), sampled in Curonian Lagoon, Lithuania, were obtained for the first time and used for comparative phylogenetic analysis. Full article

We investigated differences in circulating cell-free DNA (cfDNA) methylation between patients with cancer and those presenting with severe, nonspecific symptoms. Plasma cfDNA from 229 patients was analyzed, of whom 37 were diagnosed with a wide spectrum of cancer types within 12 months. Samples underwent enzymatic conversion, library preparation, and enrichment using the NEBNext workflow and Twist pan-cancer methylation panel, followed by sequencing. Methylation analysis was performed with nf-core/methylseq. Differentially methylated regions (DMRs) were identified with DMRichR. Machine learning with cross-validation was used to classify cancer and controls. The classifier was applied to an external validation set of 144 controls previously unseen by the model. Cancer samples showed higher overall CpG methylation than controls (1.82% vs. 1.34%, p < 0.001). A total of 162 DMRs were detected, 95.7% being hypermethylated in cancer. Machine learning identified 20 key DMRs for classification between cancer and controls. The final model achieved an AUC of 0.88 (83.8% sensitivity, 83.8% specificity), while mean cross-validation performance reached an AUC of 0.73 (57.1% sensitivity, 77.5% specificity). The specificity of the classifier on unseen control samples was 79.2%. Distinct methylation differences and DMR-based classification support cfDNA methylation as a robust biomarker for cancer detection in patients with confounding conditions. Full article

Increased seawater temperatures and nutrient loading are stressors that affect coral reefs and their microbiomes. In this study, filamentous algae were collected and exposed to different temperatures and nutrient concentrations through a laboratory experiment. Microbial DNA was extracted and analyzed using amplicon sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. In total, 1 domain, 51 phyla, 131 classes, 335 orders, 549 families, and 1905 species were identified. Proteobacteria and Bacteroidota were the dominant taxa reported. Elevated seawater temperatures and nutrient enrichment impacted microbial communities associated with turf algae under laboratory culture. Bacterial species diversity and abundance differed under different temperature and nutrient conditions. Proteobacteria and Actinobacteria were abundant in lower-temperature conditions, while Desulfobacterota, Spirochaetota, and Firmicutes were abundant in higher-temperature conditions. Ruegeria was abundant in low-temperature conditions, whereas Vibrio abundance was low. Regarding nutrient conditions, Proteobacteria and Cyanobacteria were abundant under high-nutrient conditions, while Firmicutes and Desulfobacterota were abundant under ambient-nutrient conditions. The higher nutrient concentration increased the abundance of pathogenic bacteria, such as Vibrio and Photobacterium, while Pseudoalteromonas, which is beneficial for reefs, was present under ambient nutrient conditions. This study demonstrates that temperature and nutrient enrichment can shape microbial communities under laboratory conditions, providing an experimental setting for further studies of bacterial functions and metabolic processes in natural conditions under thermal and nutrient stresses. Full article