Search Results (696)

Antibody-free nucleic acid lateral flow assays (AF-NALFA) are an established approach for rapid detection of amplified pathogens DNA but can yield inconsistent signals across targets. Since AF-NALFA depends on dual hybridization of probes to single-stranded amplicons (ssDNA), site-specific thermodynamic (Gibbs free energy-ΔG) at probe-binding regions may be crucial for performance. This study investigated how site-specific-ΔG and sequence complementarity at probe-binding regions determine Test-line signal generation, comparing native and synthetic amplicons and assessing the effects of local secondary structures and mismatches. Asymmetric PCR-generated ssDNA amplicons of Listeria monocytogenes, Mycobacterium leprae, and Leishmania amazonensis were analyzed in silico and tested in AF-NALFA prototypes with gold-labeled thiol probes and biotinylated capture probes. T-line signals were photographed, quantified (ImageJ version 1.4k), and statistically correlated with site-specific-ΔG. While native ssDNA from M. leprae and L. amazonensis failed to produce AF-NALFA T-line signals, L. monocytogenes yielded strong detection. Site-specific-ΔG below −10 kcal/mol correlated with reduced hybridization. Synthetic oligos preserved signals despite structural constraints, whereas ~3–4 mismatches, especially at capture probe regions, markedly impaired T-line intensity. The performance of AF-NALFA depends on the synergism between thermodynamic accessibility, site-specific-ΔG-induced site constraints, and sequence complementarity. Because genomic context affects hybridization, target-specific thermodynamic in silico evaluation is necessary for reliable pathogen DNA detection. Full article

Polemonium caeruleum L. (Polemoniaceae) is a perennial flowering plant native to Eurasia and North America, which is used as a fodder, medicinal, and ornamental plant. Many issues related to the taxonomy and origin of this valuable species still remain unclear. The intraspecific genetic variability of P. caeruleum and chromosomal organization of its genome are insufficiently studied. For the first time, we analyzed NGS genomic data of P. caeruleum using ReapeatExplorer2/TAREAN/DANTE Pipelines. In its repeatome, we identified 66.08% of Class I retrotransposons; 0.57% of Class II transposons; 0.42% of ribosomal DNA; and 0.87% of satellite DNA (six high-confident and three low-confident putative satellite DNAs). FISH chromosome mapping of seven tandem DNAs was carried out in two P. caeruleum varieties and two wild populations. Our results demonstrated the effectiveness of using satDNAs Pol_C 46 and Pol_C 33 in combination with 45S rDNA and 5S rDNA for precise chromosome identification. This approach allowed us to study intraspecific chromosomal variability and detect chromosomal rearrangements in the studied accessions of P. caeruleum, which could be related to the speciation process. These novel molecular markers are important for chromosome studies within Polemonium to clarify its taxonomy and phylogeny, and also, they expand the potential of different breeding programs. Full article

The conservation of native blueberries (Vaccinium spp.) from Andean and Amazonian ecosystems faces challenges from climate change, habitat fragmentation, and land use. In this context, this review article provides a comprehensive analysis of the most relevant biotechnological and genomic tools applied to the preservation of these plant genetic resources, as well as their characterization. Among the biotechnological strategies, in vitro micropropagation delivers clonal pathogen-free valuable plants, while cryopreservation offers a viable option for a long-term germplasm storage. We also summarize its protocols focus on high regeneration rates and reproducibility. In the genomic field, we show advances in the use of molecular markers (such as SNPs, SSRs, and RAPDs), DNA barcoding and next-generation sequencing that leads genetic diversity assessment and identification of species. Finally, future perspectives in native blueberry conservation are discussed that allow the integration of emerging technologies such as landscape genomics, environmental transcriptomics, and the use of artificial intelligence tools. Integrating these approaches with the active participation of local communities can substantially strengthen the sustainable conservation of native blueberries in their natural habitats. Full article

Modification of synthetic oligonucleotides and DNA is widely used in many applications in the life sciences. However, in most cases, modified DNA cannot be restored to its native state. Here, we report the preparation of a thymidine-inosine dimer building block (TID) for oligonucleotide synthesis. The TID modification supports the functionalization of synthetic oligonucleotides, which can later be removed to restore the DNA strand to its native state. The TID unit allows for a wide spectrum of postsynthetic modifications of oligonucleotides through click chemistry, including conjugation with fluorescent tags and small molecules, preparation of branched oligonucleotide scaffolds, and anchoring to a solid support. Due to the modification of the thymine base, the TID unit reduces the stability of the DNA duplex. We found that the negative effect of internal TID modification on duplex stability does not exceed the same for a single base mismatch. As long as the TID modification is present in the DNA strand, it disrupts its natural functionality. The “caging” effect of TID in the template strand with respect to DNA polymerase was demonstrated in primer extension experiments. Traceless removal of the temporary functional group occurs through oxidative cleavage of the inosine subunit, resulting in the formation of a native DNA strand with the thymine base left at the cleavage site. An anthracene-modified dodecamer oligonucleotide and a branched oligonucleotide scaffold were used to study the cleavage of the reporter group or the oligonucleotide side strand, respectively. It was shown that aqueous tetramethylguanidine efficiently cleaves the oxidized inosine subunit of TID at 37 °C, forming the native DNA strand. Full article

Fatty acid binding proteins FABP3 and FABP4 act as intracellular lipid chaperones that influence fatty acid transport and metabolism in mammary tissue, and genetic variation in these genes may affect milk composition. We examined the associations between FABP3 and FABP4 polymorphisms and milk composition and fatty acid profiles in 200 lactating Native Southern Yellow (NSY) cows. DNA from each cow was PCR-amplified and Sanger-sequenced for FABP3 and FABP4; genotypes were tested for their association with milk fatty acid concentrations and standard composition traits using linear models adjusted for relevant covariates. We detected a missense variant in FABP3 (c.3656G > A; p.Val45Met) and an intronic SNP in FABP4 (g.3509T > C). The FABP3 p.Val45Met AA genotype was associated with higher concentrations of butyric, palmitic, oleic, and α-linolenic acids. Cows with the FABP4 TC genotype exhibited elevated levels of myristoleic, γ-linolenic, conjugated linoleic, and arachidic acids, along with increased fat-free dry matter, protein, and lactose. In silico analyses provided mixed evidence for the structural effects of p.Val45Met, molecular docking suggested altered ligand affinity for several fatty acids, and splice site prediction implicated g.3509T > C in possible transcript processing changes. These variants constitute candidate markers for milk fatty acid composition in NSY cattle; replication in independent cohorts and functional validation are recommended to confirm their utility for milk quality improvement. Full article

Tall fescue-dominated pasturelands are widespread in the eastern United States and typically lack substantial plant diversity. Establishing native wildflowers into tall fescue pastures has the potential to benefit bee populations and boost pollinator ecosystem services. In this study, tall fescue pastures at five on-farm sites in Virginia, USA, were planted with wildflowers native to North America and paired with sites with conventional tall fescue pastures. Honeybee apiaries were established at the ten locations, and variables related to hive strength were measured over two years. The main study objectives were to: (1) compare metrics of hive strength between diversified and conventional pastures, (2) determine whether honeybees used native-sown wildflowers as a source of pollen, and (3) explore whether native-sown wildflowers were visited more by honeybees and other pollinators compared with nonnative, unsown forbs. Diversified pastures had many more plant species and blooms compared with conventional pastures, but this had little effect on hive parameters. Pollen DNA metabarcoding revealed that honeybee diets were similar regardless of whether hives were associated with diversified or conventional pastures. Honeybees foraged mostly on plants in the surrounding landscape—especially white clover (Trifolium repens) and less so on native wildflowers. Native-sown wildflowers received more visits from native pollinators, however. We hypothesize that the native-sown wildflowers had little impact on hive strength metrics because honeybees had access to abundant, white clover blooms and other flowering species in these landscapes. Native wildflowers that bloom in late summer/early autumn after white clover blooms diminish may be of greater value to honeybees in pasture settings. Full article

Freshwater ecosystems are a significant entity that govern the livelihood of people and are an important source of food, employment, and recreation. However, climate change is impacting freshwater ecosystems by altering their natural habitats. The purpose of this review is to highlight the vulnerability of freshwater fish to climate change. Climate change is invariably affecting natural ecosystems everywhere and in every part of the world, but these threats are more severe in Pakistan. Freshwater fish are important biotic drivers of freshwater ecosystems. Unfortunately, uncertain climate changes and anthropogenic activities have led to a decline in the diversity of these fishes. Rising temperatures, melting glaciers, changes in seasonal patterns, disturbances in the natural flow of rivers, pollution, and invasive species are major threats to native freshwater fish fauna, leading to a decline in fish diversity and population. Tor putitora, Glyptothorax kashmirensis, and Triplophysa kashmirensis are some of the species that are critically endangered in Pakistan due to these factors. In recent decades, insufficient attention has been paid to the freshwater ecosystem. This review of threats to the endemic fish species in this region is presented so that the government and policymakers can use this information as part of their management and conservation policy, thus safeguarding Pakistan’s fish industry. Environmental DNA (eDNA) biomonitoring is a new technique for assessing biodiversity and species distribution and can be useful for conserving biodiversity in this region. Another purpose of this review is to introduce this new conservation strategy to Pakistan. Full article

This study assessed the impact of untreated wastewater discharge in the Danube River on four native fish species: barbel (Barbus barbus), vimba bream (Vimba vimba), perch (Perca fluviatilis), and white bream (Blicca bjoerkna). Biomarkers of exposure and effect were evaluated, including metal and metalloid bioaccumulation in gills, liver, and gonads, DNA damage (comet assay), chromosomal abnormalities (micronucleus assay), liver enzyme activities (ALT, AST), and erythrocyte maturation. White bream showed the highest genotoxic damage (TI% = 22.57), particularly in liver tissue, indicating high sensitivity to pollution. Perch had elevated DNA damage in blood (TI% = 22.69) and strong biomarker responses, likely due to its predatory behavior. Barbel displayed notable DNA damage in gills (TI% = 30.67) and liver (TI% = 20.35), aligning with sediment exposure due to its benthic habits. Vimba bream had the lowest responses, possibly reflecting reduced exposure or resilience. Element accumulation varied across tissues and species, with perch showing the highest overall levels. Hepatic enzyme activities (highest values: ALT = 105.69 in barbel; AST = 91.25 in white bream) and changes in erythrocyte profiles supported evidence of physiological stress. Integrated Biomarker Response (IBR) analysis identified white bream as the most sensitive species, followed by perch and barbel. These results emphasize the value of multi-species biomonitoring and the importance of species-specific traits in freshwater ecotoxicology. Full article

SSB proteins play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. This study investigates the transcript levels, identification, expression and purification, subcellular localization, and immune protective potential of the SSB-like proteins of Eimeria necatrix (EnSSB), exploring its role in the development of E. necatrix and its potential as a candidate antigen for a subunit vaccine against avian coccidiosis. The level of EnSSB gene transcription was highest in unsporulated oocysts (UO), followed by gametocytes (GAM) (p < 0.05). The gene consisted of an open reading frame of 1488 nucleotides encoding a protein of 495 amino acid residues with a predicted molecular weight of 53.31 kDa. EnSSB contained a SSB domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. The molecular mass of the native protein, as determined by Western blot analysis, was ~58 kDa in second-generation merozoites (MZ-2) and UO. In addition to the 58 kDa band, four other bands (~98 kDa, ~82 kDa, ~36 kDa and ~28 kDa) were detected in GAM. No bands were detected in MZ-3. Indirect immunofluorescence and immuno-electron microscopy localized EnSSB in the cytoplasm of macrogametocytes but not in wall-forming bodies and oocyst wall. Animal challenge experiments demonstrated that rEnSSB elicited robust IgY responses, increased splenic T lymphocytes and body weight gain, reduced intestinal lesion scores and oocyst shedding, and presented anticoccidial index (ACI) more than 160. These findings not only offer a foundation for understanding the role of EnSSB protein in regulating the development of E. necatrix, but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis. Full article

Tree plantations can help reverse the negative impacts of deforestation and land degradation worldwide, and soil microbial communities play key roles in tree growth and productivity. We studied microbial communities in the bulk soil of five native species monoculture plantations in the Republic of Panamá to assess how bacteria and fungi were affected by soil chemistry and plant identity after seven years of tree growth. Relative to the other species, Terminalia amazonia accumulated over three times the aboveground biomass and had lower mortality. Soil nutrients, especially phosphorus, were low, and we found no differences in soil chemistry across the five plantation types. Similarly, there was no difference in alpha diversity of the soil microbial communities across plantation types, and the bacterial communities showed no compositional variation or enrichment of any individual taxa. However, soil fungal communities differed in T. amazonia plantations as compared to the others, exhibiting enrichment or absence of specific taxa of arbuscular mycorrhizal fungi and putative phytopathogens. Our results suggest that T. amazonia may associate with certain microbial taxa that help it overcome low nutrient availability in these habitats. Consideration of plant–soil–microbe interactions in restoration efforts may facilitate tree growth and help to promote climate resilient forested areas. Full article

Paralipsa gularis (Zeller) has become an increasingly destructive pest in both storage and field ecosystems, particularly affecting maize crops across China. As chemical control methods face limitations due to resistance development and environmental concerns, biological control presents a promising alternative. In this study, we isolated and identified a novel strain of Metarhizium sp. from naturally infected P. gularis larvae collected in Yunnan Province, China. Morphological characterization, along with ITS-rDNA and EF-1α-rDNA sequencing, confirmed the fungus as Metarhizium rileyi. The optimal growth medium for this strain was SMAY, and the optimal conditions were 25 °C under continuous light (L:D = 24:0). Laboratory bioassays showed that the strain exhibited high virulence against P. gularis larvae, with cumulative mortality reaching 82% following infestation with 5 × 10⁸ conidia/mL. Biochemical analyses revealed that fungal infection significantly inhibited the activity of the key antioxidant enzyme SOD in the host, while activities of POD, CAT, and detoxification enzymes (P450, CarE, AChE, and GSTs) were significantly increased. These results indicate that immune responses were triggered, and systemic colonization of the host was achieved. Overall, this native M. rileyi strain demonstrates strong potential as an effective biological control agent. Its ability to overcome insect defenses and induce high mortality supports its integration into pest management programs targeting P. gularis. This work advances the understanding of fungal–insect interactions and contributes to sustainable, environmentally safe strategies for managing a pest of economic importance in agricultural ecosystems. Full article

This study aimed to determine the practical efficacy of passive eDNA samplers (PEDS) for monitoring fish diversity in riverine ecosystems. It investigated the utility of environmental DNA (eDNA) in accurately depicting fish composition and diversity within the Lancang River. Environmental DNA technology, particularly PEDS, may be used as a substitute for traditional water filtration techniques. However, its effectiveness in natural water ecosystems remains to be proven. The filter materials included mixed cellulose acetate and nitrate (MCE), nylon (NL), glass fiber (GF), and polyvinyl chloride filter membrane (PVC). This study used four different types of filters, each with identical pore sizes and dimensions but constructed from various materials, to assess eDNA capture under laboratory and field conditions in the water samples. The filter materials included mixed cellulose acetate and nitrate (MCE), nylon (NL), glass fiber (GF), and polyvinyl chloride filter membrane (PVC). Environmental DNA macrobarcoding was used to analyze fish biodiversity and to understand the environmental effects on species distribution. Our study identified 50 fish species inhabiting the Lancang River, with equal representation of exotic and native species. A comparative analysis of four filter-based environmental DNA samplers and traditional environmental DNA sampling methods demonstrated comparable species richness. Redundancy analysis indicated that environmental variables, elevation, electrical conductivity, salinity, and chlorophyll-a significantly influenced the distribution patterns of both non-native and native fish species in the river. This study highlights the significance of eDNA technology in evaluating fish diversity across diverse habitats, thereby establishing a theoretical framework for the sustained monitoring and management of fish biodiversity in protected areas. Full article

Myriophyllum heterophyllum is an aquatic macrophyte that is invasive to the northeastern United States and several western European countries. Spreading by vegetative clonal propagation, especially fragmentation, extensive resources are devoted to limiting its growth and spread; however, genetic assessments are not typically included in management strategies. Reduction in genetic (clonal) diversity should accompany biomass reduction, yet without genetic assessment, the efficacy of plant removal remains unclear. This paper is the first to describe a microsatellite marker library and its use in the characterization of Myriophyllum heterophyllum. Eighty-seven tissue samples were collected across the invasive distribution of Myriophyllum heterophyllum in Maine, USA. DNA was extracted, and PCR amplification was employed to screen 13 published microsatellites. Sequencing of the amplified loci was performed to characterize repeat motifs and confirm primer binding sites. Fragment sizing of PCR amplicons was employed to determine microsatellite lengths across the 87 samples. A total of 7 of the 13 tested markers were amplified, with six of those seven found to be variable. Polyploidy was evident from allelic diversity within individuals, although precise ploidy could not be determined. Observed heterozygosity ranged from 0.16 to 1.00 across variable markers. This seven-marker library was effective in characterizing the genetic diversity of both newly discovered (<5 years) and older (>50 years) infestations and is expected to be suitable for assessment of genetic diversity in populations within the native range of M. heterophyllum. The marker library also shows potential for use in several other Myriophyllum species. Full article

Background: Hereditary transthyretin amyloidosis (ATTRv) is a systemic disorder caused by homozygosity or compound heterozygosity for pathogenic mutations in the TTR gene, leading to destabilization of the transthyretin tetramer, misfolding of monomers, and subsequent amyloid fibril deposition. Among over 150 known TTR variants, p.Val142Ile is particularly associated with late-onset cardiac involvement and is the most prevalent amyloidogenic mutation in individuals of African and, to a lesser extent, European descent. This study reports the identification and familial segregation of the p.Val142Ile mutation in a large multigenerational family from Calabria (Southern Italy). Methods: Genomic DNA was extracted from peripheral blood, and Sanger sequencing of the TTR gene was performed in the proband and extended family. Results: The proband was a 75-year-old man with clinical features suggestive of cardiac amyloidosis. Genetic testing revealed homozygosity for the TTR p.Val142Ile variant. Family screening revealed multiple heterozygous carriers across three generations, most of whom were asymptomatic. Discussion: This is the first report of a native Calabrian family carrying this variant, previously unreported in this region, where p.Phe84Leu was considered the only endemic TTR mutation. Our findings expand the mutational landscape of ATTRv in Southern Italy and highlight the presence of p.Val142Ile in a previously unrecognized geographic area. These results reinforce the importance of including TTR sequencing in the work-up of unexplained cardiomyopathy, particularly in Southern Italy, where atypical variants may be emerging. Full article

Background/Objectives: Loss and fragmentation of habitat from agricultural conversion led to the near extirpation of the pygmy rabbit (Brachylagus idahoensis Merriam, 1891) population in the Columbia Basin (CB) of Washington, USA. Recovery efforts began in 2002 and included captive breeding, translocations from other regions for genetic rescue, and reintroduction into native habitat in three sites: Sagebrush Flat (SBF), Beezley Hills (BH), and Chester Butte (CHB). Methods: We used noninvasive and invasive genetic sampling to evaluate demographic and population genetic parameters on three translocated populations of pygmy rabbits over eight years (2011–2020). For each population, our goal was to use fecal DNA sampling and 19 microsatellite loci to monitor spatial distribution, apparent survival rates, genetic diversity, reproduction, effective population size, and the persistence of CB ancestry. Over the course of this study, 1978 rabbits were reintroduced as part of a cooperative conservation effort between state and federal agencies. Results: Through winter and summer monitoring surveys, we detected 168 released rabbits and 420 wild-born rabbits in SBF, 13 released rabbits and 2 wild-born in BH, and 16 released rabbits in CHB. Observed heterozygosity (H_o) values ranged from 0.62–0.84 (SBF), 0.59–0.80 (BH), and 0.73–0.77 (CHB). Allelic richness (AR) ranged from 4.67–5.35 (SBF), 3.71–5.41 (BH), and 3.69–4.65 (CHB). Effective population (N_e) within SBF varied from 12.3 (2012) to 44.3 (2017). CB ancestry persisted in all three wild populations, ranging from 15 to 27%. CB ancestry persisted in 99% of wild-born juveniles identified in SBF. Apparent survival of juvenile rabbits differed across years (1–39%) and was positively associated with release date, release weight, and genetic diversity. Survival of adults (0–43%) was positively influenced by release day, with some evidence that genetic diversity also positively influenced adult apparent survival. Conclusions: Noninvasive genetic sampling has proven to be an effective and efficient tool in monitoring this reintroduced population, assessing both demographic and genetic factors. This data has helped managers address the goals of the Columbia Basin recovery program of establishing multiple sustainable wild populations within the sagebrush steppe habitat of Washington. Full article