Search Results (371)

The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications. Full article

Bradysia odoriphaga Yang et Zhang damages roots of 30 plant species, resulting in >50% yield loss. Heat stress can not only affect the survival but also affect the expression of heat shock proteins of B. odoriphaga. In this study, two small heat shock protein genes, Hsp21.9 and Hsp22.3, were cloned from B. odoriphaga. The full-length cDNA sequences of BoHsp21.9 and BoHsp22.3 were 749 and 941 bp in length and contained a 588 and 594 bp open reading frame (ORF), encoding a protein of 196 and 198 amino acids with a calculated molecular weight of 21.9 and 22.3 kDa and an isoelectric point of 6.84 and 6.91. Phylogenetic tree analysis showed that BoHsp21.9 and BoHsp22.3 clustered into one branch with flies. qRT-PCR analyses indicated that BoHsp21.9 and BoHsp22.3 were expressed in all tested developmental stages and body segments, especially induced by heat stress. RNAi-mediated silencing of BoHsp21.9 and BoHsp22.3 significantly decreased the survival rate of fourth-instar larvae when exposed to 38 °C. This is the first study on small heat shock proteins in B. odoriphaga, and BoHsp21.9, and BoHsp22.3 play important roles in the molecular mechanism of B. odoriphaga to theromotolerance. Full article

Since the early 19th century, linguists have collected enough linguistic data to draw a remarkably stable Uralic language family tree. However, the traditional Uralic language family tree has two main problems. First, it lacks a reliable chronology because linguistic data can suggest that some languages are closer or farther from each other, but that gives only a relative instead of a precise chronology of the branching events. Second, the extinct Mezhovskaya culture in the Ural region and the Minoan civilization on the island of Crete were not incorporated into the Uralic language family, although recent archaeogenetic and linguistic data indicate that their languages also belonged to the Uralic language family. Some recent studies took an essentially purely archaeogenetic approach to the study of the evolution of the Uralic language family. These purely archaeogenetic studies propose linguistically perplexing solutions. This is the first study of the development of the Uralic language family that fully integrates the archaeogenetic, archaeological and linguistic data and proposes a new chronology of the Uralic language family that avoids the above inconsistencies. The new chronology relies on the best current estimates of the formation of the mitochondrial DNA haplogroups that are found among present Uralic language speakers and in samples from various archaeological sites that are associated with Uralic speakers. The new chronology places the various branching events of the Uralic language family tree much earlier than usual, including the split between Proto-Finno-Permic and Proto-Ugric, which is shown to have taken place in the Mesolithic period. The new proposal makes the Bronze Age Minoan language better fit chronologically as well as linguistically into the Uralic language family. Full article

During a survey of the west coast of South Korea, two new Pseudochromadora species were recorded from Yeongjongdo Island. Descriptions of two new species, an updated list of valid species within the genus, a tabular key, partial sequences of mtCOI, near full-length SSU, and the D2–D3 region of LSU rDNA, together with phylogenetic analyses are provided. The two new species are classified as Pseudochromadora based on having a two-portioned cephalic capsule, unispiral amphidial fovea, lateral alae extending from the posterior end of the pharynx as far as the tail, and presence of copulatory thorns, as well as a short conical tail. The two species are distinguished from each other by their different types of labial regions of the cephalic capsule (round-shaped vs. hat-shaped). The two species, despite being found in the same locality, are morphologically and molecularly distinct from one another. Pairwise Kimura 2-parameter (K2P) distances between the two new species were 10.6% (18S) and 27.2% (28S), values consistent with interspecific divergence observed among congeners. Phylogenetic analyses showed both species as distinct lineages within Pseudochromadora. In the 28S rDNA tree, each was retrieved as a well-supported monophyletic clade with congeners, whereas in the 18S tree, all congeners including the two new species formed a single clade, except for P. plurichela, which branched outside the main group. These results highlight potential paraphyly within Pseudochromadora and suggest that overlooked morphological traits may hold phylogenetic significance, warranting further investigation. Full article

This study investigates the species composition and distribution of blackflies (Diptera: Simuliidae) in Kazakhstan, with a focus on two species newly recorded for the country: Simulium murmanum (Enderlein, 1935) and Simulium reptans (Linnaeus, 1758). The presence of S. murmanum in Kazakhstan is reported for the first time, supported by morphological and molecular genetic analyses. Diagnostic features of the larva, pupa, and adult stages are described in detail, including the structure and coloration of the larval head capsule, pupal cocoon, and genitalia of both sexes. Habitat preferences and pupal substrate attachment patterns are illustrated, with observations on variations in cocoon branching across different flow regimes. Species identification was conducted using the morphological keys of Rubtsov and Yankovsky, and taxonomic classification was confirmed using the framework proposed by Adler. Molecular confirmation of S. murmanum was performed via DNA analysis. The species was found to be restricted to the foothill regions of East Kazakhstan, suggesting a distribution closely associated with the Altai mountain systems and adjacent regions in Mongolia and China. Unlike its status as a dominant hematophagous species in parts of Russia, S. murmanum has not demonstrated biting activity in Kazakhstan, Mongolia, or China. Additionally, the study provides the first records of S. reptans within the fauna of Kazakhstan, initially identified in the Irtysh River (Pavlodar Region). Subsequent sampling conducted in June 2024 revealed a continuous distribution of S. reptans along the Irtysh River through to the mountain streams of East Kazakhstan. The species was found in mountainous, foothill, and lowland environments, highlighting its wide ecological plasticity. Full article

Background/Objectives: Local delivery of gene therapy products through the airways shows great promise for the treatment of a number of serious lung diseases, but its effectiveness is hampered by the mucus layer protecting the lung epithelium in the trachea and bronchi. Methods: To overcome this barrier, we engineered carbon dots (CDs) with mucus penetrating properties. Results: The CDs were synthesized by solvothermal treatment of citric acid and branched polyethyleneimine, and functionalized with maleamic acid groups to create cationic mucoinert nanoparticles with tunable charge. We characterized their interactions with a mucus model through turbidity and transport measurements, and assessed their impact on the viscoelastic properties of the biopolymer. We then demonstrated that the carriers are effective at delivering pDNA to a variety of cell models in vitro. In particular, mucus-producing Calu-3 cells cultured at the air–liquid interface (ALI) were used as a discriminating model to evaluate intracellular delivery of the genetic cargo through a thick layer of mucus at the cell surface. Conclusions: The functionalization of CDs with maleamic acid groups resulted in a 1000- to 10,000-fold increase in transfection efficiency in the mucus-producing model, offering new opportunities for lung gene therapy. Full article

Background and Objectives: Changes in the environment and physiology may be associated with an increased or decreased risk of cancer. Our study aims to evaluate the strength and the direction of the selection acting on oncosuppressor genes in association with phenotypic changes. Methods: We calculated the relative evolutionary rate (RER) using the converge method and linear regression on branches of phylogenetic trees. The association between changes in the evolutionary rate of oncosuppressors (DNA repair and cell cycle control genes) and trait selection was studied. The evolutionary rates of single oncosuppressor genes and pathways were evaluated. We studied two types of traits: those that are characteristic of vertebrates, such as homeothermy (the ability to maintain a constant body temperature), flight, and amnions; and those that are characteristic of mammals, such as high body mass and lifespan, an underground lifestyle, and hibernation. The analysis included 19,445 genes; 100 vertebrates and 46 mammalian species. We studied ancestral branches individually and all the clades having a trait. Results: Oncosuppressor genes accelerated in association with the ability to fly; p-value = 0.03 (positive or relaxed negative selection) and decelerated in homeothermic species; p-value = 0.04 (stabilizing selection). DNA repair genes were significantly accelerated in ancestral branches and in all clades of amniotic, homeothermic, and high-body-mass mammals (p-value < 0.05, FDR correction). Cell cycle control genes were under stabilizing selection in homeothermic animals, high-body-mass, long-lived, and underground mammals (p-value < 0.05, FDR correction). Data on the evolution of oncosuppressors are crucial for understanding the origin of cancer and will be important for future studies of tumor pathogenesis, pathomorphosis, and microevolution. Conclusions: The selection of traits associated with changes in cancer risk leads to positive/relaxed negative and stabilizing selection of oncosuppressor genes. Full article

Our understanding of the molecular mechanisms by which DNA meiotic recombination occurs has significantly increased in the past decades. A more representative molecular model has also undergone repeated revisions and upgrades with the continuous expansion of experimental data. Considering several apparent issues in the field, we intend to make necessary upgrades to previous models and reanalyze those data, exploring structural details and molecular mechanisms of DNA meiotic recombination. Eligible studies were identified from PubMed/Medline (up to June 2024). Key related publications and experimental data were retrieved from eligible studies, displaying five major issues. Meanwhile, the biophysical modeling method was used to establish an enlacement model. Then, the model was used to wholly reanalyze the collected data. An updated molecular model was supplemented. In the current model, a copy choice mechanism can initiate DNA meiotic recombination. The copy choice is based on a branched structure of DNA, which results from relative motion between homologous single strands. The reanalysis of previous experimental data based on this model can lead to new interpretations that can better address the discrepancies between previous experimental observations and theoretical models, including (1) the intertwinement model having embodied the particular characteristics of the SDSA model; (2) hDNA arising from JM resolution rather than being followed by a JM; (3) strand specificity of hDNA mismatch repair seeming to be an illusion and copy choice more likely to be the actual state; (4) parity in resolution patterns of a dHJ leading to parity of gene conversion; (5) the cooperation of multiple HJs readily generating a high correlation between gene conversion and crossover; and (6) transpositional recombination and site-specific recombination seeming to have a common pathway to meiotic recombination. The results indicate that both revisions and reanalysis are necessary. The novel interpretations would be critical to the understanding of the mechanisms of DNA recombination as well as its role in DNA repair. Additionally, the work could have implications for how the field views the importance of factors such as Spo11 or the mechanisms that drive meiotic pairing. Full article

Mining novel Streptomyces species from extreme environments provides a valuable strategy for the discovery of new antibiotics. Here, we report a strain of Streptomyces sp. HMX87^T, which exhibits antimicrobial activity and was isolated from desert soil collected in the Tuha Basin, China. Molecular taxonomic analysis revealed that the 16S rRNA gene sequence of strain HMX87^T shares the highest similarity with those of Streptomyces bellus CGMCC 4.1376^T (98.5%) and Streptomyces coerulescens DSM 40146^T (98.43%). In phylogenetic trees, it formed a distinct branch. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain HMX87^T and the above two type strains were below the thresholds of 95% and 70%, respectively, confirming that strain HMX87^T represents a novel species within the genus Streptomyces, for which the name Streptomyces hamibioticus sp. nov. is proposed. Physiologically, the strain HMX87^T grew at temperatures ranging from 25 to 37 °C, tolerated pH values from 5 to 12, and survived in NaCl concentrations of 0% to 8% (w/v). Chemotaxonomic characterization indicated the presence of LL-diaminopimelic acid (LL-DAP) in the cell wall, ribose and galactose as whole-cell hydrolysate sugars, MK-9(H8) (66.3%) as the predominant menaquinone, and iso-C_16:0 (25.94%) and anteiso-C_15:0 (16.98%) as the major fatty acids characteristics that clearly distinguish it from its closest relatives. Whole-genome sequencing of strain HMX87^T revealed an abundance of genes associated with high-temperature tolerance, salt-alkali resistance, and antimicrobial activity. The genomic features and secondary metabolic potential reflect its adaptation to extreme environmental conditions, including high temperature, salinity, alkalinity, strong ultraviolet radiation, and oligotrophic nutrients. The strain HMX87^T has been deposited in the Czech Collection of Microorganisms (CCM 9454^T) and the Guangdong Microbial Culture Collection Center (GDMCC 4.391^T). The 16S rRNA gene and whole-genome sequences have been submitted to GenBank under accession numbers PQ182592 and PRJNA1206124, respectively. Full article

In the present study, a new Ganoderma sp. (ZHM1939) was collected from Lincang, Yunnan, China, and described on the basis of morphological characters and multigene phylogenetic analysis of rDNA-ITS, TEF1α and RPB2 sequences. This fungus is characterized by the exceptionally large basidiomata, oval shape, a pileus measuring 63.86 cm long, 52.35 cm wide, and 21.63 cm thick, and a fresh weight of 80.51 kg. The skeleton hyphae from the basidiocarp are grayish to grayish-red in color, septate, and 1.41–2.75 μm in diameter, with frequently dichotomous branched and broadly ellipsoid basidiospores. The basidiospores are monocellular, ellipsoid, with round ends or one slightly pointed end, brown–gray in color, and measured 6.52–10.26 μm × 4.68–7.17 μm (n = 30). When cultured for 9 days at 25 ± 2 °C on PDA, the colony was white, ellipsoid or oval, with slightly ragged edges, measured Φ58.26 ± 3.05 mm (n = 5), and the growth rate = 6.47 mm/day; prosperous blast-spores formed after culturing for 21 days, making the colony surface powdery-white. The mycelia were septate, hyaline, branching at near-right angles, measured Φ1.28–3.32 μm (n = 30), and had some connections. The blast-spores were one-celled, elliptic or barley-seed shaped, and measured 6.52–10.26 μm × 4.68–7.17 μm (n = 30). Its rDNA-ITS, TEF1α and RPB2 sequences amplified through PCR were 602 bp, 550 bp and 729 bp, respectively. Blast-n comparison with these sequences showed that ZHM1939 was 99.67–100% identical to related strains of Ganoderma mutabile. A maximum likelihood phylogenic tree using the concatenated sequence of rDNA-ITS, TEF1α and RPB2 was constructed and it showed that ZHM1939 clustered on the same terminal branch of the phylogenic tree with the strains Cui1718 and YUAN 2289 of G. mutabile (Bootstrap support = 100%). ZHM1939 could grow on all the 15 original inoculum substrates tested, among which the best growth was shown on substrate 2 (cornmeal 40 g, sucrose 10 g, agar 20 g), with the fastest colony growth rate (6.79 mm/day). Of the five propagation substrates tested, substrate 1 (wheat grains 500 g, gypsum powder 6.5 g and calcium carbonate 2 g) resulted in the highest mycelium growth rate (7.78 mm/day). Among the six cultivation substrates tested, ZHM1939 grew best in substrate 2 (cottonseed hulls 75 g, rice bran 12 g, tree leaves 5 g, cornmeal 5 g, lime powder 1 g, sucrose 1 g and red soil 1 g) with a mycelium growth rate of 7.64 mm/day. In conclusion, ZHM1939 was identified as Ganoderma mutabile, which is a huge mushroom and rare medicinal macrofungus resource. The original inoculum substrate 9, propagation substrate 1 and cultivation substrate 2 were the most optimal substrates for producing the original propagation and cultivation inocula of this macrofungus. This is the first report on successful growing conditions for mycelial production, but basidiocarp production could not be achieved. The results of the present work establish a scientific foundation for further studies, resource protection and application development of G. mutabile. 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

The Kemp’s ridley sea turtle (Lepidochelys kempii) is the most critically endangered sea turtle species, with a distribution primarily restricted to the Gulf of Mexico. Its occurrence along the Iberian Peninsula is exceedingly rare. This study describes the postmortem findings of a juvenile L. kempii rescued off the Portuguese coast in 2024, which died after 11 days in rehabilitation despite intensive supportive care. Necropsy revealed severe, diffuse pneumonia. Histopathological examination showed interstitial inflammation and branching septate hyphae, while fungal culture and DNA sequencing confirmed the presence of Beauveria bassiana. Mycotic diseases in reptiles are often underrecognised but can lead to significant morbidity and mortality, particularly in immunocompromised or stressed individuals such as stranded marine turtles. This rare occurrence of a L. kempii on the Portuguese coast provides important insights into the species’ dispersal patterns and underlines the potential conservation implications of opportunistic fungal infections in endangered species. Full article

Apricots are affected by many abiotic and biotic factors that could negatively impact their vitality and yield, leading to branch and tree dieback. Knowledge of the microbiome composition is key to choosing the optimal measurement strategy. The effect of the two different growing systems, i.e., organic (ORG) and integrated pest management (IPM), on the apricot fungal microbiome was studied. The inner bark was used to isolate DNA, and the present fungi were analyzed using a metagenomics high-throughput sequencing (HTS) profiling approach of the data obtained based on the Illumina sequencing of the ITS1-ITS2 amplicons of the 18S rRNA gene. Of the 20 analyzed samples, Ascomycota was the dominant phylum, and Dothiomycetes was the most abundant. Basidiomycota was the less frequent, with Tremellomycetes being the predominant within this phylum. PCA analysis showed the complete separation of the samples obtained from the orchards grown under the ORG and IPM systems. Cladosporia, Alternaria, Aureobasidium, and Visniacozyma were detected in all samples, but they dominated the IPM samples. Filobasiadiales were recognized as an indicator species for ORG management, while Caliciales, Lecanorales, Lichinales, Mycosphaerellales, Myriangiales, Phacidiales, Teloschistales, and Thelebolales were identified as indicator species for IPM management. Based on the order and genus levels, a significantly higher fungal microbiome richness was detected in the ORG samples. This could be connected to the environmentally beneficial growing system applied in the orchard, but it is impossible to assess the risk of trunk disease development or premature apricot tree decline. Full article

Germplasm resources embody the genetic diversity of plants and form the foundation for breeding and the ongoing improvement of elite cultivars. The establishment of germplasm banks, along with their systematic evaluation, constitutes a critical step toward the conservation, sustainable use, and innovative utilization of these resources. Liriodendron, a rare and endangered tree genus with species distributed in both East Asia and North America, holds considerable ecological, ornamental, and economic significance. However, a standardized evaluation system for Liriodendron germplasm remains unavailable. In this study, 297 Liriodendron germplasm accessions were comprehensively evaluated using 34 phenotypic traits and whole-genome resequencing data. Substantial variation was observed in most phenotypic traits, with significant correlations identified among several characteristics. Cluster analysis based on phenotypic data grouped the accessions into three distinct clusters, each exhibiting unique distribution patterns. This classification was further supported by principal component analysis (PCA), which effectively captured the underlying variation among accessions. These phenotypic groupings demonstrated high consistency with subsequent population structure analysis based on SNP markers (K = 3). Notably, several key traits exhibited significant divergence (p < 0.05) among distinct genetic clusters, thereby validating the coordinated association between phenotypic variation and molecular markers. Genetic diversity and population structure were assessed using 4204 high-quality single-nucleotide polymorphism (SNP) markers obtained through stringent filtering. The results indicated that the Liriodendron sino-americanum displayed the highest genetic diversity, with an expected heterozygosity (He) of 0.18 and a polymorphic information content (PIC) of 0.14. In addition, both hierarchical clustering and PCA revealed clear population differentiation among the accessions. Association analysis between three phenotypic traits (DBH, annual height increment, and branch number) and SNPs identified 25 highly significant SNP loci (p < 0.01). Of particular interest, the branch number-associated locus SNP_17_69375264 (p = 1.03 × 10⁻⁵) demonstrated the strongest association, highlighting distinct genetic regulation patterns among different growth traits. A minimal set of 13 core SNP markers was subsequently used to construct unique DNA fingerprints for all 297 accessions. In conclusion, this study systematically characterized phenotypic traits in Liriodendron, identified high-quality and core SNPs, and established correlations between key phenotypic and molecular markers. These achievements enabled differential analysis and genetic diversity assessment of Liriodendron germplasm, along with the construction of DNA fingerprint profiles. The results provide crucial theoretical basis and technical support for germplasm conservation, accurate identification, and utilization of Liriodendron resources, while offering significant practical value for variety selection, reproduction and commercial applications of this species. Full article

Resolving the eukaryotic tree of life (eToL) remains a fundamental challenge in biology. Much of eukaryotic phylogenetic diversity is occupied by unicellular microbial eukaryotes (i.e., protists). Among these, the phylogenetic positions of a significant number of lineages remain unresolved due to limited data and ambiguous traits. To address this issue, we introduce the term “PUPAs” (protists with uncertain phylogenetic affiliations) to collectively describe these lineages, instead of using vague or inconsistent labels, such as incertae sedis or orphan taxa. Historically, protists were classified based solely on morphological features, and many with divergent cell structures were left unplaced in the eToL. With the advent of sequence-based approaches, the phylogenetic affiliations of some PUPAs have been clarified using molecular markers, such as small subunit ribosomal DNA. The combination of technological progress and continuous efforts to cultivate diverse protists, including PUPAs and novel protists, now enables phylogenetic analyses based on hundreds of proteins, providing their concrete placements in the eToL. For example, these advances have led to the discovery of new deep-branching lineages (e.g., Hemimastigophora), the resolution of relationships among major groups (e.g., Microheliella, which linked Cryptista and Archaeplastida), and insights into evolutionary innovations within specific clades (e.g., Glissandra). In this review, we summarize current consensus in eukaryotic phylogeny and highlight recent findings on PUPAs whose phylogenetic affiliations have been clarified. We also discuss a few lineages for which the phylogenetic homes remain unsettled, the evolutionary implications of these discoveries, and the remaining challenges in resolving the complete eToL. Full article