High Conservation Research Articles

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Genome-wide characterization and comparative expression profiling of dual-specificity phosphatase genes in yellow catfish (Pelteobagrus fulvidraco) after infection with exogenous Aeromonas hydrophila

IntroductionDual-specificity phosphatases (DUSPs) are crucial regulators in many mammals, managing dephosphorylation and inactivation of mitogen-activated protein kinases (MAPKs) and playing essential roles in immune responses. However, their presence and functions in teleosts, like the yellow catfish (Pelteobagrus fulvidraco), remain unexplored.MethodsIn this study, eight pfDusp genes (pfDusp1-7 and pfDusp10) were identified in yellow catfish. We characterized their molecular features, conserved protein sequences, and chromosomal localization through genome-wide analyses, and we examined their expression patterns in immune responses.ResultsOur findings reveal two conserved motifs, Leu-Phe-Leu-Gly and Ala-Tyr-Leu-Met, within the DSPc domain of DUSP proteins. The genes were mapped across seven chromosomes without evidence of duplication. Comparative analysis showed high conservation of Dusp genes across vertebrates, with evolutionary analysis suggesting Dusp3 as a potential intermediate form. Dusp transcripts were significantly upregulated in the kidney post-A. hydrophila infection.DiscussionThese results suggest the involvement of Dusp genes in the immune response of yellow catfish to bacterial pathogens, providing insights into their evolutionary significance and potential applications in aquaculture and molecular breeding.

Chromosome-Scale Genome of the Fern Cibotium barometz Unveils a Genetic Resource of Medicinal Value

Ferns represent the second-largest group of vascular plants, yet their genomic resources lag far behind. Here, we present a chromosome-scale genome assembly of Cibotium barometz (L.) J. Sm., a medicinally important fern species. The 3.49 Gb genome, assembled into 66 chromosomes with 99.41% sequence anchorage, revealed an exceptionally high proportion (83.93%) of repetitive elements, dominated by recently expanded LTR retrotransposons. We identified 30,616 protein-coding genes, providing insights into fern-specific gene families. Genomic analyses uncover the evolutionary dynamics of 513 key biosynthetic genes, particularly those involved in terpenoid and flavonoid production. Expression profiling across tissues revealed tissue-specific regulation of these pathways, with notable upregulation of chalcone synthase genes in roots. Our structural analysis of 1-deoxy-d-xylulose-5-phosphate synthase, a key enzyme in terpenoid biosynthesis, demonstrated high conservation across land plants while highlighting fern-specific adaptations. The identification of multiple isoforms for key enzymes points to potential gene-duplication events or the evolution of fern-specific variants. This genome provides a foundation for understanding fern biology, evolution, and the molecular basis of their medicinal properties. It also offers valuable resources for conservation efforts and pharmacological research, paving the way for sustainable utilization of this valuable medicinal plant and advancing our understanding of plant diversity and natural product biosynthesis.

Clinical and genetic analysis of a patient with Ataxia and vitamin E deficiency due to homozygous variant of TTPA gene

To explore the clinical phenotype and genetic characteristics of a patient with Ataxia and vitamin E deficiency syndrome (AVED) due to a variant of TTPA gene. A patient diagnosed with AVED (proband), intended for assisted reproductive technology for pregnancy in Zhongnan Hospital of Wuhan University in July 2023, was selected as research subject. Clinical data of the proband were collected, and 2 mL of peripheral venous blood samples were collected from the proband and her father and siblings for serum vitamin E level testing. Whole exome sequencing (WES) was carried out. Pathogenic variants were selected based on American public archive of interpretations of clinically relevant variants (ClinVar). Sanger sequencing was performed to validate the candidate variants detected by WES. Pathogenicity of variants was classified based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), and the impact of variants was analyzed using multiple bioinformatics tools including SIFT, Mutation Taster, CADD, and SpliceAI. Information on the protein domains was obtained from ClinVar and dbSNP databases, and a hotspot map for the variants of protein-coding region was constructed. This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (No. 2023068K). The proband has a significantly low serum level of vitamin E (5.186 μg/mL), while her father and siblings were normal. WES revealed that she has harbored a homozygous missense c.2T>A (p.0?) variant of the TTPA gene, for which her father and younger sister were heterozygous carriers. Based on the guidelines from the ACMG, the missense c.2T>A (p.0?) variant of the TTPA gene was classified as pathogenic (PVS1+PM2+PM3). Multiple bioinformatics tools had predicted this variant to be located in the initiation codon region and may lead to abnormal synthesis of the TTPA protein, indicating it was deleterious. The hotspot map based on ClinVar and dbSNP databases showed an even distribution of variants across 5 structural domains of the TTPA protein, with high conservation of the first amino acid residue across various species. The homozygous c.2T>A (p.0?) variant of the TTPA gene probably underlie the AVED in the proband. Above discovery has enriched the mutational spectrum of AVED and provided a basis for the diagnosis, genetic counseling, and assisted reproductive strategies for this family.

Description of the mitochondrial genomes of Sichuan Tetrastes sewerzowi (Galliformes: Tetraonidae) and phylogenetic relationship.

The Chinese Grouse (Tetrastes sewerzowi), a rare endemic bird in China, belongs to the Galliformes order and Tetraonidae family. Despite extensive research, subspecies classification remains inadequate, especially in Pingwu, Sichuan. This study sequenced the mitochondrial genome of T. sewerzowi in Wanglang Nature Reserve, Sichuan, analyzed its structural features, and reconstructed phylogenetic relationships within Tetraonidae. The results indicate that the high conservation in mitochondrial genome structure and sequence between the T. sewerzowi from Wanglang, Sichuan and Lianhuashan, Gansu indicates that they may belong to the same subspecies. Furthermore, we observed significant differences in the length of the mitochondrial control region between T. sewerzowi and Tetrastes bonasia. Based on the phylogenetic tree constructed using a comprehensive mitochondrial dataset, we propose the reclassification of the Hazel Grouse genus into two independent genera: Tetrastes and Bonasa. This study is the first to sequence the mitochondrial genome of T. sewerzowi from Sichuan and compare it with populations from Gansu, providing important insights into the distribution pattern of T. sewerzowi subspecies and facilitating the formulation of effective conservation strategies.

Molecular characterization, spatio-temporal expression patterns of crtc2 gene and its immune roles in yellow catfish (Pelteobagrus fulvidraco)

cAMP response element binding (CREB) protein 2 (CRTC2) is a transcriptional coactivator of CREB and plays an important role in the immune system. Thus far, the physiological roles of Crtc2 in teleost are still poorly understood. In this study, the crtc2 gene was identified and characterized from yellow catfish (Pelteobagrus fulvidraco; therefore, the gene is termed as pfcrtc2), and its evolutionary and molecular characteristics as well as potential immunity-related roles were investigated. Our results showed that the open reading frame of pfcrtc2 was 2,346 bp in length, encoding a protein with 781 amino acids. Gene structure analysis revealed its existence of 14 exons and 13 introns. A phylogenetic analysis proved that the tree of crtc2 was clustered into five groups, exhibiting a similar evolutionary topology with species evolution. Multiple protein sequence alignment demonstrated high conservation of the crtc2 in various vertebrates with similar structure. Syntenic and gene structural comparisons further established that crtc2 was highly conserved, implying its similar roles in diverse vertebrates. Tissue distribution pattern detected by quantitative real-time PCR showed that the pfcrtc2 gene was almost expressed in all detected tissues except for eyes, with the highest expression levels in the gonad, indicating that Crtc2 may play important roles in various tissues. In addition, pfcrtc2 was transcribed at all developmental stages in yellow catfish, showing the highest expression levels at 12 h after fertilization. Finally, the transcriptional profiles of crtc2 were significantly increased in yellow catfishes injected with Aeromonas hydrophila or Poly I:C, which shared a consistent change pattern with four immune-related genes including IL-17A, IL-10, MAPKp38, and NF-κBp65, suggesting pfCrtc2 may play critical roles in preventing both exogenous bacteria and virus invasion. Anyway, our findings lay a solid foundation for further studies on the functions of pfcrtc2, and provide novel genetic loci for developing new strategies to control disease outbreak in teleost.

Are Prevailing Policy Tools Effective in Conserving Ecosystem Services Under Individual Private Tenure? Challenges and Policy Gaps in a Rapidly Urbanizing Region

Forestlands worldwide provide vital ecosystem services (ES) essential for human well-being yet are facing rising threats from land-use/land-cover change. Land ownership patterns and property rights significantly influence land management practices and the protection of ES. Family forest owners (FFOs) are the targets of numerous conservation policies and programs, many of which are built around economic incentives and market-based instruments. This study combines a mail-administered survey with land use-land cover change modeling results to better understand whether FFOs plan to convert their properties, what motivates FFOs to adopt prevailing policies, and what other conservation policy models might be attractive. Our research focuses on FFOs whose parcels are identified as having high conservation values in a rapidly urbanizing watershed. Survey results show strong motivations among FFOs to protect biodiversity, wildlife habitat, and ES while expressing concerns about property taxes, regulations, and land-use threats. Our analysis indicates a relationship between anticipated land transfers or residential development and predicting factors including parcel size, government regulations, and tax concerns. Results reveal a gap between the design of existing conservation programs and FFOs’ interest in adoption. Limited interest in conservation easements and carbon markets underscores the need for more flexible conservation tools aligned with landowner preferences. Moreover, FFOs’ considerable awareness of existing conservation programs, alongside ongoing forest loss in the study region, underscores enrollment obstacles such as minimum-size eligibility requirements, thereby highlighting inadequacy of current frameworks in incentivizing long-term conservation outcomes. The findings from the studied watershed may have broader relevance for regions with similar land ownership patterns, land use dynamics, and political contexts worldwide.

Circular RNAs in coronary heart disease: From molecular mechanism to promising clinical application (Review).

Coronary heart disease (CHD) remains a leading cause of morbidity and mortality worldwide, posing a substantial public health burden. Despite advancements in treatment, the complex etiology of CHD necessitates ongoing exploration of novel diagnostic markers and therapeutic targets. Circular RNAs (circRNAs), a distinct class of non‑coding RNAs with a covalently closed loop structure, have emerged as significant regulators in various diseases, including CHD. Their high stability, tissue‑specific expression and evolutionary conservation underscore their potential as biomarkers and therapeutic agents in CHD. This review discusses the current knowledge on circRNAs in the context of CHD and explores the molecular mechanisms by which circRNAs influence the pathophysiology of CHD, including cardiomyocyte death, endothelial injury, vascular dysfunction and inflammation. It also summarizes the emerging evidence highlighting the differential expression of circRNAs in patients with CHD and their potential utilities as non‑invasive diagnostic and prognostic biomarkers and therapeutic targets for this disease.

Design, synthesis, and biological evaluation of dithiocarbamate derivatives as SARS-CoV-2 Mpro inhibitors

SARS-CoV-2 continues to mutate, spread, and impact public health and daily life. The main protease (Mpro) is essential for the replication and maturation of SARS-CoV-2, making it an ideal target for anti-coronaviral drug discovery and development due to its high conservation and lack of homologous proteases in humans. Herein, we designed and synthesized a series of dithiocarbamate derivatives as potent SARS-CoV-2 Mpro inhibitors. Notably, compound L2 exhibited an IC50 value of 9.1 ± 2.0 nM against SARS-CoV-2 Mpro, underscoring its potential as a promising candidate for anti-coronaviral therapy and justifying further research and development.

A probabilistic evaluation method on temperature and time-dependent stress intensity limit St in creep assessment of components at elevated temperatures

Abstract Creep is an important damage mode of components at elevated temperatures, and the temperature and time-dependent stress intensity limit St is an essential indicator in creep assessment of components. In general, the safety factors are directly applied to three criteria in stress intensity limit St in ASME code, but a quantitative evaluation on the failure probability covered in this indicator is rarely reported. Based on this, a probabilistic evaluation method on stress intensity limit St is proposed by correlating each criterion with mean creep rupture life data. The failure probability of three criteria in stress intensity limit St of 316 and 304 stainless steels is calculated, and the effect of safety factors on failure probability results is discussed. A probabilistic evaluation on stress intensity limit St is conducted. Results indicate that the stress intensity limit St presents a low failure probability or high conservativeness, and the minimum stress to creep rupture is the governing role in stress intensity limit St for cases involved. The failure probability of stress intensity limit St of 316 stainless steel is much lower than that of 304 stainless steel, attributed to the differences of creep deformation features and data scatter of the two stainless steels.

Self-association and multimer formation in AtLEA4-5, a desiccation-induced intrinsically disordered protein from plants.

During seed maturation, plants may experience severe desiccation, leading to the accumulation of late embryogenesis abundant (LEA) proteins. These intrinsically disordered proteins also accumulate in plant tissues under water deficit. Functional roles of LEA proteins have been proposed based on in vitro studies, where monomers are considered as the functional units. However, the potential formation of homo-oligomers has been little explored. In this work, we investigated the potential self-association of Arabidopsis thaliana group 4 LEA proteins (AtLEA4) using in vitro and in vivo approaches. LEA4 proteins represent a compelling case of study due to their high conservation throughout the plant kingdom. This protein family is characterized by a conserved N-terminal region, with a high alpha-helix propensity and invitro protective activity, as compared to the highly disordered and low-conserved C-terminal region. Our findings revealed that full-length AtLEA4 proteins oligomerize and that both terminal regions are sufficient for self-association in vitro. However, the ability of both amino and carboxy regions of AtLEA4-5 to self-associate invivo is significantly lower than that of the entire protein. Using high-resolution and quantitative fluorescence microscopy, we were able to disclose the unreported ability of LEA proteins to form high-order oligomers in planta. Additionally, we found that high-order complexes require the simultaneous engagement of both terminal regions, indicating that the entire protein is needed to attain such structural organization. This research provides valuable insights into the self-association of LEA proteins in plants and emphasizes the role of protein oligomer formation.

Dorsal fin height is not an effective tool to distinguish hatchery and wild steelhead in the field

AbstractObjectiveTo maximize harvest of hatchery steelhead Oncorhynchus mykiss and Pacific salmon Oncorhynchus spp. while reducing impacts to wild conspecifics, fishery managers rely on the ability of anglers to distinguish between the two. Fin erosion, including dorsal fin erosion, is a common result of hatchery rearing of juvenile salmonids. Since the 1980s, managers in the Pacific Northwest have utilized adult steelhead dorsal fin height as a regulatory control rule, requiring anglers to release steelhead with dorsal fins over a height threshold. This approach assumes that all or most hatchery fish have shorter dorsal fins than their wild counterparts. This study was designed to test this assumption.MethodsWe compared steelhead origin (hatchery or wild) determined using scale analysis and adipose fin clipping (hatchery n = 127, wild n = 71) with origin estimated using dorsal fin height.ResultOverall, a static dorsal fin height threshold (2.25 in, 5.72 cm) correctly identified 90% of hatchery‐origin steelhead but misidentified nearly half (44%) of wild fish as hatchery. Dorsal fin height of known hatchery fish correlated with fish length, indicating a weakness of using a static dorsal height threshold as a management tool. The accuracy of distinguishing wild fish improved, but only marginally (56–76%), using a classification model where dorsal fin height of known origin fish from this study was a continuous predictor of origin. In comparison, adipose fin status correctly identified 97% of unmarked fish as wild.ConclusionThe low accuracy of the classification model and static dorsal fin height thresholds at distinguishing hatchery and wild steelhead due to a strong correlation between dorsal fin height and fork length and the high fork length variability of hatchery origin fish. Given the low cost and accuracy of adipose fin clipping and the potentially high conservation risk associated with misidentification, we recommend discontinuing the use of dorsal fin height as a harvest control rule for steelhead.

Comparative analysis of complete chloroplast genomes of 14 Asteraceae species.

The Asteraceae family, the largest and one of the most diverse families of angiosperms, presents significant challenges in taxonomic classification and systematic research due to its vast species diversity and morphological complexity. A comprehensive understanding of the chloroplast genomes within this family is essential for refining taxonomic classifications and advancing phylogenetic studies. In this study, we sequenced the complete chloroplast genomes of 14 Asteraceae species and conducted a thorough bioinformatic analysis of their characteristics. The chloroplast genomes, ranging from 150,907bp to 152,858bp, exhibit a typical quadripartite structure: a large single-copy (LSC) region (83,044bp to 84,625bp), a small single-copy (SSC) region (18,223bp to 18,673bp), and a pair of inverted repeats (IRs) (24,806bp to 25,201bp). These genomes encode 87 to 89 protein-coding genes (PCGs), 36 to 37 tRNA genes, and 8 rRNA genes, with high conservation in size, structure, gene content, and order. Comparative analysis with other Asteraceae species' chloroplast genomes revealed notable similarities and structural variations, particularly in the IR regions. Nucleotide polymorphism analysis identified four genes-trnY-GUA, trnE-UUC, ycf1, and rrn23-with higher Pi values, suggesting potential hotspots for evolutionary studies. Phylogenetic analysis using maximum likelihood (ML) and Bayesian inference (BI) approaches provided new insights, proposing the reclassification of Himalaiella auriculata and Jacobaea raphanifolia as independent genera, distinct from Saussurea and Senecio. This study presents a comprehensive analysis of the chloroplast genome structures and phylogenetic relationships of 14 Asteraceae species, offering critical data for future molecular identification, evolutionary biology, and population genetics research. The findings hold significant implications for the ongoing refinement of Asteraceae taxonomic classifications and enhance our understanding of the evolutionary dynamics within this complex family.

Identifying complementary conservation and restoration priority areas for plant species

The coverage of protected areas (PAs) remains far from the Kunming-Montreal target and degraded ecosystems are greatly limiting the conservation efficiency of PAs. Therefore, this paper proposes a method to identify conservation and restoration priority areas to supplement existing PAs. A case study was conducted focusing on Yunnan, southwestern China, which intersects with three world biodiversity hotspots. First, the spatial ranges for 3768 representative conservation plant species were mapped using species distribution models. Subsequently, planning units were classified into three restorability categories, namely no-need restoration, potentially restorable and non-restorable units, according to land cover changes between 2000 and 2020. Then, conservation and restoration priority areas were identified by applying a two-step systematic conservation planning process. Finally, replacement cost analysis was applied to compare the effectiveness of existing PAs and the overall 30 % priority areas. Northwestern, southwestern, and southeastern Yunnan have high biodiversity conservation values. Especially in eastern and southeastern Yunnan, large amounts of restoration priority areas were identified. Conservation and restoration priority areas account for 15.80 % and 3.69 % of Yunnan's land, respectively. Compared to existing PAs, conservation priority areas can increase the number of species covered from 2461 to 3277, and further to 3566 when including restoration priority areas. Compared to existing PAs, the mean species coverage in the overall 30 % priority areas has increased from 27.28 % to 72.69 %. Notably, 12.86 % of existing PAs were identified as restoration priority areas. This study indicates that in addition to conservation measures, implementing restoration strategies in high conservation-value areas is equally important.

Spatial patterns of vascular plant species richness in Poland: Relations among species group richness and hot spot distribution

Knowledge of spatial patterns of species richness (SR) is highly relevant for theoretical research in ecology and the development of conservation plans. In Poland, despite a long tradition of botanical surveys, vascular plant SR has not been mapped, nor have the correlations in richness among different plant species groups been explored at the entire country scale. Here, we used a recently published data set to examine spatial patterns and relationships among the joined SR of vascular plant species, including native species, archaeophytes, neophytes, and species with high conservation value (i.e., red-list species). The basic spatial unit employed was a 10 × 10 km grid from the Atlas of Distribution of Vascular Plants in Poland (ATPOL). We found that SR of native species and archaeophytes, neophytes, and red-list species were positively correlated. The main patterns of SR and the percentage of particular groups in the joined SR were based on three components: (1) gradient of overall SR, (2) invasion level, and (3) peculiarity of flora in some regions resulting from the high number and proportion of rare species that often have high conservation value (red-list species). In general, northeastern Poland was species-poor, while the Carpathian Mountain range, the uplands in southern Poland, and some parts of Wisła River valley had the highest SR concentrations. e location of SR hotspots usually did not overlap with the existing national parks system. The correlations among native SR, high conservation value species, and neophyte SR suggest that biological invasions are among the most important threats to vascular plant diversity in Poland. Finally, despite likely biases in SR assessments, we demonstrated that the presented maps seem to reflect general ecological gradients influencing vascular plant distribution in Poland.

Evidence of anthropogenic subsidisation of red foxes in a national park important for breeding wading birds

AbstractThe red fox (Vulpes vulpes) is a generalist mesopredator found throughout the UK. It has been linked to national declines in native wildlife, especially ground-nesting birds such as waders. In the New Forest National Park, nest predation and poor chick survival is primarily responsible for low breeding success of Eurasian curlew (Numenius arguata), a species of high conservation concern. To reduce predation losses, foxes are lethally controlled by wildlife managers. Here, we identified the major food resources that are being exploited by foxes in the New Forest area and examined temporal and spatial patterns in the presence of specific food categories, with special reference to anthropogenic food. Stomachs from foxes culled in curlew breeding areas were collected from April 2021 - July 2022 and the contents of these stomachs were quantified. Foxes exhibited a highly varied diet with no single food category predominating. Anthropogenic food comprised 14% of the overall diet, with its presence predicted by proximity to human settlements and other infrastructure. We also estimated the total annual volume of anthropogenic food consumed by the fox population and by extension how many individual foxes this volume of food could support in isolation. According to these calculations, at present the number of foxes subsidised by anthropogenic food is approximately 64.8% (50.2–79.7%) of those removed by culling per year. Our findings highlight that better local food sanitation and education should become important parts of a more holistic management approach to reduce the burden of fox predation experienced by breeding waders.

Chloroplast genome sequencing and divergence analysis of 18 Pyrus species: insights into intron length polymorphisms and evolutionary processes.

Pears constitute an essential temperate crop and are primarily produced through interspecific hybridization owing to self-incompatibility that complicates their breeding history. To address this, we sequenced the complete chloroplast (cp) genomes of 18 Pyrus and one Malus species using the Illumina HiSeq4000 platform. The cp genomes ranged from 159,885 bp to 160,153bp and exhibited a conserved circular DNA structure with an average GC content of 36.5%. Each cp genome contained 127 genes, including 83 protein-coding, 36 tRNA, and 8 rRNA genes. Divergence analysis with mVISTA showed high conservation in the coding regions and notable variations in the non-coding regions. All species shared 17 intron-containing genes, with ycf3 and clpP each having two introns. Five intron-containing genes (ndhB, rpl2, rps12, trnA-UGC, and trnE-UUC) were located in the inverted repeat regions, while trnL-UAA was located in the large single-copy region, with conserved intron lengths across Pomoideae. We identified polymorphic intron sequences in the rpl22, petB, clpP, ndhA, and rps16 genes and designed primers for these regions. Notably, the two Pyrus ussuriensis accessions Doonggeullebae and Cheongdangrori showed intron-length polymorphisms despite being classified as the same species. Phylogenetic analysis of the cp genome sequences revealed two major clusters, indicating distinct maternal lineages and evolutionary origins. This study underscores the importance of cp gene polymorphisms in P. fauriei, P. calleryana, P. ussuriensis, and P. pyrifolia, providing valuable insights into Pyrus evolution as well as aiding in the conservation and breeding of pear germplasm.

Immune History Modifies Disease Severity to HPAI H5N1 Clade 2.3.4.4b Viral Challenge.

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity-either from LAIV vaccination and/or from Cal09 infection-showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

Identification, characterization, and distribution of novel amidase gene aphA in sphingomonads conferring resistance to amphenicol antibiotics.

Amphenicol antibiotics, such as chloramphenicol (CHL), thiamphenicol (TAP), and florfenicol (Ff), are high-risk emerging pollutants. Their extensive usage in aquaculture, livestock, and poultry farming has led to an increase in bacterial antibiotic resistance and facilitated the spread of resistance genes. Yet, limited research has been conducted on the co-resistance of CHL, TAP, and Ff. Herein, a novel amidase AphA was identified from a pure cultured strain that can concurrently mediate the hydrolytic inactivation of CHL, TAP, and Ff, yielding products p-nitrophenylserinol, thiamphenicol amine (TAP-amine), and florfenicol amine (Ff-amine), respectively. The antibacterial activity of these antibiotic hydrolysates exhibited a significant reduction or complete loss in comparison to the parent compounds. Notably, AphA shared less than 26% amino acid sequence identity with previously reported enzymes and exhibited high conservation within the sphingomonad species. Through enzymatic kinetic analysis, the AphA exhibited markedly superior affinity and catalytic activity toward Ff in comparison to CHL and TAP. Site-directed mutagenesis analysis revealed the indispensability of catalytic triad residues, particularly serine 153 and histidine 277, in forming crucial hydrogen bonds essential for AphA's hydrolytic activity. Comparative genomic analysis showed that aphA genes in some species are closely adjacent to various transposable elements, indicating that there is a high potential risk of horizontal gene transfer (HGT). This study established a hydrolysis resistance mechanism of amphenicol antibiotics in sphingomonads, which offers theoretical guidance and a novel marker gene for assessing the prevalent risk of amphenicol antibiotics in the environment.IMPORTANCEAmphenicol antibiotics are pervasive emerging contaminants that present a substantial threat to ecological systems. Few studies have elucidated resistance genes or mechanisms that can act on CHL, TAP, and Ff simultaneously. The results of this study fill this knowledge gap and identify a novel amidase AphA from the bacterium Sphingobium yanoikuyae B1, which mediates three typical amphenicol antibiotic inactivation, and the molecular mechanism is elucidated. The diverse types of transposable elements were identified in the flanking regions of the aphA gene, indicating the risk of horizontal transfer of this antibiotic resistance genes (ARG). These findings offer new insights into the bacterial resistance to amphenicol antibiotics. The gene reported herein can be utilized as a novel genetic diagnostic marker for monitoring the environmental fate of amphenicol antibiotics, thereby enriching risk assessment efforts within the context of antibiotic resistance.

Exploring voltage-gated sodium channel conformations and protein-protein interactions using AlphaFold2.

Voltage-gated sodium (Na V ) channels are vital regulators of electrical activity in excitable cells, playing critical roles in generating and propagating action potentials. Given their importance in physiology, Na V channels are key therapeutic targets for treating numerous conditions, yet developing subtype-selective drugs remains challenging due to the high sequence and structural conservation among Na V family members. Recent advances in cryo-electron microscopy have resolved nearly all human Na V channels, providing valuable insights into their structure and function. However, limitations persist in fully capturing the complex conformational states that underlie Na V channel gating and modulation. This study explores the capability of AlphaFold2 to sample multiple Na V channel conformations and assess AlphaFold Multimer's accuracy in modeling interactions between the Na V α-subunit and its protein partners, including auxiliary β-subunits and calmodulin. We enhance conformational sampling to explore Na V channel conformations using a subsampled multiple sequence alignment approach and varying the number of recycles. Our results demonstrate that AlphaFold2 models multiple Na V channel conformations, including those from experimental structures, new states not yet experimentally identified, and potential intermediate states. Furthermore, AlphaFold Multimer models Na V complexes with auxiliary β-subunits and calmodulin with high accuracy, and the presence of protein partners significantly alters the conformational landscape of the Na V α-subunit. These findings highlight the potential of deep learning-based methods to expand our understanding of Na V channel structure, gating, and modulation, with significant implications for future drug discovery efforts.