Search Results (4,907)

Prime editing (PE) is a precise genome-editing technology that avoids double-strand breaks, holding great promise for clinical and agricultural applications. However, its genome-wide off-target effects are not fully understood, raising safety concerns. Here, we systematically compared the safety profiles of four prime editor variants (PE2max, PE3max, PE4max, and PE5max) using PEM-seq and RNA-seq. We further applied an ultra-sensitive method, Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI), to assess PE5max. Our results show that PE5max did not produce detectable sgRNA-dependent off-target single-nucleotide variants (SNVs) in the GOTI assay and induced only limited large deletions and chromosomal translocations. Collectively, this side-by-side benchmarking under matched conditions demonstrates that PE5max achieves an improved specificity profile, with no detectable increase in genome-wide off-target SNVs, advancing its potential for safer therapeutic use. Full article

Background and Clinical Significance: Over the last two decades, glucagon-like peptide-1 (GLP-1) receptor agonists have dramatically improved the management of type 2 diabetes mellitus and obesity. Currently, little is known about the use of semaglutide (a second-generation GLP-1 receptor agonist) in patients with X chromosome abnormalities. Herein, we describe the therapeutic use of semaglutide in a woman with a partial deletion of the X chromosome long arm (partial Xq deletion) and comorbid obesity. We also conducted a narrative mini-review on overweight, obesity and common metabolic derangements in patients with partial Xq deletions and Turner syndrome. Case Presentation: A 65-year-old Italian woman with a partial Xq deletion, class 1 obesity, insulin resistance, prediabetes, hypercholesterolemia and metabolic dysfunction-associated steatotic liver disease (MASLD) was referred to our Institution for persistent difficulty in managing excess body weight despite regular adherence to different structured physical activity programs and hypocaloric diets. Therefore, we prescribed a combination therapy based on low-dose metformin (500 mg/day) and once-weekly subcutaneous semaglutide (as an adjunct to lifestyle intervention). At 5 months after initiation of the combination therapy, blood tests showed metabolic improvements, including improvement of prediabetes (0.3-percentage-point reduction in glycated hemoglobin [HbA1c] values) and normalization of markers of insulin sensitivity and insulin resistance (QUICKI, HOMA-IR and TyG index). At 8 months, the patient showed substantial weight loss, which amounted to 13.8 kg (percent total body weight loss: 20.95%), and was accompanied by a notable reduction in waist circumference (−14.1 cm). Moreover, body mass index (BMI)-based weight status improved from class 1 obesity to overweight: BMI value of 25.1 kg/m² at 8 months vs. 31.8 kg/m² at baseline (near-normalization of BMI values). Bioelectrical impedance analysis (BIA) revealed that the patient’s overall weight loss consisted of 74.6% fat mass (FM) loss (−10.3 kg) and 25.4% fat-free mass (FFM) loss (−3.5 kg). Despite the expected FFM reduction in absolute terms, percent FFM increased at 8 months (+9.6%). This increase in percent FFM was accompanied by a reduction in percent FM at 8 months (−9.6%), indicating an overall improvement in body composition. Normalization of percent FM and FFM values (28.6% and 71.4%, respectively) was also achieved at 8 months. These body composition changes are in line with those observed in clinical trials investigating the use of semaglutide in patients with overweight or obesity. At 6 months, an abdominal ultrasound also showed the disappearance of the sonographic characteristics suggestive of mild-to-moderate hepatic steatosis. Low-dose metformin (500 mg/day) and subcutaneous semaglutide (up to a weekly dose of 1.7 mg) were well tolerated by the patient. Conclusions: To the best of our knowledge, this is the first case documenting the effective use of once-weekly subcutaneous semaglutide plus low-dose metformin combination therapy for the treatment of obesity and prediabetes in a woman with a partial Xq deletion. Large prospective cohort studies are warranted to better investigate the safety and efficacy profile of semaglutide (alone or in combination with metformin) in patients with numerical and structural X chromosome abnormalities, comorbid overweight/obesity and related metabolic disorders. Full article

Purslane (Portulaca oleracea L.) is an important plant species that has been increasingly used in functional gene studies and molecular analyses. However, reference genes that exhibit stable expression across multiple tissues and stress conditions have not been systematically validated in purslane, which limits the accuracy of reverse transcription quantitative PCR (RT-qPCR) based gene expression analyses. In this study, ten candidate reference genes from six gene families (Actin, PP2A, CYP, eIF4A, Ubiquitin, and eIF5A) were selected based on transcriptome data. A combination of bioinformatic analyses and experimental validation was employed to comprehensively characterize these candidates, including their physicochemical properties, chromosomal localization, phylogenetic relationships, gene structures, and promoter cis-acting elements. Furthermore, the expression stability of the candidate genes was systematically evaluated across different tissues (seed, root, stem, leaf, and flower) and under multiple stress treatments, including salinity, temperature stress, drought, and hormone treatments. Based on conventional PCR amplification specificity, melting curve analysis, Ct value distribution, and amplification efficiency, ACT-2 and eIF5A-1 were identified as the most stably expressed reference genes under diverse experimental conditions. This study provides reliable reference gene candidates for accurate normalization of gene expression in purslane and establishes a systematic framework for reference gene selection in non-model plant species. Full article

Background/Objectives: Sweetness is a key determinant of the eating quality of sweet corn, primarily governed by the soluble sugar content in kernels. The soluble sugar content decreases rapidly during the postharvest shelf life, which directly affects the flavor and quality. Relatively few studies have been conducted on the shelf life of sweet corn. Methods: An F₆ recombinant inbred line (RIL) population was constructed from two super sweet inbred lines with contrasting soluble sugar degradation rates: D174 (low degradation rate) and D179 (high degradation rate). Extreme phenotype pools were established using soluble sugar content as the target trait. Based on bulked segregant analysis sequencing, we identified chromosomal segments associated with postharvest soluble sugar reduction in sweet corn, annotated the gene information within these segments, and analyzed the functions of the annotated genes using the Gene Ontology and Genomes databases. Results: Results revealed three associated regions located at 44,205,775–45,290,843 bp on chromosome 4, 6,250,656–6,744,665 bp on chromosome 2, and 135,428,709–136,732,132 bp on chromosome 10. This interval contained 195 genes. Integrated analysis of gene expression, gene annotations, and quantitative real-time PCR indicated that Zm00001eb069070, which is highly expressed in kernels with a prolonged shelf life, might be a key candidate gene regulating soluble sugar degradation in sweet corn. Conclusions: This study provides valuable genetic resources for the improvement of favorable agronomic traits and the advancement of molecular breeding strategies for sweet corn. Full article

Asparagine synthase (ASN) is a key enzyme in nitrogen metabolism. It is responsible for the synthesis of asparagine, but the function and molecular mechanism of its superfamily in the seed storage protein of soybean is not yet clear. In this study, a total of 34 members of the ASN superfamily were identified in soybean by in silico analysis (GmASNs). Phylogenetic and structural analyses show that most proteins in the GmASN superfamily contain conserved functional domains, which are predicted to be hydrophilic proteins, and are mainly located in the cytoplasm and chloroplasts, and a few are located in mitochondria. The chromosome distribution shows that they are randomly scattered on 13 chromosomes, and gene replication drives the expansion of the family. In addition, the haplotype analysis of four key GmASN superfamily genes (GmASN11, GmASN16, GmASN20, GmASN29) shows that they are significantly associated with the content of seed storage protein (SSP). Importantly, the aggregation of favorable haplotypes at these loci can significantly improve the SSP level. These results elucidate the evolutionary history of the GmASN gene superfamily and identify valuable haplotypes. These haplotypes are promising targets for the development of high-protein soybean varieties through marker-assisted breeding. Full article

Nuclear Factor Y (NF-Y) transcription factors play pivotal roles in plant adaptation to abiotic stress, yet their genomic landscape and functional mechanisms in cabbage (Brassica oleracea var. capitata L.) remain underexplored. Here, we performed a genome-wide identification of the NF-Ys in cabbage, identifying 53 BoNF-Ys classified into three subfamilies: 20 BoNF-YAs, 22 BoNF-YBs, and 11 BoNF-YCs. Phylogenetic clustering revealed evolutionary conservation with their Arabidopsis orthologs. Domain analysis revealed that all BoNF-YA members contain the CBF_NF-YA domain, while all BoNF-YB and BoNF-YC members possess the CBFD_NFYB_HMF conserved domain. The BoNF-Y genes were named according to their chromosomal locations. Bioinformatic analysis showed that BoNF-Y proteins range in size from 131 to 642 amino acids, with molecular weights of 14.82–73.18 kDa, theoretical pI values of 4.57–9.96, instability indices between 33.02 and 73.48, aliphatic indices of 45.3–86.26, and grand average of hydropathicity (GRAVY) values ranging from −1.139 to −0.367. Promoter cis-element profiling uncovered stress- and hormone-responsive motifs, including abscisic acid-responsive elements (ABREs), TC-rich repeats, and ethylene-responsive elements (EREs). RNA sequencing (RNA-seq) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) conducted under salt stress (256 mM) identified three salt-responsive candidate genes (BoNF-YA14, BoNF-YB9, and BoNF-YC8). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses highlighted significantly expressed genes’ roles in MAPK signaling, proline metabolism, and phytohormone transduction pathways. This study conducted a comprehensive survey of the BoNF-Y gene family in cabbage. It could serve as a theoretical foundation for further functional identification and utilization of BoNF-Y family members and their role in the interaction between cabbage and salt stress. Full article

Reproductive efficiency is a fundamental determinant of productivity in pig breeding programs. However, the role of the X chromosome in shaping the genetic basis of reproductive traits remains underexplored. To address this problem, Genome-Wide Association Studies were conducted to explore the genetic basis of reproductive traits in Yorkshire pigs. We collected data from 2659 Yorkshire sows, focusing on four reproductive traits across the first three parties. Statistical analyses included principal component analysis, estimation of genetic parameters, and mixed linear model association to identify SNPs and candidate genes. Heritability estimates (h²) ranged from 0.037 (TLWT_BA_P1) to 0.215 (TNB_P3). Genome-wide association analysis identified 23 significant SNPs and candidate genes, including several putatively significant X-linked genes; however, only a single X-linked locus was significantly associated with one trait. These findings demonstrate that, for 9 of the 12 parities, the proportion of total heritability attributable to X-linked variation was 0–2.7%. For the remaining three parities, the corresponding proportions were 8.4%, 21.5%, and 50.9%, while autosomes accounted for most of the genetic variation. In all but one trait, heritability estimated from X-linked GRMs was not significantly different from zero, highlighting the importance of incorporating X-linked information into genomic selection strategies for improving reproductive efficiency in pigs. Full article

Background: Microarray testing is commonly used as a screening method for phenotypic traits and common diseases and for genome-wide association studies (GWASs). Despite the known limitations, microarray services can potentially be used as a prescreening tool for chromosomal disorders, which affect approximately 0.4–0.6% of the world population, followed by further clinical diagnostic methods when appropriate. Case Presentation: Here we present a case study of a male subject in his 40s who underwent direct-to-consumer (DTC) genetic testing that utilized microarray, which revealed the absence of Y chromosome haplogroup data despite possessing a typical male phenotype. Subsequent medical consultation, whole-genome sequencing (WGS), and chromosomal analysis confirmed a diagnosis of 46,XX testicular differences of sex development (DSD, formerly XX male syndrome) characterized by the presence of Y chromosome-derived genomic material, including the SRY gene. An initial microarray test gave an indeterminate result for the Y chromosome call rate and an X chromosome heterozygosity result that aligned with the female average. These indeterminate results, coupled with the subject’s male phenotype, led to further testing—WGS, karyotyping, fluorescence in situ hybridization using an SRY Probe, and endocrine testing. From these results, the subject was diagnosed with 46,XX testicular DSD. Conclusions: To our knowledge, this represents the first reported case where 46,XX testicular DSD was diagnosed starting from a DTC test which led to medical consultation and comprehensive genomic and cytogenetic analysis. This case underscores the potential diagnostic value of consumer-initiated DTC microarray screening in the era of genomic medicine and for supporting social needs such as gender confirmation for sports. Full article

Primary Failure of Eruption (PFE) is a disorder characterized by aberrant tooth eruption, in which one or more teeth fail to follow the physiological eruptive pathway and remain partially or completely embedded within the bone or soft tissues. Although the etiopathogenesis of PFE is not yet fully elucidated, several contributing factors have been identified, including genetic alterations, hormonal disturbances, and systemic conditions. An expanding body of evidence points to the centrality of genetic determinants in the etiopathogenesis of PFE, supporting its occurrence in both syndromic contexts and non-syndromic presentations. Non-syndromic forms are closely related to heterozygous variants in the Parathyroid Hormone 1 Receptor (PTH1R) gene, located on chromosome 3p21, which encodes a receptor essential for the regulation of bone and dental growth and development. In most cases, pathogenic variants result in a non-functional receptor. To date, a substantial number 50 PTH1R variants have been documented in individuals exhibiting a phenotype consistent with PFE, underscoring the central involvement of this gene in the disorder’s molecular basis. Advances in understanding the genetic contribution to PFE emphasize the need for early diagnosis, as timely identification of the condition can prevent secondary dental complications and reduce reliance in adulthood on invasive orthodontic or surgical interventions, including extractions, orthognathic surgery, and implant-supported rehabilitation. This review aims to provide a comprehensive analysis of the spectrum of PTH1R variants implicated in PFE, examining genotype–phenotype correlations and their implications for diagnostic strategies and clinical management. Full article

Objectives: The main aim of this study consists of testing the consistency and reliability of the BIANCA (BIophysical ANalysis of Cell death and chromosome Aberrations) biophysical model across different radiation transport codes in the framework of cancer ion-therapy research. Methods: Spread-Out Bragg Peak (SOBP) profiles for protons, helium ions and carbon ions were simulated at three different depth ranges (2–3 cm, 5–8 cm, and 10–15 cm) applying two radiation transport codes, FLUKA and Geant4. While BIANCA has been interfaced to FLUKA in a previous work, an interface with Geant4 was purposely developed in this work. Cell survival along all considered SOBP profiles was predicted by BIANCA for two cell lines with very different radiosensitivities: Squamous Cell Carcinoma (SCC), with α/β = 12.68 Gy, and chordoma, with α/β = 2.37 Gy. The agreement between the predictions obtained from the two approaches was quantitatively evaluated by means of Root Mean Square Error (RMSE) and Gamma Index analysis, both for physical dose distributions and for cell survival predictions. Results: The comparison between FLUKA and Geant4 simulations demonstrated good agreement. The Gamma Index analysis yielded passing rates exceeding 94.9% for physical dose profiles (criteria: 3%/2 mm) and 96.0% for cell survival probabilities (criteria: 2%/2 mm) across all considered ion species (protons, He, C) and depths. Root Mean Square Error (RMSE) analysis confirmed average discrepancies below 2.5% for physical dose and 1% for biological survival. Conclusions: This study shows that the BIANCA model can be applied to predict cell killing along hadron therapy beams when interfaced both with FLUKA and with Geant4. Furthermore, the successful implementation of the interface with Geant4 expands the accessibility and applicability of BIANCA, paving the way for its future integration into different transport codes and/or treatment planning systems. Full article

Plant SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) constitute a large superfamily and play pivotal roles in diverse biological processes and responses to various abiotic stresses. Quinoa (Chenopodium quinoa wild.) is a nutritionally superior crop endowed with robust tolerance to environmental stresses. In this study, we identified 88 CqSNARE genes in quinoa, which are unevenly distributed across 18 chromosomes and classified into 23 subfamilies. We systematically analyzed their physicochemical properties, phylogenetic relationships, gene and protein structures, and cis-acting elements. Furthermore, transcriptome analysis of quinoa leaves under saline–alkaline stress revealed that CqSNAP30a was the most significantly upregulated. This gene is predominantly expressed in leaves and localized on the plasma membrane. Constitutive expression of CqSNAP30a enhanced plant stress resistance by regulating ion homeostasis and antioxidant capacity. Our findings provide valuable insights into the SNARE genes of stress-tolerant crops and lays a theoretical foundation for the genetic improvement of stress resilience. Full article

Gibberellins (GAs) play a crucial regulatory role in the growth and development of cotton (Gossypium hirsutum L.). Through bioinformatics analyses, we identified a total of 39 GA2ox genes (encoding gibberellin 2-oxidases) in the cotton genome, designated GhGA2ox1 to GhGA2ox39. Based on phylogenetic analysis, these genes were classified into five groups. We further examined their gene structures, conserved motifs, and chromosomal distributions, revealing that members within the same group shared similar structural and motif organizations. Collinearity and cis-element analyses provided important insights into the evolutionary history and regulatory potential of the GA2ox gene family in cotton. Notably, using nucleotide diversity (π) and population differentiation (F_ST) analyses across the entire family, we screened and identified nine candidate genes that underwent strong artificial selection during cotton domestication and improvement. Further haplotype-phenotype association analysis identified GH_D09G0919 (GhGA2ox31) as a key regulator of Plant Height (PH). To validate their regulatory roles, we analyzed the genotype distribution in accessions with extreme phenotypes. The results revealed divergent selection histories for these two loci: the favorable allele of GH_D01G0720 (GhGA2ox23) was already fixed in the tested population, whereas GH_D09G0919 maintained significant natural variation. Specifically, the Hap2 allele of GH_D09G0919 was significantly enriched in the shortest accessions compared to the tallest ones. Importantly, quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed that the Hap2 allele drives significantly higher gene expression in leaves, suggesting that enhanced GA catabolism underlies the compact phenotype. Additionally, transcriptomic profiling revealed the tissue-specific expression patterns of candidate genes, implying their functional roles in development. Furthermore, functional validation using the Arabidopsis mutant of the homologous gene (AtGA2ox8) confirmed its conserved role in regulating plant height, as the mutant exhibited a distinct short-stature phenotype. These results uncover valuable genetic resources for molecular breeding to shape compact cotton architecture. Collectively, this study aims to analyze the evolutionary patterns of the cotton GA2ox gene family and to identify key genes that regulate plant height under artificial selection, providing theoretical support for molecular breeding of compact plant types. Full article

DNA double-strand breaks (DSBs) are primarily repaired in eukaryotic cells through two pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The thermotolerant yeast Kluyveromyces marxianus is recognized for its highly active NHEJ pathway, making it a suitable model organism for studying the role of NHEJ in DSB repair. To induce DSBs in K. marxianus DMKU3-1042, an expression cassette containing the gene encoding the endonuclease RsaI was integrated into the LYS1 locus of both the wild-type and NHEJ-deficient KU70 mutant strains. This cassette is regulated by the galactose-inducible promoter GAL10. Cells expressing RsaI and grown in galactose medium exhibited an elongated, rod-shaped morphology under a microscope. Following RsaI expression, the viability of transformed KU70 cells decreased during the first three hours of culture in liquid medium and then partially recovered after six hours of incubation. In contrast, the KU70 mutant cells failed to produce viable survivors. Pulsed-field gel electrophoresis analysis revealed distinct chromosomal separation patterns among various RsaI-transformed KU70 cells. These findings demonstrate that the repair of RsaI-induced DSBs in K. marxianus DMKU3-1042 results in new strains with several forms of rearranged chromosomes. Full article

Growth Regulating Factors (GRFs) are plant-specific transcription factors that play crucial roles in regulating growth and development throughout the plant life cycle. A total of 34 Gossypium hirsutum GRF family genes were identified at the genome-wide level, which were unevenly distributed on 19 chromosomes, and were predicted to be mainly localized in the nucleus and plasma membrane. The number of GRF family genes varied greatly among different species, and they were categorized into four subfamilies (I–IV) according to their phylogenetic relationships. The G. hirsutum GRF genes possessed specific highly conserved structural domains, Trp-Arg-Cys motif (WRC) and Gln, Leu, Gln motif (QLQ), and structural analysis of the genes revealed that they contained 1–23 exons, and most of them contained UTRs. Intraspecies covariance analysis revealed that the GRF genes expanded in G. hirsutum by segmental duplication. The promoter region of the G. hirsutum GRF gene contained a large number of adversity stress response elements, as well as a small number of hormone response elements and growth and development-related response elements. Transcriptome data showed that the expression of G. hirsutum GRF genes was significantly higher in leaves than in other tissues, and some GRF genes responded to a variety of abiotic stresses. Additionally, transcriptomic sequencing revealed significantly higher expression levels of GhGRFs (e.g., GhGRF13/14/18) in embryonic callus (EC) compared to non-embryonic callus (NEC). This differential expression was validated by RT-qPCR, which confirmed that GhGRF13/14/16/20 were significantly upregulated in EC relative to NEC. These findings provide valuable candidate genes and molecular insights for improving G. hirsutum regeneration efficiency and yield-related traits through genetic manipulation, thereby accelerating the molecular breeding of elite G. hirsutum varieties. Full article

Background/Objectives: Ziziphus jujuba var. spinosa (sour jujube) is a traditional medicinal plant with remarkable tolerance to abiotic stresses, particularly salinity. However, the regulatory mechanisms underlying its salt stress tolerance remain unclear. NHX genes play a crucial role in plant adaptation to salt stress by mediating Na⁺/K⁺ transport to maintain intracellular ion homeostasis and pH balance. Although the NHX gene family has been characterized in many plant species, its functional characteristics in sour jujube have not yet been systematically investigated. Methods: In this study, using Arabidopsis thaliana as a reference, we identified NHX genes in sour jujube through genome-wide analysis and molecular approaches, and systematically analyzed their phylogenetic relationships, chromosomal locations, conserved motifs, gene structures, cis-regulatory elements in promoter regions, and expression patterns under abiotic stresses, particularly salt stress. Results: The results revealed the presence of eight NHX genes distributed across six chromosomes in sour jujube, which were classified into three subfamilies: Vac-class, Endo-class, and PM-class. Members within the same evolutionary clade exhibited high structural conservation in motif composition and gene architecture. Except for the PM-class, all other clades contained amiloride-binding sites (FF(I/L)(Y/F)LFLLPPI). Analysis of cis-regulatory elements indicated that the promoter regions of these genes were enriched with elements related to defense responses, stress adaptation, and phytohormone signaling, further supporting their role in plant environmental adaptation. Additionally, the qRT-PCR analysis showed that most of the ZjNHX genes in both roots and leaves are up-regulated by salt. Notably, ZjNHX1 expression in roots increased approximately 40-fold within 3 h, whereas ZjNHX2 and ZjNHX3 were strongly induced in leaves under prolonged salt exposure. Conclusions: Taken together, this work gives a detailed overview of the ZjNHX genes and their important roles in response to salt stress in sour jujube. Our findings also provide a foundation for further functional characterization of this gene family. Full article