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Cytological Assessment of Maize Pollen Viability Using a Simplified Staining Protocol.

Anomalies of meiosis frequently result in abnormal chromosome segregation, which leads to defects in pollen formation in maize. Thus, assessing pollen viability is an important measure for examining the overall success of male sexual reproduction. Pollen viability tests are used to characterize mutants defective in meiosis and microsporogenesis, and to determine the effects of genome instability and environmental conditions on reproduction. This protocol describes a rapid method for assessing pollen viability in maize, using a simplified cytological staining approach. Traditional pollen staining methods, such as Alexander staining, often require hazardous chemicals that are increasingly restricted due to safety concerns. The method presented here uses easily accessible reagents and avoids highly toxic substances.

Journal Article↗

Site-Specific Measurement of Meiotic Crossing-Over Rate with Droplet Digital PCR.

Understanding the frequency and distribution of meiotic crossovers (COs) is critical for both fundamental studies on meiosis and for practical applications in plant breeding, where controlling recombination can accelerate crop improvement. Determining CO rates at specific genomic loci has traditionally relied on labor-intensive methods that require the production and genotyping of large progenies. Here, we present a high-throughput protocol for site-specific quantification of meiotic COs in maize using droplet digital PCR (ddPCR). The method is based on genotyping individual pollen nuclei from hybrid plants to detect recombinant and nonrecombinant alleles at defined chromosomal intervals. By distributing several thousands of pollen nuclei into nanoliter-sized droplets and performing PCR with allele-specific fluorescent probes, this method allows precise quantification of CO frequency with high sensitivity. The protocol provides detailed guidance for nuclei isolation, probe master mix preparation, droplet generation, and data interpretation. This method can be easily adapted for use in other plants.

Journal Article↗

Analyzing Meiosis in Maize.

Meiosis is central to sexual reproduction and the main source of genetic diversity in plants. Understanding how meiotic processes are regulated has direct relevance to agriculture. As meiotic recombination is the vehicle of plant breeding, gaining the ability to influence recombination patterns can accelerate crop improvement. Maize is a powerful model for studying plant meiosis, thanks to its large chromosomes, well-developed genetics, and the availability of diverse cytogenetic and molecular tools. Insights gained from maize studies can extend to other species. In this review, we describe a variety of approaches for examining meiosis and meiotic recombination in maize. Cytological techniques, including protein immunolocalization and fluorescence in situ hybridization (FISH), enable visualization of chromosome structure and behavior, as well as crossover (CO) formation. Chromatin immunoprecipitation (ChIP) is used in meiosis research to determine locations of recombination proteins, identify recombination sites, and elucidate chromatin features, such as histone modifications. Quantification of COs at specific genomic sites through pollen typing by droplet digital PCR allows precise high-resolution measurement of recombination rates. Combining cytology, protein localization, and molecular assays provides a multiscale picture of meiosis, linking molecular mechanisms to chromosome behavior and, ultimately, to genetic variation.

Journal Article↗

A CRISPR/Cas9 gene-editing platform for the rice leaffolder, Cnaphalocrocis medinalis.

A CRISPR/Cas9 toolkit was established for the rice leaffolder (Cnaphalocrocis medinalis) using entirely endogenous regulatory elements. An all-in-one plasmid was constructed containing an IE1-EGFP marker, a CmU6 promoter driven sgRNA targeting Cmebony, and a Cmactin5C promoter driven Cas9. This platform provides a system for functional genomics in this pest.

Letter↗

Genetically Proxied Leukocyte Telomere Length and Epigenetic Age Acceleration in Relation to Healthspan: A Mendelian Randomization Study.

BACKGROUND: Leukocyte telomere length (LTL) and epigenetic age acceleration (EAA) are widely studied biomarkers of biological aging, but their potential roles in healthspan remain unclear. We evaluated whether genetically proxied LTL and EAA show evidence of potential effects on healthspan. METHODS: We conducted a two-sample Mendelian randomization study. Genetic instruments for LTL and four EAA biomarkers were obtained from published genome-wide association studies, including up to 472,174 individuals for LTL and approximately 35,000 individuals for each EAA biomarker. Summary statistics for healthspan, defined as age at first diagnosis of any of eight major chronic conditions or death, were derived from 300,447 unrelated European-ancestry participants in the UK Biobank. We used inverse-variance-weighted (IVW) models for the main analysis, with complementary MR estimators and sensitivity analyses to evaluate consistency, pleiotropy, instrument heterogeneity, and robustness. RESULTS: Genetically proxied longer LTL was associated with extended healthspan (IVW β = 0.106; 95% CI: 0.054-0.158; p = 6.9 × 10-5). The association was robust across multiple sensitivity analyses. In contrast, the four genetically proxied EAA biomarkers did not show consistent MR evidence of an association with healthspan. CONCLUSIONS: These findings provide genetic evidence consistent with a potential role of LTL in healthspan, while providing little support for comparable associations involving the genetically proxied components of the evaluated EAA biomarkers. The findings do not exclude potential associations with environmentally or physiologically acquired EAA.

Mendelian randomization↗

GPER stimulation attenuates mitochondrial dysfunction and cardiac dysfunction in ovariectomized mice with heart failure with preserved ejection fraction (HFpEF).

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is prevalent among postmenopausal women and is strongly linked to estrogen deficiency. G-protein coupled estrogen receptor (GPER) mediates non-genomic estrogen signalling and exerts cardiovascular protective effects. Its role in the pathogenesis of HFpEF remains unclear. This study aimed to explore whether GPER activation could attenuate mitochondrial dysfunction and cardiac damage in ovariectomized (OVX) mice with HFpEF. METHODS: Circulating GPER levels were measured in postmenopausal women with HFpEF and healthy controls. A correlation analysis was performed to assess the associations between GPER and cardiac function. Female C57BL/6J mice underwent ovariectomy and were fed with high-fat diet and l-NAME to induce HFpEF. Mice were treated with the GPER agonist G-1 for 4 weeks. Cardiac function, histological changes, oxidative stress, mitochondrial function and mitophagy were evaluated in vivo and in vitro. RESULTS: Serum GPER levels were significantly higher in postmenopausal women with HFpEF and correlated with NT-proBNP and E/e'. In OVX mice with HFpEF, GPER expression was up-regulated, and G-1 improved diastolic function, reduced myocardial hypertrophy and oxidative stress. Importantly, G-1 restored mitochondrial ATP production, normalized mitochondrial dynamics and promoted mitophagy in vivo and in vitro. These effects were associated with activation of the AMPK/ULK1 pathway. Inhibition of AMPK diminished the protective effects of G-1 in cardiomyocytes. CONCLUSIONS: GPER agonist G-1 ameliorated mitochondrial dysfunction, promoted mitophagy and alleviated cardiac diastolic dysfunction in OVX mice with HFpEF, partially through the AMPK/ULK1 pathway, indicating GPER as a therapeutic target for postmenopausal women with HFpEF.

AMPK/ULK1 signalling pathway↗

Spectral Transforms as a Tool to Optimize Digital Phenotyping in Biological Images.

Modern livestock breeding has mastered genotyping. Genome-wide association studies, genomic selection, and SNP arrays enable genetic merit prediction at lower cost. However, phenotyping remains the bottleneck, as manual measurement is slow, expensive, subjective, and unable to capture spatial or temporal trait organization. Digital phenotyping via artificial intelligence could resolve this, but deep learning requires thousands of labelled examples, impractical when phenotyping cost itself limits datasets to hundreds of individuals. This creates a paradox: AI could accelerate phenotyping but requires large numbers of samples to train the models. Here, we demonstrate that integrating computer vision with machine learning offers sample-efficient digital phenotyping using eggshell colour as a model system. Rather than learning features from scratch (deep learning), we engineer physically motivated features via Wavelet transforms that decompose images into multi-scale spatial components. Wavelet features captured 14.2 percentage points more variance (R2&#x2009;=&#x2009;0.976 vs. 0.834, p&#x2009;<&#x2009;0.001) than standard colorimetry, with 50% better sample efficiency (achieving at n&#x2009;=&#x2009;60 what colorimetry required n&#x2009;=&#x2009;120). Variance decomposition revealed 77% of discriminative capacity derives from spatial patterns (bands, spots, gradients) invisible to scalar averages. Additionally, we identified "cryptic phenotypes" (3.3%) where spatial patterns contradicted average colour, cases where colorimeters failed but Wavelets succeeded. The underlying principle-that spatial decomposition can recover organizational information lost by scalar averaging-may be applicable to other traits with spatial or temporal structure, such as marbling, dermatitis, or pigmentation rhythms, although whether comparable performance gains would be observed remains to be tested empirically. Hence, for breeding programs implementing genomic selection, computer vision-based digital phenotyping captures complex trait variation without massive training datasets, addressing the bottleneck that increasingly limits genetic progress as genotyping becomes trivial.

Wavelet transform↗

Genome-scale insights into metabolic streamlining and photosynthetic energy balance in the extremophile green alga Picocystis salinarum (Picocystophyceae, Chlorophyta).

Picocystis salinarum is an early-diverging chlorophyte and the sole described member of the Picocystophyceae, frequently dominating hypersaline and alkaline lakes despite extreme physicochemical constraints. To elucidate the genomic foundations of its ecological success, we generated a fully annotated, chromosome-scale nuclear genome assembly of the type strain originally isolated from a saline pond in San Francisco Bay. The 18.5-Mb genome comprises 30 chromosomal assemblies, exhibits clear diploidy, and contains multiple copies of intact Ty3/Gypsy and Ty1/Copia long terminal repeat retrotransposons encoding polyproteins with atypical accessory domains. Phylogenomic analyses reveal strong affinity with the Nephroselmidophyceae. Comparative analyses reveal extensive metabolic streamlining, including the absence of a queuosine salvage pathway, the 2-methylcitrate cycle, &#x3b2;-oxidation of propionate, and branched-chain amino acid catabolism, traits retained in several marine prasinophyte lineages. In contrast, the genome preserves multiple ancestral bacterial derived systems. Notably, P. salinarum features a complete chloroplast NADH dehydrogenase-like complex, including all membrane, electron binding, and assembly components, a configuration not previously reported in sequenced chlorophyte algae. This retention implies substantial capacity for cyclic electron flow and chlororespiration, processes expected to be critical in chronically low-light and chemically extreme environments. The genome further reveals a distinctive biochemical CO2-concentrating mechanism centered on plastid-targeted phosphoenolpyruvate carboxykinase, complete plastid peptidoglycan biosynthetic and remodeling pathways, and partial retention of lipid-A-related machinery. Conversely, P. salinarum lacks canonical non-photochemical quenching proteins while retaining xanthophyll-cycle enzymes that support slower photoprotective responses. Together, these features define a coordinated genomic architecture that underpins the specialization of P. salinarum to hypersaline, alkaline, and persistently low-light ecosystems.

3&#x2010;deoxy&#x2010;D&#x2010;manno&#x2010;octulo↗

A Mobile Glycosylation Locus Modulates Cell Wall Architecture in Lactobacillus crispatus.

Lactobacillus crispatus dominance in the vaginal microbiome is associated with beneficial health outcomes, yet strain-level variation and its implications remain poorly understood. Here, we resolve the genomic context of three glycosyltransferase gene fragments (GT1-3) previously linked with dysbiotic states. Long-read resequencing revealed that GT1-3 are part of a ~18.7&#x2009;kb Wzx/Wzy-dependent cell wall polysaccharide (CWPS) locus, containing several IS256-family transposases. Serial propagation in&#xa0;vitro produced isolates with 4.1&#x2009;kb excised via a composite transposon encompassing the GT3, UDP-galactopyranose mutase, flippase, and hypothetical protein, demonstrating structural plasticity. Transmission electron microscopy showed a ~20%-25% thinner peptidoglycan layer in the derived strains, while FT-IR and monosaccharide analysis indicated no gross changes. Molecular dynamics simulations suggest that GT3 contributes to the structural stability of the glycosyltransferase complex, without compromising catalytic function. Together, these findings establish the CWPS locus as a mobile, structurally plastic element that directly influences cell wall architecture in L. crispatus.

Lactobacillus crispatus↗

Dual Roles of RAD23b and RAD4 on the Desiccation Tolerance of Germinated Seeds.

Desiccation tolerance (DT) is a survival trait enabling orthodox seeds to withstand extremely low water content. While some protective factors are characterised, it remains mechanistically obscure. Here, based on PEG-induced DT re-establishment in germinated Brassica napus L. seeds, we investigated the dual functions of nucleotide excision repair (NER) components RAD23b and RAD4 in DNA repair and transcriptional regulation of root development. PEG pre-treatment alleviated dehydration-induced DNA damage and activated NER genes, suggesting the involvement of NER in seed DT. Unexpectedly, Arabidopsis atrad23b mutant and BnRAD23b/BnRAD4 over-expressing seeds all exhibited significantly decreased DT after dry back, which evoked a hypothesis that BnRAD23b-BnRAD4 functions beyond NER. Normally, BnRAD4 interacted with BnRAD23b and repressed the expression of root development genes NAC103, EMB1444, RRA1 by directly binding to STRE elements within their promoters. Dehydration stress alleviated this repression, drove transcriptional reprogramming and might redirect the complex to execute DNA repair. Genetic analyses revealed that germinated seeds of atnac103, atemb1444, and atrra1 single mutants all exhibited reduced DT, and double mutants under atrad23b background almost abolished DT. This study suggests that RAD23b and RAD4 may regulate DT re-establishment of germinated seeds through balancing genome integrity and radicle development, with implications for DT study broadly.

Brassica napus L.↗

Integrative Multi-Omics Analysis of Stem Growth Habit Divergence in Wild Soybean (Glycine soja).

Stem architecture is a major determinant of lodging resistance, biomass accumulation, and harvest efficiency in soybean. However, the molecular features associated with contrasting stem growth habits in wild soybean remain incompletely characterised. Here, we performed an integrated transcriptomic, metabolomic, and epigenomic analysis of stem growth-habit divergence in wild soybean, comparing the wild-type accession ZYD7068 with contrasting vining and erect mutant lines derived from carbon-ion beam mutagenesis. Pairwise transcriptomic comparisons identified between 20&#x2009;311 and 28&#x2009;705 differentially expressed genes per contrast, with a core set of 2672 genes consistently altered across the comparisons. Functional enrichment, gene set variation analysis, and gene set enrichment analysis converged on xylem and phloem pattern formation as a prominent molecular pathway associated with growth-habit divergence. Random forest analysis identified BBR-BPC and ARF transcription factor families as major molecular discriminators, while metabolomic profiling revealed distinct metabolic profiles involving amino-acid-derived and lipid-associated metabolites. Whole-genome bisulfite sequencing revealed context-specific DNA methylation differences, including substantial variation in CHG methylation among erect mutant lines. Integrated network and in silico perturbation analyses prioritised four candidate genes associated with vascular development for future functional validation. Together, these results provide a multi-layer molecular resource for investigating stem growth-habit divergence in G. soja and establish testable candidate pathways and genes for subsequent functional studies and soybean improvement.

glycine soja↗

Extensive and differential platinum chemotherapy mutagenesis in livers of children.

Childhood cancer survivors often experience late adverse effects that may be linked to chemotherapy mutagenesis. We studied chemotherapy mutagenesis in normal pediatric tissues using duplex sequencing (NanoSeq) to enable the detection of mutations from single DNA molecules. We found that platinum chemotherapeutics increased the mutation burdens of normal pediatric tissues to levels seen in adults. In the liver, platinum agents imparted a tissue-specific mutational signature that was absent from other tissues. Gene-focused duplex sequencing revealed that chemotherapy mutagenesis generates a great diversity of nonsynonymous variants, some of which may have functional potential, such as leukemogenic variants in blood. Our findings demonstrate extensive chemotherapy mutagenesis in normal tissues of children, which may provide a plausible link between chemotherapy exposure and adverse effects in later life.

Child↗

The complete telomere-to-telomere sequence of a mouse Y chromosome.

The mouse Y chromosome is essential for male reproduction, yet the GRCm39 reference contains 25 gaps, particularly in repetitive and complex regions. Here, we assembled a telomere-to-telomere Y chromosome (mT2T Y) of 95.21 Mb from a C57BL/6 mouse incorporating parental genomes. This assembly fills all gaps, corrects structural errors, and adds over 8.70 Mb of previously unassembled sequence to the reference genome. We annotated 142 previously unidentified genes, identified Y specific satellite arrays, and mapped homologous recombination loci in the pseudoautosomal region (PAR). Analysis of X Y homologous gene expression revealed a Y chromosome dosage compensation mechanism. By combining mT2T Y with T2T mhaESC, we completed the T2T assembly of all C57BL/6 chromosomes, designated T2T mhaESC+Y, providing a complete C57BL/6 reference genome.

Animals↗

Paleogenomics and habitat modeling reveal temperate Eurasian origins of woolly rhinoceroses.

The woolly rhinoceros was a prominent Ice Age megafaunal species, and there is limited knowledge regarding its origin and responses to past glacial cycles. We sequenced 29 mitochondrial and 14 nuclear genomes from Pleistocene specimens across Eurasia and modeled the species' habitats over the past 500,000 years. Our results suggest that its maternal genetic diversity mainly evolved in temperate Eurasia around 460 thousand to 420 thousand years ago during a prolonged glacial-interglacial transition. We found that a ~170-thousand-year-old East Asian individual was ancestral to all later populations, indicating East Asia as one possible origin of Late Pleistocene ancestry. We also identified the Altai region as a major climatic refugium. These findings highlight the crucial role of temperate Eurasia in the evolution of woolly rhinoceroses and the diversification of cold-adapted megafauna.

Animals↗

Manipulation of protein translation and stem cell self-renewal by CRISPR activation of rRNA transcription.

Ribosomal RNA (rRNA) transcription rates vary during development, and their dysregulation is linked to diseases such as cancer and ribosomopathies. Owing to their high abundance and genomic redundancy, the functional significance of rRNA levels remains unclear. We developed TAPIR (Targeted Activation of Protein Translation), a CRISPR-based approach to elevate rRNA levels by inducing 47S ribosomal DNA transcription. TAPIR increased nucleolar size and enhanced protein synthesis, even in rapidly proliferating cells. In neural stem cells, elevated translation promoted self-renewal and proliferation in vitro and in vivo. Furthermore, TAPIR enabled the modeling and partial rescue of associated disease phenotypes. Our findings reveal that rRNA levels directly regulate translational output and that protein synthesis capacity can act as a key determinant of mammalian stem cell behavior.

Animals↗

Noncoding transcription controls the developmental dynamics of long-range gene regulation.

The genomic regions regulating gene expression are often themselves transcribed into a variety of noncoding RNAs (ncRNAs). However, the regulatory roles of this noncoding transcription remain largely unknown. By using live imaging, we reveal that the sequential transcription of ncRNAs emanating from distinct regulatory elements underlies gene activation in Drosophila embryos. Single-allele co-visualization uncovers that optimal gene activation is achieved by only moderate levels of enhancer activity. Disrupting enhancer-associated ncRNAs causes precocious gene activation, providing evidence that ncRNAs control the timing of gene expression in development. We further show that enhancer transcription can regulate long-range interactions within complex regulatory landscapes. We propose that ncRNAs locally modulate regulatory element activity in cis to shape genome organization and orchestrate the temporal control of gene expression in development.

Journal Article↗

Repeated emergence and fitness heterogeneity of KPC-33 in ST11 Klebsiella pneumoniae under ceftazidime-avibactam pressure.

Ceftazidime-avibactam (CZA) is an important therapeutic option for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae. However, CZA exposure also selects for emergent KPC variants. Their in vivo evolutionary patterns, fitness consequences, and underlying molecular mechanisms remain unclear. We performed a longitudinal multiomics analysis of 35 clonally related ST11 KPC-producing K. pneumoniae isolates collected from eight hospitalized patients during clinical follow-up, most of whom had received CZA therapy. Whole-genome sequencing, antimicrobial susceptibility testing, in vitro competition assays, enzyme kinetic analysis, and transcriptomic sequencing were used to systematically characterize the within-host evolutionary dynamics of KPC variants and the fitness heterogeneity of KPC-33. Multiple KPC variants were identified during longitudinal follow-up, among which KPC-33 was the most frequently detected. Among the seven patients who received CZA treatment, KPC-33 was detected in longitudinal isolates from four patients. It was also identified in patient P3, who had not received CZA, whereas other variants were only sporadically identified. Biochemical analysis showed that KPC-33 exhibited an altered kinetic profile relative to KPC-2, characterized by reduced catalytic turnover and altered substrate affinity. KPC-33 did not exhibit a uniform and pronounced fitness defect but instead showed marked strain-dependent heterogeneity. Strains with higher competitive fitness generally showed only limited transcriptional changes, whereas those with lower fitness were accompanied by broader transcriptional remodeling. In this longitudinal cohort, KPC-33 was repeatedly detected, predominantly under CZA-associated selective conditions. Its fitness consequences were clearly strain background dependent and may be associated with the extent of transcriptional remodeling. These findings provide new evidence for understanding the in vivo evolution of CZA resistance.

KPC-33↗

Genomic characterization of the attenuated human cytomegalovirus strain TR-VAC developed for subviral particle vaccine production.

We report the complete genome sequence of the attenuated human cytomegalovirus strain TR-VAC, developed for subviral particle vaccine production. Oxford Nanopore duplex sequencing confirmed all engineered modifications, including UL130 repair, UL25 stop codons, ddFKBP insertion, GFP deletion, and retention of the bacterial artificial chromosome backbone, without large-scale structural rearrangements.

Human cytomegalovirus↗