The value of machine learning based on CT radiomics in the preoperative identification of peripheral nerve invasion in colorectal cancer: a two-center study.

BACKGROUND: We aimed to explore the application value of various machine learning (ML) algorithms based on multicenter CT radiomics in identifying peripheral nerve invasion (PNI) of colorectal cancer (CRC). METHODS: A total of 268 patients with colorectal cancer who underwent CT examination in two hospitals from January 2016 to December 2022 were considered. Imaging and clinicopathological data were collected through the Picture Archiving and Communication System (PACS). The Feature Explorer software (FAE) was used to identify the peripheral nerve invasion of colorectal patients in center 1, and the best feature selection and classification channels were selected. Finally, the best feature selection and classifier pipeline were verified in center 2. RESULTS: The six-feature models using RFE feature selection and GP classifier had the highest AUC values, which were 0.610, 0.699, and 0.640, respectively. FAE generated a more concise model based on one feature (wavelet-HLL-glszm-LargeAreaHighGrayLevelEmphasis) and achieved AUC values of 0.614 and 0.663 on the validation and test sets, respectively, using the "one standard error" rule. Using ANOVA feature selection, the GP classifier had the best AUC value in a one-feature model, with AUC values of 0.611, 0.663, and 0.643 on the validation, internal test, and external test sets, respectively. Similarly, when using the "one standard error" rule, the model based on one feature (wave-let-HLL-glszm-LargeAreaHighGrayLevelEmphasis) achieved AUC values of 0.614 and 0.663 on the validation and test sets, respectively. CONCLUSIONS: Combining artificial intelligence and radiomics features is a promising approach for identifying peripheral nerve invasion in colorectal cancer. This innovative technique holds significant potential for clinical medicine, offering broader application prospects in the field. CRITICAL RELEVANCE STATEMENT: The multi-channel ML method based on CT radiomics has a simple operation process and can be used to assist in the clinical screening of patients with CRC accompanied by PNI. KEY POINTS: • Multi-channel ML in the identification of peripheral nerve invasion in CRC. • Multi-channel ML method based on CT-radiomics can detect the PNI of CRC. • Early preoperative identification of PNI in CRC is helpful to improve the formulation of treatment strategies and the prognosis of patients.

Chromosomal variations of Lycoris species revealed by FISH with rDNAs and centromeric histone H3 variant associated DNAs.

Lycoris species have various chromosome numbers and karyotypes, but all have a constant total number of chromosome major arms. In addition to three fundamental types, including metacentric (M-), telocentric (T-), and acrocentric (A-) chromosomes, chromosomes in various morphology and size were also observed in natural populations. Both fusion and fission translocation have been considered as main mechanisms leading to the diverse karyotypes among Lycoris species, which suggests the centromere organization playing a role in such arrangements. We detected several chromosomal structure changes in Lycoris including centric fusion, inversion, gene amplification, and segment deletion by using fluorescence in situ hybridization (FISH) probing with rDNAs. An antibody against centromere specific histone H3 (CENH3) of L. aurea (2n = 14, 8M+6T) was raised and used to obtain CENH3-associated DNA sequences of L. aurea by chromatin immunoprecipitation (ChIP) cloning method. Immunostaining with anti-CENH3 antibody could label the centromeres of M-, T-, and A-type chromosomes. Immunostaining also revealed two centromeres on one T-type chromosome and a centromere on individual mini-chromosome. Among 10,000 ChIP clones, 500 clones which showed abundant in L. aurea genome by dot-blotting analysis were FISH mapped on chromosomes to examine their cytological distribution. Five of these 500 clones could generate intense FISH signals at centromeric region on M-type but not T-type chromosomes. FISH signals of these five clones rarely appeared on A-type chromosomes. The five ChIP clones showed similarity in DNA sequences and could generate similar but not identical distribution patterns of FISH signals on individual chromosomes. Furthermore, the distinct distribution patterns of FISH signals on each chromosome generated by these five ChIP clones allow to identify individual chromosome, which is considered difficult by conventional staining approaches. Our results suggest a different organization of centromeres of the three chromosome types in Lycoris species.

Trithorax Group Proteins Act Together with a Polycomb Group Protein to Maintain Chromatin Integrity for Epigenetic Silencing during Seed Germination in Arabidopsis.

Polycomb group (PcG) and trithorax group (trxG) proteins have been shown to act antagonistically to epigenetically regulate gene expression in eukaryotes. The trxG proteins counteract PcG-mediated floral repression in Arabidopsis, but their roles in other developmental processes are poorly understood. We investigated the interactions between the trxG genes, ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) and ULTRAPETALA1 (ULT1), and the PcG gene EMBRYONIC FLOWER 1 (EMF1) during early development. Unexpectedly, we found that mutations in the trxG genes failed to rescue the early-flowering phenotype of emf1 mutants. Instead, emf1 atx1 ult1 seedlings showed a novel swollen root phenotype and massive deregulation of gene expression. Greater ectopic expression of seed master regulatory genes in emf1 atx1 ult1 triple than in emf1 single mutants indicates that PcG and trxG factors together repress seed gene expression after germination. Furthermore, we found that the widespread gene derepression is associated with reduced levels of H3K27me3, an epigenetic repressive mark of gene expression, and with globally altered chromatin organization. EMF1, ATX1, and ULT1 are able to bind the chromatin of seed genes and ULT1 can physically interact with ATX1 and EMF1, suggesting that the trxG and EMF1 proteins directly associate at target gene loci for EMF1-mediated gene silencing. Thus, while ATX1, ULT1, and EMF1 interact antagonistically to regulate flowering, they work together to maintain chromatin integrity and prevent precocious seed gene expression after germination.

Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene.

Identification of single nucleotide polymorphism markers associated with resistance to bruchids (Callosobruchus spp.) in wild mungbean (Vigna radiata var. sublobata) and cultivated V. radiata through genotyping by sequencing and quantitative trait locus analysis.

BACKGROUND: Bruchid beetles are an important storage pest of grain legumes. Callosobruchus sp. infect mungbean (Vigna radiata) at low levels in the field, multiply during grain storage and can destroy seed stocks in a few months. Resistance against bruchid beetles has been found in wild mungbean V. radiata var. sublobata TC1966 and in cultivated mungbean line V2802. RESULTS: Bruchid resistance data were obtained from recombinant inbred line populations TC1966 (V. radiata var. sublobata) × NM92 (F12) and V2802 (V. radiata) × NM94 (F7). More than 6,000 single nucleotide polymorphic markers were generated through genotyping by sequencing (GBS) for each of these populations and were used to map bruchid resistance genes. One highly significant quantitative trait locus (QTL) associated with bruchid resistance was mapped to chromosome 5 on genetic maps of both populations, suggesting that TC1966 and V2802 contain the same resistance locus. Co-segregation of all markers associated with resistance indicated the presence of only one major resistance QTL on chromosome 5, while QTL analysis based on physical map positions of the markers suggested the presence of multiple QTLs on different chromosomes. The diagnostic capacity of the identified molecular markers located in the QTL to correctly predict resistance was up to 100 %. CONCLUSIONS: Molecular markers tightly linked to bruchid resistance loci of two different mungbean resistance sources were developed and validated. These markers are highly useful for developing resistant lines.

Genomic and transcriptomic comparison of nucleotide variations for insights into bruchid resistance of mungbean (Vigna radiata [L.] R. Wilczek).

BACKGROUND: Mungbean (Vigna radiata [L.] R. Wilczek) is an important legume crop with high nutritional value in South and Southeast Asia. The crop plant is susceptible to a storage pest caused by bruchids (Callosobruchus spp.). Some wild and cultivated mungbean accessions show resistance to bruchids. Genomic and transcriptomic comparison of bruchid-resistant and -susceptible mungbean could reveal bruchid-resistant genes (Br) for this pest and give insights into the bruchid resistance of mungbean. RESULTS: Flow cytometry showed that the genome size varied by 61 Mb (mega base pairs) among the tested mungbean accessions. Next generation sequencing followed by de novo assembly of the genome of the bruchid-resistant recombinant inbred line 59 (RIL59) revealed more than 42,000 genes. Transcriptomic comparison of bruchid-resistant and -susceptible parental lines and their offspring identified 91 differentially expressed genes (DEGs) classified into 17 major and 74 minor bruchid-resistance-associated genes. We found 408 nucleotide variations (NVs) between bruchid-resistant and -susceptible lines in regions spanning 2 kb (kilo base pairs) of the promoters of 68 DEGs. Furthermore, 282 NVs were identified on exons of 148 sequence-changed-protein genes (SCPs). DEGs and SCPs comprised genes involved in resistant-related, transposable elements (TEs) and conserved metabolic pathways. A large number of these genes were mapped to a region on chromosome 5. Molecular markers designed for variants of putative bruchid-resistance-associated genes were highly diagnostic for the bruchid-resistant genotype. CONCLUSIONS: In addition to identifying bruchid-resistance-associated genes, we found that conserved metabolism and TEs may be modifier factors for bruchid resistance of mungbean. The genome sequence of a bruchid-resistant inbred line, candidate genes and sequence variations in promoter regions and exons putatively conditioning resistance as well as markers detecting these variants could be used for development of bruchid-resistant mungbean varieties.

Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymnosperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

Previously, we showed improved shelf life for agrobacterial isopentenyltransferase (ipt) transgenic broccoli (Brassica oleracea var. italica), with yield comparable to commercial varieties, because of the protection mechanism offered by molecular chaperones and stress-related proteins. Here, we used proximate analysis to examine macronutrients, chemical and mineral constituents as well as anti-nutrient and protein changes of ipt-transgenic broccoli and corresponding controls. We also preliminarily assessed safety in mice. Most aspects were comparable between ipt-transgenic broccoli and controls, except for a significant increase in carbohydrate level and a decrease in magnesium content in ipt-transgenic lines 101, 102 and 103, as compared with non-transgenic controls. In addition, the anti-nutrient glucosinolate content was increased and crude fat content decreased in inbred control 104 and transgenic lines as compared with the parental control, "Green King". Gel-based proteomics detected more than 50 protein spots specifically found in ipt-transgenic broccoli at harvest and after cooking; one-third of these proteins showed homology to potential allergens that also play an important role in plant defense against stresses and senescence. Mice fed levels of ipt-transgenic broccoli mimicking the 120 g/day of broccoli eaten by a 60-kg human adult showed normal growth and immune function. In conclusion, the compositional and proteomic changes attributed to the transgenic ipt gene did not affect the growth and immune response of mice under the feeding regimes examined.

Proteomics and transcriptomics of broccoli subjected to exogenously supplied and transgenic senescence-induced cytokinin for amelioration of postharvest yellowing.

Previously, we investigated transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and mimics the action of exogenous supplied CK in delaying postharvest senescence of broccoli. Here, we used proteomics and transcriptomics to compare the mechanisms of ipt-transgenic and N(6)-benzylaminopurine (BA) CK treatment of broccoli during postharvest storage. The 2 treatments conferred common and distinct mechanisms. BA treatment decreased the quantity of proteins involved in energy and carbohydrate metabolism and amino acid metabolism, and ipt-transgenic treatment increased that of stress-related proteins and molecular chaperones and slightly affected levels of carbohydrate metabolism proteins. Both treatments regulated genes involved in CK signaling, sugar transport, energy and carbohydrate metabolism, amino acid metabolism and lipid metabolism, although ipt-transgenic treatment to a lesser extent. BA treatment induced genes encoding molecular chaperones, whereas ipt-transgenic treatment induced stress-related genes for cellular protection during storage. Both BA and ipt-transgenic treatments acted antagonistically on ethylene functions. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. BIOLOGICAL SIGNIFICANCE: Transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and N(6)-benzylaminopurine (BA) CK treated broccoli both showed retardation of postharvest senescence during storage. The mechanisms underlying the two treatments were compared. The combination of proteomic and transcriptomic evidences revealed that the 2 treatments conferred common and distinct mechanisms in delaying senescence of broccoli florets. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. This article is part of a Special Issue entitled: Translational Plant Proteomics.

EMBRYONIC FLOWER1 and ULTRAPETALA1 Act Antagonistically on Arabidopsis Development and Stress Response.

Epigenetic regulation of gene expression is of fundamental importance for eukaryotic development. EMBRYONIC FLOWER1 (EMF1) is a plant-specific gene that participates in Polycomb group-mediated transcriptional repression of target genes such as the flower MADS box genes AGAMOUS, APETALA3, and PISTILLATA. Here, we investigated the molecular mechanism underlying the curly leaf and early flowering phenotypes caused by reducing EMF1 activity in the leaf primordia of LFYasEMF1 transgenic plants and propose a combined effect of multiple flower MADS box gene activities on these phenotypes. ULTRAPETALA1 (ULT1) functions as a trithorax group factor that counteracts Polycomb group action in Arabidopsis (Arabidopsis thaliana). Removing ULT1 activity rescues both the abnormal developmental phenotypes and most of the misregulated gene expression of LFYasEMF1 plants. Reducing EMF1 activity increases salt tolerance, an effect that is diminished by introducing the ult1-3 mutation into the LFYasEMF1 background. EMF1 is required for trimethylating lysine-27 on histone 3 (H3K27me3), and ULT1 associates with ARABIDOPSIS TRITHORAX1 (ATX1) for trimethylating lysine-3 on histone 4 (H3K4me3) at flower MADS box gene loci. Reducing EMF1 activity decreases H3K27me3 marks and increases H3K4me3 marks on target gene loci. Removing ULT1 activity has the opposite effect on the two histone marks. Removing both gene activities restores the active and repressive marks to near wild-type levels. Thus, ULT1 acts as an antirepressor that counteracts EMF1 action through modulation of histone marks on target genes. Our analysis indicates that, instead of acting as off and on switches, EMF1 and ULT1 mediate histone mark deposition and modulate transcriptional activities of the target genes.

Molecular and functional characterization of broccoli EMBRYONIC FLOWER 2 genes.

Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development.

Structural and functional assays of AtTLP18.3 identify its novel acid phosphatase activity in thylakoid lumen.

The membrane protein AtTLP18.3 of Arabidopsis (Arabidopsis thaliana) contains a domain of unknown function, DUF477; it forms a polysome with photosynthetic apparatuses in the thylakoid lumen. To explore the molecular function of AtTLP18.3, we resolved its crystal structures with residues 83 to 260, the DUF477 only, and performed a series of biochemical analyses to discover its function. The gene expression of AtTLP18.3 followed a circadian rhythm. X-ray crystallography revealed the folding of AtTLP18.3 as a three-layer sandwich with three α-helices in the upper layer, four ß-sheets in the middle layer, and two α-helices in the lower layer, which resembles a Rossmann fold. Structural comparison suggested that AtTLP18.3 might be a phosphatase. The enzymatic activity of AtTLP18.3 was further confirmed by phosphatase assay with various substrates (e.g. p-nitrophenyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, O-phospho-L-serine, and several synthetic phosphopeptides). Furthermore, we obtained the structure of AtTLP18.3 in complex with O-phospho-L-serine to identify the binding site of AtTLP18.3. Our structural and biochemical studies revealed that AtTLP18.3 has the molecular function of a novel acid phosphatase in the thylakoid lumen. DUF477 is accordingly renamed the thylakoid acid phosphatase domain.

Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence.

Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days' post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

Constitutive components and induced gene expression are involved in the desiccation tolerance of Selaginella tamariscina.

Selaginella tamariscina, one of the most primitive vascular plants, can remain alive in a desiccated state and resurrect when water becomes available. To evaluate the nature of desiccation tolerance in this plant, we compared the composition of soluble sugars and saturation ratios of phospholipids (PLs) between hydrated and desiccated tissues of S. tamariscina using gas chromatography. In this study, differences in gene expression and ABA contents were also analyzed during dehydration. The results revealed that trehalose (at >130 mg g(-1) DW) was the major soluble sugar, and low saturated fatty acid content in PLs (0.31) was maintained in both hydrated and desiccated tissues. In addition, the ABA content of S. tamariscina increased 3-fold, and genes involved in ABA signaling and cellular protection were up-regulated while photosystem-related genes were down-regulated during dehydration. The biochemical and molecular findings suggest that both constitutive and inducible protective molecules contribute to desiccation tolerance of S. tamariscina.

The wheat TaGI1, involved in photoperiodic flowering, encodes an Arabidopsis GI ortholog.

Wheat (Triticum aestivum L.) is an important crop and requires long day and short night to flower. To study the molecular mechanism of photoperiodic regulation of flowering in this species, we isolated a wheat TaGI1 gene, an ortholog of GIGANTEA (GI) in Arabidopsis. RNA blot hybridization revealed that TaGI1 is expressed in leaves in a rhythmic manner under long day and short day conditions and its rhythmic expression is regulated by photoperiods and circadian clocks. Further study demonstrated that the TaGI1 rhythmic expression in the leaves of seedlings is initiated by photoperiods, implying that TaGI1 does not show circadian regulation until after being entrained in a light/dark cycle. Interestingly, TaGI1 mRNA was detected in adaxial epidermal cells right above the vascular bundles of leaves, suggesting that the localization of TaGI1 transcripts in leaves may function to regulate flowering in response to photoperiods. Since overexpression of TaGI1 altered flowering time in wild type and complemented the gi mutant in Arabidopsis, it confirmed that TaGI1 is an ortholog of GI in Arabidopsis.