Functional ex vivo DNA fibre assay to measure replication dynamics in breast cancer tissue.

Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = -0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = -0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Advancing Ki67 hotspot detection in breast cancer: a comparative analysis of automated digital image analysis algorithms.

AIM: Manual detection and scoring of Ki67 hotspots is difficult and prone to variability, limiting its clinical utility. Automated hotspot detection and scoring by digital image analysis (DIA) could improve the assessment of the Ki67 hotspot proliferation index (PI). This study compared the clinical performance of Ki67 hotspot detection and scoring DIA algorithms based on virtual dual staining (VDS) and deep learning (DL) with manual Ki67 hotspot PI assessment. METHODS: Tissue sections of 135 consecutive invasive breast carcinomas were immunohistochemically stained for Ki67. Two DIA algorithms, based on VDS and DL, automatically determined the Ki67 hotspot PI. For manual assessment; two independent observers detected hotspots and calculated scores using a validated scoring protocol. RESULTS: Automated hotspot detection and assessment by VDS and DL could be performed in 73% and 100% of the cases, respectively. Automated hotspot detection by VDS and DL led to higher Ki67 hotspot PIs (mean 39.6% and 38.3%, respectively) compared to manual consensus Ki67 PIs (mean 28.8%). Comparing manual consensus Ki67 PIs with VDS Ki67 PIs revealed substantial correlation (r = 0.90), while manual consensus versus DL Ki67 PIs demonstrated high correlation (r = 0.95). CONCLUSION: Automated Ki67 hotspot detection and analysis correlated strongly with manual Ki67 assessment and provided higher PIs compared to manual assessment. The DL-based algorithm outperformed the VDS-based algorithm in clinical applicability, because it did not depend on virtual alignment of slides and correlated stronger with manual scores. Use of a DL-based algorithm may allow clearer Ki67 PI cutoff values, thereby improving the clinical usability of Ki67.

Cu(II)-Catalyzed Enantioselective Aza-Friedel-Crafts Reaction of 1-Naphthols and Electron-Rich Phenols with Isatin-Derived Ketimines.

New chiral ligands could be obtained by introducing proline moieties and imidazoline moieties to binaphthyl skeletons. The chiral ligands exhibited balanced rigidity and flexibility which could allow the change of the conformations during the reactions on one hand, and could provide sufficient asymmetric induction on the other. The proline moiety could act as a linker connecting the binaphthyl skeletons and the imidazoline moieties as well as a coordinating group for the central metal, and the electronic and steric properties of the imidazoline groups could be carefully fine-tuned by the use of different substituents. In the presence of Cu(II) catalyst bearing such chiral ligands, aza-Friedel-Crafts reaction of 1-naphthols and electron-rich phenols with isatin-derived ketimines provided the desired products with good to excellent yields and up to 99 % ee. The reactions showed good scalability, and excellent ee could still be obtained when the reaction was carried out in gram-scale.

OsCAMTA3 Negatively Regulates Disease Resistance to Magnaporthe oryzae by Associating with OsCAMTAPL in Rice.

Rice (Oryza sativa) is one of the most important staple foods worldwide. However, rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, seriously affects the yield and quality of rice. Calmodulin-binding transcriptional activators (CAMTAs) play vital roles in the response to biotic stresses. In this study, we showed that OsCAMTA3 and CAMTA PROTEIN LIKE (OsCAMTAPL), an OsCAMTA3 homolog that lacks the DNA-binding domain, functioned together in negatively regulating disease resistance in rice. OsCAMTA3 associated with OsCAMTAPL. The oscamta3 and oscamtapl mutants showed enhanced resistance compared to wild-type plants, and oscamta3/pl double mutants showed more robust resistance to M. oryzae than oscamta3 or oscamtapl. An RNA-Seq analysis revealed that 59 and 73 genes, respectively, were differentially expressed in wild-type plants and oscamta3 before and after inoculation with M. oryzae, including OsALDH2B1, an acetaldehyde dehydrogenase that negatively regulates plant immunity. OsCAMTA3 could directly bind to the promoter of OsALDH2B1, and OsALDH2B1 expression was decreased in oscamta3, oscamtapl, and oscamta3/pl mutants. In conclusion, OsCAMTA3 associates with OsCAMTAPL to regulate disease resistance by binding and activating the expression of OsALDH2B1 in rice, which reveals a strategy by which rice controls rice blast disease and provides important genes for resistance breeding holding a certain positive impact on ensuring food security.

New Binaphthyl-Proline-Based Chiral Ligands Bearing Imidazoline Groups: Design, Synthesis, and Their Application in Enantioselective Conjugate Addition of 4-Hydroxycoumarin and Related Nucleophiles to ß,γ-Unsaturated α-Ketoesters.

This paper describes the design and application of new binaphthyl-proline-based chiral ligands bearing imidazoline functional groups. These chiral ligands incorporate the advantages of both the binaphthyl and proline skeletons, they are featured with regulatable electronic and steric properties for the imidazoline functional groups, and form chiral complexes with different metal salts such as cuprous acetate. In the presence of an appropriate amount of a chiral catalyst, enantioselective conjugate addition of 4-hydroxycoumarin or related nucleophiles to different ß,γ-unsaturated α-ketoesters proceeded readily, giving the desired products in high yield (up to 99%) and excellent enantiomeric excess (up to 99%).

Copper(II)-Catalyzed Enantioselective Aza-Friedel-Crafts Reaction of Indoles with Isatin-Derived N-Boc-Ketimines.

The copper(II)-catalyzed enantioselective aza-Friedel-Crafts reaction of indoles with isatin-derived N-Boc-ketimines was developed by using tunable chiral O-N-N tridentate ligands derived from BINOL and proline. In general, the reaction afforded chiral 3-indolyl-3-aminooxindoles under mild conditions in high yields (83-97%) with excellent ee (69-99%).

Enantioselective Friedel-Crafts Alkylation of Indoles with ß,γ-Unsaturated α-Ketoesters Catalyzed by New Copper(I) Catalysts.

New Cu(I) catalysts are effective in enantioselective Friedel-Crafts alkylation of a variety of indoles with different ß,γ-unsaturated α-ketoesters. A control study shows that such a catalyst system is less sensitive to air, and the reactions can be carried out without special cares such as glovebox operation or moisture/oxygen-free conditions. Preliminary computation results suggest that there exists π-π stacking between the substrate and the catalyst, and such an interaction seems to play a role in stabilizing the reaction intermediate and enhancing the stereoselectivity of the reactions. The desired products can be obtained in up to 98% yield at 99% enantiomeric excess. The same high enantioselectivity can be observed when the reaction is carried in a gram scale, indicating a good scalability of the catalyst system in enantioselective Friedel-Crafts alkylation of different indoles with ß,γ-unsaturated α-ketoesters.

Enantioselective Michael addition of malonates to ß,γ-unsaturated α-ketoesters catalysed by Cu(II) complexes bearing binaphthyl-proline hybrid ligands.

High yields (up to 96%) and high ee (up to 92%) were achieved for chiral copper(II) complex-catalysed enantioselective Michael addition of malonates to ß,γ-unsaturated-α-ketoesters. The chiral ligands took advantage of both the binaphthyl and the proline moieties, and substituents with different electronic and steric features could be tolerated. The reactions could be carried out under mild conditions, and a gram scale reaction could be realised without the loss of yield and enantioselectivity.

A novel immune subtype classification of ER-positive, PR-negative and HER2-negative breast cancer based on the genomic and transcriptomic landscape.

BACKGROUND: The diversity and plasticity behind ER+/PR-/HER2- breast cancer have not been widely explored. It is essential to identify heterogeneous microenvironment phenotypes and investigate specific genomic events driving the formation of these phenotypes. METHODS: Based on the immune-related gene expression profiles of 411 ER+/PR-/HER2- breast cancers in the METABRIC cohort, we used consensus clustering to identify heterogeneous immune subtypes and assessed their reproducibility in an independent meta-cohort including 135 patients collected from GEO database. We further analyzed the differences of cellular and molecular characteristics, and potential immune escape mechanism among immune subtypes. In addition, we constructed a transcriptional trajectory to visualize the distribution of individual patient. RESULTS: Our analysis identified and validated five reproducible immune subtypes with distinct cellular and molecular characteristics, potential immune escape mechanisms, genomic drivers, as well as clinical outcomes. An immune-cold subtype, with the least amount of lymphocyte infiltration, had a poorer prognosis. By contrast, an immune-hot subtype, which demonstrated the highest infiltration of CD8+ T cells, DCs and NK cells, and elevated IFN-γ response, had a comparatively favorable prognosis. Other subtypes showed more diverse gene expression and immune infiltration patterns with distinct clinical outcomes. Finally, our analysis revealed a complex immune landscape consisting of both discrete cluster and continuous spectrum. CONCLUSION: Overall, this study revealed five heterogeneous immune subtypes among ER+/PR-/HER2- breast cancer, also provided important implications for clinical translations.

Major Postoperative Complications in Esophageal Cancer After Minimally Invasive Esophagectomy Compared With Open Esophagectomy: An Updated Meta-analysis.

BACKGROUND: To evaluate the existing literature comparing cardiopulmonary complications after minimally invasive esophagectomy (MIE) with open esophagectomy (OE) and conduct a meta-analysis based on the relevant studies. METHODS: A systematic search for articles was performed in Medline, Embase, Wiley Online Library, and the Cochrane Library. The relative risks or odds ratios (ORs) were calculated by using fixed or random-effects models. The I2 and X2 tests were used to test for statistical heterogeneity. We performed a metaregression for the pulmonary complications with the adenocarcinoma proportion and tumor stage. Publication bias and small-study effects were assessed using Egger's test and Begg's funnel plot. RESULTS: A total of 30,850 participants were enrolled in the 63 studies evaluated in the meta-analysis. Arrhythmia, pulmonary embolism, pulmonary complications, gastric tip necrosis, anastomotic leakage, and vocal cord palsy were chosen as outcomes. The occurrence rate of arrhythmia was significantly lower in patients receiving MIE than in patients receiving OE (OR = 0.69; 95% CI = 0.53-0.89), with heterogeneity (I2 = 30.7%, P = 0.067). The incidence of pulmonary complications was significantly lower in patients receiving MIE (OR = 0.54, 95% CI = 0.45-0.63) but heterogeneity remained (I2 = 72.1%, P = 0.000). The risk of gastric tip necrosis (OR = 1.48, 95% CI = 1.07-2.05) after OE was lower than that after MIE. Anastomotic leakage, pulmonary embolism, and vocal cord palsy showed no significant differences between the two groups. CONCLUSIONS: MIE has advantages over OE, especially in reducing the incidence of arrhythmia and pulmonary complications. Thus, MIE can be recommended as the preferred alternative surgery method for resectable esophageal cancer.

Increased SIX-1 expression promotes breast cancer metastasis by regulating lncATB-miR-200s-ZEB1 axis.

Patients with advanced breast cancer (BC) showed a higher incidence of regional and distant metastases. Sine oculis homeobox homolog 1 (SIX-1) has been confirmed to be a key tumorigenic and metastatic regulator in BC progression. Yet, molecular mechanisms behind SIX-1-induced BC metastases remain largely unknown. Here we found that SIX-1 was frequently up-regulated in BC and correlated with poor outcomes when tested in human BC tissue microarray. Then, we manipulated the expression of SIX-1 by via shRNA-mediated knockdown and lentivirus-mediated overexpression. Transwell assay in vitro and lung metastases model of nude mice in vivo showed that SIX-1 promoted BC cell invasion and migration in vitro, and facilitated metastases in vivo. Mechanistically, SIX-1 could promote the transcription of lncATB, which exerts critical pro-metastatic role in BC by directly binding to the miR-200 family, especially for miR-200c, to induce EMT and promote metastases. In conclusion, SIX-1 exerts its pro-metastatic role in BC through lncATB/miR-200s axis of EMT signalling pathway and could act as an important diagnostic marker as well as a significant therapeutic target for clinically advanced BC.

Genetic analysis and gene mapping of the glabrous leaf and hull mutant glr3 in rice (Oryza sativa L.).

We obtained a glabrous leaf and hull mutant from a population of radiation mutagenesis of an indica rice cultivar R401. The mutant produced smooth leaves and hairless glumes under normal growth conditions. An F2 population was developed from a cross between a japonica cultivar Nipponbare and the glabrous leaf and hull mutant. By investigating the performance of the F2 population, we found that the mutant phenotype was controlled by a single recessive gene, temporarily designated GLR3. Bulked segregant analysis (BSA) based on the F2 mapping population revealed that GLR3 is located on chromosome 6. By analyzing 417 typical glabrous leaf F2 plants using molecular markers, GLR3 was mapped to a 0.2 cM interval between InDel markers ID27101 and ID27199, and the physical distance between the two markers is 98 kb. Thus we have mapped the gene GLR3, and our work will provide basis for future mechanistic analysis of GLR3 function.

Effect of 12-week SPARQ training on the ability of youth football players.

AIM: To evaluate the effect of a 12-week physical training regime comprising five components - speed, power, agility, reaction, quickness - on young football players' performance in terms of their capacity to move quickly in different phases. METHODS: The football players were randomly divided into an experimental group (n = 12) and a control group (n = 12). The rapid change of direction mobility was systematically investigated by using outdoor tests, the Illinois 505 Agility Test, the Square Pace Test, the Nebraska Agility Test, and the T-Shape Agility Test. RESULTS: With the intervention of training, considerable improvements were detected in all indicators, with the SPARQ training approach outperforming. CONCLUSION: The combined results indicate that SPARQ training can deliver favourable efficiency in terms of enhancing the agility of young football players.

PPM1D activity promotes the replication stress caused by cyclin E1 overexpression.

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

Effects of combined ultrasound and calcium ion pretreatments on polyphenols during mung bean germination: Exploring underlying mechanisms.

Mung beans were pretreated with a combination of ultrasonic and calcium ion to enhance the polyphenol content and antioxidant capacity during germination. Changes in polyphenol content and antioxidant capacity during germination, along with underlying mechanisms, were investigated. Both single ultrasound and combined ultrasound-Ca2+ pretreatments significantly increased the polyphenol content and enhanced the antioxidant capacity (p < 0.05) of mung beans depending on germination period. Among 74 polyphenolic metabolites identified in germinated mung beans, 50 were differential. Notably, 23 of these metabolites showed a significant positive correlation with antioxidant activity. Ultrasound pretreatment promoted flavonoid biosynthesis, whereas ultrasound-Ca2+ pretreatment favored the tyrosine synthesis pathway. Polyphenol composition and accumulation changes were mainly influenced by metabolic pathways like flavonoid, isoflavonoid, anthocyanin, and flavone/flavonol biosynthesis. The results suggest that ultrasound alone or combined with calcium ion pretreatments effectively enhance mung bean polyphenol content and antioxidant capacity during germination.

cGAS-STING pathway expression correlates with genomic instability and immune cell infiltration in breast cancer.

Genomic instability, as caused by oncogene-induced replication stress, can lead to the activation of inflammatory signaling, involving the cGAS-STING and JAK-STAT pathways. Inflammatory signaling has been associated with pro-tumorigenic features, but also with favorable response to treatment, including to immune checkpoint inhibition. In this study, we aim to explore relations between inflammatory signaling, markers of replication stress, and immune cell infiltration in breast cancer. Expression levels of cGAS-STING signaling components (STING, phospho-TBK1, and phospho-STAT1), replication stress markers (γH2AX and pRPA), replication stress-related proto-oncogenes (Cyclin E1 and c-Myc) and immune cell markers (CD20, CD4, and CD57) are determined immunohistochemically on primary breast cancer samples (n = 380). RNA-sequencing data from TCGA (n = 1082) and METABRIC (n = 1904) are used to calculate cGAS-STING scores. pTBK1, pSTAT1 expression and cGAS-STING pathway scores are all increased in triple-negative breast cancers compared to other subtypes. Expression of γH2AX, pRPA, Cyclin E1, c-Myc, and immune cell infiltration positively correlate with p-STAT1 expression (P < 0.001). Additionally, we observe significant positive associations between expression of pTBK1 and γH2AX, pRPA, c-Myc, and number of CD4+ cells and CD20+ cells. Also, cGAS-STING scores are correlated with genomic instability metrics, such as homologous recombination deficiency (P < 0.001) and tumor mutational burden (P < 0.01). Moreover, data from the I-SPY2 clinical trial (n = 71) confirms that higher cGAS-STING scores are observed in breast cancer patients who responded to immunotherapy combined with chemotherapy. In conclusion, the cGAS-STING pathway is highly expressed in TNBCs and is correlated with genomic instability and immune cell infiltration.

RAD52-dependent mitotic DNA synthesis is required for genome stability in Cyclin E1-overexpressing cells.

Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.

Effect of different milling methods on physicochemical and functional properties of mung bean flour.

There needs to be more information concerning the effect of different milling methods on the physicochemical properties of whole-grain mung bean flour. Therefore, the physicochemical properties of whole grain mung bean flour were analyzed using universal grinders (UGMB), ball mills (BMMB), and vibration mills (VMMB). The results showed that the particle size of the sample after ultrafine grinding treatment was significantly reduced to 21.34 µm (BMMB) and 26.55 µm (VMMB), and the specific surface area was increased. The particle distribution was uniform to a greater extent, and the color was white after treatment. Moreover, the water holding capacity (WHC), oil holding capacity (OHC), and swelling power (SP) increased, and the bulk density and solubility (S) decreased. The Rapid Viscosity Analyzer (RVA) indicated that the final viscosity of the sample after ultrafine grinding was high. Furthermore, rheological tests demonstrated that the consistency coefficient K, shear resistance, and viscosity were decreased. The results of functional experiments showed that the treated samples (BMMB and VMMB) increased their capacity for cation exchange by 0.59 and 8.28%, respectively, bile acid salt adsorption capacity increased from 25.56 to 27.27 mg/g and 26.38 mg/g, and nitrite adsorption capacity increased from 0.58 to 1.17 mg/g and 1.12 mg/g.

DPP-IV Inhibitory Peptides from Coix Seed Prolamins: Release, Identification, and Analysis of the Interaction between Key Residues and Enzyme Domains.

Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides can regulate type 2 diabetes by inhibiting the cleavage of glucagon-like peptide-1 and prolonging its half-life. The development of DPP-IV inhibitory peptides is still a hot topic. The primary structure of coix seed prolamins contains peptide sequence fragments that potentially inhibit DPP-IV; however, limited information is available regarding the extraction of peptides from coix seeds and the analysis of their conformational relationships. In this study, novel coix seed prolamin-derived peptides were obtained through single hydrolysis and double-enzyme stepwise hydrolysis. The inhibitory activity of these peptides against DPP-IV was evaluated to explore new functional properties of coix seeds. The results evidenced that the step-by-step enzymolysis (papain and alcalase) compared to single enzymolysis promoted the secondary structure disruption of the hydrolysates, enhanced the ß-turn structure, significantly increased the content of peptides below 1 kDa, and exhibited a substantial increase in DPP-IV inhibitory activity (97% inhibition). Three nontoxic DPP-IV inhibitory peptides, namely, LPFYPN, TFFPQ, and ATFFPQ (IC50 = 70.24, 176.87, 268.31 µM), were isolated and identified. All three peptides exhibited strong interactions with DPP-IV (all KA values >103). LPFYPN exhibited competitive inhibition, while TFFPQ and ATFFPQ demonstrated mixed competitive-noncompetitive inhibition. Hydrogen bonding and hydrophobic interactions were the main contributors to the coix seed prolamin peptides binding to DPP-IV. The central residue was a key amino acid in the parent peptide sequence, forming a more stable π-π stacking with residues in the active pocket, which may facilitate peptide activity. This study provides theoretical support for the development of coix seed-derived hypoglycemic peptides.

Zn(II)-catalysed enantioselective addition of alcohols and tert-butyl hydroperoxide to isatin-derived N-Boc ketimines.

Using binaphthyl-proline-based chiral ligands, Zn(II)-catalysed addition of alcohols and tert-butyl hydroperoxide to isatin-derived N-Boc ketimines provided the isatin-derived C3 N,O-aminals in up to 99% yield and up to 99% ee. The reactions could be carried out under mild conditions and a gram-scale reaction could be realized without the loss of the yield and enantioselectivity.