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Abstract Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.

Abstract Breast cancer has become the most prevalent cancer, globally. Adriamycin is a first-line chemotherapeutic agent, however, cancer cells acquire resistance to it, which is one of the most common causes of treatment failure. ROS and NRF2 are essential oxidative stress factors that play a key role in the oxidative stress process and are associated with cancer. Our goal is to create novel therapeutic drugs or chemical sensitizers that will improve chemotherapy sensitivity. The optimal concentration and duration for MCF-7 and MCF-7/ADR cells in ADR and CYT were determined using the CCK-8 assay. We found that ADR + CYT inhibited the activity of MCF-7 and MCF-7/ADR cells in breast cancer, as well as causing apoptosis in MCF-7 and MCF-7/ADR cells and blocking the cell cycle in the G0/G1 phase. ADR + CYT induces apoptosis in MCF-7 and MCF-7/ADR cells through ROS generation and the P62/NRF2/HO-1 signaling pathway. In breast cancer-bearing nude mice, ADR + CYT effectively suppressed tumor development in vivo. Overall, our findings showed that CYT in combination with ADR has potent anti-breast cancer cell activity both in vivo and in vitro, suggesting CYT as the main drug used to improve chemosensitivity.

BackgroundInetetamab (cipterbin) is an innovative anti-HER2 humanized monoclonal antibody. The efficacy and safety of a combination of inetetamab and vinorelbine in the first-line treatment of human epidermal receptor positive (HER2+) metastatic breast cancer (MBC) have been confirmed. We aimed to investigate real-world data of inetetamab in complex clinical practice.MethodsWe retrospectively reviewed the medical records of patients who received inetetamab as a salvage treatment at any line setting from July 2020 to June 2022. The main endpoint was progression‐free survival (PFS).ResultsA total of 64 patients were included in this analysis. The median progression‐free survival (mPFS) was 5.6 (4.6–6.6) months. Of the patients, 62.5% received two or more lines of therapy before treatment with inetetamab. The most common chemotherapy and anti-HER2 regimens combined with inetetamab were vinorelbine (60.9%) and pyrotinib (62.5%), respectively. Patients treated with inetetamab plus pyrotinib plus vinorelbine benefited the most (p=0.048), with the mPFS of 9.3 (3.1–15.5) months and an objective response rate of 35.5%. For patients with pyrotinib pretreatment, inetetamab plus vinorelbine plus pyrotinib agents resulted in mPFS of 10.3 (5.2–15.4) months. Regimens (inetetamab plus vinorelbine plus pyrotinib vs. other therapeutic agents) and visceral metastases (yes vs. no) were independent predictors of PFS. Patients with visceral metastases treated with inetetamab plus vinorelbine plus pyrotinib had a mPFS of 6.1(5.1–7.1) months. The toxicity of inetetamab was tolerable, with the most common grade 3/4 adverse event being leukopenia (4.7%).ConclusionsHER2+ MBC patients pretreated with multiple-line therapies still respond to inetetamab-based treatment. Inetetamab combined with vinorelbine and pyrotinib may be the most effective treatment regimen, with a controllable and tolerable safety profile.

BackgroundBreast cancer (BC) is the most common malignant cancer. The prognosis of patients differs according to the location of distant metastasis, with pleura being a common metastatic site in BC. Nonetheless, clinical data of patients with pleural metastasis (PM) as the only distant metastatic site at initial diagnosis of metastatic BC (MBC) are limited.Patient cohort and methodsThe medical records of patients who were hospitalized in Shandong Cancer Hospital between January 1, 2012 and December 31, 2021 were reviewed, and patients eligible for the study were selected. Survival analysis was conducted using Kaplan–Meier (KM) method. Univariate and multivariate Cox proportional-hazards models were used to identify prognostic factors. Finally, based on these selected factors, a nomogram was constructed and validated.ResultsIn total, 182 patients were included; 58 (group A), 81 (group B), and 43 (group C) patients presented with only PM, only lung metastasis (LM), and PM combined with LM, respectively. The KM curves revealed no significant difference in overall survival (OS) among the three groups. However, in terms of survival after distant metastasis (M-OS), the difference was significant: patients with only PM exhibited the best prognosis, whereas those with PM combined with LM exhibited the worst prognosis (median M-OS: 65.9, 40.5, and 32.4 months, respectively; P = 0.0067). For patients with LM in groups A and C, those with malignant pleural effusion (MPE) exhibited significantly worse M-OS than those without MPE. Univariate and multivariate analyses indicated that primary cancer site, T stage, N stage, location of PM, and MPE were independent prognostic factors for patients with PM without other distant metastasis. A nomogram prediction model incorporating these variables was created. According to the C-index (0.776), the AUC values of the 3-, 5-, and 8-year M-OS (0.86, 0.86, and 0.90, respectively), and calibration curves, the predicted and actual M-OS were in good agreement.ConclusionBC patients with PM only at the first diagnosis of MBC exhibited a better prognosis than those with LM only or PM combined with LM. We identified five independent prognostic factors associated with M-OS in this subset of patients, and a nomogram model with good predictive efficacy was established.

Previous research has shown that miR398 contributed to plant thermotolerance by silencing its target gene COPPER/ZINC SUPEROXIDE DISMUTASE1 (CSD1) in Arabidopsis thaliana. However, the phylogenesis of miR398 and CSD1 in Brassica crop and their role in regulating leaf cell death under heat stress remains unexplored. Here, we characterized the homologous genes of miR398a and CSD1 in Brassica rapa ssp. pekinensis (Chinese cabbage) and found miR398a abundance was accumulated under heat stress (38 °C and 46 °C for 1 h) in Chinese cabbage, while the expression level of its targets BraCSD1-1 and BraCSD2-1 were downregulated. To further explore their role in heat response, we constructed the transgenic plants overexpressing artificial miR398a (aBra-miR398a), Bra-miR398a target mimic (Bra-MIM398a), and BraCSD1-1 in Chinese cabbage for genetic study. Under high temperatures, p35S::aBra-miR398a lines reduced the areas of leaf cell death and delayed the leaf cell death. By contrast, p35S::Bra-MIM398a and p35S::BraCSD1-1 plants enlarged the areas of leaf cell death and displayed the earliness of leaf cell death. Finally, we found that the expression level of stress-responsive genes BraLEA76, BraCaM1, BraPLC, BraDREB2A, and BraP5CS increased in transgenic plants overexpressing aBra-miR398a, which may contribute to their resistance to heat-induced leaf cell death. Taken together, these results revealed the function of Bra-miR398a in attenuating leaf cell death to ensure plant thermotolerance, indicating that the miR398-CSD1 module could be potential candidates for heat-resistant crop breeding.

Domain of unknown function (DUF) proteins constitute a great deal of families of functionally uncharacterized proteins in eukaryotes. The DUF966 gene family is found in monocotyledons, dicotyledons, mosses, and other species. However, little is known about the functions of DUF966 genes in wheat (Triticum aestivum L.). In this study, we identified and characterized the TaDUF966 gene family members in wheat by in silico analysis. A total of 28 TaDUF966 proteins were identified in wheat. Phylogenetic analysis divided these proteins into two groups (Groups I and II). Proteins in each group showed a highly conserved DUF966 domain and conserved motif distribution, implying their functional conservation. Analysis of gene expression profiling data showed that some TaDUF966 genes were induced by salt stress. We further confirmed the role of TaDUF966-9B in salt stress using virus induced gene silencing (VIGS) assay. Compared with the empty vector control, the TaDUF966-9B knockdown plants exhibited severe leaf curling at 10 days post-inoculation with BSMV under salt stress, suggesting that TaDUF966 genes play a vital role in salt stress tolerance in wheat. Taken together, these results expand our knowledge of the evolution of the DUF966 gene family in wheat and promote the potential application of these genes in wheat genetic improvement.

Class III homeodomain-leucine zipper (HD-ZIP III) genes encode plant-specific transcription factors that play pivotal roles in plant growth and development. There is no systematic report on HD-ZIP III members in Brassica plants and their responses to stress are largely unknown. In this study, a total of 10, 9 and 16 HD-ZIP III genes were identified from B. rapa, B. oleracea and B. napus, respectively. The phylogenetic analysis showed that HD-ZIP III proteins were grouped into three clades: PHB/PHV, REV and CNA/HB8. Genes in the same group tended to have similar exon–intron structures. Various phytohormone-responsive elements and stress-responsive elements were detected in the promoter regions of HD-ZIP III genes. Gene expression levels in different tissues, as well as under different stress conditions, were investigated using public transcription profiling data. The HD-ZIP III genes were constitutively expressed among all the tested tissues and were highly accumulated in root and stem. In B. rapa, only one BrREV gene especially responded to heat stress, BrPHB and BrREV members were downregulated upon cold stress and most HD-ZIP III genes exhibited divergent responses to drought stress. In addition, we investigated the genetic variation at known miR165/166 complementary sites of the identified HD-ZIP III genes and found one single nucleotide polymorphism (SNP) in PHB members and two SNPs in REV members, which were further confirmed using Sanger sequencing. Taken together, these results provide information for the genome-wide characterization of HD-ZIP III genes and their stress response diversity in Brassica species.

14-3-3 proteins are a large multigenic family of general regulatory factors (GRF) ubiquitously found in eukaryotes and play vital roles in the regulation of plant growth, development, and response to stress stimuli. However, so far, no comprehensive investigation has been performed in the hexaploid wheat. In the present study, A total of 17 potential 14-3-3 gene family members were identified from the Chinese Spring whole-genome sequencing database. The phylogenetic comparison with six 14-3-3 families revealed that the majority of wheat 14-3-3 genes might have evolved as an independent branch and grouped into ε and non-ε group using the phylogenetic comparison. Analysis of gene structure and motif indicated that 14-3-3 protein family members have relatively conserved exon/intron arrangement and motif composition. Physical mapping showed that wheat 14-3-3 genes are mainly distributed on chromosomes 2, 3, 4, and 7. Moreover, most 14-3-3 members in wheat exhibited significantly down-regulated expression in response to alkaline stress. VIGS assay and protein-protein interaction analysis further confirmed that TaGRF6-A positively regulated slat stress tolerance by interacting with a MYB transcription factor, TaMYB64. Taken together, our findings provide fundamental information on the involvement of the wheat 14-3-3 family in salt stress and further investigating their molecular mechanism.