How does ionizing radiation affect amyloidogenesis in plants?

PURPOSE: Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation of prion-like proteins in plant tissues. The object of this study, pea (Pisum sativum L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. MATERIALS AND METHODS: Pea seeds were irradiated in the dose range 5-50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins. RESULTS: We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more ß-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology. CONCLUSION: Our data hinted that protein accumulation stimulated seedlings' growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.

Multiplexed RNA-FISH-guided Laser Capture Microdissection RNA Sequencing Improves Breast Cancer Molecular Subtyping, Prognostic Classification, and Predicts Response to Antibody Drug Conjugates.

On a retrospective cohort of 1,082 FFPE breast tumors, we demonstrated the analytical validity of a test using multiplexed RNA-FISH-guided laser capture microdissection (LCM) coupled with RNA-sequencing (mFISHseq), which showed 93% accuracy compared to immunohistochemistry. The combination of these technologies makes strides in i) precisely assessing tumor heterogeneity, ii) obtaining pure tumor samples using LCM to ensure accurate biomarker expression and multigene testing, and iii) providing thorough and granular data from whole transcriptome profiling. We also constructed a 293-gene intrinsic subtype classifier that performed equivalent to the research based PAM50 and AIMS classifiers. By combining three molecular classifiers for consensus subtyping, mFISHseq alleviated single sample discordance, provided near perfect concordance with other classifiers (κ > 0.85), and reclassified 30% of samples into different subtypes with prognostic implications. We also use a consensus approach to combine information from 4 multigene prognostic classifiers and clinical risk to characterize high, low, and ultra-low risk patients that relapse early (< 5 years), late (> 10 years), and rarely, respectively. Lastly, to identify potential patient subpopulations that may be responsive to treatments like antibody drug-conjugates (ADC), we curated a list of 92 genes and 110 gene signatures to interrogate their association with molecular subtype and overall survival. Many genes and gene signatures related to ADC processing (e.g., antigen/payload targets, endocytosis, and lysosome activity) were independent predictors of overall survival in multivariate Cox regression models, thus highlighting potential ADC treatment-responsive subgroups. To test this hypothesis, we constructed a unique 19-feature classifier using multivariate logistic regression with elastic net that predicted response to trastuzumab emtansine (T-DM1; AUC = 0.96) better than either ERBB2 mRNA or Her2 IHC alone in the T-DM1 arm of the I-SPY2 trial. This test was deployed in a research-use only format on 26 patients and revealed clinical insights into patient selection for novel therapies like ADCs and immunotherapies and de-escalation of adjuvant chemotherapy.

Differential expression of flowering genes in Arabidopsis thaliana under chronic and acute ionizing radiation.

PURPOSE: Chronic and acute irradiations have drastic effects on flowering stage that plays an important role in further seed development and can determine seed yield. The expression of the key flowering genes, AP1, CO, GI, FT, FLC, and LFY, sensitive to irradiation repair gene RAD51 and the proliferation gene PCNA2 were studied in the wild-type Arabidopsis thaliana (Columbia ecotype) under chronic and acute irradiations. MATERIALS AND METHODS: Chronic irradiation was performed using the radioactive isotope 137СsCl in two total doses of 3 cGy and 17 cGy, with the dose rate of 10-7 cGy/s and 6.8 10-6 cGy/s, respectively. The plants were grown under chronic irradiation during 6 weeks, from seeds till the 6.3 stage of flowering. For acute exposure, the plants were X-ray irradiated one time at the 5.0 development stage (20 days old) by a total dose of 15 Gy with the dose rate of 89 cGy/s. RESULTS: After chronic irradiation with the 3 cGy dose the irradiated plants demonstrated 8 ± 2.8 days earlier flowering than in the control group. However, at the 17 cGy chronic and at the 15 Gy acute doses plants showed 14 ± 3.7 and 2 ± 1.4 days later flowering, respectively. The 3 cGy chronic exposure significantly increased the expression of the CO gene by a factor of 1.152 (1.087-1.217 95% C.I.) and decreased the expression of the FT gene by a factor of 0.128 (0.021-0.396 95% C.I.). The 17 cGy chronic exposure decreased expression of the AP1 gene by a factor of 0.872 (0.803-0.940 95% C.I.) and the LFY gene by a factor of 0.471 (0.306-0.687 95% C.I.). The 15 Gy acute exposure decreased the expression of the AP1 gene by a factor of 0.104 (0.074-0.144 95% C.I.) and the PCNA2 gene by a factor of 0.346 (0.238-0.488 95% C.I.). CONCLUSIONS: The increased expression of the CO gene and decreased expression of the AP1 and FT genes under the lower dose of chronic exposure were associated with earlier flowering. The acute exposure increased the expression of the PCNA2 gene and decreased the expression of the flowering genes, except AP1. The flowering was delayed under both the higher dose of chronic exposure and under acute exposure, but it was less affected by the latter. Presumably, it was related to the activation of DNA repair under the 3 cGy chronic and 15 Gy acute irradiations.