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Targeting‐induced local lesions in genomes (TILLING) is a powerful reverse‐genetics tool that enables high‐throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis‐based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)‐mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications. [ABSTRACT FROM AUTHOR]

Abstract: Hydrophilic poly((poly(ethylene glycol) methyl ether methacrylate) (P(PEGMA)) and poly(glycidylmethacrylate) (PGMA) brushes were grafted from chloromethylated polysulfone (CMPSF) membrane surfaces via surface-initiated atom transfer radical polymerization (ATRP). Prior to ATRP, chloromethylation of PSF was performed beforehand and the obtained CMPSF was prepared into porous membranes by phase inversion process. It was demonstrated that the benzyl chloride groups on the CMPSF membrane surface afforded effective macroinitiators to graft the well-defined polymer brushes. 1H NMR was employed to confirm the structure of CMPSF. The grafting yield of P(PEGMA) and PGMA was determined by weight gain measurement. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) confirmed the grafting of P(PEGMA) and PGMA chains. Water contact angle measurements indicated that the introduction of P(PEGMA) and PGMA graft chains promoted remarkably the surface hydrophilicity of PSF membranes. The effects of P(PEGMA) and PGMA immobilization on membrane morphology, permeability and fouling resistance were investigated. It was found that P(PEGMA) and PGMA grafts brought higher pure water flux, improved hydrophilic surface and better anti-protein absorption ability to PSF membranes after modification. And evidently, macromonomer P(PEGMA) brought much better properties to the PSF membranes than PGMA macromonomer. [Copyright &y& Elsevier]

In this paper, computer simulation was used to assist in the rapid preparation of Myclobutanil (MYC) molecularly imprinted nanoparticles (MYC-MINs), which involved screening the best functional monomers and solvents, determining the optimal polymerization ratio, and estimating the MYC-2-(Trifluoromethyl)acrylic acid (MYC-TFMAA) self-assembly process and its infrared spectrum. Thermodynamic analysis showed that MYC-TFMAA self-assembly was an endothermic non-spontaneous process, and the pre-polymerization temperature was planned to be 30 °C. The MYC-MINs, prepared by solution polymerization, had a uniform particle size (the average diameter of 134.26 nm, pore volume of 2.45 mL/g, specific surface area of 143.26 m2/g, and porosity of 78.27%). The polymer got to its dynamic adsorption equilibrium in 90min, and the saturated adsorption capacity was 4.78 mg/g. Repeated adsorption experiments proved that the MYC-MINs had stable physical and chemical properties, and a high specific adsorption capacity for MYC and its structural analogs as well. The separation imprinting factors of the polymer for MYC could reach to 2.31, and it was used as a filler in the solid phase extraction tube to purify the tobacco samples spiked with six triazole fungicide, and finally the extracts were analyzed by UHPLC-MS/MS. The average recovery range of spiked tobacco samples were 77.86%–100.52%, and relative standard deviation was 1.90%–10.60% (n = 3), which realized simultaneous detection of multiple triazole fungicides in tobacco. Image 1 • The Myclobutanil Molecularly Imprinted Nanoparticles was simulated by Gaussian. • The MYC-MINs had good adsorption recovery rates for four triazole fungicides. • The MYC-MINs had stable physicochemical properties and could be recycled 5 times. • The MYC-MINs SPE-UHPLC-MS/MS could simultaneously detect multi-triazole residues. [ABSTRACT FROM AUTHOR]

Competing Interests: Declarations. Conflict of interest: The authors disclose no conflicts of interest. Ethical approval: This article is categorized under “Image of the Month; therefore, ethical approval was waived in view of the retrospective nature of the study and the fact that all the procedures performed were part of routine care.

Background: Tissue biopsy is an integral part of the diagnostic approach to lung cancer. It is however invasive and limited by heterogeneity. Liquid biopsies may complement tissue testing by providing additional molecular information and may be particularly helpful in patients from whom obtaining sufficient tissue for genomic profiling is challenging., Methods: Patients with suspected lung cancer ( n = 71) were prospectively recruited. Blood and diagnostic tissue samples were collected within 48 h of each other. Plasma cell-free DNA (cfDNA) testing was done using an ultrasensitive amplicon-based next-generation sequencing (NGS) panel (plasma NGS testing). For cases diagnosed as non-small cell lung carcinoma (NSCLC) via histology or cytology, targeted testing for epidermal growth factor receptor ( EGFR ) mutations was performed using tissue biopsy samples (tissue EGFR testing), where available. Concordance of clinically actionable mutations between methods and sample types was assessed., Results: For confirmed NSCLC cases ( n = 54), tissue EGFR test results were available only for 70.3% (38/54) due to sample inadequacies, compared to blood samples for 98.1% (53/54) cases. Tissue EGFR testing identified sensitizing EGFR (L858R or exon 19 deletion) mutation in 31.6% (12/38) of cases. Plasma NGS identified clinically actionable mutations in 37.7% (20/53) of cases, including EGFR mutations in two cases with no tissue EGFR results, and mutations in KRAS, BRAF , and MET . The overall sensitivity of sensitizing EGFR mutation detection by plasma NGS was 75% (9/12), and specificity was 100% (25/25) in patients tested in both tissue EGFR and plasma NGS ( n = 37). In this cohort of patients, tissue EGFR testing alone informed clinical decisions in 22.2% (12/54) of cases. Adding plasma NGS to tissue EGFR testing increased the detection rate of actionable mutations to 42.6% (23/54), representing a 1.9-fold increase in clinically relevant findings. The average turnaround time of plasma NGS was shorter than standard tissue testing (10 vs. 29.9 days, p < 0.05)., Conclusions: In the first-line setting, plasma NGS was highly concordant with tissue EGFR testing. Plasma NGS increases the detection of actionable findings with a shorter time to results. This study outlines the clinical utility of complementary plasma mutation profiling in the routine management of lung cancer patients., Competing Interests: YC, M-HT, JP, and KN are employees of Lucence Diagnostics Pte Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Choudhury, Tan, Shi, Tee, Ngeow, Poh, Goh and Mong.)