LsRTDv1, a reference transcript dataset for accurate transcript-specific expression analysis in lettuce.

Accurate quantification of gene and transcript-specific expression, with the underlying knowledge of precise transcript isoforms, is crucial to understanding many biological processes. Analysis of RNA sequencing data has benefited from the development of alignment-free algorithms which enhance the precision and speed of expression analysis. However, such algorithms require a reference transcriptome. Here we generate a reference transcript dataset (LsRTDv1) for lettuce (cv. Saladin), combining long- and short-read sequencing with publicly available transcriptome annotations, and filtering to keep only transcripts with high-confidence splice junctions and transcriptional start and end sites. LsRTDv1 identifies novel genes (mostly long non-coding RNAs) and increases the number of transcript isoforms per gene in the lettuce genome from 1.4 to 2.7. We show that LsRTDv1 significantly increases the mapping rate of RNA-seq data from a lettuce time-series experiment (mock- and Botrytis cinerea-inoculated) and enables detection of genes that are differentially alternatively spliced in response to infection as well as transcript-specific expression changes. LsRTDv1 is a valuable resource for investigation of transcriptional and alternative splicing regulation in lettuce.

REVEILLE2 thermosensitive splicing: a molecular basis for the integration of nocturnal temperature information by the Arabidopsis circadian clock.

Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.

Discrimination of Multidrug Resistance in Cancer Cells Achieved Using Single-Cell Analysis.

The biophysical signatures of single cells, such as multidrug resistance (MDR), may easily change during their various disease states. Therefore, there is an ever-growing need for advanced methods to study and analyze the response of cancer cells to therapeutic intervention. To determine the cancer cells and responses to various cancer therapies, from a cell mortality perspective, we report a label-free and real-time method to monitor the in situ responses of ovarian cancer cells using a single-cell bioanalyzer (SCB). The SCB instrument was used to detect different ovarian cancer cells, such as NCI/ADR-RES cells, which are multidrug resistant (MDR), and non-MDR OVCAR-8 cells. The discrimination of ovarian cells has been achieved at the single-cell level by measuring drug accumulation quantitatively in real time, in which the accumulation is high in non-MDR single cells without drug efflux but is low in MDR single cells which are not efflux-free. The SCB was constructed as an inverted microscope for optical imaging and fluorescent measurement of a single cell that was retained in a microfluidic chip. The single ovarian cancer cell retained in the chip offered sufficient fluorescent signals for the SCB to measure the accumulation of daunorubicin (DNR) in the single cell in the absence of cyclosporine A (CsA). The same cell allows us to detect the enhanced drug accumulation due to MDR modulation in the presence of CsA, which is the MDR inhibitor. The measurement of drug accumulation in a cell was achieved after it was captured in the chip for one hour, with the correction of background interference. The detection of accumulation enhancement due to MDR modulation by CsA was determined in terms of either the accumulation rate or enhanced concentration of DNR in the single cell (same cell, p < 0.01). It showed that with the effectiveness of efflux blocking by CsA, the intracellular DNR concentration in a single cell increased by threefold against its same cell control. This single-cell bioanalyzer instrument has the ability to discriminate MDR in different ovarian cells due to drug efflux in them by eliminating the interference of background fluorescence and by using the same cell control.

ProtView: A Versatile Tool for In Silico Protease Evaluation and Selection in a Proteomic and Proteogenomic Context.

Many tools have been created to generate in silico proteome digests with different protease enzymes and provide useful information for selecting optimal digest schemes for specific needs. This can save on time and resources and generate insights on the observable proteome. However, there remains a need for a tool that evaluates digest schemes beyond protein and amino acid coverages in the proteomic domain. Here, we present ProtView, a versatile in silico protease combination digest evaluation workflow that maps in silico-digested peptides to both protein and genome references, so that the potential observable portions of the proteome, transcriptome, and genome can be identified. The proteomic identification and quantification of evidence for transcriptional, co-transcriptional, post-transcriptional, translational, and post-translational regulation can all be examined in silico with ProtView prior to an experiment. Benchmarking against biological data comparing multiple proteases shows that ProtView can correctly estimate performances among the digest schemes. ProtView provides this information in a way that is easy to interpret, allowing for digest schemes to be evaluated before carrying out an experiment, in context that can optimize both proteomic and proteogenomic experiments. ProtView is available at https://github.com/SSPuliasis/ProtView.

Effects of Graphene Oxide on Plant Growth: A Review.

Several reports of graphene oxide (GO) promoting plant growth have sparked interest in its potential applications in agroforestry. However, there are still some toxicity studies that have raised concerns about the biosafety of GO. These reports show conflicting results from different perspectives, such as plant physiology, biochemistry, cytology, and molecular biology, regarding the beneficial and detrimental effects of GO on plant growth. Seemingly inconsistent studies make it difficult to effectively apply GO in agroforestry. Therefore, it is crucial to review and analyze the current literature on the impacts of GO on plant growth and its physiological parameters. Here, the biological effects of GO on plant growth are summarized. It is proposed that an appropriate concentration of GO may be conducive to its positive effects, and the particle size of GO should be considered when GO is applied in agricultural applications. This review provides a comprehensive understanding of the effects of GO on plant growth to facilitate its safe and effective use.

Second near-infrared fluorescent dye for lateral flow immunoassays rapid detection of influenza A/B virus.

Sensitive and rapid diagnostic point of care testing (POCT) system is of great significance to prevent and control human virus infection. Here reported an immunochromatographic strip technology. The second near-infrared (NIR-II) fluorescent dye encapsulated into polystyrene (PS) nanoparticles, was integrated into a lateral flow assay platform to achieve excellent detection of influenza A/B. This surface-functionalized and mono-dispersed PS nanoparticles has been conjugated with influenza nucleoprotein monoclonal antibody as targets for influenza antigen-detection. This assay achieved the detection limit of 0.015 ng/mL for influenza A nucleoprotein and 4.3*10-5 HAU/mL (102.08 TCID50/mL) influenza A virus (influenza B: 0.037 ng/mL, 9.7*10-7 HAU/mL (100.43 TCID50/mL)). Compared with an Au-based lateral flow test strip, the strip's sensitivity is about 16-fold higher than it. Strip detection properties remain stable for 6 months under 4 °C to 30 °C storage. The assay's intra assay variation is 5.14% and the inter assay variation is 7.74%. Other potential endogenous and exogenous interfering substances (whole blood, nasal mucin, saliva, antipyretics, antihistamines and neuraminidase inhibitors) showed negative results, which verified the excellent specificity of this method. This assay was successfully applied to the POCT quantitative detection of influenza A/B virus, the sensitivity to influenza A and B viruses was 70% and 87.5% respectively, and the specificity was 100%. Therefore, these microspheres can be used as an effective material for rapid POCT detection in clinical specimens.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

BaRTv2: a highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification.

Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data, and enables detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short-read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high-resolution reverse transcriptase-polymerase chain reaction and 5'-RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.

The value of genotype-specific reference for transcriptome analyses in barley.

It is increasingly apparent that although different genotypes within a species share "core" genes, they also contain variable numbers of "specific" genes and different structures of "core" genes that are only present in a subset of individuals. Using a common reference genome may thus lead to a loss of genotype-specific information in the assembled Reference Transcript Dataset (RTD) and the generation of erroneous, incomplete or misleading transcriptomics analysis results. In this study, we assembled genotype-specific RTD (sRTD) and common reference-based RTD (cRTD) from RNA-seq data of cultivated Barke and Morex barley, respectively. Our quantitative evaluation showed that the sRTD has a significantly higher diversity of transcripts and alternative splicing events, whereas the cRTD missed 40% of transcripts present in the sRTD and it only has ∼70% accurate transcript assemblies. We found that the sRTD is more accurate for transcript quantification as well as differential expression analysis. However, gene-level quantification is less affected, which may be a reasonable compromise when a high-quality genotype-specific reference is not available.