m-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides.

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.

Tlr4 Deletion Modulates Cytokine and Extracellular Matrix Expression in Chronic Spinal Cord Injury, Leading to Improved Secondary Damage and Functional Recovery.

Toll-like receptors (TLRs) play an important role in the innate immune response after CNS injury. Although TLR4 is one of the best characterized, its role in chronic stages after spinal cord injury (SCI) is not well understood. We examined the role of TLR4 signaling in injury-induced responses at 1 d, 7 d, and 8 weeks after spinal cord contusion injury in adult female TLR4 null and wild-type mice. Analyses include secondary damage, a range of transcriptome and protein analyses of inflammatory, cell death, and extracellular matrix (ECM) molecules, as well as immune cell infiltration and changes in axonal sprouting and locomotor recovery. Lack of TLR4 signaling results in reduced neuronal and myelin loss, reduced activation of NFκB, and decreased expression of inflammatory cytokines and necroptotic cell death pathway at a late time point (8 weeks) after injury. TLR4 null mice also showed reduction of scar-related ECM molecules at 8 weeks after SCI, accompanied by increase in ECM molecules associated with perineuronal nets, increased sprouting of serotonergic fibers, and improved locomotor recovery. These findings reveal novel effects of TLR4 signaling in chronic SCI. We show that TLR4 influences inflammation, cell death, and ECM deposition at late-stage post-injury when secondary injury processes are normally considered to be over. This highlights the potential for late-stage targeting of TLR4 as a potential therapy for chronic SCI.

Developmental stage-specific proteome analysis of the legume pod borer Maruca vitrata provides insights on relevant proteins.

The spotted pod borer, Maruca vitrata (Lepidoptera: Crambidae) is a destructive insect pest that inflicts significant productivity losses on important leguminous crops. Unravelling insect proteomes is vital to comprehend their fundamental molecular mechanisms. This research delved into the proteome profiles of four distinct stages -three larval and pupa of M. vitrata, utilizing LC-MS/MS label-free quantification-based methods. Employing comprehensive proteome analysis with fractionated datasets, we mapped 75 % of 3459 Drosophila protein orthologues out of which 2695 were identified across all developmental stages while, 137 and 94 were exclusive to larval and pupal stages respectively. Cluster analysis of 2248 protein orthologues derived from MaxQuant quantitative dataset depicted six clusters based on expression pattern similarity across stages. Consequently, gene ontology and protein-protein interaction network analyses using STRING database identified cluster 1 (58 proteins) and cluster 6 (25 proteins) associated with insect immune system and lipid metabolism. Furthermore, qRT-PCR-based expression analyses of ten selected proteins-coding genes authenticated the proteome data. Subsequently, functional validation of these chosen genes through gene silencing reduced their transcript abundance accompanied by a marked increase in mortality among dsRNA-injected larvae. Overall, this is a pioneering study to effectively develop a proteome atlas of M. vitrata as a potential resource for crop protection programs.

Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature.

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

prolfqua: A Comprehensive R-Package for Proteomics Differential Expression Analysis.

Mass spectrometry is widely used for quantitative proteomics studies, relative protein quantification, and differential expression analysis of proteins. There is a large variety of quantification software and analysis tools. Nevertheless, there is a need for a modular, easy-to-use application programming interface in R that transparently supports a variety of well principled statistical procedures to make applying them to proteomics data, comparing and understanding their differences easy. The prolfqua package integrates essential steps of the mass spectrometry-based differential expression analysis workflow: quality control, data normalization, protein aggregation, statistical modeling, hypothesis testing, and sample size estimation. The package makes integrating new data formats easy. It can be used to model simple experimental designs with a single explanatory variable and complex experiments with multiple factors and hypothesis testing. The implemented methods allow sensitive and specific differential expression analysis. Furthermore, the package implements benchmark functionality that can help to compare data acquisition, data preprocessing, or data modeling methods using a gold standard data set. The application programmer interface of prolfqua strives to be clear, predictable, discoverable, and consistent to make proteomics data analysis application development easy and exciting. Finally, the prolfqua R-package is available on GitHub https://github.com/fgcz/prolfqua, distributed under the MIT license. It runs on all platforms supported by the R free software environment for statistical computing and graphics.

The haplotype-resolved chromosome pairs of a heterozygous diploid African cassava cultivar reveal novel pan-genome and allele-specific transcriptome features.

BACKGROUND: Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome. FINDINGS: Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present 2 chromosome-scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. With consensus accuracy >QV46, contig N50 >18 Mb, BUSCO completeness of 99%, and 35k phased gene loci, it is the most accurate, continuous, complete, and haplotype-resolved cassava genome assembly so far. Ab initio gene prediction with RNA-seq data and Iso-Seq transcripts identified abundant novel gene loci, with enriched functionality related to chromatin organization, meristem development, and cell responses. During tissue development, differentially expressed transcripts of different haplotype origins were enriched for different functionality. In each tissue, 20-30% of transcripts showed allele-specific expression (ASE) differences. ASE bias was often tissue specific and inconsistent across different tissues. Direction-shifting was observed in <2% of the ASE transcripts. Despite high gene synteny, the HiFi genome assembly revealed extensive chromosome rearrangements and abundant intra-genomic and inter-genomic divergent sequences, with large structural variations mostly related to LTR retrotransposons. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding. CONCLUSIONS: The phased and annotated chromosome pairs allow a systematic view of the heterozygous diploid genome organization in cassava with improved accuracy, completeness, and haplotype resolution. They will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy, and continuity.

Association of proteomic markers with nutritional risk and response to nutritional support: A secondary pilot study of the EFFORT trial using an untargeted proteomics approach.

BACKGROUND: By means of a structured nutritional support intervention, EFFORT showed a risk reduction for adverse events in medical in-patients. We were interested in the prognostic and therapeutic potential of an untargeted proteomics approach to understand response to nutritional support, risk of 30-day mortality, and distinct patterns in severity of malnutrition risk as assessed by the Nutritional Risk screening (NRS 2002), respectively. METHODS: From 2,088 patients, we randomly took 120 blood samples drawn before treatment initiation on day 1 after hospital admission. Cases were selected by treatment allocation (nutritional support vs. usual nutrition), NRS 2002, and mortality at 30 days, but not on disease type. We measured proteins by untargeted liquid chromatography mass spectrometry (LC-MS/MS). RESULTS: We found 242 distinct proteins in 120 patients of which 81 (67.5%) survived until day 30. Between group analysis revealed a slight difference between the treatment groups in patients with a NRS 3, but not in those with a higher NRS. C-statistic between non-survivors and survivors at day 30 ranged from 0.60 (95% confidence interval 0.34-0.78) for a combination of 3 proteins/predictors to 0.65 (95% CI 0.53-0.78) for a combination of 32 proteins/predictors. In nutritional support non-survivors, pathway analysis found significant enrichment in pathways for signal transduction, platelet function, immune system regulation, extracellular matrix organization, and integrin cell surface interactions compared to survivors. CONCLUSION: Within this pilot study using an untargeted proteomics approach, there was only little prognostic and therapeutic potential of proteomics for phenotyping the risk of malnutrition and response to nutritional therapy. The small sample size and high heterogeneity of our population regarding comorbidity burden calls for more targeted approaches in more homogenous populations to understand the true potential of proteomics for individualizing nutritional care. TRIAL REGISTRATION: This is a pre-planned secondary analysis of the EFFORT trial (ClinicalTrials.gov NCT02517476).

Mass Spectrometry in Proteomics: Technologies, Methods, and Research Applications for the Life Sciences.

Mass spectrometry is a powerful tool in the hand of life science researchers, who constantly develop and apply new methods for the investigation of biomolecules, such as proteins, peptides, metabolites, lipids, and glycans. In this review, we will discuss the importance of mass spectrometry for the life science sector, with a special focus on the most relevant current applications in the field of proteomics. Moreover, we will comment on the factors that research groups should consider when setting up a mass spectrometry laboratory, and on the fundamental role played by academic core facilities and industrial service providers.

Quantification of the spread of SARS-CoV-2 variant B.1.1.7 in Switzerland.

BACKGROUND: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021). AIM: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland. METHODS: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale. RESULTS: We estimate B.1.1.7 had a transmission fitness advantage of 43-52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021. CONCLUSION: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.

Translational Proteomics Analysis of Anthracycline-Induced Cardiotoxicity From Cardiac Microtissues to Human Heart Biopsies.

Anthracyclines, including doxorubicin, idarubicin, and epirubicin, are common antitumor drugs as well as well-known cardiotoxic agents. This study analyzed the proteomics alteration in cardiac tissues caused by these 3 anthracyclines analogs. The in vitro human cardiac microtissues were exposed to drugs in 2 weeks; the proteomic data were measured at 7 time points. The heart biopsy data were collected from heart failure patients, in which some patients underwent anthracycline treatment. The anthracyclines-affected proteins were separately identified in the in vitro and in vivo dataset using the WGCNA method. These proteins engage in different cellular pathways including translation, metabolism, mitochondrial function, muscle contraction, and signaling pathways. From proteins detected in 2 datasets, a protein-protein network was established with 4 hub proteins, and 7 weighted proteins from both cardiac microtissue and human biopsies data. These 11 proteins, which involve in mitochondrial functions and the NF-κB signaling pathway, could provide insights into the anthracycline toxic mechanism. Some of them, such as HSPA5, BAG3, and SH3BGRL, are cardiac therapy targets or cardiotoxicity biomarkers. Other proteins, such as ATP5F1B and EEF1D, showed similar responses in both the in vitro and in vivo data. This suggests that the in vitro outcomes could link to clinical phenomena in proteomic analysis.

An improved draft genome assembly of Meloidogyne graminicola IARI strain using long-read sequencing.

The rice root-knot nematode Meloidogyne graminicola is a major biotic stress for the rice crop under upland, rain-fed lowland and irrigated cultivation conditions. Here, we present an improved draft genome assembly of M. graminicola IARI strain using the long-read sequencing approach (PacBio Sequel platform). The assembled genome size was 36.86 Mb with 514 contigs and N50 value of 105 kb. BUSCO estimated the genome to be 88.6% complete. Meloidogyne graminicola genome contained 17.83% repeat elements and showed 14,062 protein-coding gene models, 4,974 conserved orthologous genes, 561 putative secreted proteins, 49 RNAi pathway genes, 1,853 proteins involved in pathogen-host interactions, 1,575 carbohydrate-active enzymes, and 32,138 microsatellites. Five of the carbohydrate-active enzymes were found only in M. graminicola genome and were not present in any other analysed root-knot nematode genome. Together with the previous two genome assemblies, this improved genome assembly would facilitate comparative and functional genomics for M. graminicola.

Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows.

In cancer research, genomic profiles are often extracted from homogenized macrodissections of tissues, with the histological context lost and a large fraction of material underutilized. Pertinently, the spatial genomic landscape provides critical complementary information in deciphering disease heterogeneity and progression. Microscale sampling methods such as microdissection to obtain such information are often destructive to a sizeable fraction of the biopsy sample, thus showing limited multiplexability and adaptability to different assays. A modular microfluidic technology is here implemented to recover cells at the microscale from tumor tissue sections, with minimal disruption of unsampled areas and tailored to interface with genome profiling workflows, which is directed here toward evaluating intratumoral genomic heterogeneity. The integrated workflow-GeneScape-is used to evaluate heterogeneity in a metastatic mammary carcinoma, showing distinct single nucleotide variants and copy number variations in different tumor tissue regions, suggesting the polyclonal origin of the metastasis as well as development driven by multiple location-specific drivers.

Domestic animal proteomics in the 21st century: A global retrospective and viewpoint analysis.

Animal production and health are of significant economic importance, particularly regarding the world food supply. Animal and veterinary sciences have evolved immensely in the past six decades, particularly in genetics, nutrition, housing, management and health. To address major challenges such as those posed by climate change or metabolic disorders, it is of utmost importance to use state-of-the-art research tools. Proteomics and the other post-genomic tools (transcriptomics or metabolomics) are among them. Proteomics has experienced a considerable development over the last decades. This brought developments to different scientific fields. The use and adoption of proteomics tools in animal and veterinary sciences has some limitations (database availability or access to proteomics platforms and funding). As a result, proteomics' use by animal science researchers varies across the globe. In this viewpoint article, we focus on the developments of domestic animal proteomics over the last decade in different regions of the globe and how the researchers have coped with such challenges. In the second part of the article, we provide examples of funding, educational and laboratory establishment initiatives designed to foster the development of (animal-based) proteomics. International scientific collaboration is a definitive and key feature in the development and advancement of domestic animal proteomics. SIGNIFICANCE: Animal production and health are very important for food supply worldwide particularly as a source of proteinaceous foods. Animal and veterinary sciences have evolved immensely in the last decades. In order to address the major contemporary challenges facing animal and veterinary sciences, it is of utmost importance to use state-of-the-art research tools such as Proteomics and other Omics. Herein, we focus on the major developments in domestic animal proteomics worldwide during the last decade and how different regions of the world have used the technology in this specific research field. We address also major international efforts aiming to increase the research output in this area and highlight the importance of international cooperation to address specific problems inherent to domestic animal proteomics.

A Natural Low Phytic Acid Finger Millet Accession Significantly Improves Iron Bioavailability in Indian Women.

Iron deficiency and anemia are common in low- and middle-income countries. This is due to a poor dietary iron density and low iron absorption resulting from the high inhibitory phytic acid content in cereal and millet-based diets. Here, we report that a naturally occurring low phytic acid finger millet accession (571 mg 100 g-1), stable across three growing seasons with normal iron content (3.6 mg 100 g-1), increases iron absorption by 3-folds in normal Indian women. The accessions differing in grain phytic acid content, GE 2358 (low), and GE1004 (high) were selected from a core collection of 623 accessions. Whole genome re-sequencing of the accessions revealed significant single nucleotide variations segregating them into distinct clades. A non-synonymous mutation in the EcABCC phytic acid transporter gene between high and low accessions could affect gene function and result in phytic acid differences. The highly sensitive dual stable-isotope erythrocyte incorporation method was adopted to assess the fractional iron absorption. The low phytic acid accession resulted in a significantly higher iron absorption compared with the high phytic acid accession (3.7 vs. 1.3%, p < 0.05). The low phytic acid accession could be effective in preventing iron deficiency in regions where finger millet is habitually eaten. With its low water requirement, finger millet leaves low environmental footprints and hence would be an excellent sustainable strategy to mitigate iron deficiency.

Gas-Phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and Their Implication in Database Searches.

ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and particularly of their acceptor amino acids remains a major challenge. The attachment sites of the modification are difficult to localize by mass spectrometry (MS) because of the labile nature of the linkage and the complex fragmentation pattern of the ADP-ribose in MS/MS experiments. In this study we performed a comprehensive analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine, or lysine residues. In addition to the fragmentation of the peptide backbone, various cleavages of the ADP-ribosylated amino acid side chains were investigated. We focused on gas-phase fragmentations that were specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid, or aspartic acid was the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine was relatively stable. The main cleavage site was the inner bond of the guanidine group, which resulted in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking peptide fragment ions resulting from this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches. Furthermore, the presence of diagnostic ions and of losses of fragments from peptide ions allowed us, in most cases, to distinguish between ADP-ribosylated arginine and serine residues.

Dependence of Fluorescence Quenching of CY3 Oligonucleotide Conjugates on the Oxidation Potential of the Stacking Base Pair.

To understand the complex fluorescence properties of astraphloxin (CY3)-labelled oligonucleotides, it is necessary to take into account the redox properties of the nucleobases. In oligonucleotide hybrids, we observed a dependence of the fluorescence intensity on the oxidation potential of the neighbouring base pair. For the series I < A < G < 8-oxoG, the extent of fluorescence quenching follows the trend of decreasing oxidation potentials. In a series of 7 nt hybrids, stacking interactions of CY3 with perfect match and mismatch base pairs were found to stabilise the hybrid by 7-8 kJ/mol. The fluorescence measurements can be explained by complex formation resulting in fluorescence quenching that prevails over the steric effect of a reduced excited state trans-cis isomerisation, which was expected to increase the fluorescence efficiency of the dye when stacking to a base pair. This can be explained by the fact that, in a double strand, base pairing and stacking cause a dramatic change in the oxidation potential of the nucleobases. In single-molecule fluorescence measurements, the oxidation of G to 8-oxoG was observed as a result of photoinduced electron transfer and subsequent chemical reactions. Our results demonstrate that covalently linked CY3 is a potent oxidant towards dsDNA. Sulfonated derivatives should be used instead.

Network integration and modelling of dynamic drug responses at multi-omics levels.

Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data.

Unraveling the proteomic changes involved in the resistance response of Cajanus platycarpus to herbivory by Helicoverpa armigera.

The pigeonpea wild relative Cajanus platycarpus is resistant to Helicoverpa armigera, one of the major pests responsible for yield losses in Cajanus cajan. Deciphering the molecular mechanism underlying host plant resistance is pertinent to identify proteins that aid in the mitigation of the insect pest. The present study adopted comparative proteomics as a tool to interpret the resistance mechanism(s) in C. platycarpus vis-à-vis C. cajan during continued herbivory (up to 96 h). Over-representation analysis of the differentially expressed proteins implicated a multi-dimensional resistance response accomplished by both physical and chemical barriers in C. platycarpus. While the chemical basis for resistance was depicted by the upregulation of proteins playing a rate limiting role in the phenylpropanoid pathway, the physical basis was provided by the regulation of proteins involved in microtubule assembly and synthesis of lignins. Upregulation of proteins in the polyamine pathway indicated the role of metabolite conjugates to be negatively affecting herbivore growth. Reallocation of resources and diversion of metabolic flux to support the production of secondary metabolites could be the probable approach in the wild relative against herbivory. Our study provided deeper insights into the pod borer resistance mechanism in C. platycarpus for utility in crop improvement. KEY POINTS: • Pod borer resistance in Cajanus platycarpus is multi-dimensional. • Pod borer resistance has been arbitrated to cell wall rigidity and secondary metabolites. • Phenylpropanoid pathway derivatives apparently shaped the plant chemical defense against pod borer.

Single-molecule chemistry. Part I: monitoring oxidation of G in oligonucleotides using CY3 fluorescence.

Single-molecule hybridisation of CY3 dye labelled short oligonucleotides to surface immobilised probes was investigated in zero-mode waveguide nanostructures using a modified DNA sequencer. At longer measuring times, we observed changes of the initial hybridisation fluorescence pulse pattern which we attribute to products created by chemical reactions at the nucleobases. The origin is a charge separated state created by a photoinduced electron transfer from nucleobases to the dye followed by secondary reactions with oxygen and water, respectively. The positive charge can migrate through the hybrid resulting in base modifications at distant sites. Static fluorescence spectra were recorded in order to determine the properties of CY3 stacking to different base pairs, and compared to pulse intensities. A characteristic pulse pattern change was assigned to the oxidation of G to 8-oG besides the formation of a number of secondary products that are not yet identified. Further, we present a method to visualise the degree of chemical reactions to gain an overview of ongoing processes. Our study demonstrates that CY3 is able to oxidise nucleobases in ds DNA, and also in ss overhangs. An important finding is the correlation between nucleobase oxidation potential and fluorescence quenching which explains the intensity changes observed in single molecule measurements. The analysis of fluorescence traces provides the opportunity to track complete and coherent reaction sequences enabling to follow the fate of a single molecule over a long period of time, and to observe chemical reactions in real-time. This opens up the opportunity to analyse reaction pathways, to detect new products and short-lived intermediates, and to investigate rare events due to the large number of single molecules observed in parallel.