ShinyMetID: An R shiny package for metabolite identification by mass spectral matching.

We present metabolite identification software in the form of R Shiny. Metabolite identification by mass spectral matching in gas chromatography (GC-MS)-based untargeted metabolomics can be done by using the easy-to-use software. Various similarity measures are given and toy example using graphical user interface is presented.

Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. × ananassa).

Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently used to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. None of the mutations associated with mefenoxam resistance found in this study were in the RNA polymerase subunit genes, the hypothesized target of this compound in oomycetes. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes since serves as a foundation to validate the candidate genes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.

A multi-omics framework reveals strawberry flavor genes and their regulatory elements.

Flavor is essential to consumer preference of foods and is an increasing focus of plant breeding programs. In fruit crops, identifying genes underlying volatile organic compounds has great promise to accelerate flavor improvement, but polyploidy and heterozygosity in many species have slowed progress. Here we use octoploid cultivated strawberry to demonstrate how genomic heterozygosity, transcriptomic intricacy and fruit metabolomic diversity can be treated as strengths and leveraged to uncover fruit flavor genes and their regulatory elements. Multi-omics datasets were generated including an expression quantitative trait loci map with 196 diverse breeding lines, haplotype-phased genomes of a highly-flavored breeding selection, a genome-wide structural variant map using five haplotypes, and volatile genome-wide association study (GWAS) with > 300 individuals. Overlaying regulatory elements, structural variants and GWAS-linked allele-specific expression of numerous genes to variation in volatile compounds important to flavor. In one example, the functional role of anthranilate synthase alpha subunit 1 in methyl anthranilate biosynthesis was supported via fruit transient gene expression assays. These results demonstrate a framework for flavor gene discovery in fruit crops and a pathway to molecular breeding of cultivars with complex and desirable flavor.

Functional role of formate dehydrogenase 1 (FDH1) for host and nonhost disease resistance against bacterial pathogens.

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In the present study, the metabolic enzyme formate dehydrogenase 1 (FDH1) was identified to associate with nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. The Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that both SA and JA are involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 mainly localized to mitochondria, and the expression level of FDH1 was drastically increased upon infection with host or nonhost pathogens. Furthermore, we identified the potential co-localization of mitochondria expressing FDH1 with chloroplasts after the infection with nonhost pathogens in Arabidopsis. This finding suggests the possible role of FDH1 in mitochondria and chloroplasts during defense responses against bacterial pathogens in plants.

Comparative Transcriptome Analysis to Identify Candidate Genes for FaRCg1 Conferring Resistance Against Colletotrichum gloeosporioides in Cultivated Strawberry (Fragaria × ananassa).

Colletotrichum crown rot (CCR) caused by Colletotrichum gloeosporioides is a serious threat to the cultivated strawberry (Fragaria × ananassa). Our previous study reported that a major locus, FaRCg1, increases resistance. However, the genomic structure of FaRCg1 and potential candidate genes associated with the resistance remained unknown. Here, we performed comparative transcriptome analyses of resistant 'Florida Elyana' and susceptible 'Strawberry Festival' after infection and identified candidate genes potentially involved in resistance. In 'Florida Elyana', 6,099 genes were differentially expressed in response to C. gloeosporioides. Gene ontology analysis showed that the most upregulated genes were functionally associated with signaling pathways of plant defense responses. Three genes in the genomic region of FaRCg1 were highly upregulated: a von Willebrand Factor A domain-containing protein, a subtilisin-like protease, and a TIFY 11A-like protein. Subgenome-specific markers developed for the candidate genes were tested with a diverse panel of 219 accessions from University of Florida and North Carolina State University breeding programs. Significant and positive associations were found between the high-resolution melting (HRM) marker genotypes and CCR phenotypes. These newly developed subgenome-specific functional markers for FaRCg1 can facilitate development of resistant varieties through marker-assisted selection.

Genetic Analysis of Methyl Anthranilate, Mesifurane, Linalool, and Other Flavor Compounds in Cultivated Strawberry (Fragaria × ananassa).

The cultivated strawberry (Fragaria × ananassa) is an economically important fruit crop that is intensively bred for improved sensory qualities. The diversity of fruit flavors and aromas in strawberry results mainly from the interactions of sugars, acids, and volatile organic compounds (VOCs) that are derived from diverse biochemical pathways influenced by the expression of many genes. This study integrates multiomic analyses to identify QTL and candidate genes for multiple aroma compounds in a complex strawberry breeding population. Novel fruit volatile QTL was discovered for methyl anthranilate, methyl 2-hexenoate, methyl 2-methylbutyrate, mesifurane, and a shared QTL on Chr 3 was found for nine monoterpene and sesquiterpene compounds, including linalool, 3-carene, ß-phellandrene, α-limonene, linalool oxide, nerolidol, α-caryophellene, α-farnesene, and ß-farnesene. Fruit transcriptomes from a subset of 64 individuals were used to support candidate gene identification. For methyl esters including the grape-like methyl anthranilate, a novel ANTHANILIC ACID METHYL TRANSFERASE-like gene was identified. Two mesifurane QTL correspond with the known biosynthesis gene O-METHYL TRANSFERASE 1 and a novel FURANEOL GLUCOSYLTRANSFERASE. The shared terpene QTL contains multiple fruit-expressed terpenoid pathway-related genes including NEROLIDOL SYNTHASE 1 (FanNES1). The abundance of linalool and other monoterpenes is partially governed by a co-segregating expression-QTL (eQTL) for FanNES1 transcript variation, and there is additional evidence for quantitative effects from other terpenoid-pathway genes in this narrow genomic region. These QTLs present new opportunities in breeding for improved flavor in commercial strawberry.

Genomic Characterization of the Fruity Aroma Gene, FaFAD1, Reveals a Gene Dosage Effect on γ-Decalactone Production in Strawberry (Fragaria × ananassa).

Strawberries produce numerous volatile compounds that contribute to the unique flavors of fruits. Among the many volatiles, γ-decalactone (γ-D) has the greatest contribution to the characteristic fruity aroma in strawberry fruit. The presence or absence of γ-D is controlled by a single locus, FaFAD1. However, this locus has not yet been systematically characterized in the octoploid strawberry genome. It has also been reported that the volatile content greatly varies among the strawberry varieties possessing FaFAD1, suggesting that another genetic factor could be responsible for the different levels of γ-D in fruit. In this study, we explored the genomic structure of FaFAD1 and determined the allele dosage of FaFAD1 that regulates variations of γ-D production in cultivated octoploid strawberry. The genome-wide association studies confirmed the major locus FaFAD1 that regulates the γ-D production in cultivated strawberry. With the hybrid capture-based next-generation sequencing analysis, a major presence-absence variation of FaFAD1 was discovered among γ-D producers and non-producers. To explore the genomic structure of FaFAD1 in the octoploid strawberry, three bacterial artificial chromosome (BAC) libraries were developed. A deletion of 8,262 bp was consistently found in the FaFAD1 region of γ-D non-producing varieties. With the newly developed InDel-based codominant marker genotyping, along with γ-D metabolite profiling data, we revealed the impact of gene dosage effect for the production of γ-D in the octoploid strawberry varieties. Altogether, this study provides systematic information of the prominent role of FaFAD1 presence and absence polymorphism in producing γ-D and proposes that both alleles of FaFAD1 are required to produce the highest content of fruity aroma in strawberry fruit.

Comprehensive Comparative Analysis of Local False Discovery Rate Control Methods.

Due to the advance in technology, the type of data is getting more complicated and large-scale. To analyze such complex data, more advanced technique is required. In case of omics data from two different groups, it is interesting to find significant biomarkers between two groups while controlling error rate such as false discovery rate (FDR). Over the last few decades, a lot of methods that control local false discovery rate have been developed, ranging from one-dimensional to k-dimensional FDR procedure. For comparison study, we select three of them, which have unique and significant properties: Efron et al. (2001), Ploner et al. (2006), and Kim et al. (2018) in chronological order. The first approach is one-dimensional approach while the other two are two-dimensional ones. Furthermore, we consider two more variants of Ploner's approach. We compare the performance of those methods on both simulated and real data.

A Novel Role of Salt- and Drought-Induced RING 1 Protein in Modulating Plant Defense Against Hemibiotrophic and Necrotrophic Pathogens.

Many plant-encoded E3 ligases are known to be involved in plant defense. Here, we report a novel role of E3 ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) in plant immunity. Even though SDIR1 is reasonably well-characterized, its role in biotic stress response is not known. The silencing of SDIR1 in Nicotiana benthamiana reduced the multiplication of the virulent bacterial pathogen Pseudomonas syringae pv. tabaci. The Arabidopsis sdir1 mutant is resistant to virulent pathogens, whereas SDIR1 overexpression lines are susceptible to both host and nonhost hemibiotrophic bacterial pathogens. However, sdir1 mutant and SDIR1 overexpression lines showed hypersusceptibility and resistance, respectively, against the necrotrophic pathogen Erwinia carotovora. The mutant of SDIR1 target protein, i.e., SDIR-interacting protein 1 (SDIR1P1), also showed resistance to host and nonhost pathogens. In SDIR1 overexpression plants, transcripts of NAC transcription factors were less accumulated and the levels of jasmonic acid (JA) and abscisic acid were increased. In the sdir1 mutant, JA signaling genes JAZ7 and JAZ8 were downregulated. These data suggest that SDIR1 is a susceptibility factor and its activation or overexpression enhances disease caused by P. syringae pv. tomato DC3000 in Arabidopsis. Our results show a novel role of SDIR1 in modulating plant defense gene expression and plant immunity.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Allelic Variation of MYB10 Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry (Fragaria spp.) Fruit.

The fruits of diploid and octoploid strawberry (Fragaria spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in MYB10 cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsy-transposon in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional loss-of-function mutations in MYB10 were identified among geographically diverse white-fruited F. vesca ecotypes. Genetic and transcriptomic analyses of octoploid Fragaria spp revealed that FaMYB10-2, one of three MYB10 homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in FaEnSpm-2 are responsible for enhanced MYB10-2 expression and anthocyanin biosynthesis in strawberry fruit flesh.

Mussel-inspired enzyme immobilization and dual real-time compensation algorithms for durable and accurate continuous glucose monitoring.

Blood glucose sensing is very important for diabetic management. It is shifting towards a continuous glucose monitoring because such a system can alleviate patient suffering and provide a large number of glucose measurements. Here, we proposed a novel approach for the development of durable and accurate enzymatic continuous glucose monitoring system (CGMS). For the long-term durable and selective immobilization of glucose oxidase on a microneedle electrode, a biocompatible engineered mussel adhesive protein was employed through efficient electrochemical oxidation strategy. For the accurate performance in in vivo environments, we also suggested dual real-time compensated algorithms to cover both temperature and time-lag differences. After pre-clinical and pilot-clinical evaluations, we confirmed that our proposed CGMS has an outstanding performance compared with various commercially available continuous systems and achieves comparable performance to disposable glucose sensors.

Observations on significant hemodynamic changes caused by a high concentration of epidurally administered ropivacaine: correlation and prediction study of stroke volume variation and central venous pressure in thoracic epidural anesthesia.

BACKGROUND: Thoracic epidural anesthesia (TEA) exacerbates hypotension due to peripheral vasodilator effects following the use of general anesthetics. This study aimed to compare the hemodynamic changes caused by three different concentrations of epidural ropivacaine and to evaluate the performance of the stroke-volume variation (SVV) and central venous pressure (CVP) during TEA with general anesthesia. METHODS: A total of 120 patients were administered 8 mL of ropivacaine solution via epidural injection, following randomization into one of three groups based on the concentration of ropivacaine in the study solution: 0.75%, 0.375%, or 0.2%. Hemodynamics were monitored for 30 min after loading. We analyzed the hemodynamic changes in the subgroups according to an age cutoff of 60 years. Receiver operating characteristic (ROC) analysis was performed to characterize the relationship of the SVV, CVP, and a 20% decrease in the mean arterial pressure (MAP) following TEA. RESULTS: Data from 109 patients were analyzed. MAP and systemic vascular resistance index were significantly decreased, and SVV was significantly increased after epidural loading only in the 0.75% ropivacaine group. There was a significant difference in hemodynamics between young and elderly subgroups in the 0.75% ropivacaine group. SVV showed a negative correlation with MAP, whereas CVP showed no correlation. The ROC analysis of SVV demonstrated a weak predictive ability of a 20% decrease in MAP at 10 min after the loading dose, with an area-under-the-curve of 0.687 and a 9.5% optimal cutoff value (sensitivity, 60.6%; specificity, 68.9%). CONCLUSIONS: A high concentration of ropivacaine through TEA caused a significant decrease in the systemic vascular resistance and blood pressure. More significant decreases were shown in the elderly patients. Though the change of SVV showed a negative correlation with hypotension and indicated functional hypovolemia after TEA, the predictability was limited. CLINICAL TRIALS REGISTRATION: Number: NCT01559285 , date: January 24, 2013.

Plasmon enhanced photoacoustic generation from volumetric electromagnetic hotspots.

This work reports plasmon enhanced photoacoustic generation by using a three dimensional plasmonic absorber. The 3D plasmonic absorber comprises a thin polymer film on glass nanopillar arrays with nanogap-rich silver nanoislands. The 3D plasmonic absorber clearly shows 24.6 times higher enhancement of photoacoustic signals at an excitation wavelength of 630 nm than a simple polymeric absorber. The photoacoustic enhancement results from the volumetric electromagnetic field enhancement on a light-absorbing polymer through 3D plasmonic nanostructures. This novel photoacoustic absorber provides a new direction for highly efficient ultrasonic generation.

Electrokinetic preconcentration of small molecules within volumetric electromagnetic hotspots in surface enhanced Raman scattering.

The on-chip integration of a preconcentration chamber for ultrasensitive surface-enhanced Raman scattering (SERS) is shown. Small molecules are preconcentrated using 3D volumetric electromagnetic hotspots. The experimental results demonstrate an enhancement of the SERS signals of over two orders of magnitude, which allows the fingerprinting of neurotransmitter molecules at the nanomolar level and furthers the selective detection of oppositely charged molecules. This on-chip integration will provide new directions for ultrasensitive SERS applications.

Differences in cold hardiness, carbohydrates, dehydrins and related gene expressions under an experimental deacclimation and reacclimation in Prunus persica.

To boost our understanding of a recent outbreak of freezing injury, we sought to confirm distinctive features between the shoot tissues of the peach (Prunus persica) cultivars Daewol and Kiraranokiwami by mimicking unseasonable changes of temperatures that occur in the early spring through repeated deacclimation and reacclimation treatments. Patterns of cold hardiness declined dramatically during the deacclimation and rose during the reacclimation in both cultivars. Our results indicated that 'Daewol' possessed higher capacity in response to repeated deacclimation and reacclimation treatments than 'Kiraranokiwami'. 'Daewol' showed more sensitive changes in the carbohydrates in response to warm and low temperatures compared with 'Kiraranokiwami'. 'Daewol' indicated almost similar repeated down- and up-patterns in soluble sugar content in response to repeated deacclimation and reacclimation, whereas it indicated repeated up- and down-patterns in starch content. However, 'Kiraranokiwami' showed a progressive increase in the soluble sugar content and a progressive decrease in starch content. Notably, patterns of accumulation of a 60-kDa dehydrin protein encoded by the PpDhn1 gene were confirmed through western blotting and paralleled fluctuations of cold hardiness in both cultivars. Expression of this dehydrin was weak in both cultivars during deacclimation but its band intensity increased during reacclimation. Changes in related genes (ß-amylase, PpDhn1, PpDhn2 and PpDhn3) were positively correlated with changes in cold hardiness throughout the experiment. Our results indicate that recent repeated warm periods may cause premature deacclimation in the early spring, and that more cold-tolerant cultivar may be more resilient to freezing injury caused by unstable temperature conditions.

A deformable nanoplasmonic membrane reveals universal correlations between plasmon resonance and surface enhanced Raman scattering.

A quantitative correlation between plasmon resonance and surface enhanced Raman scattering (SERS) signals is revealed by using a novel active plasmonic method, that is, a deformable nanoplasmonic membrane. A single SERS peak has the maximum gain at the corresponding plasmon resonance wavelength, which has the maximum extinction product of an excitation and the corresponding Raman scattering wavelengths.

Optofluidic SERS chip with plasmonic nanoprobes self-aligned along microfluidic channels.

This work reports an optofluidic SERS chip with plasmonic nanoprobes self-aligned along microfluidic channels. Plasmonic nanoprobes with rich electromagnetic hot spots are selectively patterned along PDMS microfluidic channels by using a Scotch tape removal and oxygen plasma treatment, which also provide the permanent bonding between PDMS and a glass substrate. A silver film with an initial thickness of 30 nm after oxygen plasma treatment creates nanotips and nanodots with a maximum SERS performance, which were successfully implanted with microfluidic concentration gradient generators. The novel device enables the label-free and solution-phase SERS detection of small molecules with low Raman activity such as dopamine at micromolar level in flow. This optofluidic SERS chip can be readily expanded for microfluidic networks with diverse functions for advanced optical biochemical assays.

Terahertz photoconductive antenna with metal nanoislands.

This work presents a nanoplasmonic photoconductive antenna (PCA) with metal nanoislands for enhancing terahertz (THz) pulse emission. The whole photoconductive area was fully covered with metal nanoislands by using thermal dewetting of thin metal film at relatively low temperature. The metal nanoislands serve as plasmonic nanoantennas to locally enhance the electric field of an ultrashort pulsed pump beam for higher photocarrier generation. The plasmon resonance of metal nanoislands was achieved at an excitation laser wavelength by changing the initial thickness of metal film. This nanoplasmonic PCA shows two times higher enhancement for THz pulse emission power than a conventional PCA. This work opens up a new opportunity for plasmon enhanced large-aperture THz photoconductive antennas.