Transcriptome Profiling of a Salt Excluder Hybrid Grapevine Rootstock 'Ruggeri' throughout Salinity.

Salinity is one of the substantial threats to plant productivity and could be escorted by other stresses such as heat and drought. It impairs critical biological processes, such as photosynthesis, energy, and water/nutrient acquisition, ultimately leading to cell death when stress intensity becomes uncured. Therefore, plants deploy several proper processes to overcome such hostile circumstances. Grapevine is one of the most important crops worldwide that is relatively salt-tolerant and preferentially cultivated in hot and semi-arid areas. One of the most applicable strategies for sustainable viticulture is using salt-tolerant rootstock such as Ruggeri (RUG). The rootstock showed efficient capacity of photosynthesis, ROS detoxification, and carbohydrate accumulation under salinity. The current study utilized the transcriptome profiling approach to identify the molecular events of RUG throughout a regime of salt stress followed by a recovery procedure. The data showed progressive changes in the transcriptome profiling throughout salinity, underpinning the involvement of a large number of genes in transcriptional reprogramming during stress. Our results established a considerable enrichment of the biological process GO-terms related to salinity adaptation, such as signaling, hormones, photosynthesis, carbohydrates, and ROS homeostasis. Among the battery of molecular/cellular responses launched upon salinity, ROS homeostasis plays the central role of salt adaptation.

E-cig vapor condensate alters proteome and lipid profiles of membrane rafts: impact on inflammatory responses in A549 cells.

Electronic cigarettes (e-cigs) are battery-operated heating devices that aerosolize e-liquid, typically containing nicotine and several other chemicals, which is then inhaled by a user. Over the past decade, e-cigs have gained immense popularity among both smokers and non-smokers. One reason for this is that they are advertised as a safe alternative to conventional cigarettes. However, the recent reports of e-cig use associated lung injury have ignited a considerable debate about the relative harm and benefits of e-cigs. The number of reports about e-cig-induced inflammation and pulmonary health is increasing as researchers seek to better understand the effects of vaping on human health. In line with this, we investigated the molecular events responsible for the e-cig vapor condensate (ECVC)-mediated inflammation in human lung adenocarcinoma type II epithelial cells (A549). In an attempt to limit the variables caused by longer ingredient lists of flavored e-cigs, tobacco-flavored ECVC (TF-ECVC±nicotine) was employed for this study. Interestingly, we observed significant upregulation of cytokines and chemokines (IL-6, IL-8, and MCP-1) in A549 cells following a 48 h TF-ECVC challenge. Furthermore, there was a significant increase in the expression of pattern recognition receptors TLR-4 and NOD-1, lipid raft-associated protein caveolin-1, and transcription factor NF-кB in TF-ECVC with and/or without nicotine-challenged lung epithelial cells. Our results further demonstrate the harboring of TLR-4 and NOD-1 in the caveolae of TF-ECVC-challenged A549 cells. Proteomic and lipidomic analyses of lipid raft fractions from control and challenged cells revealed a distinct protein and lipid profile in TF-ECVC (w/wo nicotine)-exposed A549 cells. Interestingly, the inflammatory effects of TF-ECVC (w/wo nicotine) were inhibited following the caveolin-1 knockdown, thus demonstrating a critical role of caveolae raft-mediated signaling in eliciting inflammatory responses upon TF-ECVC challenge. Graphical Abstract Graphical Abstract.

Binding of cloned Cel enzymes on clay minerals related to the pI of the enzymes and database survey of cellulases of soil bacteria for pI.

The Cel genes from Bacillus licheniformis MSB03 were cloned and expressed to investigate binding ability on clay minerals and sea sand at pH ranging 3 to 9. FTIR analysis has been done to characterize bound enzymes on clay minerals. Subsequent, surveying of NCBI database for extracellular enzymes of soil bacteria was carried out. Among the five cloned Cel enzymes assayed for binding to clay minerals, only Cel5H enzyme had the binding ability. Enzyme Cel5H exhibited highest binding to montmorillonite followed by kaolinite and sea sand. Interestingly, Cel5H had higher pI value of 9.24 than other proteins (5.2-5.7). Cel5H binding to montmorillonite was shown to be negatively affected below pH 3 and above pH 9. Infrared absorption spectra of the Cel5H-montmorillonite complexes showed distinct peaks for clay minerals and bound proteins. Furthermore, database survey of soil bacterial extracellular enzymes revealed that Bacillus species enzymes had higher pI than other soil bacterial enzymes.

Multi-year Quantitative Evaluation of Stilbenoids Levels Among Selected Muscadine Grape Cultivars.

Stilbenoids such as t-piceid, t-resveratrol, ε-viniferins, and t-pterostilbene can differ significantly among grape cultivars and years due to variation in environmental conditions and subsequent stressors encountered during a year. This study evaluated diverse muscadine grape cultivars for their ability to consistently produce four major stilbenoids such as t-piceid, t-resveratrol, ε-viniferins, and t-pterostilbene irrespective of environmental changes that can impact their production. Berries from forty-two muscadine grape cultivars were collected for three years (2013, 2014, and 2015) to measure stilbenoids. Results showed significant differences in the composition of four stilbenoids among the muscadine cultivars. The highest level of stilbenoids was observed in 'Fry Seedless' (270.20 µg/g fresh weight) in each of the three consecutive years tested followed by 'Pride' (46.18 µg/g fresh weight) while 'Doreen' produced the lowest level of stilbenoids (1.73 µg/g fresh weight). Results demonstrated that certain muscadine grape cultivars consistently produced varied levels of the four major stilbenoids year after year. Based on the total content of stilbenoids, the 42 muscadine cultivars studied were grouped into three categories such as High, Medium and Low stilbenoid-containing cultivars. This information will help establish new vineyards with cultivars that are less prone to variations in environmental conditions and can consistently produce stilbenoid-rich muscadine grape berries with enhanced market value to promote consumer health.

Modeling the clay minerals-enzyme binding by fusion fluorescent proteins and under atomic force microscope.

In the present study, binding of cellulase protein to different clay minerals were tested using fluorescent-protein complex and microscopic techniques. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H-mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H-fusion enzymes, green-fusion enzyme showed higher intensity compared with other red and yellow fusion-proteins. Intensity of fusion-proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free-state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Even surface of kaolinite also exerted some strain on protein molecular conformation after binding to surface. Our results provide further evidence for 3D visualization of enzyme-soil complex and encourage furthering study of the force involved interactions. Therefore, our results indicate that binding of proteins to clay minerals was external and provides a molecular method to observe the interaction of clay minerals-enzyme complex.

Proteome Profile of American Hybrid Grape cv. Blanc du Bois during Ripening Reveals Proteins Associated with Flavor Volatiles and Ethylene Production.

The study of key control points in ripening is essential to improve grape wine quality. Molecular basis of ripening is still far from being understood from the Pierce's disease (PD)-tolerant grapes predominantly grown in the southeastern United States. To identify proteins expressed during Blanc du Bois grape berry green and ripening stages, proteome analysis from five different stages revealed 1091, 1131, 1078, 1042, and 1066 proteins. Differential expression analysis revealed 551 common proteins across different stages of maturity that are involved in various biochemical and metabolic pathways. The proteins identified were associated with phenylpropanoids, isoquinoline alkaloids, fatty acids, unsaturated fatty acids, and furanones. Our data provide the first step to understand the complex biochemical changes during ripening of PD-tolerant American hybrid grapes that are popular for their aroma and flavor profile in the southeastern United States. Proteomics data are deposited to the ProteomeXchange PXD004157.

In Depth Proteome Analysis of Ripening Muscadine Grape Berry cv. Carlos Reveals Proteins Associated with Flavor and Aroma Compounds.

Ripening in nonclimacteric fruits such as grape involves complex chemical changes that have a profound influence on the accumulation of flavor and aroma compounds distinct to a particular grape genotype. In this study, proteome characterization of wine type bronze muscadine grape (Vitis rotundifolia cv. Carlos), primarily grown in the Southeastern United States was performed during berry ripening. Stage-specific protein expression was obtained among different stages of berries. Differential analysis showed the expression of 522 proteins that regulate diverse biological processes and metabolic pathways. Of these, 30 proteins are associated with the production of key phenolic compounds, whereas 25 are associated with the production of muscadine aroma compounds. These proteins are involved in the phenylpropanoid pathway, terpene synthesis, fatty acid derived volatiles and esters that affect muscadine berry flavor and aroma characteristics. Further, gene expression analysis during ripening validated the expression pattern of 12 proteins. Catechin, epicatechin, and four stilbenes were quantified to correlate observed proteome changes. This study not only revealed biochemical changes during muscadine berry ripening but also offers indicators for marker-assisted breeding to enhance organoleptic properties of muscadine grape to improve its flavor and aroma properties.

Comparative leaf proteomics of drought-tolerant and -susceptible peanut in response to water stress.

UNLABELLED: Water stress (WS) predisposes peanut plants to fungal infection resulting in pre-harvest aflatoxin contamination. Major changes during water stress including oxidative stress, lead to destruction of photosynthetic apparatus and other macromolecules within cells. Two peanut cultivars with diverse drought tolerance characteristics were subjected to WS, and their leaf proteome was compared using two-dimensional electrophoresis complemented with MALDI-TOF/TOF mass spectrometry. Ninety-six protein spots were differentially abundant to water stress in both cultivars that corresponded to 60 non-redundant proteins. Protein interaction prediction analysis suggests that 42 unique proteins showed interactions in tolerant cultivar while 20 showed interactions in the susceptible cultivar, activating other proteins in directed system response networks. Four proteins: glutamine ammonia ligase, chitin class II, actin isoform B, and beta tubulin, involved in metabolism, defense and cellular biogenesis, are unique in tolerant cultivar and showed positive interactions with other proteins. In addition, four proteins: serine/threonine protein phosphate PP1, choline monooxygenase, peroxidase 43, and SNF1-related protein kinase regulatory subunit beta-2, that play a role as cryoprotectants through signal transduction, were induced in drought tolerant cultivar following WS. Eleven interologs of these proteins were found in Arabidopsis interacting with several proteins and it is believed that similar mechanisms/pathways exist in peanut. SIGNIFICANCE: Peanuts (Arachis hypogaea L.) are a major source of plant protein grown in subtropical and tropical regions of the world. Pre-harvest aflatoxin contamination is a major problem that affects peanut crop yield and food safety. Poor understanding of molecular and cellular mechanisms associated with aflatoxin resistance is largely responsible for the lack of progress in elucidating a process/methodology for reducing aflatoxin contamination in peanuts. Drought perturbs the invasion of the aflatoxin producing fungus and thus affects the quality and yield of peanut. Therefore, more studies involving the effects of drought stress to determine the molecular changes will enhance our understanding of the key metabolic pathways involved in the combined stresses. The changes associated with the biotic and abiotic interactions within the peanut will be used to determine the metabolic pathways involved in the stress tolerance. This research would be beneficial in identifying the tolerant molecular signatures and promoting food safety and consumer health through breeding superior quality peanut cultivars.

A novel pathogenic Mammalian orthoreovirus from diarrheic pigs and Swine blood meal in the United States.

UNLABELLED: Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence. IMPORTANCE: Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea.

iTRAQ-based quantitative proteomics of developing and ripening muscadine grape berry.

Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening.

Resistance to Elsinoë ampelina and expression of related resistant genes in Vitis rotundifolia Michx. grapes.

Muscadine grapes (Vitis rotundifolia Michx) are considered as excellent genetic resources for grape breeding programs as they are known for their hardiness and resistance to pests and diseases. However, contrary to popular belief, our study indicated that not all muscadine cultivars are resistant to anthracnose disease. In order to identify a source of genetic tolerance towards anthracnose among muscadine cultivars, a series of in-situ and ex-situ experiments were conducted through strict and sensitive screening processes. Two consecutive years of field evaluation of 54 grape cultivars showed various levels of anthracnose incidence among the cultivars between a scale of 0 (tolerant) to 5 (highly-susceptible). Resistance bioassay by inoculation of different spore densities of Elsinoë ampelina on 40 cultivars presented similar results and was consistent with those obtained from the field test. A real-time PCR analysis was conducted to investigate differences of gene expression between susceptible and tolerant cultivars and to confirm results by phenotypic identification. Expression of genes encoding chalcone synthase, stilbene synthase, polygalacturonase-inhibiting protein, chitinase and lipid transfer-protein was only detected in tolerant cultivars. Resistant muscadine cultivars identified in this study could be excellent candidates for grape disease resistance breeding programs.

Fungal diversity in composting process of pig manure and mushroom cultural waste based on partial sequence of large subunit rRNA.

Fungal diversity during composting was investigated by culture-independent rDNA sequence analysis. Composting was carried out with pig manure and mushroom cultural waste using a field-scale composter (Hazaka system), and samples were collected at various stages. Based on partial sequence analysis of large subunit (LSU) ribosomal RNA (rRNA) and sequence identity values, a total of 12 different fungal species were found at six sampling sites; Geotrichum sp., Debaryomyces hansenii, Monographella nivalis, Acremonium strictum, Acremonium alternatum, Cladosporium sphaerospermum, Myriangium durosai, Pleurotus eryngii, Malassezia globosa, Malassezia restricta, Rhodotorula glutinis, and Fusarium sporotrichioides. Geotrichum sp. of the class Saccharomycetes was the most predominant fungal species throughout the composting process (185 out of a total of 236 identified clones, or 78.4%), followed by Acremonium strictum (7.6%), Monographella nivalis (5.1%), and Pleurotus eryngii (3.8%). The prevalence of Geotrichum sp. was the lowest (61.1%) at the beginning of composting, and then gradually increased to 92.5% after 10 days of composting.

Culture-independent analysis of microbial succession during composting of Swine slurry and mushroom cultural wastes.

Bacterial diversity and the composition of individual communities during the composting process of swine and mushroom cultural wastes in a field-scale composter (Hazaka system) were examined using a PCR-based approach. The composting process was divided into six stages based on recorded temperature changes. Phylogenetic analysis of eighty 16S rRNA sequences from uncultured composting bacterial groups revealed the presence of representatives from three divisions, including plant pathogenic bacteria, high-molecule-degrading bacteria and spore-forming bacteria. The plant pathogen A. tumefaciens gradually decreased in abundance during the composting process and eventually disappeared during the thermophilic and cooling stage. A bacterium homologous to Bacillus humi first appeared at the early thermophilic stage and was established at the intermediate thermophilic, post-thermophilic, and cooling stages. It was not possible to isolate the B. humi during any of the stages using general culture techniques.