Genome-wide identification of the class III peroxidase gene family of sugarcane and its expression profiles under stresses.

Introduction: Plant-specific Class III peroxidases (PRXs) play a crucial role in lignification, cell elongation, seed germination, and biotic and abiotic stresses. Methods: The class III peroxidase gene family in sugarcane were identified by bioinformatics methods and realtime fluorescence quantitative PCR. Results: Eighty-two PRX proteins were characterized with a conserved PRX domain as members of the class III PRX gene family in R570 STP. The ShPRX family genes were divided into six groups by the phylogenetic analysis of sugarcane, Saccharum spontaneum, sorghum, rice, and Arabidopsis thaliana. The analysis of promoter cis-acting elements revealed that most ShPRX family genes contained cis-acting regulatory elements involved in ABA, MeJA, light responsiveness, anaerobic induction, and drought inducibility. An evolutionary analysis indicated that ShPRXs was formed after Poaceae and Bromeliaceae diverged, and tandem duplication events played a critical role in the expansion of ShPRX genes of sugarcane. Purifying selection maintained the function of ShPRX proteins. SsPRX genes were differentially expressed in stems and leaves at different growth stages in S. spontaneum. However, ShPRX genes were differentially expressed in the SCMV-inoculated sugarcane plants. A qRT-PCR analysis showed that SCMV, Cd, and salt could specifically induce the expression of PRX genes of sugarcane. Discussion: These results help elucidate the structure, evolution, and functions of the class III PRX gene family in sugarcane and provide ideas for the phytoremediation of Cd-contaminated soil and breeding new sugarcane varieties resistant to sugarcane mosaic disease, salt, and Cd stresses.

Comparative Analysis of Sugar Metabolites and Their Transporters in Sugarcane Following Sugarcane mosaic virus (SCMV) Infection.

Sugarcane mosaic virus (SCMV) is one of the major pathogens of sugarcane. SCMV infection causes dynamic changes in plant cells, including decreased photosynthetic rate, respiration, and sugar metabolism. To understand the basics of pathogenicity mechanism, we performed transcriptome and proteomics analysis in two sugarcane genotypes (Badila: susceptible to SCMV and B-48: SCMV resistant). Using Saccharum spontaneum L. genome as a reference, we identified the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) that participate in sugar metabolism, transport of their metabolites, and Carbohydrate Activating enZYmes (CAZymes). Sequencing data revealed 287 DEGs directly or indirectly involved in sugar metabolism, transport, and storage, while 323 DEGs are associated with CAZymes. Significant upregulation of glucose, sucrose, fructose, starch, and SWEET-related transcripts was observed in the Badila after infection of SCMV. B-48 showed resistance against SCMV with a limited number of sugar transcripts up-regulation at the post-infection stage. For CAZymes, only glycosyltransferase (GT)1 and glycosyl hydrolase (GH)17 were upregulated in B-48. Regulation of DEGs was analyzed at the proteomics level as well. Starch, fructose, glucose, GT1, and GH17 transcripts were expressed at the post-translational level. We verified our transcriptomic results with proteomics and qPCR data. Comprehensively, this study proved that Badila upregulated sugar metabolizing and transporting transcripts and proteins, which enhance virus multiplication and infectionl.

Experimental evaluation of genomic selection prediction for rust resistance in sugarcane.

The total sugarcane (Saccharum L.) production has increased worldwide; however, the rate of growth is lower compared with other major crops, mainly due to a plateauing of genetic gain. Genomic selection (GS) has proven to substantially increase the rate of genetic gain in many crops. To investigate the utility of GS in future sugarcane breeding, a field trial was conducted using 432 sugarcane clones using an augmented design with two replications. Two major diseases in sugarcane, brown and orange rust (BR and OR), were screened artificially using whorl inoculation method in the field over two crop cycles. The genotypic data were generated through target enrichment sequencing technologies. After filtering, a set of 8,825 single nucleotide polymorphic markers were used to assess the prediction accuracy of multiple GS models. Using fivefold cross-validation, we observed GS prediction accuracies for BR and OR that ranged from 0.28 to 0.43 and 0.13 to 0.29, respectively, across two crop cycles and combined cycles. The prediction ability further improved by including a known major gene for resistance to BR as a fixed effect in the GS model. It also substantially reduced the minimum number of markers and training population size required for GS. The nonparametric GS models outperformed the parametric GS suggesting that nonadditive genetic effects could contribute genomic sources underlying BR and OR. This study demonstrated that GS could potentially predict the genomic estimated breeding value for selecting the desired germplasm for sugarcane breeding for disease resistance.

Photosynthetic characterization and expression profiles of sugarcane infected by Sugarcane mosaic virus (SCMV).

Sugarcane mosaic virus (SCMV), belonging to genus Potyvirus, family Potyviridae, is a severe pathogen of several agricultural important crops, mainly sugarcane. Due to complex nature of sugarcane, the effect of SCMV pathogenicity on sugarcane photosynthetic systems remains to be explored. In this study, we investigated the alterations occurring in the photosynthetic system in the sugarcane genotypes at the cytopathological, physiological and biological, transcriptome and proteome level. We generated the transcriptome assembly of two genotypes (susceptible Badila and resistant B-48) using Saccharum spontaneum L. as a reference genome. RNA-sequencing data revealed the significant upregulation of NAD(P)H, RubisCO, oxygen-evolving complex, chlorophyll a and b binding protein, Psb protein family, PSI reaction center subunit II, and IVgenes in B-48, as compared to its counterparts. Upregulated genes in B-48 are associated with various processes such as stability and assembly of photosystem, protection against photoinhibition and antiviral defense. The expression pattern of differentially abundant genes were further verified at the proteomics level. Overall, differentially expressed genes/proteins (DEGs/DEPs) showed the consistency of expression at both transcriptome and proteome level in B-48 genotype. Comprehensively, these data supported the efficiency of B-48 genotype under virus infection conditions and provided a better understanding of the expression pattern of photosynthesis-related genes in sugarcane.

Gene expression profiling of reactive oxygen species (ROS) and antioxidant defense system following Sugarcane mosaic virus (SCMV) infection.

BACKGROUND: Viruses are infectious pathogens, and plant virus epidemics can have devastating consequences to crop yield and quality. Sugarcane mosaic virus (SCMV, belonging to family Potyviridae) is one of the leading pathogens that affect the sugarcane crop every year. To combat the pathogens' attack, plants generate reactive oxygen species (ROS) as the first line of defense whose sophisticated balance is achieved through well-organized antioxidant scavenging pathways. RESULTS: In this study, we investigated the changes occurring at the transcriptomic level of ROS associated and ROS detoxification pathways of SCMV resistant (B-48) and susceptible (Badila) sugarcane genotypes, using Saccharum spontaneum L. genome assembly as a reference genome. Transcriptomic data highlighted the significant upregulation of ROS producing genes such as NADH oxidase, malate dehydrogenase and flavin-binding monooxygenase, in Badila genotype after SCMV pathogenicity. To scavenge the ROS, the Badila genotype illustrated a substantial enhancement of antioxidants i.e. glutathione s-transferase (GST), as compared to its resistant counterpart. GST is supposed to be a key indicator of pathogen attacks on the plant. A remarkably lower GST expression in B-48, as compared to Badila, indicated the development of resistance in this genotype. Additionally, we characterized the critical transcription factors (TFs) involved in endowing resistance to B-48. Among these, WRKY, AP2, NAC, bZIP, and bHLH showed enhanced expression in the B-48 genotype. Our results also confirmed the linkage of transcriptomic data with the enzymatic and qPCR data. The estimation of enzymatic activities for superoxide dismutase, catalase, ascorbate peroxidase, and phenylalanine ammonia-lyase supported the transcriptomic data and evinced higher resistance in B-48 genotype. CONCLUSION: The current study supported the efficiency of the B-48 genotype under SCMV infection. Moreover, comparative transcriptomic data has been presented to highlight the role of significant transcription factors conferring resistance to this genotype. This study provides an in-depth knowledge of the expression profiling of defense mechanisms in sugarcane.

Overexpression of UHRF1 and its potential role in the development of invasive ductal breast cancer validated by integrative bioinformatics and immunohistochemistry analyses.

BACKGROUND: Increasing evidence has highlighted the role of ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) in the development of cancers, including hepatocellular carcinoma, pancreatic cancer, and bladder cancer. However, the correlation between UHRF1 and breast cancer remains unclear. The present study aimed to analyze the expression of UHRF1 and its role in the development of invasive ductal breast cancer (IDC) by integrating multilevel expression data and immunohistochemistry analysis. METHODS: The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were used to gather UHRF1 expression data on IDC. Additionally, immunohistochemistry analysis was used to investigate the correlations between UHRF1 expression and the clinical characteristics of IDC. RESULTS: The GEO and TCGA databases indicated that UHRF1 was up-regulated in IDC. Consistently, the immunohistochemical specimens showed that the significant overexpression of UHRF1 in IDC, and its expression level showed an increasing trend from ductal carcinomas in situ to IDC. Notably, the increased levels of UHRF1 were closely correlated with estrogen receptor expression, pathological grade, and the prognosis of the disease. In addition, patients with a high UHRF1 expression had a poorer prognosis. CONCLUSIONS: In conclusion, our findings suggested that UHRF1 plays a promoting role in breast tumorigenesis, and the over-expression of UHRF1 could serve as a biomarker for the prognosis in invasive ductal carcinomas in breast cancer.

A novel design to screen chlorogenic acid-producing microbial strains from the environment.

The present study aimed to develop a plate-screening method, based on the specific color development of complexes formed between chlorogenic acid, a valuable plant-derived compound, and aluminum (III), to detect chlorogenic acid-producing microbial strains. Modified media with 0.75 mM aluminum chloride were developed to identify CGA-producing bacteria (based on beef extract agar medium) or fungi (based on the potato dextrose agar medium). Compared with conventional screening, the modified media let to 3.3 times more CGA producers from plants, at 90.9% selective accuracy. Novel chlorogenic acid-biosynthesizing strains included Brevibacillus borstelensis B14, Bacillus amyloliquefaciens B17, Bacillus badius B19, Sphingomonas yabuuchiae N21, Enterobacter tabaci N22, and Lodderomyces elongisporus S216 and P212. Strain S216 produced the highest chlorogenic acid yield (23.39 mg L-1). This study provides a highly efficient and low-cost tool for quick detection and subsequent identification of several newly isolated strains with chlorogenic acid-producing potential.

Field Performance of Transgenic Sugarcane Lines Resistant to Sugarcane Mosaic Virus.

Sugarcane mosaic disease is mainly caused by the sugarcane mosaic virus (SCMV), which can significantly reduce stalk yield and sucrose content of sugarcane in the field. Coat protein mediated protection (CPMP) is an effective strategy to improve virus resistance. A 2-year field study was conducted to compare five independent transgenic sugarcane lines carrying the SCMV-CP gene (i.e., B2, B36, B38, B48, and B51) with the wild-type parental clone Badila (WT). Agronomic performance, resistance to SCMV infection, and transgene stability were evaluated and compared with the wild-type parental clone Badila (WT) at four experimental locations in China across two successive seasons, i.e., plant cane (PC) and 1st ratoon cane (1R). All transgenic lines derived from Badila had significantly greater tons of cane per hectare (TCH) and tons of sucrose per hectare (TSH) as well as lower SCMV disease incidence than those from Badila in the PC and 1R crops. The transgenic line B48 was highly resistant to SCMV with less than 3% incidence of infection. The recovery phenotype of transgenic line B36 was infected soon after virus inoculation, but the subsequent leaves showed no symptoms of infection. Most control plants developed symptoms that persisted and spread throughout the plant with more than 50% incidence. B48 recorded an average of 102.72 t/ha, which was 67.2% more than that for Badila. The expression of the transgene was stable over many generations with vegetative propagation. These results show that SCMV-resistant transgenic lines derived from Badila can provide resistant germplasm for sugarcane breeding and can also be used to study virus resistance mechanisms. This is the first report on the development and field performance of transgenic sugarcane plants that are resistant to SCMV infection in China.

Bioavailability of oral methylnaltrexone increases with a phosphatidylcholine-based formulation.

OBJECTIVE: Methylnaltrexone (MNTX), a peripherally restricted opioid antagonist with mu-opioid receptor selectivity, can reduce opioid activity in the gastrointestinal tract while sparing the pain relief afforded by opioids. Since the bioavailability of oral MNTX is low, it is necessary to explore the oral formulations of MNTX that increase its bioavailability. MATERIALS AND METHODS: An MNTX-phosphatidylcholine complex (MNTX-PC) formulation was prepared. The physicochemical properties of MNTX-PC were analyzed, and its bioavailability was evaluated in rats. After 250 mg/kg of oral MNTX-PC, plasma samples were collected up to 9 h. The concentrations of the compound in rat plasma were quantified using LC/MS/MS. RESULTS: Two MNTX plasma concentration peaks were observed at 120 and 180 min for the MNTX-PC group and control (MNTX in a water solution). Tmax was 180 min, C(max) was 1083.7 ± 293.9 ng/mL, and T(½) was 496 min for the MNTX-PC group. For control, T(max) was 180 min, C(max) was 448.4 ± 126.0 ng/mL, and T(½) was 259 min. The AUC0₋540 min for the MNTX-PC group was 5758.2 ± 1474.2 ngh/mL; for control, 1405.9 ± 447.8 ngh/mL. Thus, the relative bioavailability after the oral administration of MNTX-PC was 410% compared to that of control. CONCLUSION: MNTX-PC formulation significantly enhanced the oral bioavailability of MNTX.

Preparation, characterization and uptake of PEG-coated, muco-inert nanoparticles in HGC-27 cells, a mucin-producing, gastric-cancer cell line.

To prepare polyethylene glycol (PEG)-coated, muco-inert nanoparticles, we investigated the interaction between nanoparticles and mucin, and analyzed the cellular uptake of nanoparticles in HGC-27 cells, which are a mucin-producing, gastric cancer cell line. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were manufactured through a nanoprecipitation/emulsion evaporation technique, and coumarin-6 was encapsulated as a fluorescent marker. We characterized and evaluated the nanoparticles. We examined the cellular uptake of PLGA nanoparticles in HGC-27 cells with confocal laser scanning microscopy and flow cytometry. Nanoparticles prepared using a nanoprecipitation method were 120-150 nm in size, while nanoparticles prepared using an emulsion evaporation method were 400-450 nm in size. EE% of coumarin-6 in the nanoparticles was more than 80%. The anti-adhesive ability of nanoparticles to pig-gastric mucin (PM) was notably affected by PEG coating-density, 10% PEG2000-PLGA nanoparticles and 15% PEG2000-PLGA nanoparticles possessed the strongest resistance to PM. However, the PLGA nanoparticles were strong while the chitosan-loaded nanoparticles were very weak. Our results from both confocal laser scanning microscopy as well as flow cytometry showed that mPEG-PLGA-NPs are rapidly uptaken by mucin-producing HGC-27 cells. The number of 10% mPEG-PLGA-NPs taken up by cells was 1.6- to 2.1-fold higher than PLGA-NPs incubated for the same amount of time. In conclusion, mPEG-PLGA-NPs possess hydrophilic and near-neutrally-charged surfaces that minimize mucoadhesion by reducing hydrophobic or electrostatic interactions. Both the low-PEG MW and the high density of PEG surface coverage are critical for the uptake of mPEG-PLGA-NPs in HGC-27 cells. At the same time, the size of nanoparticles was also very important.

[Transport of mPEG-PLGA nanoparticles across the rat nasal mucosa].

To investigate the effects of particle size, mPEG molecular weight, coating density and zeta potential of monomethoxyl poly(ethylene glycol)-poly(lactic-co-glycolic acid) (mPEG-PLGA) nanoparticles on their transportation across the rat nasal mucosa, mPEG-PLGA-NPs with different mPEG molecular weights (M(r) 1 000, 2 000) and coating density (0, 5%, 10%, 15%) and chitosan coated PLGA-NP, which loaded coumarin-6 as fluorescent marker, were prepared with the nanoprecipitation method and emulsion-solvent evaporation method, and determine their particle size, zeta potential, the efficiency of fluorescent labeling, in vitro leakage rate and the stability with the lysozyme were determined. The effects of physical and chemical properties on the transmucosal transport of the fluorescent nanoparticles were investigated by confocal laser scanning microscopy (CLSM). The result showed that the size of nanoparticles prepared with nanoprecipitation method varied between 120 and 200 nm; the size of nanoparticles prepared with emulsion-solvent evaporation method varied between 420 and 450 nm. Nanoparticles dispersed uniformly; the zeta potential of PLGA-NPs was negative; mPEG-PLGA-NPs was close to neutral; chitosan coated PLGA-NPs was positive; and the efficiency of fluorescent labeling were higher than 80%. In vitro leak was less than 5% within 4 h and nanoparticles were basically stable with lysozyme. The CLSM results show that the transportation efficiency of mPEG-PLGA-NPs with a high PEG coating density and high mPEG molecular weight was significantly higher than that of uncoated PLGA nanoparticles and also that of chitosan coated PLGA-NPs (P < 0.05). The hydrophilcity, zeta potential and particle size of nanoparticles play important roles on the efficiency of mPEG-PLGA nanoparticles to transport across the rat nasal mucosa.

[Transport of PLGA nanoparticles across Caco-2/HT29-MTX co-cultured cells].

The present study is to establish Caco-2/HT29-MTX co-cultured cells and investigate the transport capability of PLGA nanoparticles with different surface chemical properties across Caco-2/HT29-MTX co-cultured cells. PLGA-NPs, mPEG-PLGA-NPs and chitosan coated PLGA-NPs were prepared by nanoprecipitation method using poly(lactic-co-glycolic acid) as carrier material with surface modified by methoxy poly(ethylene glycol) and chitosan. The particle size and zeta potential of nanoparticles were measured by dynamic light scattering. Coumarin 6 was used as a fluorescent marker in the transport of nanoparticles investigated by confocal laser scanning microscopy. The transport of furanodiene (FDE) loaded nanoparticles was quantitively determined by high performance liquid chromatography. Colchicine and nocodazole were used in the transport study to explore the involved endocytosis mechanisms of nanoparticles. Distribution of the tight junction proteins ZO-1 was also analyzed by immunofluorescence staining. The results showed that the nanoparticles dispersed uniformly. The zeta potential of PLGA-NPs was negative, the mPEG-PLGA-NPs was close to neutral and the CS-PLGA-NPs was positive. The entrapment efficiency of FDE in all nanoparticles was higher than 75%. The transport capability of mPEG-PLGA-NPs across Caco-2/HT29-MTX co-cultured cells was higher than that of PLGA-NPs and CS-PLGA-NPs. Colchicine and nocodazole could significantly decrease the transport amount of nanoparticles. mPEG-PLGA-NPs could obviously reduce the distribution of ZO-1 protein than PLGA-NPs and CS-PLGA-NPs. The transport mechanism of PLGA-NPs and mPEG-PLGA-NPs were indicated to be a combination of endocytosis and paracellular way, while CS-PLGA-NPs mainly relied on the endocytosis way. PEG coating could shield the surface charge and enhance the hydrophilicity of PLGA nanoparticles, which leads mPEG-PLGA-NPs to possess higher anti-adhesion activity. As a result, mPEG-PLGA-NPs could penetrate the mucus layer rapidly and transport across Caco-2/HT29-MTX co-cultured cells.