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Whole-genome sequencing technologies provide opportunities to further understand genetic variation among different varieties. Some related genes that are useful for the breeding process could be identified by sequencing technologies. In this study, two peach varieties, Green No. 9 (LH) and its bud mutant Daifei (DT), were analyzed with whole-genome re-sequencing. Approximately 109 million total reads were generated, which covered ∼89% of the peach reference genome. A total of 1 143 757 single nucleotide polymorphisms (SNPs), 169 827 insertion–deletion mutations (InDels), 17 132 structural variations (SVs) and 5 040 copy number variations (CNVs) were detected in Green No. 9 and Daifei. Variant genes were classified by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database. Detected genes such as LOC18768059 (carbohydrate metabolism) and LOC18785045 (anthocyanin biosynthesis), were good candidate genes for exploring the phenotypic variations between Green No. 9 and Daifei. Green No. 9 and its bud mutant Daifei showed obvious differences in phenotypes and variant genetic loci. The detected genomic variations will contribute to explorations of important functional genes and to understanding the genetic basis of peach bud mutations.

Background MCM3AP-AS1 is a recently characterized lncRNA playing an oncogenic role in several cancers. However, its role in lung cancer remains unknown. Here, we aimed to explore the functions of MCM3AP-AS1 in small cell lung cancer (SCLC) and the possible underlying mechanisms. Methods MCM3AP-AS1 and ROCK1 levels in SCLC patients were analyzed by qPCR. RNA pull-down and luciferase assays were performed to analyze the interaction between MCM3AP-AS1 and miR-148a. ROCK1 mRNA and protein levels were detected by qPCR and Western blot, respectively. Cell invasion and migration were analyzed by Transwell assays. Results MCM3AP-AS1 was upregulated in patients with SCLC, and a high MCM3AP-AS1 level was accompanied by a low survival rate. The binding of MCM3AP-AS1 to miR-148a predicted by bioinformatics analysis was verified by RNA pull-down and luciferase assays. However, MCM3AP-AS1 and miR-148a did not affect each other’s expression. ROCK1 was upregulated in SCLC tissues and positively correlated with MCM3AP-AS1. In SCLC cells, MCM3AP-AS1 overexpression increased ROCK1 and promoted cancer cell invasion and migration, while miR-148a overexpression showed the opposite effects and attenuated the effects of MCM3AP-AS1 overexpression on ROCK1 expression and cell behaviors. Conclusions MCM3AP-AS1 sponges miR-148a, thereby increasing SCLC cell invasion and migration via upregulating ROCK1 expression.

AINTEGUMENTA-like (AIL) proteins are members of the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain family of transcription factors involved in plant growth, development, and abiotic stress responses. However, the biological functions of AIL members in pumpkin (Cucurbita moschata Duch.) remain unknown. In this study, we identified 12 AIL genes in the pumpkin genome encoding proteins predicted to be localized in the nucleus. Phylogenetic analysis showed that the AIL gene family could be classified into six major subfamilies, with each member encoding two AP2/ERF domains separated by a linker region. CmoAIL genes were expressed at varying levels in the examined tissues, and CmoANT genes showed different expression patterns under auxin (IAA), 1-naphthylphthalamic acid (NPA), and abscisic acid (ABA) treatments. Ectopic overexpression of CmoANT1.2 in Arabidopsis increased organ size and promoted growth of grafted plants by accelerating graft union formation. However, there was no significant difference at the graft junction for WT/WT and WT/ANT under IAA or NPA treatments. Taken together, the results of this study provide critical information about CmoAIL genes and their encoded proteins, and suggest future work should investigate the functions of CmoANT1.2 in the grafting process in pumpkin.

Long non-coding RNAs (lncRNAs) are crucial in chronic liver diseases, but the specific molecular mechanism of lncRNAs in alcoholic fatty liver (AFL) remains unclear. In this study, we investigated the in-depth regulatory mechanism of mTOR affected by AIRN non-protein coding RNA (lncRNA-AIRN) in the development of AFL. LncRNA-AIRN was highly expressed in the liver tissues of AFL C57BL/6 mice and oleic acid+alcohol (O+A) treated AML-12 cells by using quantitative real-time PCR. RNA pull-down and RNA immunoprecipitation experiments demonstrated that there was an interaction between lncRNA-AIRN and mTOR, and that interference with lncRNA-AIRN could promote the mTOR protein level. Results of cycloheximide-chase assay showed that the protein level of mTOR was decreased with the treatment time after the knockdown of lncRNA-AIRN. Furthermore, the knockdown of lncRNA-AIRN reduced mTOR protein level by promoting the E3 ubiquitin ligase FBXW7-mediated ubiquitination. The lncRNA-AIRN/mTOR axis was involved in the regulation of the mitophagy of O+A treated hepatocytes, which was confirmed by the cell transfection and the MTT assay. SPSS 16.0 was used for analyzing data. The difference between the two groups was analyzed by performing Student’s t-test, and ANOVA was used to analyze the difference when more than two groups. P values < 0.05 were considered to be significantly different. Our findings demonstrated that the knockdown of lncRNA-AIRN influenced mitophagy in AFL by promoting mTOR ubiquitination.

The current dogma in ophthalmology and vision research presumes the intraocular environment to be sterile. However, recent evidence of intestinal bacterial translocation into the bloodstream and many other internal organs including the eyes, found in healthy and diseased animal models, suggests that the intraocular cavity may also be inhabited by a microbial community. Here, we tested intraocular samples from over 1000 human eyes. Using quantitative PCR, negative staining transmission electron microscopy, direct culture, and high-throughput sequencing technologies, we demonstrated the presence of intraocular bacteria. The possibility that the microbiome from these low-biomass communities could be a contamination from other tissues and reagents was carefully evaluated and excluded. We also provide preliminary evidence that a disease-specific microbial signature characterized the intraocular environment of patients with age-related macular degeneration and glaucoma, suggesting that either spontaneous or pathogenic bacterial translocation may be associated with these common sight-threatening conditions. Furthermore, we revealed the presence of an intraocular microbiome in normal eyes from non-human mammals and demonstrated that this varied across species (rat, rabbit, pig, and macaque) and was established after birth. These findings represent the first-ever evidence of intraocular microbiota in humans.

Enterovirus A71 (EV-A71) is an important pathogen of severe hand, foot, and mouth disease (HFMD) in young children. This study aimed to retrospectively analyze the molecular epidemiology and recombination of EV-A71 in mainland China during 1987–2017. Phylogenetic tree showed that besides the previously reported subgenotypes A, B5, C0, C2, C3, and C4, a new subgenotype C6 emerged in mainland China. Recombination analysis indicated that C4 EV-A71 was derived from a common ancestor as a “double-recombinant” virus by intertypic recombination between C EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains in P3 region and intratypic recombination between C and B EV-A71 strains in P2 region. The B5 EV-A71 shared high similarity with C EV-A71 in P1 region while it contained an unidentified sequence in P2 and P3 regions with two possible recombination patterns: one occurred between C4 EV-A71 and CVA3, CVA5, CVA6, CVA10, and CVA12 stains with one breakpoint in 3C, and the other occurred between C1, C2, C3, and C5 EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains with two breakpoints in the 2A/2B junction and 3C. The C2 EV-A71 was probably a recombinant virus between C4 EV-A71 and CVA8 strains with two breakpoints located in the 5′UTR and 2A/2B junction. Moreover, an incredible recombination of C6 EV-A71 occurred between C4 and C2 EV-A71 with multiple breakpoints. Thus, continuous studies on EV-A71 genome characteristics are still useful and essential for monitoring emergence of new viruses and preventing HFMD outbreaks. Supplementary Information The online version contains supplementary material available at 10.1007/s10123-021-00164-2.

Quorum sensing inhibitors (QSIs) can block the quorum sensing system and might be useful in therapy of drug-resistant bacterial infections. To explore the anti-quorum sensing actinomycetes from marine habitats, in this study, a marine actinobacterium exhibiting strong anti-quorum sensing activity was isolated from marine sediment and identified as Nocardiopsis dassonvillei JS106. Effects of different culture media on the bioactivity and metabolites production of JS106 were investigated by using single-factor and orthogonal experiments. The results showed that soybean meal and NaCl were two important factors in the culture medium, and could significantly affect both the bioactivity and metabolites production of JS106. By culturing in the optimized medium containing 10 g/L soluble starch, 10 g/L soybean meal, and 15 g/L NaCl at pH 6.5, both metabolites production and anti-quorum sensing activity of JS106 were significantly increased (302 and 241%, respectively) compared to the original condition. The culture liquid of JS106 also showed strong anti-biofilm activity against Staphylococcus aureus and Pseudomonas aeruginosa with the inhibition rate of 77.94 and 50.56% at the concentration of 20 vol %, while it did not inhibit the growth of planktonic cells. Questiomycin A and a new compound 2-hydroxyacetate-3-hydroxyacetamido-phenoxazine (HHP) were isolated and identified from the cultures of JS106. The anti-quorum sensing activities (IC50) of questiomycin A and HHP against violacein production by Chromobacterium violaceum 12472 were 6.82 and 23.59 μg/mL, respectively. The strong anti-quorum sensing activity of N. dassonvillei JS106 indicated that this strain and its active phenoxazine metabolites could have good potential in anti-quorum sensing related applications.

Rectal cancer is the most common malignant tumor in the digestive system with rapidly metastasis and highly recurrence. Agrin (AGRN) is a proteoglycan involving in a large number of human cancers. However, how AGRN regulates the progression of rectal cancer remains largely unknown. We aimed to determine the biological role of AGRN and its mechanism in rectal cancer. AGRN expression in rectal cancer tissues was detected based on TCGA. The survival curve was plotted using the Kaplan-Meier method. qRT-PCR and western blot were utilized to examine the expression level of AGRN in cells. Cell proliferation, clonogenic ability, invasion, and migration of rectal cancer cells were analyzed by CCK-8, colony formation and transwell experiments. GSEA was employed for the analysis of the potential pathways-related with AGRN in rectal cancer. The activity of WNT pathway was determined by western blot. AGRN expression was dramatically increased in rectal cancer, and its up-regulation was associated with poorer prognosis of rectal cancer patients. AGRN expression was an independent factor for the prognosis of rectal cancer. AGRN inhibition suppressed rectal cancer cell growth, invasion, and migration, whereas AGRN overexpression facilitated these behaviors of rectal cancer cells

Accurate knowledge of species distributions and population dynamics is the basis for conservation biology. However, for certain species with unique life histories and very small populations, monitoring species distributions and population dynamics is extremely difficult. Recently, newly developed eDNA technology has been widely used in species monitoring, biodiversity assessments, and biomass assessments. In this research, we studied Fenneropenaeus chinensis in the Bohai Sea. We used a 0.45 μm glass fiber filter combined with a vacuum to filter 2 L seawater samples for eDNA enrichment from 54 stations in June and 60 stations in August. A DNeasy Blood and Tissue kit was used to extract the eDNA. After DNA extraction, the specific primers and probes of the mitochondrial DNA COI gene of F. chinensis were designed. Real-time quantitative PCR was used to qualitatively and quantitatively analyze the eDNA in the Bohai Sea. The temporal and spatial distribution of F. chinensis was detected by the eDNA technique. The relationship between the eDNA copy number and trawl-netted F. chinensis biomass was explored. The applicability and sensitivity of eDNA technology in marine crustacean research were verified. The results showed that the mtDNA COI gene of F. chinensis was successfully amplified in the 54 water samples collected in June, and the detection rate reached 100%. Detections occurred in only 23 of the 60 stations sampled in August. F. chinensis had a detection rate of only 38%. The copy number of eDNA obtained by real-time PCR was fitted to the density of F. chinensis captured by bottom trawling. There was not a significant positive correlation between copy number and biomass. The results obtained through this research will provide a method and theoretical basis for the application of eDNA technology in marine crustacean research. Keywords: Bohai Sea, eDNA, Fenneropenaeus chinensis, Spatial and temporal distribution, Biomass

Nitrate accumulation causes long-time threat to aquatic animals in recirculating aquaculture system (RAS); thus, nitrate removal is also required in RASs. However, the lack of carbon sources makes denitrification difficult to function. Nitrate removal performance of an aerobic denitrifying and extracellular polyhydroxyalkanoate depolymerase–producing bacterium, Pseudomonas sp. AOB-7, using polyhydroxyalkanoate (PHA) granules as a solid sustained-release carbon source in RAS was evaluated. With the initial nitrate-N concentration of 140 mg/L, the high denitrification rates of 0.056 g NO3−-N L−1 day−1 and 0.035 g NO3−-N L−1 day−1 were achieved in denitrification medium containing poly-β-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), respectively. Significant erosions and pits formed on the surface of the granules made them a good biofilm carrier for AOB-7, and 3-hydroxybutyrate (3-HB) monomer was the major product released to aquatic phase, which was benefit to animals. SEM photos showed that AOB-7 entered and attached on the inside of the PHA particle holes. A 4-week application trial was conducted to reveal the effects of PHB (AOB-7) denitrifying agent and 3-HB produced on growth of zebrafish (Brachydanio rerio) by adding 0.1% (w/v) PHB (AOB-7) denitrifying agent. Result indicated that PHB (AOB-7) denitrifying agent can significantly reduce nitrate-N content in RASs. Compared with the control group, feed coefficient ratio reduced by 18% and weight gain ratio increased by 29% in the PHB (AOB-7) denitrifying agent group. 3-HB monomer produced during the denitrification was speculated to function as a prebiotic and promote zebrafish growth. • AOB-7 showed a good aerobic denitrifying ability on PHA granules as sustained-release C source. • PHB (AOB-7) denitrifying agent can significantly reduce nitrate content in RAS. • R-3-HB monomer was the major product released to aquatic phase and function as a prebiotic.

BackgroundGlioblastoma multiforme (GBM) is characterized by widespread genetic and transcriptional heterogeneity. Aberrant DNA methylation plays a vital role in GBM progression by regulating gene expression. However, little is known about the role of methylation and its association with prognosis in GBM. Our aim was to explore DNA methylation-driven genes (DMDGs) and provide evidence for survival prediction and individualized treatment of GBM patients.MethodsUse of the MethylMix R package identified DMDGs in GBM. The prognostic signature of DMDGs based on the risk score was constructed by multivariate Cox regression analysis. Receiver operating characteristics (ROC) curve and C-index were applied to assess the predictive performance of the DMDG prognostic signature. The predictive ability of the multigene signature model was validated in TCGA and CGGA cohorts. Finally, the role of DMDG β-Parvin (PARVB) was explored in vitro.ResultsThe prognostic signature of DMDGs was constructed based on six genes (MDK, NMNAT3, PDPN, PARVB, SERPINB1, and UPP1). The low-risk cohort had significantly better survival than the high-risk cohort (p < 0.001). The area under the curve of the ROC of the six-gene signature was 0.832, 0.927, and 0.980 within 1, 2, and 3 years, respectively. The C-index of 0.704 indicated superior specificity and sensitivity. The six-gene model has been demonstrated to be an independent prognostic factor for GBM. In addition, joint survival analysis indicated that the MDK, NMNAT3, PARVB, SERPINB1, and UPP1 genes were significantly associated with prognosis and therapeutic targets for GBM. Importantly, our DMDG prognostic model was more suitable and accurate for low-grade gliomas. Finally, we verified that PARVB induced epithelial-mesenchymal transition partially through the JAK2/STAT3 pathway, which in turn promoted GBM cell proliferation, migration, and invasion.ConclusionThis study demonstrated the potential value of the prognostic signature of DMDGs and provided important bioinformatic and potential therapeutic target data to facilitate individualized treatment for GBM, and to elucidate the specific mechanism by which PARVB promotes GBM progression.

IntroductionOx-LDL (oxidized low densitylipoprotein)-induced endothelial cell injury and dysfunction of vascular smooth muscle cell played critical roles in the development of atherosclerosis (AS). Tropomyosin 2 (TPM2) was indicated to be implicated in cardiac diseases, but its critical role and regulation mechanism in AS progression had not yet been elucidated.Material and methodsThe expression of TPM2 was investigated in AS tissues. Ox-LDL was used to construct an AS in vitro model based on endothelial and vascular smooth muscle cells (HAEC and VSMC). Overexpression assay was performed to evaluate the role of TPM2 in AS. Meanwhile, Narciclasine treatment analysed the involvement of RhoA pathway.ResultsTPM2 was dramatically upregulated both in AS tissues and ox-LDL-induced HAEC cells. The overexpression of TPM2 attenuated ox-LDL-stimulated cell growth depression, inflammatory and adhesive responses in HAEC, as well as oxidative stress and mitochondrial dysfunction. Besides, VSMC, impacted by TPM2-overexpressed HAEC, showed alleviated cellular processes which was abnormally activation induced by ox-LDL. Furthermore, the depressed activation of RhoA pathway was found in TPM2-overexpressed HAEC and activating the signaling rescued these effects of TPM2 exerted on ox-LDL-stimulated HAEC and VSMC.ConclusionsTPM2 had an advantageous impact on ox-LDL-induced AS progression in vitro via mediating RhoA pathway. This evidence might be contributed to the therapy of AS.

The use of heterografts is widely applied for the production of several important commercial crops, but the molecular mechanism of graft union formation remains poorly understood. Here, cucumber grafted onto pumpkin was used to study graft union development, and genome-wide tempo-spatial gene expression at the graft interface was comprehensively investigated. Histological analysis suggested that resumption of the rootstock growth occurred after both phloem and xylem reconnection, and the scion showed evident callus production compared with the rootstock 3 days after grafting. Consistently, transcriptome data revealed specific responses between the scion and rootstock in the expression of genes related to cambium development, the cell cycle, and sugar metabolism during both vascular reconnection and healing, indicating distinct mechanisms. Additionally, lower levels of sugars and significantly changed sugar enzyme activities at the graft junction were observed during vascular reconnection. Next, we found that the healing process of grafted etiolated seedlings was significantly delayed, and graft success, xylem reconnection, and the growth of grafted plants were enhanced by exogenous glucose. This demonstrates that graft union formation requires the correct sugar content. Furthermore, we also found that graft union formation was delayed with a lower energy charge by the target of rapamycin (TOR) inhibitor AZD-8055, and xylem reconnection and the growth of grafted plants were enhanced under AZD-8055 with exogenous glucose treatment. Taken together, our results reveal that sugars play a positive role in graft union formation by promoting the growth of cucumber/pumpkin and provide useful information for understanding graft union healing and the application of heterografting in the future.

BackgroundDepression, one of the most frequently-occurring psychiatric disorders worldwide, is a significant inflammatory disorder. The polyphenol curcumin (Cur), which is extracted from Curcuma longa, has marked anti-inflammatory and anti‑oxidative effects against inflammatory diseases. However, whether Cur has antidepressant effects and the possible mechanisms, are unclear. The present study aimed to assess Cur’s beneficial effects on depressive-like behaviors using a chronic unpredictable mild stress (CUMS) model and its possible molecular mechanisms. MethodsWe performed CUMS treated Sprague Dawley (SD) rats as a model of depression. Behavioral observations were performed by sucrose preference test (SPT), force swimming test (FST) and tail suspension test (TST). Hippocampal expression of oxidative stress markers and inflammatory cytokines were measured with ELISA. Hippocampal expression of high-mobility group box 1 (HMGB1), IL-1β, TNF-α and IL-6 were determined with quantitative PCR analyses and immunofluorescent staining. Hippocampal Toll-like receptor 4 (TLR4) and NF-κB activation were examined with Western blotting analysis.ResultsRats subjected to CUMS demonstrated marked depressive-like behavior (decreased locomotor activity and sucrose intake, and prolonged immobility). Their levels of oxidative stress and inflammatory cytokines increased significantly, and their levels of phosphorylated nuclear factor kappa-B (NF-κB), toll-like receptor 4 (TLR4), and HMGB1, also increased in the hippocampus. The changes were ameliorated significantly by treatment with Cur (50, 100 mg/kg) to varying degrees.Conclusion This study demonstrated that Cur has a potent antidepressant effect via the HMGB1/TLR4/NF-κB pathway, suggesting that Cur might be a promising therapeutic drug for depression.

Background Nuclear Factor Y (NF-Y) is a heterotrimeric complex composed of three unique subunits: NF-YA, NF-YB, and NF-YC. The NF-Y transcription factor complex binds to the CCAAT box of eukaryotic promoters, playing a vital role in various biological processes in plants. However, the NF-Y gene family has not yet been reported from the peach genome. The current study identified and classified candidate peach NF-Y genes for further functional analysis of this family. Results The current study identified 24 Nuclear Factor Y (NF-Y) transcription factor subunits (6 NF-YA, 12 NF-YB, and 6 NF-YC subunits) in peach. These NF-Y subunits were described with respect to basic physicochemical characteristics, chromosome locations, gene structures, and conserved domains. Based on an analysis of the phylogenetic relationships among peach NF-Ys, six pairs of paralogous NF-Ys were detected. The expansion of the peach NF-Y family occurred by segmental and tandem duplication. Phylogenetic gene synteny of NF-Y proteins was observed between peach and Arabidopsis, and five pairs of paralogous NF-Y proteins from peach and Arabidopsis were identified. Twenty-four peach NF-Ys displayed a diversity of tissue expression patterns. In addition, drought-responsive cis-elements were observed in peach NF-Y promoters, and 9 peach NF-Y genes were shown to distinctly increase their transcript abundances under drought stress. Conclusions This study identified 24 NF-Y genes in the peach genome and analysed their properties at different levels, providing a foundation for researchers to understand this gene family in peach. The up-regulation of 9 NF-Y genes under drought stress indicates that they can serve as candidate functional genes to further study drought resistance in peach. Electronic supplementary material The online version of this article (10.1186/s12864-019-5968-7) contains supplementary material, which is available to authorized users.

Celastrus orbiculatus is a medicinal plant belonging to the Celastraceae family. In this survey on the secondary metabolites of plants for obtaining antitumor substances, the chemical constituents of the stems of C. orbiculatus were investigated. Nortriptonoterpene (1), a new C19-norabietane diterpenoid, together with six other known abietane-type diterpenoids (2–7) and five known kaurane-type diterpenoids (8–12) were isolated and identified from the EtOAc extract of C. orbiculatus. Their structures were elucidated on the basis of extensive spectroscopic methods, including UV, IR, HR-ESI–MS, ECD, and NMR experiments, and by comparison with literature data. Compound 1 is a new C19-norabietane diterpenoid with 19 carbons. All compounds except for 10 and 11 were isolated from C. orbiculatus for the first time. The NMR data of 9 were reported for the first time. Compounds 1, 7 and 11 showed cytotoxicities against SGC-7901 with IC50 values of 63.2, 80.9 and 56.7 μM, respectively.

Objective Irisin is a myokine that greatly affects energy expenditure and systemic metabolism. While thyroid hormone is likely associated with irisin, a direct relationship remains to be fully elucidated. This study aimed to investigate plasma irisin levels in Chinese patients with hypothyroidism. Methods A total of 155 subjects were divided into the hypothyroidism group or the control group. Fifty-seven patients in the hypothyroidism group received levothyroxine treatment. Baseline irisin levels were measured in the two groups and post-treatment levels were measured in the hypothyroidism group. Results Irisin levels were significantly lower in the hypothyroidism group than in the control group. In the hypothyroidism group, irisin levels were positively associated with free triiodothyronine and free thyroxine levels, and negatively associated with thyrotropin levels. In the hypothyroidism group, irisin levels were significantly increased after levothyroxine treatment. Multiple linear regression models showed that total cholesterol and free thyroxine levels were the only significant predictors of serum irisin levels. Conclusions Irisin levels are decreased in patients with hypothyroidism. Our results suggest that decreased irisin levels are directly associated with reduced thyroid hormone levels. These values may be restored after levothyroxine treatment in Chinese patients with hypothyroidism.

To determine the effect of grafting method (hole insertion grafting, HIG; tongue approach grafting, TAG; and spliced grafting, SG) on graft union formation in cucumber grafted onto bottle gourd rootstock, an anatomical and physiological study was carried out during the healing stage. The results showed that the TAG method resulted in an obviously higher daily growth rate than the other grafting methods 7 days after grafting (DAG), with significantly higher values in the scion than in the rootstock. The isolation layer, callus formation, and vascular connection all occurred at the graft junction when using HIG, TAG, and SG. Vascular bridging first occurred between scion and rootstock using HIG or TAG at 5 DAG; this process was delayed with SG. The activities of superoxide dismutase (SOD), POD (peroxidase), CAT (catalase), PAL (Phenylalanine ammonia-lyase), and PPO (polyphenol oxidase) were significantly enhanced in the graft union during the healing process when different grafting methods were used. Compared with HIG and SG, the POD and CAT activities and total phenolic compounds were higher with TAG at 7 DAG; a reduction in PPO activity and lignin content were also overserved at the graft with TAG at 7 DAG. These results suggest that the association of TAG with a higher growth rate during the healing period is linked with the earlier improvement in vascular bundle connection that may be caused by increased antioxidant activities and lower lignin content at the graft junction. These morphological and physiological differences may provide valuable information for revealing the graft union healing process.

The coexistence of mycotoxins in agricultural products poses a serious threat to food safety. This study developed a dual immunochromatographic test strips (DICTS) method based on double antibodies labeled with time-resolved fluorescent microspheres (TRFM) to realize simultaneous rapid detection of aflatoxin M1 (AFM1) and ochratoxin A (OTA) in milk. As bridge antibody, the polyclonal antibody (pAb) was first conjugated with the TRFM and then with the monoclonal antibody (mAb). Meanwhile, a biotin-streptavidin system was introduced to replace the traditional goat anti-mouse Immunoglobulin G, thus providing a stable signal on the control line. After optimization, the detection limit of AFM1 and OTA in milk was respectively 0.018 and 0.036 ng/mL. The recoveries of intraassay and interassay experiments ranged from 89.65% to 103.99%. The accuracy, repeatability, and specificity of the developed TRFM-DICTS were estimated. The results of TRFM-DICTS showed a high consistency with those of the ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15N2 from (15NH4)2SO4. Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2.

Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature. Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bo nd of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave AP-containing ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. Phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs. KEY POINTS: We first reported a novel DNA glycosylase with bifunctionality. Tba UDG247 possesses an AP lyase activity.

Background Low temperature severely depresses the uptake, translocation from the root to the shoot, and metabolism of nitrate and ammonium in thermophilic plants such as cucumber (Cucumis sativus). Plant growth is inhibited accordingly. However, the availability of information on the effects of low temperature on nitrogen transport remains limited. Results Using non-invasive micro-test technology, the net nitrate (NO3−) and ammonium (NH4+) fluxes in the root hair zone and vascular bundles of the primary root, stem, petiole, midrib, lateral vein, and shoot tip of cucumber seedlings under normal temperature (NT; 26 °C) and low temperature (LT; 8 °C) treatment were analyzed. Under LT treatment, the net NO3− flux rate in the root hair zone and vascular bundles of cucumber seedlings decreased, whereas the net NH4+ flux rate in vascular bundles of the midrib, lateral vein, and shoot tip increased. Accordingly, the relative expression of CsNRT1.4a in the petiole and midrib was down-regulated, whereas the expression of CsAMT1.2a–1.2c in the midrib was up-regulated. The results of 15N isotope tracing showed that NO3−-N and NH4+-N uptake of the seedlings under LT treatment decreased significantly compared with that under NT treatment, and the concentration and proportion of both NO3−-N and NH4+-N distributed in the shoot decreased. Under LT treatment, the actual nitrate reductase activity (NRAact) in the root did not change significantly, whereas NRAact in the stem and petiole increased by 113.2 and 96.2%, respectively. Conclusions The higher net NH4+ flux rate in leaves and young tissues may reflect the higher NRAact in the stem and petiole, which may result in a higher proportion of NO3− being reduced to NH4+ during the upward transportation of NO3−. The results contribute to an improved understanding of the mechanism of changes in nitrate transportation in plants in response to low-temperature stress.

The pollution caused by estrogens in the environment and food has received increasing attention. It is still challenging for on-site immunochromatographic assay (ICA) detection of estrogens. The performance of the prepared probes plays a decisive role in the sensitivity and stability of the ICA system. The published probes usually directly couple the detection antibody to the label, ignoring the influence of the label on the activity of the antibody. In this study, 17β-estradiol (E2) was used as a model analyte for the ICA system. Two universal probes were constructed based on quantum dot nanobeads (QBs), recombinant protein A (SPA, from Staphylococcus aureus), and rabbit anti-mouse immunoglobulin G antibody (anti-IgG). The probes were prepared by coupling QBs with SPA, releasing anti-E2 monoclonal antibody (mAb), and maintaining its activity. The prepared universal probes can orient recognize the Fc region of mAb and fully expose its Fab region, improving the detection sensitivity of the ICA system. The free anti-E2 mAb and the universal probe (QBs@SPA or QBs@SPA@anti-IgG) were used as the detection antibodies and signal donors, respectively. The results show that the proposed ICA based on QBs@SPA and QBs@SPA@anti-IgG probes could detect E2 with IC50 of 8.83 and 0.93 ng/mL, respectively, within 15 min under optimal conditions. The recovery results of ICA based on QBs@SPA and QBs@SPA@anti-IgG probes showed good agreement with the findings of the high-performance liquid chromatography (HPLC) analysis for spiked samples. The developed ICA system based on universal probes was superior in terms of sensitivity, rapidity, and applicability, and held great promise for its implementation in detecting environmental and food small-molecule pollutants.

Nowadays, contamination by polycyclic aromatic hydrocarbons (PAHs) has become a serious problem all over the world; in particular, high-molecular-weight PAHs (HWM PAHs, four to seven rings) are more harmful to human health and environment due to their more complex structure and metabolic pathway. Biodegradation of PAHs with six or more rings, such as indeno[1,2,3-cd]pyrene (IcdP), was rarely described. An IcdP-degrading strain, Rhodococcus aetherivorans IcdP1, was isolated from HWM PAH-contaminated soil. It could grow on and efficiently degrade various HWM PAHs, such as IcdP, benzo[a]pyrene, and benzo[j]fluoranthene. It showed highest degrading ability toward IcdP (> 70% within 10 days). The IcdP degradation was initiated by ring hydroxylation with multiple pathways, including the hydroxylation at the 1,2 and 7,8 positions, according to the relevant metabolites detected, e.g., cyclopenta[cd]pyrene-3,4-dicarboxylic acid and 2,3-dimethoxy-2,3-dihydrofluoranthene. The transcriptional patterns of the genes encoding ring-hydroxylating oxygenases (RHOs) and cytochrome P450 monooxygenases (CYP450s) under the induction of IcdP, pyrene, and benzo[b]fluoranthene (BbF) were compared to determine the key initial RHOs in the conversion of IcdP. The expression of genes encoding RHOs 1892–1894, 1917–1920, and 4740–4741 was induced strictly by IcdP, and the amino acid sequences of these proteins showed very low identities with their homologs. These results suggested that IcdP was degraded through a dioxygenation-initiated metabolism pattern, and RHOs 1892–1894, 1917–1920, and 4740–4741 responded to the initial ring cleavage of IcdP through 1,2-dihydrodiol or 7,8-dihydrodiol. The studies would contribute to the understanding of the molecular mechanism of initial degradation of IcdP.

Background This study aims to investigate clinical significance of HMGB1 in neonatal patients with necrotizing enterocolitis (NEC). Methods This observational study enrolled a total of 106 stage II-III NEC neonatal patients, who were admitted in our hospital from March 2014 to March 2019. In addition, 99 suspected NEC patients and 200 healthy controls were included. The serum levels of HMGB1, I-FABP, and inflammatory factors CRP, IL-1β, IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). Then, the demographic data and clinical characteristics of all patients were collected. Statistical analysis was conducted to determine the correlation between HMGB1 and the clinical characteristics. Results No significant difference was found in the basic characteristics of NEC patients and healthy controls, except for birth weight and gestational age. The expression levels of HMGB1, I-FABP, and inflammatory factors IL-1β, IL-6 and TNF-α were significantly higher in NEC patients, when compared to healthy controls. The serum levels of HMGB1, I-FABP, IL-1β and IL-6 markedly increased in stage II-III NEC patients, when compared to stage I NEC patients. The Pearson’s analysis revealed a positive correlation between HMGB1 and I-FABP, HMGB1 and IL-1β, and HMGB1 and IL-6. The ROC curve revealed that both HMGB1 and I-FABP can potentially be used as diagnostic factors for NEC. The logistic multivariate regression revealed that I-FABP, IL-1β and IL-6 are independent risk factors for mortality in neonatal NEC patients. Conclusions Serum HMGB1 levels are upregulated in neonatal NEC patients, and these are correlated with the patient’s prognosis.

Hydroxylamine is a key intermediate of microbial ammonia oxidation and plays an important role in the biogeochemical cycling of N-compounds. Hydroxylamine is oxidized to NO or N2O by hydroxylamine oxidases or cytochrome P460 from heterotrophic or autotrophic bacteria, but its enzymatic oxidation to N2 has not yet been observed. Here, we report on the discovery of a novel oxidase that converts hydroxylamine to N2 from the newly isolated heterotrophic nitrifier Alcaligenes strain HO-1. Strain HO-1 accumulated hydroxylamine and produced N2 from ammonia oxidation. Using transcriptome analysis and heterologous expression via fosmid library screening, we identified three genes (dnfABC) of strain HO-1 that enabled E. coli cells not only to produce hydroxylamine from 15N-labelled ammonium but also to further convert it to 15N2. The three genes were individually cloned and expressed, and their translational products DnfA, DnfB, and DnfC were purified. In vitro DnfA bound to hydroxylamine and catalyzed the conversion of hydroxylamine to N2 in the presence of FAD, NADH and O2. Thus, DnfA was identified as a novel hydroxylamine oxidase and catalyzed a previously unknown N-N bond forming reaction with a yet-to-be discovered mechanism. DnfA homologs were detected in different bacterial groups, suggesting that hydroxylamine oxidation to nitrogen might occur in additional microbial taxa.

Summary Glucosinolates (GSs) are sulfur-containing secondary metabolites characteristic of cruciferous plants [ 1 , 2 ]. Their breakdown products, isothiocyanates (ITCs), are released following tissue disruption by insect feeding or other mechanical damages [ 3 , 4 ]. ITCs repel and are toxic to generalist herbivores, while specialist herbivores utilize the volatile ITCs as key signals for localizing host plants [ 5 , 6 ]. However, the molecular mechanisms underlying detection of ITCs remain open. Here, we report that in the diamondback moth Plutella xylostella, a crucifer specialist, ITCs indeed drive the host preference for Arabidopsis thaliana, and the two olfactory receptors Or35 and Or49 are essential for this behavior. By performing gene expression analyses, we identified 12 (out of 59 in total) female-biased Ors, suggesting their possible involvement in oviposition choice. By ectopically expressing these Ors in Xenopus oocytes and screening their responses with 49 odors (including 13 ITCs, 25 general plant volatiles, and 11 sex pheromone components), we found that Or35 and Or49 responded specifically to three ITCs (iberverin, 4-pentenyl ITC, and phenylethyl ITC). The same ITCs also exhibited highest activity in electroantennogram recordings with female antennae and were the strongest oviposition stimulants. Knocking out either Or35 or Or49 via CRISPR-Cas9 resulted in a reduced oviposition preference for the ITCs, while double Or knockout females lost their ITC preference completely and were unable to choose between wild-type A. thaliana and a conspecific ITC knockout plant. We hence conclude that the ITC-based oviposition preference of the diamondback moth for its host A. thaliana is governed by the cooperation of two highly specific olfactory receptors.

A sponge-associated bacterium, which was identified as Psychrobacter sp. in this study, was found with high activity against biofilm formation of benthic diatoms, including Amphora sp., Nitzschia closterium, Nitzschia frustulum, and Stauroneis sp. The activity against diatom biofilm formation by the tested strain was confirmed mostly in the culture supernatant and could be extracted using organic solvents. Treatment with its supernatant crude extract significantly reduced the cells of Stauroneis sp. forming biofilm and slightly increased the cells floating in the culture medium, which results in the ratio of biofilm cell/floating cell altering from 0.736 in control to 0.414 in treatment. Use of the supernatant crude extract led to increased production of extracellular polymeric substances (EPSs) by diatom Stauroneis sp. from 16.66 to 41.59 (g/g cell dry weight). The increase in EPS production was mainly contributed by soluble EPS (SL-EPS) and followed by the EPS that was tightly bound to biofilm cells (BF-TB-EPS). In addition, the supernatant crude extract caused significant changes in the monosaccharides composition of the EPS of Stauroneis sp. Specifically, glucuronic acid (Glc-A) and N-acetyl-D-glucosamine (Glc-NAc) in BF-TB-EPS were 55% fold decreased and 1219% fold increased, respectively. Based on our findings, we proposed that these changes in monosaccharides composition might lead to a decreased biofilm formation efficiency of diatom.

Haemophilus parasuis infection is of considerable economic importance in the swine industry due to high morbidity and mortality in naive swine populations. Accurate detection and identification of the causative agent are difficult, yet necessary, for disease control. In this study, a simple and rapid method of cross-priming amplification (CPA) with a vertical flow (VF) visualization strip was established to detect H. parasuis. The reaction can specifically identify 15 serovar reference strains and 57 clinically isolated strains of H. parasuis, with a detection limit of 14CFU. The performance of the CPA-VF assay was evaluated and compared with that of species-specific PCR by testing 62 clinical culture-positive specimens of H. parasuis. The entire process, from specimen processing to analysis of the results, can be completed in 2h without a complicated apparatus. The convenience and speed of the CPA-VF assay in this study make it a suitable choice for epidemiological investigation and point-of-care testing (POCT) for H. parasuis infection.

Clonal integration facilitates the growth and reproduction of clonal plants by providing the ability to share resources among ramets in heterogeneous environments. The benefits of clonal integration for plant growth may depend on a contrast in resource availability and may encounter costs, especially when a young part of the clone is growing across a border between richer and poorer conditions than the old part. We studied a clonal amphibian plant growing across a border between an aquatic and a terrestrial ecosystem, which typically differ in the availability of resources. We asked whether the young part of the clone is supporting the old part with phosphorus and whether this support has costs. We performed an experiment with Alternanthera philoxeroides where plants grow from water to a terrestrial habitat. The terrestrial habitat had either a low or high phosphorus supply, and the connection between the old and young parts of the clone was either left intact or split. We determined that the young part of the clone growing in a terrestrial habitat supported the old part with phosphorus when growing on a substrate rich in phosphorus. We have found no cost of this resource translocation; on the contrary, whole clones increased not only their accumulation of phosphorus, but also of nitrogen. Our study shows how an amphibian plant may profit from heterogeneous habitats by resource sharing in a clonal network.

Since bacterial quorum sensing system has been verified to regulate many microbial physiological activities, such as virulence factor production and biofilm formation in pathogens, blocking quorum sensing has being recognized as a viable approach for the development of novel therapeutics in treating bacterial infections. In this study, quorum quenching activities of marine actinomycetes isolated from seawater sample were investigated. Out of 56 morphological different strains, 5 strains had been found exhibiting anti-quorum sensing activity against the violacein production of Chromobacterium violaceum 12472. One highly active strain HY026 was identified as Streptomyces parvulus based on the 16S rDNA sequence analysis and morphological features. Spent culture medium of HY026 could also significantly inhibit the biofilm formation of four bacteria. One major active compound named F1-4, accounting for 26.3% of crude extracts, was obtained from HY026 cultures and identified as actinomycin D based on the analysis of the NMR and MS. F1-4 could significantly inhibit the violacein production of C. violaceum with the inhibition rate of 64.9% but not affect the bacterial growth at the concentration of 12.5 µg/mL. F1-4 could also inhibit the prodigiosin production of Serratia proteamaculans 657 with the pigment inhibition zone of 13.5 mm at concentration of 25 µg/disc without affecting the bacterial growth. Another pure compound, cyclic (4-hydroxy-Pro-Phe) was also isolated and identified from this actinomycetes strain. The higher production and easier isolation of actinomycin D from S. parvulus HY026 indicated that this strain could be a potential source of quorum sensing inhibitors.

Decoquinate ( 1 ), an old and inexpensive coccidiostat, exhibited potent antimalarial activity, however, its antischistosomal activity against Schistosoma japonicum has not yet been evaluated. Based on decoquinate, a series of decoquinate derivatives was designed, synthesized, evaluated as a new class of antischistosomal agents against S. japonicum adult worms in vitro . Among them, compound 15 killed 100% of adult S. japonicum in 72 h at the concentration of 10 μmol/L in vitro , exhibited stronger worm-killing activity than PZQ in vitro and could serve as a promising lead compound to develop new antischistosomal agents.

The bacterium Aeromonas salmonicida is the causative agent of furunculosis, a systemic, ubiquitous disease of fish in the salmon family, characterized by high mortality and morbidity. Probiotics are a promising approach for prevention of furunculosis in aquaculture. A bacterial strain with anti-A. salmonicida properties, Bacillus velezensis V4, was isolated and the mechanisms underlying these properties were investigated. Anti-A. salmonicida compounds present in cell-free supernatant of V4 were purified and structurally identified as members of the iturin, macrolactin, and difficidin groups. The compounds contributed jointly to inhibition of A. salmonicida, and the diversity of the compounds was related to the versatility of their mode of action. Addition of the compounds to A. salmonicida cell suspensions reduced cell density. Analyses by confocal microscopy and scanning electron microscopy revealed cell membrane disruption, deletion of cellular content, and cell lysis of A. salmonicida. The V4 genome was sequenced, and gene clusters involved in synthesis of anti-Aeromonas compounds were detected and identified. A possible probiotic effect on growth performance of Oncorhynchus mykiss (rainbow trout) was investigated by addition of 0, 1, and 3 % (v/w) V4. Relative to control, mortality was reduced 27.25 % in the 1 % addition group and 81.86 % in the 3 % addition group. Feed coefficient ratio was reduced 19.49 % and weight gain ratio was increased 71.22 % in the 1 % addition group. Our findings demonstrate that V4 is an effective probiotic strain in O. mykiss and has clear potential for both control of furunculosis and growth promotion of aquaculture animals.

Aim The present study was conducted to determine whether two important signalling molecules of store-operated channel (SOC), stromal interaction molecule 1 (STIM1) and Orai1, were involved in glomerular podocyte injury. We explored the effects of STIM1/Orai1 overexpression on podocyte associated proteins and cell permeability. Methods The expression of STIM1 and Orai1 were examined in the renal cortex of adriamycin-induced nephropathy mice by real-time RT-PCR. The recombinant plasmid of STIM1/Orai1, identified by restriction enzyme digestion and PCR, was transfected into MPC5 cells via lipofectamine 2000. The transfecting efficiency was observed by a fluorescence microscope. RT-PCR and Western blotting were used to evaluate the expression levels of STIM1, Orai 1 and some podocyte-associated molecules in the transfected MPC5 cells. In addition, we examined the diffusion of FITC-dextran across the podocyte monolayer to investigate whether STIM1/Orai1 overexpression could affect cell permeability. Results We found that the mRNA levels of STIM1 and Orai1 were increased in adriamycin-induced nephropathy mice. STIM1/Orai1 overexpression significantly decreased the expression of podocin and CD2-associated protein (CD2AP), whereas it increased the expression of α-actinin-4. The permeability was significantly increased in the STIM1/Orai1 overexpression group. Conclusion Our findings suggested that STIM1/Orai1 overexpression could affect the cell permeability and the expression of partial podocyte-associated proteins, which may ultimately result in podocyte injury.

OBJECTIVE Metabolic activity and tumor burden are significant for prognosis and metastasis of non-small cell lung cancer (NSCLC), including maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Chemotherapy resistance is a great challenge for treating NSCLC patients and is also closely related with several biomarkers such as epidermal growth factor receptor (EGFR), p53, and excision repair cross-complementing group 1 protein (ERCC1). Our purpose was to determine the correlation between positron emission tomography/computed tomography (PET/CT) parameters and tumor markers-related chemotherapy resistance in NSCLC. METHODS Forty-six NSCLC chemotherapy-naive patients were enrolled. The SUVmax, MTV, and TLG were calculated by PET/CT imaging, and expression of EGFR, p53, and ERCC1 were analyzed by immunohistochemistry on tissues. SUVmax, MTV, and TLG compared for their performance in predicting the expression of EGFR, p53, and ERCC1 were illustrated with statistical analysis. RESULTS SUVmax was significantly correlated with p53 expression (P=0.001), and MTV and TLG were significantly associated with ERCC1 (P=0.000; P=0.000). Furthermore, multiple stepwise regression analysis revealed that SUVmax was the primary predictor for p53, MTV and TLG was the primary predictor for ERCC1. SUVmax had a sensitivity of 91% and specificity of 50% for the detection of p53 positive. The sensitivities of MTV and TLG were 83% and 80%, and specificities were 69% and 75% for the detection of ERCC1 positive, respectively. When we suggested p53 or ERCC1 positive, the cutoff value of SUVmax, MTV, and TLG were 7.68, 23.62, and 129.65 cm3, respectively. CONCLUSION SUVmax, MTV, and TLG were closely associated with p53 and ERCC1' expressions. Therefore, 18F-fludrodeoxyglucose PET/CT could be a new way of predicting p53 or ERCC1-related chemotherapy effect in NSCLC patients with more convenience.

Gustatory receptors (GRs) are crucial in the peripheral coding of the non-volatile compounds in insects, and thus play important roles in multiple behaviors including feeding, mating, and oviposition. However, little research has been done on GRs in lepidopteran pests. In the current work with Spodoptera litura, an important worldwide crop&rsquo, s pest, a candidate fructose GR gene (SlitGR8) was cloned in full length, and its spatial and temporal expression profiles were determined by quantitative real-time PCR (qPCR). It revealed that SlitGR8 was highly expressed in antennae of both male and female adults, as well as in larva of first, fifth and sixth instar. Functional analyses were further conducted using the Xenopus oocyte system. SlitGR8 responded specifically to D-fructose among 12 tested sugar compounds. In addition, the behavioral assay demonstrated that both female and male moths could respond with proboscis extension behavior to D-fructose applied onto the antenna, but females showed higher sensitivity than males. The results provide an important base for further elucidation of molecular mechanisms of gustation, and a potential target for development of feeding interfering technique in S. litura.

Rootstocks frequently exert detrimental effects on the fruit quality of grafted cucumber (Cucumis sativus L.) plants. To understand and ultimately correct this deficiency, a transcriptomic and metabolomic comparative analysis was performed among cucumber fruits from non-grafted plants (NG), and fruits from plants grafted onto different rootstocks of No.96 and No.45 (Cucurbita moschata. Duch), known to confer a different aroma and taste. We found remarkable changes in the primary metabolites of sugars, organic acids, amino acids, and alcohols in the fruit of the grafted cucumber plants with different rootstocks, compared to the non-grafted ones, especially No.45. We identified 140, 131, and 244 differentially expressed genes (DEGs) in the comparisons of GNo.96 vs. NG, GNo.45 vs. NG, and GNo.45 vs. GNo.96. The identified DEGs have functions involved in many metabolic processes, such as starch and sucrose metabolism, the biosynthesis of diterpenoid, carotenoid, and zeatin compounds, and plant hormone signal transduction. Members of the HSF, AP2/ERF-ERF, HB-HD-ZIP, and MYB transcription factor families were triggered in the grafted cucumbers, especially in the cucumber grafted on No.96. Based on a correlation analysis of the relationships between the metabolites and genes, we screened 10 candidate genes likely to be involved in sugar metabolism (Fructose-6-phosphate and trehalose), linoleic acid, and amino-acid (isoleucine, proline, and valine) biosynthesis in grafted cucumbers, and then confirmed the gene expression patterns of these genes by qRT-PCR. The levels of TPS15 (Csa3G040850) were remarkably increased in cucumber fruit with No.96 rootstock compared with No.45, suggesting changes in the volatile chemical production. Together, the results of this study improve our understanding of flavor changes in grafted cucumbers, and identify the candidate genes involved in this process.

Uracil DNA glycosylases (UDGs) play an important role in removing uracil from DNA to initiate DNA base excision repair. Here, we characterized biochemically a thermostable UDG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba UDG), and probed its mechanism by mutational analysis. The recombinant Tba UDG cleaves exclusively uracil-containing ssDNA and dsDNA at 65°C. The enzyme displays an optimal cleavage activity at 70-75°C. Tba UDG cleaves DNA over a wide pH spectrum ranging from 4.0 to 11.0 with an optimal pH of 7.0-9.0. In addition, Tba UDG activity is independent on a divalent metal ion; however, both Zn2+ and Cu2+ completely inhibit the enzyme activity. Tba UDG activity is also inhibited by high NaCl concentration. Tba UDG removes uracil from DNA with the following preference: U≈U/G>U/T≈U/C>U/A. Kinetic results showed that Tba UDG cleaves uracil-containing ssDNA and dsDNA at a similar rate. The mutational studies showed that the E118A, N159A and H216A mutants completely abolish cleavage activity and retain compromised binding activity while the Y127A mutant displays similar cleavage and binding activities with the wild-type protein, suggesting that residues E118, N159 and H216 are essential for uracil removal and necessary for uracil recognition.

Background A major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels. Results Total carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24–72 h was only 14–17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose. Conclusion We conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus . Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature ( T m ). Mutation of His299 to Tyr increased the optimal temperature, the T m , and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.

Three new alkaloids, namely, (−)-(S)-variecolortide D (1a), (+)-(R)-variecolortide D (1b) and brevicompanine I (3), along with three known alkaloids (2, 4 and 5) were isolated from Aspergillus ruber WH4-1. Variecolortide D (1) is a racemic natural product composed of anthraquinone and isoechinulin alkaloid, separation of 1 by chiral HPLC, provided two optically pure enantioners (1a and 1b). The structures of all compounds were determined by extensive analysis of their spectroscopic data as well as by comparison with literature. 2 showed weak inhibitory activity against the production of NO in RAW264.7 cells stimulated by lipopolysaccharide (LPS), with inhibitory rate of 53.2 ± 3.8% at 30 μM.

Purpose Microbial ecosystems interact with the human body and affect human health. The microbial community on the ocular surface remains an underexplored territory despite its importance as the first line of defense barrier that protects the eye and ultimately sight. We investigated how age and sex affected human ocular surface microbiome, and in the present study wanted to understand how geographic difference shaped the microbiome in the ocular surface. Methods We collected conjunctival specimens of 172 eyes from 86 healthy volunteers living in three Chinese cities, namely, Guangzhou, Wenzhou, and Beijing. Using the direct metagenomic shotgun sequencing approach, we characterized how geographic difference affected the human ocular microbiome. Results We surveyed the taxonomic composition and metabolic function of the microbiota on human ocular surface. We showed that the ocular surface microbiota was composed of bacteria, viruses, and fungi. A geographical difference in both composition and function of the conjunctival microbiome suggests that the environment people lived in shapes their conjunctival microbiome, especially the dominate species. Conclusions Our study provides a reference catalog of the healthy conjunctival metagenome and raises a concern for environmental influences on the ocular surface microbiome.

Two new unsaturated fatty acids, 6R,8R-dihydroxy-9Z,12Z-octadecadienoic acid (1) and methyl-6R,8R-dihydroxy-9Z,12Z-octadecadienoate (2), and two known 9Z,12Z-octadecadienoic acid analogues (3, 4) together with a known sesquiterpenoid (5) were isolated from the mangrove rhizosphere soil-derived fungus Penicillium javanicum HK1-22. An acetonide derivative (1a) from 1 was also prepared. The relative configuration of 1 was determined by analysis of the 1D and 2D NOE spectra of 1a. The absolute configuration of 1 was assigned on the basis of biogenetic considerations. The antifungal activity of the high yield compound 5 was evaluated against four strains of crop pathogens and it showed significant antifungal activities against all the tested strains.

BASIC PENTACYSTEINE (BPC) is a small transcription factor family that functions in diverse growth and development processes in plants. However, the roles of BPCs in plants, especially cucumber (Cucumis sativus L.), in response to abiotic stress and exogenous phytohormones are still unclear. Here, we identified four BPC genes in the cucumber genome, and classified them into two groups according to phylogenetic analysis. We also investigated the gene structures and detected five conserved motifs in these CsBPCs. Tissue expression pattern analysis revealed that the four CsBPCs were expressed ubiquitously in both vegetative and reproductive organs. Additionally, the transcriptional levels of the four CsBPCs were induced by various abiotic stress and hormone treatments. Overexpression of CsBPC2 in tobacco (Nicotiana tabacum) inhibited seed germination under saline, polyethylene glycol, and abscisic acid (ABA) conditions. The results suggest that the CsBPC genes may play crucial roles in cucumber growth and development, as well as responses to abiotic stresses and plant hormones. CsBPC2 overexpression in tobacco negatively affected seed germination under hyperosmotic conditions. Additionally, CsBPC2 functioned in ABA-inhibited seed germination and hypersensitivity to ABA-mediated responses. Our results provide fundamental information for further research on the biological functions of BPCs in development and abiotic stress responses in cucumber and other plant species.

Background: Recent evidence has shown that some long non-coding RNAs (lncRNAs) play important roles in various biological processes. However, the regulatory mechanism of lncRNA in coronary artery disease (CAD) remains unclear. Methods: We reannotated the disease related to CAD gene expression profile into a lncRNA-mRNA biphasic profile and integrated the microRNA target data to construct a global CAD triple network. A further clustering and function enrichment analysis was performed on the triple network from the level of topology analyses. Quantitative real-time PCR was used to determine the expression of four non-coding RNAs (ncRNAs). Receiver operating characteristic (ROC) curve was performed to evaluate the prognostic value of three ncRNAs. Findings: We constructed a CAD lncRNA-miRNA-mRNA network including one lncRNAs, 3 miRNAs, 15 mRNAs and 19 edges. After RT-PCR validation in four ncRNAs between CAD and non-CAD samples, only three ncRNAs with significant meaning for further analysis. ROC curve showed that TWF1 presented an AUC of 0.862, the AUC of hsa -miR-142-3p was 0.856 and hsa -miR126-5p was 0.822. After the pairwise comparison, we found that TWF1 has significant statistical significance (PTWF1-142 < 0.05 and PTWF1-126 < 0.01). For the shared lncRNA TWF1, the interacting gene and microRNA functional enrichment studies implied that lncRNA TWF1 might function as a new regulator in CAD. Interpretation: This study focused on CAD lncRNA-miRNA-mRNA regulatory networks, and found that lncRNA TWF1 was a new CAD risk lncRNA, which might provide novel insight into a better understanding of the pathogenesis of CAD. Funding Statement: The authors acknowledge the essential role of the funding of the National Natural Science Foundation of China (No: 81460169) and the Innovation Project of Guangxi Graduate Education in this motif. Declaration of Interests: The authors have no potential conflicts of interest to report Ethics Approval Statement: The study design was approved by the Ethics Committee of the First Affiliated Hospital, Guangxi Medical University (No: Lunshen-2011-KY-Guoji-001; Mar. 7, 2011). All procedures were performed in accordance with ethical standards. Informed consent was obtained from all subjects after receiving a full explanation of the study.

Purpose A growing body of evidence suggests that the microbiome of the ocular surface confers potent immunoregulatory functions and has a key role in the physiologic maintenance of healthy eyes and in the pathogenesis of ocular diseases. Although the microbiome is known to be affected by age and sex, the influence of these factors on ocular surface microbiota in healthy adults remains largely unknown. Methods Ocular surface microbiome samples were obtained from the inferior bulbar conjunctiva of 48 young and 42 old adults at Zhongshan Ophthalmic Center. Using metagenomic shotgun sequencing, we characterized the sex- and age-differences in conjunctival microbiome profiles of healthy adults. Results Male and female groups differed only in the β diversity of bacterial communities, while there were significant differences in bacterial composition, metabolic functions, and the abundance of antibiotic resistance genes between young and old adult groups. Conclusions Our findings suggest that age and sex collectively shape the conjunctival microbiome, and may change the immune homeostasis of the ocular surface through alterations of its commensal microbiome.

Sight threatening immune responses that damage the eye characterize intraocular inflammatory diseases. These diseases including uveitis and age-related macular degeneration are worryingly common and quality of life shattering. Genetic studies in past decades significantly advanced our understanding of the etiology of these devastating diseases. Unfortunately, patient genetics alone failed to adequately explain disease origin, susceptibility, and progression. Non-genetic factors such as the epigenetic regulation of ocular diseases and the environmental factors triggering intraocular inflammation offer new insight into intraocular inflammatory disorders. Importantly, mounting evidence is signaling that dysbiosis of human microbiota leads to rapid epigenomic reprograming of host cells and results in the onset of many diseases. In this review, we discuss how epigenetic mechanisms and microbiota may cooperate to initiate and perpetuate ocular inflammation. Lastly, we propose that the discovery of intraocular microbiota presents a significant shift in thought affecting current approaches to the diagnosis, treatment, and prevention of intraocular inflammatory diseases such as uveitis and age-related macular degeneration. The geographical and genetic background difference in both disease presentation and genetic association of intraocular inflammatory diseases may be due to the variation of intraocular microbiota.

Supports for the molecular evolution of host pathogen interactions on enemy release hypothesis are rare. According to the theory of plant immunity and the coevolution of hosts and pathogens, we hypothesized that the evolutionary rate (d(N)/d(S)) of resistance genes (R-genes) in invasive plants would be greater than in non-invasive plants, assuming that based on the enemy release hypothesis, the former would suffer less selection stress from co-evolutionary specialist pathogens. To test our hypothesis, we isolated and analyzed the conserved nucleotide-binding sites (NBS) of resistance gene analogues (RGAs) of an invasive weed, Wedelia trilobata (WTRGA). We then used the information in GenBank to compare the d(N)/d(S) of the NBS R-gene/RGAs in invasive and homologous non-invasive plants. Three W. trilobata NBS RGA sequences were obtained, belonging to the Toll/Interleukin-1 receptor (TIR) (WTRGA1 and WTRGA2) and non-TIR subclasses (WTRGA3). Compared with the homologous non-invasive plants, the invasive plants showed a significantly greater d(N)/d(S) for TIR NBS R-gene/RGAs (p < 0.0001), supporting our hypothesis. Future research should include an examination of R-genes/RGAs from more invasive plants on a population level to understand diversity and R-gene functions in invasive plant species, as well as to explore how disease resistance allows plants to adapt to changing pathogen stresses. (C) 2015 Elsevier Ltd. All rights reserved.

Penicillin G is a widely used antimicrobial in food-producing animals, and one of the most predominant drug residues in animal-derived food products. Due to reduced sensitivity of bacteria to penicillin, extralabel use of penicillin G is common, which may lead to violative residues in edible tissues and cause adverse reactions in consumers. This study aimed to develop a physiologically based pharmacokinetic (PBPK) model to predict drug residues in edible tissues and estimate extended withdrawal intervals for penicillin G in swine and cattle. A flow-limited PBPK model was developed with data from Food Animal Residue Avoidance Databank using Berkeley Madonna. The model predicted observed drug concentrations in edible tissues, including liver, muscle, and kidney for penicillin G both in swine and cattle well, including data not used in model calibration. For extralabel use (5× and 10× label dose) of penicillin G, Monte Carlo sampling technique was applied to predict times needed for tissue concentrations to fall below established tolerances for the 99th percentile of the population. This model provides a useful tool to predict tissue residues of penicillin G in swine and cattle to aid food safety assessment, and also provide a framework for extrapolation to other food animal species.

Glasser's disease is an economically important infectious disease of pigs caused by Haemophilus parasuis. Few vaccines are currently available that could provide effective cross-protection against various serovars of H. parasuis. In this study, five OMPs (OppA, TolC, HxuC, LppC, and HAPS_0926) identified by bioinformatic approaches, were cloned and expressed as recombinant proteins. Antigenicity of the purified proteins was verified through Western blotting, and primary screening for protective potential was evaluated in vivo. Recombinant TolC (rTolC), rLppC, and rHAPS_0926 proteins showing marked protection of mice against H. parasuis infection, and were further evaluated individually or in combination. Mice treated with these three OMPs produced humoral and host cell-mediated responses, with a significant rise in antigen-specific IgG titer and lymphoproliferative response in contrast with the mock-immunized group. Significant increases were noted in CD4+, CD8+ T cells, and three cytokines (IL-2, IL-4, and IFN-γ) in vaccinated animals. The antisera against candidate antigens could efficiently impede bacterial survival in whole blood bactericidal assay against H. parasuis infection. The multi-protein vaccine induced more pronounced immune responses and offered better protection than individual vaccines. Our findings indicate that these three OMPs are promising antigens for the development of multi-component subunit vaccines against Glasser's disease.