Comparative Genomic Analysis of Asian Cultivated Rice and Its Wild Progenitor (Oryza rufipogon) Has Revealed Evolutionary Innovation of the Pentatricopeptide Repeat Gene Family through Gene Duplication.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in land plants. However, current knowledge about the evolution of the PPR gene family remains largely limited. In this study, we performed a comparative genomic analysis of the PPR gene family in O. sativa and its wild progenitor, O. rufipogon, and outlined a comprehensive landscape of gene duplications. Our findings suggest that the majority of PPR genes originated from dispersed duplications. Although segmental duplications have only expanded approximately 11.30% and 13.57% of the PPR gene families in the O. sativa and O. rufipogon genomes, we interestingly obtained evidence that segmental duplication promotes the structural diversity of PPR genes through incomplete gene duplications. In the O. sativa and O. rufipogon genomes, 10 (~33.33%) and 22 pairs of gene duplications (~45.83%) had non-PPR paralogous genes through incomplete gene duplication. Segmental duplications leading to incomplete gene duplications might result in the acquisition of domains, thus promoting functional innovation and structural diversification of PPR genes. This study offers a unique perspective on the evolution of PPR gene structures and underscores the potential role of segmental duplications in PPR gene structural diversity.

Structural and functional characterization of turbot pparγ: Activation during high temperature and regulation of lipid metabolism.

Transcription factors of the peroxisome proliferator-activated receptor gamma (pparγ) is a ligand-activated receptor that plays key roles in lipid metabolism and inflammation. In this study, we cloned the pparγ cDNA of turbot (Scophthalmus maximus). It has a 1659 bp coding sequence (CDS) and encodes 552 amino acids. The deduced PPARγ protein of turbot contains two highly conserved domains, a C4-type zinc finger, a nuclear hormone receptor DNA-binding region signature, and a HOLI domain (ligand-binding domain of hormone receptors) identical to that of in other species. qPCR results showed that the expression level of pparγ and the expression of the fatty acid transporters fatp and cd36 were significantly increased under high-temperature stress. This indicates that high temperatures activate the transcriptional activity of pparγ, and lipid metabolism plays an important role in turbot under high-temperature stress. In addition, RNA interference was used to explore the regulation of pparγ on lipid metabolism of turbot at high temperatures. The results showed that the mRNA level of pparγ was significantly decreased. After the expression level of pparγ was inhibited, the expression levels of fatp and cd36 were significantly decreased. At the same time, GW9662 (a pparγ antagonist) was used to inhibit pparγ activity in turbot kidney cells, and fatp and cd36 gene expressions were detected. The mRNA expression levels of pparγ, fatp, and cd36 were significantly decreased. Our results suggest that high temperatures activate pparγ in turbot and that pparγ regulates lipid transport and maintains lipid metabolism homeostasis through positive regulation of the expression of downstream genes fatp and cd36. This study further elucidates that the pparγ-mediated signaling pathway may play an important role in regulating lipid metabolism during heat stress in teleost fish.

Differential binding of LuxR in response to temperature gauges switches virulence gene expression in Vibrio alginolyticus.

Vibrio pathogens must cope with temperature changes for proper thermo-adaptation and virulence gene expression. LuxR is a quorum-sensing (QS) master regulator of vibrios, playing roles in response to temperature alteration. However, the molecular mechanisms how LuxR is involved in adapting to different temperatures in bacteria have not been precisely elucidated. In this study, using chromatin immunoprecipitation and nucleotide sequencing (ChIP-seq), we identified 272 and 22 enriched loci harboring LuxR-binding peaks at ambient temperature (30 ËšC) and heat shock (42 ËšC) in the Vibrio alginolyticus genome, respectively. Analysis with the MEME (multiple EM for motif elicitation) algorithm indicated that the binding motifs of LuxR varied from temperatures. Three novel binding regions (the promoter of orf00292, orf00397 and fadD) of LuxR were identified and verified that the rising temperature causes the decreasing binding affinity of LuxR to these promoters. Meanwhile, the expression of orf00292, orf00397 and fadD were regulated by LuxR. Moreover, the weak binding of LuxR to the promoter of extracellular protease (Asp) was attributed to the attenuated Asp expression at thermal stress conditions. Taken together, our study demonstrated distinct binding characteristics of LuxR in response to temperature changes, thus highlighting LuxR as a thermo-sensor to switch and control virulence gene expression in V. alginolyticus.

Coordinate regulation of carbohydrate metabolism and virulence by PtsH in pathogen Edwardsiella piscicida.

Carbohydrate metabolism of bacterial pathogens conducts crucial roles in regulating pathogenesis but the molecular mechanisms by which metabolisms and virulence are been modulated and coordinated remain to be illuminated. Here, we investigated in this regard Edwardsiella piscicida, a notorious zoonotic pathogen previously named E. tarda that could ferment very few PTS sugars including glucose, fructose, mannose, N-acetylglucosamine, and N-acetylgalactosamine. We systematically characterized the roles of each of the predicted 23 components of phosphotransferase system (PTS) with the respective in-frame deletion mutants and defined medium containing specific PTS sugar. In addition, PtsH was identified as the crucial PTS component potentiating the utilization of all the tested PTS sugars. Intriguingly, we also found that PtsH while not Fpr was involved in T3SS gene expression and was essential for the pathogenesis of E. piscicida. To corroborate this, His15 and Ser46, the two established PtsH residues involved in phosphorylation cascade, showed redundant roles in regulating T3SS yields. Moreover, PtsH was shown to facilitate mannose uptake and transform it into mannose-6-phosphate, an allosteric substrate established to activate EvrA to augment bacterial virulence. Collectively, our observations provide new insights into the roles of PTS reciprocally regulating carbohydrate metabolism and virulence gene expression. KEY POINTS: • PTS components' roles for sugar uptake are systematically determined in Edwardsiella piscicida. • PtsH is involved in saccharides uptake and in the regulation of E. piscicida's T3SS expression. • PtsH phosphorylation at His15 and Ser46 is essential for the T3SS expression and virulence.

Molecular cloning, characterization and expression analysis of p53 from turbot Scophthalmus maximus and its response to thermal stress.

The tumor suppressor protein, p53 plays a crucial role in protecting genetic integrity. Once activated by diverse cell stresses, p53 reversibly activates downstream target genes to regulate cell cycle and apoptosis. However, few studies have investigated the effects of thermal stress in turbot, specifically the p53 signaling pathway. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot p53 gene (Sm-p53) and perform bioinformatics analysis. The results showed that the cDNA of the Sm-p53 gene was 2928 bp in length, encoded a 381 amino acid protein, with a theoretical isoelectric point of 6.73. It was composed of a DNA binding and a tetramerization domain. Expression of Sm-p53 in different tissues was detected and quantified by qRT-PCR, and was highest in the liver. We also investigated the expression profiles of Sm-p53 in different tissue and TK cells after thermal stress. These result suggested that Sm-p53 plays a key role, and provides a theoretical basis for Sm-p53 changes in environmental stress responses in the turbot.

Transcriptome analysis and weighted gene co-expression network reveals potential genes responses to heat stress in turbot Scophthalmus maximus.

Turbot (Scophthalmus maximus) is an economically important marine fish cultured in China. In this study, we performed transcriptome gene expression profiling of kidney tissue in turbot exposed to heat stress (20, 23, 25 and 28 °C); control fish were maintained at 14 °C. We investigated gene relationships based on weighted gene co-expression network analysis (WGCNA). Accordingly, enrichment analyses of GO terms and KEGG pathways showed that several pathways (e.g., fat metabolism, cell apoptosis, immune system, and insulin signaling) may be involved in the response of turbot to heat stress. Moreover, via WGCNA, we identified 19 modules: the dark grey module was mainly enriched in pathways associated with fat metabolism and the FOXO and Jak-STAT signaling pathways. The ivory module was significantly enriched in the P53 signaling pathway. Furthermore, the key hub genes CBP, AKT3, CCND2, PIK3r2, SCOS3, mdm2, cyc-B, and p48 were enriched in the FOXO, Jak-STAT and P53 signaling pathways. This is the first study reporting co-expression patterns of a gene network after heat stress in marine fish. Our results may contribute to our understanding of the underlying molecular mechanism of thermal tolerance.

PepA binds to and negatively regulates esrB to control virulence in the fish pathogen Edwardsiella piscicida.

As an important marine fish pathogen, Edwardsiella piscicida infects a broad range of fish species and causes substantial economic losses. The EsrA-EsrB two-component system is essential for the expression of type III and type VI secretion systems (T3/T6SSs), the key virulence determinants in the bacterium. In this study, a pull-down assay with the esrB promoter as bait was performed to identify the upstream regulators of esrB. As a result, PepA, a leucyl aminopeptidase, was identified as a repressor of EsrB and T3/T6SS expression. PepA bound to the esrB promoter region and negatively regulated the production of T3/T6SS proteins in early stages. Moreover, PepA was found to affect the in vivo colonization of E. piscicida in turbot livers through the regulation of EsrB expression. Collectively, our results enhance the understanding of the virulence regulatory network and in vivo colonization mechanism of E. piscicida. One sentence summary: PepA regulates EsrB expression in Edwardsiella piscicida.

Alternative Sigma Factor RpoX Is a Part of the RpoE Regulon and Plays Distinct Roles in Stress Responses, Motility, Biofilm Formation, and Hemolytic Activities in the Marine Pathogen Vibrio alginolyticus.

Vibrio alginolyticus is one of the most abundant microorganisms in marine environments and is also an opportunistic pathogen mediating high-mortality vibriosis in marine animals. Alternative sigma factors play essential roles in bacterial pathogens in the adaptation to environmental changes during infection and the adaptation to various niches, but little is known about them for V. alginolyticus Our previous investigation indicated that the transcript level of the gene rpoX significantly decreased in an RpoE mutant. Here, we found that rpoX was highly expressed in response to high temperature and low osmotic stress and was under the direct control of the alternative sigma factor RpoE and its own product RpoX. Moreover, transcriptome sequencing (RNA-seq) results showed that RpoE and RpoX had different regulons, although they coregulated 105 genes at high temperature (42°C), including genes associated with biofilm formation, motility, virulence, regulatory factors, and the stress response. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) analyses as well as electrophoretic mobility shift assays (EMSAs) revealed the distinct binding motifs of RpoE and RpoX proteins. Furthermore, quantitative real-time reverse transcription-PCR (qRT-PCR) analysis also confirmed that RpoX can upregulate genes associated with flagella, biofilm formation, and hemolytic activities at higher temperatures. rpoX abrogation does not appear to attenuate virulence toward model fish at normal temperature. Collectively, data from this study demonstrated the regulatory cascades of RpoE and an alternative sigma factor, RpoX, in response to heat and osmotic stresses and their distinct and overlapping roles in pathogenesis and stress responses in the marine bacterium V. alginolyticusIMPORTANCE The alternative sigma factor RpoE is essential for the virulence of Vibrio alginolyticus toward marine fish, coral, and other animals in response to sea surface temperature increases. In this study, we characterized another alternative sigma factor, RpoX, which is induced at high temperatures and under low-osmotic-stress conditions. The expression of rpoX is under the tight control of RpoE and RpoX. Although RpoE and RpoX coregulate 105 genes, they are programming different regulatory functions in stress responses and virulence in V. alginolyticus These findings illuminated the RpoE-RpoX-centered regulatory cascades and their distinct and overlapping regulatory roles in V. alginolyticus, which facilitates unraveling of the mechanisms by which the bacterium causes diseases in various sea animals in response to temperature fluctuations as well as the development of appropriate strategies to tackle infections by this bacterium.

Pattern analysis of conditional essentiality (PACE)-based heuristic identification of an in vivo colonization determinant as a novel target for the construction of a live attenuated vaccine against Edwardsiella piscicida.

Edwardsiella piscicida is the aetiological agent of fish edwardsiellosis, causing huge economic losses in aquaculture industries. The use of a live attenuated vaccine (LAV) will be an effective strategy to control the disease in farmed fish. Thus, methods facilitating exploration of targets used for construction of an LAV will be of great significance. Previously, we devised an algorithm termed pattern analysis of conditional essentiality (PACE) to perform genome-wide analysis of the temporal dynamic behaviour of E. piscicida mutants colonizing turbot. Here, we correlated the conditional essentiality patterns of the PACE-derived colonization determinants with that of the aroC gene encoding chorismate synthase, the established target for LAV construction in E. piscicida, and identified ETAE_0023 as a novel valuable LAV target. ETAE_0023 encodes an uncharacterized DcrB family protein. Deletion of ETAE_0023 dramatically impaired E. piscicida invasion capability in ZF4 cells as well as colonization in fish and resulted in in vivo clearance at ∼30 days post-infection. ΔETAE_0023 showed an ∼2500-fold higher 50% lethal dose (LD50) than that of the wild type strain. Vaccination with ΔETAE_0023 by intraperitoneal (i.p.) injection upregulated expression of immune factors, i.e., IL-1ß, IgM, MHC-I and MHC-II, and produced significantly high levels of E. piscicida-specific IgM as well as serum bactericidal capacities in turbot. Moreover, a single i.p. inoculation with ΔETAE_0023 generated significant protection comparable to the established WED LAV strain in turbot against challenge with the wild type strain after 5 weeks of vaccination. Taken together, we demonstrated a PACE-based method for heuristic identification of targets for LAV construction and presented ΔETAE_0023 as a new LAV candidate against edwardsiellosis.

Genome-Wide Identification of Fitness Factors in Seawater for Edwardsiella piscicida.

Marine pathogens are transmitted from one host to another through seawater. Therefore, it is important for marine pathogens to maintain survival or growth in seawater. However, little is known about how marine pathogens adapt to living in seawater environments. Here, transposon insertion sequencing was performed to explore the genetic determinants of Edwardsiella piscicida survival in seawater at 16 and 28°C. Seventy-one mutants with mutations mainly in metabolism-, transportation-, and type III secretion system (T3SS)-related genes showed significantly increased or impaired fitness in 16°C water. In 28°C seawater, 63 genes associated with transcription and translation, as well as energy production and conversion, were essential for optimal survival of the bacterium. In particular, 11 T3SS-linked mutants displayed enhanced fitness in 16°C seawater but not in 28°C seawater. In addition, 13 genes associated with oxidative phosphorylation and 4 genes related to ubiquinone synthesis were identified for survival in 28°C seawater but not in 16°C seawater, which suggests that electron transmission and energy-producing aerobic respiration chain factors are indispensable for E. piscicida to maintain survival in higher-temperature seawater. In conclusion, we defined genes and processes related to metabolism and virulence that operate in E. piscicida to facilitate survival in low- and high-temperature seawater, which may underlie the infection outbreak mechanisms of E. piscicida and facilitate the development of improved vaccines against marine pathogens.IMPORTANCEEdwardsiella piscicida is one of the most important marine pathogens and causes serious edwardsiellosis in farmed fish during the summer-autumn seasonal changes, resulting in enormous losses to aquaculture industries worldwide. Survival and transmission of the pathogen in seawater are critical steps that increase the risk of outbreaks. To investigate the mechanism of survival in seawater for this marine pathogen, we used transposon insertion sequencing analysis to explore the fitness determinants in summer and autumn seawater. Approximately 127 genes linked to metabolism and virulence, as well as other processes, were revealed in E. piscicida to contribute to better adaptations to the seasonal alternations of seawater environments; these genes provide important insights into the infection outbreak mechanisms of E. piscicida and potential improved treatments or vaccines against marine pathogens.

VqsA controls exotoxin production by directly binding to the promoter of asp in the pathogen Vibrio alginolyticus.

Quorum sensing (QS) system is an important bacterial cell-to-cell signaling system controlling expression of various genes in response to cell densities. In vibrios, LuxR/AphA are two established master QS regulators (MQSRs), and VqsA is recently identified to be the third putative MQSR. As a novel LysR-type regulator, the regulon and the underlying regulation mechanisms of VqsA remains to be elucidated. Here our investigation indicated that the yields of alkaline serine protease (Asp), the exotoxin in Vibrio alginolyticus was dependent on both LuxR and VqsA in growth phase dependent manner. Various in vivo and in vitro analyses including electrophoretic mobility shift assays (EMSA) along with DNase I footprinting investigations demonstrated that VqsA positively controls asp expression through directly binding to the partially palindromic 29 bp binding motif in the promoter region of asp. Moreover, RNA-seq analysis validated the regulatory roles of VqsA in various processes in the organism. Collectively, our data showed that VqsA positively regulates the expression of exotoxin and other virulence-associated genes and is essential for the QS regulation in V. alginolyticus.

[Determinants of Drinking Relapse after Treatments in Patients with Alcohol Dependence in Sichuan Province].

OBJECTIVE: To identify factors that influent drinking relapse after treatments in patients with alcohol dependence in Sichuan province. METHODS: Data were collected in 10 cities of Sichuan province from September 2014 to June 2015,involving 599 patients who received treatments for alcohol dependence. A questionnaire survey was administered on these patients one year after discharge through face to face interviews,collecting data in relation to their demographic characteristics,drinking over the past year,smoking,mood and level of stress. Ordinal polytomous logistic regression analyses were performed to determine the association of these factors with drinking relapse. RESULTS: All of the 599 patients started drinking again after treatments: 18 having low-risk drinking,92 having hazardous drinking,103 having harmful drinking,and 386 having alcohol dependence. Younger patients [odds ratio (OR)=0.978,P=0.009],those who experienced less positive events (OR=0.978,P<0.001) or more negative events (OR=1.014,P=0.003),and those with depression (OR=1.121,P=0.001) were more likely to resume a higher level of alcohol drinking than their counterparts. CONCLUSION: High relapse with alcohol dependence is evident. So does hazardous and harmful drinking. Negative life events and depression are risk factors of drinking relapse,while older age and positive life events are protective factors.

VqsA, a Novel LysR-Type Transcriptional Regulator, Coordinates Quorum Sensing (QS) and Is Controlled by QS To Regulate Virulence in the Pathogen Vibrio alginolyticus.

The quorum sensing (QS) system controls bacterial group behaviors in response to cell density. In vibrios, LuxR and AphA are two master QS regulators (MQSRs) controlling gene expression in response to high or low cell density. Other regulators involved in the regulation of these two MQSRs and QS pathways remain to be determined. Here, we performed bacterial one-hybrid (B1H)-assay-based screens of transcriptional factors (TFs) to identify TFs that can directly regulate the expression of luxR and aphA from a library of 285 TFs encoded by the fish pathogen Vibrio alginolyticus A total of 7 TFs were identified to bind to the promoters of both luxR and aphA Among these TFs, the novel LysR-type transcriptional regulator (LTTR) VqsA could activate LuxR and repress AphA transcription. Meanwhile, LuxR and AphA exerted feedback inhibition and activation of vqsA expression, respectively, indicating that VqsA coordinates QS and is also regulated by QS. In addition, VqsA inhibited its own expression by directly binding to its own promoter region. The VqsA-binding sites in the promoter regions of luxR and aphA as well as the binding sites of LuxR, AphA, and VqsA in the vqsA gene were uncovered by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis. Finally, VqsA was verified to play essential roles in QS-regulated phenotypes, i.e., type VI secretion system 2 (T6SS2)-dependent interbacterial competition, biofilm formation, exotoxin production, and in vivo virulence of V. alginolyticus Collectively, our data showed that VqsA is an important QS regulator in V. alginolyticusIMPORTANCE Investigation of the mechanism of regulation of quorum sensing (QS) systems will facilitate an understanding of bacterial pathogenesis and the identification of effective QS interference (QSI) targets. Here, we systematically screened transcriptional factors (TFs) that modulate the expression of the master QS regulators (MQSRs) LuxR and AphA, and a novel LysR-type transcriptional regulator, VqsA, was identified. Our data illuminated the mechanisms mediating the interaction among LuxR, AphA, and VqsA as well as the effects of these regulators on the expression and output of QS. The impaired expression of virulence genes as a result of vqsA disruption demonstrated that VqsA is an important player in QS regulation and pathogenesis and may be the third MQSR involved in sensing environmental signals by vibrios to coordinate QS responses. This study will facilitate the development of strategies to interfere with QS and effectively control this pathogen that plagues the aquaculture industry.

Pharmacokinetics and bioequivalence evaluation of two different atorvastatin calcium 10-mg tablets: A single-dose, randomized-sequence, open-label, two-period crossover study in healthy fasted Chinese adult males.

BACKGROUND: Atorvastatin calcium is a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor indicated for the prevention of cardiovascular disease and for the treatment of dyslipidemia. Information on the pharmacokinetics of atorvastatin in a Chinese population is lacking, and regulatory requirements necessitate a bioequivalence study for the marketing of a generic product in China. OBJECTIVE: The aim of the present study was to assess the pharmacokinetics and bioequivalence of a test and branded reference formulation of atorvastatin calcium 10-mg tablets in healthy fasted Chinese male volunteers. METHODS: This was a single-dose, randomized-sequence, open-label, 2-period crossover study with a 2-week washout period between doses. Healthy Chinese males were randomly assigned to receive 20 mg of either the test or reference formulation, and 13 blood samples were obtained over a 48-hour interval. Plasma concentrations of parent atorvastatin and ortho-hydroxy-atorvastatin (primary active metabolite) were simultaneously determined using a validated liquid chromatography-isotopic dilution mass spectrometry method. Pharmacokinetic parameters, including C(max), T(max), t((1/2)), AUC(0-t), and AUC(0-infinity)), were calculated. The 2 formulations were to be considered bioequivalent if 90% CIs for the log transformed ratios of AUC and C(max) of atorvastatin were within the predetermined bioequivalence range (0.80-1.25 for AUC and 0.70-1.43 for C(max)) as established by the State Food and Drug Administration of China. Tolerability was evaluated throughout the study by vital signs monitoring, physical examinations, 12-lead ECGs, and subject interviews on adverse events (AEs). RESULTS: A total of 66 subjects were assessed for inclusion; 20 were excluded prior to study initiation. Of the 46 healthy subjects (mean [SD] age, 24.1 [2.5] years; height, 170.8 [5.1] cm; weight, 64.6 [6.4] kg; body mass index (BMI), 22.1 [1.7] kg/m(2)) who completed the study, 45 subjects (mean [SD] age, 24.1 [2.5] years; height, 171.1 [4.9] cm; weight, 64.8 [6.3] kg; BMI, 22.1 [1.7] kg/m(2)) were included in the pharmacokinetic and bioequivalence analyses; 1 subject was excluded from these analyses because he mistakenly received the same formulation in both periods. No period or sequence effect was observed. The mean values of C(max), AUC(0-t), and AUC(0-infinity)) for the test and reference formulations of atorvastatin (8.78 and 10.76 ng/mL, 38.22 and 40.02 ng/mL/h, 42.73 and 44.51 ng/mL/h, respectively) and ortho-hydroxy-atorvastatin (5.78 and 5.77 ng/mL, 47.32 and 48.47 ng/mL/h, 52.36 and 53.14 ng/mL/h) were not significantly different. The 90% CIs for natural log-transformed ratios of C(max), AUC(0-t), and AUC(0-infinity)) of both atorvastatin (0.73-0.91, 0.92-1.02, and 0.91-1.01, respectively) and ortho-hydroxy-atorvastatin (0.83-1.05, 0.92-1.02, and 0.93-1.02) were within the bioequivalence acceptance limits. Three subjects (6.5%) reported a total of 4 mild AEs (1 abdominal discomfort and 3 venipuncture syncope), which were not considered to be associated with administration of the study drug. CONCLUSIONS: This single-dose (20 mg) study found that the test and reference formulations of atorvastatin calcium 10-mg tablet met the regulatory definition for assuming bioequivalence in these healthy fasted Chinese male volunteers. Both formulations were generally well tolerated in the population studied. Chinese National Registry Code: 2007L02512.