Genetic and Environmental Investigation of a Novel Phenylamino Acetamide Inhibitor of the Pseudomonas aeruginosa Type III Secretion System.

Traditional antibiotics target essential cellular components or metabolic pathways conserved in both pathogenic and nonpathogenic bacteria. Unfortunately, long-term antibiotic use often leads to antibiotic resistance and disruption of the overall microbiota. In this work, we identified a phenylamino acetamide compound, named 187R, that strongly inhibited the expression of the type III secretion system (T3SS) encoding genes and the secretion of the T3SS effector proteins in Pseudomonas aeruginosa. T3SS is an important virulence factor, as T3SS-deficient strains of P. aeruginosa are greatly attenuated in virulence. We further showed that 187R had no effect on bacterial growth, implying a reduced selective pressure for the development of resistance. 187R-mediated repression of T3SS was dependent on ExsA, the master regulator of T3SS in P. aeruginosa. The impact of 187R on the host-associated microbial community was also tested using the Arabidopsis thaliana phyllosphere as a model. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the application of 187R had little impact on the composition and function of microbial community compared to the antibiotic streptomycin. Together, these results suggested that compounds that target virulence factors could serve as an alternative strategy for disease management caused by bacterial pathogens. IMPORTANCE New antimicrobial therapies are urgently needed, since antibiotic resistance in human pathogens has become one of the world's most urgent public health problems. Antivirulence therapy has been considered a promising alternative for the management of infectious diseases, as antivirulence compounds target only the virulence factors instead of the growth of bacteria, and they are therefore unlikely to affect commensal microorganisms. However, the impacts of antivirulence compounds on the host microbiota are not well understood. We report a potent synthetic inhibitor of the P. aeruginosa T3SS, 187R, and its effect on the host microbiota of Arabidopsis. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the impacts of the antivirulence compound on the composition and function of host microbiota were limited. These results suggest that antivirulence compounds can be a potential alternative method to antibiotics.

Insufficient or excessive dietary carbohydrates affect gut health through change in gut microbiota and regulation of gene expression of gut epithelial cells in grass carp (Ctenopharyngodon idella).

Dietary carbohydrate levels can affect gut health, but the roles played by gut microbiota and gut epithelial cells, and their interactions remain unclear. In this experiment, we investigated gut health, gut microbiota, and the gene expression profiles of gut epithelial cells in grass carp consuming diets with different carbohydrate levels. Compared to the moderate-carbohydrate diet, low-carbohydrate diet significantly increased the relative abundance of pathogenic bacteria (Ralstonia and Elizabethkingia) and decreased the abundance of metabolism in cofactors and vitamins, implying a dysregulated gut microbiota and compromised metabolic function. Moreover, low-carbohydrate diet inhibited the expression levels of key genes in autophagy-related pathways in gut epithelial cells, which might directly lead to reduced clearance of defective organelles and pathogenic microorganisms. These aforementioned factors may be responsible for the imperfect organization of the intestinal tract. High-carbohydrate diet also significantly increased the abundance of pathogenic bacteria (Flavobacterium), which directly contributed to a decrease in the abundance of immune system of the microbiota. Furthermore, the active pathways of staphylococcus aureus infection and complement and coagulation cascades, as well as the inhibition of the glutathione metabolism pathway were observed. Above results implied that high-carbohydrate diet might ultimately cause severe gut damage by affecting immune function of microbiota, mentioned immune-related pathways, and the antioxidant capacity. Finally, the correlation network diagram revealed strong correlations of the differentially immune-related gene major histocompatibility complex class I antigen (MR1) with Enhydrobacter and Ruminococcus_gnavus_group in low-carbohydrate diet group, and Arenimonas in high-carbohydrate diet group, respectively, suggesting that MR1 might be a central target for immune responses in gut epithelial cells induced by gut microbiota at different levels of dietary carbohydrate. All these results provided insight in the development of antagonistic probiotics and target genes to improve the utilization of carbohydrate.

Evaluation of Plant Defense Inducers and Plant Growth Regulators for Fire Blight Management Using Transcriptome Studies and Field Assessments.

Fire blight, caused by Erwinia amylovora, is a destructive disease of pome fruit trees. In the United States, apple and pear growers rely on applications of copper and antibiotics during bloom to control fire blight, but such methods have already led to regional instances of resistance. In this study, we used transcriptome analyses and field trials to evaluate the effectiveness of three commercially available plant defense elicitors and one plant growth regulator for fire blight management. Our data indicated that foliar applications of acibenzolar-S-methyl (ASM; Actigard 50WG) triggered a strong defense-related response in apple leaves, whereas applications of Bacillus mycoides isolate J (LifeGard WG) or Reynoutria sachalinensis extract (Regalia) did not. Genes upregulated by ASM were enriched in the biological processes associated with plant immunity, such as defense response and protein phosphorylation. The expression of several pathogenesis-related (PR) genes was induced by ASM as well. Surprisingly, many differentially expressed genes in ASM-treated apple leaves overlapped with those induced by treatment with prohexadione-calcium (ProCa; Apogee), a plant growth regulator that suppresses shoot elongation. Further analysis suggested that ProCa likely acts similarly to ASM to stimulate plant immunity because genes involved in plant defense were shared and significantly upregulated (more than twofold) by both treatments. Our field trials agreed with the transcriptome study, demonstrating that ASM and ProCa exhibit the best control performance relative to the other biopesticides. Taken together, these data are pivotal for the understanding of plant response and shed light on future improvements of strategies for fire blight management.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

SPA-Net: A Deep Learning Approach Enhanced Using a Span-Partial Structure and Attention Mechanism for Image Copy-Move Forgery Detection.

With the wide application of visual sensors and development of digital image processing technology, image copy-move forgery detection (CMFD) has become more and more prevalent. Copy-move forgery is copying one or several areas of an image and pasting them into another part of the same image, and CMFD is an efficient means to expose this. There are improper uses of forged images in industry, the military, and daily life. In this paper, we present an efficient end-to-end deep learning approach for CMFD, using a span-partial structure and attention mechanism (SPA-Net). The SPA-Net extracts feature roughly using a pre-processing module and finely extracts deep feature maps using the span-partial structure and attention mechanism as a SPA-net feature extractor module. The span-partial structure is designed to reduce the redundant feature information, while the attention mechanism in the span-partial structure has the advantage of focusing on the tamper region and suppressing the original semantic information. To explore the correlation between high-dimension feature points, a deep feature matching module assists SPA-Net to locate the copy-move areas by computing the similarity of the feature map. A feature upsampling module is employed to upsample the features to their original size and produce a copy-move mask. Furthermore, the training strategy of SPA-Net without pretrained weights has a balance between copy-move and semantic features, and then the module can capture more features of copy-move forgery areas and reduce the confusion from semantic objects. In the experiment, we do not use pretrained weights or models from existing networks such as VGG16, which would bring the limitation of the network paying more attention to objects other than copy-move areas.To deal with this problem, we generated a SPANet-CMFD dataset by applying various processes to the benchmark images from SUN and COCO datasets, and we used existing copy-move forgery datasets, CMH, MICC-F220, MICC-F600, GRIP, Coverage, and parts of USCISI-CMFD, together with our generated SPANet-CMFD dataset, as the training set to train our model. In addition, the SPANet-CMFD dataset could play a big part in forgery detection, such as deepfakes. We employed the CASIA and CoMoFoD datasets as testing datasets to verify the performance of our proposed method. The Precision, Recall, and F1 are calculated to evaluate the CMFD results. Comparison results showed that our model achieved a satisfactory performance on both testing datasets and performed better than the existing methods.

Exopolysaccharides amylovoran and levan contribute to sliding motility in the fire blight pathogen Erwinia amylovora.

Erwinia amylovora, the causative agent of fire blight, uses flagella-based motilities to translocate to host plant natural openings; however, little is known about how this bacterium migrates systemically in the apoplast. Here, we reveal a novel surface motility mechanism, defined as sliding, in E. amylovora. Deletion of flagella assembly genes did not affect this movement, whereas deletion of biosynthesis genes for the exopolysaccharides (EPSs) amylovoran and levan resulted in non-sliding phenotypes. Since EPS production generates osmotic pressure that potentially powers sliding, we validated this mechanism by demonstrating that water potential positively contributes to sliding. In addition, no sliding was observed when the water potential of the surface was lower than -0.5 MPa. Sliding is a passive motility mechanism. We further show that the force of gravity plays a critical role in directing E. amylovora sliding on unconfined surfaces but has a negligible effect when cells are sliding in confined microcapillaries, in which EPS-dependent osmotic pressure acts as the main force. Although amylovoran and levan are both required for sliding, we demonstrate that they exhibit different roles in bacterial communities. In summary, our study provides fundamental knowledge for a better understanding of mechanisms that drive bacterial sliding motility.

The RNA-Binding Protein ProQ Impacts Exopolysaccharide Biosynthesis and Second Messenger Cyclic di-GMP Signaling in the Fire Blight Pathogen Erwinia amylovora.

Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in many economically important plants, including apples and pears. This bacterium produces three exopolysaccharides (EPSs), amylovoran, levan, and cellulose, and forms biofilms in host plant vascular tissues, which are crucial for pathogenesis. Here, we demonstrate that ProQ, a conserved bacterial RNA chaperone, was required for the virulence of E. amylovora in apple shoots and for biofilm formation in planta. In vitro experiments revealed that the deletion of proQ increased the production of amylovoran and cellulose. Prc is a putative periplasmic protease, and the prc gene is located adjacent to proQ. We found that Prc and the associated lipoprotein NlpI negatively affected amylovoran production, whereas Spr, a peptidoglycan hydrolase degraded by Prc, positively regulated amylovoran. Since the prc promoter is likely located within proQ, our data showed that proQ deletion significantly reduced the prc mRNA levels. We used a genome-wide transposon mutagenesis experiment to uncover the involvement of the bacterial second messenger c-di-GMP in ProQ-mediated cellulose production. The deletion of proQ resulted in elevated intracellular c-di-GMP levels and cellulose production, which were restored to wild-type levels by deleting genes encoding c-di-GMP biosynthesis enzymes. Moreover, ProQ positively affected the mRNA levels of genes encoding c-di-GMP-degrading phosphodiesterase enzymes via a mechanism independent of mRNA decay. In summary, our study revealed a detailed function of E. amylovora ProQ in coordinating cellulose biosynthesis and, for the first time, linked ProQ with c-di-GMP metabolism and also uncovered a role of Prc in the regulation of amylovoran production. IMPORTANCE Fire blight, caused by the bacterium Erwinia amylovora, is an important disease affecting many rosaceous plants, including apple and pear, that can lead to devastating economic losses worldwide. Similar to many xylem-invading pathogens, E. amylovora forms biofilms that rely on the production of exopolysaccharides (EPSs). In this paper, we identified the RNA-binding protein ProQ as an important virulence regulator. ProQ played a central role in controlling the production of EPSs and participated in the regulation of several conserved bacterial signal transduction pathways, including the second messenger c-di-GMP and the periplasmic protease Prc-mediated systems. Since ProQ has recently been recognized as a global posttranscriptional regulator in many bacteria, these findings provide new insights into multitiered regulatory mechanisms for the precise control of virulence factor production in bacterial pathogens.

A Novel Signaling Pathway Connects Thiamine Biosynthesis, Bacterial Respiration, and Production of the Exopolysaccharide Amylovoran in Erwinia amylovora.

Erwinia amylovora is a plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. This bacterium colonizes host vascular tissues via the production of exopolysaccharides (EPSs) including amylovoran. It is well-established that the nearly ubiquitous plasmid pEA29 of E. amylovora is an essential virulence factor, but the underlying mechanism remains uncharacterized. Here, we demonstrated that pEA29 was required for E. amylovora to produce amylovoran and to form a biofilm, and this regulation was dependent on the thiamine biosynthesis operon thiOSGF. We then conducted carbohydrate and genetic analyses demonstrating that the thiamine-mediated effect on amylovoran production was indirect, as cells lacking thiOSGF produced an EPS that did not contain glucuronic acid, one of the key components of amylovoran, whereas the transcriptional activity and RNA levels of the amylovoran biosynthesis genes were not altered. Alternatively, addition of exogenous thiamine restored amylovoran production in the pEA29-cured strain of E. amylovora and positively impacted amylovoran production in a dose-dependent manner. Individual deletion of several chromosomal thiamine biosynthesis genes also affected amylovoran production, implying that a complete thiamine biosynthesis pathway is required for the thiamine-mediated effect on amylovoran production in E. amylovora. Finally, we determined that an imbalanced tricarboxylic acid cycle negatively affected amylovoran production, which was restored by addition of exogenous thiamine or overexpression of the thiOSGF operon. In summary, our report revealed a novel signaling pathway that impacts E. amylovora virulence in which thiamine biosynthesis enhances bacterial respiration that provides energetic requirements for the biosynthesis of EPS amylovoran.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cell-length heterogeneity: a population-level solution to growth/virulence trade-offs in the plant pathogen Dickeya dadantii.

Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10µm), the rest elongated into filamentous cells (>10µm). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed upregulated metabolic genes and increased growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential production of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.

Detecting rare haplotype association with two correlated phenotypes of binary and continuous types.

Multiple correlated traits/phenotypes are often collected in genetic association studies and they may share a common genetic mechanism. Joint analysis of correlated phenotypes has well-known advantages over one-at-a-time analysis including gain in power and better understanding of genetic etiology. However, when the phenotypes are of discordant types such as binary and continuous, the joint modeling is more challenging. Another research area of current interest is discovery of rare genetic variants. Currently there is no method available for detecting association of rare (or common) haplotypes with multiple discordant phenotypes jointly. Our goal is to fill this gap specifically for two discordant phenotypes. We consider a rare haplotype association method for a binary phenotype, logistic Bayesian LASSO (univariate LBL) and its extension for two correlated binary phenotypes (bivariate LBL-2B). Under this framework, we propose a haplotype association test with binary and continuous phenotypes jointly (bivariate LBL-BC). Specifically, we use a latent variable to induce correlation between the two phenotypes. We carry out extensive simulations to investigate bivariate LBL-BC and compare it with univariate LBL and bivariate LBL-2B. In most settings, bivariate LBL-BC performs the best. In only two situations, bivariate LBL-BC has similar performance-when the two phenotypes are (1) weakly or not correlated and the target haplotype affects the binary phenotype only and (2) strongly positively correlated and the target haplotype affects both phenotypes in positive direction. Finally, we apply the method to a data set on lung cancer and nicotine dependence and detect several haplotypes including a rare one.

Differential expression of lncRNAs in hypertension-induced pericytes.

Pericytes facilitate vessel maturation and endothelial barrier functions closely related with the pathogenesis of organ damage from cardiovascular and cerebrovascular diseases such as hypertension. We used a computational-based strategy to first screen for differentially expressed genes and lncRNAs and characterized associations between lncRNAs of microvascular pericytes and hypertension. In total, 22 lncRNAs were upregulated and 46 were downregulated in the rats afflicted with spontaneous hypertension. Expression profiles for lncRNAs were significantly altered in the hypertension afflicted tissue samples and the transcripts have good potential for use as molecular targets to inhibit the progression of hypertension.

The feedback regulation of carbohydrates intake on food intake and appetite in grass carp (Ctenopharyngodon idella).

Improving carbohydrate utilization can contribute to sustainability of aquaculture. In order to explore the feedback mechanism of glucose homeostasis in fish, one control diet (25% carbohydrate and 40% protein), one relatively high carbohydrate diet named HG (42% carbohydrate and 40% protein), and one high dietary carbohydrate coupled with relatively low protein diet named HGP (42% carbohydrate and 25% protein) were fed to grass carp for 40 days. After the feeding trial, HG group impeded the food intake and growth performance of fish compared with the other two groups. Meanwhile, the serum glucose and insulin level were both significantly elevated under the condition of high carbohydrates intake when compared HG with control group. However, although no significant difference was observed in peripheral glucose or insulin between HG and HGP groups, fish fed with HGP diet increased growth performance and food intake compared with the HG group. Gene expression data indicated that fish selectively regulated the expressions of the cerebral anorexigenic genes (cart and pomc) to adapt to the HG and HGP intake. Therefore, the HGP diet with high carbohydrate and low protein was more suitable for grass carp feeding and growth when compared with the other two diets, possibly because the diet composition was closer to the natural food of this fish. In addition, the serum leptin level was highly consistent with changes in food intake and anorexigenic gene expressions when comparing the three experimental diets, indicating that leptin might be the key to mediate the feedback regulation of carbohydrates intake on food intake and appetite in fish.

Bivariate logistic Bayesian LASSO for detecting rare haplotype association with two correlated phenotypes.

In genetic association studies, joint modeling of related traits/phenotypes can utilize the correlation between them and thereby provide more power and uncover additional information about genetic etiology. Moreover, detecting rare genetic variants are of current scientific interest as a key to missing heritability. Logistic Bayesian LASSO (LBL) has been proposed recently to detect rare haplotype variants using case-control data, that is, a single binary phenotype. As there is currently no haplotype association method that can handle multiple binary phenotypes, we extend LBL to fill this gap. We develop a bivariate model by using a latent variable to induce correlation between the two outcomes. We carry out extensive simulations to investigate the bivariate LBL and compare with the univariate LBL. The bivariate LBL performs better or similar to the univariate LBL in most settings. It has the highest gain in power when a haplotype is associated with both traits and it affects at least one trait in a direction opposite to the direction of the correlation between the traits. We analyze two data sets-Genetic Analysis Workshop 19 sequence data on systolic and diastolic blood pressures and a genome-wide association data set on lung cancer and smoking and detect several associated rare haplotypes.

Expansion of sweet taste receptor genes in grass carp (Ctenopharyngodon idellus) coincided with vegetarian adaptation.

BACKGROUND: Taste is fundamental to diet selection in vertebrates. Genetic basis of sweet taste receptor in the shaping of food habits has been extensively studied in mammals and birds, but scarcely studied in fishes. Grass carp is an excellent model for studying vegetarian adaptation, as it exhibits food habit transition from carnivory to herbivory. RESULTS: We identified six sweet taste receptors (gcT1R2A-F) in grass carp. The four gcT1R2s (gcT1R2C-F) have been suggested to be evolved from and paralogous to the two original gcT1R2s (gcT1R2A and gcT1R2B). All gcT1R2s were expressed in taste organs and mediated glucose-, fructose- or arginine-induced intracellular calcium signaling, revealing they were functional. In addition, grass carp was performed to prefer fructose to glucose under a behavioral experiment. Parallelly, compared with gcT1R2A-F/gcT1R3 co-transfected cells, gcT1R2C-F/gcT1R3 co-transfected cells showed a higher response to plant-specific fructose. Moreover, food habit transition from carnivory to herbivory in grass carp was accompanied by increased gene expression of certain gcT1R2s. CONCLUSIONS: We suggested that the gene expansion of T1R2s in grass carp was an adaptive strategy to accommodate the change in food environment. Moreover, the selected gene expression of gcT1R2s might drive the food habit transition from carnivory to herbivory in grass carp. This study provided some evolutional and physiological clues for the formation of herbivory in grass carp.

Tricarboxylic Acid (TCA) Cycle Enzymes and Intermediates Modulate Intracellular Cyclic di-GMP Levels and the Production of Plant Cell Wall-Degrading Enzymes in Soft Rot Pathogen Dickeya dadantii.

Dickeya dadantii is a plant-pathogenic bacterium that causes soft-rot in a wide range of plants. Although we have previously demonstrated that cyclic bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial secondary messenger, plays a central role in virulence regulation in D. dadantii, the upstream signals that modulate c-di-GMP remain enigmatic. Using a genome-wide transposon mutagenesis approach of a Δhfq mutant strain that has high c-di-GMP and reduced motility, we uncovered transposon mutants that recovered the c-di-GMP-mediated repression on swimming motility. A number of these mutants harbored transposon insertions in genes encoding tricarboxylic acid (TCA) cycle enzymes. Two of these TCA transposon mutants were studied further by generating chromosomal deletions of the fumA gene (encoding fumarase) and the sdhCDAB operon (encoding succinate dehydrogenase). Disruption of the TCA cycle in these deletion mutants resulted in reduced intracellular c-di-GMP and enhanced production of pectate lyases (Pels), a major plant cell wall-degrading enzyme (PCWDE) known to be transcriptionally repressed by c-di-GMP. Consistent with this result, addition of TCA cycle intermediates such as citrate also resulted in increased c-di-GMP levels and decreased production of Pels. Additionally, we found that a diguanylate cyclase GcpA was solely responsible for the observed citrate-mediated modulation of c-di-GMP. Finally, we demonstrated that addition of citrate induced not only an overproduction of GcpA protein but also a concomitant repression of the c-di-GMP-degrading phosphodiesterase EGcpB which, together, resulted in an increase in the intracellular concentration of c-di-GMP. In summary, our report demonstrates that bacterial respiration and respiration metabolites serve as signals for the regulation of c-di-GMP signaling.

Differential miRNA expression analysis of extracellular vesicles from brain microvascular pericytes in spontaneous hypertensive rats.

OBJECTIVE: This study is to explore the exact roles of extracellular vesicle (EVs) miRNAs from brain microvascular pericytes in the pathogenesis of hypertension. RESULTS: Forty-eight significantly differentially expressed miRNAs were identified, of which 17 were found to be upregulated and 31 were found to be downregulated in brain microvascular pericytes of spontaneous hypertensive rats, compared with that of normotension Wistar Kyoto rats. The GO enrichment analysis verified that the target genes were enriched in signaling pathways and molecular functions, such as metal ion binding, nucleotide binding and ATP binding. The KEGG analysis indicated that the target genes were enriched in Linoleic acid, alpha-linolenic acid and sphingolipid metabolism pathways. CONCLUSIONS: Several EV derived miRNAs, such as miR-21-5p, let-7c-5p and let-7a-5p, were found to be abnormally expressed in brain microvascular pericytes obtained from spontaneous hypertensive rats, compared with that of normotension Wistar Kyoto rats. The results of our research provide more insights into the functional link between brain microvascular pericytes and the pathogenesis of hypertension.

Xanthomonas oryzae pv. oryzae Response Regulator TriP Regulates Virulence and Exopolysaccharide Production Via Interacting With c-di-GMP Phosphodiesterase PdeR.

PdeR, a response regulator of the two-component system (TCS) with the cognate histidine kinase PdeK, has been shown to be an active phosphodiesterase (PDE) for intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) turnover and positively regulates the virulence of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice. To further reveal the key components and pathways involved in the PdeR-mediated c-di-GMP regulation of virulence, 16 PdeR-interacting proteins were identified, using the yeast two-hybrid (Y2H) assay. Among them, PXO_04421 (named as TriP, a putative transcriptional regulator interacting with PdeR) was verified via Y2H and glutathione-S-transferase pull-down assays, and its regulatory functions in bacterial virulence and exopolysaccharide (EPS) production were assessed by biochemical and genetic analysis. The REC domain of TriP specifically interacted with the EAL domain of PdeR. TriP promoted the PDE activity of PdeR to degrade c-di-GMP in the presence of PdeK. In-frame deletion in triP abolished the polar localization of PdeR in the cell. Notably, the ∆triP mutant showed significantly reduced virulence on susceptible rice leaves and impaired EPS production compared with wild type, whereas the double mutant ∆triP∆pdeR, like ∆pdeR, caused shorter lesion lengths and produced less EPS than ∆triP. In addition, cross-complementation showed in trans expression of pdeR in ∆triP restored its EPS production to near wild-type levels but not vice versa. Taken together, our results suggest that TriP is a novel regulator that is epistatic to PdeR in positively regulating virulence expression in X. oryzae pv. oryzae.

A feed-forward signalling circuit controls bacterial virulence through linking cyclic di-GMP and two mechanistically distinct sRNAs, ArcZ and RsmB.

Dickeya dadantii is a plant pathogen that causes soft rot disease on vegetable and potato crops. To successfully cause infection, this pathogen needs to coordinately modulate the expression of genes encoding several virulence determinants, including plant cell wall degrading enzymes (PCWDEs), type III secretion system (T3SS) and flagellar motility. Here, we uncover a novel feed-forward signalling circuit for controlling virulence. Global RNA chaperone Hfq interacts with an Hfq-dependent sRNA ArcZ and represses the translation of pecT, encoding a LysR-type transcriptional regulator. We demonstrate that the ability of ArcZ to be processed to a 50 nt 3'- end fragment is essential for its regulation of pecT. PecT down-regulates PCWDE and the T3SS by repressing the expression of a global post-transcriptional regulator- (RsmA-) associated sRNA encoding gene rsmB. In addition, we show that the protein levels of two cyclic di-GMP (c-di-GMP) diguanylate cyclases (DGCs), GcpA and GcpL, are repressed by Hfq. Further studies show that both DGCs are essential for the Hfq-mediated post-transcriptional regulation on RsmB. Overall, our report provides new insights into the interplays between ubiquitous signalling transduction systems that were most studied independently and sheds light on multitiered regulatory mechanisms for a precise disease regulation in bacteria.

Intraspinal administration of interleukin-7 promotes neuronal apoptosis and limits functional recovery through JAK/STAT5 pathway following spinal cord injury.

It has been previously reported that the blockade of interleukin-7 receptor (IL-7R) promotes functional recovery following spinal cord injury (SCI), however, the direct function and molecular mechanism of IL-7 involved in this pathogenic process are unclear. Here, we report that, contrary to IL-7R blockade, the intraspinal administration of IL-7 limits functional recovery following SCI. In addition, IL-7 treatment promotes neuronal apoptosis in spinal cord lesions, which may be attributed to exacerbated focal inflammatory response, as shown by increased accumulation of activated microglia/macrophage and production of proinflammatory mediators. Moreover, IL-7 treatment activates JAK/STAT5 pathway following SCI. At last, more importantly, the pharmacological inhibition of STAT5 abrogates the effects of IL-7 treatment on functional recovery, neuronal apoptosis and focal inflammatory response, suggesting that the effects of IL-7 treatment following SCI are dependent on activating the JAK/STAT5 pathway. Overall, this study reveals the JAK/STAT5 pathway-dependent detrimental role of IL-7 following SCI, and also implies that targeting the IL-7/JAK/STAT5 axis may represent a potential therapeutic approach for SCI treatment.

A comparison of the cutaneous microvascular properties of the Spontaneously Hypertensive and the Wistar-Kyoto rats by Spectral analysis of Laser Doppler.

This work was aimed to study skin blood perfusion, vasomotion and vascular responses of the Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) in different stages of age using spectral. Laser-Doppler flowmetry (LDF) was used to examine the ears and limbs of WKY (12 and 48 weeks old) and SHR (12 and 48 weeks old). The skin blood flow oscillations (SBFOs) were studied by wavelet spectral analysis of LDF tracings. Then, we observed that old groups showed decreased perfusion and SBFO in the ears of both SHR and WKY. The SHR showed obviously lower postocclusive reactive hypera (PORH) ratio at the same age. A decreased peak-time occurred in the SHR of old age group. After PORH test, a statistically significant increase was observed within all subintervals in the absolute amplitude of 12-week WKY and only within IV and III subintervals in the absolute amplitude of 12-week SHR. But, the absolute amplitude of 48-week WKY and SHR showed no statistically significant increase within all subintervals. Results indicated that local regulating function of peripheral vascular was impaired in rat with hypertension and aging. Abbreviations LDF: Laser-Doppler flowmetry; SBF: Skin blood flow; SBFO: Skin blood flow oscillation; PORH: Postocclusive reactive hyperemia; SHR: Spontaneously hypertensive rats; WKY: Wistar-Kyoto rats; LDF: Laser-Doppler flowmetry; LDI: Laser Doppler Imaging; BP: Blood pressure.