Epigenetic upregulation of carotid body angiotensin signaling increases blood pressure.

Epigenetic changes can be shaped by a wide array of environmental cues as well as maternal health and behaviors. One of the most detrimental behaviors to the developing fetus is nicotine exposure. Perinatal nicotine exposure remains a significant risk factor for cardiovascular health and in particular, hypertension. Increased basal carotid body activity and excitation are significant contributors to hypertension. This study investigated the epigenetic changes to carotid body activity induced by perinatal nicotine exposure resulting in carotid body-mediated hypertension. Using a rodent model of perinatal nicotine exposure, we show that angiotensin II type 1 receptor signaling is upregulated in the carotid bodies of nicotine-exposed offspring. These changes were attributed to an upregulation of genetic promotion as DNA methylation of AT1r and PKC occurred within intron regions, exemplifying an upregulation of genetic transcription for these genes. Nicotine increased angiotensin signaling in vitro . Carotid body reactivity to angiotensin was increased in perinatal nicotine-exposed offspring compared to control offspring. Further, carotid body denervation reduced arterial pressure as a result of suppressed efferent sympathetic activity in perinatal nicotine-exposed offspring. Our data demonstrate that perinatal nicotine exposure adversely affects carotid body afferent sensing, which augments efferent sympathetic activity to increase vasoconstrictor signaling and induce hypertension. Targeting angiotensin signaling in the carotid bodies may provide a way to alleviate hypertension acquired by adverse maternal uterine environments in general and perinatal nicotine exposure in particular.

The Purine Biosynthesis Repressor, PurR, Contributes to Vancomycin Susceptibility of Methicillin-resistant Staphylococcus aureus in Experimental Endocarditis.

BACKGROUND: Staphylococcus aureus is the most common cause of life-threatening endovascular infections, including infective endocarditis (IE). These infections, especially when caused by methicillin-resistant strains (MRSA), feature limited therapeutic options and high morbidity and mortality rates. METHODS: Herein, we investigated the role of the purine biosynthesis repressor, PurR, in virulence factor expression and vancomycin (VAN) treatment outcomes in experimental IE due to MRSA. RESULTS: The PurR-mediated repression of purine biosynthesis was confirmed by enhanced purF expression and production of an intermediate purine metabolite in purR mutant strain. In addition, enhanced expression of the transcriptional regulators, sigB and sarA, and their key downstream virulence genes (eg, fnbA, and hla) was demonstrated in the purR mutant in vitro and within infected cardiac vegetations. Furthermore, purR deficiency enhanced fnbA/fnbB transcription, translating to increased fibronectin adhesion versus the wild type and purR-complemented strains. Notably, the purR mutant was refractory to significant reduction in target tissues MRSA burden following VAN treatment in the IE model. CONCLUSIONS: These findings suggest that the purine biosynthetic pathway intersects the coordination of virulence factor expression and in vivo persistence during VAN treatment, and may represent an avenue for novel antimicrobial development targeting MRSA.

Sensory neurons regulate stimulus-dependent humoral immunity.

Sensory neurons sense pathogenic infiltration, serving to inform immune coordination of host defense. However, sensory neuron-immune interactions have been predominantly shown to drive innate immune responses. Humoral memory, whether protective or destructive, is acquired early in life - as demonstrated by both early exposure to streptococci and allergic disease onset. Our study further defines the role of sensory neuron influence on humoral immunity in the lung. Using a murine model of Streptococcus pneumonia pre-exposure and infection and a model of allergic asthma, we show that sensory neurons are required for B-cell and plasma cell recruitment and antibody production. In response to S. pneumoniae , sensory neuron depletion resulted in a larger bacterial burden, reduced B-cell populations, IgG release and neutrophil stimulation. Conversely, sensory neuron depletion reduced B-cell populations, IgE and asthmatic characteristics during allergen-induced airway inflammation. The sensory neuron neuropeptide released within each model differed. With bacterial infection, vasoactive intestinal polypeptide (VIP) was preferentially released, whereas substance P was released in response to asthma. Administration of VIP into sensory neuron-depleted mice suppressed bacterial burden and increased IgG levels, while VIP1R deficiency increased susceptibility to bacterial infection. Sensory neuron-depleted mice treated with substance P increased IgE and asthma, while substance P genetic ablation resulted in blunted IgE, similar to sensory neuron-depleted asthmatic mice. These data demonstrate that the immunogen differentially stimulates sensory neurons to release specific neuropeptides which specifically target B-cells. Targeting sensory neurons may provide an alternate treatment pathway for diseases involved with insufficient and/or aggravated humoral immunity.

Gp05, a Prophage-Encoded Virulence Factor, Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a serious public health threat. We recently demonstrated that the presence of a novel prophage ϕSA169 was associated with vancomycin (VAN) treatment failure in experimental MRSA endocarditis. In this study, we assessed the role of a ϕSA169 gene, ϕ80α_gp05 (gp05), in VAN-persistent outcome using gp05 isogenic MRSA strain sets. Of note, Gp05 significantly influences the intersection of MRSA virulence factors, host immune responses, and antibiotic treatment efficacy, including the following: (i) activity of the significant energy-yielding metabolic pathway (e.g., tricarboxylic acid cycle); (ii) carotenoid pigment production; (iii) (p)ppGpp (guanosine tetra- and pentaphosphate) production, which activates the stringent response and subsequent downstream functional factors (e.g., phenol-soluble modulins and polymorphonuclear neutrophil bactericidal activity); and (iv) persistence to VAN treatment in an experimental infective endocarditis model. These data suggest that Gp05 is a significant virulence factor which contributes to the persistent outcomes in MRSA endovascular infection by multiple pathways. IMPORTANCE Persistent endovascular infections are often caused by MRSA strains that are susceptible to anti-MRSA antibiotics in vitro by CLSI breakpoints. Thus, the persistent outcome represents a unique variant of traditional antibiotic resistance mechanisms and a significant therapeutic challenge. Prophage, a critical mobile genetic element carried by most MRSA isolates, provides their bacterial host with metabolic advantages and resistance mechanisms. However, how prophage-encoded virulence factors interact with the host defense system and antibiotics, driving the persistent outcome, is not well known. In the current study, we demonstrated that a novel prophage gene, gp05, significantly impacts tricarboxylic acid cycle activity, stringent response, and pigmentation, as well as vancomycin treatment outcome in an experimental endocarditis model using isogenic gp05 overexpression and chromosomal deletion mutant MRSA strain sets. The findings significantly advance our understanding of the role of Gp05 in persistent MRSA endovascular infection and provide a potential target for development of novel drugs against these life-threatening infections.

Transcriptome profiling of transcription factors in Ganoderma lucidum in response to methyl jasmonate.

Ganoderma lucidum is a traditional Chinese medicine and its major active ingredients are ganoderma triterpenoids (GTs). To screen for transcription factors (TFs) that involved in the biosynthetic pathway of GTs in G. lucidum, the chemical composition in mycelia, primordium and fruiting body were analyzed, and the transcriptomes of mycelia induced by methyl jasmonate (MeJA) were analyzed. In addition, the expression level data of MeJA-responsive TFs in mycelia, primordia and fruiting body were downloaded from the database, and the correlation analysis was carried out between their expression profiles and the content of total triterpenoids. The results showed that a total of 89 components were identified, and the content of total triterpenoids was the highest in primordium, followed by fruiting body and mycelia. There were 103 differentially expressed TFs that response to MeJA-induction including 95 upregulated and 8 downregulated genes. These TFs were classified into 22 families including C2H2 (15), TFII-related (12), HTH (9), fungal (8), bZIP (6), HMG (5), DADS (2), etc. Correlation analysis showed that the expression level of GL23559 (MADS), GL26472 (HTH), and GL31187 (HMG) showed a positive correlation with the GTs content, respectively. While the expression level of GL25628 (fungal) and GL26980 (PHD) showed a negative correlation with the GTs content, respectively. Furthermore, the over expression of the Glmhr1 gene (GL25628) in Pichia pastoris GS115 indicated that it might be a negative regulator of GT biosynthesis through decreasing the production of lanosterol. This study provided useful information for a better understanding of the regulation of TFs involved in GT biosynthesis and fungal growth in G. lucidum.

Transcriptome Analyses of Prophage in Mediating Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant subset of S. aureus infections and correlate with exceptionally high mortality. We have recently demonstrated that the lysogenization of prophage ϕSA169 from a clinical persistent MRSA bacteremia isolate (300-169) into a clinical resolving bacteremia MRSA isolate (301-188) resulted in the acquisition of well-defined in vitro and in vivo phenotypic and genotypic profiles related to persistent outcome. However, the underlying mechanism(s) of this impact is unknown. In the current study, we explored the genetic mechanism that may contribute to the ϕSA169-correlated persistence using RNA sequencing. Transcriptomic analyses revealed that the most significant impacts of ϕSA169 were: (i) the enhancement of fatty acid biosynthesis and purine and pyrimidine metabolic pathways; (ii) the repression of galactose metabolism and phosphotransferase system (PTS); and (iii) the down-regulation of the mutual prophage genes in both 300-169 and 301-188 strains. In addition, the influence of different genetic backgrounds between 300-169 and 301-188 might also be involved in the persistent outcome. These findings may provide targets for future studies on the persistence of MRSA.

New Mechanistic Insights into Purine Biosynthesis with Second Messenger c-di-AMP in Relation to Biofilm-Related Persistent Methicillin-Resistant Staphylococcus aureus Infections.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinically challenging subset of invasive, life-threatening S. aureus infections. We have recently demonstrated that purine biosynthesis plays an important role in such persistent infections. Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger that regulates many cellular pathways in bacteria. However, whether there is a regulatory connection between the purine biosynthesis pathway and c-di-AMP impacting persistent outcomes was not known. Here, we demonstrated that the purine biosynthesis mutant MRSA strain, the ΔpurF strain (compared to its isogenic parental strain), exhibited the following significant differences in vitro: (i) lower ADP, ATP, and c-di-AMP levels; (ii) less biofilm formation with decreased extracellular DNA (eDNA) levels and Triton X-100-induced autolysis paralleling enhanced expressions of the biofilm formation-related two-component regulatory system lytSR and its downstream gene lrgB; (iii) increased vancomycin (VAN)-binding and VAN-induced lysis; and (iv) decreased wall teichoic acid (WTA) levels and expression of the WTA biosynthesis-related gene, tarH. Substantiating these data, the dacA (encoding diadenylate cyclase enzyme required for c-di-AMP synthesis) mutant strain (dacAG206S strain versus its isogenic wild-type MRSA and dacA-complemented strains) showed significantly decreased c-di-AMP levels, similar in vitro effects as seen above for the purF mutant and hypersusceptible to VAN treatment in an experimental biofilm-related MRSA endovascular infection model. These results reveal an important intersection between purine biosynthesis and c-di-AMP that contributes to biofilm-associated persistence in MRSA endovascular infections. This signaling pathway represents a logical therapeutic target against persistent MRSA infections. IMPORTANCE Persistent endovascular infections caused by MRSA, including vascular graft infection syndromes and infective endocarditis, are significant and growing public health threats. A particularly worrisome trend is that most MRSA isolates from these patients are "susceptible" in vitro to conventional anti-MRSA antibiotics, such as VAN and daptomycin (DAP), based on Clinical and Laboratory Standards Institute breakpoints. Yet, these antibiotics frequently fail to eliminate these infections in vivo. Therefore, the persistent outcomes in MRSA infections represent a unique and important variant of classic "antibiotic resistance" that is only disclosed during in vivo antibiotic treatment. Given the high morbidity and mortality associated with the persistent infection, there is an urgent need to understand the specific mechanism(s) of this syndrome. In the current study, we demonstrate that a functional intersection between purine biosynthesis and the second messenger c-di-AMP plays an important role in VAN persistence in experimental MRSA endocarditis. Targeting this pathway may represent a potentially novel and effective strategy for treating these life-threatening infections.

[Construction of yeast one-hybrid library and screening of transcription factors regulating LS expression in Ganoderma lucidum].

Lanosterol synthase( LS) is a key enzyme involving in the mevalonate pathway( MVA pathway) to produce lanosterol,which is a precursor of ganoderma triterpenoid. And the transcriptional regulation of LS gene directly affects the content of triterpenes in Ganoderma lucidum. In order to study the transcriptional regulation mechanism of LS gene,yeast one-hybrid technique was used to screen the transcription regulators which interact withthe promoter of LS. The bait vector was constructed by LS promoter,then the vector was transformed yeast cells to construct bait yeast strain. One-hybrid c DNA library was constructed via SMART technology. Then the c DNA and p GADT7-Rec vector were co-transformed into the bait yeast strain to screen the upstream regulatory factors of the promoter region of LS by homologous recombination. Total of 23 positive clones were screened. After sequencing,blast was performed against the whole-genome sequence of G. lucidum. As a result,8 regulatory factors were screened out including the transcription initiation TFIIB,the alpha/beta hydrolase super family,ALDH-SF superfamily,60 S ribosomal protein L21,ATP synthase ß-subunit,microtubule associated protein Cript,prote asome subunit ß-1,and transaldolase. Until now,the regulation effect of these 8 regulatory factors in G.lucidum has not been reported. This study provides candidate proteins for in-depth study on the expression regulation of LS.

Increased activity of the orexin system in the paraventricular nucleus contributes to salt-sensitive hypertension.

The orexin system is involved in arginine vasopressin (AVP) regulation, and its overactivation has been implicated in hypertension. However, its role in salt-sensitive hypertension (SSHTN) is unknown. Here, we tested the hypothesis that hyperactivity of the orexin system in the paraventricular nucleus (PVN) contributes to SSHTN via enhancing AVP signaling. Eight-week-old male Dahl salt-sensitive (Dahl S) and age- and sex-matched Sprague-Dawley (SD) rats were placed on a high-salt (HS; 8% NaCl) or normal-salt (NS; 0.4% NaCl) diet for 4 wk. HS intake did not alter mean arterial pressure (MAP), PVN mRNA levels of orexin receptor 1 (OX1R), or OX2R but slightly increased PVN AVP mRNA expression in SD rats. HS diet induced significant increases in MAP and PVN mRNA levels of OX1R, OX2R, and AVP in Dahl S rats. Intracerebroventricular infusion of orexin A (0.2 nmol) dramatically increased AVP mRNA levels and immunoreactivity in the PVN of SD rats. Incubation of cultured hypothalamus neurons from newborn SD rats with orexin A increased AVP mRNA expression, which was attenuated by OX1R blockade. In addition, increased cerebrospinal fluid Na+ concentration through intracerebroventricular infusion of NaCl solution (4 µmol) increased PVN OX1R and AVP mRNA levels and immunoreactivity in SD rats. Furthermore, bilateral PVN microinjection of the OX1R antagonist SB-408124 resulted in a greater reduction in MAP in HS intake (-16 ± 5 mmHg) compared with NS-fed (-4 ± 4 mmHg) anesthetized Dahl S rats. These results suggest that elevated PVN OX1R activation may contribute to SSHTN by enhancing AVP signaling.NEW & NOTEWORTHY To our best knowledge, this study is the first to investigate the involvement of the orexin system in salt-sensitive hypertension. Our results suggest that the orexin system may contribute to the Dahl model of salt-sensitive hypertension by enhancing vasopressin signaling in the hypothalamic paraventricular nucleus.

New Findings on the Effects of Tannic Acid: Inhibition of L-Type Calcium Channels, Calcium Transient and Contractility in Rat Ventricular Myocytes.

Tannic acid (TA) is a group of water-soluble polyphenolic compounds that occur mainly in plant-derived feeds, food grains and fruits. Many studies have explored its biomedical properties, such as anticancer, antibacterial, antimutagenic, antioxidant, antidiabetic, antiinflammatory and antihypertensive activities. However, the effects of TA on the L-type Ca(2+) current (ICa-L) of cardiomyocytes remain undefined. The present study examined the effects of TA on ICa-L using the whole-cell patch-clamp technique and on intracellular Ca(2+) handling and cell contractility in rat ventricular myocytes with the aid of a video-based edge detection system. Exposure to TA resulted in a concentration- and voltage-dependent blockade of ICa-L, with the half maximal inhibitory concentration of 1.69 µM and the maximal inhibitory effect of 46.15%. Moreover, TA significantly inhibited the amplitude of myocyte shortening and peak value of Ca(2+) transient and increased the time to 10% of the peak. These findings provide new experimental evidence for the cellular mechanism of action of TA and may help to expand clinical treatments for cardiovascular disease.

Salvia miltiorrhiza (Danshen) inhibits L-type calcium current and attenuates calcium transient and contractility in rat ventricular myocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza (SM, Danshen), a traditional Chinese herbal drug, has been widely used for hundreds of years to treat coronary artery disease. MATERIALS AND METHODS: We studied the effects of SM on the L-type Ca(2+) current (ICa-L) with whole-cell patch-clamp technique in rat ventricular myocytes, and its influence on Ca(2+) transient and contractility using video-based edge detection and dual excitation fluorescence photomultiplier systems as well. RESULTS: Exposure to SM solution caused a concentration- and voltage-dependent blockade of ICa-L, and the dose of SM solution (10g/l) decreased the maximal inhibitory effect of 35.2±1.2%. However, SM solution did not significantly change the current-voltage relationship or reversal potential of ICa-L, nor did it altered the activation and inactivation gating properties of cardiac Ca(2+) channels. Meanwhile, SM decreased the amplitude of myocyte shortening and the peak value of Ca(2+) transient with a significant decrease in the time to 90% of the baseline (Tr), but the time to 10% of the peak (Tp) was not dramatically prolonged. CONCLUSIONS: The results indicated that SM significantly inhibited L-type Ca(2+) channels, decreased [Ca(2+)]i and contractility in adult rat cardiac myocytes. These findings may be relevant to the cardioprotective efficacy of SM.