Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Factors important to the secretion and matrix deposition of tissue transglutaminase

Data suggest that for TG2 to be secreted, an intact N-terminal FN binding site (for which TG2 has high affinity) is required, however interaction of TG2 with its high affinity binding partners presents both in the intracellular and extracellular space as well as with specific cell surface receptors may also be involved in this process. Using a site-directed mutagenesis approach, the effects of specific mutations of TG2 on its translocation to the cell surface and secretion into the ECM have been investigated. Mutations include those affecting FN binding (FN1), HSPGs binding (HS1, HS2) GTP/GDP binding site (GTP1, 2) as well as N-terminal and C-terminal domains (TG2 deletion mutants N, and C). By performing transglutaminase activity assays, cell surface protein biotinylation and verifying distribution of TG2 mutants in the ECM we demonstrated that one of the potential heparan sulfate binding site mutants (HS2 mutant) is secreted at the cell surface in a much reduced manner and is less deposited into the ECM than the HS1 mutant. The HS2 mutant showed a low affinity for binding to a heparin sepharose column demonstrating this mutation site may be a potential heparan binding site of TG2. Analogous peptides to this site were shown to have some efficiency in the inhibition of the binding of the FN-TG2 complex to cell surface heparan sulfates in a cell adhesion assay indicating the peptide to be representative of the novel heparin binding site within TG2. The GTP binding site mutants GTP1 and GTP2 exhibited low specific activity however, GTP2 showed more secretion to the cell surface in comparison to GTP1. The FN1 binding mutant did not greatly affect TG2 activity nor did it alter TG2 secretion at the cell surface and deposition into the ECM indicating that fibronectin binding at this site on the enzyme is not an important factor. Interestingly an intact N-terminus (?1-15) appeared to be essential for enzyme externalisation. Removal of the first 15 amino acids (N-terminal mutant) abolished TG2 secretion to the cell surface as well as deposition into the ECM. In addition it reduced the enzymes affinity for binding to heparin. In contrast, deletion of the C-terminal TG2 domain (?594-687) increased enzyme secretion to the cell surface. Consistent with the data presented in this thesis we speculate that TG2 must fulfill two requirements to be successfully secreted from cells. The findings indicate that the closed conformation of the enzyme as well as intact N-terminal tail and a novel HS binding site within the TG2 molecule are key elements for the enzyme’s localisation at the cell surface and its deposition into the extracellular matrix. The importance of understanding the interactions between TG2, heparan sulfates and other TG2 binding partners at the cell surface could have an impact on the design of novel strategies for enzyme inhibition which could be important in the control of extracellular TG2 related diseases.

Adrenomedullin and calcitonin gene-related peptide receptors in endocrine-related cancers:opportunities and challenges

Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the 'ADMR'), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.

Factors preventing myopia progression with orthokeratology correction

PURPOSE: To examine which baseline measurements constitute predictive factors for axial length growth over 2 years in children wearing orthokeratology contact lenses (OK) and single-vision spectacles (SV). METHODS: Sixty-one children were prospectively assigned to wear either OK (n = 31) or SV (n = 30) for 2 years. The primary outcome measure (dependent variable) was axial length change at 2 years relative to baseline. Other measurements (independent variables) were age, age of myopia onset, gender, myopia progression 2 years before baseline and baseline myopia, anterior chamber depth, corneal power and shape (p value), and iris and pupil diameters as well as parental refraction. The contribution of all independent variables to the 2-year change in axial length was assessed using univariate and multivariate regression analyses. RESULTS: After univariate analyses, smaller increases in axial length were found in the OK group compared to the SV group in children who were older, had earlier onset of myopia, were female, had lower rate of myopia progression before baseline, had less myopia at baseline, had longer anterior chamber depth, had greater corneal power, had more prolate corneal shape, had larger iris diameter, had larger pupil sizes, and had lower levels of parental myopia (all p < 0.05). In multivariate analyses, older age and greater corneal power were associated with smaller increases in axial length in the OK group (both p < 0.05), whereas in SV wearers, smaller iris diameter was associated with smaller increases in axial length (p = 0.021). CONCLUSIONS: Orthokeratology is a successful treatment option in controlling axial elongation compared to SV in children of older age, had earlier onset of myopia, were female, had lower rate of myopia progression before baseline, had lower myopia at baseline, had longer anterior chamber depth, had greater corneal power, had more prolate corneal shape, had larger iris and pupil diameters, and had lower levels of parental myopia. © American Academy of Optometry.

Water network dynamics at the critical moment of a peptide's ß-turn formation:a molecular dynamics study

All-atom molecular dynamics simulations for a single molecule of Leu-Enkephalin in aqueous solution have been used to study the role of the water network during the formation of ß-turns. We give a detailed account of the intramolecular hydrogen bonding, the water-peptide hydrogen bonding, and the orientation and residence times of water molecules focusing on the short critical periods of transition to the stable ß-turns. These studies suggest that, when intramolecular hydrogen bonding between the first and fourth residue of the ß-turn is not present, the disruption of the water network and the establishment of water bridges constitute decisive factors in the formation and stability of the ß-turn. Finally, we provide possible explanations and mechanisms for the formations of different kinds of ß-turns.

Laminar distribution of β-amyloid (Aβ) peptide deposits in the frontal lobe in familial and sporadic Alzheimer's disease

To determine whether genetic factors influence frontal lobe degeneration in Alzheimer's disease (AD), the laminar distributions of diffuse, primitive, and classic β-amyloid (Aβ) peptide deposits were compared in early-onset familial AD (EO-FAD) linked to mutations of the amyloid precursor protein (APP) or presenilin 1 (PSEN1) gene, late-onset familial AD (LO-FAD), and sporadic AD (SAD). The influence of apolipoprotein E (Apo E) genotype on laminar distribution was also studied. In the majority of FAD and SAD cases, maximum density of the diffuse and primitive Aβ deposits occurred in the upper cortical layers, whereas the distribution of the classic Aβ deposits was more variable, either occurring in the lower layers, or a double-peaked (bimodal) distribution was present, density peaks occurring in upper and lower layers. The cortical layer at which maximum density of Aβ deposits occurred and maximum density were similar in EO-FAD, LO-FAD and SAD. In addition, there were no significant differences in distributions in cases expressing Apo E ε4 alleles compared with cases expressing the ε2 or ε3 alleles. These results suggest that gene expression had relatively little effect on the laminar distribution of Aβ deposits in the frontal lobe of the AD cases studied. Hence, the pattern of frontal lobe degeneration in AD is similar regardless of whether it is associated with APP and PSEN1, mutation, allelic variation in Apo E, or with SAD.

Influence of diabetes on natriuretic peptide thresholds in screening for Stage B heart failure

CONTEXT: Natriuretic peptide (NP) has been shown to be an effective screening tool to identify patients with Stage B heart failure and to have clinical value in preventing heart failure progression. The impact of associated metabolic confounders on the screening utility of NP needs clarification.

OBJECTIVE: To assess the impact of diabetes mellitus (DM) on NP screening for asymptomatic Stage B heart failure.

MATERIALS AND METHODS: The study population consisted of 1368 asymptomatic patients with cardiovascular risk factors recruited from general practice as part of the STOP-HF trial. B-type NP (BNP) was quantified at point-of-care.

RESULTS: BNP was found to be as accurate for detecting Stage B heart failure in DM patients compared to non-DM patients (AUC 0.75 [0.71,0.78] and 0.77 [0.72,0.82], respectively). However, different BNP thresholds are required to achieve the same level of diagnostic sensitivity in DM compared with non-DM patients. To achieve 80% sensitivity a difference of 5-ng/L lower is required for patients with DM.

CONCLUSION: Although a significantly different BNP threshold is detected for patients with DM, the BNP concentration difference is small and unlikely to warrant a clinically different diagnostic threshold.

Cost-effectiveness of natriuretic peptide-based screening and collaborative care: a report from the STOP-HF (St Vincent's Screening TO Prevent Heart Failure) study

AIMS: Prevention of cardiovascular disease and heart failure (HF) in a cost-effective manner is a public health goal. This work aims to assess the cost-effectiveness of the St Vincent's Screening TO Prevent Heart Failure (STOP-HF) intervention.

METHODS AND RESULTS: This is a substudy of 1054 participants with cardiovascular risk factors [median age 65.8 years, interquartile range (IQR) 57.8:72.4, with 4.3 years, IQR 3.4:5.2, follow-up]. Annual natriuretic peptide-based screening was performed, with collaborative cardiovascular care between specialist physicians and general practitioners provided to patients with BNP levels >50 pg/mL. Analysis of cost per case prevented and cost-effectiveness per quality-adjusted life year (QALY) gained was performed. The primary clinical endpoint of LV dysfunction (LVD) with or without HF was reduced in intervention patients [odds ratio (OR) 0.60; 95% confidence interval (CI) 0.38-0.94; P = 0.026]. There were 157 deaths and/or emergency hospitalizations for major adverse cardiac events (MACE) in the control group vs. 102 in the intervention group (OR 0.68; 95% CI 0.49-0.93; P = 0.01). The cost per case of LVD/HF prevented was €9683 (sensitivity range -€843 to €20 210), whereas the cost per MACE prevented was €3471 (sensitivity range -€302 to €7245). Cardiovascular hospitalization savings offset increased outpatient and primary care costs. The cost per QALY gain was €1104 and the intervention has an 88% probability of being cost-effective at a willingness to pay threshold of €30 000.

CONCLUSION: Among patients with cardiovascular risk factors, natriuretic peptide-based screening and collaborative care reduced LVD, HF, and MACE, and has a high probability of being cost-effective.

TRIAL REGISTRATION: NCT00921960.

Associates of an elevated natriuretic peptide level in stable heart failure patients:implications for targeted management

BACKGROUND: Persistently elevated natriuretic peptide (NP) levels in heart failure (HF) patients are associated with impaired prognosis. Recent work suggests that NP-guided therapy can improve outcome, but the mechanisms behind an elevated BNP remain unclear. Among the potential stimuli for NP in clinically stable patients are persistent occult fluid overload, wall stress, inflammation, fibrosis, and ischemia. The purpose of this study was to identify associates of B-type natriuretic peptide (BNP) in a stable HF population.

METHODS: In a prospective observational study of 179 stable HF patients, the association between BNP and markers of collagen metabolism, inflammation, and Doppler-echocardiographic parameters including left ventricular ejection fraction (LVEF), left atrial volume index (LAVI), and E/e prime (E/e') was measured.

RESULTS: Univariable associates of elevated BNP were age, LVEF, LAVI, E/e', creatinine, and markers of collagen turnover. In a multiple linear regression model, age, creatinine, and LVEF remained significant associates of BNP. E/e' and markers of collagen turnover had a persistent impact on BNP independent of these covariates.

CONCLUSION: Multiple variables are associated with persistently elevated BNP levels in stable HF patients. Clarification of the relative importance of NP stimuli may help refine NP-guided therapy, potentially improving outcome for this at-risk population.

Natriuretic peptide-based screening and collaborative care for heart failure:the STOP-HF randomized trial

IMPORTANCE: Prevention strategies for heart failure are needed.

OBJECTIVE: To determine the efficacy of a screening program using brain-type natriuretic peptide (BNP) and collaborative care in an at-risk population in reducing newly diagnosed heart failure and prevalence of significant left ventricular (LV) systolic and/or diastolic dysfunction.

DESIGN, SETTING, AND PARTICIPANTS: The St Vincent's Screening to Prevent Heart Failure Study, a parallel-group randomized trial involving 1374 participants with cardiovascular risk factors (mean age, 64.8 [SD, 10.2] years) recruited from 39 primary care practices in Ireland between January 2005 and December 2009 and followed up until December 2011 (mean follow-up, 4.2 [SD, 1.2] years).

INTERVENTION: Patients were randomly assigned to receive usual primary care (control condition; n=677) or screening with BNP testing (n=697). Intervention-group participants with BNP levels of 50 pg/mL or higher underwent echocardiography and collaborative care between their primary care physician and specialist cardiovascular service.

MAIN OUTCOMES AND MEASURES: The primary end point was prevalence of asymptomatic LV dysfunction with or without newly diagnosed heart failure. Secondary end points included emergency hospitalization for arrhythmia, transient ischemic attack, stroke, myocardial infarction, peripheral or pulmonary thrombosis/embolus, or heart failure.

RESULTS: A total of 263 patients (41.6%) in the intervention group had at least 1 BNP reading of 50 pg/mL or higher. The intervention group underwent more cardiovascular investigations (control, 496 per 1000 patient-years vs intervention, 850 per 1000 patient-years; incidence rate ratio, 1.71; 95% CI, 1.61-1.83; P<.001) and received more renin-angiotensin-aldosterone system-based therapy at follow-up (control, 49.6%; intervention, 56.5%; P=.01). The primary end point of LV dysfunction with or without heart failure was met in 59 (8.7%) of 677 in the control group and 37 (5.3%) of 697 in the intervention group (odds ratio [OR], 0.55; 95% CI, 0.37-0.82; P = .003). Asymptomatic LV dysfunction was found in 45 (6.6%) of 677 control-group patients and 30 (4.3%) of 697 intervention-group patients (OR, 0.57; 95% CI, 0.37-0.88; P = .01). Heart failure occurred in 14 (2.1%) of 677 control-group patients and 7 (1.0%) of 697 intervention-group patients (OR, 0.48; 95% CI, 0.20-1.20; P = .12). The incidence rates of emergency hospitalization for major cardiovascular events were 40.4 per 1000 patient-years in the control group vs 22.3 per 1000 patient-years in the intervention group (incidence rate ratio, 0.60; 95% CI, 0.45-0.81; P = .002).

CONCLUSION AND RELEVANCE: Among patients at risk of heart failure, BNP-based screening and collaborative care reduced the combined rates of LV systolic dysfunction, diastolic dysfunction, and heart failure.

TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00921960.

Attenuation of monocyte chemotaxis--a novel anti-inflammatory mechanism of action for the cardio-protective hormone B-type natriuretic peptide

B-type natriuretic peptide (BNP) is a prognostic and diagnostic marker for heart failure (HF). An anti-inflammatory, cardio-protective role for BNP was proposed. In cardiovascular diseases including pressure overload-induced HF, perivascular inflammation and cardiac fibrosis are, in part, mediated by monocyte chemoattractant protein (MCP)1-driven monocyte migration. We aimed to determine the role of BNP in monocyte motility to MCP1. A functional BNP receptor, natriuretic peptide receptor-A (NPRA) was identified in human monocytes. BNP treatment inhibited MCP1-induced THP1 (monocytic leukemia cells) and primary monocyte chemotaxis (70 and 50 %, respectively). BNP did not interfere with MCP1 receptor expression or with calcium. BNP inhibited activation of the cytoskeletal protein RhoA in MCP1-stimulated THP1 (70 %). Finally, BNP failed to inhibit MCP1-directed motility of monocytes from patients with hypertension (n = 10) and HF (n = 6) suggesting attenuation of this anti-inflammatory mechanism in chronic heart disease. We provide novel evidence for a direct role of BNP/NPRA in opposing human monocyte migration and support a role for BNP as a cardio-protective hormone up-regulated as part of an adaptive compensatory response to combat excess inflammation.

Long-term statin therapy in patients with systolic heart failure and normal cholesterol:effects on elevated serum markers of collagen turnover, inflammation, and B-type natriuretic peptide

BACKGROUND: The role of statin therapy in heart failure (HF) is unclear. The amino-terminal propeptide of procollagen type III (PIIINP) predicts outcome in HF, and yet there are conflicting reports of statin therapy effects on PIIINP.

OBJECTIVES: This study determined whether there was an increase in serum markers of inflammation, fibrosis (including PIIINP), and B-type natriuretic peptide (BNP) in patients with systolic HF and normal total cholesterol and determined the effects of long-term treatment with atorvastatin on these markers.

METHODS: Fifty-six white patients with systolic HF and normal cholesterol levels (age 72 [13] years; 68% male; body mass index 27.0 [7.3] kg/m(2); ejection fraction 35 [13]%; 46% with history of smoking) were randomly allocated to atorvastatin treatment for 6 months, titrated to 40 mg/d (A group) or not (C group). Age- and/or sex-matched subjects without HF (N group) were also recruited. Biomarkers were measured at baseline (all groups) and 6 months (A and C groups).

RESULTS: Serum markers of collagen turnover, inflammation, and BNP were significantly elevated in HF patients compared with normal participants (all P < 0.05). There were correlations between these markers in HF patients but not in normal subjects. Atorvastatin treatment for 6 months caused a significant reduction in the following biomarkers compared with baseline: BNP, from median (interquartile range) 268 (190-441) pg/mL to 185 (144-344) pg/mL; high-sensitivity C-reactive protein (hs-CRP), from 5.26 (1.95 -9.29) mg/L to 3.70 (2.34-6.81) mg/L; and PIIINP, from 4.65 (1.86) to 4.09 (1.25) pg/mL (all P < 0.05 baseline vs 6 months). Between-group differences were significant for PIIINP only (P = 0.027). There was a positive interaction between atorvastatin effects and baseline hs-CRP and PIIINP (P < 0.01).

CONCLUSIONS: Long-term statin therapy reduced PIIINP in this small, selected HF population with elevated baseline levels. Further evaluation of statin therapy in the management of HF patients with elevated PIIINP is warranted.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2-6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0-10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) or the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs.