In Timor-Leste, cardiac interventions and surgical procedures are largely provided by the East Timor Hearts Fund. Since March 2020, no Timorese patients have been able to travel to Australia for humanitarian cardiac procedures.

Abstract Background The Rheumatic Heart disease in Timor Leste school students (RHD-TL) study identified Timor Leste as having some of the highest rates of definite rheumatic heart disease (RHD) in the world. The RHD-TL follow-up study aimed to assess the delivery and outcomes of the secondary prophylaxis program in known patients with echocardiographic screen detected definite and borderline RHD. Methods School-students in Timor Leste where reassessed over a 3-year period since the initial study in 2016. Prospective assessments included adherence to secondary prophylaxis, complications of prophylaxis, follow-up clinical assessment and serial echocardiography. Of the 48 patients, 25 Definite and 23 Borderline, 38 (79%) of all patients, and 92% of definite RHD cases have had one or more follow-up assessments including full datasets for adherence, recurrence rates and progression of disease. Follow-up is provided by the volunteer paediatric cardiology team and rheumatic heart disease team of two NGOs in collaboration with local clinics. Results The median duration of follow-up of the 38 patients was of 1.6 years. The median age was 13 years (range 8-22) and 75% were female. Adherence rates in patients with definite RHD was on average greater than 95% during the follow-up period. Of the 23 patients with mild or moderate RHD one case with documented acute rheumatic fever (ARF) recurrence progressed whilst 8 cases improved on benzathine-penicillin G (BPG) therapy. There was no progression of the 6 borderline cases who were not prescribed BPG. Out of the 9 borderline cases in whom BPG was prescribed, one, with 67% adherence, had a documented episode of ARF leading to echocardiographic progression and moderate definite RHD. Conclusion This was the first follow-up study to look at disease natural history, both in treated and untreated groups, in Timor-Leste and brought practical insights into the efficacy of the Timor Leste RHD monitoring and prophylaxis programs. Its ongoing project will enable advocacy and quality assessment for the program as it expands. Abstract 225 Figure 1.

Apocynin (APO) was shown to be the major constituent of early herbal medicines used to reduce painful suffering from many ailments. Because of its proven utility in rat models, it is now an active candidate for treating humans for several major autoimmune diseases such as Alzheimer's, arthritis, and cardiovascular diseases. In plant research, APO is referred to as acetovanillone, a secondary metabolite that is induced in the leaf apoplast during bacterial invasion. Since its role in plants is unknown, we examined whether any of the attributes found in medical studies could found in plants. In medical studies, APO is considered to be an NADPH oxidase (NOX) inhibitor, however when tested in tobacco suspension cells, APO failed to inhibit NOX activity. In addition, when APO was used to pretreat plants against virulent or avirulent pathovars of Pseudomonas syringae, there was no reduction of symptoms. When pathovars were incubated with 100 mu M APO for 90 min, there was only a slight decrease in the bacterial population, suggesting that it was not toxic. In animals, a key step for the inhibition of NOX by APO appears to be its oxidation by myeloperoxidase, yielding dimers and trimers. In vitro studies using pH 6, which is more comparable the plant environment, APO oxidation by horseradish peroxidase yielded similar products. We monitored the redox potential during some of these experiments, trying to begin looking at this parameter, which like pH, can have a more global effect on closed systems such as the apoplast. Phenolics such as APO that are induced upon bacterial ingress, seem likely to play a role in the plant bacterial interaction, but it will require additional and creative research to find and fully understand this role.

This study demonstrates that the accumulation of apoplastic phenolics is stimulated in planta in response to bacterial inoculation. Past studies have shown that levels of extracellular phenolics are elicited in plant cell suspensions in response to bacteria, and that tomato plants infected with viroids showed changes in apoplastic phenolics. The method described here monitored changes in apoplastic phenolics in tobacco leaves following bacterial inoculation of the same tissue. Inoculation with a saprophyte, Pseudomonas fluorescens, which does not cause visible symptoms or physical damage, was used to elicit phenolics and examine the effects of variable parameters on phenolic composition. Location of the inoculation on the leaf, position, or developmental age of the leaf on the plant, and inoculum concentration were standardized for further experiments. The patterns of phenolic change in the apoplast were compared for tobacco inoculated with P. syringae pathovars, pv. syringae, which causes a resistant HR reaction within 15 h, and pv. tabaci, which causes a susceptible reaction with delayed visible symptoms. Both pathogens elicited lower increased levels of acetosyringone compared to the saprophyte, P. fluorescens but had greatly increased levels of the chlorogenic acid derivatives. The latter metabolites appear to have come from the intracellular stores, which could indicate a weakening of the apoplast/symplast barrier. This unexpected aspect will require further study of intracellular phenolics.

The plant apoplast is one of the first battlegrounds for pathogens invading via leaf stomata. The apoplast is the border just outside the cell membrane and in a leaf, much of the apoplast is in direct contact or near the air space within the leaf. It is one of the most accessible parts of the plant for the study of early events in the bacteria/plant interactions and likewise it offers lucrative possibilities for plant disease control. Recent studies have shown that two simple phenolics, acetovanillone and acetosyringone, respond to bacterial infection within the first few hours after inoculation, but their timing and concentrations vary depending on the type of interaction: saprophytic, susceptible, or resistant. Here we describe a second phase of the interaction, about 10h post inoculation, in which chlorogenic acids increase dramatically in pathogenic interactions, susceptible and resistant. Chlorogenic acid is stored in the plant vacuole and its appearance indicates leakage across the apoplast/membrane barrier, which in turn is likely to affect bacterial growth. Therefore we have tried to correlate bacterial multiplication with these chemical events in the three types of interactions. The oxidative environment of the apoplast during these early periods suggests that these phenolic compounds may influence the redox environment and directly inhibit bacterial multiplication during certain periods of the interaction.

Most methods used for the quantification of chlorogenic acids (CGA) in plant tissue use a multi-step organic solvent extraction, which improves accuracy but is laborious and time-consuming. We needed a simpler technique that would allow us to compare the effects of various bacterial treatments on leaf tissue CGA levels over time. Here we describe such a technique that was rapid, used less tissue, and was highly reproducible. The final technique involved grinding a small amounts of leaf tissue in acidified water, which helped stabilize the phenolics, and after clarification by centrifugation could directly be analyzed by Ultra High Performance Liquid Chromatography with Ultraviolet and Mass spectrophotometer detectors (UHPLC-UV-MS). Using this method, we found that CGA levels can vary greatly with leaf age, location on the leaf, and between plants. Therefore in order to observe the more subtle changes caused by bacterial treatments, it was essential to first be aware of the CGA variations in plants to find the most homogenous tissue to use. Ultimately, we found that leaf halves were most homogenous and adjacent panels on a same side of the midrib could be used for multiple sampling of a treatment over time. During this study we also found some interesting relationships regarding CGA distribution in the plant.

The leaf apoplast is the first battleground for plant pathogens invading via stomata. The apoplast is the border just outside the plant cell membrane and in a leaf, much of the apoplast is in direct contact or near the air space within the leaf. It is one of the most accessible parts of the plant for the study of early events in the bacteria/plant interactions and likewise it offers lucrative possibilities for plant disease control. Recent studies have shown that several apoplast phenolics respond to bacterial infection within the first few hours after inoculation. Two phenolics in particular, acetovanillone and acetosyringone, vary in their timing and concentrations depending on the type of interaction: saprophytic (Pseudomonas fluorescens), susceptible (P. syringae pv. tabaci), or resistant (P. s. pv. syringae). In pathogenic interactions, a second phase occurs which involves a dramatic increase of chlorogenic acid into the apoplast. Chlorogenic acid is stored in the plant vacuole and its appearance indicates leakage across the apoplast/symplast (A/S) barrier. All of these changes in apoplast phenolics could affect bacterial growth. Therefore we have tried to correlate bacterial multiplication with these chemical events in the three types of interactions. The oxidative environment of the apoplast that is thought to exist during these early periods suggests that these phenolic compounds would influence the redox environment and could directly affect bacterial multiplication. Published by Elsevier Ltd.

Pseudomonas syringae infects diverse crop plants and comprises at least 50 different pathovar strains with different host ranges. More information on the physiological and molecular effects of the host inhibitory environment on the pathogen is needed to develop resistant cultivars. Recently, we reported an in vitro model system that mimics the redox pulse associated with the oxidative burst in plant cells inoculated with Pseudomonas syringae pv. syringae. Using this system, we demonstrated that oxidation of acetosyringone, a major extracellular phenolic compound induced in some plants in response to bacteria, rendered Pseudomonas syringae pv. syringae to a "viable but nonculturable" (VBNC) state. Here we performed a large scale transcriptome profiling of P s. pv syringae in the VBNC state induced by acetosyringone treatment and identified bacterial genes and pathways presumably associated with this condition. The findings offer insight into what events occur when bacterial pathogens are first encountered and host defense responses are triggered. The acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance. We believe that this is the first work on global gene expression profiling of VBNC cells in plant pathogenic bacteria.

Acetosyringone is a phenolic metabolite often found in plant apoplasts. Its oxidation by hydrogen peroxide and peroxidase results in a prolonged increase in the redox potential of the reaction mixture, similar to redox increases observed in tobacco suspension cells upon treatment with incompatible bacteria. Since high redox potentials, being oxidative, are generally detrimental to bacteria, the effect of acetosyringone oxidation on bacterial viability was examined. Pseudomonas syringae pv. syringae was added to reaction mixtures containing acetosyringone, hydrogen peroxide and peroxidase and samples were removed to determine viability by dilution plating. Initial studies were done with low bacterial concentrations, 10(5) CFU ml(-1), to ensure that scavenging of H2O2 was negligible and did not interfere with the reaction mixture. No colonies were formed by bacteria that had been added to reaction mixtures with acetosyringone ranging from 25 to 100 mu M. Examination of the bacteria by microscopy and flow cytometry, using fluorescent stains that indicate bacterial membrane integrity, suggested that these bacteria had maintained their membrane integrity. In addition they were able to respire based on oxygen uptake. When bacteria were added to on-going reaction mixtures at a time point after the prolonged redox response, the CFU ml(-1) increased indicating that a stable reaction product was not responsible for the non-culturability bioactive effect. Other bacterial isolates, P. s. pv. tabaci and Pseudomonas fluorescens, were less susceptible to the bioactive effect of the acetosyringone oxidation. Other phenolics were tested and had lesser degrees of bioactivity and in some cases reduced the bioactivity of acetosyringone oxidation. The 'viable but non-culturable' (VBNC) state of the bacteria in this study is compared to that described for other medical and plant pathogens. Published by Elsevier Ltd.

Previous work demonstrated that when tobacco cell suspensions were inoculated with certain bacterial strains, the redox potential of the suspensions would increase (oxidative), as much as 100 mV, and in some cases last more than an hour. To discover possible contributors to this prolonged redox potential burst, we examined the oxidation of various plant phenolics that were similar to those found in the cell suspensions. Acetosyringone, one of the major extracellular phenolics in tobacco cell suspensions, was unique in causing a prolonged increase in redox potential of more than 100 mV when incubated with H2O2 and peroxidase. The increase in potential appears to be due to a relatively stable radical intermediate. The length of time that the redox potential increased was dependent on the ratio of H2O2 and acetosyringone. The most favorable was a 1:1 ratio. In vitro characterization showed that production of this intermediate is favored by relatively low pH (around 6) and cooler temperatures possibly due to slower kinetics. Phosphate buffer was substituted for MES buffer, which had been used initially but was found to interfere with the oxidation. The results of this study demonstrate that apoplastic phenolics, in addition to often playing a role as antioxidants that reduce H2O2, can also cause rapid transient periods of extreme redox potential creating a local environment hostile to pathogen ingress. Published by Elsevier Ltd.

In both plants and animals, there has been a strong focus on reactive oxygen species and antioxidants in regard to stress responses. This has led to an awareness of the importance of 'redox status' as a prime regulatory determinant of cellular function and responses to internal and external stimuli. It has been difficult to study the effect of or fluctuation in the redox potential during interactions, since the frequency of sampling is limiting. We tested a method of using redox electrodes with bacterial and plant cell suspensions to monitor the extracellular redox potential during different plant/bacterial interactions. The advantage of the electrodes is that they provide continuous and nonintrusive monitoring of the redox potential, and interact with a larger array of metabolites. We found four different responses of redox potential to different plant/bacterial interactions. The redox responses are coincident with phenolic changes in the extracellular fluid of the suspensions. Further investigation of the mechanisms and parameters that affect the technique will provide insight into using electrodes to measure redox potential in planta.

The plant leaf apoplast is one of the first lines of defense against many foliar pathogens. The aqueous layer lining the airspace within leaves is enriched with secondary metabolites that can serve many roles including protection against environmental hazards, both biotic and abiotic. The constituents and their concentration change as the leaf matures or undergoes stress. To monitor and quantify changes in these metabolites during pathogen stress, we needed a more sensitive technique. We were able to modify the infiltration-centrifugation technique to use smaller samples, individual tomato leaflets, plus use an internal standard which indicated the amount of dilution in each sample. Dinotefuran, a neonicotinoid used as a systemic pesticide, proved to be resistant to the redox environment of the apoplast and had similar chemical properties allowing it to be analyzed under the same UPLC conditions as the other phenolic metabolites. Water soluble metabolites on the leaf surface were found to be a major source of contamination that could be avoided by rinsing leaves with water prior to infiltration. The improved sample efficiency and accuracy provided by this technique, along with the use of Dinotefuran as an internal standard, will provide the sensitivity needed to monitor apoplast metabolites during pathogen stress.

To measure the impact of the catastrophic 2008 earthquake in the Sichuan province of China on HRQOL among survivors. The study employed a two-wave longitudinal study of survivors 8 months and three years after the earthquake, using a probability cluster sample design stratified according to three levels of earthquake impact. Twelve shelters out of nine townships were selected during the first-wave sampling, which included three shelters out of three severely affected townships, six shelters out of three moderately affected townships, and three shelters out of three mildly affected townships. The study was restricted to individuals between 16 and 89 years of age. A total of 1617 survivors returned the study survey. The SF-36 instrument was used to measure HRQOL and compared with the provincial norm. The second wave was a random sample of 323 respondents from the initial 1617 first-wave respondents, who were interviewed with an identical instrument 3 years after the earthquake. ANOVA and t-tests were used to compare SF-36 scores among residents by earthquake impact levels; against a provincial norm; and between 8 months and 3 years. The SF-36 subscale scores differed by the impact level of earthquake except for RP. Compared with the Sichuan provincial norm, all subscale SF-36 scores of the first-wave respondents were lower at month 8 (all p-values<0.001). Seven subscale scores of RP, BP, GH, VT, SF, RE, and MH at 3 years were statistically lower than the provincial norm (all p-values<0.05). Among the second-wave respondents, all the 3-year SF-36 subscale scores improved in comparison to those taken 8 months after the earthquake except for RP and SF. The HRQOL declined compared with the norm, especially the psychological aspects. Furthermore, the HRQOL 3 years after the earthquake were persistently lower than the norm level, despite its recovery from 8-month level.

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2-4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR-), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili. Published by Elsevier Ltd.

Plants are capable of producing a wide array of secondary metabolites that serve a variety of functions, due to their bioactive, redox or structural properties. Subtle changes in the external or internal environment of the plant can cause significant changes in the array of secondary metabolites present in the tissue. During the last 10 years the critical roles that these metabolites play both in plant development as well as plant defense against biological and abiotic stresses have begun to become more widely appreciated. Here we describe changes in apoplastic phenolics associated with systemic infections of Potato spindle tuber viroid (PSTVd) as it spreads through inoculated tomato plants. Apoplast fluids from leaves at different distances from the infection site were examined by HPLC-UV over a 5 week period. The phenolic composition of apoplasts in healthy plants was highly dynamic and depended on leaf position as well as plant age. Differences in the apoplast composition of PSTVd infected plants were detected only in leaves in which the viroid was present and multiplied. In those leaves, apoplast phenolics that were similar to the control generally decreased although some increased compared to control plants. Another set of phenolics that were unique to the viroid infected plants appeared simultaneously with the onset of viroid replication and then disappeared. An important basic finding of this study is that the composition of apoplastic metabolites is dynamic, changing with time and leaf development. In addition we demonstrate that the secondary metabolites of the leaf apoplast respond to the presence of the viroid in the symplast of the cell. This study provides a basis for a more detailed investigation of the dynamics and identification of the apoplastic phenolics involved in the viroide-host interaction. Published by Elsevier Ltd.

BACKGROUNDG-protein 133 subunit (GNB3) gene C825T and endothelial nitric oxide (eNCS) gene G894T polymorphisms both influence arterial structure and function. However, information is scant regarding the interaction of these genes on arterial wall thickness.METHODSThis aspect was examined in 654 white and black subjects, aged 25-43 years (72.9% white, 39.3% male). Arterial wall thickness was assessed in terms of the average intima-media thickness (IMT) of common carotid, internal carotid, and carotid bulb segments by B-mode ultrasonography.RESULTSFrequencies of T allele of the GNB3 C825T polymorphism (0.718 vs. 0.304, P < 0.0001) and G allele of the eNOS G894T polymorphism (0.868 vs. 0.661, P < 0.0001) were higher in blacks compared to whites. In a multivariate model including gender, age, mean arterial pressure, body mass index, triglycerides/HDL cholesterol ratio, insulin resistance index, smoking, and/or race, there was no significant genotypic effect on carotid IMT with respect to GNB3 C825T or eNOS G894T polymorphisms among whites, blacks, and total sample. However, the carriers of TT genotype of the GNB3 C825T and T allele of the eNOS G894T had a significantly lower carotid IMT among blacks (P =0.003) and the total sample (P=0.006).CONCLUSIONThese results indicate that the genetic variations of the eNOS gene in combination with the GNB3 gene jointly influence carotid artery wall thickening process in young adults, especially in blacks.

This study focuses on the differential induction of extracellular phenolic amides that accumulate in potato cell suspensions during the first few hours of the interaction between these plant cells and either bacterial pathogens or pathogen-related elicitors. Using suspension cells of Solanum tuberosum we identified 4 hydroxycinnamic acid amides that accumulate in the extracellular environment. Treatment of the suspension cells with pathovars of the plant pathogens Pseudomonas syringae or Ralstonia solanacearum or with pathogen-related elicitors changed the composition of the extracellular phenolic amides within hours and the composition differed for each treatment. Some of the phenolic amides were sensitive to oxidative stress; when suspension cells were treated with bacterial strains or elicitors that triggered an oxidative burst, the phenolics were oxidized and depleted for the duration of the burst. Other critical parameters that affected the qualitative and quantitative makeup of these phenolic amides were plant cell age and density.

While characterizing the kinetic parameters of apoplastic phenolic oxidation by peroxidase, we found anomalies caused by the Mes [2-(4-morpholino)ethanesulfonic acid] buffer being used. In the presence of Mes, certain phenolics appeared not to be oxidized by peroxidase, yet the oxidant, H(2)O(2), was utilized. This anomaly seems to be due to the recycling of the phenolic substrate. The reaction is relatively inefficient, but at buffer concentrations of 10 mM or greater the recycling effect is nearly 100% with substrate concentrations less than 100 microM. The recycling effect is dependent on substrate structure, occurring with 4'-hydroxyacetophenone but not with 3',5'-dimethoxy-4'-hydroxyacetophenone (acetosyringone). Characterization of the reaction parameters suggests that the phenoxyl radical from the peroxidase reaction interacts with Mes, causing the reduction and regeneration of the phenol. Similar responses occurred with related buffers such as Hepes [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] and Pipes [piperazine-1,4-bis(2-ethanesulfonic acid)]. Results from this work and other reports in the literature indicate that great care is required in interpreting any results involving these buffers under oxidizing conditions.

The plant apoplast is an important mediator of communication between the cell cytoplasm and its surroundings. Plant cell suspensions offer a convenient model system to gain insight into apoplastic physiology. Here, we describe a novel phenomenon that took place when two naturally occurring phenolics were added together to either soybean or tobacco cell suspensions. Acetosyringone (AS) and/or hydroxyacetophenone (HAP), phenolics found in the extracellular/apoplast of tobacco cells, were added to soybean or tobacco cell suspensions undergoing an oxidative burst. Individually, AS appeared to be utilized as a typical peroxidase substrate to scavenge hydrogen peroxide, while HAP was utilized at a much lower rate. However, when added together the rate of utilization of both phenolics increased and surprisingly resulted in the production of hydrogen peroxide. We have further characterized this novel phenomenon in suspension cells. This study demonstrates that certain phenolics in plants can cause co-oxidation which, as in animals, could alter the structure and bioactivity of surrounding phenolics.