Dead reckoning of protist viability with propidium monoazide (PMA)-quantitative PCR; a case study using Neoparamoeba perurans.

The ability to distinguish between viable and non-viable protozoan parasites is central to improved human and animal health management. While conceptually simple, methods to differentiate cell viability in situ remain challenging. Amoebic gill disease, caused by Neoparamoeba perurans is a parasitic disease impacting Atlantic salmon aquaculture globally. Although commercial freshwater treatments alleviate AGD, viable amoebae remain on gills or in used treatment water. Existing PCR-based assays are able to quantify N. perurans abundance but cannot discriminate amoeba viability. We investigated the use of propidium monoazide (PMA) application, prior to real-time PCR, to distinguish between alive and dead cells. We demonstrate that 200 µM PMA can significantly reduce amplification from non-viable (isopropanol treated) cultured amoebae across at least three logs of cell concentrations. Using a serial dilution of viable and non-viable cells, we show that non-PMA PCR amplifies both viable and non-viable amoebae, while PMA exposure suppresses (but does not completely inhibit) amplification from non-viable amoebae. The effect of freshwater treatment on N. perurans viability was assessed using the PMA-PCR. Following PMA exposure, amplification from freshwater treated amoebae was reduced by approximately 94-97 %. Taken together this study demonstrates that PMA combined with traditional real-time PCR can estimate amoeba viability.

Map-based cloning identifies velvet A as a critical component of virulence in Fusarium pseudograminearum during infection of wheat heads.

Although better known as a pathogen of wheat stem bases, Fusarium pseudograminearum also causes Fusarium head blight. A natural isolate of F. pseudograminearum was identified that showed severely reduced virulence towards wheat heads and a map-based cloning approach was undertaken to identify the genetic basis of this phenotype. Using a population of 95 individuals, a single locus on chromosome 1 was shown to be responsible for the low virulence. Fine mapping narrowed the region to just five possible SNPs of which one was in the F. pseudograminearum homologue of velvet A. Knockout mutants of velvet A, which were non-pathogenic towards wheat, confirmed that velvet A regulates virulence in this pathogen. The mutation in velvet A was only found in a single field isolate and the origin of the mutation is unknown.

A High Throughput Viability Screening Method for the Marine Ectoparasite Neoparamoeba perurans.

The marine protozoan parasite Neoparamoeba perurans has been established as the causative agent for amoebic gill disease (AGD) in Atlantic salmon (Salmo salar). Freshwater bathing is the only routinely used treatment for AGD in Australia while hydrogen peroxide (H2O2) is used in countries with cooler water temperatures. The identification of new treatments that do not rely on either freshwater or H2O2 bathing is highly sought. However, in vitro based methods for high throughput screening of antiparasitic compounds have not been established for this parasite. To this end the present study evaluated two in vitro bioassays based on metabolic energy production and cellular membrane integrity to distinguish between amoebistatic (crenated or pseudocyst forms with recovery possible) and amoebicidal (death) activity. Amoebae were subject to either freshwater, H2O2 or chloramine-T for 4h treatment and assessed 24h after recovery. Visualization by microscopy and bioassay assessment 24h post-treatment confirmed that H2O2 and freshwater are 95% amoebicidal albeit due to different mechanisms of action. These data are consistent with other studies where amoebae have been observed to recover following exposure to these compounds and provide evidence for the inclusion of a recovery component to differentiate between the mechanism of action of amoebicidal and amoebistatic treatments. Together these bioassays are a critical tool for high throughput screening of novel and more effective treatments against AGD.

Can natural gene drives be part of future fungal pathogen control strategies in plants?

Globally, fungal pathogens cause enormous crop losses and current control practices are not always effective, economical or environmentally sustainable. Tools enabling genetic management of wild pathogen populations could potentially solve many problems associated with plant diseases. A natural gene drive from a heterologous species can be used in the globally important cereal pathogen Fusarium graminearum to remove pathogenic traits from contained populations of the fungus. The gene drive element became fixed in a freely crossing population in only three generations. Repeat-induced point mutation (RIP), a natural genome defence mechanism in fungi that causes C to T mutations during meiosis in highly similar sequences, may be useful to recall the gene drive following release, should a failsafe mechanism be required. We propose that gene drive technology is a potential tool to control plant pathogens once its efficacy is demonstrated under natural settings.

Fusaristatin A production negatively affects the growth and aggressiveness of the wheat pathogen Fusarium pseudograminearum.

Fusarium pseudograminearum (Fp), the causative fungal pathogen of the diseases Fusarium crown rot, is an important constraint to cereals production in many countries including Australia. Fp produces a number of secondary metabolites throughout its life cycle. One of these metabolites, the cyclic lipopeptide fusaristatin A, is encoded by a specific gene cluster containing a polyketide synthase and a three-module non-ribosomal peptide synthetase. However, a recent survey of Fp populations across Australia suggests that this cluster may only be present in a subset of isolates from Western Australia (WA). In this study, we screened 319 Fp isolates from WA and 110 Fp isolates from the Australian eastern states of New South Wales, Victoria, Queensland and South Australia to examine the distribution of this gene cluster among Australian Fp populations. The fusaristatin A gene cluster was found to be present in ~50% of Fp isolates from WA but completely absent in Fp isolates from eastern states. To determine its potential function, mutants of the fusaristatin A gene cluster were generated by disrupting the non-ribosomal peptide synthetase and polyketide synthase genes simultaneously in two different parental backgrounds. The mutants showed increased growth rates and were significantly more aggressive than their respective parental strains on wheat in crown rot pathogenicity assays. This suggested that fusaristatin A has a negative effect on fungal development and aggressiveness. The possible reasons for the geographically restricted presence of the fusaristatin A gene cluster and its role in fungal biology are discussed.

ESAM predicts cardiovascular mortality in diabetic hemodialysis patients.

AIM: To assess endothelial cell selective adhesion molecule (ESAM) as predictor of cardiovascular mortality in diabetic dialysis patients (DDPs). METHODS: ESAM, clinical and laboratory parameters were assessed in 73 DDP. Cardiovascular mortality was recorded in a 2 years' prospective observational study. RESULTS: Baseline ESAM was 17.1 (10.05-24.8) ng/ml and was correlated to phosphate (r = -0.42, p = 0.008), parathormone (r = -0.36, p = 0.048), albumin (r = -0.24, p = 0.048). ESAM significantly predicted cardiovascular death in univariate [HR = 1.03, 95% CI (1.006-1.054), p = 0.01] and multivariate [HR = 1.034, 95% CI (1.003-1.066), p = 0.03] Cox analysis. Time to cardiovascular death was shorter for patients with ESAM >12.44 ng/ml, p = 0.0045. CONCLUSION: ESAM is an independent predictor of cardiovascular mortality in DDP.

ESAM predicts cardiovascular mortality in diabetic hemodialysis patients.

AIM: To assess endothelial cell selective adhesion molecule (ESAM) as predictor of cardiovascular mortality in diabetic dialysis patients (DDPs). METHODS: ESAM, clinical and laboratory parameters were assessed in 73 DDP. Cardiovascular mortality was recorded in a 2 years' prospective observational study. RESULTS: Baseline ESAM was 17.1 (10.05-24.8) ng/ml and was correlated to phosphate (r = -0.42, p = 0.008), parathormone (r = -0.36, p = 0.048), albumin (r = -0.24, p = 0.048). ESAM significantly predicted cardiovascular death in univariate [HR = 1.03, 95% CI (1.006-1.054), p = 0.01] and multivariate [HR = 1.034, 95% CI (1.003-1.066), p = 0.03] Cox analysis. Time to cardiovascular death was shorter for patients with ESAM >12.44 ng/ml, p = 0.0045. CONCLUSION: ESAM is an independent predictor of cardiovascular mortality in DDP.

Investigating the Association between Flowering Time and Defense in the Arabidopsis thaliana-Fusarium oxysporum Interaction.

Plants respond to pathogens either by investing more resources into immunity which is costly to development, or by accelerating reproductive processes such as flowering time to ensure reproduction occurs before the plant succumbs to disease. In this study we explored the link between flowering time and pathogen defense using the interaction between Arabidopsis thaliana and the root infecting fungal pathogen Fusarium oxysporum. We report that F. oxysporum infection accelerates flowering time and regulates transcription of a number of floral integrator genes, including FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT) and GIGANTEA (GI). Furthermore, we observed a positive correlation between late flowering and resistance to F. oxysporum in A. thaliana natural ecotypes. Late-flowering gi and autonomous pathway mutants also exhibited enhanced resistance to F. oxysporum, supporting the association between flowering time and defense. However, epistasis analysis showed that accelerating flowering time by deletion of FLC in fve-3 or fpa-7 mutants did not alter disease resistance, suggesting that the effect of autonomous pathway on disease resistance occurs independently from flowering time. Indeed, RNA-seq analyses suggest that fve-3 mediated resistance to F. oxysporum is most likely a result of altered defense-associated gene transcription. Together, our results indicate that the association between flowering time and pathogen defense is complex and can involve both pleiotropic and direct effects.

Fusarium oxysporum triggers tissue-specific transcriptional reprogramming in Arabidopsis thaliana.

Some of the most devastating agricultural diseases are caused by root-infecting pathogens, yet the majority of studies on these interactions to date have focused on the host responses of aerial tissues rather than those belowground. Fusarium oxysporum is a root-infecting pathogen that causes wilt disease on several plant species including Arabidopsis thaliana. To investigate and compare transcriptional changes triggered by F. oxysporum in different Arabidopsis tissues, we infected soil-grown plants with F. oxysporum and subjected root and leaf tissue harvested at early and late timepoints to RNA-seq analyses. At least half of the genes induced or repressed by F. oxysporum showed tissue-specific regulation. Regulators of auxin and ABA signalling, mannose binding lectins and peroxidases showed strong differential expression in root tissue. We demonstrate that ARF2 and PRX33, two genes regulated in the roots, promote susceptibility to F. oxysporum. In the leaves, defensins and genes associated with the response to auxin, cold and senescence were strongly regulated while jasmonate biosynthesis and signalling genes were induced throughout the plant.

Fluorescent polymeric aggregates for selective response to sarin surrogates.

By combining the sensitivity of fluorescent units with the response of "smart" polymers to environmental changes, we propose a new approach for chemical detection applications. The system proved to be sensitive to 12 ppb of diethyl chlorophosphate (DCP), a Sarin surrogate, and to discriminate between the interfering molecules.

Relationship of endothelial cell selective adhesion molecule to markers of oxidative stress in type 2 diabetes.

BACKGROUND: Endothelial dysfunction is an important contributor to micro and macrovascular complications of type 2 diabetes (T2D) and is reflected by increased systemic oxidative stress. Endothelial cell selective adhesion molecule (ESAM) influences endothelial function. We aimed to assess, for the first time to our knowledge, the relationship of soluble ESAM to markers of systemic oxidative stress. MATERIALS AND METHODS: ESAM, malondialdehyde (MDA) level and catalase activity were determined in 54 T2D patients and 43 controls. RESULTS: T2D patients had significantly higher ESAM when compared to controls (16.07 ± 5.77 µg/L versus 8.57 ± 5.28 µg/L, p < 0.0001), they also had higher MDA level (3.88 ± 1.50 µmol/L vs. 1.58 ± 0.72 µmol/L, p < 0.0001) and lower catalase activity (3.07 (2.63-3.44) U/mg vs. 8.72 (4.55-10.46) U/mg, p < 0.0001). In T2D patients ESAM was inversely related to catalase activity (r = -0.27, p = 0.04), relationship to MDA level was direct but not significant (r = 0.16, p = 0.24). MDA concentration correlated inversely to catalase activity (r = -0.28, p = 0.04). In multiple regression catalase activity remained significantly correlated to ESAM (p = 0.02) and MDA level was significantly related to glycated hemoglobin (p = 0.01); there was trend towards a positive correlation of MDA level to ESAM (p = 0.08). When patients were divided according to oxidative stress, those with increased oxidative stress (defined as MDA concentration > 2.98 µmol/L and catalase activity < 3.38 U/mg) had higher ESAM than the rest of the patients (17.99 ± 5.02 µg/L vs. 14.29 ± 5.94 µg/L p = 0.01). CONCLUSION: ESAM is higher in T2D than in controls and parallels oxidative stress: ESAM is inversely related to catalase activity and higher ESAM is found in T2D patients with increased oxidative stress.

The influence of vitamin E supplementation on erythropoietin responsiveness in chronic hemodialysis patients with low levels of erythrocyte superoxide dismutase.

About 12-15 % of hemodialysis patients have a poor response to recombinant human erythropoietin (rHuEPO). The aim of this prospective study was to examine the influence of oxidative stress and vitamin E supplementation on rHuEPO responsiveness in chronic hemodialysis patients. Sixty-five hemodialysis patients treated with rHuEPO were studied. Those with iron deficiency, blood loss, malignancy, vitamin B12 and folate deficiency, severe hyperparathyroidism, liver cirrhosis, and congestive heart failure were excluded. Twenty-one healthy volunteers served as a control group. Malondialdehyde, carbonyl proteins, erythrocyte superoxide dismutase (SOD), ceruloplasmin, and serum antioxidant capacity were measured. Values of SOD > 150 U/ml were considered as normal. Patients with SOD < 150 U/ml were divided in two groups: group A (n = 11): treated with vitamin E 400 mg/day (600 IU/day) for 8 weeks; group B (n = 13): not treated. A third, group C consisted of patients with normal SOD. rHuEPO doses (U/kg/week) were recorded. rHuEPO responsiveness index was calculated as rHuEPO U/week/hematocrit. A poor response was defined as a rHuEPO responsiveness index >200. SOD positively correlated with hemoglobin (p = 0.0018, R = 0.337) and negatively with rHuEPO responsiveness index (p = 0.0122, R = 0.319). Vitamin E-treated patients from group A exhibited significantly increased hemoglobin levels as compared to initial values (10.5 ± 0.3 vs. 8.6±0.4, p = 0.002). In comparison with group B, the vitamin E-treated patients displayed a higher hemoglobin (10.5 ± 0.3 vs. 9.4 ± 0.3, p = 0.04), had a lower rHuEPO dose (85.7 ± 7.4 vs. 136.8 ± 13.8, p = 0.025), and a significantly improved rHuEPO responsiveness (rHuEPO responsiveness index 177.9 ± 28.6 vs. 314.1 ± 34.0, p = 0.006). Patients from group A significantly improved their rHuEPO responsiveness after vitamin E therapy as compared to baseline (rHuEPO responsiveness index 177.9 ± 28.6 vs. 271.7 ± 30.3, p = 0.034). We conclude that lower values of SOD correlate with lower hemoglobin, higher rHuEPO dose and poor response to rHuEPO in chronic hemodialysis patients. Vitamin E supplementation significantly improves rHuEPO responsiveness, increases hemoglobin level, and decreases rHuEPO dose.

Comparative pathogenomics reveals horizontally acquired novel virulence genes in fungi infecting cereal hosts.

Comparative analyses of pathogen genomes provide new insights into how pathogens have evolved common and divergent virulence strategies to invade related plant species. Fusarium crown and root rots are important diseases of wheat and barley world-wide. In Australia, these diseases are primarily caused by the fungal pathogen Fusarium pseudograminearum. Comparative genomic analyses showed that the F. pseudograminearum genome encodes proteins that are present in other fungal pathogens of cereals but absent in non-cereal pathogens. In some cases, these cereal pathogen specific genes were also found in bacteria associated with plants. Phylogenetic analysis of selected F. pseudograminearum genes supported the hypothesis of horizontal gene transfer into diverse cereal pathogens. Two horizontally acquired genes with no previously known role in fungal pathogenesis were studied functionally via gene knockout methods and shown to significantly affect virulence of F. pseudograminearum on the cereal hosts wheat and barley. Our results indicate using comparative genomics to identify genes specific to pathogens of related hosts reveals novel virulence genes and illustrates the importance of horizontal gene transfer in the evolution of plant infecting fungal pathogens.

[Tonic pupil, pupil Adie syndrome Adie Holmes: current reassessment of terminology -- a clinical case]. / Pupila tonica, pupila Adie sindromul Adie Holmes reevaluarea actuala a terminologiei - prezentare de caz clinic.

The benign syndrome of pupillotonia and absence of deep reflexes is not uncommon. It was clearly and accurately described by Adie (1932), although incompletely recognized many years, before. The pupillary abnormality was reported by ophthalmologists at the turn of the century (Saenger, 1902, Strasburger 1902), and the associated deep reflex change was described by Markus (1906), Roemheld (1921) and Parkes Weber (1923). Holmes (1932) was fully aware of the association of "partial iridoplegia" with diminished reflexes. Tonic pupils react poorly to light but constrict during viewing of a near stimulus. Adie's name is typically used in association with tonic pupils, but a review of Adie's articles reveals that he described the syndrome of tonic pupils and absent reflexes and not the pupillary abnormality per se. Therefore, it would be more appropriate to refer to a tonic pupil as simply a 'tonic pupil" and leave Adie's name for the syndrome. We report a typical case of tonic pupil.

Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production.

BACKGROUND: The fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB) disease on wheat which can lead to trichothecene mycotoxin (e.g. deoxynivalenol, DON) contamination of grain, harmful to mammalian health. DON is produced at low levels under standard culture conditions when compared to plant infection but specific polyamines (e.g. putrescine and agmatine) and amino acids (e.g. arginine and ornithine) are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during FHB are unknown, but plant derived polyamines could contribute to DON induction in infected heads. However, the temporal and spatial accumulation of polyamines and amino acids in relation to that of DON has not been studied. RESULTS: Following inoculation of susceptible wheat heads by F. graminearum, DON accumulation was detected at two days after inoculation. The accumulation of putrescine was detected as early as one day following inoculation while arginine and cadaverine were also produced at three and four days post-inoculation. Transcripts of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), two key biosynthetic enzymes for putrescine biosynthesis, were also strongly induced in heads at two days after inoculation. These results indicated that elicitation of the polyamine biosynthetic pathway is an early response to FHB. Transcripts for genes encoding enzymes acting upstream in the polyamine biosynthetic pathway as well as those of ODC and ADC, and putrescine levels were also induced in the rachis, a flower organ supporting DON production and an important route for pathogen colonisation during FHB. A survey of 24 wheat genotypes with varying responses to FHB showed putrescine induction is a general response to inoculation and no correlation was observed between the accumulation of putrescine and infection or DON accumulation. CONCLUSIONS: The activation of the polyamine biosynthetic pathway and putrescine in infected heads prior to detectable DON accumulation is consistent with a model where the pathogen exploits the generic host stress response of polyamine synthesis as a cue for production of trichothecene mycotoxins during FHB disease. However, it is likely that this mechanism is complicated by other factors contributing to resistance and susceptibility in diverse wheat genetic backgrounds.

Hepcidin and multiple myeloma related anemia.

Multiple myeloma (MM) is a B cell lymphoproliferative disorder in which malignant plasma cells accumulate in the bone marrow and usually produce monoclonal immunoglobulin in excess. Interleukin-6 (IL-6), is known to be an essential survival factor of myeloma cells, high IL-6 levels being correlated with an adverse prognosis. IL-6 modulates the transcription of several liver-specific acute phase protein genes, including C-reactive protein and hepcidin. Anemia is one of the prominent features of MM, along with recurrent osteolytic lesions, bacterial infections and renal insufficiency. The current treatment strategies of MM related anemia are often inadequate and many patients rely on transfusions. Several causes have been implicated, but anemia of chronic disease (ACD) related to the inflammatory cytokines appears to be one of the main culprits. The pathogenesis of ACD had been poorly understood, but recently it has been shown that increased Il-6 upregulates the hepatic production of hepcidin, which, by binding to its cellular receptor, ferroportin, causes anemia by blocking iron export from enterocytes and macrophages. We hereby argue that by virtue of its biological characteristics, multiple myeloma should be an ideal clinical setting to test the role of hepcidin in the pathogenesis of ACD. Hepcidin levels should be higher in MM patients and might correlate with prognosis. Anemic MM patients should also be among those who would benefit mostly from hepcidin targeted therapies.

The SEN1 gene of Arabidopsis is regulated by signals that link plant defence responses and senescence.

Plant defence and senescence share many similarities as evidenced by extensive co-regulation of many genes during these responses. To better understand the nature of signals that are common to plant defence and senescence, we studied the regulation of SEN1 encoding a senescence-associated protein during plant defence responses in Arabidopsis. Pathogen inoculations and treatments with defence-related chemical signals, salicylic acid and methyl jasmonate induced changes in SEN1 transcript levels. Analysis of transgenic plants expressing the SEN1 promoter fused to uidA reporter gene confirmed the responsiveness of the SEN1 promoter to defence- and senescence-associated signals. Expression analysis of SEN1 in a number of defence signalling mutants indicated that activation of this gene by pathogen occurs predominantly via the salicylic and jasmonic acid signalling pathways, involving the functions of EDS5, NPR1 and JAR1. In addition, in the absence of pathogen challenge, the cpr5/hys1 mutant showed elevated SEN1 expression and displayed an accelerated senescence response following inoculation with the necrotrophic fungal pathogen Fusarium oxysporum. Although the analysis of the sen1-1 knock-out mutant did not reveal any obvious role for this gene in defence or senescence-associated events, our results presented here show that SEN1 is regulated by signals that link plant defence and senescence responses and thus represents a useful marker gene to study the overlap between these two important physiological events.