Immuno-Assessment of «Pseudomonas syringae» Lipodepsipeptides (Syringomycins and Syringopeptins)

Following a previous work on the immunological detection of syringopeptins (SPs), polyclonal antibodies with a high specificity for syringomycins (SRs) were raised in rabbits and purified. Assayed in a competitive ELISA, the most common forms of SR, i.e. SR-E and SR-G, were recognised with a detection limit of 0.1 mg per well, whereas other structurally related bacterial lipodepsipeptides (LDP), such as SPs, pseudomycins (PSs) and syringotoxins (STs) were not recognised. The immuno-assay (competitive ELISA) method developed in this work is about 100 times more sensitive than the current chromatographic (HPLC) method and requires no previous extraction of the toxin. The production of LDP in culture by strains of three pathovars of Pseudomonas syringae (pv. aptata, pv. lachrymans and pv. syringae) was found to range from 0.026 to 0.055 mg ml-1 for SRs and from 0.02 to 0.06 mg ml-1 for SPs. Both the concentration of LDP in aqueous extracts from zucchini cotyledons infected by P. syringae pv. lachrymans and the severity of symptoms were shown to increase progressively after infection. The immunologically estimated concentration of SRs in the infected cotyledons averaged 0.22 mg g-1 f wt after 12 hours, and 0.39 mg g-1 after 4 days. The corresponding values for SPs were 0.11 and 0.37 mg g-1. In a recovery experiment, solutions of pure toxins (0.22 mg SR-E and 0.14 mg SP25A g-1 f wt) were injected in healthy cotyledons. After 2 days, overestimation due to toxin complexing in planta was of 10% for (SR-E) and 40% for (SP25A). Applying these percentages to the values estimated for infected cotyledons, the net concentrations were as follows: 12 h after inoculation: 0.20 (SRs) and 0.07 (SPs) mg g-1 f wt; four days after inoculation: 0.35 (SRs) and 0.22 (SPs) mg g-1 f wt. The values obtained with aqueous extracts from infected plants are relatively high if compared to the figures of the in vitro experiments. It is assumed that the high reactivity of ELISA to the immune-LDP-related compounds present in the water extracts from infected plants is due to the presence of high molecular weight LDP complexes having a cross-reactivity with antibodies substantially higher than that of free toxins

Immuno-assessment of Pseudomonas syringae lipodepsipeptides (syringomycins and syringopeptins)

Following a previous work on the immunological detection of syringopeptins (SPs), polyclonal antibodies with a high specificity for syringomycins (SRs) were raised in rabbits and purified. Assayed in a competitive ELISA, the most common forms of SR, i.e. SR-E and SR-G, were recognised with a detection limit of 0.1 mg per well, whereas other structurally related bacterial lipodepsipeptides (LDP), such as SPs, pseudomycins (PSs) and syringotoxins (STs) were not recognised. The immuno-assay (competitive ELISA) method developed in this work is about 100 times more sensitive than the current chromatographic (HPLC) method and requires no previous extraction of the toxin. The production of LDP in culture by strains of three pathovars of Pseudomonas syringae (pv. aptata, pv. lachrymans and pv. syringae) was found to range from 0.026 to 0.055 mg ml-1 for SRs and from 0.02 to 0.06 mg ml-1 for SPs. Both the concentration of LDP in aqueous extracts from zucchini cotyledons infected by P. syringae pv. lachrymans and the severity of symptoms were shown to increase progressively after infection. The immunologically estimated concentration of SRs in the infected cotyledons averaged 0.22 mg g-1 f wt after 12 hours, and 0.39 mg g-1 after 4 days. The corresponding values for SPs were 0.11 and 0.37 mg g-1. In a recovery experiment, solutions of pure toxins (0.22 mg SR-E and 0.14 mg SP25A g-1 f wt) were injected in healthy cotyledons. After 2 days, overestimation due to toxin complexing in planta was of 10% for (SR-E) and 40% for (SP25A). Applying these percentages to the values estimated for infected cotyledons, the net concentrations were as follows: 12 h after inoculation: 0.20 (SRs) and 0.07 (SPs) mg g-1 f wt; four days after inoculation: 0.35 (SRs) and 0.22 (SPs) mg g-1 f wt. The values obtained with aqueous extracts from infected plants are relatively high if compared to the figures of the in vitro experiments. It is assumed that the high reactivity of ELISA to the immune-LDP-related compounds present in the water extracts from infected plants is due to the presence of high molecular weight LDP complexes having a cross-reactivity with antibodies substantially higher than that of free toxins

A new syringopeptin produced by bean strains of Pseudomonas syringae pv. syringae

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP22Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP22Phv did not differ substantially from the previously characterized syringopeptin 22 (SP22) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium

Role of CLU, PICALM, and TNK1 Genotypes in Aging With and Without Alzheimerâ\u80\u99s Disease

Healthy and impaired cognitive aging may be associated to different prevalences of single-nucleotide polymorphisms (SNPs). In a multicenter case-control association study, we studied the SNPs rs11136000 (clusterin, CLU), rs541458 (phosphatidylinositol binding clatrin assembly protein, PICALM), and rs1554948 (transcription factor A, and tyrosine kinase, non-receptor, 1, TNK1) according to the three age groups 50â\u80\u9365 years (group 1), 66â\u80\u9380 years (group 2), and 80+ years (group 3) in 569 older subjects without cognitive impairment (NoCI) and 520 Alzheimerâ\u80\u99s disease (AD) patients. In NoCI subjects, a regression analysis suggested a relationship between age and TNK1 genotypes, with the TNK1-A/A genotype frequency that increased with higher age, and resulting in a different distribution of the TNK1-A allele. In AD patients, a regression analysis suggested a relationship between age and PICALM genotypes and TNK1 genotypes, with the PICALM-T/C and TNK1-A/A genotype frequencies that decreased with increasing age. A resulting difference in the distribution of PICALM-C allele and TNK1-A allele was also observed. The TNK1-A allele was overrepresented in NoCI subjects than in AD patients in age groups 2 and 3. These results confirmed after adjustment for apolipoprotein E polymorphism, which suggested a different role of PICALM and TNK1 in healthy and impaired cognitive aging. More studies, however, are needed to confirm the observed associations

Prenatal predictors of adverse perinatal outcome in congenital cytomegalovirus infection: a retrospective multicenter study

Objectives To identify predictors of adverse perinatal outcome in congenital cytomegalovirus (CMV) infection. Methods In a multicenter study fetuses with congenital CMV infection diagnosed by PCR on amniotic fluid and normal prenatal imaging at the time of diagnosis were included. Primary outcome was the occurrence of structural anomalies at follow-up ultrasound or prenatal magnetic resonance imaging (MRI). Secondary outcomes were the occurrence of anomalies detected exclusively postnatally and the rate of symptomatic infection. Results One hundred and four fetuses with congenital CMV were included in the study. Anomalies were detected at follow-up ultrasound or MRI in 18.3% (19/104) cases. Additional anomalies were found after birth in 11.9% (10/84) of cases and 15.5% (13/85) of newborns showed clinical symptoms related to CMV infection. There was no difference in either maternal age (p=0.3), trimester (p=0.4) of infection and prenatal therapy (p=0.4) between fetuses with or whiteout anomalies at follow-up. Conversely, median viral load in the amniotic fluid was higher in fetuses with additional anomalies at follow-up (p=0.02) compared to those without. At multivariate logistic regression analysis, high viral load in the amniotic fluid, defined as >= 100,000 copies/mL was the only independent predictor for the occurrence of anomalies detected exclusively at follow-up ultrasound assessment or MRI, with an OR of 3.12. Conclusions Viral load in the amniotic fluid is a strong predictor of adverse perinatal outcome in congenital CMV infection. The results of this study emphasize the importance of adequate follow up even in case of negative neurosonography to better predict postnatal adverse outcomes of infected newborns, especially in amniotic fluid high viral load

Prognostic Value of Amniotic Fluid Viral Load to Predict Adverse Outcome in Pregnancies Complicated by Congenital Cytomegalovirus Infection: A Multicenter Study

Introduction: To report the prognostic value of Cytomegalovirus (CMV) viral load in the amniotic fluid (AF) in predicting the outcome of infected pregnancies.Methods: Multicenter retrospective study involving 11 Italian referral centers from 2012 to 2021. Inclusion criteria were fetuses with confirmed congenital CMV infection. The primary outcome was the prognostic value accuracy of CMV qPCR in AF in predicting the risk of additional anomalies detected either at follow-up ultrasound or fetal MRI. The secondary outcome was prediction of post-natal clinical symptoms related CMV infection. Multivariate logistic regression and area under the curve (AUC) analyses were used to analyze the data.Results: 104 fetuses were included. Associated anomalies detected at follow-up ultrasound or fetal MRI were detected in 14.4% of cases (15/104). Mean AF CMV viral load was significantly higher in fetuses with compared to those without additional anomalies at follow-up ultrasound or fetal MRI (3346634.27 +/- 402582.95 vs 761934 +/- 222513,2 p1310520 copies/ml, a sensitivity of 66.7% and a specificity of 84.3% a positive likelihood ratio of 4.24. Once excluding fetuses with anomalies at ultrasound or MRI, the diagnostic performance of qPCR in identifying fetuses with symptomatic infection after birth was low, with an AUC of 0.586,Conclusion: CMV viral load at second trimester amniocentesis has a moderate accuracy for the occurrence of CMV related anomalies in fetuses with congenital infection and normal ultrasound at the initial diagnosis. Conversely, prediction of symptomatic infection is low

Role of fetal magnetic resonance imaging in fetuses with congenital cytomegalovirus infection: a multicenter study

Objective: To investigate the role of fetal brain MRI in detecting anomalies in fetuses with congenital CMV infection undergoing neurosonography. Methods: Multicenter, retrospective, cohort study involving 11 referral fetal medicine centers in Italy from 2012. The inclusion criteria were fetuses with congenital CMV infection diagnosed by PCR analysis of amniotic fluid, detailed multiplanar assessment of the fetal brain as recommended by the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG), normal karyotype and MRI performed within 3 weeks from the last ultrasound examination. The primary outcome was the rate of central nervous system (CNS) anomalies detected exclusively on MRI and confirmed after birth or autopsy in fetuses with a prenatal diagnosis of congenital CMV infection and normal neurosonographic assessment at diagnosis. Additional CNS anomalies were classified into anomalies of the ventricular and the periventricular zone, intra-cranial calcifications in the basal ganglia or geminal matrix, destructive encephalopathy in the white matter, malformations of cortical development, delayed myelinization, midline, posterior and complex brain anomalies. Univariate and multivariate logistic regression analysis were used to identify and adjust for potential confounders. Results: The analysis included 95 fetuses with a prenatal diagnosis of congenital CMV infection and normal neurosonography at first examination. The rate of structural anomalies detected exclusively at fetal MRI was 10.5% (10/95). When considering the type of anomaly, malformations of cortical development were detected at MRI in 40% (4/10) of fetuses, destructive encephalopathy in 20% (2/10), intracranial calcifications in the germinal matrix in 10% (1/10), and complex anomalies in 30% (3/10). At the multivariate logistic regression analysis, only CMV viral load in the amniotic fluid >100,000 copies/ml (OR: 12.0, 95% CI 1.2-124.7, p=0.04) was independently associated with the likelihood of detecting fetal anomalies at MRI, while maternal age (p=0.62), maternal body mass index (BMI) (p=0.73), maternal primary CMV infection (p=0.31), first trimester infection (p=0.685), prenatal therapy (p=0.11), or the interval between ultrasound and MRI (p=0.27) were not. Associated anomalies were detected exclusively at birth and missed at both types of prenatal imaging in 3.8% (3/80) of fetuses with congenital CMV infection. Conclusions: Fetal brain MRI can detect additional anomalies in a significant proportion of fetuses with congenital CMV infection and negative neurosonography. Viral load in the amniotic fluid was an independent predictor of the risk of associated anomalies in these fetuses. The findings from the study support a longitudinal evaluation using fetal MRI in congenital CMV infection even in cases with negative neurosonography at diagnosis. This article is protected by copyright. All rights reserved. Keywords: CMV; Cytomegalovirus; MRI; hearing loss; infection; neurosonography; ultrasound