Impedance Spectroscopy in Frost Hardiness Evaluation of Rhododendron Leaves

Impedance spectroscopy was used in studying frost hardinessof leaves of two diploid rhododendron cultivars, RhododendronL. ‘PJM’ and R. ‘Cunningham's White’,and their tetraploid derivatives, R. ‘Northern Starburst’(NSB) and CW4. After the growing season and initial hardeningin a greenhouse, plants were subjected to an acclimation regimein a phytotron: 3 consecutive weeks at +5, +1 and -2°C each.Hardiness was studied with controlled freezing tests beforeeach decrease in temperature and at the end of the experiment,based on data of extracellular resistance r_eand relaxation time of the frost-exposed leaves. The correlation of the two estimateswas 0.92. Generally, the diploid clones had better frost hardinessthan the tetraploid clones. At the end of the experiment, frosthardiness of the diploid ‘PJM’ was -28.7°C andthat of the tetraploid NSB -20.6°C. Leaves of the diploid‘Cunningham's White’ and of the tetraploid CW4 hardenedto -32.0°C and -20.9°C, respectively. Frost hardinessestimated by impedance spectroscopy correlated well with earlierresults based on visual scoring (r = 0.81–0.86) and electrolyteleakage tests (r = 0.84–0.90), but results from impedancespectroscopy indicated weaker hardiness than the other tests.The difference between the results from impedance spectroscopyand the other tests was smaller and more coherent within the‘Cunningham's White’ clones than within ‘PJM’and NSB. Changes in extracellular and intracellular resistanceof non-frozen leaves during the acclimation correlated withthe changes in frost hardiness of ‘Cunningham's White’clones, but not with those of ‘PJM’ and NSB, whichbelong to another subspecies.Copyright 2000 Annals of BotanyCompany Cold resistance, evergreen, frost hardiness, impedance spectroscopy, polyploid, Rhododendron, tetraploid     

Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has not been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of HSF2 have been characterized in hemin-treated human K562 erythroleukemia cells. Here, we demonstrate that a stress signal for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) upon shift to the nonpermissive temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory mechanism to respond to changes in the protein-degradative machinery.     

Identification and characterization of a unique cysteine residue proximal to the catalytic site of Arabidopsis thaliana carotenoid cleavage enzyme 1

AtCCD1 and AtNCED3 are related carotenoid cleavage enzymes from Arabidopsis thaliana that catalyze the oxidative cleavage of, respectively, the 9,10 (9',10') double bonds of carotenoid substrates such as beta-carotene, and the 11,12 double bond of 9-cis epoxycarotenoids. Although the cellular and cleavage functionalities of these enzymes have been reported, their mechanisms and related structural environments mediating these disparate specificities in homologous enzymes have not been well characterized. By relating the differences observed in UV and visible light absorption and Cu(II) electron paramagnetic signals to variations in sequence alignments and 3-D homology models of the two A. thaliana enzymes, we identified a putatively proximal cysteine residue (Cys352) in AtCCD1 that is not conserved in AtNCED3. Spectral analysis of the Cys to Ala mutant confirmed its uniqueness and proximity to the metal binding site, but precluded any role for the residue in the mediation of the observed metal binding affinity or associated steric constraint differences. Further analysis of kinetic substrate cleavage properties indicated a decrease in Vmax and a subtle increase in Km for the C352A mutant compared with those observed for the wild-type, thus confirming catalytic site proximity and suggesting possible roles for the unique cysteine in the modulation of substrate affinity and (or) the reaction rate of AtCCD1.     

Characterization of Early Disease Status in Treatment-Naive Male Paediatric Patients with Fabry Disease Enrolled in a Randomized Clinical Trial

Trial Design

This analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.

Methods

Males aged 5–18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13–17 years), renal function, and glycolipid levels (plasma, urine).

Results

Plasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m² (range 90.4–161.0 mL/min/1.73 m²) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0–27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.

Conclusions

These data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.

Trial Registration

ClinicalTrials.gov NCT00701415     

A Genome-Wide Association Study of a Biomarker of Nicotine Metabolism

Individuals with fast nicotine metabolism typically smoke more and thus have a greater risk for smoking-induced diseases. Further, the efficacy of smoking cessation pharmacotherapy is dependent on the rate of nicotine metabolism. Our objective was to use nicotine metabolite ratio (NMR), an established biomarker of nicotine metabolism rate, in a genome-wide association study (GWAS) to identify novel genetic variants influencing nicotine metabolism. A heritability estimate of 0.81 (95% CI 0.70–0.88) was obtained for NMR using monozygotic and dizygotic twins of the FinnTwin cohort. We performed a GWAS in cotinine-verified current smokers of three Finnish cohorts (FinnTwin, Young Finns Study, FINRISK2007), followed by a meta-analysis of 1518 subjects, and annotated the genome-wide significant SNPs with methylation quantitative loci (meQTL) analyses. We detected association on 19q13 with 719 SNPs exceeding genome-wide significance within a 4.2 Mb region. The strongest evidence for association emerged for CYP2A6 (min p = 5.77E-86, in intron 4), the main metabolic enzyme for nicotine. Other interesting genes with genome-wide significant signals included CYP2B6, CYP2A7, EGLN2, and NUMBL. Conditional analyses revealed three independent signals on 19q13, all located within or in the immediate vicinity of CYP2A6. A genetic risk score constructed using the independent signals showed association with smoking quantity (p = 0.0019) in two independent Finnish samples. Our meQTL results showed that methylation values of 16 CpG sites within the region are affected by genotypes of the genome-wide significant SNPs, and according to causal inference test, for some of the SNPs the effect on NMR is mediated through methylation. To our knowledge, this is the first GWAS on NMR. Our results enclose three independent novel signals on 19q13.2. The detected CYP2A6 variants explain a strikingly large fraction of variance (up to 31%) in NMR in these study samples. Further, we provide evidence for plausible epigenetic mechanisms influencing NMR.     

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.     

Micro-Economic Impact of Congenital Heart Surgery: Results of a Prospective Study from a Limited-Resource Setting

Introduction

The microeconomic impact of surgery for congenital heart disease is unexplored, particularly in resource limited environments. We sought to understand the direct and indirect costs related to congenital heart surgery and its impact on Indian households from a family perspective.

Methods

Baseline and first follow-up data of 644 consecutive children admitted for surgery for congenital heart disease (March 2013 – July 2014) in a tertiary referral hospital in Central Kerala, South India was collected prospectivelyfrom parents through questionnaires using a semi-structured interview schedule.

Results

The median age was 8.2 months (IQR: 3.0– 36.0 months). Most families belonged to upper middle (43.0%) and lower middle (35.7%) socioeconomic class. Only 3.9% of families had some form of health insurance. The median expense for the admission and surgery was INR 201898 (IQR: 163287–266139) [I$ 11989 (IQR: 9696–15804)], which was 0.93 (IQR: 0.52–1.49) times the annual family income of affected patients. Median loss of man-days was 35 (IQR: 24–50) and job-days was 15 (IQR: 11–24). Surgical risk category and hospital stay duration significantly predicted higher costs. One in two families reported overwhelming to high financial stress during admission period for surgery. Approximately half of the families borrowed money during the follow up period after surgery.

Conclusion

Surgery for congenital heart disease results in significant financial burden for majority of families studied. Efforts should be directed at further reductions in treatment costs without compromising the quality of care together with generating financial support for affected families.     

Clinical,Biological and Genetic Analysis of Prepubertal Isolated Ovarian Cyst in 11 Girls

Background

The cause of isolated gonadotropin-independent precocious puberty (PP) with an ovarian cyst is unknown in the majority of cases. Here, we describe 11 new cases of peripheral PP and, based on phenotypes observed in mouse models, we tested the hypothesis that mutations in the GNAS1, NR5A1, LHCGR, FSHR, NR5A1, StAR, DMRT4 and NOBOX may be associated with this phenotype.

Methodology/Principal Findings

11 girls with gonadotropin-independent PP were included in this study. Three girls were seen for a history of prenatal ovarian cyst, 6 girls for breast development, and 2 girls for vaginal bleeding. With one exception, all girls were seen before 8 years of age. In 8 cases, an ovarian cyst was detected, and in one case, suspected. One other case has polycystic ovaries, and the remaining case was referred for vaginal bleeding. Four patients had a familial history of ovarian anomalies and/or infertility. Mutations in the coding sequences of the candidate genes GNAS1, NR5A1, LHCGR, FSHR, NR5A1, StAR, DMRT4 and NOBOX were not observed.

Conclusions/Significance

Ovarian PP shows markedly different clinical features from central PP. Our data suggest that mutations in the GNAS1, NR5A1, LHCGR, FSHR StAR, DMRT4 and NOBOX genes are not responsible for ovarian PP. Further research, including the identification of familial cases, is needed to understand the etiology of ovarian PP.     

Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.