Assessment of frost damage in mycorrhizal and non-mycorrhizal roots of Scots pine seedlings using classification analysis of their electrical impedance spectra

Key message

A novel non-destructive method is presented for studying the frost hardiness of roots. Principal component analysis from the electrical impedance spectra revealed differences between freezing temperatures, but no clear differences between the mycorrhizal treatments as regards freezing stress.

Abstract

We present a novel non-destructive method for the classification of root systems with different degrees of freezing injuries based on the measurement of electrical impedance spectra (EIS). Roots of Scots pine (Pinus sylvestris L.) seedlings, raised in perlite with nutrient solution, were colonized by Hebeloma sp. or Suillus luteus or left non-mycorrhizal, and exposed to a series of low temperatures (5, ?5, ?12 and ?18 °C) after cultivation with and without cold acclimation regimes. In EIS measurements, we ran a small-amplitude electric current to the root system at 44 frequencies between 5 Hz and 100 kHz through electrodes set in the stem and in perlite at the bottom of the container. The normalized (Euclidian) electrical impedance spectra were classified using the CLAFIC-method (CLAss-Featuring Information Compression) that is based on a subspace method with two variants where the longest projection vector defines the sample class. The current delivery through the root system was affected by freezing injuries in the roots. The most remarkable change, indicating the threshold for cold tolerance, took place between ?5 and ?12 °C for non-acclimated and between ?12 and ?18 °C for cold acclimated roots. No difference was found between the mycorrhizal treatments in the response to the freezing temperatures. The results on the effects of both the low-temperature exposure and mycorrhizas agree with freezing damage assessments done by other methods.

     

Angeli's salt induces neurotoxicity in dopaminergic neurons in vivo and in vitro

In this study, we investigated the hypothesis that the pro-oxidative properties of Angeli's salt (AS), a nitroxyl anion (HNO/NO -) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 μM) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 μM, while inhibiting it at 1000 μM concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro . Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced when measured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [ 3 H] dopamine uptake with concentrations over 200 μM confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro , infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity. These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.     

A preliminary evaluation of a new enzyme immunoassay to detect Chlamydia pneumoniae-specific antibodies

New enzyme immunoassays (EIAs) for determination of specific IgG, IgA, and IgM antibody titers to Chlamydia pneumoniae were evaluated independently in three research laboratories. Specificity of the EIAs was enhanced by removing LPS from the chlamydial antigen. The performance of these EIAs was evaluated in comparison with the microimmunofluorescence (MIF) test using specimens from: (i) a group of adult patients with community-acquired pneumonia (CAP) previously diagnosed as having an acute chlamydial infection by the complement fixation test or the whole inclusion fluorescence test; (ii) from a group of adult patients with acute respiratory tract infections; and (iii) from a group of young children consecutively presenting with acute respiratory tract infections. The MIF test and the EIAs detected acute infections in paired serum specimens from 12 of 14 patients from the first group. Eleven of these 12 patients were positive in both tests. The MIF test detected seven acute infections in single convalescence serum specimens from eight patients. Two of these were also positive in the EIAs. Paired serum specimens from the second group of adult patients (n=12) were collected during an epidemic of C. pneumoniae. The EIAs detected six acute infections. The MIF test detected two additional patients with acute infections. From the group of young children (n=30), the EIAs detected two patients with acute infections. Our conclusion from this preliminary evaluation is that these EIAs could be useful for laboratory diagnosis of acute C. pneumoniae infections. Comprehensive prospective studies should provide suitable data to calculate the sensitivity, specificity, and predictive values.     

Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.     

The role of land cover in bioclimatic models depends on spatial resolution

Aim We explored the importance of climate and land cover in bird species distribution models on multiple spatial scales. In particular, we tested whether the integration of land cover data improves the performance of pure bioclimatic models. Location Finland, northern Europe. Methods The data of the bird atlas survey carried out in 1986–89 using a 10 × 10 km uniform grid system in Finland were employed in the analyses. Land cover and climatic variables were compiled using the same grid system. The dependent and explanatory variables were resampled to 20‐km, 40‐km and 80‐km resolutions. Generalized additive models (GAM) were constructed for each of the 88 land bird species studied in order to estimate the probability of occurrence as a function of (1) climate and (2) climate and land cover variables. Model accuracy was measured by a cross‐validation approach using the area under the curve (AUC) of a receiver operating characteristic (ROC) plot. Results In general, the accuracies of the 88 bird–climate models were good at all studied resolutions. However, the inclusion of land cover increased the performance of 79 and 78 of the 88 bioclimatic models at 10‐km and 20‐km resolutions, respectively. There was no significant improvement at the 40‐km resolution. In contrast to the finer resolutions, the inclusion of land cover variables decreased the modelling accuracy at 80km resolution. Main conclusions Our results suggest that the determinants of bird species distributions are hierarchically structured: climatic variables are large‐scale determinants, followed by land cover at finer resolutions. The majority of the land bird species in Finland are rather clearly correlated with climate, and bioclimate envelope models can provide useful tools for identifying the relationships between these species and the environment at resolutions ranging from 10 km to 80 km. However, the notable contribution of land cover to the accuracy of bioclimatic models at 10–20‐km resolutions indicates that the integration of climate and land cover information can improve our understanding and model predictions of biogeographical patterns under global change.     

Body electrical loss analysis (BELA) in the assessment of visceral fat: a demonstration

Background

Parkinson's disease (PD) is a neurodegenerative disorder resulting in motor disturbances that can impact normal gait. Although PD initially responds well to pharmacological treatment, as the disease progresses efficacy often fluctuates over the course of the day, and clinical management would benefit from long-term objective measures of gait. We have previously described a small device worn on the shank that uses acceleration and angular velocity sensors to calculate stride length and identify freezing of gait in PD patients. In this study we extend validation of the gait monitor to 24-h using simultaneous video observation of PD patients.

Methods

A sleep laboratory was adapted to perform 24-hr video monitoring of patients while wearing the device. Continuous video monitoring of a sleep lab, hallway, kitchen and conference room was performed using a 4-camera security system and recorded to hard disk. Subjects (3) wore the gait monitor on the left shank (just above the ankle) for a 24-h period beginning around 5 pm in the evening. Accuracy of stride length measures were assessed at the beginning and end of the 24-h epoch. Two independent observers rated the video logs to identify when subjects were walking or lying down.

Results

The mean error in stride length at the start of recording was 0.05 m (SD 0) and at the conclusion of the 24 h epoch was 0.06 m (SD 0.026). There was full agreement between observer coding of the video logs and the output from the gait monitor software; that is, for every video observation of the subject walking there was a corresponding pulse in the monitor data that indicated gait.

Conclusions

The accuracy of ambulatory stride length measurement was maintained over the 24-h period, and there was 100% agreement between the autonomous detection of locomotion by the gait monitor and video observation.     

High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel “tyrosine” finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a K_i of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease.     

Psychrotolerant Paenibacillus tundrae isolates from barley grains produce new cereulide-like depsipeptides (paenilide and homopaenilide) that are highly toxic to mammalian cells

Paenilide is a novel, heat-stable peptide toxin from Paenibacillus tundrae, which colonizes barley. P. tundrae produced 20 to 50 ng of the toxin mg(-1) of cells (wet weight) throughout a range of growth temperatures from +5°C to +28°C. Paenilide consisted of two substances of 1,152 Da and 1,166 Da, with masses and tandem mass spectra identical to those of cereulide and a cereulide homolog, respectively, produced by Bacillus cereus NS-58. The two components of paenilide were separated from those of cereulide by high-performance liquid chromatography (HPLC), showing a structural difference suggesting the replacement of O-Leu (cereulide) by O-Ile (paenilide). The exposure of porcine spermatozoa and kidney tubular epithelial (PK-15) cells to subnanomolar concentrations of paenilide resulted in inhibited motility, the depolarization of mitochondria, excessive glucose consumption, and metabolic acidosis. Paenilide was similar to cereulide in eight different toxicity endpoints with porcine and murine cells. In isolated rat liver mitochondria, nanomolar concentrations of paenilide collapsed respiratory control, zeroed the mitochondrial membrane potential, and induced swelling. The toxic effect of paenilide depended on its high lipophilicity and activity as a high-affinity potassium ion carrier. Similar to cereulide, paenilide formed lipocations, i.e., lipophilic cationic compounds, with K(+) ions already at 4 mM [K(+)], rendering lipid membranes electroconductive. Paenilide-producing P. tundrae was negative in a PCR assay with primers specific for the cesB gene, indicating that paenilide was not a product of plasmid pCER270, encoding the biosynthesis of cereulide in B. cereus. Paenilide represents the first potassium ionophoric compound described for Paenibacillus. The findings in this paper indicate that paenilide from P. tundrae is a potential food-poisoning agent.