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[Display omitted] • Pyrochroa coccinea larvae increase supercooling capacity upon cold hardening. • The cold hardening is associated with an increase in hemolymph osmolality. • All osmotically relevant contributors during the summer state was identified. • The concentration change in these osmolytes did not explain the osmolality increase. • Non-colligative effects of up-regulated proteins are speculated to be the cause. The larvae of the black headed cardinal beetle Pyrochroa coccinea, overwinters under the bark of dead logs in northern European dioecious forests, and are thus exposed to temperatures below the melting point of their bodily fluids. Here we explore the mechanisms behind their seasonal cold hardening by characterising field samples collected monthly throughout the year. Both the lower lethal temperature and supercooling point dropped as much as 10℃ in the second half of November, reaching values around –15℃ by the beginning of December. This change was accompanied by a 320 mosmol/kg increase in hemolymph osmolality, which is a doubling compared to the summer levels. We used NMR metabolomics to identify and measure the absolute concentrations of the responsible cryoprotective C-H containing metabolites in the hemolymph. The largest increase was found to be in either glucose or trehalose, with an average total increase of 120 mM. Proline, alanine, and choline concentrations were found to increase by around 10 mM each. Contrarily, phosphocholine and phosphoethanolamine were halved, resulting in a total decrease of around 50 mM. These measurements were complemented with ion exchange chromatography measurements. This allowed us to account for all the osmotic pressure in the summer hemolymph, and the measured concentration changes explained as much as 40 % of the observed osmolality increase upon cold hardening. Preliminary results indicate that the remainder may be explained by non-colligative protein contributions. [ABSTRACT FROM AUTHOR]

Abstract: A short exposure to a mild cold stress is sufficient to increase cold tolerance in many insects. This phenomenon, termed rapid cold hardening (RCH) expands the thermal interval that can be exploited by the insect. To investigate the possible role of altered metabolite levels during RCH, the present study used untargeted 1H NMR metabolomic profiling to examine the metabolomic response in Drosophila melanogaster during the 72h following RCH and cold shock treatment. These findings are discussed in relation to the costs and benefits of RCH that are measured in terms of survival and reproductive output. Cold shock caused a persistent disturbance of the metabolite profile that correlated well with a delayed onset of cold shock mortality. The disruption of metabolite homeostasis was smaller following RCH, where control levels were fully recovered after 72h. RCH improved both survival and reproductive output after a subsequent cold shock but the RCH treatment alone was associated with costs in terms of reduced survival and reproductive output. The most pronounced changes following the RCH treatment were elevated levels of glucose and trehalose. Although, it is difficult to discern if a change in a specific metabolite is linked to physiological processes of adaptive, neutral or detrimental nature we observed that the onset and magnitude of the increased sugar levels correlated tightly with the improved chill tolerance following RCH. These findings suggest a putative role of cryoprotectants during RCH which are discussed in the light of the existing literature on the mechanistic background of RCH. [Copyright &y& Elsevier]

Calcium-dependent protein kinases (CDPKs) are structurally unique Ser/Thr kinases found in plants and certain protozoa. They are distinguished by a calmodulin-like regulatory apparatus (calmodulin-like domain (CaM-LD)) that is joined via a junction (J) region to the C-terminal end of the kinase catalytic domain. Like CaM, the CaM-LD is composed of two globular EF structural domains (N-lobe, C-lobe), each containing a pair of Ca2+ binding sites. Spectroscopic analysis shows that the CaM-LD is comprised of helical elements, but the isolated CaM-LD does not form a conformationally homogeneous tertiary structure in the absence of Ca2+. The addition of substoichiometric amounts of Ca2+ is sufficient to stabilize the C-terminal lobe in a construct containing J and CaM-LD (JC) but not in the CaM-LD alone. Moreover, as J is titrated into Ca2+-saturated CaM-LD, interactions are stronger with the C-lobe than the N-lobe of the CaM-LD. Measurements of Ca2+ affinity for JC reveal two cooperatively interacting high affinity binding sites (Kd,mean = 5.6 nM at 20 mM KCl) in the C-lobe and two weaker sites in the N-lobe (Kd,mean = 110 nM at 20 mM KCl). The corresponding Ca2+ binding constants in the isolated CaM-LD are lower by more than 2 orders of magnitude, which indicates that the J region has an essential role in stabilizing the structure of the CDPK regulatory apparatus. The large differential affinity between the two domains together with previous studies on a plasmodium CDPK (Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M., and Kappes, B. (1994) Biochemistry 33, 3714-3721) suggests a model whereby even at normally low cytosolic levels of Ca2+, the C-lobe interacts with the junction, but the kinase remains in an autoinhibited state. Activation then occurs when Ca2+ levels rise to fill the two weaker affinity binding sites in the N-lobe, thereby triggering a conformational change that leads to release of the autoinhibitory region. [ABSTRACT FROM AUTHOR]

Amyloid beta (Aβ) peptide aggregation is linked to the initiation of Alzheimer's disease; accordingly, aggregation-prone isoforms of Aβ, expressed in the brain, shorten the lifespan of Drosophila melanogaster . However, the lethal effects of Aβ are not apparent until after day 15. We used shibire TS flies that exhibit a temperature-sensitive paralysis phenotype as a reporter of proteostatic robustness. In this model, we found that increasing age but not Aβ expression lowered the flies' permissive temperature, suggesting that Aβ did not exert its lethal effects by proteostatic disruption. Instead, we observed that chemical challenges, in particular oxidative stressors, discriminated clearly between young (robust) and old (sensitive) flies. Using nuclear magnetic resonance spectroscopy in combination with multivariate analysis, we compared water-soluble metabolite profiles at various ages in flies expressing Aβ in their brains. We observed 2 genotype-linked metabolomic signals, the first reported the presence of any Aβ isoform and the second the effects of the lethal Arctic Aβ. Lethality was specifically associated with signs of oxidative respiration dysfunction and oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: The perception of odour and flavour of foods is a complicated physiological and psychological process that cannot be explained by simple models. Unfortunately, taste is not objective, but partially subjective and it depends also on the mood of the taster. Generally, sensory analysis is used to describe sensory features. The availability of a number of instrumental techniques has opened up the possibility to calibrate the sensory perception. Here we have tested the potentiality of nuclear magnetic resonance spectroscopy as “magnetic tongue” to measure sensory descriptors in extra-virgin olive oil. We were able to correlate the NMR metabolomic fingerprints of extra-virgin olive oil to the sensory descriptors: tomato, bitter, pungent, rosemary, artichoke, sweet, grassy and leaf. [Copyright &y& Elsevier]

Abstract: In the present study the joint transcriptomic and metabolomic responses in Folsomia candida (Collembola) to temperature changes on a short-term scale were studied. Change in heat tolerance was examined as survival after a 35°C heat shock (2h) in the course of either a fluctuating temperature regime (8 to 32°C; pre-treated) or a constant temperature (20°C; control) over a period of 24h. Exposure to a temperature increase from 20 to 32°C (2.4°Cmin− 1) induced a significantly increased heat tolerance which continued throughout the experiment. Expression of the gene encoding heat shock protein Hsp70 was assessed at the mRNA level using real time quantitative polymerase chain reaction (QPCR). Hsp70 was rapidly induced and significantly increased by the temperature increase. The relative concentrations of low molecular weight metabolites were analysed in F. candida using nuclear magnetic resonance spectroscopy (1H NMR). A significant metabolomic divergence between pre-treated and control collembolans was evident; partly due to a significantly reduced relative concentration of five free amino acids (arginine, leucine, lysine, phenylalanine and tyrosine) in pre-treated collembolans. These results, obtained from ecological transcriptomics and metabolomics jointly generated insight on various levels into the combined responses to a changing environment. [Copyright &y& Elsevier]

Cocaine is the most widely used illicit drug, and its origin is always the focus of intense investigation aimed at identifying the trafficking routes. Since NMR represents a unique methodology for performing chemical identification and quantification, here it is proposed a strategy based on ¹H NMR spectral analysis in conjunction with multivariate analysis to identify the chemical ''fingerprint'' of cocaine samples, and to link cocaine samples based on this information. The most relevant spectral regions containing the fingerprint have been identified: δH 0.86-0.96, 1.50-1.56, 5.90-5.93, 6.48-6.52, 7.31- 7.34, 7.61-7.63, 7.68-7.72 ppm. The strategy has been applied on samples seized in different times and places in Naples (Italy). The chemical ''fingerprint'' depend on what plant they were extracted from, where it was cultivated, and which procedures were used for extraction and purification, thus adding significant information in the process toward identification of the trafficking routes for this drug. [ABSTRACT FROM AUTHOR]

Abstract: Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE. [Copyright &y& Elsevier]

Abstract: Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about −4°C when cooled under dry conditions, but freezing of body fluids was readily induced at −1°C when in contact with external ice crystals. All three species survived freezing for 2days at −1°C, and some A. rufus and A. lusitanicus also survived freezing at −2°C. 1H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates. [Copyright &y& Elsevier]

Abstract: The metabolic profile of calf rumen epithelial tissue was for the first time characterized by 1H NMR spectroscopy of chloroform/methanol extracts. The metabolite profiles comprised a number of amino acids, creatine, taurine, short-chain fatty acids and triglycerides. The effects of two dietary interventions; i) four levels of milk allowance with a concomitant different uptake of starter concentrate, and ii) two diets with varying content of starch and fibre in the concentrate, were elucidated. Partial least square regression analysis revealed that the intensity of NMR signals assigned to leucine, isoleucine and valine (0.90  ppm), propionate (1.06 and 2.18  ppm), lactate (1.32  ppm), butyrate (1.56  ppm), acetate (1.93  ppm), glutamine/glutamate (2.18, 2.35 and 3.75  ppm), creatine (3.04 and 3.94  ppm) and glycine (3.55  ppm) decreased with increasing milk allowance. In addition, the analysis revealed that the main difference between the two diets was the content of propionate in the epithelia tissue extracts. Previous morphological analyses of the same rumen epithelia were not able to detect any significant effects of either milk allowance or dietary starch content. Accordingly, the present study demonstrated that 1H NMR spectroscopy applied on extracts is a useful tool for metabolite profiling of epithelial tissue and for following the development of epithelia tissue in young calves, and that the technique may be more sensitive to dietary effects than morphological studies. [Copyright &y& Elsevier]