A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.     

Mapping of quantitative adult plant field resistance to leaf rust and stripe rust in two European winter wheat populations reveals co-location of three QTL conferring resistance to both rust pathogens

Key message

We detected several, most likely novel QTL for adult plant resistance to rusts. Notably three QTL improved resistance to leaf rust and stripe rust simultaneously indicating broad spectrum resistance QTL.

Abstract

The rusts of wheat (Puccinia spp.) are destructive fungal wheat diseases. The deployment of resistant cultivars plays a central role in integrated rust disease management. Durability of resistance would be preferred, but is difficult to analyse. The Austrian winter wheat cultivar Capo was released in the 1989 and grown on a large acreage during more than two decades and maintained a good level of quantitative leaf rust and stripe rust resistance. Two bi-parental mapping populations: Capo × Arina and Capo × Furore were tested in multiple environments for severity of leaf rust and stripe rust at the adult plant stage in replicated field experiments. Quantitative trait loci associated with leaf rust and stripe rust severity were mapped using DArT and SSR markers. Five QTL were detected in multiple environments associated with resistance to leaf rust designated as QLr.ifa-2AL, QLr.ifa-2BL, QLr.ifa-2BS, QLr.ifa-3BS, and QLr.ifa-5BL, and five for resistance to stripe rust QYr.ifa-2AL, QYr.ifa-2BL, QYr.ifa-3AS, QYr.ifa-3BS, and QYr.ifa-5A. For all QTL apart from two (QYr.ifa-3AS, QLr.ifa-5BL) Capo contributed the resistance improving allele. The leaf rust and stripe rust resistance QTL on 2AL, 2BL and 3BS mapped to the same chromosome positions, indicating either closely linked genes or pleiotropic gene action. These three multiple disease resistance QTL (QLr.ifa-2AL/QYr.ifa-2AL, QLr.ifa.2BL/QYr.ifa-2BL, QLr.ifa-3BS/QYr.ifa.3BS) potentially contribute novel resistance sources for stripe rust and leaf rust. The long-lasting resistance of Capo apparently rests upon a combination of several genes. The described germplasm, QTL and markers are applicable for simultaneous resistance improvement against leaf rust and stripe rust.     

Functional Significance of Labellum Pattern Variation in a Sexually Deceptive Orchid (Ophrys heldreichii): Evidence of Individual Signature Learning Effects

Mimicking female insects to attract male pollinators is an important strategy in sexually deceptive orchids of the genus Ophrys, and some species possess flowers with conspicuous labellum patterns. The function of the variation of the patterns remains unresolved, with suggestions that these enhance pollinator communication. We investigated the possible function of the labellum pattern in Ophrys heldreichii, an orchid species in which the conspicuous and complex labellum pattern contrasts with a dark background. The orchid is pollinated exclusively by males of the solitary bee, Eucera berlandi. Comparisons of labellum patterns revealed that patterns within inflorescences are more similar than those of other conspecific plants. Field observations showed that the males approach at a great speed and directly land on flowers, but after an unsuccessful copulation attempt, bees hover close and visually scan the labellum pattern for up to a minute. Learning experiments conducted with honeybees as an accessible model of bee vision demonstrated that labellum patterns of different plants can be reliably learnt; in contrast, patterns of flowers from the same inflorescence could not be discriminated. These results support the hypothesis that variable labellum patterns in O. heldreichii are involved in flower-pollinator communication which would likely help these plants to avoid geitonogamy.     

Proteins related to lipoprotein profile were identified using a pharmaco-proteomic approach as markers for growth response to growth hormone (GH) treatment in short prepubertal children

Background  

The broad range in growth observed in response to growth hormone (GH) treatment is mainly caused by individual variations in both GH secretion and GH sensitivity. Individual GH responsiveness can be estimated using evidence-based models that predict the response to GH treatment; however, these models can be improved. High-throughput proteomics techniques can be used to identify proteins that may potentially be used as variables in such models in order to improve their predictive ability. Previously we have reported that proteomic analyses can identify biomarkers that discriminate between short prepubertal children with idiopathic short stature (ISS) who show good or poor growth in response to GH treatment. In this study we used a pharmaco-proteomic approach to identify novel factors that correlate with the growth response to GH treatment in prepubertal children who are short due to GH deficiency or ISS. The study included 128 short prepubertal children receiving GH treatment, of whom 39 were GH-deficient and 89 had ISS. Serum protein expression profiles at study start and after 1 year of GH treatment were analyzed using SELDI-TOF. Cross-validated regression and random permutation analyses were performed to identify significant correlations between protein expression patterns and the 2-year growth response to GH treatment.     

Different HLA-DRB1 allele distributions in distinct clinical subgroups of sarcoidosis patients

Background

A strong genetic influence by the MHC class II region has been reported in sarcoidosis, however in many studies with different results. This may possibly be caused by actual differences between distinct ethnic groups, too small sample sizes, or because of lack of accurate clinical subgrouping.

Subjects and methods

In this study we HLA typed a large patient population (n = 754) recruited from one single centre. Patients were sub-grouped into those with Löfgren''s syndrome (LS) (n = 302) and those without (non-Löfgren''s) (n = 452), and the majority of them were clinically classified into those with recovery within two years (resolving) and those with signs of disease for more than two years (non-resolving). PCR was used for determination of HLA-DRB1 alleles. Swedish healthy blood donors (n = 1366) served as controls.

Results

There was a dramatic difference in the distribution of HLA alleles in LS compared to non-LS patients (p = 4 × 10^-36). Most notably, DRB1*01, DRB1*03 and DRB1*14, clearly differed in LS and non-LS patients. In relation to disease course, DRB1*07, DRB1*14 and DRB1*15 generally associated with, while DRB1*01 and DRB1*03 protected against, a non-resolving disease. Interestingly, the clinical influence of DRB1*03 (good prognosis) dominated over that of DRB1*15 (bad prognosis).

Conclusions

We found several significant differences between LS and non-LS patients and we therefore suggest that genetic association studies in sarcoidosis should include a careful clinical characterisation and sub-grouping of patients, in order to reveal true genetic associations. This may be particularly accurate to do in the heterogeneous non-LS group of patients.     

The Brichos domain-containing C-terminal part of pro-surfactant protein C binds to an unfolded poly-val transmembrane segment

Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane alpha-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to beta-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a approximately 100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in beta-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind alpha-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated beta-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents beta-sheet aggregation and that mutations in CTproSP-C can interfere with this function.     

Importance of the N-distal AP-2 binding element in Nef for simian immunodeficiency virus replication and pathogenicity in rhesus macaques

Point mutations in SIVmac239 Nef disrupting CD4 downmodulation and enhancement of virion infectivity attenuate viral replication in acutely infected rhesus macaques, but changes selected later in infection fully restore Nef function (A. J. Iafrate et al., J. Virol. 74:9836-9844, 2000). To further evaluate the relevance of these Nef functions for viral persistence and disease progression, we analyzed an SIVmac239 Nef mutant containing a deletion of amino acids Q64 to N67 (delta64-67Nef). This mutation inactivates the N-distal AP-2 clathrin adaptor binding element and disrupts the abilities of Nef to downregulate CD4, CD28 and CXCR4 and to stimulate viral replication in vitro. However, it does not impair the downmodulation of CD3 and class I major histocompatibility complex (MHC-I) or MHC-II and the upregulation of the MHC-II-associated invariant chain, and it has only a moderate effect on the enhancement of virion infectivity. Replication of the delta64-67Nef variant in acutely infected macaques was intermediate between grossly nef-deleted and wild-type SIVmac239. Subsequently, three of six macaques developed moderate to high viral loads and developed disease, whereas the remaining animals efficiently controlled SIV replication and showed a more attenuated clinical course of infection. Sequence analysis revealed that the deletion in nef was not repaired in any of these animals. However, some changes that slightly enhanced the ability of Nef to downmodulate CD4 and moderately increased Nef-mediated enhancement of viral replication and infectivity in vitro were observed in macaques developing high viral loads. Our results imply that both the Nef functions that were disrupted by the delta64-67 mutation and the activities that remained intact contribute to viral pathogenicity.     

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

Interaction with transducin depletes metarhodopsin III: a regulated retinal storage in visual signal transduction?

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (G(t)). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of G(t) with Meta III, which was previously considered inactive. G(t) thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.