Cryo X-ray nano-tomography of vaccinia virus infected cells

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.     

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.     

Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover

Protein turnover through cullin-3 is tightly regulated by posttranslational modifications, the COP9 signalosome, and BTB/POZ-domain proteins that link cullin-3 to specific substrates for ubiquitylation. In this paper, we report how potassium channel tetramerization domain containing 6 (KCTD6) represents a novel substrate adaptor for cullin-3, effectively regulating protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5). Binding of sAnk1.5 to KCTD6, and its subsequent turnover is regulated through posttranslational modification by nedd8, ubiquitin, and acetylation of C-terminal lysine residues. The presence of the sAnk1.5 binding partner obscurin, and mutation of lysine residues increased sAnk1.5 protein levels, as did knockdown of KCTD6 in cardiomyocytes. Obscurin knockout muscle displayed reduced sAnk1.5 levels and mislocalization of the sAnk1.5/KCTD6 complex. Scaffolding functions of obscurin may therefore prevent activation of the cullin-mediated protein degradation machinery and ubiquitylation of sAnk1.5 through sequestration of sAnk1.5/KCTD6 at the sarcomeric M-band, away from the Z-disk-associated cullin-3. The interaction of KCTD6 with ankyrin-1 may have implications beyond muscle for hereditary spherocytosis, as KCTD6 is also present in erythrocytes, and erythrocyte ankyrin isoforms contain its mapped minimal binding site.     

β-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.     

Influence of Soil Physical Properties on Plants of the Mussununga Ecosystem, Brazil

Distribution ranges of plant species are related to physical variables of ecosystems that limit plant growth. Therefore, each plant species response to physical factors builds up the functional diversity of an ecosystem. The higher the species richness of an ecosystem, the larger the probability of maintaining functions and the higher the potential number of plant functional groups (FGs). Thus, the richness potentially increases the number of functions of the highly diverse Atlantic Rainforest domain in Brazil. Severe plant growth limitations caused by stress, however, decrease species richness. In the Spodosols of the Mussununga, an associated ecosystem of Atlantic Rainforest, the percentage of fine sand is directly related to water retention. Moreover, the depth of the cementation layer in the Mussununga??s sandy soil is a physical factor that can affect the plants?? stress gradients. When a shallow cementation layer depth is combined with low water retention in soils and with low fine sand percentage, the double stresses of flooding in the rainy season and water scarcity in the dry season result. This study aimed to identify FGs among Mussununga plant species responding to water stress gradients of soil and to verify the effects of the gradients on plant species richness of the Mussununga. A canonical correspondence analysis (CCA) of species abundance and soil texture variables was performed on 18 plots in six physiognomies of the Mussununga. Species richness rarefactions were calculated for each vegetation form to compare diversity. The two main axes of the CCA showed two FGs responding to soil texture and cementation layer depth: stress tolerator species and mesic species. Physical variables affect plant diversity, with species richness rising as the fine sand proportion also rises in the Mussununga. The effect of the cementation layer is not significantly related to species richness variation.     

Subterranean Sympatry: An Investigation into Diet Using Stable Isotope Analysis

In the Western Cape three species of mole-rat occur in sympatry, however, little is known about differences in their dietary preferences. Dietary composition of the three species; the common mole-rat (Cryptomys hottentotus hottentotus), the Cape mole-rat (Georychus capensis) and the Cape dune mole-rat (Bathyergus suillus) were examined using stable isotope analysis. Blood, fur and claw samples were collected from 70 mole-rats, in addition to several potential food items, to assess food selection of the three species under natural conditions. Overall there was a significant difference in the isotopic composition (δ¹³C and δ¹⁵N) between all three species and significant differences in their diet composition. There were also significant differences between tissues in all three species suggesting temporal variation in diet. The small size and colonial lifestyle of C. h. hottentotus allows it to feed almost 100% on bulbs, while the solitary and larger species G. capensis and B. suillus fed to a greater extent on other resources such as grasses and clover. B. suillus, the largest of the species, had the most generalized diet. However, overall all species relied most heavily upon geophytes and consumed the same species suggesting competition for resources could exist. We also showed a high level of individual variation in diet choices. This was most pronounced in B. suillus and G. capensis and less so in C. h. hottentotus. We demonstrate that stable isotope analysis can successfully be applied to examine dietary patterns in subterranean mammals and provide insights into foraging patterns and dietary variation at both the inter and intra population level.     

X-Linked Adrenoleukodystrophy: Molecular and Functional Analysis of the ABCD1 Gene in Argentinean Patients

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disease associated with mutations in the ABCD1 gene that encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long-chain fatty acids (VLCFAs) in plasma and in adrenal, testicular and nervous tissues, due to a defect in peroxisomal VLCFA β-oxidation. In the present study, we analyzed 10 male patients and 17 female carriers from 10 unrelated pedigrees with X-ALD from Argentina. By sequencing the ABCD1 we detected 9 different mutations, 8 of which were novel. These new mutations were verified by a combination of methods that included both functional (western blot and peroxisomal VLCFA β-oxidation) and bioinformatics analysis. The spectrum of novel mutations consists of 3 frameshift (p.Ser284fs*16, p.Glu380Argfs*21 and p.Thr254Argfs*82); a deletion (p.Ser572_Asp575del); a splicing mutation (c.1081+5G>C) and 3 missense mutations (p.Ala341Asp, p.His420Pro and p.Tyr547Cys). In one patient 2 changes were found: a known missense (p.His669Arg) and an unpublished amino acid substitution (p.Ala19Ser). In vitro studies suggest that p.Ala19Ser is a polymorphism. Moreover, we identified two novel intronic polymorphisms and two amino acid polymorphisms. In conclusion, this study extends the spectrum of mutation in X-ALD and facilitates the identification of heterozygous females.     

Maternal Vitamin C Deficiency during Pregnancy Persistently Impairs Hippocampal Neurogenesis in Offspring of Guinea Pigs

While having the highest vitamin C (VitC) concentrations in the body, specific functions of VitC in the brain have only recently been acknowledged. We have shown that postnatal VitC deficiency in guinea pigs causes impairment of hippocampal memory function and leads to 30% less neurons. This study investigates how prenatal VitC deficiency affects postnatal hippocampal development and if any such effect can be reversed by postnatal VitC repletion. Eighty pregnant Dunkin Hartley guinea pig dams were randomized into weight stratified groups receiving High (900 mg) or Low (100 mg) VitC per kg diet. Newborn pups (n = 157) were randomized into a total of four postnatal feeding regimens: High/High (Control); High/Low (Depleted), Low/Low (Deficient); and Low/High (Repleted). Proliferation and migration of newborn cells in the dentate gyrus was assessed by BrdU labeling and hippocampal volumes were determined by stereology. Prenatal VitC deficiency resulted in a significant reduction in postnatal hippocampal volume (P<0.001) which was not reversed by postnatal repletion. There was no difference in postnatal cellular proliferation and survival rates in the hippocampus between dietary groups, however, migration of newborn cells into the granular layer of the hippocampus dentate gyrus was significantly reduced in prenatally deficient animals (P<0.01). We conclude that a prenatal VitC deficiency in guinea pigs leads to persistent impairment of postnatal hippocampal development which is not alleviated by postnatal repletion. Our findings place attention on a yet unrecognized consequence of marginal VitC deficiency during pregnancy.     

Deimatic Display in the European Swallowtail Butterfly as a Secondary Defence against Attacks from Great Tits

Background

Many animals reduce the risk of being attacked by a predator through crypsis, masquerade or, alternatively, by advertising unprofitability by means of aposematic signalling. Behavioural attributes in prey employed after discovery, however, signify the importance of also having an effective secondary defence if a predator uncovers, or is immune to, the prey’s primary defence. In butterflies, as in most animals, secondary defence generally consists of escape flights. However, some butterfly species have evolved other means of secondary defence such as deimatic displays/startle displays. The European swallowtail, Papilio machaon, employs what appears to be a startle display by exposing its brightly coloured dorsal wing surface upon disturbance and, if the disturbance continues, by intermittently protracting and relaxing its wing muscles generating a jerky motion of the wings. This display appears directed towards predators but whether it is effective in intimidating predators so that they refrain from attacks has never been tested experimentally.

Methodology/Principal Findings

In this study we staged encounters between a passerine predator, the great tit, Parus major, and live and dead swallowtail butterflies in a two-choice experiment. Results showed that the dead butterfly was virtually always attacked before the live butterfly, and that it took four times longer before a bird attacked the live butterfly. When the live butterfly was approached by a bird this generally elicited the butterfly’s startle display, which usually caused the approaching bird to flee. We also performed a palatability test of the butterflies and results show that the great tits seemed to find them palatable.

Conclusions/Significance

We conclude that the swallowtail’s startle display of conspicuous coloration and jerky movements is an efficient secondary defence against small passerines. We also discuss under what conditions predator-prey systems are likely to aid the evolution of deimatic behaviours in harmless and palatable prey.