A Comparative Analysis of Genetic Differentiation across Six Shared Willow Host Species in Leaf- and Bud-Galling Sawflies

Genetic divergence and speciation in plant-feeding insects could be driven by contrasting selection pressures imposed by different plant species and taxa. While numerous examples of host-associated differentiation (HAD) have been found, the overall importance of HAD in insect diversification remains unclear, as few studies have investigated its frequency in relation to all speciation events. One promising way to infer the prevalence and repeatability of HAD is to estimate genetic differentiation in multiple insect taxa that use the same set of hosts. To this end, we measured and compared variation in mitochondrial COI and nuclear ITS2 sequences in population samples of leaf-galling Pontania and bud-galling Euura sawflies (Hymenoptera: Tenthredinidae) collected from six Salix species in two replicate locations in northern Fennoscandia. We found evidence of frequent HAD in both species complexes, as individuals from the same willow species tended to cluster together on both mitochondrial and nuclear phylogenetic trees. Although few fixed differences among the putative species were found, hierarchical AMOVAs showed that most of the genetic variation in the samples was explained by host species rather than by sampling location. Nevertheless, the levels of HAD measured across specific pairs of host species were not correlated in the two focal galler groups. Hence, our results support the hypothesis of HAD as a central force in herbivore speciation, but also indicate that evolutionary trajectories are only weakly repeatable even in temporally overlapping radiations of related insect taxa.     

Effects of epidural morphine and transdermal fentanyl analgesia on physiology and behaviour after abdominal surgery in pigs

The objective of this work was to evaluate the physiological and behavioural effects of opioid analgesic treatment in pigs subjected to abdominal surgery. Ten Swedish Landrace x Yorkshire pigs (20 +/- 4 kg b.w.) were submitted for intestinal cannulation. The pigs were allocated into two groups during one preoperative, one surgical and two postoperative days. All pigs were anaesthetized with medetomidine, tiletamine and zolazepam. One group was treated with epidural morphine (0.1 mg/kg) preoperatively, and transdermal fentanyl patches (50 microg/kg/h) were applied behind the ear immediately after surgery. The other group received epidural saline (equivalent volume) and placebo patches. All pigs were regularly weighed and clinically examined and repeated blood samples were analysed for serum concentrations of cortisol, beta-endorphin and fentanyl. Pre- and postoperative behaviours were evaluated by a swine specialist blinded to the treatment, three times a day, and were also videotape recorded for a total of 84 h per pig. No differences in behaviour were noted by the observer. During the first postoperative 12 h, treated pigs did not differ in activity compared with preoperative recordings, while untreated pigs were found to be less active. The treated group started to show interest in eating immediately after anaesthesia recovery, whereas the placebo group did not. During the 12-60 h postoperative period, the treated group had lower activity levels compared with the preoperative levels, which were similar to those in the placebo group. Treated pigs gained 0.5 +/- 0.2 kg during the subsequent two postoperative days, whereas the untreated pigs lost weight throughout the experiment. Cortisol concentration differed immediately after the surgery: Group P had 325 +/- 120 nmol/L and Group M 159 +/- 49 nmol/L. beta-endorphin concentration did not differ between groups. The highest serum fentanyl concentration (0.37 +/- 0.3 ng/mL) was measured 24 h postoperatively. Preoperative epidural morphine in combination with postoperative transdermal fentanyl resulted in earlier return to normal activity levels and an immediate weight gain after surgery.     

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome,global protein secretion,and T cells

Introduction: The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses.

Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses.

Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.      

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

A hierarchical whole-body modeling approach elucidates the link between in Vitro insulin signaling and in Vivo glucose homeostasis

Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.     

Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.

In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.

Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.

These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and—possibly to a lesser extent—by some other amino acids taken up from the endosperm.

     

Intranuclear localization of a new snRNP-related antigen.

The intranuclear distribution of a new antigen (F78) associated with U snRNPs (small nuclear RNA-protein complexes) was compared with that of the RNP and Sm protein antigens previously identified on individual snRNP particles. Human and rat cells were double stained with human autoantisera and mouse monoclonal antibodies. The binding of the human and mouse antibodies was detected with secondary antibodies conjugated with fluorescein and rhodamine, respectively. The resulting immunofluorescence patterns were compared by digital image analysis. The F78, RNP, and Sm antigens show speckled fluorescence patterns which overlap to a great extent. The F78 pattern, however, also contains two classes of structural elements not present in the RNP pattern. Furthermore, during mitosis expression of the F78 antigen is completely suppressed from early prophase to telophase, while the RNP and Sm antigens are found evenly distributed throughout the cytoplasm of the dividing cells.     

Heterogeneous response to biostimulation for U(VI) reduction in replicated sediment microcosms

A field-scale experiment to assess biostimulation of uranium reduction is underway at the Natural and Accelerated Bioremediation Research Field Research Center (FRC) in Oak Ridge, Tennessee. To simulate the field experiment, we established replicate batch microcosms containing well-mixed contaminated sediment from a well within the FRC treatment zone, and we added an inoculum from a pilot-scale fluidized bed reactor representing the inoculum in the field experiment. After reduction of nitrate, both sulfate and soluble U(VI) concentration decreased. X-ray absorption near edge structure (XANES) spectroscopy confirmed formation of U(IV) in sediment from biostimulated microcosms, but did not detect reduction of solid-phase Fe(III). Two to three fragments dominated terminal restriction fragment length polymorphism (T-RFLP) profiles of the 16S rDNA gene. Comparison to a clone library indicated these fragments represented denitrifying organisms related to Acidovorax, and Acidovorax isolates from the inoculum were subsequently shown to reduce U(VI). Investigation using the T-RFLP Analysis Program (TAP T-RFLP) and chemical analyses detected the presence and activity of fermenting and sulfate-reducing bacteria after 2 weeks. These organisms likely contributed to uranium reduction. In some microcosms, soluble U(VI) concentration leveled off or rebounded, indicating microbial and/or mineralogical heterogeneity among samples. Sulfate, acetate, and ethanol were depleted only in those microcosms exhibiting a rebound in soluble U(VI). This suggests that rates of U(VI) desorption can exceed rates of U(VI) reduction when sulfate-reducing bacteria become substrate-limited. These observations underscore the importance of effective chemical delivery and the role of serial and parallel processes in uranium reduction.