Immunoproteasomes are essential for clearance of Listeria monocytogenes in nonlymphoid tissues but not for induction of bacteria-specific CD8+ T cells

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8(+) T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8(+) T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8(+) T cell epitopes. I-proteasome-deficient mice (beta5i(-/-) mice) exhibited normal frequencies of L. monocytogenes-specific CD8(+) T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8(+) T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.     

Interactive effects of warming,eutrophication and size structure: impacts on biodiversity and food‐web structure

Warming and eutrophication are two of the most important global change stressors for natural ecosystems, but their interaction is poorly understood. We used a dynamic model of complex, size‐structured food webs to assess interactive effects on diversity and network structure. We found antagonistic impacts: Warming increases diversity in eutrophic systems and decreases it in oligotrophic systems. These effects interact with the community size structure: Communities of similarly sized species such as parasitoid–host systems are stabilized by warming and destabilized by eutrophication, whereas the diversity of size‐structured predator–prey networks decreases strongly with warming, but decreases only weakly with eutrophication. Nonrandom extinction risks for generalists and specialists lead to higher connectance in networks without size structure and lower connectance in size‐structured communities. Overall, our results unravel interactive impacts of warming and eutrophication and suggest that size structure may serve as an important proxy for predicting the community sensitivity to these global change stressors.     

Neonatal outcomes after influenza immunization during pregnancy: a randomized controlled trial

Background:

There are limited data about the effect of maternal influenza infection on fetuses and newborns. We performed a secondary analysis of data from the Mother’s Gift project, a randomized study designed to test the effectiveness of inactivated influenza and pneumococcal vaccines during pregnancy.

Methods:

In the Mother’s Gift project, 340 pregnant women in Bangladesh received either inactivated influenza vaccine or 23-valent pneumococcal polysaccharide vaccine (control). This study was performed from August 2004 through December 2005. We performed a secondary analysis of outcomes following maternal influenza immunization during two periods: when influenza virus was not circulating (September 2004 through January 2005) and when influenza virus was circulating (February through October 2005). We assessed gestational age, mean birth weight and the proportion of infants who were small for gestational age.

Results:

During the period with no circulating influenza virus, there were no differences in the incidence of respiratory illness with fever per 100 person-months among mothers and infants in the two groups (influenza vaccine: 3.9; control: 4.0; p > 0.9). The proportion of infants who were small for gestational age and the mean birth weight were similar between groups (small for gestational age: influenza vaccine 29.1%, control 34.3%; mean birth weight: influenza vaccine 3083 g, control 3053 g). During the period with circulating influenza virus, there was a substantial reduction in the incidence per 100 person-months of respiratory illness with fever among the mothers and infants who had received the influenza vaccine (influenza vaccine: 3.7; control: 7.2; p = 0.0003). During this period, the proportion of infants who were small for gestational age was lower in the influenza vaccine group than in the control group (25.9% v. 44.8%; p = 0.03). The mean birth weight was higher among infants whose mothers received the influenza vaccine than among those who received the control vaccine during this period (3178 g v. 2978 g; p = 0.02).

Interpretation:

During the period with circulating influenza virus, maternal immunization during pregnancy was associated with a lower proportion of infants who were small for gestational age and an increase in mean birth weight. These data need confirmation but suggest that prevention of influenza infection in pregnancy can influence intrauterine growth.

Trial Registration:

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT 00142389","term_id":"NCT00142389"}}NCT 00142389Influenza infection in young infants is common and results in high rates of hospital admission,¹ but infection can be prevented by immunization of the mother during pregnancy.² There are few prospective studies of the effect of antenatal vaccination against influenza on fetal and neonatal outcomes.³ There is conflicting information about the effect of maternal influenza infection on the fetus and newborn,³ though other antepartum maternal infections have well-described adverse effects on the fetus.⁴The Mother’s Gift project is a randomized trial designed to assess the safety and efficacy of maternal pneumococcal and influenza immunization in Bangladesh. The primary outcomes of this study have been reported.² In the current article, we report the results of a secondary analysis to assess the hypothesis that influenza immunization influenza the outcomes of infants whose mothers were exposed to influenza during pregnancy.     

Expression Profiling of Primary and Metastatic Ovarian Tumors Reveals Differences Indicative of Aggressive Disease

The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.     

Calculation of electron paramagnetic resonance spectra from Brownian dynamics trajectories: application to nitroxide side chains in proteins.

We present a method to simulate electron paramagnetic resonance spectra of spin-labeled proteins that explicitly includes the protein structure in the vicinity of the attached spin label. The method is applied to a spin-labeled polyleucine alpha-helix trimer. From short (6 ns) stochastic dynamics simulations of this trimer, an effective potential energy function is calculated. Interaction with secondary and tertiary structures determine the reorientational motion of the spin label side chains. After reduction to a single particle problem, long stochastic dynamic trajectories (700 ns) of the spin label side-chain reorientation are calculated from which the Lamor frequency trajectory and subsequently the electron paramagnetic resonance spectrum is determined. The simulated spectra agree well with experimental electron paramagnetic resonance spectra of bacteriorhodopsin mutants with spin labels in similar secondary and tertiary environments as in the polyleucine.     

Zechstein 2 carbonate platform subfacies and grain-type distribution (Upper Permian,Northwest Germany)

Summary The Upper Permian Zechstein 2 Carbonate (Stassfurt Carbonate, or Ca2) platform facies of Northwest Germany can be subdivided into twelve subfacies types using slabbed cores from fifteen representative wells. Thin section and scanning microscopic analysis further provide subfacies-specific characteristics, based on distribution, size, shape, and spatial arrangement of the grains contained in the different subfacies types. Thirteen grain types can be distinguished within the different subfacies types on the Ca2-platform: 1) one type of oncoid, 2) one type of grapestone, 3) three types of peloids, 4) four types of ooids and 5) four types of aggregate grains. Both presence and composition of grains are indicative of the different subfacies types. There is also a relation between grain composition and porosity of the Ca2-subfacies types. The size and quantity of ooids correlate positively with increasing porosity, whereas an increasing amount of algal structures (algal-lamination) correlates negatively with porosity. The Ca2-platform carbonates almost exclusively represent highstand systems tract and lowstand systems tract deposits. The presence or absence of type-3 aggregate grains within the grainy shoal and algal-laminated shoal subfacies allows the assignment of these subfacies to highstand (grains absent) or lowstand (grains present) systems tracts deposits. The Ca2-highstand deposits can be subdivided into four shallowing-upward parasequences (PS3 to PS7) bounded by parasequence boundaries (PSB3 to PSB6) and Zechstein sequence boundary ZSB4. In contrast to macroscopic core studies, microscopic studies to identify Ca2-subfacies types can utilize cutting material. This allows reconstruction of the subfacies distribution on the Ca2-platform, and delineation of potentially porous zones in uncored Ca2 intervals.     

Light-induced switching of HAMP domain conformation and dynamics revealed by time-resolved EPR spectroscopy

HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain’s dynamics into the current model.     

Time-resolved detection of transient movement of helix F in spin-labelled pharaonis sensory rhodopsin II

Sensory rhodopsin II (also called phoborhodopsin) from the archaeal Natronobacterium pharaonis (pSRII) functions as a repellent phototaxis receptor. The excitation of the receptor by light triggers the activation of a transducer molecule (pHtrII) which has close resemblance to the cytoplasmic domain of bacterial chemotaxis receptors. In order to elucidate the first step of the signal transduction chain, the accessibility as well as static and transient mobility of cytoplasmic residues in helices F and G were analysed by electron paramagnetic resonance spectroscopy. The results indicate an outward tilting of helix F during the early steps of the photocycle which is sustained until the reformation of the initial ground state. Co-expression of pSRII with a truncated fragment of pHtrII affects the accessibility and/or the mobility of certain spin-labelled residues on helices F and G. The results suggest that these sites are located within the binding surface of the photoreceptor with its transducer.     

Evidence for a role of ADAM17 (TACE) in the regulation of platelet glycoprotein V

Glycoprotein V (GPV) is a subunit of the GPIb-IX-V receptor for von Willebrand factor and thrombin and has been shown to modulate platelet responses to the two strongest physiological agonists, thrombin and collagen. Thrombin directly cleaves GPV from the platelet surface, yielding a 69-kDa fragment GPV f1 of unknown function. We show here that a approximately 82-kDa fragment of GPV is shed from the platelet surface upon cellular activation with phorbol 12-myristate 13-acetate or the collagen-related peptide. This shedding was inhibited by the broad range metalloproteinase inhibitor GM6001, the two potent ADAM17 inhibitors GW280264X and TAPI-2, and was absent in mice lacking functional ADAM17 (ADAM17 lacking Zn-binding domain; ADAM17(DeltaZn/DeltaZn)). Furthermore, we show that recombinant ADAM17 ectodomain efficiently releases GPV from the platelet surface. GPV is known to be associated with the intracellular regulatory protein calmodulin, which has previously been shown to be involved in ADAM17-mediated shedding of l-selectin from the surface of leukocytes. As in these reports, inhibition of calmodulin led to rapid GPV shedding from the platelet surface, a process that was again blocked by GM6001 or ADAM17 inhibitors and that was absent in ADAM17(DeltaZn/DeltaZn) mice. Inhibition of outside-in signaling through GPIIb/IIIa did not significantly affect GPV shedding, excluding an essential role of this pathway for the regulation of ADAM17 activity. These results demonstrate that GPV is cleaved upon agonist-induced platelet activation and show that ADAM17 is the major enzyme mediating this process.     

Substrate-Induced Conformational Changes in the S-Component ThiT from an Energy Coupling Factor Transporter

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