ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15−/− macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.     

Oxygen-dependent niche formation of a pyrite-dependent acidophilic consortium built by archaea and bacteria

Biofilms can provide a number of different ecological niches for microorganisms. Here, a multispecies biofilm was studied in which pyrite-oxidizing microbes are the primary producers. Its stability allowed not only detailed fluorescence in situ hybridization (FISH)-based characterization of the microbial population in different areas of the biofilm but also to integrate these results with oxygen and pH microsensor measurements conducted before. The O₂ concentration declined rapidly from the outside to the inside of the biofilm. Hence, part of the population lives under microoxic or anoxic conditions. Leptospirillum ferrooxidans strains dominate the microbial population but are only located in the oxic periphery of the snottite structure. Interestingly, archaea were identified only in the anoxic parts of the biofilm. The archaeal community consists mainly of so far uncultured Thermoplasmatales as well as novel ARMAN (Archaeal Richmond Mine Acidophilic Nanoorganism) species. Inductively coupled plasma analysis and X-ray absorption near edge structure spectra provide further insight in the biofilm characteristics but revealed no other major factors than oxygen affecting the distribution of bacteria and archaea. In addition to catalyzed reporter deposition FISH and oxygen microsensor measurements, microautoradiographic FISH was used to identify areas in which active CO₂ fixation takes place. Leptospirilla as well as acidithiobacilli were identified as primary producers. Fixation of gaseous CO₂ seems to proceed only in the outer rim of the snottite. Archaea inhabiting the snottite core do not seem to contribute to the primary production. This work gives insight in the ecological niches of acidophilic microorganisms and their role in a consortium. The data provided the basis for the enrichment of uncultured archaea.     

Male-Female Differences in Upregulation of Vasoconstrictor Responses in Human Cerebral Arteries

Background and purpose

Male-female differences may significantly impact stroke prevention and treatment in men and women, however underlying mechanisms for sexual dimorphism in stroke are not understood. We previously found in males that cerebral ischemia upregulates contractile receptors in cerebral arteries, which is associated with lower blood flow. The present study investigates if cerebral arteries from men and women differ in cerebrovascular receptor upregulation.

Experimental approach

Freshly obtained human cerebral arteries were placed in organ culture, an established model for studying receptor upregulation. 5-hydroxtryptamine type 1B (5-HT_1B), angiotensin II type 1 (AT₁) and endothelin-1 type A and B (ET_A and ET_B) receptors were evaluated using wire myograph for contractile responses, real-time PCR for mRNA and immunohistochemistry for receptor expression.

Key results

Vascular sensitivity to angiotensin II and endothelin-1 was markedly lower in cultured cerebral arteries from women as compared to men. ET_B receptor-mediated contraction occurred in male but not female arteries. Interestingly, there were similar upregulation in mRNA and expression of 5-HT_1B, AT₁, and ET_B receptors and in local expression of Ang II after organ culture.

Conclusions and Implications

In spite of receptor upregulation after organ culture in both sexes, cerebral arteries from women were significantly less responsive to vasoconstrictors angiotensin II and endothelin-1 as compared to arteries from men. This suggests receptor coupling and/or signal transduction mechanisms involved in cerebrovascular contractility may be suppressed in females. This is the first study to demonstrate sex differences in the vascular function of human brain arteries.     

The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway

The NO reductase from Paracoccus denitrificans reduces NO to N₂O (2NO + 2H⁺ + 2e⁻ → N₂O + H₂O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O₂-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O₂ reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O₂. We further show that electron transfer during single-turnover reduction of O₂ is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.     

Mutations in B3GALNT2 Cause Congenital Muscular Dystrophy and Hypoglycosylation of α-Dystroglycan

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.     

New insights into plastid nucleoid structure and functionality

Investigations over many decades have revealed that nucleoids of higher plant plastids are highly dynamic with regard to their number, their structural organization and protein composition. Membrane attachment and environmental cues seem to determine the activity and functionality of the nucleoids and point to a highly regulated structure–function relationship. The heterogeneous composition and the many functions that are seemingly associated with the plastid nucleoids could be related to the high number of chromosomes per plastid. Recent proteomic studies have brought novel nucleoid-associated proteins into the spotlight and indicated that plastid nucleoids are an evolutionary hybrid possessing prokaryotic nucleoid features and eukaryotic (nuclear) chromatin components, several of which are dually targeted to the nucleus and chloroplasts. Future studies need to unravel if and how plastid–nucleus communication depends on nucleoid structure and plastid gene expression.     

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

Mycobacterium avium subsp. paratuberculosis causes Johne''s disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne''s disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.     

Thermal tolerance,net CO2 exchange and growth of a tropical tree species, Ficus insipida, cultivated at elevated daytime and nighttime temperatures

Global warming and associated increases in the frequency and amplitude of extreme weather events, such as heat waves, may adversely affect tropical rainforest plants via significantly increased tissue temperatures. In this study, the response to two temperature regimes was assessed in seedlings of the neotropical pioneer tree species, Ficus insipida. Plants were cultivated in growth chambers at strongly elevated daytime temperature (39 °C), combined with either close to natural (22 °C) or elevated (32 °C) nighttime temperatures. Under both growth regimes, the critical temperature for irreversible leaf damage, determined by changes in chlorophyll a fluorescence, was approximately 51 °C. This is comparable to values found in F. insipida growing under natural ambient conditions and indicates a limited potential for heat tolerance acclimation of this tropical forest tree species. Yet, under high nighttime temperature, growth was strongly enhanced, accompanied by increased rates of net photosynthetic CO₂ uptake and diminished temperature dependence of leaf-level dark respiration, consistent with thermal acclimation of these key physiological parameters.     

Partitioning of space, habitat, and timing of activity by large felids in an enclosed South African system

Reintroductions of large carnivores into enclosed reserves that confine movements may fail due to intensive intra-guild interspecific conflict. To assess conflict potential, in winter 2006 we used direct observations aided by radio-tracking to focally monitor continuously one female cheetah with cub (Acinonyx jubatus), a female leopard (Panthera pardus), and a lion pride (Panthera leo) at Shamwari Private Game Reserve, South Africa. Home ranges of all individuals/social groups overlapped, whereas core areas had little overlap. The cheetah core area had no overlap with the lion core area, with lion avoidance also recorded for a radio-tracked single female cheetah and a male leopard. The female cheetah with cub selected thicket habitat which was avoided by lions, the latter preferring naturally revegetated areas that were also selected by the female leopard. Lions also selected low elevations, which were avoided by the smaller felids. Habitat preference differences occurred at study area and home range levels, suggesting a broad-scale feline avoidance strategy to minimize intra-guild conflict. In addition, the focally monitored cheetah and leopard were often stationary when the lions were active, especially during nocturnal lion hunts. These intra-guild mechanisms of reintroduced carnivore coexistence should be tested with longer-term studies across enclosed systems of different sizes, and hosting varying carnivore guilds.     

Diagnosis of rheumatoid arthritis: multivariate analysis of biomarkers

Objective. To test if a combination of biomarkers can increase the classification power of autoantibodies to cyclic citrullinated peptides (anti-CCP) in the diagnosis of rheumatoid arthritis (RA) depending on the diagnostic situation. Methods. Biomarkers were subject to three inclusion/exclusion criteria (discrimination between RA patients and healthy blood donors, ability to identify anti-CCP-negative RA patients, specificity in a panel with major non-rheumatological diseases) before univariate ranking and multivariate analysis was carried out using a modelling panel (n=906). To enable the evaluation of the classification power in different diagnostic settings the disease controls (n=542) were weighted according to the admission rates in rheumatology clinics modelling a clinic panel or according to the relative prevalences of musculoskeletal disorders in the general population seen by general practitioners modelling a GP panel. Results. Out of 131 biomarkers considered originally, we evaluated 32 biomarkers in this study, of which only seven passed the three inclusion/exclusion criteria and were combined by multivariate analysis using four different mathematical models. In the modelled clinic panel, anti-CCP was the lead marker with a sensitivity of 75.8% and a specificity of 94.0%. Due to the lack in specificity of the markers other than anti-CCP in this diagnostic setting, any gain in sensitivity by any marker combination is off-set by a corresponding loss in specificity. In the modelled GP panel, the best marker combination of anti-CCP and interleukin (IL)-6 resulted in a sensitivity gain of 7.6% (85.9% vs. 78.3%) at a minor loss in specificity of 1.6% (90.3% vs. 91.9%) compared with anti-CCP as the best single marker. Conclusions. Depending on the composition of the sample panel, anti-CCP alone or anti-CCP in combination with IL-6 has the highest classification power for the diagnosis of established RA.