Chasing iron bioavailability in the Southern Ocean: Insights from Phaeocystis antarctica and iron speciation.

Dissolved iron (dFe) availability limits the uptake of atmospheric CO2 by the Southern Ocean (SO) biological pump. Hence, any change in bioavailable dFe in this region can directly influence climate. On the basis of Fe uptake experiments with Phaeocystis antarctica, we show that the range of dFe bioavailability in natural samples is wider (<1 to ~200% compared to free inorganic Fe') than previously thought, with higher bioavailability found near glacial sources. The degree of bioavailability varied regardless of in situ dFe concentration and depth, challenging the consensus that sole dFe concentrations can be used to predict Fe uptake in modeling studies. Further, our data suggest a disproportionately major role of biologically mediated ligands and encourage revisiting the role of humic substances in influencing marine Fe biogeochemical cycling in the SO. Last, we describe a linkage between in situ dFe bioavailability and isotopic signatures that, we anticipate, will stimulate future research.

Benthic Dissolved Silicon and Iron Cycling at Glaciated Patagonian Fjord Heads.

Glacier meltwater supplies silicon (Si) and iron (Fe) sourced from weathered bedrock to downstream ecosystems. However, the extent to which these nutrients reach the ocean is regulated by the nature of the benthic cycling of dissolved Si and Fe within fjord systems, given the rapid deposition of reactive particulate fractions at fjord heads. Here, we examine the benthic cycling of the two nutrients at four Patagonian fjord heads through geochemical analyses of sediment pore waters, including Si and Fe isotopes (δ30Si and δ56Fe), and reaction-transport modeling for Si. A high diffusive flux of dissolved Fe from the fjord sediments (up to 0.02 mmol m-2 day-1) compared to open ocean sediments (typically <0.001 mmol m-2 day-1) is supported by both reductive and non-reductive dissolution of glacially-sourced reactive Fe phases, as reflected by the range of pore water δ56Fe (-2.7 to +0.8‰). In contrast, the diffusive flux of dissolved Si from the fjord sediments (0.02-0.05 mmol m-2 day-1) is relatively low (typical ocean values are >0.1 mmol m-2 day-1). High pore water δ30Si (up to +3.3‰) observed near the Fe(II)-Fe(III) redox boundary is likely associated with the removal of dissolved Si by Fe(III) mineral phases, which, together with high sedimentation rates, contribute to the low diffusive flux of Si at the sampled sites. Our results suggest that early diagenesis promotes the release of dissolved Fe, yet suppresses the release of dissolved Si at glaciated fjord heads, which has significant implications for understanding the downstream transport of these nutrients along fjord systems.

Measuring the quality of scientific references in Wikipedia: an analysis of more than 115M citations to over 800 000 scientific articles.

Wikipedia is a widely used online reference work which cites hundreds of thousands of scientific articles across its entries. The quality of these citations has not been previously measured, and such measurements have a bearing on the reliability and quality of the scientific portions of this reference work. Using a novel technique, a massive database of qualitatively described citations, and machine learning algorithms, we analyzed 1 923 575 Wikipedia articles which cited a total of 824 298 scientific articles in our database and found that most scientific articles cited by Wikipedia articles are uncited or untested by subsequent studies, and the remainder show a wide variability in contradicting or supporting evidence. Additionally, we analyzed 51 804 643 scientific articles from journals indexed in the Web of Science and found that similarly most were uncited or untested by subsequent studies, while the remainder show a wide variability in contradicting or supporting evidence.

Age-specific transcriptional response to stroke.

Increased age is a major risk factor for stroke incidence and post-ischemic mortality. To develop age-adjusted therapeutic interventions, a clear understanding of the complexity of age-related post-ischemic mechanisms is essential. Transient occlusion of the middle cerebral artery--a model that closely resembles human stroke--was used to induce cerebral infarction in mice of 4 different ages (2, 9, 15, 24 months). By using Illumina cDNA microarrays and quantitative PCR we detected a distinct age-dependent response to stroke involving 350 differentially expressed genes. Our analyses also identified 327 differentially expressed genes that responded to stroke in an age-independent manner. These genes are involved in different aspects of the inflammatory and immune response, oxidative stress, cell cycle activation and/or DNA repair, apoptosis, cytoskeleton reorganization and/or astrogliosis, synaptic plasticity and/or neurotransmission, and depressive disorders and/or dopamine-, serotonin-, GABA-signaling. In agreement with our earlier work, aged brains displayed an attenuated inflammatory and immune response (Sieber et al., 2011) and a reduced impairment of post-stroke synaptic plasticity. Our data also revealed a distinct age-related susceptibility for post-ischemic depression, the most common neuropsychiatric consequence of stroke, which has a major influence on functional outcome.

Attenuated inflammatory response in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice following stroke.

BACKGROUND: Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear. METHODS AND RESULTS: As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1ß, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed. CONCLUSIONS: Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.

Feeding of the probiotic bacterium Enterococcus faecium NCIMB 10415 differentially affects shedding of enteric viruses in pigs.

Effects of probiotic bacteria on viral infections have been described previously. Here, two groups of sows and their piglets were fed with or without feed supplementation of the probiotic bacterium Enterococcus faecium NCIMB 10415. Shedding of enteric viruses naturally occurring in these pigs was analyzed by quantitative real-time RT-PCR. No differences between the groups were recorded for hepatitis E virus, encephalomyocarditis virus and norovirus. In contrast, astrovirus was exclusively detected in the non-supplemented control group. Rotavirus was shedded later and with lower amounts in the probiotic piglet group (p < 0.05); rotavirus-shedding piglets gained less weight than non-infected animals (p < 0.05). Serum titres of anti-rotavirus IgA and IgG antibodies were higher in piglets from the control group, whereas no difference was detected between sow groups. Phenotype analysis of immune cell antigens revealed significant differences of the CD4 and CD8ß (p < 0.05) as well as CD8α and CD25 (p < 0.1) T cell populations of the probiotic supplemented group compared to the non-supplemented control group. In addition, differences were evident for CD21/MHCII-positive (p < 0.05) and IgM-positive (p < 0.1) B cell populations. The results indicate that probiotic bacteria could have effects on virus shedding in naturally infected pigs, which depend on the virus type. These effects seem to be caused by immunological changes; however, the distinct mechanism of action remains to be elucidated.

Attenuated inflammatory response in aged mice brains following stroke.

BACKGROUND: Increased age is a major risk factor for stroke incidence, post-ischemic mortality, and severe and long-term disability. Stroke outcome is considerably influenced by post-ischemic mechanisms. We hypothesized that the inflammatory response following an ischemic injury is altered in aged organisms. METHODS AND RESULTS: To that end, we analyzed the expression pattern of pro-inflammatory cytokines (TNF, IL-1α, IL-1ß, IL-6), anti-inflammatory cytokines (IL-10, TGFß1), and chemokines (Mip-1α, MCP-1, RANTES) of adult (2 months) and aged (24 months) mice brains at different reperfusion times (6 h, 12 h, 24 h, 2 d, 7 d) following transient occlusion of the middle cerebral artery. The infarct size was assessed to monitor possible consequences of an altered inflammatory response in aged mice. Our data revealed an increased neuro-inflammation with age. Above all, we found profound age-related alterations in the reaction to stroke. The response of pro-inflammatory cytokines (TNF, and IL-1ß) and the level of chemokines (Mip-1α, and MCP-1) were strongly diminished in the aged post-ischemic brain tissue. IL-6 showed the strongest age-dependent decrease in its post-ischemic expression profile. Anti-inflammatory cytokines (TGFß1, and IL-10) revealed no significant age dependency after ischemia. Aged mice brains tend to develop smaller infarcts. CONCLUSION: The attenuated inflammatory response to stroke in aged animals may contribute to their smaller infarcts. The results presented here highlight the importance of using aged animals to investigate age-associated diseases like stroke, and should be considered as a major prerequisite in the development of age-adjusted therapeutic interventions.

Substantial performance discrepancies among commercially available kits for reverse transcription quantitative polymerase chain reaction: a systematic comparative investigator-driven approach.

Reverse transcription followed by quantitative polymerase chain reaction (rt-qPCR) has become the state-of-the-art tool for quantification of nucleic acids. However, there are still significant problems associated with its sensitivity, reproducibility, and efficiency and the choice of an appropriate rt-qPCR kit. The purpose of this article is to give insights into strategies to optimize and validate the performance of currently available kits for rt-qPCR and to provide up-to-date information about the benefits, potentials, and pitfalls of rt-qPCR assays. A selection of 9 complementary DNA (cDNA) synthesis and 12 qPCR kits were tested using samples obtained from three species (mouse, rat, and human) and three transcripts (Gapdh, Actb, and Hmbs) under highly standardized conditions. Kits with outstanding performance were further analyzed to identify the dynamic range for a reliable quantification of messenger RNA (mRNA). Reverse transcription efficiency varied up to 90-fold depending on the choice of reverse transcriptase, priming strategy, and assay volume. The qPCR kit test revealed variations in mean relative amplification efficiency ranging from 54% to 171%. We conclude that currently available kits for rt-qPCR vary considerably. However, with an appropriate validation strategy and knowledge about capabilities of a particular kit, sensitivity, efficiency, and reliability could be improved significantly.

Novel inhibitors of the calcineurin/NFATc hub - alternatives to CsA and FK506?

The drugs cyclosporine A (CsA) and tacrolimus (FK506) revolutionized organ transplantation. Both compounds are still widely used in the clinic as well as for basic research, even though they have dramatic side effects and modulate other pathways than calcineurin-NFATc, too. To answer the major open question - whether the adverse side effects are secondary to the actions of the drugs on the calcineurin-NFATc pathway - alternative inhibitors were developed. Ideal inhibitors should discriminate between the inhibition of (i) calcineurin and peptidyl-prolyl cis-trans isomerases (PPIases; the matchmaker proteins of CsA and FK506), (ii) calcineurin and the other Ser/Thr protein phosphatases, and (iii) NFATc and other transcription factors. In this review we summarize the current knowledge about novel inhibitors, synthesized or identified in the last decades, and focus on their mode of action, specificity, and biological effects.

Inhibition of calcineurin-NFAT signaling by the pyrazolopyrimidine compound NCI3.

Dephosphorylation of NFAT by the Ca(2+)-calmodulin-dependent Ser/Thr protein phosphatase calcineurin is a bottleneck of T cell receptor-dependent activation of T cells. In dimeric complexes with immunophilins, the immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) block this process by inhibition of the enzymatic activity of calcineurin. We have identified the pyrazolopyrimidine compound NCI3 as a novel inhibitor of calcineurin-NFAT signaling. Similar to CsA and FK506, NCI3 inhibits dephosphorylation and nuclear translocation of NFAT, IL-2 production and proliferation of stimulated human primary T cells with IC(50) values from 2 to 4.5 microM. However, contrary to CsA and FK506, NCI3 neither blocks calcineurin;s phosphatase activity nor requires immunophilins for inhibiting NFAT activation. Our data suggest that NCI3 binds to calcineurin and causes an allosteric change interfering with NFAT dephosphorylation in vivo but not in vitro. NCI3 acts not only on the endogenous calcineurin but also on a C-terminally truncated, constitutively active version of calcineurin. The novel inhibitor described herein will be useful in better defining the cellular regulation of calcineurin activation and may serve as a lead for the development of a new type of immunosuppressants acting not by direct inhibition of the calcineurin phosphatase activity.

Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins.

Disintegrins represent a group of disulfide-rich peptides ranging in size from 41 to over 80 residues and are antagonists of several integrin receptors. Disintegrins containing an RGD or KGD sequence are potent inhibitors of platelet aggregation as they block the binding of fibrinogen to alpha(IIb)beta(3) integrin. The high affinity binding to alpha(IIb)beta(3) in comparison to short linear peptides has been attributed to the localisation of the RGD or KGD sequence within a defined three-dimensional structure. Cystine knot microproteins are members of another family of small disulfide-rich peptides that consist of only 28-40 amino acid residues. They display numerous biological activities depending on the peptide sequence of loop regions that are fixed on a structural scaffold that is stabilised by three knot-forming disulfide bonds. In the present study we grafted RGD and KGD containing peptide sequences with seven and 11 amino acids, respectively, into two cystine knot microproteins, the trypsin inhibitor EETI-II and the melanocortin receptor binding domain of the human agouti-related protein AGRP, as well as into the small disintegrin obtustatin. The engineered proteins were much more potent to inhibit the fibrinogen binding, alpha(IIb)beta(3) activation and platelet aggregation when compared to the grafted peptides. Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences.

Transcriptional regulation of ADAMTS13.

The metalloproteinase ADAMTS13 cleaves VWF multimers instantaneously when they are released from endothelial cells. Absent or manifestly diminished proteolytic activity of ADAMTS13 results in the appearance and accumulation of ultralarge VWF multimers (ULVWFM) in plasma, characterised by the manifestation of Thrombotic Thrombocytopenic Purpura (TTP). Despite congenital defects, infections and the actions of drugs such as cyclosporine A, doxycycline and corticosteroids apparently are involved in its development. To examine the possibility of transcriptional regulation of ADAMTS13 activity, we analyzed RNA levels in various cell culture systems and the response to known and assumed modulators of gene expression. We demonstrate the expression of ADAMTS13 in liver homogenates and a parenchyma liver cell culture system Hep3B, supporting the hypothesis that liver is an important source of plasma ADAMTS13, whereas there was no alteration in gene expression after stimulation of liver cells with proinflammatory stimuli such as endotoxin, TNF-alpha, IL-6, IL-1beta as well as immuno-suppressive agents, such as cyclosporine A, a variety of steroids as well as doxycycline. Therefore, we analysed the ADAMTS13 gene for binding sites of transcription factors in silico and compared the data with those found in two sets of 24 genes considered either as differentially regulated by prototypic inflammatory regulation or as unvaried under various conditions. On the basis of these data, the promotor of ADAMTS13 features the characteristics of a gene, which remains unvaried under a variety of conditions. To our knowledge, the current data demonstrate for the first time, that an alteration in transcriptional activity is negligible in accounting for diminished proteolytic activity as observed under various experimental and, in particular, clinical conditions.