A divergent transcriptional landscape underpins the development and functional branching of MAIT cells.

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing-based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 "mature" MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ-producing MAIT1 cells and interleukin-17-producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)-deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.

Evidence for shared broad-scale climatic niches of diploid and polyploid plants.

Whole-genome duplication (polyploidy) occurs frequently and repeatedly within species, often forming new lineages that contribute to biodiversity, particularly in plants. Establishment and persistence of new polyploids may be thwarted by competition with surrounding diploids; however, climatic niche shifts, where polyploids occupy different niches than diploid progenitors, may help polyploids overcome this challenge. We tested for climatic niche shifts between cytotypes using a new ordination approach and an unprecedentedly large data set containing young, conspecific diploids and polyploids. Despite expectations of frequent niche shifts, we show evidence for alternative patterns, such as niche conservatism and contraction, rather than a prevalent pattern of niche shifts. In addition, we explore how interpreting climatic niches plotted on environmental niche (principal component) axes can generate hypotheses about processes underlying niche dynamics. Dispersal capabilities or other life-history traits, rather than shifts to new climatic niches, could better explain polyploid persistence in the long term.

Considerations for the processing and analysis of GoldenGate-based two-colour Illumina platforms.

Illumina's GoldenGate technology is a two-channel microarray platform that allows for the simultaneous interrogation of about 1,500 locations in the genome. GoldenGate has proved a flexible platform not only in the choice of those 1,500 locations, but also in the choice of the property being measured at them. It retains the desirable properties of Illumina's BeadArrays in that the probes (in this case 'beads') are randomly arranged across the microarray, there are multiple instances of each probe and many samples can be processed simultaneously. As for other Illumina technologies, however, these properties are not exploited as they might be. Here we review the various common adaptations of the GoldenGate platform, review the analysis methods that are associated with each adaptation and then, with the aid of a number of example data sets we illustrate some of the improvements that can be made over the default analysis.

BASH: a tool for managing BeadArray spatial artefacts.

SUMMARY: With their many replicates and their random layouts, Illumina BeadArrays provide greater scope fordetecting spatial artefacts than do other microarray technologies. They are also robust to artefact exclusion, yet there is a lack of tools that can perform these tasks for Illumina. We present BASH, a tool for this purpose. BASH adopts the concepts of Harshlight, but implements them in a manner that utilizes the unique characteristics of the Illumina technology. Using bead-level data, spatial artefacts of various kinds can thus be identified and excluded from further analyses. AVAILABILITY: The beadarray Bioconductor package (version 1.10 onwards), www.bioconductor.org

Functional and metabolic remodelling in GLUT4-deficient hearts confers hyper-responsiveness to substrate intervention.

Impaired glucose uptake is associated with both cardiac hypertrophy and contractile dysfunction, but whether there are common underlying mechanisms linking these conditions is yet to be determined. Using a 'gene dose' Cre-Lox GLUT4-deficient murine model, we examined the effect of suppressed glucose availability on global myocardial gene expression and glycolysis substrate bypass on the function of isolated perfused hearts. Performance of hearts from 22- to 60-week-old male GLUT4 knockout (KO, >95% reduction in GLUT4), GLUT4 knockdown (KD, 85% reduction in cardiac GLUT4) and C57Bl/6 wild-type (WT) controls was measured ex vivo in Langendorff mode perfusion. DNA microarray was used to profile mRNA expression differences between GLUT4-KO and GLUT4-KD hearts. At 22 weeks, GLUT4-KO hearts exhibited cardiac hypertrophy and impaired contractile function ex vivo, characterized by a 40% decrease in developed pressure. At 60 weeks, dysfunction was accentuated in GLUT4-KO hearts and evident in GLUT4-KD hearts. Exogenous pyruvate (5 mM) restored systolic pressure to a level equivalent to WT (GLUT4-KO, 176.8+/-13.2 mmHg vs. WT, 146.4+/-9.56 mmHg) in 22-week-old GLUT4-KO hearts but not in 60-week-old GLUT4-KO hearts. In GLUT4-KO, DNA microarray analysis detected downregulation of a number of genes centrally involved in mitochondrial oxidation and upregulation of other genes indicative of a shift to cytosolic beta-oxidation of long chain fatty acids. A direct link between cardiomyocyte GLUT4 deficiency, hypertrophy and contractile dysfunction is demonstrated. These data provide mechanistic insight into the myocardial metabolic adaptations associated with short and long-term insulin resistance and indicate a window of opportunity for substrate intervention and functional 'rescue'.

Effects of macrophyte species richness on wetland ecosystem functioning and services.

Wetlands provide many important ecosystem services to human society, which may depend on how plant diversity influences biomass production and nutrient retention. Vascular aquatic plant diversity may not necessarily enhance wetland ecosystem functioning, however, because competition among these plant species can be strong, often resulting in the local dominance of a single species. Here we have manipulated the species richness of rooted, submerged aquatic plant (macrophyte) communities in experimental wetland mesocosms. We found higher algal and total plant (algal plus macrophyte) biomass, as well as lower loss of total phosphorus, in mesocosms with a greater richness of macrophyte species. Greater plant biomass resulted from a sampling effect; that is, the increased chance in species mixtures that algal production would be facilitated by the presence of a less competitive species-in this case, crisped pondweed. Lower losses of total phosphorus resulted from the greater chance in species mixtures of a high algal biomass and the presence of sago pondweed, which physically filter particulate phosphorus from the water. These indirect and direct effects of macrophyte species richness on algal production, total plant biomass and phosphorus loss suggest that management practices that maintain macrophyte diversity may enhance the functioning and associated services of wetland ecosystems.

Three separate coronary artery ostia arising from the right coronary cusp: a case report.

Coronary artery anomalies are uncommon in the general population and most are asymptomatic. We report a rare congenital coronary anomaly and discuss diagnosis and management strategies as well as review the pertinent literature.

Refractory no-reflow successfully treated with local infusion of high-dose adenosine and verapamil--a case report.

No-reflow is an unpredictable complication following percutaneous coronary intervention. No-reflow is associated with myocardial ischemia and infarction and increased mortality. A case of refractory no-reflow is described that was rapidly and successfully treated with multiple infusions of high doses of verapamil and adenosine applied directly at the site of no-reflow through a perfusion catheter.

Incidence of thrombocytopenia following coronary stent placement using abciximab plus clopidogrel or ticlopidine.

The incidence of thrombocytopenia with ticlopidine and clopidogrel when used in conjunction with abciximab has not been systematically addressed. We evaluated the rate of thrombocytopenia in patients undergoing intracoronary stent implantation receiving bolus plus infusion of abciximab and either ticlopidine or clopidogrel. We noted an incidence of 24% with the combination of 300-mg clopidogrel and abciximab. Other doses of ticlopidine (250 and 500 mg) and clopidogrel (75 mg) did not result in a statistically significant increase in thrombocytopenia over that of the 2.5%-5.2% reported incidence with abciximab alone. Length of hospital stay was 2.3 vs. 6.4 days in those developing thrombocytopenia (P = 0.06). Four (25%) developed thrombocytopenia requiring blood transfusion. Eight (50%) had no sequelae. The combination of 300-mg clopidogrel and abciximab results in a significant increase in the incidence of thrombocytopenia. This is an important clinical observation that merits further study.

Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo.

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation appear important in conferring hypertrophy in vitro. However, the response of PKC and MAP kinase to stimuli known to induce hypertrophy in vivo has not been determined. We recently demonstrated that pressure-overload hypertrophy induced a transiently transfected gene driven by an hypertrophy responsive enhancer (HRE) through a marked increase in binding activity of its interacting nuclear factor (HRF). These data suggested that the HRE/HRF could serve as a target for evaluating the signal transduction events responsible for hypertrophy in vivo. Accordingly, we characterized MAP kinase and PKC isoform activation, injected HRE driven reporter gene expression, and HRF binding activity in rat hearts subjected to ascending aortic clipping or sham operation in the presence of the angiotensin-converting enzyme (ACE) inhibitor fosinopril, hydralazine, or no treatment. Analyses showed that PKC-epsilon and MAP kinase were acutely activated following ascending aortic ligature and that fosinopril significantly inhibited but did not completely abrogate PKC-epsilon and MAP kinase activation. However, fosinopril completely prevented pressure overload-mediated induction of HRE containing constructs and obviated increased HRF binding activity. These results suggest a direct relationship between ACE activity and HRE/HRF-mediated gene activation and imply that PKC-epsilon and MAP kinase may be involved in transducing this signal.

Spatial scaling laws yield a synthetic theory of biodiversity.

Ecologists still search for common principles that predict well-known responses of biological diversity to different factors. Such factors include the number of available niches in space, productivity, area, species' body size and habitat fragmentation. Here we show that all these patterns can arise from simple constraints on how organisms acquire resources in space. We use spatial scaling laws to describe how species of different sizes find food in patches of varying size and resource concentration. We then derive a mathematical rule for the minimum similarity in size of species that share these resources. This packing rule yields a theory of species diversity that predicts relations between diversity and productivity more effectively than previous models. Size and diversity patterns for locally coexisting East African grazing mammals and North American savanna plants strongly support these predictions. The theory also predicts relations between diversity and area and between diversity and habitat fragmentation. Thus, spatial scaling laws provide potentially unifying first principles that may explain many important patterns of species diversity.

Effects of plant species richness on invasion dynamics, disease outbreaks, insect abundances and diversity.

Declining biodiversity represents one of the most dramatic and irreversible aspects of anthropogenic global change, yet the ecological implications of this change are poorly understood. Recent studies have shown that biodiversity loss of basal species, such as autotrophs or plants, affects fundamental ecosystem processes such as nutrient dynamics and autotrophic production. Ecological theory predicts that changes induced by the loss of biodiversity at the base of an ecosystem should impact the entire system. Here we show that experimental reductions in grassland plant richness increase ecosystem vulnerability to invasions by plant species, enhance the spread of plant fungal diseases, and alter the richness and structure of insect communities. These results suggest that the loss of basal species may have profound effects on the integrity and functioning of ecosystems.

Nuclear factor-kappaB is selectively and markedly activated in humans with unstable angina pectoris.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) resides inactive in the cytoplasm of lymphocytes, monocytes, endothelial cells, and smooth muscle cells, where, after stimulation, it transcriptionally activates interleukins, interferon, tumor necrosis factor-alpha, and adhesion molecules. Because acute inflammation may play a role in coronary artery plaque rupture, it was hypothesized that NF-kappaB activation correlated with coronary artery disease (CAD) activity. METHODS AND RESULTS: Evidence of NF-kappaB activation in the circulation of 102 consecutive patients without an acute myocardial infarction who were undergoing cardiac catheterization was determined. Of these, 19 had unstable angina (USA) and were within 24 hours of the last episode of chest pain. The remaining 83 were being evaluated for stable angina (53), valvular heart disease (8), atypical chest pain (12), or congestive heart failure (10). Evidence of NF-kappaB activation was determined by electromobility shift assays (EMSAs) with the NF-kappaB binding-site-specific probe and nuclear proteins isolated from the buffy coat of blood obtained at the beginning of the procedure. Specificity of this DNA-protein interaction was confirmed by competition and supershift EMSAs. Analyses showed that 17 of 19 patients with USA had marked activation of NF-kappaB. Despite a significant number of patients with severe CAD (69%), only 2 of the 83 without USA showed marked NF-kappaB activation. A lack of NF-kappaB activation was not due to a lack of functional cell/protein because NF-kappaB was appropriately activated by lipopolysaccharide ex vivo in all patients. NF-kappaB activation was not a nonspecific response of all transcription factors because neither Sp1 or Oct1 was activated in patients with activated NF-kappaB. There was no relationship between drugs used, hemodynamic status, or other clinical characteristics and state of NF-kappaB activation. CONCLUSIONS: These data show that NF-kappaB is specifically and significantly activated in unstable angina pectoris and is not affected by severity of CAD or medical therapy. Furthermore, because NF-kappaB is activated before a clinical event, it may be mechanistically involved in the plaque disruption that produces acute coronary artery syndromes.

Use of transesophageal echocardiography to detect unsuspected massive pulmonary emboli.

This report describes three patients in whom unsuspected large central pulmonary emboli were identified by transesophageal echocardiography. We discuss the utility and limitations of transesophageal echocardiography in diagnosing pulmonary emboli and its potential beneficial impact on the management of patients in the intensive care unit, particularly those with unexplained hypotension.

Pseudo-pseudoaneurysm of the left ventricle.

Left ventricular pseudoaneurysms are an uncommon complication of myocardial infarction and need urgent surgical repair. Though it is critical that they be accurately identified, pseudoaneurysms are occasionally misdiagnosed. We report an abnormality which may be mislabeled a pseudoaneurysm which we term a pseudo-pseudoaneurysm. The approach to accurate diagnosis of pseudoaneurysms is discussed.

Ergonovine-testing-directed therapy and long-term outcome of sudden-death survivors with no apparent heart disease.

Coronary artery vasospasm, the most common cause of sudden death in patients with structurally normal hearts, is not well recognized. We describe 2 survivors of sudden death with ergonovine-inducible coronary artery vasospasm successfully treated long term with calcium channel blockers. Of the reported (17 worldwide) patients with documented vasospasm-mediated sudden cardiac death treated with calcium blockers, none have had a recurrent event. This is substantially less than the 6-month sudden-death rate of 18 % of untreated vasospasm. We advocate that ergonovine provocative testing of sudden-death survivors with structurally normal hearts, followed by appropriate therapy with calcium channel blockers, be the standard approach for these highly treatable patients.

Effects of herbivores on grassland plant diversity.

The role of herbivores in controlling plant species richness is a critical issue in the conservation and management of grassland biodiversity. Numerous field experiments in grassland plant communities show that herbivores often, but not always, increase plant diversity. Recent work suggests that the mechanisms of these effects involve alteration of local colonization of species from regional species pools or local extinction of species, and recent syntheses and models suggest that herbivore effects on plant diversity should vary across environmental gradients of soil fertility and precipitation.

bFGF induces BCK promoter-driven expression in muscle via increased binding of a nuclear protein.

Changes in gene expression occurring during skeletal muscle differentiation are exemplified by downregulation of brain creatine kinase (BCK) and induction of muscle creatine kinase (MCK). Although both are transcriptionally regulated, there appears to be no transcription factor-element overlap, suggesting that their coordinate expression results from culture medium-related influences. Basic fibroblast growth factor (bFGF) prevents myogenesis and represses MCK expression by inhibiting transcriptional activation. It was hypothesized that bFGF similarly influenced BCK by inducing its expression. Accordingly, BCK promoter constructs were transiently transfected into C2C12 cells and, after a switch to differentiation medium, were treated with bFGF, bFGF plus herbimycin, adenosine 3',5'-cyclic monophosphate (cAMP), or phorbol 12-myristate 13-acetate (PMA). Analyses demonstrated that bFGF responsiveness was contained within a 33-base pair element. Electromobility shift assays showed that bFGF induction increased the abundance of the nuclear factor binding the element. Both effects were prevented by herbimycin. Neither cAMP nor PMA specifically induced the construct containing the bFGF-responsive element. The induced factor required phosphorylation to bind, implying that bFGF-mediated increases in binding may be due to transcription factor phosphorylation.

Characterization of human B creatine kinase gene regulation in the heart in vitro and in vivo.

During cardiogenesis, genes indicative of the adult phenotype are transcriptionally activated while genes characteristic of the embryonic phenotype are down-regulated. The regulation of embryonic genes such as the brain isoform of creatine kinase (BCK) during cardiac development has not been characterized. Accordingly, the transcriptional regulation of BCK in the developing heart was determined. In vitro and in vivo promoter analyses of the human BCK gene identified an element between +25 and +57 that functioned as an enhancer. Electromobility shift assays using adult and neonatal nuclear extracts identified a specific complex binding this element, the abundance of which correlated with the developmental level of endogenous cardiac BCK expression. Mutations at +47 and +53 led to a loss of activity in transfected cells and obviated binding in electromobility shift assays. These data show that a nuclear factor in cardiocytes interacts with an enhancer element (+25 and +57), via nucleotides +47 and +53, to drive BCK expression in the heart and suggest that developmental BCK expression is via abundance of this factor. The nuclear factor has not been identified but as described previously binding sites are not present in the enhancer, it is either a known factor interacting with a new recognition site or a new factor.

Human B creatine kinase gene expression in C2C12 cells is regulated by protein interactions involving the first exon.

While the activation of muscle specific genes is well characterized, the mechanism mediating the regulation during muscle development of embryonic genes is poorly understood. To begin to investigate this area, the transcriptional regulation of the human brain creatine kinase (BCK) gene was characterized during in vitro myogenesis of C2C12 muscle cells. Initial analyses identified the first exon as important for high level expression in general and for the decrease in expression in response to C2C12 differentiation. In vivo competition confirmed the functional importance of the first exon. Electrophoretic mobility shift assays using portions of the exon showed that two separate proteins bound to sequences +1 to +27 and +25 to +57, respectively. The +1 to +27 interacting factor is present in skeletal but not cardiac muscle and thus may function as a skeletal muscle determining factor. The +25 to +57 interacting factor functions as a positive effector in myoblasts. This region also imparts the differentiation dependent decrease in expression via either modification of the myoblast factor or due to the presence of an additional factor. Site directed mutagenesis show that base pair +26 is critical for appropriate down regulation of BCK.