Epinephrine infusion increases adipose interleukin-6 gene expression and systemic levels in humans.

Exercise increases IL-6 mRNA in subcutaneous adipose tissue; however, the immediate signal for the IL-6 induction is unknown. We, therefore, explored the possible role of epinephrine in the induction of IL-6 in adipose tissue. Subcutaneous adipose tissue biopsies and blood samples were obtained from eight healthy men (mean age 27 yr, mean height 184 cm, mean weight 83 kg) in response to epinephrine infusion or in response to saline infusion. The rate of epinephrine infusion was such that circulating epinephrine concentrations mimicked that typically seen during exercise. The level of IL-6 mRNA in subcutaneous adipose tissue increased 26-fold (95% confidence interval, 9- to 166-fold) at 3 h of epinephrine infusion compared with controls (P=0.028). In addition, plasma levels of IL-6 increased in response to epinephrine infusion (P <0.001). However, epinephrine did not affect the IL-6 receptor mRNA. In conclusion, epinephrine acutely increases IL-6 mRNA levels in subcutaneous adipose tissue as well as circulating IL-6 levels in healthy men.     

Alignment-independent comparisons of human gastrointestinal tract microbial communities in a multidimensional 16S rRNA gene evolutionary space

We present a novel approach for comparing 16S rRNA gene clone libraries that is independent of both DNA sequence alignment and definition of bacterial phylogroups. These steps are the major bottlenecks in current microbial comparative analyses. We used direct comparisons of taxon density distributions in an absolute evolutionary coordinate space. The coordinate space was generated by using alignment-independent bilinear multivariate modeling. Statistical analyses for clone library comparisons were based on multivariate analysis of variance, partial least-squares regression, and permutations. Clone libraries from both adult and infant gastrointestinal tract microbial communities were used as biological models. We reanalyzed a library consisting of 11,831 clones covering complete colons from three healthy adults in addition to a smaller 390-clone library from infant feces. We show that it is possible to extract detailed information about microbial community structures using our alignment-independent method. Our density distribution analysis is also very efficient with respect to computer operation time, meeting the future requirements of large-scale screenings to understand the diversity and dynamics of microbial communities.     

Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice

The N -acylethanolamines (NAEs) and 2-arachidonoylglycerol (2-AG) are bioactive lipids that can modulate inflammatory responses and protect neurons against glutamatergic excitotoxicity. We have used a model of focal cerebral ischemia in young adult mice to investigate the relationship between focal cerebral ischemia and endogenous NAEs. Over the first 24 h after induction of permanent middle cerebral artery occlusion, we observed a time-dependent increase in all the investigated NAEs, except for anandamide. Moreover, we found an accumulation of 2-AG at 4 h that returned to basal level 12 h after induction of ischemia. Accumulation of NAEs did not depend on regulation of N -acylphosphatidylethanolamine-hydrolyzing phospholipase D or fatty acid amide hydrolase. Treatment with the fatty acid amide hydrolase inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester; 1 mg/kg; i.p.) 1.5 h before arterial occlusion decreased the infarct volume in our model system. Our results suggest that NAEs and 2-AG may be involved in regulation of neuroprotection during focal cerebral ischemia in mice.     

Distribution of neuropeptides in the primary olfactory center of the heliothine moth <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.     

Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of <6, C-SNARF-4 stained 15 bacterial species frequently isolated from dental biofilm and visualized the entire bacterial biomass in in vivo-grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo-grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.     

Deep‐branching Novel Lineages and High Diversity of Haptophytes in the Skagerrak (Norway) Uncovered by 454 Pyrosequencing

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high‐throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano‐ and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta‐specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep‐branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity.     

A pilot study testing the feasibility of skin temperature monitoring to reduce recurrent foot ulcers in patients with diabetes – a randomized controlled trial

Background

Although monitoring foot skin temperatures has been associated with diabetic foot ulcer recurrence, no studies have been carried out to test the feasibility among European Caucasians. Moreover, the educational and/or motivational models that promote cognitive or psychosocial processes in these studies are lacking. Thus, we conducted a pilot randomized controlled trial to test the feasibility of monitoring foot skin temperatures in combination with theory-based counselling to standard foot care to reduce diabetic foot ulcer recurrence.

Methods

In a single-blinded nurse-led 1-year controlled trial, conducted at a hospital setting in Norway, 41 patients with diabetic neuropathy and previous foot ulcer were randomized to the intervention (n?=?21) or control groups (n?=?20). All participants were instructed in foot care and recording observations daily. Additionally, the intervention group was taught how to monitor and record skin temperature at baseline, and received counselling every third month supporting them to use the new treatment. Subjects observing temperature differences >2.0 °C between corresponding sites on the left and right foot on two consecutive days were asked to contact the study nurse and reduce physical activity. Fisher exact test was used to evaluate the effect of the intervention on the proportion of subjects with a foot ulcer. Kaplan-Meier survival analysis was performed to compare the two groups in regard to the time to development of a foot ulcer.

Results

In the intervention group, 67 % (n?=?14/21) monitored and recorded skin temperatures ≥80 % of the time while 70 % (n?=?14/20) of the controls recorded foot inspections. Foot ulcer incidence was 39 % (7/21) vs. 50 % (10/20) in the intervention and control groups, respectively (ns).

Conclusions

This feasibility study showed that the addition of counselling to promote self-monitoring of skin temperature to standard care to prevent recurrence of foot ulcer is feasible in patients with diabetes in Norway. Home skin temperature monitoring was performed as frequently by the intervention group as usual foot observations in the controls despite the extra effort required. We did not detect a difference in foot ulcer recurrence between groups, but our study may inform future full scale studies.

Trial registration

Clinicaltrials.gov NCT01269502

     

The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1

The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH₂]-Ser(Bz)-X_aa-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived β-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K_i-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [¹⁴C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.     

Lack of CCR7 induces pulmonary hypertension involving perivascular leukocyte infiltration and inflammation

The chemokine receptor CCR7 regulates lymphocyte trafficking, and CCR7 deficiency induces infiltration of T and B cells adjacent to vessels in mouse lungs. Perivascular infiltration of T and B cells has also been found in human pulmonary arterial hypertension, and downregulation of the CCR7 receptor in circulating leukocytes of such patients has been observed. To investigate whether changes in the CCR7 system contribute to the pathogenesis of pulmonary hypertension, we utilized mice deficient of the CCR7 receptor. The cardiopulmonary and inflammatory responses of CCR7 depletion were evaluated in CCR7-deficient and wild-type mice. Measurements of cytokines upregulated in the animal model were also performed in patients with pulmonary hypertension and controls and in vascular smooth muscle cells. We found that mice lacking CCR7 had increased right ventricular systolic pressure, reduced pulmonary artery acceleration time, increased right ventricular/tibial length ratio, Rho kinase-mediated pulmonary vasoconstriction, and increased muscularization of distal arteries, indicating pulmonary hypertension. These mice also showed increased perivascular infiltration of leukocytes, consisting mainly of T and B cells, and increased mRNA levels of the inflammatory cytokines interleukin-12 and CX3CL1 within pulmonary tissue. Increased serum levels of interleukin-12 and CX3CL1 were also observed in patients with pulmonary hypertension, particularly in those with pulmonary hypertension associated with connective tissue disorder. In smooth muscle cells, interleukin-12 induced secretion of the angiogenic cytokine interleukin-8. We conclude that these results suggest a role for CCR7 in the development of pulmonary arterial hypertension, at least in some subgroups, possibly via pulmonary infiltration of lymphocytes and secretion of interleukin-12 and CX3CL1.