Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopy was used to quantitate changes in gap junctions. We found that at 30 days post-LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post-LPS. We conclude that a single LPS injection exerts long-term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia.     

IL-12 induction of mRNA encoding substance P in murine macrophages from the spleen and sites of inflammation

Substance P (SP), a neuropeptide, interacts with the neurokinin 1 receptor (NK-1R) on immune cells to help control IFN-gamma production. In murine schistosomiasis mansoni, schistosome worms produce ova that incite focal Th2-type granulomatous inflammation within the liver and intestines. Normal gut is characterized by a controlled state of inflammation. IL-10 knockout mice develop chronic Th1-type colitis spontaneously. Both schistosome granulomas and gut mucosa display an SP immune regulatory circuit. However, the origin and regulation of SP production at these sites of inflammation are poorly understood. Macrophages are a potential source of SP. We therefore studied macrophages (F4/80(+)) from these models of inflammation. SP mRNA (preprotachykinin A (PPT A)) was detected within the schistosome granuloma, spleen, and lamina propria macrophages. Compared with those from wild-type mice, granuloma macrophages from STAT6(-/-) mice had 10-fold higher PPT A mRNA expression, whereas in STAT4(-/-) animals, PPT A mRNA expression was nearly abolished. IL-12 signals via STAT4 to induce Th1-type inflammation. It was demonstrated that IL-12, but not IL-18, induces SP mRNA expression in resting splenic macrophages from Schistosoma-infected mice and in wild-type lamina propria mononuclear cells. Thus, macrophages are a source for SP at these sites of chronic inflammation, and IL-12 and STAT4 are regulators of macrophage SP mRNA expression.     

Transcriptional profiling of CD133(+) cells in coronary artery disease and effects of exercise on gene expression

Background aimsBone marrow (BM)-derived progenitor cells are under investigation for cardiovascular repair but may be altered by disease. Our aim was to identify differences in gene expression in CD133⁺ cells of patients with coronary artery disease (CAD) and healthy controls, and determine whether exercise modifies gene expression.MethodsCD133⁺ cells were flow-sorted from 10 CAD patients and four controls, and total RNA was isolated for microarray-based gene expression profiling. Genes that were found to be differentially regulated in patients were analyzed further to investigate whether exercise had any normalizing effect on CD133⁺ cells in CAD patients following 3 months of an exercise program.ResultsImprovement in effort tolerance and increases in the number of CD133⁺ cells were observed in CAD patients after 3 months of exercise. Gene expression analysis of the CD133⁺ cells identified 82 differentially expressed genes (2-fold cut-off, 25% false-discovery rate and % present calls) in patients compared with controls, of which 59 were found to be up-regulated and 23 down-regulated. These genes were found to be involved in carbohydrate metabolism, cell cycle, cellular development and signaling, and molecular transport. Following completion of the exercise program, gene expression patterns resembled those of controls in seven of 10 patients.ConclusionsAlterations in gene expression of BM-derived CD133⁺ progenitor cells were found in CAD patients, which in part may be normalized by exercise.     

Efficacy,Safety, and Dose of Pafuramidine,a New Oral Drug for Treatment of First Stage Sleeping Sickness,in a Phase 2a Clinical Study and Phase 2b Randomized Clinical Studies

Background

Sleeping sickness (human African trypanosomiasis [HAT]) is caused by protozoan parasites and characterized by a chronic progressive course, which may last up to several years before death. We conducted two Phase 2 studies to determine the efficacy and safety of oral pafuramidine in African patients with first stage HAT.

Methods

The Phase 2a study was an open-label, non-controlled, proof-of-concept study where 32 patients were treated with 100 mg of pafuramidine orally twice a day (BID) for 5 days at two trypanosomiasis reference centers (Angola and the Democratic Republic of the Congo [DRC]) between August 2001 and November 2004. The Phase 2b study compared pafuramidine in 41 patients versus standard pentamidine therapy in 40 patients. The Phase 2b study was open-label, parallel-group, controlled, randomized, and conducted at two sites in the DRC between April 2003 and February 2007. The Phase 2b study was then amended to add an open-label sequence (Phase 2b-2), where 30 patients received pafuramidine for 10 days. The primary efficacy endpoint was parasitologic cure at 24 hours (Phase 2a) or 3 months (Phase 2b) after treatment completion. The primary safety outcome was the rate of occurrence of World Health Organization Toxicity Scale Grade 3 or higher adverse events. All subjects provided written informed consent.

Findings/Conclusion

Pafuramidine for the treatment of first stage HAT was comparable in efficacy to pentamidine after 10 days of dosing. The cure rates 3 months post-treatment were 79% in the 5-day pafuramidine, 100% in the 7-day pentamidine, and 93% in the 10-day pafuramidine groups. In Phase 2b, the percentage of patients with at least 1 treatment-emergent adverse event was notably higher after pentamidine treatment (93%) than pafuramidine treatment for 5 days (25%) and 10 days (57%). These results support continuation of the development program for pafuramidine into Phase 3.     

Nuclear magnetic resonance evidence for a structural intermediate at an early stage in the refolding of ribonuclease A

The kinetics of refolding of heat-unfolded ribonuclease A have been studied by Fourier transform proton nuclear magnetic resonance at 10 °C, pH 2. A single refolding reaction is observed: it corresponds to the slow-refolding reaction seen in stopped-flow studies of refolding at higher temperatures. There are two results of interest for the mechanism of protein folding. (1) A new resonance (X) is observed that shows the presence of a structural intermediate in refolding. (2) The α-helix close to the N-terminal end of ribonuclease A apparently forms rapidly when the unfolded protein is brought to refolding conditions.The folding intermediate has been studied by monitoring the C-2 protons of the four histidine residues. The intermediate contains one residue (X) in a partly folded environment and the other three residues in unfolded environments. The composite resonance (U) of these three protons at 10 °C agrees with the average chemical shift of the histidine residues in heat-unfolded ribonuclease A at high temperatures. During refolding at 10 °C, the resonance intensities of U and X disappear at the same rate that the spectrum of native ribonuclease A is regained.Partial deuteration experiments show that X is either histidine 12 or 119. Comparative studies of the amino-terminal fragment 1–20 of ribonuclease A indicate that X is histidine 12. The appearance of structure in this peptide can be followed by temperature-dependent changes in the chemical shift of histidine 12. At 10 °C the chemical shifts of histidine 12 and X agree closely. These results are consistent with the circular dichroism study of peptide 1–13 by Brown &; Klec (1971), who concluded that helix formation occurs at low temperatures.     

Symmetry breakage in the vertebrate embryo: When does it happen and how does it work?

Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.     

Metabolism of viprostol — A synthetic vasolidator PGE2 analog

Metabolic studies were done with ¹⁴C-Viprostol (I) administered by various routes (I.V., oral and topical) to six animal species and to man. Total radioactivity and metabolic profiles were analyzed in plasma, tissues and excreta. The main metabolites were isolated and identified by capillary GC/MS.Plasma and urinary metabolic profiles were qualitatively similar across species, with two major metabolic reactions being predominant: rapid hydrolysis to the pharmacologically active free acid (II)_ and oxidation of the alpha-chain to dinor and tetranor acids (III, IV). In the monkey and man, reduction of the 9-keto group lead to PGF₂ type metabolites (VI–VIII). In the rat, omega oxidation of the beta-chain occurred as well, resulting in the formation of dicarboxylic acids (V).     

Desulfohalophilus alkaliarsenatis gen. nov., sp. nov., an extremely halophilic sulfate- and arsenate-respiring bacterium from Searles Lake, California

A haloalkaliphilic sulfate-respiring bacterium, strain SLSR-1, was isolated from a lactate-fed stable enrichment culture originally obtained from the extreme environment of Searles Lake, California. The isolate proved capable of growth via sulfate-reduction over a broad range of salinities (125-330?g/L), although growth was slowest at salt-saturation. Strain SLSR-1 was also capable of growth via dissimilatory arsenate-reduction and displayed an even broader range of salinity tolerance (50-330?g/L) when grown under these conditions. Strain SLSR-1 could also grow via dissimilatory nitrate reduction to ammonia. Growth experiments in the presence of high borate concentrations indicated a greater sensitivity of sulfate-reduction than arsenate-respiration to this naturally abundant anion in Searles Lake. Strain SLSR-1 contained genes involved in both sulfate-reduction (dsrAB) and arsenate respiration (arrA). Amplicons of 16S rRNA gene sequences obtained from DNA extracted from Searles Lake sediment revealed the presence of close relatives of strain SLSR-1 as part of the flora of this ecosystem despite the fact that sulfate-reduction activity could not be detected in situ. We conclude that strain SLSR-1 can only achieve growth via arsenate-reduction under the current chemical conditions prevalent at Searles Lake. Strain SLSR-1 is a deltaproteobacterium in the family Desulfohalobiacea of anaerobic, haloalkaliphilic bacteria, for which we propose the name Desulfohalophilus alkaliarsenatis gen. nov., sp. nov.     

A promising genomic transfectant into Xeroderma pigmentosum group A with highly amplified mouse DNA and intermediate UV resistance turns revertant

Following transfection of genomic mouse DNA into an SV40 transformed fibroblast cell line from a patient with Xeroderma pigmentosum (complementation group A, XPA), a single UV resistant cell clone was isolated out of a total of 10(4) independent transfectants. The recipient XPA cell line has as yet not produced spontaneous revertants among 2.2 x 10(8) cells. The isolated cell clone contains 50-70 kb of mouse sequences which are heavily amplified (500-fold), and has acquired both intermediate resistance to UV killing and intermediate unscheduled DNA synthesis (UDS) capacity. By continued passage without selective pressure, cells were generated, which had lost both the dominant marker gene and repetitive mouse sequences. Single colonies of these cells were still intermediately resistant to UV suggesting that either undetected unique mouse DNA had segregated from the bulk of repetitive DNA, or, more likely, that the initially isolated transfectant was a spontaneous revertant. This documents that a persuasive clone isolated can still be a false positive (spontaneous revertant) and that an extremely laborious approach may lead into a dead end.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.