Ten years of life in compost: temporal and spatial variation of North German Caenorhabditis elegans populations

The nematode Caenorhabditis elegans is a central laboratory model system in almost all biological disciplines, yet its natural life history and population biology are largely unexplored. Such information is essential for in‐depth understanding of the nematode's biology because its natural ecology provides the context, in which its traits and the underlying molecular mechanisms evolved. We characterized natural phenotypic and genetic variation among North German C. elegans isolates. We used the unique opportunity to compare samples collected 10 years apart from the same compost heap and additionally included recent samples for this and a second site, collected across a 1.5‐year period. Our analysis revealed significant population genetic differentiation between locations, across the 10‐year time period, but for only one location a trend across the shorter time frame. Significant variation was similarly found for phenotypic traits of likely importance in nature, such as choice behavior and population growth in the presence of pathogens or naturally associated bacteria. Phenotypic variation was significantly influenced by C. elegans genotype, time of isolation, and sampling site. The here studied C. elegans isolates may provide a valuable, genetically variable resource for future dissection of naturally relevant gene functions.     

Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols

Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP(ow) (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of > or =2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested P. putida DOT-T1E tolerance to different aliphatic alcohols with a logP(ow) value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP(ow) around 1.5 are produced. P. putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow P. putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP(ow) of aliphatic alcohols correlates with their toxic effects, as octanol (logP(ow) = 2.9) has more negative effects in P. putida cells than 1-nonanol (logP(ow) = 3.4) or 1-decanol (logP(ow) = 4). A P. putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP(ow) = 3.2) into 3-methylcatechol (logP(ow) = 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.     

R-wave synchronised atrial pacing in pediatric patients with postoperative junctional ectopic tachycardia: the atrioventricular interval investigated by computational analysis and clinical evaluation

Background

R-wave synchronised atrial pacing is an effective temporary pacing therapy in infants with postoperative junctional ectopic tachycardia. In the technique currently used, adverse short or long intervals between atrial pacing and ventricular sensing (AP–VS) may be observed during routine clinical practice.

Objectives

The aim of the study was to analyse outcomes of R-wave synchronised atrial pacing and the relationship between maximum tracking rates and AP–VS intervals.

Methods

Calculated AP–VS intervals were compared with those predicted by experienced pediatric cardiologist.

Results

A maximum tracking rate (MTR) set 10 bpm higher than the heart rate (HR) may result in undesirable short AP–VS intervals (minimum 83 ms). A MTR set 20 bpm above the HR is the hemodynamically better choice (minimum 96 ms). Effects of either setting on the AP–VS interval could not be predicted by experienced observers. In our newly proposed technique the AP–VS interval approaches 95 ms for HR > 210 bpm and 130 ms for HR < 130 bpm. The progression is linear and decreases strictly (? 0.4 ms/bpm) between the two extreme levels.

Conclusions

Adjusting the AP–VS interval in the currently used technique is complex and may imply unfavorable pacemaker settings. A new pacemaker design is advisable to allow direct control of the AP–VS interval.

     

Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering

The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates.     

Characterization of Early Disease Status in Treatment-Naive Male Paediatric Patients with Fabry Disease Enrolled in a Randomized Clinical Trial

Trial Design

This analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.

Methods

Males aged 5–18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13–17 years), renal function, and glycolipid levels (plasma, urine).

Results

Plasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m² (range 90.4–161.0 mL/min/1.73 m²) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0–27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.

Conclusions

These data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.

Trial Registration

ClinicalTrials.gov NCT00701415     

Homing of intravenously and intralymphatically injected human dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells

Dendritic cells (DC) are professional antigen-presenting cells that can be generated in vitro from CD34⁺ peripheral blood progenitor cells by recombinant cytokines. These cells have potential implications for immunotherapeutic approaches in the treatment of cancer and other diseases. Physiologically, immature DC in the periphery capture and process antigens, then mature to interdigitating DC and migrate to lymphoid organs, where they activate lymphocytes. However, it is not known if DC generated in vitro have the capacity to traffic in vivo to the lymphoid tissues, such as spleen and lymph nodes. We have investigated whether human radiolabeled DC differentiated in vitro migrate and localize to lymphoid tissues after intravenous and intralymphatic injection. The distribution and localization of the DC were evaluated in five patients with malignant melanoma using serial whole-body gamma camera imaging. Intravenously infused DC demonstrated transient lung uptake followed by localization in the spleen and liver for at least 7 days. DC injected into a lymphatic vessel at the dorsal foot were rapidly detected in the draining lymph nodes where they remained for more than 24 h. These data suggest that DC differentiated in vitro localize preferentially to lymphoid tissue, where they could induce specific immune responses. Received: 28 January 1999 / Accepted: 4 March 1999     

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

Increased concentrations of circulating vitamin E in carriers of the apolipoprotein A5 gene - 1131T>C variant and associations with plasma lipids and lipid peroxidation

The aim of this study was to investigate the effects of the apolipoprotein A5 (APOA5) 1131T>C gene variant on vitamin E status and lipid profile. The gene variant was determined in 297 healthy nonsmoking men aged 20-75 years and recruited in the VITAGE Project. Effects of the genotype on vitamin E in plasma, LDL, and buccal mucosa cells (BMC) as well as on cholesterol and triglyceride (TG) concentrations in plasma and apolipoprotein A-I (apoA-I), apoB, apoE, apoC-III, and plasma fatty acids were determined. Plasma malondialdehyde concentrations as a marker of in vivo lipid peroxidation were determined. C allele carriers showed significantly higher TG, VLDL, and LDL in plasma, higher cholesterol in VLDL and intermediate density lipoprotein, and higher plasma fatty acids. Plasma alpha-tocopherol (but not gamma-tocopherol, LDL alpha- and gamma-tocopherol, or BMC total vitamin E) was increased significantly in C allele carriers compared with homozygote T allele carriers (P = 0.02), but not after adjustment for cholesterol or TG. Plasma malondialdehyde concentrations did not differ between genotypes. In conclusion, higher plasma lipids in the TC+CC genotype are efficiently protected against lipid peroxidation by higher alpha-tocopherol concentrations. Lipid-standardized vitamin E should be used to reliably assess vitamin E status in genetic association studies.