Chip calorimetry and biomagnetic separation: Fast detection of bacterial contamination at low cell titers

We present a new chip calorimeter for fast and quantitative measurement of metabolic heat rates of microorganisms attached to magnetic beads. In biomagnetic separation (BMS) experiments, Escherichia coli K12 immobilized on nonspecifically functionalized beads has a specific heat rate of around 1 pW per cell at 37°C. Therefore, at least 2 × 10⁴ bacteria are required to exceed the calorimetric signal resolution of 20 nW. If the samples to be analyzed have the original volume of 4 mL, bacteria at less than 10⁴ cells mL^?1 should be detectable. In practice, we achieved the detection of approximately 2 × 10⁴ cells mL^?1. The method presented here might also find some applications in the investigation of biofilms and study of biomolecular interactions.     

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Purpose

Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.

Methods

We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.

Results

The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.

Conclusions

We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.     

On the Effect of Prevalent Carbazole Homocoupling Defects on the Photovoltaic Performance of PCDTBT:PC71BM Solar Cells

The photophysical properties and solar cell performance of the classical donor–acceptor copolymer PCDTBT (poly(N‐9′‐heptadecanyl‐2,7‐carbazole‐alt ‐5,5‐(4′,7′‐di‐2‐thienyl‐2′,1′,3′‐benzothiadiazole))) in relation to unintentionally formed main chain defects are investigated. Carbazole–carbazole homocouplings (Cbz hc) are found to significant extent in PCDTBT made with a variety of Suzuki polycondensation conditions. Cbz hc vary between 0 and 8 mol% depending on the synthetic protocol used, and are quantified by detailed nuclear magnetic resonance spectroscopy including model compounds, which allows to establish a calibration curve from optical spectroscopy. The results are corroborated by extended time‐dependent density functional theory investigations on the structural, electronic, and optical properties of regularly alternating and homocoupled chains. The photovoltaic properties of PCDTBT:fullerene blend solar cells significantly depend on the Cbz hc content for constant molecular weight, whereby an increasing amount of Cbz hc leads to strongly decreased short circuit currents J_SC. With increasing Cbz hc content, J_SC decreases more strongly than the intensity of the low energy absorption band, suggesting that small losses in absorption cannot explain the decrease in J_SC alone, rather than combined effects of a more localized LUMO level on the TBT unit and lower hole mobilities found in highly defective samples. Homocoupling‐free PCDTBT with optimized molecular weight yields the highest efficiency up to 7.2% without extensive optimization.     

The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.     

Growth and protection against oxidative stress in young clones and mature spruce trees (Picea abies L.) at high altitudes

Clones of Norway spruce (Picea abies L.) were grown for several years on an altitudinal gradient (1750 m, 1150 m and 800 m above sea level) to study the effects of environmental × genetic interactions on growth and foliar metabolites (protein, pigments, antioxidants). Clones at the tree line showed 4.3-fold lower growth rates and contained 60% less chlorophyll (per gram of dry matter) than those at valley level. The extent of growth reduction was clone-dependent. The mortality of the clones was low and not altitude-dependent. At valley level, but not at high altitude, needles of mature spruce trees showed lower pigment and protein concentrations than clones. In general, antioxidative systems in needles of the mature trees and young clones did not increase with increasing altitude. Needles of all trees at high altitude showed higher concentrations of dehydroascorbate than at lower altitudes, indicating higher oxidative stress. In one clone, previously identified as sensitive to acute ozone doses, this increase was significantly higher and the growth reduction was stronger than in the other genotypes. This clone also displayed a significant reduction in glutathione reductase activity at high altitude. These results suggest that induction of antioxidative systems is apparently not a general prerequisite to cope with altitude in clones whose mother plants originated from higher altitudes (about 650–1100 m above sea level, Hercycnic-Carpathian distribution area), but that the genetic constitution for maintenance of high antioxidative protection is important for stress compensation at the tree line. Received: 13 October 1998 / Accepted: 22 June 1999     

GENETIC DATA HINT AT A COMMON DONOR REGION FOR INVASIVE ATLANTIC AND PACIFIC POPULATIONS OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA)1

Gracilaria vermiculophylla (Ohmi) Papenf., an agar‐producing red alga introduced from northeast Asia to Europe and North America, is often highly abundant in invaded areas. To assay its genetic diversity and identify the putative source of invasive populations, we analyzed the mitochondrial cytochrome c oxidase subunit I (cox1) gene from 312 individuals of G. vermiculophylla collected in 37 native and 32 introduced locations. A total of 19 haplotypes were detected: 17 in northeast Asia and three in Europe and eastern and western North America, with only one shared among all regions. The shared haplotype was present in all introduced populations and in ～99% of individuals in the introduced areas. This haplotype was also found at three native locations in east Korea, west Japan, and eastern Russia. Both haplotype and nucleotide diversities were extremely low in Europe and North America compared to northeast Asia. Our study indicates that the East Sea/Sea of Japan is a likely donor region of the invasive populations of G. vermiculophylla in the east and west Atlantic and the east Pacific.     

The role of osteopontin (OPN/SPP1) haplotypes in the susceptibility to Crohn's disease

Background

Osteopontin represents a multifunctional molecule playing a pivotal role in chronic inflammatory and autoimmune diseases. Its expression is increased in inflammatory bowel disease (IBD). The aim of our study was to analyze the association of osteopontin (OPN/SPP1) gene variants in a large cohort of IBD patients.

Methodology/Principal Findings

Genomic DNA from 2819 Caucasian individuals (n = 841 patients with Crohn''s disease (CD), n = 473 patients with ulcerative colitis (UC), and n = 1505 healthy unrelated controls) was analyzed for nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138). Considering the important role of osteopontin in Th17-mediated diseases, we performed analysis for epistasis with IBD-associated IL23R variants and analyzed serum levels of the Th17 cytokine IL-22. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. rs4754 was protective in male CD patients (p = 0.0004, OR = 0.69). None of the other investigated OPN SNPs was associated with CD or UC susceptibility. However, several OPN haplotypes showed significant associations with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 (omnibus p-value = 2.07×10⁻⁸). Overall, the mean IL-22 secretion in the combined group of OPN minor allele carriers with CD was significantly lower than that of CD patients with OPN wildtype alleles (p = 3.66×10⁻⁵). There was evidence for weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10⁻²) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10⁻²) but none of these associations remained significant after Bonferroni correction.

Conclusions/Significance

Our study identified OPN haplotypes as modifiers of CD susceptibility, while the combined effects of certain OPN variants may modulate IL-22 secretion.     

IncP-1-beta plasmid pGNB1 isolated from a bacterial community from a wastewater treatment plant mediates decolorization of triphenylmethane dyes

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.