Fluorescence image-guided brain tumour resection with adjuvant metronomic photodynamic therapy: pre-clinical model and technology development.

Fluorescence-guided resection (FGR) and photodynamic therapy (PDT) have previously been investigated separately with the objectives, respectively, of increasing the extent of brain tumour resection and of selectively destroying residual tumour post-resection. Both techniques have demonstrated trends towards improved survival, pre-clinically and clinically. We hypothesize that combining these techniques will further delay tumour re-growth. In order to demonstrate technical feasibility, we here evaluate fluorescence imaging and PDT treatment techniques in a specific intracranial tumour model. The model was the VX2 carcinoma grown by injection of tumour cells into the normal rabbit brain. An operating microscope was used for white light imaging and a custom-built fluorescence imaging system with co-axial excitation and detection was used for FGR. PDT treatment light was applied by intracranially-implanted light emitting diodes (LED). The fluorescent photosensitizer used for both FGR and PDT was ALA-induced PpIX. For PDT, ALA (100 mg kg(-1)) and low light doses (15 and 30 J) were administered over extended periods, which we refer to as metronomic PDT (mPDT). Eighteen tumour bearing rabbits were divided equally into three groups: controls (no resection); FGR; and FGR followed by mPDT. Histological whole brain sections (H&E stain) showed primary and recurrent tumours. No bacteriological infections were found by Gram staining. Selective tumour cell death through mPDT-induced apoptosis was demonstrated by TUNEL stain. These results demonstrate that the combined treatment is technically feasible and this model is a candidate to evaluate it. Further optimization of mPDT treatment parameters (drug/light dose rates) is required to improve survival.     

Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns: an ecological perspective

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes--while playing an essential role in fern colonization and reproduction--generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology.     

Overexpression of the Endothelial Protein C Receptor Is Detrimental during Pneumonia-Derived Gram-negative Sepsis (Melioidosis)

Background

The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia.

Methodology/Principal Findings

Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR^−/−). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR^−/− mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma.

Conclusion/Significance

Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.     

Fern Spore Longevity in Saline Water: Can Sea Bottom Sediments Maintain a Viable Spore Bank?

Freshwater and marine sediments often harbor reservoirs of plant diaspores, from which germination and establishment may occur whenever the sediment falls dry. Therewith, they form valuable records of historical inter- and intraspecific diversity, and are increasingly exploited to facilitate diversity establishment in new or restored nature areas. Yet, while ferns may constitute a considerable part of a vegetation’s diversity and sediments are known to contain fern spores, little is known about their longevity, which may suffer from inundation and - in sea bottoms - salt stress. We tested the potential of ferns to establish from a sea or lake bottom, using experimental studies on spore survival and gametophyte formation, as well as a spore bank analysis on sediments from a former Dutch inland sea. Our experimental results revealed clear differences among species. For Asplenium scolopendrium and Gymnocarpium dryopteris, spore germination was not affected by inundated storage alone, but decreased with rising salt concentrations. In contrast, for Asplenium trichomanes subsp. quadrivalens germination decreased following inundation, but not in response to salt. Germination rates decreased with time of storage in saline water. Smaller and less viable gametophytes were produced when saline storage lasted for a year. Effects on germination and gametophyte development clearly differed among genotypes of A. scolopendrium. Spore bank analyses detected no viable spores in marine sediment layers. Only two very small gametophytes (identified as Thelypteris palustris via DNA barcoding) emerged from freshwater sediments. Both died before maturation. We conclude that marine, and likely even freshwater sediments, will generally be of little value for long-term storage of fern diversity. The development of any fern vegetation on a former sea floor will depend heavily on the deposition of spores onto the drained land by natural or artificial means of dispersal.     

Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

Background

Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells.

Methodology/Principal Findings

The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength.

Conclusions/Significance

We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.     

Population admixture,biological invasions and the balance between local adaptation and inbreeding depression

When previously isolated populations meet and mix, the resulting admixed population can benefit from several genetic advantages, including increased genetic variation, the creation of novel genotypes and the masking of deleterious mutations. These admixture benefits are thought to play an important role in biological invasions. In contrast, populations in their native range often remain differentiated and frequently suffer from inbreeding depression owing to isolation. While the advantages of admixture are evident for introduced populations that experienced recent bottlenecks or that face novel selection pressures, it is less obvious why native range populations do not similarly benefit from admixture. Here we argue that a temporary loss of local adaptation in recent invaders fundamentally alters the fitness consequences of admixture. In native populations, selection against dilution of the locally adapted gene pool inhibits unconstrained admixture and reinforces population isolation, with some level of inbreeding depression as an expected consequence. We show that admixture is selected against despite significant inbreeding depression because the benefits of local adaptation are greater than the cost of inbreeding. In contrast, introduced populations that have not yet established a pattern of local adaptation can freely reap the benefits of admixture. There can be strong selection for admixture because it instantly lifts the inbreeding depression that had built up in isolated parental populations. Recent work in Silene suggests that reduced inbreeding depression associated with post-introduction admixture may contribute to enhanced fitness of invasive populations. We hypothesize that in locally adapted populations, the benefits of local adaptation are balanced against an inbreeding cost that could develop in part owing to the isolating effect of local adaptation itself. The inbreeding cost can be revealed in admixing populations during recent invasions.     

Herbivore-associated elicitors: FAC signaling and metabolism

The recognition of insect and pathogen attack requires the plant's ability to perceive chemical cues generated by the attacker. In contrast to the recognition of microbe-associated molecular patterns and effectors, little is known about the molecular recognition of herbivore-associated elicitors (HAEs) and the signaling mechanisms operating in plants after their perception. HAE perception depends strongly on the natural history of both plants and insects and it is therefore expected that many of the responses induced by different HAEs are specific to the species involved in the interaction. The interaction between Nicotiana attenuata and the specialist lepidopteran Manduca sexta presents a relevant biological system to understand HAE perception and signal transduction systems in plants.     

Complexities of Particulate Matter Measurement in Parenteral Formulations of Small-Molecule Amphiphilic Drugs

Reconstituted parenteral solutions of three surface-active anti-infective small-molecule drugs and solutions of sodium dodecyl sulfate (SDS, a model surfactant) were studied to quantify the impact of sample preparation and handling on particle counts. Turbidimetry and light obscuration profiles were recorded as a function of agitation and shearing with and without the introduction of foam into the solutions. SDS solutions at concentrations above the critical micelle concentration (CMC) show significantly greater sensitivity to shear and foam presence than SDS solution below the CMC: Values of >10 μm particles increased 8 fold over control (an unsheared sample) in the micellar solution vs. 4 fold particle count increase over control at a sub-micellar concentration. An even more significant increase in the ratio of particle count in sheared/unsheared solution is seen for >25 μm unit counts, due to the increased interference of foam with the measurement. Two commercial products, injection formulations of teicoplanin and cefotaxime sodium, as well as an investigational compound 1, showed an increase in scattering as a function of foam production. The impact of foaming was significant, resulting in an increase of turbidity and light obscuration measurements in all solutions. The results illustrate some of the challenges that are inherent to optically clear, homogeneous pharmaceutical injections containing compounds which have a tendency toward self-association and surfactant-like behavior.