Investigation of optical attenuation imaging using optical coherence tomography for monitoring of scars undergoing fractional laser treatment

We demonstrate the use of the near‐infrared attenuation coefficient, measured using optical coherence tomography (OCT), in longitudinal assessment of hypertrophic burn scars undergoing fractional laser treatment. The measurement method incorporates blood vessel detection by speckle decorrelation and masking, and a robust regression estimator to produce 2D en face parametric images of the attenuation coefficient of the dermis. Through reliable co‐location of the field of view across pre‐ and post‐treatment imaging sessions, the study was able to quantify changes in the attenuation coefficient of the dermis over a period of ～20 weeks in seven patients. Minimal variation was observed in the mean attenuation coefficient of normal skin and control (untreated) mature scars, as expected. However, a significant decrease (13 ± 5%, mean ± standard deviation) was observed in the treated mature scars, resulting in a greater distinction from normal skin in response to localized damage from the laser treatment. By contrast, we observed an increase in the mean attenuation coefficient of treated (31 ± 27%) and control (27 ± 20%) immature scars, with numerical values incrementally approaching normal skin as the healing progressed. This pilot study supports conducting a more extensive investigation of OCT attenuation imaging for quantitative longitudinal monitoring of scars.

En face 2D OCT attenuation coefficient map of a treated immature scar derived from the pre‐treatment (top) and the post‐treatment (bottom) scans. (Vasculature (black) is masked out.) The scale bars are 0.5 mm.     

Synthesis of indirubin-N'-glycosides and their anti-proliferative activity against human cancer cell lines

The first indirubin-N'-glycosides were prepared based on reactions of isatin-N'-glycosides with indoxyls. The products show a significant anti-proliferative activity against various human cancer cell lines. Good results were observed for an indirubin-N'-mannoside which was shown to have medium to high anti-proliferative activity against all investigated cell lines. The highest activities and selectivities against the MCF-7 breast cancer cell line were observed for indirubin-N'-rhamnosides.     

Community dynamics within a bacterial consortium during growth on toluene under sulfate-reducing conditions

A sulfate-reducing bacterial consortium was enriched from an anoxic aquifer contaminated with BTEX compounds, using toluene as a growth substrate. Total cell counts, protein contents and sulfide production were determined to follow growth at the in situ temperature (14 °C) and at 25 °C, respectively. Community members were identified by 16S rRNA gene cloning and sequencing. Phylogenetic analysis revealed 12 sequence types belonging to Deltaproteobacteria (several groups) , Epsilonproteobacteria, Bacteroidetes, Spirochaetaceae and an unclassified bacterial clade. The most prominent phylotype comprising 34% of all clones was affiliated to the Desulfobulbaceae and closely related to environmental clones retrieved from hydrocarbon-contaminated aquifers. Flow-cytometric methods were applied to analyze the community dynamics and to identify key organisms involved in toluene assimilation. Flow-cytometric measurement of DNA contents and scatter behavior served to detect and quantify dominant and newly emerging clusters of subcommunities. Up to seven subcommunities, two of them dominant, were distinguished. Cell sorting was used to facilitate the analysis of conspicuous clusters for phylogenetic identity by terminal restriction fragment length polymorphism profiling of the 16S rRNA genes. The Desulfobulbaceae phylotype accounted for up to 87% in proliferating subcommunities, indicating that it represents the key organism of toluene degradation within this complex anaerobic consortium.     

Detection of single quantum dots in model organisms with sheet illumination microscopy

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 μm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.     

Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.     

Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation

Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre-treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin-binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up-regulated gelsolin messenger RNA and protein expression in a dose- and time-dependent manner. Double-label confocal immunocytochemistry visualized the up-regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up-regulation, TSA pre-treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.     

Phylogenetic and Functional Diversity Within Toluene-Degrading,Sulphate-Reducing Consortia Enriched from a Contaminated Aquifer

Three toluene-degrading microbial consortia were enriched under sulphate-reducing conditions from different zones of a benzene, toluene, ethylbenzene and xylenes (BTEX) plume of two connected contaminated aquifers. Two cultures were obtained from a weakly contaminated zone of the lower aquifer, while one culture originated from the highly contaminated upper aquifer. We hypothesised that the different habitat characteristics are reflected by distinct degrader populations. Degradation of toluene with concomitant production of sulphide was demonstrated in laboratory microcosms and the enrichment cultures were phylogenetically characterised. The benzylsuccinate synthase alpha-subunit (bssA) marker gene, encoding the enzyme initiating anaerobic toluene degradation, was targeted to characterise the catabolic diversity within the enrichment cultures. It was shown that the hydrogeochemical parameters in the different zones of the plume determined the microbial composition of the enrichment cultures. Both enrichment cultures from the weakly contaminated zone were of a very similar composition, dominated by Deltaproteobacteria with the Desulfobulbaceae (a Desulfopila-related phylotype) as key players. Two different bssA sequence types were found, which were both affiliated to genes from sulphate-reducing Deltaproteobacteria. In contrast, the enrichment culture from the highly contaminated zone was dominated by Clostridia with a Desulfosporosinus-related phylotype as presumed key player. A distinct bssA sequence type with high similarity to other recently detected sequences from clostridial toluene degraders was dominant in this culture. This work contributes to our understanding of the niche partitioning between degrader populations in distinct compartments of BTEX-contaminated aquifers.     

A kinetic model for simultaneous saccharification and fermentation of Avicel with Saccharomyces cerevisiae

This work describes a numerical model for predicting simultaneous saccharification and fermentation of Avicel, an insoluble crystalline cellulose polymer. Separate anoxic cultivations of 40 g/L glucose and 100 g/L Avicel were conducted to verify model predictions and obtain parameters to describe the reaction kinetics. Saccharification of Avicel was achieved with Trichoderma reesei cellulases from the enzyme preparation Spezyme CP with an enzyme loading of 10 FPU/g cellulose. Cultivations were supplemented with 50 IU/g cellulose of β‐glucosidase from Novozym 188 to prevent product inhibition by cellobiose. Saccharomyces cerevisiae MH‐1000 is a robust industrial strain and was used to ferment glucose to ethanol, glycerol, and carbon dioxide. The numerical model presented in this paper differs from previous models by separating the endoglucanase and exoglucanase enzyme kinetics and allowing for inhibitive site competition. Assuming all enzymes remain active and that each enzyme complex has a corresponding constant specific activity, the model is capable of predicting adsorbed enzyme concentrations with reasonable accuracy. Comparison of predicted values to experimental measurements indicated that the numerical model was capable of capturing the significant elements involved with cellulose conversion to ethanol. Biotechnol. Bioeng. 2011; 108:924–933. © 2010 Wiley Periodicals, Inc.