Quantitative full-colour transmitted light microscopy and dyes for concentration mapping and measurement of diffusion coefficients in microfluidic architectures

International audienceA simple and versatile methodology has been developed for the simultaneous measurement of multiple concentration profiles of colourants in transparent microfluidic systems, using a conventional transmitted light microscope, a digital colour (RGB) camera and numerical image processing combined with multicomponent analysis. Rigorous application of the Beer-Lambert law would require monochromatic probe conditions, but in spite of the broad spectral bandwidths of the three colour channels of the camera, a linear relation between the measured optical density and dye concentration is established under certain conditions. An optimised collection of dye solutions for the quantitative optical microscopic characterisation of microfluidic devices is proposed. Using the methodology for optical concentration measurement we then implement and validate a simplified and robust method for the microfluidic measurement of diffusion coefficients using an H-filter architecture. It consists of measuring the ratio of the concentrations of the two output channels of the H-filter. It enables facile determination of the diffusion coefficient, even for non-fluorescent molecules and nanoparticles, and is compatible with non-optical detection of the analyte

Fluorescence correlation spectroscopy reveals strong fluorescence quenching of FITC adducts on PEGylated gold nanoparticles in water and the presence of fluorescent aggregates of desorbed thiolate ligands

Colloidal gold particles functionalised with oligoethylene-glycolated disulfide ligands and fluorescent moieties derived from fluorescein isothiocyanate (FITC) have been prepared and studied in aqueous suspension using fluorescence correlation spectroscopy (FCS). FCS probes the dynamics of the particles at the single object level, and reveals the desorption of fluorescent ligands which subsequently aggregate into larger (slower diffusing) objects. Cross-correlation spectroscopy of the FITC fluorescence and the Rayleigh-Mie scattering (RM-FCCS) of the gold cores shows that the only detectable fluorescent objects are free ligands and aggregates not associated with a gold particle. The fluorescence of bound fluorophores is quenched making their fluorescence too weak to be detected. FCS and RM-FCCS are useful tools for characterising functionalised noble metal particles in solution, under conditions similar to those used in optical bio-imaging. Desorption of thiolates from gold nanoparticles needs to be taken into account when working with these materials at low concentration

Structural and Luminescence Properties of Silica-Based Hybrids Containing New Silylated-Diketonato Europium(III) Complex

A new betadiketonate ligand displaying a trimethoxysilyl group as grafting function and a diketone moiety as complexing site (TTA-Si = 4,4,4-trifluoro-2-(3-trimethoxysilyl)propyl)-1-3-butanedione (C4H3S)COCH[(CH2)3Si(OCH3)3]COCF3) and its highly luminescent europium(III) complex [Eu(TTA-Si)3] have been synthesized and fully characterized. Luminescent silica-based hybrids have been prepared as well with this new complex grafted on the surface of dense silica nanoparticles (28 (+/-3 nm) or on mesoporous silica particles. The covalent bonding of Eu(TTA-Si)3 inside the core of uniform silica nanoparticles (40 (+/- 5 nm) was also achieved. Luminescence properties are discussed in relation to the europium chemical environment involved in each of the three hybrids. The general methodology proposed allowed high grafting ratios and overcame chelate release and tendency to agglomeration, and it could be applied to any silica matrix (in the core or at the surface, nanosized or not, dense or mesoporous) and therefore numerous applications such as luminescent markers and luminophors could be foreseen

Optical extinction and scattering cross sections of plasmonic nanoparticle dimers in aqueous suspension

Absolute extinction and scattering cross sections for gold nanoparticle dimers were determined experimentally using a chemometric approach involving singular-value decomposition of the extinction and scattering spectra of slowly aggregating gold nanospheres in aqueous suspension. Quantitative spectroscopic data on plasmonic nanoparticle assemblies in liquid suspension are rare, in particular for particles larger than 40 nm, and in this work we demonstrate how such data can be obtained directly from the aggregating suspension. Our method can analyse, non invasively, the evolution of several sub-populations of nanoparticle assemblies. It may be applied to other self-assembling nanoparticle systems with an evolving optical response. The colloidal systems studied here are based on 20, 50 and 80 nm gold nanospheres in aqueous solutions containing sodium lipoate. In these systems, the reversible dimerisation process can be controlled using pH and ionic strength, and this control is rationalised in terms of DLVO theory. The dimers were identified in suspension by their translational and rotational diffusion through scattering correlation spectroscopy. Moreover, their gigadalton molecular weight was measured using electrospray charge-detection mass spectrometry, demonstrating that mass spectrometry can be used to study nanoparticles assemblies of very high molecular mass. The extinction and scattering cross sections calculated in the discrete-dipole approximation (DDA) agree very well with those obtained experimentally using our approach

Chemoinformatic-guided engineering of polyketide synthases

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A central challenge in PKS design is replacing a partially reductive module with a fully reductive module through a reductive loop exchange, thereby generating a saturated β-carbon. In this work, we sought to establish an engineering strategy for reductive loop exchanges based on chemoinformatics, a field traditionally used in drug discovery. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). These results demonstrated that chemical similarity between the substrate of the donor loops and recipient LipPKS1 correlated with product titers. Consequently, we identified donor loops with substrates chemically similar to LipPKS1 for further reductive loop exchanges, and we observed a statistically significant correlation with production. Reductive loops with the highest chemical similarity resulted in production of branched, short-chain fatty acids reaching a titer of 165 mg/L in Streptomyces albus J1074. Collectively, our work formulizes a new chemoinformatic paradigm for de novo PKS biosynthesis which may accelerate the production of valuable bioproducts

Examining provider perceptions and practices for comprehensive geriatric assessment among cancer survivors: a qualitative study with an implementation science focus

Introduction: Cancer rates increase with age, and older cancer survivors have unique medical care needs, making assessment of health status and identification of appropriate supportive resources key to delivery of optimal cancer care. Comprehensive geriatric assessments (CGAs) help determine an older person’s functional capabilities as cancer care providers plan treatment and follow-up care. Despite its proven utility, research on implementation of CGA is lacking.Methods: Guided by a qualitative description approach and through interviews with primary care providers and oncologists, our goal was to better understand barriers and facilitators of CGA use and identify training and support needs for implementation. Participants were identified through Cancer Prevention and Control Research Network partner listservs and a national cancer and aging organization. Potential interviewees, contacted via email, were provided with a description of the study purpose. Eight semi-structured interviews were conducted via Zoom, recorded, and transcribed verbatim by a professional transcription service. The interview guide explored providers’ knowledge and use of CGAs. For codebook development, three representative transcripts were independently reviewed and coded by four team members. The interpretive process involved reflecting, transcribing, coding, and searching for and identifying themes.Results: Providers shared that, while it would be ideal to administer CGAs with all new patients, they were not always able to do this. Instead, they used brief screening tools or portions of CGAs, or both. There was variability in how CGA domains were assessed; however, all considered CGAs useful and they communicated with patients about their benefits. Identified facilitators of implementation included having clinic champions, an interdisciplinary care team to assist with implementation and referrals for intervention, and institutional resources and buy-in. Barriers noted included limited staff capacity and competing demands on time, provider inexperience, and misaligned institutional priorities.Discussion: Findings can guide solutions for improving the broader and more systematic use of CGAs in the care of older cancer patients. Uptake of processes like CGA to better identify those at risk of poor outcomes and intervening early to modify treatments are critical to maximize the health of the growing population of older cancer survivors living through and beyond their disease

Chemoinformatic-Guided Engineering of Polyketide Synthases.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated β-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates  into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products

Mycoplasma hyorhinis-contaminated cell lines activate primary innate immune cells via a protease-sensitive factor

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments

Venous Graft-Derived Cells Participate in Peripheral Nerve Regeneration

Background: Based on growing evidence that some adult multipotent cells necessary for tissue regeneration reside in the walls of blood vessels and the clinical success of vein wrapping for functional repair of nerve damage, we hypothesized that the repair of nerves via vein wrapping is mediated by cells migrating from the implanted venous grafts into the nerve bundle. Methodology/Principal Findings: To test the hypothesis, severed femoral nerves of rats were grafted with venous grafts from animals of the opposite sex. Nerve regeneration was impaired when decellularized or irradiated venous grafts were used in comparison to untreated grafts, supporting the involvement of venous graft-derived cells in peripheral nerve repair. Donor cells bearing Y chromosomes integrated into the area of the host injured nerve and participated in remyelination and nerve regeneration. The regenerated nerve exhibited proper axonal myelination, and expressed neuronal and glial cell markers. Conclusions/Significance: These novel findings identify the mechanism by which vein wrapping promotes nerve regeneration. © 2011 Lavasani et al