Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.¹In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.^2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.     

Protection of melanized Cryptococcus neoformans from lethal dose gamma irradiation involves changes in melanin's chemical structure and paramagnetism

Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans), which uses L-3,4-dihydroxyphenylalanine (L-DOPA) to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50 to 80% of the cells did not lead to a detectable loss of melanin integrity according to EPR spectra of melanin radicals. Moreover, upon UV-visible (Xe-lamp) illumination of melanized cells, the increase in radical population was unchanged after γ-irradiation. Gamma-irradiation of frozen cell suspensions and storage of samples for several days at 77 K however, produced melanin modification noted by a reduced radical population and reduced photoresponse. More direct evidence for structural modification of melanin came from the detection of soluble products with absorbance maxima near 260 nm in supernatants collected after γ-irradiation of cells and cell-free melanin. These products, which include thiobarbituric acid (TBA)-reactive aldehydes, were also generated by Fenton reagent treatment of cells and cell-free melanin. In an assay of melanin integrity based on the metal (Bi(+3)) binding capacity of cells, no detectable loss in binding was detected after γ-irradiation. Our results show that melanin in C. neoformans cells is susceptible to some damage by hydroxyl radical formed in lethal radioactive aqueous environments and serves a protective role in melanized fungi that involves sacrificial breakdown.     

Phage display library derived peptides that bind to human tumor melanin as potential vehicles for targeted radionuclide therapy of metastatic melanoma

Metastatic melanoma remains an incurable disease, and there is a great need for novel therapeutic modalities. We have recently identified melanin as a target for radionuclide therapy of melanoma and demonstrated the feasibility of this approach using a 188-rhenium ( (188)Re)-radiolabeled melanin-binding decapeptide to fungal melanin known as 4B4. Although the results indicated that radiolabeled melanin-binding decapeptide had activity against melanoma, that peptide also manifested high kidney uptake and this might become a concern during clinical trials. We hypothesized that by identifying peptides with different amino acid composition against tumor melanin we might be able to decrease their kidney uptake. Using the Heptapeptide Ph.D.-7 Phage Display Library, we identified three heptapeptides that bind to human tumor melanin. These peptides were radiolabeled with (188)Re via HYNIC ligand, and their comprehensive biodistribution in A2058 human metastatic melanoma tumor-bearing nude mice was compared to that of (188)Re-4B4 decapeptide. While tumor uptake of heptapeptides was quite similar to that of (188)Re-4B4 decapeptide, there was dramatically less uptake in the kidneys at both 3 h (6% ID/g vs 38%) and 24 h (2% ID/g vs 15%) postinjection. Administration of one of the generated heptapeptides, (188)Re-HYNIC-AsnProAsnTrpGlyProArg, to A2058 human metastatic melanoma-bearing nude mice resulted in significant retardation of the tumor growth. Immunofluorescence showed that in spite of their relatively small size heptapeptides were not able to penetrate through the membranes of viable melanoma cells and bound only to extracellular melanin, which provides assurance that they will be safe to healthy melanin-containing tissues during radionuclide therapy. Thus, these heptapeptides appear to have potentially significant advantages for targeted therapy of melanoma relative to existing melanin-binding peptides.     

Platelet-activating Factor Contributes to Bacillus anthracis Lethal Toxin-associated Damage

The lethal toxin (LeTx) of Bacillus anthracis plays a central role in the pathogenesis of anthrax-associated shock. Platelet-activating factor (PAF) is a potent lipid mediator that has been implicated in endotoxin-associated shock. In this study, we examined the contribution of PAF to the manifestations of lethal toxin challenge in WT mice. LeTx challenge resulted in transient increase in serum PAF levels and a concurrent decrease in PAF acetylhydrolase activity. Inhibition of PAF activity using PAF antagonists or toxin challenge of PAF receptor negative mice reversed or ameliorated many of the pathologic features of LeTx-induced damage, including changes in vascular permeability, hepatic necrosis, and cellular apoptosis. In contrast, PAF inhibition had minimal effects on cytokine levels. Findings from these studies support the continued study of PAF antagonists as potential adjunctive agents in the treatment of anthrax-associated shock.     

A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.     

Toward Developing a Universal Treatment for Fungal Disease Using Radioimmunotherapy Targeting Common Fungal Antigens

Background

Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall.

Methods

MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth (²¹³Bi) using the chelator CHXA”. B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium (¹⁸⁸Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls.

Results

²¹³Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80–100%. The ²¹³Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing.

Conclusion

Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

     

Fc gamma receptors regulate immune activation and susceptibility during Mycobacterium tuberculosis infection

The critical role of cellular immunity during tuberculosis (TB) has been extensively studied, but the impact of Abs upon this infection remains poorly defined. Previously, we demonstrated that B cells are required for optimal protection in Mycobacterium tuberculosis-infected mice. FcgammaR modulate immunity by engaging Igs produced by B cells. We report that C57BL/6 mice deficient in inhibitory FcgammaRIIB (RIIB-/-) manifested enhanced mycobacterial containment and diminished immunopathology compared with wild-type controls. These findings corresponded with enhanced pulmonary Th1 responses, evidenced by increased IFN-gamma-producing CD4+ T cells, and elevated expression of MHC class II and costimulatory molecules B7-1 and B7-2 in the lungs. Upon M. tuberculosis infection and immune complex engagement, RIIB-/- macrophages produced more of the p40 component of the Th1-promoting cytokine IL-12. These data strongly suggest that FcgammaRIIB engagement can dampen the TB Th1 response by attenuating IL-12p40 production or activation of APCs. Conversely, C57BL/6 mice lacking the gamma-chain shared by activating FcgammaR had enhanced susceptibility and exacerbated immunopathology upon M. tuberculosis challenge, associated with increased production of the immunosuppressive cytokine IL-10. Thus, engagement of distinct FcgammaR can divergently affect cytokine production and susceptibility during M. tuberculosis infection.     

Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.     

The rocker bone: a new kind of mineralised tissue?

In some Ophidiiform fishes, the anterior part of the swimbladder is thickened into a hard structure called the "rocker bone", which is thought to play a role in sound production. Although this structure has been described as cartilage or bone, its nature is still unknown. We have made a thorough analysis of the rocker bone in Ophidion barbatum and compared it with both classical bone and cartilage. The rocker bone appears to be a new example of mineralisation. It consists of (1) a ground substance mainly composed of proteoglycans (mucopolysaccharide acid) and fibres and (2) a matrix containing small mineralised spherules composed of a bioapatite and fibrils. These spherules are embedded in mineralised cement of a similar composition to the spherules themselves. The rocker bone grows via the apposition of new apatite spherules at its periphery. These spherules are first secreted by the innermost fibroblast layer of the capsule contained in the rocker bone and then grow extracellularly. Blood vessels, which represent the only means of transport for matrix and mineral material, are numerous. They enter the rocker bone via the hyle and ramify towards the capsule. We propose to call this new kind of mineralised tissue constituting the rocker bone "frigolite" (the Belgian name for styrofoam) in reference to the presence of spherules of different sizes and the peculiarity of the rocker bone in presenting a smooth surface when fractured.