Neuronal basis and diverse mechanisms of pathogen avoidance in Caenorhabditis elegans

Pathogen avoidance behaviour has been observed across animal taxa as a vital host-microbe interaction mechanism. The nematode Caenorhabditis elegans has evolved multiple diverse mechanisms for pathogen avoidance under natural selection pressure. We summarise the current knowledge of the stimuli that trigger pathogen avoidance, including alterations in aerotaxis, intestinal bloating, and metabolites. We then survey the neural circuits involved in pathogen avoidance, transgenerational epigenetic inheritance of pathogen avoidance, signalling crosstalk between pathogen avoidance and innate immunity, and C. elegans avoidance of non-Pseudomonas bacteria. In this review, we highlight the latest advances in understanding host-microbe interactions and the gut-brain axis

Superior structure stability and selectivity of hairpin nucleic acid probes with an l-DNA stem

Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2′-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications

Subwavelength spectoscopy, exciton supertips and mesoscopic light-matter interactions

Subwavelength optical light sources and probes are leading to new devices, technologies and techniques such as near-field microscopy, spectroscopy and biochemical sensing. We have prepared nanometer light sources as small as 40 nm in diameter and exciton sources that are even smaller by adding various inorganic and organic crystals and molecularly doped polymers to micropipettes and nanofabricated fiber-optic tips. We demonstrate subwavelength spectroscopy with highly spatially resolved flourescence spectral of nanocrystals (perylene) in polymeric glasses (PMMA). We also demontrate an interfacial Kasha (external heavy-atom) effect on a perylene nanocrystal surface. We give results on near-field optics and energy transfer at interfaces of nanocrystals (anthracene or diphenyl-anthracene) and dyes (rhodamine B) or dyes embedded in Langmuir-Blodgett films. We discuss the rise of new phenomena when the light source and the sample form a combined mesoscopic system, with subsequent quantum optics effects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31927/1/0000880.pd

Fluorescent Nanoparticle-Based Indirect Immunofluorescence Microscopy for Detection of Mycobacterium tuberculosis

A method of fluorescent nanoparticle-based indirect immunofluorescence microscopy (FNP-IIFM) was developed for the rapid detection of Mycobacterium tuberculosis. An anti-Mycobacterium tuberculosis antibody was used as primary antibody to recognize Mycobacterium tuberculosis, and then an antibody binding protein (Protein A) labeled with Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (RuBpy)-doped silica nanoparticles was used to generate fluorescent signal for microscopic examination. Prior to the detection, Protein A was immobilized on RuBpy-doped silica nanoparticles with a coverage of ∼5.1×102 molecules/nanoparticle. With this method, Mycobacterium tuberculosis in bacterial mixture as well as in spiked sputum was detected. The use of the fluorescent nanoparticles reveals amplified signal intensity and higher photostability than the direct use of conventional fluorescent dye as label. Our preliminary studies have demonstrated the potential application of the FNP-IIFM method for rapid detection of Mycobacterium tuberculosis in clinical samples

Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX

Ovarian cancer is the most lethal gynecological malignancy, and the ovarian clear cell carcinoma subtype (OCCA) demonstrates a particularly poor response to standard treatment. Improvements in ovarian cancer outcomes, especially for OCCA, could be expected from a clearer understanding of the molecular pathology that might guide strategies for earlier diagnosis and more effective treatment.Cell-SELEX technology was employed to develop new molecular probes for ovarian cancer cell surface markers. A total of thirteen aptamers with K(d)'s to ovarian cancer cells in the pico- to nanomolar range were obtained. Preliminary investigation of the targets of these aptamers and their binding characteristics was also performed.We have selected a series of aptamers that bind to different types of ovarian cancer, but not cervical cancer. Though binding to other cancer cell lines was observed, these aptamers could lead to identification of biomarkers that are related to cancer

Selection of Aptamers Specific for Adipose Tissue

Obesity has reached epidemic proportions, affecting more than one tenth of the world's population. As such, adipose tissue is being increasingly recognized as an important therapeutic target for obesity and related metabolic disorders. While many potential targets of adipose tissue have been established and drugs developed, very few of those drugs specifically target adipose tissue without affecting other tissue. This results from a limited knowledge of both cell-surface markers and physicochemical traits specific to adipocytes that might otherwise be exploited by circulating drugs.Here we report the use of cell-SELEX technology to select two aptamers that can specifically recognize mature adipocytes: adipo-1 and adipo-8. Adipo-8 shows high affinity for differentiated, mature 3T3-L1 adipocytes with a K(d) value of 17.8±5.1 nM. The binding was sustained upon incubation at 37°C and insulin stimulation, but was lost upon trypsin treatment. The binding ability was also verified on frozen tissue slides with low background fluorescence and isolated adipocytes.Aptamer adipo-8 selected from a random library appears to bind to mature differentiated adipocytes specifically. This aptamer holds great promise as a molecular recognition tool for adipocyte biomarker discovery or for targeted delivery of molecules to adipocytes

Evanescent luminescence and nanometer-size light source

Subwavelength light sources have been constructed with the aid of luminescent materials. These EXCITOR (exciton transmitted optical radiation) sources are of both theoretical and practical interest. The production of evanescent luminescence may test some of the predictions of quantum electrodynamics concerning the interactions of matter and radiation (e.g., when the emission process and the "subsequent" absorption process are no longer independent). Furthermore, the scannable luminescent tip can be applied to near-field optical microscopy, to scanning exciton microscopy, and to sub-microspectroscopy. An example of an EXCITOR consists of a gold-plated glass micropipette with an inner diameter tapering down to 50 nanometer, "plugged" with an anthracene microcrystal or a polymeric matrix doped with a laser dye. Design considerations involve optical, excitonic, photochemical, and mechanical properties of the luminescent point source. In the ideal limit, the luminescent source consists of a single active chromophore (analogous to the photosynthetic reaction center). As it is scanned over a sample, it senses a variety of perturbations on the atomic or molecular scale such as quenching or external heavy atom effect.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29648/1/0000737.pd

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons

To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies