Nobiletin affects circadian rhythms and oncogenic characteristics in a cell-dependent manner.

The natural product nobiletin is a small molecule, widely studied with regard to its therapeutic effects, including in cancer cell lines and tumors. Recently, nobiletin has also been shown to affect circadian rhythms via their enhancement, resulting in protection against metabolic syndrome. We hypothesized that nobiletin's anti-oncogenic effects, such as prevention of cell migration and formation of anchorage independent colonies, are correspondingly accompanied by modulation of circadian rhythms.

Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock.

Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence are tightly linked to cell fate. Several recent studies have used small molecules to affect circadian oscillations; however, their concomitant cellular effects were not assessed, and they have not been compared under similar experimental conditions. In this work, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS) and determine their influences on cellular behaviors, including motility and colony formation.

Chemical modulation of circadian rhythms and assessment of cellular behavior via indirubin and derivatives.

Circadian rhythms are critical regulators of many physiological and behavioral functions. The use and abilities of small molecules to affect oscillations have recently received significant attention. These manipulations can be reversible and tunable, and have been used to study various biological mechanisms and molecular properties.

A novel Vibrio beta-glucosidase (LamN) that hydrolyzes the algal storage polysaccharide laminarin.

The metabolic versatility, tractability and rapid growth potential of the Vibrio spp. have made them increasingly attractive systems for investigating carbon cycling in the marine environment. In this study, an in silico subtractive proteomic strategy was used to identify a novel 101 kDa GH3 family β-glucosidase (LamN) that was found in bioluminescent Vibrio campbellii strains capable of utilizing the algal storage glucan laminarin.

Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression.

Packaging specific exogenous active proteins and DNAs together within a single viral-nanocontainer is challenging. The bacteriophage T4 capsid (100 × 70 nm) is well suited for this purpose, because it can hold a single long DNA or multiple short pieces of DNA up to 170 kb packed together with more than 1,000 protein molecules. Any linear DNA can be packaged in vitro into purified procapsids.

Engineered bacteriophage T4 nanoparticles for cellular imaging.

Tailless T4 nanoparticles (NPs) have large surface areas consisting of more than 10(5) diverse surface reactive groups and offer great flexibility in chemical modification for tailoring the desired functionality. Dye-conjugated T4 NPs exhibiting bright fluorescence are biocompatible and can be internalized by various eukaryotic cells which land themselves as excellent cellular imaging agents. Here, we describe the preparation of engineered T4 NPs including dye-conjugation and characterization, and the procedure for cellular uptake and confocal microscopy.

3-substituted indole inhibitors against Francisella tularensis FabI identified by structure-based virtual screening.

In this study, we describe novel inhibitors against Francisella tularensis SchuS4 FabI identified from structure-based in silico screening with integrated molecular dynamics simulations to account for induced fit of a flexible loop crucial for inhibitor binding. Two 3-substituted indoles, 54 and 57, preferentially bound the NAD(+) form of the enzyme and inhibited growth of F. tularensis SchuS4 at concentrations near that of their measured Ki.

Adaptation of the black yeast Wangiella dermatitidis to ionizing radiation: molecular and cellular mechanisms.

Observations of enhanced growth of melanized fungi under low-dose ionizing radiation in the laboratory and in the damaged Chernobyl nuclear reactor suggest they have adapted the ability to survive or even benefit from exposure to ionizing radiation. However, the cellular and molecular mechanism of fungal responses to such radiation remains poorly understood. Using the black yeast Wangiella dermatitidis as a model, we confirmed that ionizing radiation enhanced cell growth by increasing cell division and cell size.

Function and regulation of Vibrio campbellii proteorhodopsin: acquired phototrophy in a classical organoheterotroph.

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii.

Engineered viral nanoparticles for flow cytometry and fluorescence microscopy applications.

Viral nanoparticles (VNPs) are attractive platforms for use in the biotechnology and biomedical fields because of their biological nature. A wide variety of these particles, labeled with fluorescent reporters, have been characterized using flow cytometry and cellular imaging techniques. Fluorescence microscopy allows the direct observation of VNPs on the cell surface or inside the membrane as well as the cellular localization of the nanoparticles while flow cytometry allows the statistical quantification of nanoparticle uptake and targeting specificity.

Locked nucleic acid flow cytometry-fluorescence in situ hybridization (LNA flow-FISH): a method for bacterial small RNA detection.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses.

Probing the donor and acceptor substrate specificity of the γ-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined.

Locked nucleic acid and flow cytometry-fluorescence in situ hybridization for the detection of bacterial small noncoding RNAs.

We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter.

Engineered T4 viral nanoparticles for cellular imaging and flow cytometry.

Viruses are of particular interest as scaffolds for biotechnology applications given their wide range of shapes and sizes and the possibility to modify them with a variety of functional moieties to produce useful virus-based nanoparticles (VNPs). In order to develop functional VNPs for cell imaging and flow cytometry applications, we used the head of the T4 bacteriophage as a scaffold for bioconjugation of fluorescent dyes. Bacteriophage T4 is a double-stranded DNA virus with an elongated icosahedron head and a contractile tail.

Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments.

Fluoromodules are complexes formed upon the noncovalent binding of a fluorogenic dye to its cognate biomolecular partner, which significantly enhances the fluorescence quantum yield of the dye. Previously, several single-chain, variable fragment (scFv) antibodies were selected from a yeast cell surface-displayed library that activated fluorescence from a family of unsymmetrical cyanine dyes covering much of the visible and near-IR spectrum. The current work expands our repertoire of genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate fluorogenic violet-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles.

A hard microflow cytometer using groove-generated sheath flow for multiplexed bead and cell assays.

With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ~35 μm × 40 μm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 μm × 125 μm) successfully minimized the risk of clogging.

Monitoring viral RNA in infected cells with LNA flow-FISH.

We previously showed the feasibility of using locked nucleic acid (LNA) for flow cytometric-fluorescence in situ hybridization (LNA flow-FISH) detection of a target cellular mRNA. Here we demonstrate how the method can be used to monitor viral RNA in infected cells. We compared the results of the LNA flow-FISH with other methods of quantifying virus replication, including the use of an enhanced green fluorescent protein (EGFP) viral construct and quantitative reverse-transcription polymerase chain reaction.

Identification of non-coding RNAs in environmental vibrios.

The discovery of non-coding RNA (ncRNA) has been mainly limited to laboratory model systems and human pathogenic bacteria. In this study, we begin to explore the ncRNA diversity in four recently sequenced environmental Vibrio species (Vibrio alginolyticus 40B, Vibrio communis 1DA3, Vibrio mimicus VM573 and Vibrio campbellii BAA-1116) by performing in silico searches using Infernal and Rfam for the identification of putative ncRNA-encoding genes. This search method resulted in the identification of 31-38 putative ncRNA genes per species and the total ncRNA catalogue spanned an assortment of regulatory mechanisms (riboswitches, cis-encoded ncRNAs, trans-encoded ncRNAs, modulators of protein activity, ribonucleoproteins, transcription termination ncRNAs and unknown).

LNA flow-FISH: a flow cytometry-fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes.

We present a novel method using flow cytometry-fluorescence in situ hybridization (flow-FISH) to detect specific messenger RNA (mRNA) in suspended cells using locked nucleic acid (LNA)-modified oligonucleotide probes. beta-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to beta-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry.

Synthesis of new fluorogenic cyanine dyes and incorporation into RNA fluoromodules.

A new fluorogenic cyanine dye was synthesized and found to have low fluorescence quantum yield in fluid solution and in the presence of double-stranded DNA but 80-fold enhanced fluorescence in viscous glycerol solution. An RNA aptamer selected for binding to the new dye exhibits K(d) = 87 nM and 60-fold fluorescence enhancement. The dye-aptamer pair is a fluoromodule that can be incorporated into fluorescent sensors and labels.

Fluorescent PNA probes as hybridization labels for biological RNA.

Fluorescent labeling of biological RNA is complicated by the narrow range of nucleoside triphosphates that can be used for biological synthesis (i.e., transcription) as well as the inability to site-specifically incorporate them into long RNA transcripts.