Emmanuel A. Theodorakis

HIF-1α expression regulates the bactericidal capacity of phagocytes

Hypoxia is a characteristic feature of the tissue microenvironment during bacterial infection. Here we report on our use of conditional gene targeting to examine the contribution of hypoxia-inducible factor 1, alpha subunit (HIF-1alpha) to myeloid cell innate immune function. HIF-1alpha was induced by bacterial infection, even under normoxia, and regulated the production of key immune effector molecules, including granule proteases, antimicrobial peptides, nitric oxide, and TNF-alpha. Mice lacking HIF-1alpha in their myeloid cell lineage showed decreased bactericidal activity and failed to restrict systemic spread of infection from an initial tissue focus. Conversely, activation of the HIF-1alpha pathway through deletion of von Hippel-Lindau tumor-suppressor protein or pharmacologic inducers supported myeloid cell production of defense factors and improved bactericidal capacity. HIF-1alpha control of myeloid cell activity in infected tissues could represent a novel therapeutic target for enhancing host defense.

Anti-TNF-α therapies: the next generation

The functioning of the immune system is finely balanced by the activities of pro-inflammatory and anti-inflammatory mediators or cytokines. Unregulated activities of these mediators can lead to the development of serious inflammatory diseases. In particular, enhanced tumour-necrosis factor-α (TNF-α) synthesis is associated with the development of rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease. Inhibiting TNF-α activities in these diseases has been remarkably successful. However, the current injectable protein therapies have associated risks and limitations. An oral, small molecule that regulates TNF-α biology could either replace the injectables or provide better disease control when used alone or in conjunction with existing therapies. In this review, we discuss briefly the present understanding of TNF-α-mediated biology and the current injectable therapies in clinical use, and focus on some of the new therapeutic approaches with oral, small-molecule inhibitors.

Molecular rotors—fluorescent biosensors for viscosity and flow

Viscosity is a measure of the resistance of a fluid against gradients in flow (shear rate). Both flow and viscosity play an important role in all biological systems from the microscopic (e.g., cellular) to the systemic level. Many methods to measure viscosity and flow have drawbacks, such as the tedious and time-consuming measurement process, expensive instrumentation, or the restriction to bulk sample sizes. Fluorescent environment-sensitive dyes are known to show high sensitivity and high spatial and temporal resolution. Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation and therefore exhibit two competing deexcitation pathways: fluorescence emission and non-radiative deexcitation from the TICT state. Since TICT formation is viscosity-dependent, the emission intensity of molecular rotors depends on the solvents viscosity. Furthermore, shear-stress dependency of the emission intensity was recently described. Although the photophysical processes are widely explored, the practical application of molecular rotors as sensors for viscosity and the fluid flow introduce additional challenges. Intensity-based measurements are influenced by fluid optical properties and dye concentration, and solvent-dye interaction requires calibration of the measurement system to a specific solvent. Ratiometric dyes and measurement systems help solve these challenges. In addition, the combination of molecular rotors with specific recognition groups allows them to target specific sites, for example the cell membrane or cytoplasm. Molecular rotors are therefore emerging as new biosensors for both bulk and local microviscosity, and for flow and fluid shear stress on a microscopic scale and with real-time response.

Environment-sensitive behavior of fluorescent molecular rotors

Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation. When intramolecular twisting occurs, the molecular rotor returns to the ground state either by emission of a red-shifted emission band or by nonradiative relaxation. The emission properties are strongly solvent-dependent, and the solvent viscosity is the primary determinant of the fluorescent quantum yield from the planar (non-twisted) conformation. This viscosity-sensitive behavior gives rise to applications in, for example, fluid mechanics, polymer chemistry, cell physiology, and the food sciences. However, the relationship between bulk viscosity and the molecular-scale interaction of a molecular rotor with its environment are not fully understood. This review presents the pertinent theories of the rotor-solvent interaction on the molecular level and how this interaction leads to the viscosity-sensitive behavior. Furthermore, current applications of molecular rotors as microviscosity sensors are reviewed, and engineering aspects are presented on how measurement accuracy and precision can be improved.

New fluorescent probes for the measurement of cell membrane viscosity

BACKGROUND Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility. RESULTS Longer hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses. CONCLUSIONS The modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

Significance A mechanistic understanding of how calcium phosphate (CaP) minerals contribute to osteogenic commitment of stem cells and bone tissue formation is a necessary requirement for developing efficient CaP-based synthetic matrices to treat bone defects. This study unravels a previously unknown mechanism, phosphate-ATP-adenosine metabolic signaling, by which the CaP-rich mineral environment in bone tissues promotes osteogenic differentiation of human mesenchymal stem cells. In addition to a mechanical perspective on how biomaterials can influence stem cell differentiation through metabolic pathways, this discovery opens up new avenues for treating critical bone defects and bone metabolic disorders. Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

The relationship of brevetoxin ‘length’ and A-ring functionality to binding and activity in neuronal sodium channels

BACKGROUND Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. RESULTS Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. CONCLUSIONS Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.

Nature-inspired total synthesis of (-)-fusarisetin A.

A concise, protecting group-free total synthesis of (-)-fusarisetin A (1) was efficiently achieved in nine steps from commercially available (S)-(-)-citronellal. The synthetic approach was inspired by our proposed biosynthesis of 1. Key transformations of our strategy include a facile construction of the decalin moiety that is produced via a stereoselective IMDA reaction and a one-pot TEMPO-induced radical cyclization/aminolysis that forms the C ring of 1. Our route is amenable to analogue synthesis for biological evaluation.

Chemistry and Biology of the Caged Garcinia Xanthones

Natural products have been a great source of many small molecule drugs for various diseases. In spite of recent advances in biochemical engineering and fermentation technologies that allow us to explore microorganisms and the marine environment as alternative sources of drugs, more than 70 % of the current small molecule therapeutics derive their structures from plants used in traditional medicine. Natural-product-based drug discovery relies heavily on advances made in the sciences of biology and chemistry. Whereas biology aims to investigate the mode of action of a natural product, chemistry aims to overcome challenges related to its supply, bioactivity, and target selectivity. This review summarizes the explorations of the caged Garcinia xanthones, a family of plant metabolites that possess a unique chemical structure, potent bioactivities, and a promising pharmacology for drug design and development.

A central strategy for converting natural products into fluorescent probes

A Central Strategy for Converting Natural Products into Fluorescent Probes Matthew D. Alexander, Michael D. Burkart, Michael S. Leonard, Padma Portonovo, Bo Liang, Xiaobin Ding, Madeleine M. Joulli!, Brian M. Gulledge, James B. Aggen, A. Richard Chamberlin, Joel Sandler, William Fenical, Jian Cui, Santosh J. Gharpure, Alexei Polosukhin, Hai-Ren Zhang, P. Andrew Evans, Adam D. Richardson, Mary Kay Harper, Chris M. Ireland, Binh G. Vong, Thomas P. Brady, Emmanuel A. Theodorakis, and James J. La Clair*