Hydrodynamic efficiency in sharks: the combined role of riblets and denticles.

We investigate the influence of smooth and ribletted shark skin on a turbulent boundary layer flow. Through laser Doppler anemometry (LDA) the role of riblets in combination with the shark skin denticle is established for the first time. Our results show that smooth denticles behave like a typical rough surface when exposed to an attached boundary layer. Drag is increased for the full range of tested dimensionless denticle widths,w+≈ 25-80, wherew+is the denticle width,w, scaled by the friction velocity,uτ, and the kinematic viscosity,ν. However, when riblets are added to the denticle crown we demonstrate there is a significant reduction in drag, relative to the smooth denticles. We obtain a modest maximum drag reduction of 2% for the ribletted denticles when compared to the flat plate, but when compared to the smooth denticles the difference in drag is in excess of 20% forw+≈ 80. This study enables a new conclusion that riblets have evolved as a mechanism to reduce or eliminate the skin friction increase due to the presence of scales (denticles). The combination of scales and riblets is hydrodynamically efficient in terms of skin-friction drag, while also acting to maintain flow attachment, and providing the other advantages associated with scales, e.g. anti-fouling, abrasion resistance, and defence against parasites.

High throughput drug screening for human immunodeficiency virus type 1 reactivating compounds.

The ability of human immunodeficiency virus type 1 (HIV-1) to persist in a latent stage in memory T cells in the presence of antiretroviral therapy poses a major obstacle to the development of an HIV-1 therapy with curative intent. As latently infected cells are phenotypically not distinguishable from uninfected cells, therapeutic reactivation of the latent infection, followed by the death of the host cell induced by viral cytopathicity, is considered the only means to eliminate this viral reservoir. To identify compounds with the potential to reactivate latent HIV-1, we have developed a series of latently HIV-1-infected reporter cell lines that allow for high throughput drug screening (HTS) in a 384-well plate-based format. The latent reporter cell lines use enhanced green fluorescence protein (eGFP) as a direct and quantitative marker of HIV-1 expression. To aid identification of specific compounds, the cells are engineered to constitutively express a second, red fluorescent protein that has no spectral overlap with eGFP, which allows for the simultaneous quantification of cell viability (inversely correlated to compound toxicity). Thus, these reporters enable prioritization of compounds most likely to have a favorable therapeutic window. The high dynamic signal range and the excellent reproducibility of the primary screening assay result in a Z' -factor of 0.89, which characterizes the HTS system as very robust. The assay has been implemented for automated drug screening, and we here discuss the advantages and limitations of the HTS system based on the data obtained for 1,600 compounds during a limited proof-of-concept drug screen.

A novel inhibitor of Mycobacterium tuberculosis pantothenate synthetase.

Pantothenate synthetase (PS; EC 6.3.2.1), encoded by the panC gene, catalyzes the essential adenosine triphosphate (ATP)-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast, and plants; pantothenate is a key precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). Because the enzyme is absent in mammals and both CoA and ACP are essential cofactors for bacterial growth, PS is an attractive chemotherapeutic target. An automated high-throughput screen was developed to identify drugs that inhibit Mycobacterium tuberculosis PS. The activity of PS was measured spectrophotometrically through an enzymatic cascade involving myokinase, pyruvate kinase, and lactate dehydrogenase. The rate of PS ATP utilization was quantitated by the reduction of absorbance due to the oxidation of NADH to NAD+ by lactate dehydrogenase, which allowed for an internal control to detect interference from compounds that absorb at 340 nm. This coupled enzymatic reaction was used to screen 4080 compounds in a 96-well format. This discussion describes a novel inhibitor of PS that exhibits potential as an antimicrobial agent.

Development and application of a high-throughput screening assay for HIV-1 integrase enzyme activities.

Integrase (IN) mediates the covalent insertion of the retroviral genome into its host chromosomal DNA. This enzymatic activity can be reconstituted in vitro with short DNA oligonucleotides, which mimic a single viral DNA end, and purified IN. Herein we report a highly efficient and sensitive high-throughput screen, HIV Integrase Target SRI Assay (HITS), for HIV-1 IN activity using 5' biotin-labeled DNA (5' BIO donor) and 3' digoxygenin-labeled DNA (3' DIG target). Following 3' processing of the 5' BIO donor, strand transfer proceeds with integration of the 5' BIO donor into the 3' DIG target. Products were captured on a streptavidin-coated microplate and the amount of DIG retained in the well was measured. The end point values, measured as absorbance, ranged from 0.9 to 1.5 for IN-mediated reactions as compared with background readings of 0.05 to 0.12. The Z factor for the assay ranged from 0.7 to 0.85. The assay was used to screen drugs in a high-throughput format, and furthermore, we adapted the assay to study mechanistic questions regarding the integration process. For example, using variations of the assay format, we showed high preference of E strand of the long terminal repeat (LTR) viral DNA as a target strand compared with its complementary A strand. The E strand is the strand processed by IN. Furthermore, we explored the reported inhibitory effect of reverse transcriptase on integration.