The molecular and cellular basis of bitter taste in Drosophila

The extent of diversity among bitter-sensing neurons is a fundamental issue in the field of taste. Data are limited and conflicting as to whether bitter neurons are broadly tuned and uniform, resulting in indiscriminate avoidance of bitter stimuli, or diverse, allowing a more discerning evaluation of food sources. We provide a systematic analysis of how bitter taste is encoded by the major taste organ of the Drosophila head, the labellum. Each of 16 bitter compounds is tested physiologically against all 31 taste hairs, revealing responses that are diverse in magnitude and dynamics. Four functional classes of bitter neurons are defined. Four corresponding classes are defined through expression analysis of all 68 gustatory taste receptors. A receptor-to-neuron-to-tastant map is constructed. Misexpression of one receptor confers bitter responses as predicted by the map. These results reveal a degree of complexity that greatly expands the capacity of the system to encode bitter taste.     

An acquired defect in IgG-dependent phagocytosis explains the impairment in antibody-mediated cellular depletion in Lupus

B cells play important roles in autoimmune diseases ranging from multiple sclerosis to rheumatoid arthritis. B cells have also long been considered central players in systemic lupus erythematosus. However, anti-CD20-mediated B cell depletion was not effective in two clinical lupus studies, whereas anti-B lymphocyte stimulator, which inhibits B cell survival, was effective. Others and we previously found that anti-CD20-based depletion was surprisingly ineffective in tissues of lupus-prone mice, but that persistent high doses eventually led to depletion and ameliorated lupus. Lupus patients might also have incomplete depletion, as suggested in several studies, and which could have led to therapeutic failure. In this study, we investigated the mechanism of resistance to Ab-mediated cellular depletion in murine lupus. B cells from lupus-prone mice were easily depleted when transferred into normal environments or in lupus-prone mice that lacked serum Ig. Serum from lupus-prone mice transferred depletion resistance, with the active component being IgG. Because depletion is FcγR-dependent, we assayed macrophages and neutrophils exposed to lupus mouse serum, showing that they are impaired in IgG-mediated phagocytosis. We conclude that depletion resistance is an acquired, reversible phagocytic defect depending on exposure to lupus serum IgG. These results have implications for optimizing and monitoring cellular depletion therapy.     

Bacterial Diversity of Lonar Soda Lake of India

Total seventy four bacteria were isolated from Lonar soda lake of Maharashtra state, India. Eleven isolates were identified using morphological, biochemical and molecular analysis. The bacteria isolated belonged to phylum firmicutes and proteobacteria. Majorities (eight) were firmicutes and three were proteobacteria. For the first time we are reporting Alcanivorax spp. which is a genus well known for its oil degradation capacity, indicate the probable existence of oil reservoir in vicinity of Lonar lake. In addition all the eleven bacteria are potential producers of industrially important enzymes, pigments, antibiotics as well.     

A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water

The specific 1,3 dipolar Hüisgen cycloaddition reaction known as ‘click-reaction’ between azide and alkyne groups is employed for the synthesis of peptide–oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, ~3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products.     

High‐throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high‐throughput small‐scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two‐step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two‐step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:516–520, 2014     

A cell-based computational modeling approach for developing site-directed molecular probes

Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.     

Design and docking studies of [diethylenetriaminepentaacetic acid-(amino acid)2] with acetylcholine receptor as a molecular imaging agent for single-photon emission computed tomographic application

The acetylcholine receptor is an essential link between the brain and the muscles, so it is a sensitive location for attack. In this study, some reversible [diethylenetriaminepentaacetic acid-(amino acid)2] have been docked computationally to the active site of the acetylcholine receptor. The induced fit method was employed to perform the automolecular docking for these systems. The result of docking studies generated thermodynamic properties, such as free energy of bindings (Glide score) and their weak electrostatic interactions. On the basis of these results, scintigraphic imaging studies were performed in mice. Among the radiotracers evaluated in this study, compound derived from 5-hydroxytryptophan/tryptophan exhibited remarkable localization in the brain, whereas radiotracer derived from l-histidine shows moderate accumulation in the brain. Preliminary studies with these amino acid-based ligands are encouraging to carrying out further in vivo experiments for targeted imaging.