Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B–natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility.     

Neural Substrates of Drosophila Larval Anemotaxis

1. Download high-res image (205KB)
2. Download full-size image

     

The evolution of microendemism in a reef fish (Hypoplectrus maya)

Marine species tend to have extensive distributions, which are commonly attributed to the dispersal potential provided by planktonic larvae and the rarity of absolute barriers to dispersal in the ocean. Under this paradigm, the occurrence of marine microendemism without geographic isolation in species with planktonic larvae poses a dilemma. The recently described Maya hamlet (Hypoplectrus maya, Serranidae) is exactly such a case, being endemic to a 50‐km segment of the Mesoamerican Barrier Reef System (MBRS). We use whole‐genome analysis to infer the demographic history of the Maya hamlet and contrast it with the sympatric and pan‐Caribbean black (H. nigricans), barred (H. puella) and butter (H. unicolor) hamlets, as well as the allopatric but phenotypically similar blue hamlet (H. gemma). We show that H. maya is indeed a distinct evolutionary lineage, with genomic signatures of inbreeding and a unique demographic history of continuous decrease in effective population size since it diverged from congeners just ~3,000 generations ago. We suggest that this case of microendemism may be driven by the combination of a narrow ecological niche and restrictive oceanographic conditions in the southern MBRS, which is consistent with the occurrence of an unusually high number of marine microendemics in this region. The restricted distribution of the Maya hamlet, its decline in both census and effective population sizes, and the degradation of its habitat place it at risk of extinction. We conclude that the evolution of marine microendemism can be a fast and dynamic process, with extinction possibly occurring before speciation is complete.     

The role of GDF11 in aging and skeletal muscle,cardiac and bone homeostasis

GDF11 is a secreted factor in the TGFß family of cytokines. Its nearest neighbor evolutionarily is myostatin, a factor discovered as being a negative regulator of skeletal muscle growth. High profile studies several years ago suggested that GDF11 declines with age, and that restoration of systemic GDF11 to ‘youthful’ levels is beneficial for several age-related conditions. Particularly surprising was a report that supplementation of GDF11 aided skeletal muscle regeneration, as its homolog, myostatin, has the opposite role. Given this apparent contradiction in functionality, multiple independent labs sought to discern differences between the two factors and better elucidate age-related changes in circulating GDF11, with most failing to reproduce the initial finding of declining GDF11 levels, and, importantly, all subsequent studies examining the effects of GDF11 on skeletal muscle described an inhibitory effect on regeneration – and that higher doses induce skeletal muscle atrophy and cachexia. There have also been several studies examining the effect of GDF11 and/or the downstream ActRII pathway on cardiac function, along with several interesting reports on bone. A review of the GDF11 literature, as it relates in particular to aging and skeletal muscle, cardiac and bone biology, is presented.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

Inhibition of the dopamine D1 receptor signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.