A Guide to Simple and Informative Binding Assays

The aim of binding assays is to measure interactions between two molecules, such as a protein binding another protein, a small molecule, or a nucleic acid. Hard work is required to prepare reagents, but flaws in the design of many binding experiments limit the information obtained. In particular many experiments fail to measure the affinity of the reactants for each other. This essay describes simple methods to get the most out of valuable reagents in binding experiments

Cocaine- and Amphetamine-Regulated Transcript (CART) Signaling within the Paraventricular Thalamus Modulates Cocaine-Seeking Behaviour

Background: Cocaine- and amphetamine-regulated transcript (CART) has been demonstrated to play a role in regulating the rewarding and reinforcing effects of various drugs of abuse. A recent study demonstrated that i.c.v. administration of CART negatively modulates reinstatement of alcohol seeking, however, the site(s) of action remains unclear. We investigated the paraventricular thalamus (PVT) as a potential site of relapse-relevant CART signaling, as this region is known to receive dense innervation from CART-containing hypothalamic cells and to project to a number of regions known to be involved in mediating reinstatement, including the nucleus accumbens (NAC), medial prefrontal cortex (mPFC) and basolateral amygdala (BLA). Methodology/Principal Findings: Male rats were trained to self-administer cocaine before being extinguished to a set criterion. One day following extinction, animals received intra-PVT infusions of saline, tetrodotoxin (TTX; 2.5 ng), CART (0.625 µg or 2.5 µg) or no injection, followed by a cocaine prime (10 mg/kg, i.p.). Animals were then tested under extinction conditions for one hour. Treatment with either TTX or CART resulted in a significant attenuation of drug-seeking behaviour following cocaine-prime, with the 2.5 µg dose of CART having the greatest effect. This effect was specific to the PVT region, as misplaced injections of both TTX and CART resulted in responding that was identical to controls. Conclusions/Significance: We show for the first time that CART signaling within the PVT acts to inhibit drug-primed reinstatement of cocaine seeking behaviour, presumably by negatively modulating PVT efferents that are important for drug seeking, including the NAC, mPFC and BLA. In this way, we identify a possible target for future pharmacological interventions designed to suppress drug seeking

The Early Pliocene extinction of the mega-toothed shark Otodus megalodon: a view from the eastern North Pacific

The extinct giant shark Otodus megalodon is the last member of the predatory megatoothed lineage and is reported from Neogene sediments from nearly all continents. The timing of the extinction of Otodus megalodon is thought to be Pliocene, although reports of Pleistocene teeth fuel speculation that Otodus megalodon may still be extant. The longevity of the Otodus lineage (Paleocene to Pliocene) and its conspicuous absence in the modern fauna begs the question: when and why did this giant shark become extinct? Addressing this question requires a densely sampled marine vertebrate fossil record in concert with a robust geochronologic framework. Many historically important basins with stacked Otodus-bearing Neogene marine vertebrate fossil assemblages lack well-sampled and well-dated lower and upper Pliocene strata (e.g., Atlantic Coastal Plain). The fossil record of California, USA, and Baja California, Mexico, provides such an ideal sequence of assemblages preserved within well-dated lithostratigraphic sequences. This study reviews all records of Otodus megalodon from post-Messinian marine strata from western North America and evaluates their reliability. All post-Zanclean Otodus megalodon occurrences from the eastern North Pacific exhibit clear evidence of reworking or lack reliable provenance; the youngest reliable records of Otodus megalodon are early Pliocene, suggesting an extinction at the early-late Pliocene boundary (∼3.6 Ma), corresponding with youngest occurrences of Otodus megalodon in Japan, the North Atlantic, and Mediterranean. This study also reevaluates a published dataset, thoroughly vetting each occurrence and justifying the geochronologic age of each, as well as excluding several dubious records. Reanalysis of the dataset using optimal linear estimation resulted in a median extinction date of 3.51 Ma, somewhat older than a previously proposed Pliocene-Pleistocene extinction date (2.6 Ma). Post-middle Miocene oceanographic changes and cooling sea surface temperature may have resulted in range fragmentation, while alongside competition with the newly evolved great white shark (Carcharodon carcharias) during the Pliocene may have led to the demise of the megatoothed shark. Alternatively, these findings may also suggest a globally asynchronous extinction of Otodus megalodon

A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors

The Pan-STARRS 1 Photometric Reference Ladder, Release 12.0

As of 2012 Jan 21, the Pan-STARRS1 $3\pi$ Survey has observed the 3/4 of the sky visible from Hawaii with a minimum of 2 and mean of 7.6 observations in 5 filters, $g_{\rm P1},r_{\rm P1},i_{\rm P1},z_{\rm P1},y_{\rm P1}$. Now at the end of the second year of the mission, we are in a position to make an initial public release of a portion of this unprecedented dataset. This article describes the PS1 Photometric Ladder, Release 12.01 This is the first of a series of data releases to be generated as the survey coverage increases and the data analysis improves. The Photometric Ladder has rungs every hour in RA and at 4 intervals in declination. We will release updates with increased area coverage (more rungs) from the latest dataset until the PS1 survey and the final re-reduction are completed. The currently released catalog presents photometry of $\sim 1000$ objects per square degree in the rungs of the ladder. Saturation occurs at $g_{\rm P1}, r_{\rm P1}, i_{\rm P1} \sim 13.5; z_{\rm P1} \sim 13.0;$ and $y_{\rm P1} \sim 12.0$. Photometry is provided for stars down to $g_{\rm P1}, r_{\rm P1}, i_{\rm P1} \sim 19.1$ in the AB system. This data release depends on the rigid `Ubercal' photometric calibration using only the photometric nights, with systematic uncertainties of (8.0, 7.0, 9.0, 10.7, 12.4) millimags in $(g_{\rm P1},r_{\rm P1},i_{\rm P1},z_{\rm P1},y_{\rm P1})$. Areas covered only with lower quality nights are also included, and have been tied to the Ubercal solution via relative photometry; photometric accuracy of the non-photometric regions is lower and should be used with caution.Physic

Legitimating space: art and the politics of place

Rock art, graffiti, and other emplaced works of art bring people together at specific places. This type of art allows for encounters between people in their absence, and thus presents a range of possibilities for making statements about specific places and those who occupy or visit. This opens the possibility for issues of legitimation to become implicitly or explicitly articulated. However, the legitimate use of space, and the legitimate employment of art, can vary drastically across different contexts. Here, the paper discusses a range of different strategies of art and legitimation in three case studies from India, California, and Spai