Detection of G-Quadruplex DNA Using Primer Extension as a Tool

DNA sequence and structure play a key role in imparting fragility to different regions of the genome. Recent studies have shown that non-B DNA structures play a key role in causing genomic instability, apart from their physiological roles at telomeres and promoters. Structures such as G-quadruplexes, cruciforms, and triplexes have been implicated in making DNA susceptible to breakage, resulting in genomic rearrangements. Hence, techniques that aid in the easy identification of such non-B DNA motifs will prove to be very useful in determining factors responsible for genomic instability. In this study, we provide evidence for the use of primer extension as a sensitive and specific tool to detect such altered DNA structures. We have used the G-quadruplex motif, recently characterized at the BCL2 major breakpoint region as a proof of principle to demonstrate the advantages of the technique. Our results show that pause sites corresponding to the non-B DNA are specific, since they are absent when the G-quadruplex motif is mutated and their positions change in tandem with that of the primers. The efficiency of primer extension pause sites varied according to the concentration of monovalant cations tested, which support G-quadruplex formation. Overall, our results demonstrate that primer extension is a strong in vitro tool to detect non-B DNA structures such as G-quadruplex on a plasmid DNA, which can be further adapted to identify non-B DNA structures, even at the genomic level.

Separation index of graphs and stacked 2-spheres

In 1987, Kalai proved that stacked spheres of dimension d >= 3 are characterised by the fact that they attain equality in Barnette's celebrated Lower Bound Theorem. This result does not extend to dimension d = 2. In this article, we give a characterisation of stacked 2-spheres using what we call the separation index. Namely, we show that the separation index of a triangulated 2-sphere is maximal if and only if it is stacked. In addition, we prove that, amongst all n-vertex triangulated 2-spheres, the separation index is minimised by some n-vertex flag sphere for n >= 6. Furthermore, we apply this characterisation of stacked 2-spheres to settle the outstanding 3-dimensional case of the Lutz-Sulanke-Swartz conjecture that ``tight-neighbourly triangulated manifolds are tight''. For dimension d >= 4, the conjecture has already been proved by Effenberger following a result of Novik and Swartz. (C) 2015 Elsevier Inc. All rights reserved.

Temperature-Dependent Photoluminescence of g-C3N4: Implication for Temperature Sensing

We report the temperature-dependent photoluminescence (PL) properties of polymeric graphite-like carbon nitride (g-C3N4) and a methodology for the determination of quantum efficiency along with the activation energy. The PL is shown to originate from three different pathways of transitions: sigma*-LP, pi*-LP, and pi*-pi, respectively. The overall activation energy is found to be similar to 73.58 meV which is much lower than the exciton binding energy reported theoretically but ideal for highly sensitive wide-range temperature sensing. The quantum yield derived from the PL data is 23.3%, whereas the absolute quantum yield is 5.3%. We propose that the temperature-dependent PL can be exploited for the evaluation of the temperature dependency of quantum yield as well as for temperature sensing. Our analysis further indicates that g-C3N4 is well-suited for wide-range temperature sensing.

Numerical simulation of g-jitter effects on half floating zone convection under microgravity environment

The g-jitter effects on the thermocapillary convection in liquid bridge of floating half zone were studied by numerical simulation for unsteady and axi-symmetric model in the cylindrical coordinate system. The g-jitter field was given by a steady microgravity field in addition to an oscillatory low-gravity field, and the effects on the flow field, temperature distribution and free surface deformation were analyzed numerically.

Effects of g-jitter on the critical marangoni number

A half floating zone is fixed on a vibrational deck, which supports a periodical applied acceleration to simulate the effect of g-jitter. This paper deals with the effects of g-jitter on the fluid fields and the critical Marangoni number, which describes the transition from a forced oscillation of thermocapillary convection into an instability oscillatory convection in a liquid bridge of half floating zone with top rod heated. The responses of g-jitter field on the temperature profiles and flow pattern in the liquid bridge were obtained experimentally. The results indicated that the critical Marangoni number decreases with the increasing of g-jitter effect and is slightly smaller for higher frequency of g-jitter with fixed strength of applied gravity.

Fixed Points of Closed and Compact Composite Sequences of Operators and Projectors in a Class of Banach Spaces

Some results on fixed points related to the contractive compositions of bounded operators in a class of complete metric spaces which can be also considered as Banach's spaces are discussed through the paper. The class of composite operators under study can include, in particular, sequences of projection operators under, in general, oblique projective operators. In this paper we are concerned with composite operators which include sequences of pairs of contractive operators involving, in general, oblique projection operators. The results are generalized to sequences of, in general, nonconstant bounded closed operators which can have bounded, closed, and compact limit operators, such that the relevant composite sequences are also compact operators. It is proven that in both cases, Banach contraction principle guarantees the existence of unique fixed points under contractive conditions.

An examination of fluctuations in the “concentration index” of purse seiners and baitboats in the fishery for tropical tunas in the Eastern Pacific, 1951-1961

ENGLISH: In the eastern Pacific Ocean nearly all of the commercial catches of yellowfin tuna (Thunnus albacares) and skipjack (Katsuwonus pelamis) are taken by two types of vessels, baitboats, which use pole and line in conjunction with live-bait, and purse-seiners. From its inception until very recently (1959), this fishery was dominated by baitboats. This method of fishing has been described by Godsil (1938) and Shimada and Schaefer (1956). From 1951 through 1958 baitboats caught between 66.4 and 90.8 per cent of the yellowfin and between 87.2 and 95.3 per cent of the skipjack landed by the California-based fleet. These vessels fished for tuna throughout the year and covered virtually all of the area from southern California to northern Chile. The purse-seine fishery for tunas developed out of the round-haul net fisheries for California sardines and other species. Scofield (1951) gives a detailed description of the development of gear and fishing methods. Prior to 1959 many of the seiners engaged in other fisheries during the fall and early winter months and consequently most of the fishing effort for tuna occurred in the period February-August. The vessels were quite small, averaging approximately 120 tons carrying capacity (Broadhead and Marshall, 1960), in comparison to the baitboats, of which the most numerous size-class was 201-300 tons. The seiners were naturally more restricted in range than the baitboats and most of their effort was restricted to the northern grounds. During the period 1959-61 most of the large baitboats were converted for purse-seining and the existing seiner fleet was modernized. These developments increased the range of the seiner fleet and resulted in a wider and more nearly even spatial and temporal distribution of effort. By the early part of 1961, the purse-seine fleet approximated the level of the preconversion baitboat fleet in amount of effort applied and area covered. The changes in the purse-seine fishery and the fishing methods employed in the modernized fleet are described by Orange and Broadhead (1959), Broadhead and Marshall (1960), McNeely (1961) and Broadhead (1962). The change in the relative importance of the two gears is illustrated by the decline in the proportion of the total logged tonnage landed by California-based baitboats, in comparison to the proportion landed by seiners. In 1959 baitboats landed 49.5 per cent of the yellowfin and 87.8 per cent of the skipjack. In 1960 these percentages were 22.9 and 74.7 respectively and in 1961 the decline continued to 12.6 per cent of the yellowfin and 30.0 per cent of the skipjack (Schaefer, 1962). In previous Bulletins of this Commission (Griffiths, 1960; Calkins, 1961) the baitboat catch and effort statistics were used to compute two indices of population density and an index of concentration of fishing effort and the fluctuations of these indices were analyzed in some detail. Due to the change in the relative importance of the two gears it is appropriate to extend this investigation to include the purse-seine data. The objectives of this paper are to compute two indices of population density and an index of concentration of fishing effort and to examine the fluctuations in these indices before and after the changes in the fishery. A further objective is to compare the purse-seine indices with those of the baitboats for the same time periods. SPANISH: En el Océano Pacífico Oriental casi todas las capturas comerciales del atún aleta amarilla (Thunnus albacares) y del barrilete (Katsuwonus pelamis) son efectuadas por dos tipos de barcos, los barcos de carnada que emplean la caña y el anzuelo en conjunto con la carnada viva, y los barcos rederos. Desde su comienzo hasta hace poco tiempo (1959), esta pesquería estaba dominada por los barcos de carnada. El método de pesca usado por estos barcos ha sido descrito por Godsil (1938) y por Shimada y Schaefer (1956). De 1951 a 1958, los barcos de carnada pescaron entre el 66.4 y el 90.8 por ciento del atún aleta amarilla y entre el 87.2 y el 95.3 por ciento del barrilete descargados por la flota que tiene su base en California. Estos barcos pescaron atún durante todo el año y cubrieron virtualmente toda el área de California meridional hasta la parte norte de Chile. La pesquería del atún con redes de cerco se originó en las pesquerías de las sardinas de California y otras especies, con redes que se remolcaban circularmente. Scofield (1951) dá una descripción detallada del desarrollo de los métodos y del equipo de pesca. Antes de 1959 muchos de los rederos se dedicaban a otras pesquerías durante los meses del otoño y a principios del invierno y consecuentemente, la mayor parte del esfuerzo depesca para la producción del atún ocurría en el período febrero-agosto. Las embarcaciones eran bastante pequeñas, con un promedio de aproximadamente 120 toneladas de capacidad para el transporte (Broadhead y Marshall, 1960) en comparación con los barcos de carnada, de los cuales la clase de tamaño más numerosa era de 201 a 300 toneladas. Los rederos estaban naturalmente más restringidos en su radio de acción que los barcos de carnada y la mayor parte de su esfuerzo se limitaba a las localidades del norte. Durante el período 1959-61, la mayoría de los grandes barcos de carnada fueron convertidos al sistema de pesca con redes de cerco, y se modernizó la flota existente de los rederos. Estos cambios aumentaron el alcance de la flota de los barcos rederos dando como resultado una distribución más amplia y casi más uniforme del esfuerzo espaciado y temporal. En la primera parte del año 1961, la flota de rederos se aproximó al nivel de la preconversión de la flota de clipers, en la cantidad de esfuerzo aplicado y al área comprendida. Los cambios en la pesquería con red y los métodos de pesca empleados en la flota modernizada, han sido descritos por Orange y Broadhead (1959), Broadl1ead y Marshall (1960), McNeely (1961) y Broadhead (1962). El cambio en la importancia relativa de los dos sistemas de pesca está ilustrado por la declinación en la proporción del tonelaje total registrado, como descargado por los barcos de carnada que tienen su base en California, comparado con la proporción desembarcada por los barcos rederos. En 1959 los clipers descargaron el 49.5 por ciento del atún aleta amarilla y el 87.8 por ciento del barrilete. En 1960 estos porcentajes fueron del 22.9 y 74.7 respectivamente, y en 1961 continuó la reducción hasta el 12.6 por ciento del atún aleta amarilla y el 30.0 por ciento del barrilete (Schaefer, 1962). En Boletines anteriores de la Comisión (Griffiths, 1960; Calkins, 1961) las estadísticas de la pesca y el esfuerzo de los clipers se utilizaron para computar dos índices de la densidad de población y un índice de la concentración del esfuerzo de pesca, y se analizaron algo detalladamente las fluctuaciones de estos índices. Debido al cambio en la importancia relativa de los dos sistemas de pesca, es conveniente extender esta investigación para incluir los datos correspondientes a los barcos rederos. Los objetivos del presente estudio son de computar dos índices de la densidad de población y un índice de la concentración del esfuerzo de pesca, y examinar las fluctuaciones en estos índices, antes y después de los cambios en la pesquería. Otro objetivo es de comparar los índices de los barcos rederos, con aquellos de los clipers en los mismos períodos de tiempo.

Role of G protein coupled inwardly rectifier K+ channels (GIRK) on the neurobiology depression

353 págs.

Chemical-scale studies of G protein-coupled receptors and ligand-gated ion channels

This dissertation describes studies of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) using unnatural amino acid mutagenesis to gain high precision insights into the function of these important membrane proteins.

Chapter 2 considers the functional role of highly conserved proline residues within the transmembrane helices of the D2 dopamine GPCR. Through mutagenesis employing unnatural α-hydroxy acids, proline analogs, and N-methyl amino acids, we find that lack of backbone hydrogen bond donor ability is important to proline function. At one proline site we additionally find that a substituent on the proline backbone N is important to receptor function.

In Chapter 3, side chain conformation is probed by mutagenesis of GPCRs and the muscle-type nAChR. Specific side chain rearrangements of highly conserved residues have been proposed to accompany activation of these receptors. These rearrangements were probed using conformationally-biased β-substituted analogs of Trp and Phe and unnatural stereoisomers of Thr and Ile. We also modeled the conformational bias of the unnatural Trp and Phe analogs employed.

Chapters 4 and 5 examine details of ligand binding to nAChRs. Chapter 4 describes a study investigating the importance of hydrogen bonds between ligands and the complementary face of muscle-type and α4β4 nAChRs. A hydrogen bond involving the agonist appears to be important for ligand binding in the muscle-type receptor but not the α4β4 receptor.

Chapter 5 describes a study characterizing the binding of varenicline, an actively prescribed smoking cessation therapeutic, to the α7 nAChR. Additionally, binding interactions to the complementary face of the α7 binding site were examined for a small panel of agonists. We identified side chains important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh.

Chapter 6 describes efforts to image nAChRs site-specifically modified with a fluorophore by unnatural amino acid mutagenesis. While progress was hampered by high levels of fluorescent background, improvements to sample preparation and alternative strategies for fluorophore incorporation are described.

Chapter 7 describes efforts toward a fluorescence assay for G protein association with a GPCR, with the ultimate goal of probing key protein-protein interactions along the G protein/receptor interface. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other.

Structure Prediction of G-Protein Coupled Receptors

G-protein coupled receptors (GPCRs) form a large family of proteins and are very important drug targets. They are membrane proteins, which makes computational prediction of their structure challenging. Homology modeling is further complicated by low sequence similarly of the GPCR superfamily.

In this dissertation, we analyze the conserved inter-helical contacts of recently solved crystal structures, and we develop a unified sequence-structural alignment of the GPCR superfamily. We use this method to align 817 human GPCRs, 399 of which are nonolfactory. This alignment can be used to generate high quality homology models for the 817 GPCRs.

To refine the provided GPCR homology models we developed the Trihelix sampling method. We use a multi-scale approach to simplify the problem by treating the transmembrane helices as rigid bodies. In contrast to Monte Carlo structure prediction methods, the Trihelix method does a complete local sampling using discretized coordinates for the transmembrane helices. We validate the method on existing structures and apply it to predict the structure of the lactate receptor, HCAR1. For this receptor, we also build extracellular loops by taking into account constraints from three disulfide bonds. Docking of lactate and 3,5-dihydroxybenzoic acid shows likely involvement of three Arg residues on different transmembrane helices in binding a single ligand molecule.

Protein structure prediction relies on accurate force fields. We next present an effort to improve the quality of charge assignment for large atomic models. In particular, we introduce the formalism of the polarizable charge equilibration scheme (PQEQ) and we describe its implementation in the molecular simulation package Lammps. PQEQ allows fast on the fly charge assignment even for reactive force fields.

Computational Predictions of G Protein-Coupled Receptor Structures and Binding Sites

G protein-coupled receptors (GPCRs) are the largest family of proteins within the human genome. They consist of seven transmembrane (TM) helices, with a N-terminal region of varying length and structure on the extracellular side, and a C-terminus on the intracellular side. GPCRs are involved in transmitting extracellular signals to cells, and as such are crucial drug targets. Designing pharmaceuticals to target GPCRs is greatly aided by full-atom structural information of the proteins. In particular, the TM region of GPCRs is where small molecule ligands (much more bioavailable than peptide ligands) typically bind to the receptors. In recent years nearly thirty distinct GPCR TM regions have been crystallized. However, there are more than 1,000 GPCRs, leaving the vast majority of GPCRs with limited structural information. Additionally, GPCRs are known to exist in a myriad of conformational states in the body, rendering the static x-ray crystal structures an incomplete reflection of GPCR structures. In order to obtain an ensemble of GPCR structures, we have developed the GEnSeMBLE procedure to rapidly sample a large number of variations of GPCR helix rotations and tilts. The lowest energy GEnSeMBLE structures are then docked to small molecule ligands and optimized. The GPCR family consists of five subfamilies with little to no sequence homology between them: class A, B1, B2, C, and Frizzled/Taste2. Almost all of the GPCR crystal structures have been of class A GPCRs, and much is known about their conserved interactions and binding sites. In this work we particularly focus on class B1 GPCRs, and aim to understand that family’s interactions and binding sites both to small molecules and their native peptide ligands. Specifically, we predict the full atom structure and peptide binding site of the glucagon-like peptide receptor and the TM region and small molecule binding sites for eight other class B1 GPCRs: CALRL, CRFR1, GIPR, GLR, PACR, PTH1R, VIPR1, and VIPR2. Our class B1 work reveals multiple conserved interactions across the B1 subfamily as well as a consistent small molecule binding site centrally located in the TM bundle. Both the interactions and the binding sites are distinct from those seen in the more well-characterized class A GPCRs, and as such our work provides a strong starting point for drug design targeting class B1 proteins. We also predict the full structure of CXCR4 bound to a small molecule, a class A GPCR that was not closely related to any of the class A GPCRs at the time of the work.