Adenosine Triphosphate Binding Research Articles

A novel aptasensor for the ultra-sensitive detection of adenosine triphosphate via aptamer/quantum dot based resonance energy transfer

We designed a novel aptamer based biosensor (aptasensor) for ultrasensitive detection of adenosine triphosphate (ATP) through resonance energy transfer (RET). The ATP aptamer was modified with Cy3 at the 3' end, and a green quantum dot (525) was attached to the 5' end of its complementary sequence respectively. The ATP aptamer and its complementary sequence could assemble into a duplex structure in the absence of target ATP, and then decrease the distance between the quantum dot and Cy3 which could produce significant RET signal. Upon ATP binding, the ATP aptamer could dissociate with its complementary sequence and then increase the distance between the quantum dot and Cy3 which would significantly decrease the RET signal. Therefore, the ATP detection could be easily achieved through detection of the fluorescence intensity ratio between 525 nm and 560 nm. The results show that the emission fluorescence intensity ratio of 525/560 is linearly related to the logarithmic concentration of ATP. The linear range of this aptasensor is from 0.1 nM to 1 μM, and the detection limit is lower down to 0.01 nM. Excellent selectivity of this aptasensor for ATP has been demonstrated through the detection of thymidine triphosphate (TTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and adenosine diphosphate (ADP) respectively as control. The method we described here could easily detect ATP with excellent selectivity, linearity and sensitivity down to the nanomolar range, as well as avoid photobleaching.

Dynamic Nucleotide-dependent Interactions of Cysteine- and Histidine-rich Domain (CHORD)-containing Hsp90 Cochaperones Chp-1 and Melusin with Cochaperones PP5 and Sgt1

Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca(2+), which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca(2+). Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones.

Nanoapertures for AFM-based single-molecule force spectroscopy

Simultaneous single–molecule force spectroscopy and microfluorescence binding measurements are often hampered by background fluorescence from the bulk. Zero–Mode Waveguides (ZMW) restrict the excited volume but require a special design, which allows the tip of the force probing cantilever to protrude into the nanoaperture. Here, we describe the design and fabrication of such ZMW and report the first experiments where binding of fluorescent adenosine triphosphate to the force–activated enzyme titin kinase was measured while the enzyme was subjected to mechanical forces.

New insights into the pathogenesis of pseudoxanthoma elasticum and related soft tissue calcification disorders by identifying genetic interactions and modifiers

Screening of the adenosine triphosphate binding cassette transporter protein subfamily C member 6 gene (ABCC6) in pseudoxanthoma elasticum (PXE) revealed a mutation detection rate of approximately 87%. Although 25% of the unidentified disease alleles underlie deletions/insertions, there remain several PXE patients with no clear genotype. The recent identification of PXE-related diseases and the high intra-familiar and inter-individual clinical variability of PXE led to the assumption that secondary genetic co-factors exist. Here, we summarize current knowledge of the genetics underlying PXE and PXE-related disorders based on human and animal studies. Furthermore, we discuss the role of genetic interactions and modifier genes in PXE and PXE-related diseases characterized by soft tissue calcification.

JAK2 inhibitors for myelofibrosis: why are they effective in patients with and without JAK2V617F mutation?

An activating mutation (V617F) in the pseudokinase domain of the Janus kinase (JAK)-2 tyrosine kinase has been described in 90% of patients with polycythemia vera (PV) and 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (MF). The discovery of JAK2V617F stirred the development of JAK2 inhibitors for treatment of patients with MF, ET and PV. Similar to other tyrosine kinase (TK) inhibitors in current use, JAK2 inhibitors target the adenosine triphosphate (ATP) binding site at the TK domain and not the pseudokinase domain, thus affecting both mutated and wild-type kinases. In fact, clinical trials of these compounds have demonstrated improvements in constitutional symptoms and splenomegaly in patients with both mutated and wild-type JAK2 MF. It is believed that these drugs may act not only through inhibition of neoplastic cell proliferation, but also by downregulating signaling through proinflammatory cytokine receptors. In this article, we review the current state of JAK2 inhibitors and discuss why these drugs could be a valuable addition to the treatment armamentarium for patients with and without the JAK2V617F mutation.

Lipid-Sensing High-Throughput ApoA-I Assays

Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647–labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.

P2X receptor-mediated ATP purinergic signaling in health and disease

P2X receptor-mediated ATP purinergic signaling in health and disease Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP), an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

Computer simulation based selection of optimal monomer for imprinting of tri-O-acetiladenosine in polymer matrix: vacuum calculations

Molecularly imprinted polymers can be anticipated as synthetic imitation of natural antibodies, receptors and enzymes. In case of successful imprinting the selectivity and affinity of the imprint for substrate molecules are comparable with those of natural counterparts. The selection of the optimal functional monomer, monomer/template ratio as well as choosing of polymerization solvent is crucial determinants of the successful imprinting. In the present study the simulation approach to the development of molecular imprinting polymers for the extraction of new protein kinase ATP-competitive inhibitors is presented. By imprinting tri-O-acetyladenosine into polymer matrix the synthetic reproduction of adenosine triphosphate binding site to protein kinases can be fabricated and further used for adenosine triphosphate analogs screening in different sources. The optimized geometrical structure and energy of the pre-polymerization complexes of tri-O-acetyladenosine (template) with three different monomers-methacrylic acid, 3-vinyl benzoic acid and acrylamide in vacuum were calculated using hybrid quantum mechanical/molecular mechanical (QM/MM) approach. These calculations demonstrate that methacrylic acid forms the most stable complex with template, the next is 3-vinyl benzoic acid complex and the third-acrylamide one. The bond energies of the complexes are shown to increase monotonically as more monomers are linked to the template. The same conclusions are made from purely quantum self-consistent field calculations of pre-polymerization complex energy and structure. Hybrid calculation is shown to be effective and can substantially accelerate the development of the imprinting technology.

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Pathogen inactivation of platelets using ultraviolet C light: effect on in vitro function and recovery and survival of platelets

We evaluated the effect of treating platelets (PLTs) using ultraviolet (UV)C light without the addition of any photosensitizing chemicals on PLT function in vitro and PLT recovery and survival in an autologous radiolabeled volunteer study. For in vitro studies, pooled or single buffy coat-derived PLT concentrates (PCs) were pooled and split to obtain identical PCs that were either treated with UVC or untreated (n = 6 each) and stored for 7 days. PLT recovery and survival were determined in a two-arm parallel autologous study in healthy volunteers performed according to BEST guidelines. UVC-treated or untreated PCs (n = 6 each) were stored for 5 days and were compared to fresh PLTs from the same donor. There were no significant differences on Day 7 of storage between paired UVC-treated and control PC units for pH, adenosine triphosphate, lactate dehydrogenase, CD62P, CD63, PLT microparticles, and JC-1 binding, but annexin V binding, lactate accumulation, and expression of CD41/61 were significantly higher in treated units (p < 0.05). Compared with control units, the recovery and survival of UVC-treated PC were reduced after 5 days of storage (p < 0.05) and when expressed as a percentage of fresh values, survival was reduced by 20% (p = 0.005) and recovery by 17% (p = 0.088). UVC-treated PLTs stored for 5 days showed marginal changes in PLT metabolism and activation in vitro and were associated with a degree of reduction in recovery and survival similar to other pathogen inactivation systems that are licensed and in use.

Graphene Enhanced Electron Transfer at Aptamer Modified Electrode and Its Application in Biosensing

Graphene (GN), a two-dimensional and one-atom thick carbon sheet, is showing exciting applications because of its unique morphology and properties. In this work, a new electrochemical biosensing platform by taking advantage of the ultrahigh electron transfer ability of GN and its unique GN/ssDNA interaction was reported. Adenosine triphosphate binding aptamer (ABA) immobilized on Au electrode could strongly adsorb GN due to the strong π-π interaction and resulted in a large decrease of the charge transfer resistance (R(ct)) of the electrode. However, the binding reaction between ABA and its target adenosine triphosphate (ATP) inhibited the adsorption of GN, and R(ct) could not be decreased. On the basis of this, we developed a new GN-based biosensing platform for the detection of small molecule ATP. The experimental results confirmed that the electrochemical aptasensor we developed possessed a good sensitivity and high selectivity for ATP. The detection range for ATP was from 15 × 10(-9) to 4 × 10(-3) M. The method here was label-free and sensitive and did not require sophisticated fabrication. Furthermore, we can generalize this strategy to detect Hg(2+) using a thymine (T)-rich, mercury-specific oligonucleotide. Therefore, we expected that this method may offer a promising approach for designing high-performance electrochemical aptasensors for the sensitive and selective detection of a spectrum of targets.

Mechanism of flexibility control for ATP access of hepatitis C virus NS3 helicase

Hepatitis C virus (HCV) NS3 helicase couples adenosine triphosphate (ATP) binding and hydrolysis to polynucleotide unwinding. Understanding the regulation mechanism of ATP binding will facilitate targeting of the ATP-binding site for potential therapeutic development for hepatitis C. T324, an amino acid residue connecting domains 1 and 2 of NS3 helicase, has been suggested as part of a flexible hinge for opening of the ATP-binding cleft, although the detailed mechanism remains largely unclear. We used computational simulation to examine the mutational effect of T324 on the dynamics of the ATP-binding site. A mutant model, T324A, of the NS3 helicase apo structure was created and energy was minimized. Molecular dynamics simulation was conducted for both wild type and the T324A mutant apo structures to compare their differences. For the mutant structure, histogram analysis of pairwise distances between residues in domains 1 and 2 (E291-Q460, K210-R464 and R467-T212) showed that separation between the two domains was reduced by ∼10% and the standard deviation by ∼33%. Root mean square fluctuation (RMSF) analysis demonstrated that residues in close proximity to residue 324 have at least 30% RMSF value reductions in the mutant structure. Solvent RMSF analysis showed that more water molecules were trapped near D290 and H293 in domain 1 to form an extensive interaction network constraining cleft opening. We also demonstrated that the T324A mutation established a new atomic interaction with V331, revealing that an atomic interaction cascade from T324 to residues in domains 1 and 2 controls the flexibility of the ATP-binding cleft.

Gefitinib Enhances Cytotoxicities of Antimicrotubule Agents in Non–Small-Cell Lung Cancer Cells Exhibiting No Sensitizing Epidermal Growth Factor Receptor Mutation

Although randomized clinical trials showed no benefit from combining epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) with standard chemotherapy for advanced non-small-cell lung cancer (NSCLC), better results might be obtained by combining EGFR-TKI with individual agents that are substrates for the adenosine triphosphate binding cassette transporters (ABCTs) because EGFR-TKIs can inhibit their efflux. The combination effects deserved to be further examined in vitro. The combination effects of gefitinib with three antimicrotubule agents (AMTAs), paclitaxel, docetaxel or vinorelbine, or with gemcitabine were tested in 17 NSCLC cell lines using the tetrazolium colorimetric assay and classical isobole method. The effects of drug combinations, identified by the values of mean combination index (mCIs), were correlated with the expression levels of ABCTs. Dose-versus-log-response curves were analyzed to further evaluate the possible mechanisms of drug interactions. Synergistic gefitinib/AMTA interactions were observed in the tested cell lines. The synergism was more robust in the four lines overexpressing de novo or acquired P-glycoprotein (Pgp; individual mCIs range, 0.484-0.859; all p values were < 0.05), or in 12 cell lines exhibiting no sensitizing EGFR mutations (group mCIs for gefitinib/paclitaxel, gefitinib/docetaxel, and gefitinib/vinorelbine were 0.869, 0.82, and 0.853, respectively. All p values were < 0.02). The synergism could be observed in cells expressing nearly undetectable Pgp and other ABCTs tested in this study. The combination of gefitinib/gemcitabine was additive (mCI = 1.027). Combined gefitinib/AMTAs showed synergism in NSCLC cells harboring no sensitizing EGFR mutations. Gefitinib could enhance AMTA effects through mechanisms not restricted to Pgp blockage.

Graphene-Based Aptamer Logic Gates and Their Application to Multiplex Detection

In this work, a GO/aptamer system was constructed to create multiplex logic operations and enable sensing of multiplex targets. 6-Carboxyfluorescein (FAM)-labeled adenosine triphosphate binding aptamer (ABA) and FAM-labeled thrombin binding aptamer (TBA) were first adsorbed onto graphene oxide (GO) to form a GO/aptamer complex, leading to the quenching of the fluorescence of FAM. We demonstrated that the unique GO/aptamer interaction and the specific aptamer-target recognition in the target/GO/aptamer system were programmable and could be utilized to regulate the fluorescence of FAM via OR and INHIBIT logic gates. The fluorescence changed according to different input combinations, and the integration of OR and INHIBIT logic gates provided an interesting approach for logic sensing applications where multiple target molecules were present. High-throughput fluorescence imagings that enabled the simultaneous processing of many samples by using the combinatorial logic gates were realized. The developed logic gates may find applications in further development of DNA circuits and advanced sensors for the identification of multiple targets in complex chemical environments.

Donor age and ABCB1 1199G>A genetic polymorphism are independent factors affecting long-term renal function after kidney transplantation

BackgroundIn renal tubular cells, cytochrome P4503A enzyme and adenosine triphosphate–binding cassette transporter activities result in intracellular drug or metabolite exposure variability, depending on genetic polymorphisms. Our aim was to establish whether long-term renal function is affected by genetic polymorphisms in biotransformation enzymes and drug transporters of the donor after kidney transplantation. Materials and methodsThe study was conducted in a selected cohort of 97 kidney recipients. Genotyping of donors was performed on renal biopsy samples obtained before transplantation. Serum creatinine levels and Cockcroft–Gault estimated glomerular filtration rate were considered 1 y after transplantation and at the last follow-up. ResultsLong-term function was significantly better in recipients of an organ from donors carrying the ABCB1 1199A mutated allele (median and range creatinine values were 1.1 mg/dL [0.8–1.5mg/dL] in case of at least one ABCB1 1199A allele versus 1.5 mg/dL [0.7–3.7 mg/dL] for homozygous carriers of wild-type allele, P < 0.01). ABCB1 1199G>A polymorphism and donor age had an independent impact on both serum creatinine and estimated glomerular filtration rate. Unlike donor age, the mutated ABCB1 1199A allele was found to have a protective effect on renal function. ConclusionsDonor age and ABCB1 1199G>A polymorphism affect long-term renal function after transplantation. Analysis of genetic factors offers a promising approach to calcineurin inhibitor toxicity risk assessment.

Current and emerging strategies for the management of imatinib-refractory advanced gastrointestinal stromal tumors

Since its approval by the US Food and Drug Administration in February 2002, the tyrosine kinase inhibitor, imatinib, has become the standard of care for patients with metastatic or unresectable KIT-positive gastrointestinal stromal tumors (GISTs). Imatinib functions by blocking the adenosine triphosphate binding site of the constitutively activated mutant KIT or platelet-derived growth factor receptor α, effectively shutting down the oncogenic signal that drives up to 90% of these tumors. In doing so, it has transformed the management of a condition previously refractory to systemic treatments and established GIST as a model for the use of targeted therapies and oncogene addiction in solid tumors. However, while more than 80% of patients will receive clinical benefit from imatinib monotherapy, more than half will develop progressive disease by 2 years. In this article we review the mechanism and patterns of imatinib resistance in GIST; attempt to offer a practical schema for managing imatinib-refractory patients; and lastly, offer some insight as to future directions and emerging therapeutics for the management of this highly interesting and challenging disease.

Multitargeted Tyrosine Kinase Inhibitors in Unselected Patients With Advanced Non–Small-Cell Lung Cancer (NSCLC): Impressions From MONET (the Motesanib NSCLC Efficacy and Tolerability Study)

Angiogenesis is a complex and tightly regulated process that is defined as the growth of new blood vessels, especially those that supply oxygen and nutrients to cancerous tissue, from existing vasculature. Because of the critical role of angiogenesis in the growth and metastases from solid tumors, including non–small-cell lung cancer (NSCLC), its inhibition represents a rational therapeutic strategy. Vascular endothelial growth factor (VEGF) is a key ligand and regulator of both physiologic and pathologic angiogenesis, including that of tumors, with signaling occurring mainly through the VEGF receptor 2 (VEGFR-2). Bevacizumab, a humanized monoclonal antibody against circulating VEGF, was the first antiangiogenesis drug to enter clinical practice for NSCLC. Because of an increased risk of life-threatening bleeding among patients with squamous cell carcinoma in the phase II trial, subsequent trials were limited to nonsquamous histology. The addition of bevacizumab to chemotherapy was associated with improved response rate (RR) and progression-free survival (PFS) in two large randomized trials, Eastern Cooperative Oncology Group (ECOG) 4599 and Avastin in Lung (AVAiL), although only ECOG 4599 was associated with improved overall survival (OS). Unfortunately, the benefit from bevacizumab in patients with advanced NSCLC is modest and transient, with essentially all responding patients developing secondary resistance. One of the mechanisms that is implicated in the development of resistance is the stimulation of compensatory proangiogenic factors such as fibroblast growth factor and platelet-derived growth factor(PDFGR). Tyrosine kinase inhibitors (TKIs) compete with adenosine triphosphate for the active site of the kinase domain. Because of the well-conserved adenosine triphosphate binding sites of the kinases, many TKIs (unlike bevacizumab, which exclusively targets the VEGF ligand) inhibit multiple receptors. This ability to inhibit multiple angiogenesis pathways offers the potential for increased efficacy and decreased development of secondary resistance through redundant pathways. Nevertheless, the decreased specificity may be associated with increased toxicity. Given that single-agent TKIs directed against similar targets such as sorafenib (VEGF-R2, VEGFR-3, PDGFR, RAF-1, BRAF, c-KIT, FLT-3), sunitinib (VEGFR-2, PDGFR, FLT-3, c-KIT, RET), and axitinib (VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, c-KIT) have been well tolerated in NSCLC, the next step would be to pursue their combination with chemotherapy or other biologic agents. Several randomized clinical trials comparing chemotherapy alone or with the addition of TKIs have been conducted (Table 1). In phase II studies, neither the addition of vandetanib nor cediranib improved the median PFS or OS compared with chemotherapy alone using carboplatin and paclitaxel. Lack of OS improvement was also observed in the two randomized phase III studies in the first-line setting, the Evaluation of Sorafenib, Carboplatin, and Paclitaxel Efficacy (ESCAPE) and NSCLC Research Experience Utilizing Sorafenib (NExUS), as well as in previously treated patients. Motesanib is a small-molecule targeted antagonist of VEGF receptors 1, 2, and 3, PDGFR, and KIT. In the phase IB study, five (17%) of 29 patients who were treated with motesanib (50 mg per day, 125 mg per day, or 75 mg twice per day) in combination with carboplatin and paclitaxel achieved partial response. Because of increased toxicity, the twice-daily cohort was discontinued. The recommended motesanib dose for subsequent studies was 125 mg once per day, which had already been established as the maximum-tolerated dose for singleagent therapy. In the phase II study, there were no significant differences in outcomes between patients treated with carboplatin plus paclitaxel and either bevacizumab or motesanib. In the article that accompanies this editorial, Scagliotti et al present the results of the Motesanib NSCLC Efficacy and Tolerability (MONET1) trial, a multicenter, randomized, double-blind phase III trial comparing carboplatin and paclitaxel alone or in combination with motesanib 125 mg per day as first-line therapy for NSCLC. The primary end point was OS. All tumor types were initially included, but squamous cell carcinoma was excluded after increasing gross hemoptysis and mortality JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 30 NUMBER 23 AUGUST 1

Retinal remodeling

Retinal photoreceptor degeneration takes many forms. Mutations in rhodopsin genes or disorders of the retinal pigment epithelium, defects in the adenosine triphosphate binding cassette transporter, ABCR gene defects, receptor tyrosine kinase defects, ciliopathies and transport defects, defects in both transducin and arrestin, defects in rod cyclic guanosine 3',5'-monophosphate phosphodiesterase, peripherin defects, defects in metabotropic glutamate receptors, synthetic enzymatic defects, defects in genes associated with signaling, and many more can all result in retinal degenerative disease like retinitis pigmentosa (RP) or RP-like disorders. Age-related macular degeneration (AMD) and AMD-like disorders are possibly due to a constellation of potential gene targets and gene/gene interactions, while other defects result in diabetic retinopathy or glaucoma. However, all of these insults as well as traumatic insults to the retina result in retinal remodeling. Retinal remodeling is a universal finding subsequent to retinal degenerative disease that results in deafferentation of the neural retina from photoreceptor input as downstream neuronal elements respond to loss of input with negative plasticity. This negative plasticity is not passive in the face of photoreceptor degeneration, with a phased revision of retinal structure and function found at the molecular, synaptic, cell, and tissue levels involving all cell classes in the retina, including neurons and glia. Retinal remodeling has direct implications for the rescue of vision loss through bionic or biological approaches, as circuit revision in the retina corrupts any potential surrogate photoreceptor input to a remnant neural retina. However, there are a number of potential opportunities for intervention that are revealed through the study of retinal remodeling, including therapies that are designed to slow down photoreceptor loss, interventions that are designed to limit or arrest remodeling events, and optogenetic approaches that target appropriate classes of neurons in the remnant neural retina.

An ATP-Site On-Off Switch That Restricts Phosphatase Accessibility of Akt

The protein serine-threonine kinase Akt undergoes a substantial conformational change upon activation, which is induced by the phosphorylation of two critical regulatory residues, threonine 308 and serine 473. Paradoxically, treating cells with adenosine 5'-triphosphate (ATP)-competitive inhibitors of Akt results in increased phosphorylation of both residues. We show that binding of ATP-competitive inhibitors stabilized a conformation in which both phosphorylated sites were inaccessible to phosphatases. ATP binding also produced this protection of the phosphorylated sites, whereas interaction with its hydrolysis product adenosine 5'-diphosphate (ADP) or allosteric Akt inhibitors resulted in increased accessibility of these phosphorylated residues. ATP-competitive inhibitors mimicked ATP by targeting active Akt. Forms of Akt activated by an oncogenic mutation or myristoylation were more potently inhibited by the ATP-competitive inhibitors than was wild-type Akt. These data support a new model of kinase regulation, wherein nucleotides modulate an on-off switch in Akt through conformational changes, which is disrupted by ATP-competitive inhibitors.

Aptamer-conjugated silver nanoparticles for electrochemical detection of adenosine triphosphate

The capability of silver nanoparticles (SNP) as redox tag in the construction of an electrochemical aptasensor for the detection of adenosine triphosphate (ATP) is investigated in the present manuscript. To construct the aptasensor, a well-known ATP binding aptamer (ABA) splits into two segments. The first amino-labeled segment of the aptamer was covalently immobilized on 3-mercaptopropionic acid modified gold electrode surface by the formation of carbodiimide bond. The second segment was modified by SNPs and associated with the first segment in the presence of ATP. The direct oxidation signal of SNPs is followed as the analytical signal to detect ATP. The sandwich assay shows a suitable signal gain and importantly, a good response time. The sensor can detect the concentrations of ATP as low as micromolar scales with a desirable stability under optimum conditions. Furthermore, analog nucleotides including GTP, UTP and CTP, do not show serious interferences and this sensor readily detects its target in a complex media such as human blood plasma.