Slow Efflux Research Articles

18F- or 177Lu-labeled bivalent ligand of fibroblast activation protein with high tumor uptake and retention.

Fibroblast activation protein (FAP) has become a promising cancer-related target for diagnosis and therapy. The aim of this study was to develop a bivalent FAP ligand for both diagnostic PET imaging and endoradiotherapy. We synthesized a bivalent FAP ligand (ND-bisFAP) and labeled it with 18F or 177Lu. FAP-positive A549-FAP cells were used to study competitive binding to FAP, cellular internalization, and efflux properties in vitro. Micro-PET imaging with [18F]AlF-ND-bisFAPI was conducted in mice bearing A549-FAP or U87MG tumors. Biodistribution and therapeutic efficacy of [177Lu]Lu-ND-bisFAPI were conducted in mice bearing A549-FAP tumors. The FAP binding affinity of ND-bisFAPI is 0.25 ± 0.05nM, eightfold higher in potency than the monomeric DOTA-FAPI-04 (IC50 = 2.0 ± 0.18nM). In A549-FAP cells, ND-bisFAPI showed specific uptake, a high internalized fraction, and slow cellular efflux. Compared to the monomeric [18F]AlF-FAPI-42, micro-PET imaging with [18F]AlF-ND-bisFAPI showed higher specific tumor uptake and retention for at least 6h. Biodistribution studies showed that [177Lu]Lu-ND-bisFAPI had higher tumor uptake than [177Lu]Lu-FAPI-04 at the 24, 72, 120, and 168h time points (all P < 0.01). [177Lu]Lu-ND-bisFAPI delivered fourfold higher radiation than [177Lu]Lu-FAPI-04 to A549-FAP tumors. For the endoradiotherapy study, 37MBq of [177Lu]Lu-ND-bisFAPI significantly reduced tumor growth compared to the same dose of [177Lu]Lu-FAPI-04. Half of the dose of [177Lu]Lu-ND-bisFAPI (18.5MBq) has comparable median survival as 37MBq of [177Lu]Lu-FAPI-04 (37 vs 36days). The novel bivalent FAP ligand was developed as a theranostic radiopharmaceutical and showed promising properties including higher tumor uptake and retention compared to the established radioligands [18F]AlF-FAPI-42 and [177Lu]Lu-FAPI-04. Preliminary experiments with 18F- or 177Lu-labeled ND-bisFAPI showed promising imaging properties and favorable anti-tumor responses.

The Whole Is Bigger than the Sum of Its Parts: Drug Transport in the Context of Two Membranes with Active Efflux.

Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis-Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux.

Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette – based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

Dry spinal tap during real-time ultrasound-guided paramedian spinal injection with patient in the lateral decubitus position: A single-centre retrospective study.

Real-time ultrasound-guided (USG) spinal injection is generally performed via the paramedian sagittal oblique (PMSO) ultrasound window. The aim of this retrospective study was to draw attention to the occurrence of 'dry tap' during real-time USG spinal injection. Single-centre retrospective study. University teaching hospital, Hong Kong, China. Data from 113 patients (aged 69.2 ± 18.0 years and BMI 22.3 ± 3.6 kg m-2) of American Society of Anesthesiologists physical status 1 to 3 scheduled for surgery under neuraxial blockade between 2007 to 2017 were reviewed. Real-time USG spinal injections or combined spinal-epidural (CSE) using the PMSO ultrasound window with the patient in the lateral decubitus position and the spinal needle inserted from the nondependent side were studied. 'Dry tap' was defined as a failure of cerebrospinal fluid (CSF) to efflux from the hub of the needle, within 3 min, with the spinal needle visualised sonographically within the thecal sac. 'Slow CSF efflux' was defined as efflux of CSF within 1 to 3 min. Irrespective of whether it was a 'dry tap' or 'slow CSF efflux', the planned dose of local anaesthetic was injected through the spinal needle. The combined incidence of 'dry tap' and 'slow CSF efflux' was 23.8% (27/113) with an individual incidence for each event of 9.7% (11/113) and 14.2% (16/113), respectively. Under the conditions of this study, successful spinal anaesthesia developed in all patients. 'Dry tap' occurs in 9.7% of cases during real-time USG spinal injection using the PMSO ultrasound window, with the patient in the lateral decubitus position and the spinal needle inserted from the nondependent side. ChiCTR-IOR-1800019011, Chinese Clinical Trials Registry (www.chictr.org.cn).

Therapeutic Considerations for Docetaxel and Paclitaxel in Metastatic Breast Cancer

Breast cancer is the main source of death among women. Currently, 77% of women diagnosed with breast cancer are age 50 and older; however, it is projected that approximately 66% of the new cases diagnosed will occur in women younger than 65. Several clinical trials have assessed the wellbeing and adequacy of taxanes along with their tolerability in patients with metastatic cancer (MBC) The overview of these Paclitaxel and Docetaxel, the mechanism of action, pharmacokinetics and pharmacodynamics, dose and administration, adverse effects, clinical potency, and sufferable profiles combination therapies, the pathological complete response of these taxanes are included. The different novel formulations of taxanes are formulated from nanoparticles, polyglutamate, liposomes to improve the wellbeing and adequacy taxanes to reduce their toxicities. Single-agent research located with docetaxel and paclitaxel in metastatic breast most cancers show clinically huge antitumor motion even in the advanced stage, heavily pretreated, safe, as properly as in refractory diseases. This action is likewise clear with taxane-based combination regimens. Serious hematologic and nonhematologic toxicities are incompatible, with different toxicities noted dependent on the portion and weekly regimen selected. Weekly docetaxel and paclitaxel regimens speak to important helpful treatment options for women suffering from metastatic breast cancer and have entered assessment as a major aspect of adjuvant treatment for this disease Toxicity associated with taxanes chemotherapy are based totally on the dose schedules and weekly regimen selected and the most frequent toxicities related with these marketers include myalgia, peripheral neuropathy, neutropenia, etc Docetaxel retains in tumor cells for longer duration when compared to paclitaxel because of its slow efflux and large amounts of uptake into the cell which explains its more benefits when compared to paclitaxel. Clinical studies conducted so far suggested a more benefit to risk ratio for docetaxel when compared to paclitaxel. This article reviews mainly different actions exhibited by taxanes in the therapy of metastatic breast cancer and others on stages of cancer along with the toxicities associated with these agents.&#x0D;

194 - Mitochondrial matrix H2O2 release in INS-1E cells monitored by the H2O2-selective fluorescent probe Mito-HyPer

Mitochondria of pancreatic β-cells act as the perfect glucose sensor substantiating glucose-stimulated insulin secretion (GSIS). Despite controversies, whether this leads or not to the increased superoxide release into the matrix, it has been recently indicated that H2O2 degradation is favored in the matrix upon GSIS [1]. We attempted to study this in model β-cells, INS-1E cells [2], using confocal microscopy time-resolved monitoring with Mito-HyPer. We found a substantial negative drift in fluorescence F485/F405 ratio, indicating degradation and possible slow H2O2 efflux in INS-1E cells pre-incubated with 3 mM or 11 mM glucose. After glucose addition to 25 mM, the H2O2 decrease was enhanced by several folds, which was prevented by inhibition of mitochondrial redox shuttles exporting NADPH to the cell cytosol on the expense of matrix NADH. We conclude that pancreatic β-cells do not create mitochondrial oxidative stress upon GSIS.

Nucleopeptide Assemblies Selectively Sequester ATP in Cancer Cells to Increase the Efficacy of Doxorubicin

AbstractHerein, we report that assemblies of nucleopeptides selectively sequester ATP in complex conditions (for example, serum and cytosol). We developed assemblies of nucleopeptides that selectively sequester ATP over ADP. Counteracting enzymes interconvert ATP and ADP to modulate the nanostructures formed by the nucleopeptides and the nucleotides. The nucleopeptides, sequestering ATP effectively in cells, slow down efflux pumps in multidrug‐resistant cancer cells, thus boosting the efficacy of doxorubicin, an anticancer drug. Investigation of 11 nucleopeptides (including d‐ and l‐enantiomers) yields five more nucleopeptides that differentiate ATP and ADP through either precipitation or gelation. As the first example of assemblies of nucleopeptides that interact with ATP and disrupt intracellular ATP dynamics, this work illustrates the use of supramolecular assemblies to interact with small and essential biological molecules for controlling cell behavior.

Nucleopeptide Assemblies Selectively Sequester ATP in Cancer Cells to Increase the Efficacy of Doxorubicin.

Herein, we report that assemblies of nucleopeptides selectively sequester ATP in complex conditions (for example, serum and cytosol). We developed assemblies of nucleopeptides that selectively sequester ATP over ADP. Counteracting enzymes interconvert ATP and ADP to modulate the nanostructures formed by the nucleopeptides and the nucleotides. The nucleopeptides, sequestering ATP effectively in cells, slow down efflux pumps in multidrug-resistant cancer cells, thus boosting the efficacy of doxorubicin, an anticancer drug. Investigation of 11 nucleopeptides (including d- and l-enantiomers) yields five more nucleopeptides that differentiate ATP and ADP through either precipitation or gelation. As the first example of assemblies of nucleopeptides that interact with ATP and disrupt intracellular ATP dynamics, this work illustrates the use of supramolecular assemblies to interact with small and essential biological molecules for controlling cell behavior.

Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae.

Some hyperthermophilic heterotrophs in the genus Thermococcus produce H2 in the absence of S° and have up to seven hydrogenases, but their combined physiological roles are unclear. Here, we show which hydrogenases in Thermococcus paralvinellae are affected by added H2 during growth without S°. Growth rates and steady-state cell concentrations decreased while formate production rates increased when T. paralvinallae was grown in a chemostat with 65 µM of added H2(aq) . Differential gene expression analysis using RNA-Seq showed consistent expression of six hydrogenase operons with and without added H2 . In contrast, expression of the formate hydrogenlyase 1 (fhl1) operon increased with added H2 . Flux balance analysis showed H2 oxidation and formate production using FHL became an alternate route for electron disposal during H2 inhibition with a concomitant increase in growth rate relative to cells without FHL. T. paralvinellae also grew on formate with an increase in H2 production rate relative to growth on maltose or tryptone. Growth on formate increased fhl1 expression but decreased expression of all other hydrogenases. Therefore, Thermococcus that possess fhl1 have a competitive advantage over other Thermococcus species in hot subsurface environments where organic substrates are present, S° is absent and slow H2 efflux causes growth inhibition.

Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation

ObjectiveTo investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). MethodsFifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration–response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. ResultsAll 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 μg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 μg/mL. ConclusionsThe present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

Evaluation of DOTA-chelated neurotensin analogs with spacer-enhanced biological performance for neurotensin-receptor-1-positive tumor targeting

IntroductionNeurotensin receptor 1 (NTR1) is overexpressed in many cancer types. Neurotensin (NT), a 13 amino acid peptide, is the native ligand for NTR1 and exhibits high (nM) affinity to the receptor. Many laboratories have been investigating the development of diagnostic and therapeutic radiopharmaceuticals for NTR1-positive cancers based on the NT peptide. To improve the biological performance for targeting NTR1, we proposed NT analogs with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelation system and different lengths of spacers. MethodsWe synthesized four NTR1-targeted conjugates with spacer lengths from 0 to 9 atoms (null (N0), β-Ala-OH (N1), 5-Ava-OH (N2), and 8-Aoc-OH (N3)) between the DOTA and the pharmacophore. In vitro competitive binding, internalization and efflux studies were performed on all four NT analogs. Based on these findings, metabolism studies were carried out on our best performing conjugate, 177Lu-N1. Lastly, in vivo biodistribution and SPECT/CT imaging studies were performed using 177Lu-N1 in an HT-29 xenograft mouse model. ResultsAs shown in the competitive binding assays, the NT analogs with different spacers (N1, N2 and N3) exhibited lower IC50 values than the NT analog without a spacer (N0). Furthermore, N1 revealed higher retention in HT-29 cells with more rapid internalization and slower efflux than the other NT analogs. In vivo biodistribution and SPECT/CT imaging studies of 177Lu-N1 demonstrated excellent accumulation (3.1±0.4%ID/g) in the NTR1-positive tumors at 4h post-administration. ConclusionsThe DOTA chelation system demonstrated some modest steric inhibition of the pharmacophore. However, the insertion of a 4-atom hydrocarbon spacer group restored optimal binding affinity of the analog. The in vivo assays indicated that 177Lu-N1 could be used for imaging and radiotherapy of NTR1-positive tumors.

Comparing trace metal bioaccumulation characteristics of three freshwater decapods of the genus Macrobrachium

Potential sources and kinetics of metal bioaccumulation by the three Macrobrachium prawn species M. australiense, M. rosenbergii and M. latidactylus were assessed in laboratory experiments. The prawns were exposed to two scenarios: cadmium in water only; and exposure to metal-rich mine tailings in the same water. The cadmium accumulation from the dissolved exposure during 7 days, followed by depuration in cadmium-free water for 7 days, was compared with predictions from a biokinetic model that had previously been developed for M. australiense. M. australiense and M. latidactylus accumulated significant tissue cadmium during the exposure phase, albeit with different uptake rates. All three species retained >95% of the bioaccumulated cadmium during the depuration phase, indicating very slow efflux rates. Following exposure to tailings, there were significant (p<0.05) differences in tissue arsenic, cadmium, lead and zinc concentrations among species. Cadmium and zinc concentrations were increased relative to controls for all three species but were not different between treatments (direct/indirect contact with tailings), suggesting these metals were primarily accumulated via the dissolved phase. All species bioaccumulated significantly greater arsenic and lead when in direct contact with mine tailings, demonstrating the importance of an ingestion pathway for these metals. Copper was not bioaccumulated above control concentrations for any species. The differences between the metal accumulation of the three prawns indicated that a biokinetic model of cadmium bioaccumulation for M. australiense could potentially be used to describe the metal bioaccumulation of the other two prawn species, albeit with an over-prediction of 3–9 times. Despite these being the same genus of decapod crustacean, the study highlights the issues with using surrogate species, even under controlled laboratory conditions. It is recommended that future studies using surrogate species quantify the metal bioaccumulation characteristics of each species in order to account for any differences between species.

Smart pH‐Responsive Nanocarriers Based on Nano‐Graphene Oxide for Combined Chemo‐ and Photothermal Therapy Overcoming Drug Resistance

A pH-responsive nanocarrier is developed by coating nanoscale graphene oxide (NGO) with dual types of polymers, polyethylene glycol (PEG) and poly(allylamine hydrochloride) (PAH), the latter of which is then modified with 2,3-dimethylmaleic anhydride (DA) to acquire pH-dependent charge reversibility. After loading with doxorubicin (DOX), a chemotherapy drug, the obtained NGO-PEG-DA/DOX complex exhibits a dual pH-responsiveness, showing markedly enhanced cellular uptake under the tumor microenvironmental pH, and accelerated DOX release under a further lowered pH inside cell lysosomes. Combining such a unique behavior with subsequently slow efflux of DOX, NGO-PEG-DA/DOX offers remarkably improved cell killing for drug-resistant cancer cells under the tumor microenvironmental pH in comparison with free DOX. Exploiting its excellent photothermal conversion ability, combined chemo- and photothermal therapy is further demonstrated using NGO-PEG-DA/DOX, realizing a synergistic therapeutic effect. This work presents a novel design of surface chemistry on NGO for the development of smart drug delivery systems responding to the tumor microenvironment and external physical stimulus, with the potential to overcome drug resistance.

ECG Response: August 20, 2013

HomeCirculationVol. 128, No. 8ECG Response: August 20, 2013 Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBECG Response: August 20, 2013 Originally published20 Aug 2013https://doi.org/10.1161/CIRCULATIONAHA.113.005120Circulation. 2013;128:869ECG Challenge: A 67-year-old man recently underwent radical neck surgery for pharyngeal cancer. Three days later, he developed severe muscle twitching and spasms and sharp chest pain. An ECG was obtained.The rhythm is regular at a rate of 78 bpm. There is a P wave (+) before each QRS complex with a stable PR interval (0.16 second). The P waves are positive in leads I, II, aVF, and V4 through V6. Hence, this is a normal sinus rhythm. The QRS complex duration is normal (0.08 second) and has a normal axis (between 0° and 90°, positive QRS complex in leads I and aVF). The QRS complex has a normal morphology, although there is poor R-wave progression in leads V1 and V2. This is likely a normal variant, although an old anteroseptal myocardial infarction should be considered. The QT/QTc intervals are prolonged (480/540 milliseconds). There are 2 different patterns for QT prolongation. The first is delayed repolarization in which the long-QT interval is the result of a long ST segment (^) and the T wave is normal in duration. This pattern is seen with either hypocalcemia or hypomagnesemia, which results in a prolongation in phase 2 of the action potential and hence a delay in the onset of repolarization. This type of QT prolongation is not associated with any cardiac abnormalities; specifically, it is not associated with torsade des pointes. The second pattern is called prolonged repolarization in which there is a widening of the T wave (prolongation in the T-wave duration). This is often the result of a slow efflux of potassium during phase 3 of the action potential, which prolongs repolarization or the refractoriness of the membrane. This is a long-QT pattern that is associated with the development of torsade des pointes, or a long-QT syndrome. The long-QT interval in this situation may be acquired, a result of therapy with a drug that interferes with the efflux of potassium during repolarization (eg, the class III antiarrhythmic drugs), or congenital, a result of a channelopathy that causes a prolongation in membrane refractoriness. In this ECG, the pattern is one of delayed repolarization as there is a long ST segment (^) and a normal T wave. The patient likely developed hypocalcemia as a result of hypoparathyroidism due to the recent neck surgery.Download figureDownload PowerPoint Previous Back to top Next FiguresReferencesRelatedDetails August 20, 2013Vol 128, Issue 8 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.113.005120PMID: 23960258 Originally publishedAugust 20, 2013 PDF download Advertisement SubjectsElectrocardiology (ECG)

Neuronoprotective action of cortexin andcortagen

The changes of both intracellular resting potential and action potential of the identified neurons of pedal ganglia of the mollusk Planorbarius corneus CNS registered by means of intracellular microelectrodes, and ionic currents of isolated neurons under fixed potential after administration of officinal peptide drug cortexine (cortexine 0.5–1000 µg/ ml and glycine 4–4000 µM), cortexine peptides (0.5–1000 µg/ ml), glycine (0.5 and 5 µM) and tetrapeptide cortagen (0.1–10000 µM) were studied. Both the drug cortexine and cortexine peptides were shown to modulate the electrical activity of neurons by the same manner: moderately changed the resting potential, the action potential and impulse frequency that was interpreted as activating action. Glycine activated neuronal activity as cortexine but in less degree. The mixture of cortexine and glycine (drug cortexine) did not inhibit and did not disturb the functional state of neurons of action, and the hyperpolarization was always observed after administration of peptides as well as the shortening of the action potential, increase of their amplitude and reduction of impulse frequency of neurons. Tetrapeptide cortagen in concentrations 0.1–100 µM also hyperpolarized neurons by 2–3 mV and reduced their spontaneous activity that indicated on its activating (neuronoprotective) action. Cortagen in concentration 1000 µM depolarized the neurons moderately (by 2–4 mV), the increase of impulse activity was registered, and in concentration 10 mM it depolarized neurons significantly and reversible, increasing the frequency and inhibiting the generation of the action potential. Cortagen activated neurons in more degree than cortexine. Cortagen in concentration 0.1 µM also increased the amplitude of slow efflux current by 3–5 %. We did not observe the increased amplitude (activation) of influx sodium and calcium channels after administration of cortagen. A dose-dependent and reversible inhibition of amplitudes of these currents began after administration of cortagen in concentrations 100 µM and more up to 80–90 % inhibition after peptide concentration 10 mM. The inhibition of sodium channels was more intensive than calcium channels. These effects of action of the peptide administered in high concentrations can be qualified as nonspecific and toxic ones.

Pharmacologic differences in taxanes leading to difference in clinical activity and toxicity.

e13567 Background: Paclitaxel and docetaxel share major parts of their structures and mechanism of action, but differ in several aspects. Both paclitaxel and docetaxel act at microtubule causing tubulin polymer generation but exhibit pharmacodynamic differences. These pharmacological differences may account for the differences observed between taxanes in their clinical activity and toxicity. Methods: Review of articles providing details of taxane pharmacology was conducted to evaluate pharmacological basis. Results: Greater uptake and slower efflux of docetaxel from tumor cells leads to longer retention time; whereas equal uptake and efflux of paclitaxel shorter retention time for paclitaxel. Hence, apoptotic and mitotic responses of paclitaxel is complete within 4 days implying that more frequent (weekly) administration of paclitaxel will be superior. High affinity to β-tubulin with docetaxel (1.9) than with paclitaxel (1.0) allows higher inhibition of tumor growth. Secondly, drug concentration causing maximum polymerization is 0.2 µM and 0.4 µM for docetaxel and paclitaxel, respectively; indicating that docetaxel is twice as potent as paclitaxel in causing microtubule polymerization. Docetaxel arrests cells in the radiosensitive G2/M phase of the cell cycle making it a potent radiosensetizer than paclitaxel. Mechanisms consistent with allosteric modulation of the reductases increase doxorubicinol formation at clinically relevant concentrations of paclitaxel when it is administered with doxorubicin. Enhanced formation of doxorubicinol aglycone, a metabolite potentially involved in the reversible phase of cardiotoxicity causes higher cardiotoxicty when doxorubicin is administered with paclitaxel than with docetaxel. Non linear pharmacokinetics and hypersensitivity with paclitaxel and fluid retention with docetaxel is attributed to excepient of the drug rather than drug itself. Conclusions: Influx and efflux mechanism support superior weekly administration of Paclitaxel. Pharmacological differences between taxanes justify the incomplete resistance seen between taxanes. The incomplete cross resistance is specifically related to docetaxelactivity in paclitaxel resistant cases.

An Image Based Microtiter Plate Reader System for 96-well Format Fluorescence Assays

Background: 96-well microtiter plate assay are becoming popular analytical procedures in laboratory and clinical practices generating a demand for microtiter plate readers. The present colorimetric and fluorimetric based microtiter plate reader are efficient in general applications such as measuring reflectance, absorbance, and optical density of reaction mixtures. However, a dedicated system incorporating automation and computational techniques in specific microtiter plate readers for Drug discovery and Microbial viability, provides portability and compatibility with highthroughput analysis at affordable cost. Objective: To develop image-based reader system for semi-quantitative measurements of transport assay performed in microtiter plate. In this work, we focus on deduction of illumination source and classification of fluorescence emitted by transport assay using this microtiter plate reader. Method: We have taken the images of 96-well fluorescence efflux assay and developed an appropriate image analysis system to read the individual well images and categorize the results as fast efflux and slow efflux by the classifier. The images exhibited non-uniform illumination due to UV transilluminator, which then replaced by the circuit of blue LED after performing a study on its classification performance. Statistical descriptive features extracted from the image are classified using k-means clustering technique. The proposed system can be adapted to read any kind of microtiter plate assay. Results: For uniform illumination of the source, of both UV transilluminator and blue LED, the color profile varied in accord to reaction mixture concentration. For classification performance of the source, out of four different images at various intensities of illumination from blue LED circuit controlled by potentiometer, the classification of efflux assay for image with maximum intensity gave high accuracy than the rest. For classification performance of the classifier, out of 1920-sub-images, the classifier misclassifies none. Conclusion: For the microtiter plate reader, which classifies the liquid transport assay as fast efflux or slow efflux, the usage of UV transilluminator as illumination source is limited due to its non-uniform illumination and blue LED is apt for this application due its uniformity. The k-means clustering technique had been effective for this application when fed with statistical features describing the clusters.

Evolution of Bombesin Conjugates for Targeted PET Imaging of Tumors

Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel 64/67Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC50: 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle14 by Met, and deletion of D-Tyr6, further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6th, 11th, and 14th position of BN induced only slight influences on affinity to mouse GRPr. [CuII]-CPTA-[βAla11] BN(7–14) ([CuII]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [64/67Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [64/67Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [64/67Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes.

Bioaccumulation Dynamics and Modeling in an Estuarine Invertebrate Following Aqueous Exposure to Nanosized and Dissolved Silver

Predicting the environmental impact of engineered nanomaterials (ENMs) is increasingly important owing to the prevalence of emerging nanotechnologies. We derived waterborne uptake and efflux rate constants for the estuarine snail, Peringia ulvae, exposed to dissolved Ag (AgNO(3)) and silver nanoparticles (Ag NPs), using biodynamic modeling. Uptake rates demonstrated that dissolved Ag is twice as bioavailable as Ag in nanoparticle form. Biphasic loss dynamics revealed the faster elimination of Ag from Ag NPs at the start of depuration, but similar slow efflux rate constants. The integration of biodynamic parameters into our model accurately predicted Ag tissue burdens during chronic exposure with 85% of predicted values within a factor of 2 of observed values. Zeta potentials for the Ag NPs were lower in estuarine waters than in waters of less salinity; and uptake rates in P. ulvae were slower than reported for the freshwater snail Lymnaea stagnalis in similar experiments. This suggests aggregation of Ag NPs occurs in estuarine waters and reduces, but does not eliminate, bioavailability of Ag from the Ag NPs. Biodynamic modeling provides an effective methodology to determine bioavailable metal concentrations (originating from metal and metal-oxide nanoparticles) in the environment and may aid future ENM risk assessment.

Cytosolic K+ and extracellular ATP as regulators of NLRP3 inflammasome activation and the IL-1β secretion response of macrophages to crystalline stimuli. (114.9)

Abstract The crystalline particulate Alum has widely been used as an adjuvant in vaccines. Alum and other crystalline particulates also stimulate NLRP3/caspase-1 inflammasome assembly and secretion of mature IL-1β via poorly understood mechanisms that involve decreases in cytosolic [K+]. This study characterized mechanisms for K+ efflux in LPS-primed murine bone marrow-derived macrophages (BMDM) stimulated with either Imject Alum or the Na+, K+-ATPase inhibitor ouabain. Treatment with Alum for 3 h induced robust secretion of IL-1β that was similar in the absence or presence of antagonists of the P2X7 receptor, an ATP-gated K+ efflux channel known to regulate NLRP3 inflammasome assembly. However, the ATP-scavenging enzyme apyrase markedly reduced this IL-1β secretion, suggesting that NLRP3 activation and IL-1β release induced by Alum crystals is at least partially dependent on a P2X receptor subtype(s) other than P2X7. Current experiments are testing possible involvement of P2X4 receptor channels. Treatment of LPS-primed BMDMs with ouabain for 6 h also induced significant release of IL-1β. We hypothesize that ouabain causes NLRP3 activation and IL-1β secretion by inhibiting the steady-state Na+, K+ATPase activity which counteracts the slow efflux of cytosolic K+ via leak/rectifier-type K+ channels.