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The SARS-Cov-2 virion responsible for the current world-wide pandemic Covid-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD). The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions (charge, partial charge, and van der Waals) of the SARS-Cov-2 Spike protein in its static prefusion state based on recent structural information. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences (different so-called “glue” points) between residues in the Up and Down state protomers. In general, the S2 or fusion machinery domain of S is relatively rigid with strong noncovalent interactions facilitated by helical secondary structures, whereas the S1 domain, which contains the RBD and N-terminal domain (NTD), is relatively more flexible and characterized by beta strand structural motifs. The S2 domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer’s intrachain, S1-S2 interactions. However, the S1 domain interactions across neighboring protomers, which include the RBD-NTD cross chain interactions, showed significant energetic differences between Up-Down and Down-Down neighboring protomers. Surprisingly, the Up-Down, RBD-NTD interactions were overall stronger and more numerous than the Down-Down cross chain interactions, including the appearance of the three residue sequence ALA520-PRO521-ALA522 associated with a turn structure in the RBD of the Up state protomer. Additionally, our intrachain dominant energy mappings within each protomer, identified a significant “glue” point or possible “latch” for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-Cov-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. To help verify the potential latch for the Down state protomer, we conducted some preliminary molecular dynamic simulations that effectively turn off this specific latch glue point via a single point mutation of GLN564. Interestingly, the single point mutation lead to the latch releasing in less than a few nanoseconds, but the latch remained fixed in the wild state protomer for up to 0.1 microseconds that were simulated. Many more detailed studies are needed to understand the dynamics of the Up and Down states of the Spike protein, including the stabilizing chain-chain interactions and the mechanisms of transition from Down to Up state protomers. Nonetheless, static dominant energy landscape mappings and preliminary molecular dynamic studies given here may represent a useful starting point for more detailed dynamic analyses and hopefully an improved understanding of the structure-function relationship of this highly complex protein associated with COVID-19.

Bedaquiline, clofazimine and linezolid are pertinent drugs for drug-resistant tuberculosis. Drug-resistant mutants provide insight into important resistance acquisition mechanisms. Methods for in vitro Mycobacterium tuberculosis mutant generation are poorly described.Induction (serial passaging) and spontaneous (adapted Luria-Delbruck assay) approaches using M. tuberculosis ATCC reference strains (one fully-susceptible, four unique mono-resistant) were performed.Mutant MIC values were confirmed (MGIT960) and resultant RAVs compared between approaches and to a catalog of previously published RAVs. Mutant MIC values showed a 3-4-fold (induced) and a 1-4-fold (spontaneous) increase compared to baseline. The pyrazinamide-resistant strain had higher baseline MIC values and acquired resistance (>= 4-fold) in fewer passages than other strains (induction approach) for bedaquiline. Previously described and novel RAVs in atpE (8 vs. 1) and rv0678 (4 vs. 12) genes were identified in bedaquiline- and clofazimine-resistant mutants. No rv1979c and rv2535c RAVs were identified. Previously described RAVs were identified in TIC and rrl genes for linezolid-resistant mutants.Both approaches successfully led to in vitro mutants with novel RAVs being described in atpE and rv0678 genes. It was observed that pre-existing resistance may influence mutant phenotypic and genotypic characteristics and warrants further attention.

We describe and characterize a multi-micron silicon photonics platform that was designed to combine performance, power efficiency, manufacturability, and versatility for integrated photonic applications ranging from data communications to sensors. We outline the attributes needed for broad applicability, high-volume manufacturing, and large-scale deployment of silicon photonics, and describe how the platform is favorable with respect to these attributes. We present demonstrations of key technologies needed for the communications and sensing applications, including low-loss fiber attach, compact low-loss filters, efficient hybrid wavelength division multiplexed lasers, and high-speed electro-absorption modulators and integrated photodetectors.

Six in vitro clofazimine-resistant spontaneous mutants obtained from a wild-type or pyrazinamide-resistant ATCC reference strain were selected to evaluate bedaquiline cross-resistance. The reverse was conducted for bedaquiline mutants. All clofazimine mutants harboring an rv0678 mutation displayed phenotypic cross-resistance. We observed the same for rv0678 bedaquiline mutants; however, atpE bedaquiline mutants showed no phenotypic cross-resistance. This confirms that upfront clofazimine usage may impact subsequent bedaquiline use due to a shared efflux resistance pathway.

High speed back-to-back transmission of NRZ data at 12.5 Gbit/s was achieved over a repeaterless optical network without the use of forward error correction or optical clock recovery using a hybrid integrated silicon photonics optical interconnect. The interconnect comprises an electroabsorption modulator based on dilute nitride multiple quantum well material on GaAs substrate optically coupled to large core silicon waveguide using passive alignment and flip-chip bonding.

Skeletal muscle channelopathies, many of which are inherited as autosomal dominant mutations, include both myotonia and periodic paralysis. Myotonia is defined by a delayed relaxation after muscular contraction, whereas periodic paralysis is defined by episodic attacks of weakness. One sub-type of periodic paralysis, known as hypokalemic periodic paralysis (hypoPP), is associated with low potassium levels. Interestingly, the P1158S missense mutant, located in the third domain S4-S5 linker of the ‘‘skeletal muscle’’ voltage-gated sodium channel, Nav1.4, has been implicated in causing both myotonia and hypoPP. A common trigger for these conditions is physical activity. We previously reported that Nav1.4 is relatively insensitive to changes in extracellular pH compared to Nav1.2 and Nav1.5. Given that intense exercise is often accompanied by blood acidosis, we decided to test whether changes in pH would push gating in P1158S towards either phenotype. Our results indicate that, unlike in WT Nav1.4, low pH depolarizes the voltage-dependence of activation and steady-state fast inactivation, decreases current density, and increases late currents in P1185S. Thus, P1185S turns the normally pH-insensitive Nav1.4 into a proton-sensitive channel. Using action potential modeling we also predict a pH-to-phenotype correlation in patients with P1158S. We conclude that activities which alter blood pH may trigger myotonia or periodic paralysis in P1158S patients.SIGNIFICANCE STATEMENTVoltage-gated sodium channels (Nav) contribute to the physiology and pathophysiology of electrical signaling in excitable cells. Nav subtypes are expressed in a tissue-specific manner, thus they respond differently to physiological modulators. For instance, the cardiac subtype, Nav1.5, can be modified by changes in blood pH; however, the skeletal muscle subtype, Nav1.4, is mostly pH-insensitive. Nav1.4 mutants can mostly cause either hyper-or hypo-excitability in skeletal muscles, leading to conditions such as myotonia or periodic paralysis. P1158S uniquely causes both phenotypes. This study investigates pH-sensitivity in P1158S, and describes how physiological pH changes can push P1158S to cause myotonia and periodic paralysis.

In the brainstem nucleus of the solitary tract (NTS), primary vagal afferent neurons express the transient receptor potential vanilloid subfamily member 1 (TRPV1) at their central terminals where it contributes to quantal forms of glutamate release. The endogenous membrane lipid anandamide (AEA) is a putative TRPV1 agonist in the brain, yet the extent to which AEA activation of TRPV1 has a neurophysiological consequence is not well established. We investigated the ability of AEA to activate TRPV1 in vagal afferent neurons in comparison to capsaicin (CAP). Using ratiometric calcium imaging and whole-cell patch clamp recordings we confirmed that AEA excitatory activity requires TRPV1, binds competitively at the CAP binding site, and has low relative affinity. While AEA-induced increases in peak cytosolic calcium were similar to CAP, AEA-induced membrane currents were significantly smaller. Removal of bath calcium increased the AEA current with no change in peak CAP currents revealing a calcium sensitive difference in specific ligand activation of TRPV1. Both CAP- and AEA-activated TRPV1 currents maintained identical reversal potentials, arguing against a major difference in ion selectivity to resolve the AEA differences in signaling. In contrast with CAP, AEA did not alter spontaneous glutamate release at NTS synapses. We conclude: (1) AEA activation of TRPV1 is markedly different from CAP and produces different magnitudes of calcium influx from whole-cell current; and (2) exogenous AEA does not alter spontaneous glutamate release onto NTS neurons. As such, AEA may convey modulatory changes to calcium-dependent processes, but does not directly facilitate glutamate release.

The pathology of Alzheimer’s disease can ultimately be traced to the increased aggregation stability of Aβ42 peptides which possess two extra residues (Ile 41 & Ala 42) that the non-pathological strain (Aβ40) lacks. We have found Aβ42 fibrils to exhibit stronger energies in inter-chain interactions and we have also identified the cause for this increase to be the result of different Ramachandran angle values in certain residues of the Aβ42 strain compared to Aβ40. These unique angle configurations result in the peptide planes in the fibril structures to be more vertical along the fibril axis for Aβ42 which thus reduces the inter-atomic distance between interacting atoms on vicinal peptide chains thereby increasing the electrostatic interaction energies. We lastly postulate that these different Ramachandran angle values could possibly be traced to the unique conformational folding avenues sampled by the Aβ42 peptide owing to the presence of its two extra residues.

Dravet syndrome is the prototype of SCN1A-mutation associated epilepsies. It is characterised by prolonged seizures, typically provoked by fever. We describe the evaluation of an SCN1A mutation in a child with early-onset temperature-sensitive seizures.The patient carries a heterozygous miss-sense variant (c3818C>T; pAla1273Val) in the NaV1.1 brain sodium channel. We characterised the functional effects of the variant using patch clamp recordings on Chinese Hamster Ovary Cells at different temperatures (32, 37, and 40°C).The variant channels produced a temperature-dependent destabilization of channel activation and fast inactivation. Implementing these empirical abnormalities in a computational model produces a higher threshold for depolarization block in the variant, particularly at 40°C, suggesting a failure to autoregulate at high-input states.These results reveal direct effects of abnormalities in biophysical NaV1.1 channel properties on neuronal dynamics. They illustrate the value of combining cellular measurements with computational models to integrate different scales (gene/channel to patient) of observations.

We analyze the composition profiles within intermixed and non-intermixed AlInGaAs-based multiple quantum wells structures by secondary ion mass spectrometry and observe that the band gap blue shift is mainly attributed to the interdiffusion of In and Ga atoms between the quantum wells and the barriers. Based on these results, several AlInGaAs-based single quantum well (SQW) structures with various compressive strain (CS) levels were grown and their photoluminescence spectra were investigated after the intermixing process involving the encapsulation of thin SiNx dielectric films on the surface followed by rapid thermal annealing. In addition to the annealing temperature, we report that the band gap shift can be also enhanced by increasing the CS level in the SQW. For instance, at an annealing temperature of 850 degrees C, the photoluminescence blue shift is found to reach more than 110 nm for the sample with 1.2%-CS SQW, but only 35 nm with 0.4%-CS SQW. We expect that this relatively larger atomic compositional gradient of In (and Ga) between the compressively strained quantum well and the barrier can facilitate the atomic interdiffusion and it thus leads to the larger band gap shift. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4764856]

Cholecystokinin (CCK) facilitates the process of satiation via activation of vagal afferent neurons innervating the upper gastrointestinal tract. Recent findings indicate CCK acts on these neurons via a ruthenium red (RuR) sensitive pathway that involves members of the vanilloid (V) subfamily of transient receptor potential (TRP) channels. To further test this mechanism, the mouse provides an ideal model in which genetic tools could be applied. However, whether CCK acts by similar mechanism(s) in mice has not been determined. In the present study we explored the actions of CCK on nodose neurons isolated from Sprague Dawley (SD) rat and two strains of mice; C57BL/6 and BalbC using fluorescence-based calcium imaging. With minor exceptions nodose neurons isolated from all species/strains behaved similarly. They all respond to brief depolarization with a large calcium transient. A significant subset of neurons responded to capsaicin (CAP), a TRPV1 agonist, although neurons from C57BL/6 were 10-fold more sensitive to CAP than SD rats or BalbC mice, and a significantly smaller fraction of neurons from BalbC mice responded to CAP. CCK-8 dose-dependently activated a subpopulation of neurons with similar dose dependency, percent responders, and overlap between CCK and CAP responsiveness. In all species/strains CCK-8 induced activation was significantly attenuated (but not completely blocked) by pretreatment with the TRPV channel blocker RuR. Surprisingly, the CCK analogue JMV-180, which is reported to have pure antagonistic properties in rat but mixed agonist/antagonist properties in mice, behaved as a pure antagonist to CCK in both rat and mouse neurons. The pure antagonistic action of JMV-180 in this in vitro preparation suggests that prior reported differential effects of JMV-180 on satiation in rats versus mouse must be mediated by a site other than vagal afferent activation.

The volatile anesthetic isoflurane poses a number of experimental challenges in the laboratory. Due to its rapid evaporation, the open conditions of most in vitro electrophysiological recording systems make the determination of actual isoflurane concentrations a challenge. Since the absolute anesthetic concentration in solution is directly related to efficacy, concentration measurements are important to allow comparisons between laboratory and clinical studies. In this study we quantify the sources of isoflurane loss during experimentation and describe a method for the measurement of isoflurane concentrations using gas chromatography and mass spectrometry simultaneous to in vitro electrophysiological measurements. Serial samples of perfused bath solution allowed correlation of isoflurane concentrations with ongoing biological effects. Saturated physiological solutions contained 13.4 +/- 0.2 mM isoflurane and were diluted to desired "nominal" concentrations for experiments. The perfusion system established stable isoflurane concentrations within the bath by 2 minutes. However, bath isoflurane concentrations varied substantially and unpredictably between experiments. The magnitudes of such discrepancies in isoflurane concentrations spanned clinically important levels. Our studies suggest that, despite countermeasures, solution handling significantly impacted the isoflurane content in the tissue bath. The magnitude of these discrepancies appears to necessitate systematic direct measurement of bath isoflurane concentrations during most in vitro conditions.

New techniques have emerged for the detection of bacteria in blood, because the blood culture as gold standard is slow and insufficiently sensitive when the patient has previously received antibiotics or in the presence of fastidious organisms. DNA-based techniques, hybridisation probes, and PCR-based detection or protein-based detection by mass spectroscopy are aimed at rapid identification of bacteria and provide results within 2 h after the first signal of growth in conventional blood cultures. Also, detection of microorganisms directly in blood by pathogen-specific or broad-range PCR assays (eubacterial or panfungal) shows promising results. Interpretation is complex, however, because of detection of DNA rather than living pathogens, the risk of interfering contamination, the presence of background DNA in blood, and the lack of a gold standard. As these techniques are emerging, clinical value and cost-effectiveness have to be assessed. Nevertheless, molecular assays are expected eventually to replace the current conventional microbiological techniques for detection of bloodstream infections.

Purpose: The aim of this study was to characterize and to validate noninvasive 19 F-magnetic resonance relaxometry for the measurement of oxygen tensions in human glioma xenografts in nude mice. The following three questions were addressed: 1. When perfluorocarbon compounds (PFCs) are administrated intravenously, which tumor regions are assessed by 19 F-MR relaxometry? 2. Are oxygen tension as detected by 19 F-MR relaxometry (pO 2/relaxo ) comparable to Eppendorf O 2 -electrode measurements (pO 2/electrode )? 3. Can 19 F-MR relaxometry be used to detect oxygen tension changes in tumor tissue during carbogen breathing? Methods and Materials: Slice-selective 19 F-MR relaxometry was carried out with perfluoro-15-crown-5-ether as oxygen sensor. The PFC was injected i.v. 3 days before the 19 F-MR experiments. Two datasets were acquired before and two after the start of carbogen breathing. The distribution of PFCs and necrotic areas were analyzed in 19 F-Spin Echo (SE) density MR images and T 2 -weighted 1 H-SE MR images, respectively. One day after the MR investigations, oxygen tensions were measured by oxygen electrodes in the same slice along two perpendicular tracks. These measurements were followed by (immuno)histochemical analysis of the 2D distribution of perfused microvessels, hypoxic cells, necrotic areas, and macrophages. Results: The PFCs mainly became sequestered in perfused regions at the tumor periphery; thus, 19 F-MR relaxometry probed mean oxygen tensions in these regions throughout the selected MR slice. In perfused regions of the tumor, mean pO 2/relaxo values were comparable to mean pO 2/electrode values, and varied from 0.03 to 9 mmHg. Median pO 2/electrode values of both tracks were lower than mean pO 2/relaxo values, because low pO 2/electrode values that originate from hypoxic and necrotic areas were also included in calculations of median pO 2/electrode values. After 8-min carbogen breathing, the average pO 2/relaxo increase was 3.3 ± 0.8 (SEM) mmHg and 2.1 ± 0.6 (SEM) after 14 min breathing. Conclusions: We have demonstrated that PFCs mainly became sequestered in perfused regions of the tumor. Here, mean pO 2/relaxo values were comparable to mean pO 2/electrode values. In these areas, carbogen breathing was found to increase the pO 2/relaxo values significantly.

An l -aminopeptidase of Pseudomonas putida , used in an industrial process for the hydrolysis of d,l -amino acid amide racemates, was purified to homogeneity. The highly l -enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and α-H amino acid amides, e.g., l -phenylglycine amide.

Objective: This study was undertaken to investigate the role of the angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor in the development and progression of Barrett esophagus and adenocarcinomas of the esophagus and gastroesophageal junction. Methods: Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels, relative to the control gene encoding β-actin, were measured by using a quantitative reverse transcription-polymerase chain reaction method (ABI 7700 Sequence Detector system) in specimens of Barrett intestinal metaplasia (n = 16), dysplasia (n = 11), adenocarcinoma (n = l 5), and matching normal squamous esophageal tissues (n = 35). Vascular endothelial growth factor and basic fibroblast growth factor protein expression and CD31+ microvessel density were assessed by means of immunohistochemistry in 25 tissue sections that included representative areas for each of these Barrett stages. Results: Expression levels were significantly increased in adenocarcinoma compared with in either normal squamous mucosa (P

Fibronectin (Fn) is produced by cells in blood vessels at inflammatory sites in vivo. Fn release into the circulation thus may be a marker for vascular injury. In support of this, we found that oxidant-induced vascular injury of isolated perfused rabbit lungs caused elevated circulating Fn levels. Western blot analysis indicated that Fn released from the injured blood vessels was intact, dimeric, and possessed electrophoretic mobility identical with Fn produced by fibroblasts. Unlike Fn isolated from rabbit plasma, Fn derived from lung perfusate or produced by fibroblasts reacted with antibodies raised to a synthetic peptide containing sequences from the extra type III Fn domain that is transcribed in fibroblasts but not hepatocytes. Vascular injury by protease was also associated with intravascular release of Fn, but with cleavage. Oxidant-induced vascular injury causes release of tissue-derived Fn, which can be distinguished from plasma Fn by its size and content of antigenic determinants of the extra type III domain.

Dapsone (4,4′-diaminodiphenylsulfone [DDS]) was administered intravenously to anesthetized dogs; urine was collected, heparinized venous blood was obtained, and bile was collected from some of the dogs. A constant infusion of inulin was maintained, and isosmotic or hypoosmotic fluids were administered. Dogs were studied under conditions of standardized, increased or decreased urine flow, and before and after plasmapheresis. Plasma, urine, and bile samples were analyzed for DDS and DDS conjugates; the degree of binding of DDS by plasma proteins was also determined. The renal clearances of inulin and DDS were calculated. No monoacetyldapsone (MADDS) was detected in the plasma, and only negligible quantities were found in the urine. Small quantities of DDS and DDS conjugates were detected in the bile in 4 h following the dose. Between 10 and 30% of the administered drug could be identified as DDS plus DDS conjugates in the urine in 8 h after the dose. Renal clearance of unbound DDS was proportional to the urine flow rate, and the clearance ratio of DDS to inulin approached the same maximal value as that for urea. Although the rate of urinary excretion of DDS conjugates was the same in the dog as in man, the rates of excretion of DDS and of DDS plus DDS conjugates were greater in the dog than in man, suggesting that the acetylation of DDS to MADDS by man but not by the dog and the greater degree of plasma protein binding of DDS and MADDS by man account for the longer half-time of disappearance of DDS in man compared to that in the dog.

Haptoglobin is an a2 plasma glycoprotein that forms a stable complex with hemoglobin (1-5). The complex is cleared from the plasma much more rapidly than free haptoglobin so that marked hypoor anhaptoglobinemia is a concomitant of hemolysis (6, 7). Since free hemoglobin passes readily into the urine, whereas the complex, perhaps owing to its much greater size, does not, haptoglobin may play a role in the conservation of iron by the body (5, 7). Haptoglobin concentration increases markedly in man (8-12) and animals (13-15) in the presence of inflammation or tissue necrosis. Estrogen administration depresses the haptoglobin concentration in the serum of man (10). The site of synthesis of this protein is unknown although the liver has been suggested (16), for it is known that in some instances of parenchymatous liver disease haptoglobin concentration is reduced (10, 17). At least theoretically this might be the result of decreased synthesis by a damaged organ. However, increased hemoglobin

EAG, ERG and ELK are members of the Ether-a-go-go voltage-gated potassium channel family. Each channel possesses a C-terminal domain that is homologous to the cyclic nucleotide-binding domain (CNBD) of CNG/HCN channels but insensitive to cyclic nucleotides. X-ray crystallography of EAG suggests the side chains of two residues, Y699 and L701, interact in a manner analogous to the two moieties of a cyclic nucleotide in the binding pocket of bona fide CNBDs. Using two-electrode voltage clamp of channel proteins expressed in Xenopus oocytes, we found that alanine substitutions of Y699 and L701 (“AA”) dramatically slowed activation and shifted the g-V curve by +15 mV. Mutagenesis of both residues were required to elicit an effect, as if the “apo” configuration required the loss of both side chains. Thus, the intrinsic ligand of the CNB homology domain (CNBhD) reduces the stability of the closed state relative to the open state. To determine whether the AA mutant phenotype represents a loss of function, we deleted the CNBhD. In contrast to the AA mutant, the deletion gave rise to faster activation kinetics but a more dramatic g-V shift of +40 mV. This finding indicates that, unlike HCN channel, in which the unliganded CNBD mimics the CNBD deletion, the AA mutant phenotype is not a loss of function of the CNBhD. Instead, both the intrinsically-liganded and apo conformations communicate with the gating machinery. The current properties can be recapitulated by a 7-state allosteric model, in which the AA mutant alters multiple transitions during channel gating whereas CNBhD deletion affects only the last opening step. We conclude that the EAG1 CNBhD serves a function mechanistically distinct from those of the corresponding domains in channels gated by cyclic nucleotides despite extensive sequence and structural homology.

Previously, we reported that the multidrug resistance proteins MRP1, MRP2 and MRP3 confer resistance to therapeutic antifolates by mediating their cellular extrusion. We now determined whether MRPs also play a role in controlling cellular homeostasis of natural folates. In MRP1, MRP2 and MRP3-transfected 2008 human ovarian carcinoma cells total cellular folate content was 32-38% lower than in 2008 cells (105+/-14pmolfolate/mgprotein) when grown in medium containing 2.3 microM folic acid (FA). Under these conditions cellular growth rates were not compromised. However, when cells were challenged under folate-depleted conditions with a short exposure (4 hr) to FA or leucovorin, MRP1 and MRP3 overexpressing cells were impaired in their growth. In contrast to wild-type cells, MRP1 transfected cells retained only 60% of the maximum growth when exposed to 500 nM leucovorin or 500 microM FA. For 2008/MRP1 and 2008/MRP3 cells FA growth stimulation capacity was dramatically decreased when, during a 4 hr exposure, metabolism into rapidly polyglutamatable and retainable dihydrofolate was blocked by the dihydrofolate reductase inhibitor trimetrexate. To retain growth under such conditions MRP1 overexpressing cells required much higher concentrations of FA (EC(50) > 500 microM) compared to 2008 cells (EC(50): 12 microM). These results suggest that down- and up-regulation of MRP1 (and MRP3) expression can influence cellular folate homeostasis, in particular when cellular retention by polyglutamylation of folates is attenuated.

Background: Gemcitabine (20,20-difluoro-20-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors. Several studies have shown a relationship between gemcitabine peak plasma concentration (Cmax) and hematological toxicity. An immunoassay for gemcitabine in plasma was developed and validated to facilitate therapeutic drug monitoring (TDM) by providing an economical, robust method for automated chemistry analyzers. Methods: A monoclonal antibody was coated on nanoparticles to develop a homogenous agglutination inhibition assay. To prevent ex vivo degradation of gemcitabine in blood, tetrahydrouridine was used as a sample stabilizer. Validation was conducted for precision, recovery, cross-reactivity, and linearity on a Beckman Coulter AU480. Verification was performed on an AU5800 in a hospital laboratory. A method comparison was performed with (LC-MS/MS) liquid chromatography tandem mass spectrometry using clinical samples. Selectivity was demonstrated by testing cross-reactivity of the major metabolite, 20,20-difluorodeoxyuridine. Results: Coefficients of variation for repeatability and withinlaboratory precision were ,8%. The deviation between measured and assigned values was ,3%. Linear range was from 0.40 to 33.02 m/mL (1.5-125.5 mM). Correlation with validated LC-MS/ MS methods was R2 = 0.977. The assay was specific for gemcitabine: there was no cross-reactivity to 20,20-difluorodeoxyuridine, chemotherapeutics, concomitant, or common medications tested. Tetrahydrouridine was packaged in single-use syringes. Gemcitabine stability in whole blood was extended to 8 hours (at room temperature) and in plasma to 8 days (2-88C). Conclusions: The assay demonstrated the selectivity, test range, precision, and linearity to perform reliable measurements of gemcitabine in plasma. The addition of stabilizer improved the sample handling. Using general clinical chemistry analyzers, gemcitabine could be measured for TDM.

Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these cells irresponsive to EGF. Using an activator protein (AP)-1 sensitive reporter construct, we show that AP-1 activity is strongly decreased in density-arrested NRK cells, but is restored after relaxation of densitydependent growth inhibition by removing neighboring cells. EGF could not induce AP-1 activity or S-phase entry in density-arrested cells, but could do so after pretreatment with retinoic acid, which enhances EGF receptor expression. Our results support a model in which the EGF receptor regulates density-dependent growth control in NRK fibroblasts, which is reflected by EGF-induced mitogenic signaling and consequent AP-1 activity. © 2006 Humana Press Inc. All rights of any nature whatsoever reserved.

An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.

Coastal areas of the North Sea are commercially important for fishing and tourism, and are subject to the increasingly adverse effects of harmful algal blooms, eutrophication and climate change. Monitoring phytoplankton in these areas using Ocean Colour Remote Sensing is hampered by the high spatial and temporal variations in absorption and scattering properties. In this paper we demonstrate a clustering method based on specific-absorption properties that gives accurate water quality products from the Medium Resolution Imaging Spectrometer (MERIS). A total of 468 measurements of Chlorophyll a (Chla), Total Suspended Material (TSM), specific- (sIOP) and inherent optical properties (IOP) were measured in the North Sea between April 1999 and September 2004. Chla varied from 0.2 to 35 mg m− 3, TSM from 0.2 to 75 g m− 3 and absorption properties of coloured dissolved organic material at 442 nm (aCDOM(442)) was 0.02 to 0.26 m− 1. The variation in absorption properties of phytoplankton (aph) and non-algal particles (aNAP) were an order of magnitude greater than that for aph normalized to Chla (aph*) and aNAP normalized to TSM (aNAP*). Hierarchical cluster analysis on aph*, aNAP* and aCDOM reduced this large data set to three groups of high aNAP*–aCDOM, low aph* situated close to the coast, medium values further offshore and low aNAP*–aCDOM, high aph* in open ocean and Dutch coastal waters. The median sIOP of each cluster were used to parameterize a semi-analytical algorithm to retrieve concentrations of Chla, TSM and aCDOM(442) from MERIS data. A further 60 measurements of normalized water leaving radiance (nLw), Chla, TSM, aCDOM(442) and aNAP(442) collected between 2003 and 2006 were used to assess the accuracy of the satellite products. The regionalized MERIS algorithm showed improved performance in Chla and aCDOM(442) estimates with relative percentage differences of 29 and 8% compared to 34 and 134% for standard MERIS Chla and adg(442) products, and similar retrieval for TSM at concentrations > 1 g− 3.

The pigment phycocyanin (PC) is a marker for cyanobacterial presence in eutrophic inland water. We present a reflectance band‐ratio algorithm for retrieval of cyanobacterial PC. The model conforms to the band settings of the Medium Resolution Imaging Spectrometer. The parameters of the algorithm were optimized using reflectance and absorption data from two highly eutrophic lakes. Using measured specific absorption coefficients for PC [ a (620)] * for every sample, the error in the predicted PC concentrations was 19.7% (r 2 5 0.94, n 5 34) for measured PC concentrations up to 80 mg m 23 . Applying a fixed value of a (620) caused an overestimation of the PC content * that increased toward lower PC concentrations. The PC prediction best matched observed values during periods of high relative abundance of cyanobacteria in the plankton community. The results suggest strong seasonal variation in a (620). The presence of pigments other than PC and chlorophyll a (Chl a) and a variable influence of Chl a * on retrieved absorption at 620 nm are potential causes of errors in PC retrieval. The algorithm in its current form is considered to be suitable for detection of the PC concentration in turbid, cyanobacteria-dominated waters. Eutrophic inland waters often exhibit blooms of cyanobacteria. Notorious for their negative impact on water quality, cyanobacterial blooms have been increasingly subject of water management and scientific studies. The hazards of toxic cyanobacterial blooms call for frequent and rapid monitoring of water bodies. Remote sensing provides insights into the distribution of blooms for a large number of lakes or reservoirs simultaneously. The concentration of chlorophyll a (Chl a) as a general indicator for plankton biomass can be assessed using imagery from a wide range of air- and spaceborn sensors (Vos et al. 2003). Recent advances in spaceborn remote sensing technology broaden the perspectives of monitoring toward the identification and quantification of plankton groups. Algorithms for the retrieval of Chl a from turbid water reflectance were already being developed (Gons et al. 2002). Now, the retrieval of the pigment phycocyanin (PC), which is characteristic of the presence of cyanobacterial, is being attempted. It is known that the presence of PC can be detected from spectral reflectance (Dekker et al. 1