Multiplexed drug testing of tumor slices using a microfluidic platform.

Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine.

A microfluidic platform for functional testing of cancer drugs on intact tumor slices.

Present approaches to assess cancer treatments are often inaccurate, costly, and/or cumbersome. Functional testing platforms that use live tumor cells are a promising tool both for drug development and for identifying the optimal therapy for a given patient, i.e. precision oncology. However, current methods that utilize patient-derived cells from dissociated tissue typically lack the microenvironment of the tumor tissue and/or cannot inform on a timescale rapid enough to guide decisions for patient-specific therapy. We have developed a microfluidic platform that allows for multiplexed drug testing of intact tumor slices cultured on a porous membrane. The device is digitally-manufactured in a biocompatible thermoplastic by laser-cutting and solvent bonding. Here we describe the fabrication process in detail, we characterize the fluidic performance of the device, and demonstrate on-device drug-response testing with tumor slices from xenografts and from a patient colorectal tumor.

Resolution analysis in computational imaging with patterned illumination and bucket detection.

In computational imaging by pattern projection, a sequence of microstructured light patterns codified onto a programmable spatial light modulator is used to sample an object. The patterns are used as generalized measurement modes where the object information is expressed. In this Letter, we show that the resolution of the recovered image is only limited by the numerical aperture of the projecting optics regardless of the quality of the collection optics. We provide proof-of-principle experiments where the single-pixel detection strategy outperforms the resolution achieved using a conventional optical array detector for optical imaging. It is advantageous in the presence of real-world conditions, such as optical aberrations and optical imperfections in between the sample and the sensor. We provide experimental verification of image retrieval even when an optical diffuser prevents imaging with a megapixel array camera.

Perceptual characteristics of Parkinsonian speech: a comparison of the pharmacological effects of levodopa across speech and non-speech motor systems.

The purpose of this study was to: (1) define perceptual speech characteristics of idiopathic Parkinson disease (IPD) across 35 speech dimensions adapted from Darley et al. [19] and grouped under six speech-sign clusters (respiration, phonation, resonance, articulation, prosody and rate); (2) examine the effects of levodopa on the 35 perceptual speech dimensions and speech-sign clusters; and (3) to compare the relative effectiveness of levodopa on global motor functioning vs. speech production. Sixteen patients with IPD read the 'Grandfather Passage' both 'on' and 'off' levodopa. Three blinded speech-language pathologists performed perceptual speech analyses using a seven-point scale. The diagnosis of IPD was made by a movement disorders fellowship trained neurologist who applied UK Brain bank criteria and administered the Unified Parkinson Disease Rating Scale. Concordant with previous studies, the results of this experiment indicated that IPD disrupted multiple speech production subsystems, with prosody being the most severely affected domain. The perceptual dimensions that were most severely affected included: (1) sound imprecision; (2) mono-loudness; (3) mono-pitch; (4) reduced stress and (5) harsh voice. No significant differences were obtained between medicated states ('on'/'off') for any of the 35 individual speech dimensions and speech-sign clusters. Global motor function significantly improved following dopaminergic medications.

Mode of cembranoid action on embryonic muscle acetylcholine receptor.

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 microM EUAC with 50 microM, 100 microM, or 500 microM carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 microM CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 microM) obtained with 100 microM and 500 microM CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations.

Mechanisms of potentiation of the mammalian GABAA receptor by the marine cembranoid eupalmerin acetate.

BACKGROUND AND PURPOSE: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor. EXPERIMENTAL APPROACH: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. KEY RESULTS: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. CONCLUSIONS AND IMPLICATIONS: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.

Effects of restricted nursing on milk production and collection, kid growth and plasma prolactin and growth hormone concentrations in dairy goats.

The milk production of dairy goats under various regimes of mother-young contact from day 4 post partum were studied during the first 2 months of lactation, together with the prolactin (PRL) and growth hormone (GH) responses to udder stimulation. In the control group, 13 goats and their kids were left in permanent contact and did not undergo milking. In two additional groups, goats were machine milked once a day in the morning (at 0800 h) and kids were allowed 10 hours (from 1000 to 2000 h; 10H group, n = 11) or 5 h (from 1000 to 2000 h; 5H group, n = 11) of mother-young interaction per day. In the last group (MO, n = 10), mothers were permanently separated from their kids on day 4 post partum and milked once a day. Milk production during a 24-h period at 37 days post partum performed by controlled nursing and weighing of the kids (groups with kids) or by two machine milking 12 h apart (milking only group) revealed a higher production in the three groups with some mother-young contact than in the MO group. Total milk collected by milking over the 2 months of the study did not differ between the three groups that underwent milking. Kid weights at 2 months were 3.4 to 4.8 kg. lighter in the groups that underwent milking than in the control group. Hormonal profiles were significantly affected by restricted mother-young contact, with highest pre-stimulation concentrations of PRL and GH in the 5H group. Restricting mother-young contact from the first week postpartum can permit an early collection of milk without major effects on kid growth, when compared with one daily milking in goats totally separated from their young.

[Clinical experience and results of ESWL treatment for 1.000 consecutive patients with the Siemens Lithostar Modularis]. / Lithostar Modularis de Siemens: exposición de los resultados de los 1.000 primeros pacientes.

PATIENTS AND METHODS: We describe the outcome of the treatment of 1.000 consecutive patients treated since may 2000 to december 2003 with Siemens Lithostar Modularis to evaluate the efficacy of this new lithotripter RESULTS: 60.5% male and 39.5% female's patients were treated. Average age of 53.5 years old. 44.1% of the urinary stones were located on the right side and 55.9% on the left. The mean size of the stones was 1.22 +/- 0.74 cm with 59% lesser than 1 cm, 27.6% between 1 and 2 cm and 13.4% bigger than 2 cm. Mean number of sessions required were 1.6. There were differences in the number of sessions related to the size of the lithiasis, 1.3 session in the group of lithiasis smaller than 1 cm and 2.01 sessions in the group bigger than 1 cm. The stones that required less number of sessions were located in the ureter and upper calyx. 71.2% of the patients were stone free after one treatment. CONCLUSIONS: Siemens Lithostar Modularis has the adventages of a great confort of the patient during the procedure, better image because of a very good cuality of the fluoroscopy and the main point is a great comminution of the stone doing very easy the elimination of the fragments achieving a stone free rate of more than 90% in our experience, decreasing the need of auxiliary manoeuvres and the leng of the treatment.

Calyculaglycosides D and E, novel cembrane glycosides from the Caribbean gorgonian octocoral Eunicea species and structural revision of the aglycon of calyculaglycosides A-C.

A recent investigation of the constituents of Eunicea sp. from Colombia and Puerto Rico has provided two novel cembrane glycosides, calyculaglycosides D (6) and E (7), and a new cembranoid diterpene in free form, (+)-nephthenol (4). Metabolites 6 and 7 possess a glycosyl-fused cembrane skeleton with a 1S configuration. Biogenetic considerations have led to a revision of the previously assigned dilophol skeleton of calyculaglycosides A-C, which was confirmed by extensive 2D NMR investigation and a chemical degradation study. Consequently, the true structures for calyculaglycosides A-C are 8-10, respectively, not 1-3. This is the first report of the occurrence of marine diterpene glycosides having a cembrane aglycon.

New diterpenes from the Caribbean sponge Epipolasis reiswigi.

Two diterpenes, epipolone (1) and epipolol (3), produced by terpenoid pathways leading to a tricarbocyclic structure with an irregular "head to tail" isoprene configuration, have been isolated from the Caribbean marine sponge Epipolasis reiswigi collected in Puerto Rico. The structures of 1 and 3 were elucidated largely by 1D and 2D NMR methods and chemical conversion.

Cembranoid and long-chain alkanol sites on the nicotinic acetylcholine receptor and their allosteric interaction.

Long-chain alkanols are general anesthetics which can also act as uncharged noncompetitive inhibitors of the peripheral nicotinic acetylcholine receptor (AChR) by binding to one or more specific sites on the AChR. Cembranoids are naturally occurring, uncharged noncompetitive inhibitors of peripheral and neuronal AChRs, which have no demonstrable general anesthetic activity in vivo. In this study, [3H]tenocyclidine ([3H]TCP), an analogue of the cationic noncompetitive inhibitor phencyclidine (PCP), was used to characterize the cembranoid and long-chain alkanol sites on the desensitized Torpedo californica AChR and to investigate if these sites interact. These studies confirm that there is a single cembranoid site which sterically overlaps the [3H]TCP channel site. This cembranoid site probably also overlaps the sites for the cationic noncompetitive inhibitors, procaine and quinacrine. Evidence is also presented for one or more allosteric cembranoid sites which negatively modulate cembranoid affinity for the inhibitory site. In contrast, long-chain alkanols inhibit [3H]TCP binding through an allosteric mechanism involving two or more alkanol sites which display positive cooperativity toward each other. Double inhibitor studies show that the cembranoid inhibitory site and the alkanol sites are not independent of each other but interfere allosterically with each other's inhibition of [3H]TCP binding. The simplest models consistent with the observed data are presented and discussed.

Synthesis of analogues of Eunicea gamma-cembranolides containing cyclic ethers via saponification.

A method for the synthesis of derivatives of the lead structures euniolide (1), 12,13-bisepieupalmerin (2), and eupalmerin acetate (3) containing tetrahydrofuran and tetrahydropyran ring systems was developed on the basis of alkali-induced intramolecular oxacyclizations. Representatives of the new analogues were submitted to the in vitro antitumor cell-line-screening program of the National Cancer Institute (NCI). While it was shown that a variety of structural modifications are possible, these transformations led typically to nontoxic synthetic cembranoids.

Tobacco cembranoids block behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptor function.

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.

Serrulatane diterpenes with antimycobacterial activity isolated from the West Indian sea whip Pseudopterogorgia elisabethae.

Two new antimycobacterial serrulatane diterpenes, erogorgiaene (3) and 7-hydroxyerogorgiaene (4), and a novel C(40) bisditerpene (5), have been isolated from the West Indian gorgonian octocoral Pseudopterogorgia elisabethae. The structures of compounds 3-5 were determined by spectral (1D and 2D NMR, IR, UV, and HREIMS) analysis.

New metabolites from the West Indian sea feather Pseudopterogorgia bipinnata.

Two diterpenes, a novel seco-furanocembranolide (3) and the highly oxygenated cembranolide bipinnatolide K (4), have been isolated from the West Indian gorgonian octocoral Pseudopterogorgia bipinnata. The chemical structures of these compounds were determined by 1D and 2D NMR spectroscopy in combination with IR, UV, and HRFABMS analyses.

The cumbiasins, structurally novel diterpenes possessing intricate carbocyclic skeletons from the West Indian sea whip Pseudopterogorgia elisabethae (Bayer).

From the hexane extract of the West Indian gorgonian Pseudopterogorgia elisabethae, two diterpenes, cumbiasins A (1) and B (2), having a novel tetracyclic carbon skeleton named cumbiane, have been isolated. In addition, we have isolated cumbiasin C (3), a ring cleavage product of cumbiasin B that possesses an unusual carbocyclic framework named seco-cumbiane. The structures and relative configurations of metabolites 1-3 were elucidated by interpretation of overall spectral data, which included 2D NMR correlation methods, IR, UV, and accurate mass measurements (HREI-MS and HRFAB-MS). The carbocyclic skeletons of the cumbiasins are unprecedented and represent new classes of C20 rearranged diterpenes. Cumbiasins A and B display mild in vitro anti-tuberculosis activity.

Novel terpenoids from the West Indian sea whip Pseudopterogorgia elisabethae (Bayer). Elisapterosins A and B: rearranged diterpenes possessing an unprecedented cagelike framework.

Four diterpenes and a nor-diterpenoid, all of which possess unusual carbocyclic skeletons, were isolated from the hexane solubles of the West Indian gorgonian Pseudopterogorgia elisabethae. The structures and relative configurations of novel metabolites elisabethin D (2), elisabethin D acetate (3), 3-epi-elisabanolide (5), elisapterosin A (6), and elisapterosin B (7) were elucidated by interpretation of overall spectral data, which included 2D NMR correlation methods, IR, UV, and accurate mass measurements (HREI-MS and HRFAB-MS), chemical reactions, and X-ray diffraction analyses. The tetracyclic carbon skeleton of the elisapterosins is undescribed and constitutes a new class of C(20) rearranged diterpenes. Elisapterosin B displays strong in vitro anti-tuberculosis activity.

Isolation, structural characterization, and synthesis of a naturally occurring bisfuranopseudopterane ether: biskallolide A. Evidence for a carbocation intermediate during the facile conversion of kallolide A and isokallolide A into various solvolysis products.

The West Indian alcyonacean Pseudopterogorgia bipinnata (Verrill, 1864) is shown to contain a novel bisditerpenoid ether: biskallolide A (2). The structural assignment of 2 was mainly based on 1D and 2D NMR and MS spectral data and was further confirmed by synthesis. The 2-C-alkoxylation of furanopseudopteranes kallolide A (1) and isokallolide A (8) occurs spontaneously in some solvents and involves replacement of the C2 hydroxyl with an alkoxyl group to yield solvolysis products that display net retention of configuration. The facile solvolytic 2-C-acyloxylation of kallolide A was achieved readily under similar circumstances to afford kallolide A acetate (4) as the sole product. Mechanistic details in conversion of alcohols 1 and 8 into various solvolysis products, including dimeric ethers 2 and 9, were investigated in this study. Solvolysis of kallolide A and isokallolide A in [(18)O]-labeled solvent demonstrated that the C2 alkoxyl of the solvolysis products originated from the solvent, suggesting that these conversions may proceed through an S(N)1 mechanism with generation of a carbocation intermediate. The chemical structures of kallolide A derivatives 3-7and those of isokallolide A congeners 9-11 were established by detailed analysis of the spectral data.