Pharmaceutical Screening Research Articles

Image-Based Phenotypic Screening with Human Primary T Cells Using One-Dimensional Imaging Cytometry with Self-Tuning Statistical-Gating Algorithms

The parallel microfluidic cytometer (PMC) is an imaging flow cytometer that operates on statistical analysis of low-pixel-count, one-dimensional (1D) line scans. It is highly efficient in data collection and operates on suspension cells. In this article, we present a supervised automated pipeline for the PMC that minimizes operator intervention by incorporating multivariate logistic regression for data scoring. We test the self-tuning statistical algorithms in a human primary T-cell activation assay in flow using nuclear factor of activated T cells (NFAT) translocation as a readout and readily achieve an average Z′ of 0.55 and strictly standardized mean difference of 13 with standard phorbol myristate acetate/ionomycin induction. To implement the tests, we routinely load 4 µL samples and can readout 3000 to 9000 independent conditions from 15 mL of primary human blood (buffy coat fraction). We conclude that the new technology will support primary-cell protein-localization assays and “on-the-fly” data scoring at a sample throughput of more than 100,000 wells per day and that it is, in principle, consistent with a primary pharmaceutical screen.

Term amniotic fluid: an unexploited reserve of mesenchymal stromal cells for reprogramming and potential cell therapy applications

BackgroundMesenchymal stromal cells (MSCs) are currently being evaluated in numerous pre-clinical and clinical cell-based therapy studies. Furthermore, there is an increasing interest in exploring alternative uses of these cells in disease modelling, pharmaceutical screening, and regenerative medicine by applying reprogramming technologies. However, the limited availability of MSCs from various sources restricts their use. Term amniotic fluid has been proposed as an alternative source of MSCs. Previously, only low volumes of term fluid and its cellular constituents have been collected, and current knowledge of the MSCs derived from this fluid is limited. In this study, we collected amniotic fluid at term using a novel collection system and evaluated amniotic fluid MSC content and their characteristics, including their feasibility to undergo cellular reprogramming.MethodsAmniotic fluid was collected at term caesarean section deliveries using a closed catheter-based system. Following fluid processing, amniotic fluid was assessed for cellularity, MSC frequency, in-vitro proliferation, surface phenotype, differentiation, and gene expression characteristics. Cells were also reprogrammed to the pluripotent stem cell state and differentiated towards neural and haematopoietic lineages.ResultsThe average volume of term amniotic fluid collected was approximately 0.4 litres per donor, containing an average of 7 million viable mononuclear cells per litre, and a CFU-F content of 15 per 100,000 MNCs. Expanded CFU-F cultures showed similar surface phenotype, differentiation potential, and gene expression characteristics to MSCs isolated from traditional sources, and showed extensive expansion potential and rapid doubling times. Given the high proliferation rates of these neonatal source cells, we assessed them in a reprogramming application, where the derived induced pluripotent stem cells showed multigerm layer lineage differentiation potential.ConclusionsThe potentially large donor base from caesarean section deliveries, the high yield of term amniotic fluid MSCs obtainable, the properties of the MSCs identified, and the suitability of the cells to be reprogrammed into the pluripotent state demonstrated these cells to be a promising and plentiful resource for further evaluation in bio-banking, cell therapy, disease modelling, and regenerative medicine applications.

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

Isoelectric focusing (IEF) is a powerful tool for amphoteric protein separations because of high sensitivity, bio-compatibility, and reduced complexity compared to chromatography or mechanical separation techniques. IEF miniaturization is attractive because it enables rapid analysis, easier adaptation to point of care applications, and smaller sample demands. However, existing small-scale IEF tools have not yet been able to analyze single protein spots from array libraries, which are ubiquitous in many pharmaceutical discovery and screening protocols. Thus, we introduce an in situ, novel, miniaturized protein analysis approach that we have termed surface isoelectric focusing (sIEF). Low volume printed sIEF gels can be run at length scales of ∼300μm, utilize ∼0.9ng of protein with voltages below 10V. Further, the sIEF device platform is so simple that it can be integrated with protein library arrays to reduce cost; devices demonstrate reusability above 50 uses. An acrylamide monomer solution containing broad-range carrier ampholytes was microprinted with a Nano eNablerTM between micropatterned gold electrodes spaced 300μm apart on a glass slide. The acrylamide gel was polymerized in situ followed by protein loading via printed diffusional exchange. A pH gradient formed via carrier ampholyte stacking when electrodes were energized; the gradient was verified using ratiometric pH-sensitive FITC/TRITC dyes. Green fluorescent protein (GFP) and R-phycoerythrin (R-PE) were utilized both as pI markers and to test sIEF performance as a function of electric field strength and ampholyte concentration. Factors hampering sIEF included cathodic drift and pH gradient compression, but were reduced by co-printing non-ionic Synperonic® F-108 surfactant to reduce protein-gel interactions. sIEF gels achieved protein separations in <10min yielding bands < 50μm wide with peak capacities of ∼8 and minimum pI differences from 0.12 to 0.14. This new sIEF technique demonstrated comparable focusing at ∼100 times smaller dimensions than any previous IEF. Further, sample volumes required were reduced four orders of magnitude from 20μL for slab gel IEF to 0.002μL for sIEF. In summary, sIEF advantages include smaller volumes, reduced power consumption, and microchip surface accessibility to focused bands along with equivalent separation resolutions to prior IEF tools. These attributes position this new technology for rapid, in situ protein library analysis in clinical and pharmaceutical settings.

Agilent buys into Raman spectroscopy

Agilent Technologies has entered the Raman spectroscopy market with the acquisition of the British firm Cobalt Light Systems for about $52 million. Based in Oxfordshire, England, Cobalt was formed in 2008 as a spin-off from the U.K. Science & Technology Facilities Council. Today it has 52 employees and claims as customers 20 of the 25 largest drug companies and more than 75 airports across Europe and Asia. Cobalt’s products are benchtop and portable Raman spectrometers that allow identification of chemicals and materials without opening containers. Applications include materials identification in pharmaceutical quality control and screening of containers for explosives in airport security checks. Conventional spectroscopy is poor at detecting materials through sealed, nontransparent containers. With the acquisition, Agilent gets Cobalt’s spatially offset Raman spectroscopy (SORS) technology. Invented by a group at the Science & Technology Facilities Council that included Cobalt’s chief scientific officer,...

Advances in research on combining the opotogenetics with bionic olfaction sensor

There are plenty of amazing wonders existing in nature, and the crisis-crossing connections between them are performing their best duties. For example, biological olfactory system is an ingenious biosensor, since the olfactory sensory neurons in peripheral nervous respond to the external stimuli, then the signals are transmitted to the olfactory bulb to be integrated and processed, the axonal connection is precisely organized signals from about 1000 different types of odorant receptors, then sorted out in 1800 glomeruli in the mouse olfactory bulb, after delivered to the most advanced central nervous system, the information can be distinguished eventually. Cortical regions underlying vision, audition, and somatosens- ation receive sensory information from the thalamus and also make corticothalamic feedback projections that influence thalamic sensory processing. This process is responding with particularly high sentivity, specificity and rapidity. The ability to detect odorants is crucial for survival as it informs an organism with its environment: food, approaching predators, mates nearby, etc. During recently year, researchers have employed optogenetics technology to explore deeper in the mechanism of olfactory system. Microbial proteins that can be rapidly activated by light have been adapted for research in neuroscience, including ChR2 and NpHR, which permit millisecond-precision optical control of genetically defined cell types in intact neural tissue. This technology allows the use of light to alter neural processing at the level of single spikes and synaptic events, yielding a widely applicable tool for neuroscientists and biomedical engineers. An important question in the study of the olfactory system is the role of spatial factors in olfactory processing, but traditional methods have been relatively unsuccessful in elucidating their theories, odor stimulation evokes complex spatiotemporal activity in the olfactory bulb, suggesting that both the identity of activated neurons and the timing of their activity convey information about odors, the emergency of optogenetics technology can solve this problem efficiently. Biological olfactory systems can recognize and discriminate thousands of distinct odors with very high sensitivity and specificity, with the progress of research on olfactory mechanisms and the advancements of olfaction, many types of biomimetic olfactory-based biosensors have been developed by the combination of olfactory functional components with various secondary sensors. The bioinspired olfaction sensor is an innovative chemical sensing bionic system with far-reaching significance, it contains various biological components as the sensitive elements including olfactory receptors, olfactory cells and olfactory tissues, which has great applicable prospects in many fields for detecting specific odorants with high sensitivity, also by chronically coupling multiple microelectrodes to olfactory bulb of behaving rats, we extract an array of mitral/tufted cells which could generate odor-specific temporal patterns of neural discharge. They have great potential commercial prospects and promising applications in such fields as biomedicine, environmental monitoring, pharmaceutical screening and the quality control of food and water. This review interpreted the transferring mechanism of biological olfactory system and the applications of optogenetics in researches concerning the foundamental research of olfactory system. In addition, a combination of the work of our laboratory with other domestic and international researchers were presented here to predict the future scenario and the promising applications in disease diagnosis, environmental monitoring, military and food quality control.

PubChem BioAssay: A Decade’s Development toward Open High-Throughput Screening Data Sharing

High-throughput screening (HTS) is now routinely conducted for drug discovery by both pharmaceutical companies and screening centers at academic institutions and universities. Rapid advance in assay development, robot automation, and computer technology has led to the generation of terabytes of data in screening laboratories. Despite the technology development toward HTS productivity, fewer efforts were devoted to HTS data integration and sharing. As a result, the huge amount of HTS data was rarely made available to the public. To fill this gap, the PubChem BioAssay database (https://www.ncbi.nlm.nih.gov/pcassay/) was set up in 2004 to provide open access to the screening results tested on chemicals and RNAi reagents. With more than 10 years’ development and contributions from the community, PubChem has now become the largest public repository for chemical structures and biological data, which provides an information platform to worldwide researchers supporting drug development, medicinal chemistry study, and chemical biology research. This work presents a review of the HTS data content in the PubChem BioAssay database and the progress of data deposition to stimulate knowledge discovery and data sharing. It also provides a description of the database’s data standard and basic utilities facilitating information access and use for new users.

Next generation human skin constructs as advanced tools for drug development.

Many diseases, as well as side effects of drugs, manifest themselves through skin symptoms. Skin is a complex tissue that hosts various specialized cell types and performs many roles including physical barrier, immune and sensory functions. Therefore, modeling skin invitro presents technical challenges for tissue engineering. Since the first attempts at engineering human epidermis in 1970s, there has been a growing interest in generating full-thickness skin constructs mimicking physiological functions by incorporating various skin components, such as vasculature and melanocytes for pigmentation. Development of biomimetic invitro human skin models with these physiological functions provides a new tool for drug discovery, disease modeling, regenerative medicine and basic research for skin biology. This goal, however, has long been delayed by the limited availability of different cell types, the challenges in establishing co-culture conditions, and the ability to recapitulate the 3D anatomy of the skin. Recent breakthroughs in induced pluripotent stem cell (iPSC) technology and microfabrication techniques such as 3D-printing have allowed for building more reliable and complex invitro skin models for pharmaceutical screening. In this review, we focus on the current developments and prevailing challenges in generating skin constructs with vasculature, skin appendages such as hair follicles, pigmentation, immune response, innervation, and hypodermis. Furthermore, we discuss the promising advances that iPSC technology offers in order to generate invitro models of genetic skin diseases, such as epidermolysis bullosa and psoriasis. We also discuss how future integration of the next generation human skin constructs onto microfluidic platforms along with other tissues could revolutionize the early stages of drug development by creating reliable evaluation of patient-specific effects of pharmaceutical agents. Impact statement Skin is a complex tissue that hosts various specialized cell types and performs many roles including barrier, immune, and sensory functions. For human-relevant drug testing, there has been a growing interest in building more physiological skin constructs by incorporating different skin components, such as vasculature, appendages, pigment, innervation, and adipose tissue. This paper provides an overview of the strategies to build complex human skin constructs that can faithfully recapitulate human skin and thus can be used in drug development targeting skin diseases. In particular, we discuss recent developments and remaining challenges in incorporating various skin components, availability of iPSC-derived skin cell types and invitro skin disease models. In addition, we provide insights on the future integration of these complex skin models with other organs on microfluidic platforms as well as potential readout technologies for high-throughput drug screening.

Experience in the Use of Solid-Phase Extraction in the Screening of Pharmaceuticals and Narcotics in the Blood by Gas Chromatography with Mass Spectrometric Detection

Studies using 224 blood samples as examples showed that the use of solid-phase extraction and gas chromatography with mass spectrometric detection for screening blood provides reliable identification of a wide range of narcotic and pharmaceutical substances of forensic medical interest, including “designer” synthetic narcotics – methylenedioxypyrovalerone (MDPV), pyrrolidinovalerophenone (PVP), and their metabolites; cannabimimetic metabolites N-(1-carbamoyl-2-methylpropyl)-1-pentyl-1H-indazole-3-carboxamide (ABPINACA) and/or N-[(1-pentyl-1H-indazol-3-yl)carbonyl]valine methyl ester (AMB). The distribution of substances in different elusion fractions is given and dissociation constants (pKa) and coefficients of distribution in an octanol-water system (log P) are calculated.

Rapid Nanogram Scale Screening Method of Microarrays to Evaluate Drug-Polymer Blends Using High-Throughput Printing Technology.

A miniaturized, high-throughput assay was optimized to screen polymer-drug solid dispersions using a 2-D Inkjet printer. By simply printing nanoliter amounts of polymer and drug solutions onto an inert surface, drug/polymer microdots of tunable composition were produced in an easily addressable microarray format. The amount of material printed for each dried spot ranged from 25 ng to 650 ng. These arrays were used to assess the stability of drug/polymer dispersions with respect to recrystallization, using polarized light microscopy. One array with a panel of 6 drugs formulated at different ratios with a poly(vinylpyrrolidone-vinyl acetate) (PVPVA) copolymer was developed to estimate a possible bulk (gram-scale) approximation threshold from the final printed nanoamount of formulation. Another array was printed at a fixed final amount of material to establish a literature comparison of one drug formulated with different commercial polymers for validation. This new approach may offer significant efficiency in pharmaceutical formulation screening, with each experiment in the nanomicro-array format requiring from 3 up to 6 orders of magnitude lower amounts of sample than conventional screening methods.

Extrusion Bioprinting of Shear-Thinning Gelatin Methacryloyl Bioinks.

Bioprinting is an emerging technique for the fabrication of 3D cell-laden constructs. However, the progress for generating a 3D complex physiological microenvironment has been hampered by a lack of advanced cell-responsive bioinks that enable bioprinting with high structural fidelity, particularly in the case of extrusion-based bioprinting. Herein, this paper reports a novel strategy to directly bioprint cell-laden gelatin methacryloyl (GelMA) constructs using bioinks of GelMA physical gels (GPGs) achieved through a simple cooling process. Attributed to their shear-thinning and self-healing properties, the GPG bioinks can retain the shape and form integral structures after deposition, allowing for subsequent UV crosslinking for permanent stabilization. This paper shows the structural fidelity by bioprinting various 3D structures that are typically challenging to fabricate using conventional bioinks under extrusion modes. Moreover, the use of the GPG bioinks enables direct bioprinting of highly porous and soft constructs at relatively low concentrations (down to 3%) of GelMA. It is also demonstrated that the bioprinted constructs not only permit cell survival but also enhance cell proliferation as well as spreading at lower concentrations of the GPG bioinks. It is believed that such a strategy of bioprinting will provide many opportunities in convenient fabrication of 3D cell-laden constructs for applications in tissue engineering, regenerative medicine, and pharmaceutical screening.

Modifiers of prion protein biogenesis and recycling identified by a highly parallel endocytosis kinetics assay

The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endolysosomal organelles. Understanding the intracellular trafficking of PrPC may, therefore, help elucidate the conversion process. Here we describe a time-resolved fluorescence energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrPC The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC (Z' factor >0.5). RNA interference assays showed that suppression of AP2M1 (AP-2 adaptor protein), RAB5A, VPS35 (vacuolar protein sorting 35 homolog), and M6PR (mannose 6-phosphate receptor) blocked PrPC internalization, whereas down-regulation of GIT2 and VPS28 increased PrPC internalization. PrPC cell-surface expression was reduced by down-regulation of RAB5A, VPS28, and VPS35 and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrPC trafficking and demonstrate the power of this assay for identifying modulators of PrPC trafficking.

Precision Plating of Electrogenic Cells on Microelectrodes Enhanced with Nano-Porous Platinum for Cell-Based Biosensing Applications

Microelectrode Array (MEA) devices are well established platforms for biosensing applications, however, limitations in electrode materials and cell coupling to electrodes still exist. Signal-to-noise ratio (SNR), a fabrication limitation, can be improved by reducing the impedance of the microelectrodes without increasing their physical size using conductive nanomaterials. Platinum is a well-studied conductive material that has been determined to be compatible with MEA devices, contains high electronic conductivity, is non-toxic to cells and has excellent catalytic activity. Nano-porous platinum, as compared to pure platinum, has larger surface area enabled by nano-scale definition, and as a result can lower the impedance of microelectrodes. Therefore, in this study nano-porous platinum was electroplated onto thin film gold electrodes to increase SNR in a single-well MEA device. Numerous commercial and research grade MEAs are available and have been subject to for various applications such as disease modeling and pharmaceutical screening. Although high densities of cells can increase the probability of cell-electrode coupling, only signals near electrodes will be detected. Our approach introduces a method of precise patterning of cells on electrodes to increase the probability of cell-electrode coupling and reducing inaccuracies and expenses associated with cell plating. The single-well MEA glass die was designed and fabricated to contain 64 microelectrodes (30 µm diameter with a pitch of 200 µm) and 4 internal on-chip reference electrodes. The MEA glass die fabrication was performed on a 4-inch, 500 µm thick glass wafer. First, metal traces composed of titanium and gold were deposited using lift-off lithography. Subsequently, 5 µm of SU-8 was spin coated and defined as an insulation layer. A separate printed circuit board (PCB) was designed and fabricated to contain 72 contact pads and routing traces. The biocompatible epoxy 353ND was used to package the glass die and PCB in specific orientations determined by alignment markers. Ball bonding with 20 µm diameter gold wire was used to wirebond the glass die to the PCB. A packaging technique was developed that would allow the characterization of key properties of the MEA. This technique involved using copper wire, 100 µm in diameter, and attaching it to the contact pads on the backside of PCBs using a conductive epoxy. Finally, a polystyrene culture well and lid were attached to the MEA using the epoxy, 353ND. Nano-Porous platinum electroplating solution was prepared with a combination of chloroplantinic acid, HCl and lead acetate, all diluted in DI water. Fully packaged devices containing approximately 3 mL of electrolyte were placed within the Axion BioSystems Muse for nano-porous platinum electrodeposition, utilizing the custom Application Specific Integrated Circuit (ASIC) in the system at room temperature. The nano-porous platinum electroplated devices were subsequently characterized using electrical impedance spectroscopy and cyclic voltammetry against the control devices, which contained gold electrodes. The impedance of each device was measured across a scanned frequency range of 10 Hz-100 kHz between the microelectrode, the internal reference electrode and cellular conductive media. The impedance of the nano-porous platinum devices is an order of magnitude lower than the devices with gold electrodes. Cyclic voltammograms were obtained at a scan rate of 20 mV/s in the range from -0.28 to 1.2 V vs. Ag/AgCl. RMS noise comparison between nano-porous platinum electroplated devices and control devices (gold electrodes) was determined using the Muse system and Axion’s Integrated Studio (AxIS) software. Biosensing applications for the devices were explored using electrogenic cells in combination with a precise plating technique. As depicted in Figure 1, cells were positioned in a pattern on and around electrodes to increase the probability of cell-electrode coupling. The electrical response was obtained and analyzed using the Muse system and AxIS software. SNR of action potentials recorded from cells growing on devices enhanced with nano-porous platinum electrodeposition and cells on control devices with gold electrodes were compared. Activity within neuronal networks / beat frequencies of cell patches on electrodes were monitored over the course of 2-3 weeks. The methods explored in this study not only decreases impedance of microelectrodes to increase SNR but also increases cell-electrode coupling by warranting the placement of cells next or on the electrodes by precision patterning. Future studies will aim to optimize electrode sensitivity using novel electrode materials, the porosity of different materials, and characterization using emerging analytical methods. Eventually these devices will provide in vitro systems to monitor both acute and chronic effects of drugs and toxins as well as to perform functional studies under physiological or induced pathophysiological conditions that mimic in vivo damages. Figure 1: Electrogenic cells patterned near nano-porous platinum electrode Figure 1

Transfer of silica gel TLC screening methods for clarithromycin, azithromycin, and amodiaquine + artesunate to HPTLC–densitometry with detection by reagentless thermochemical activation of fluorescence quenching

ABSTRACTA previously published model process was used for the transfer of silica gel thin-layer chromatography (TLC) screening methods for clarithromycin, azithromycin, and amodiaquine + artesunate pharmaceutical formulations published in the Global Pharma Health Fund E.V. Minilab manual for the identification of counterfeit drugs to high-performance TLC–densitometry quantitative methods that can be used in support of regulatory compliance actions. In these new methods, detection of clarithromycin, azithromycin, and artesunate was achieved by thermochemical activation involving simple reagent-free heating of the layer to produce derivatives of the drugs that quench fluorescence under 254 nm ultraviolet light. Additional drugs with TLC screening methods published in the Minilab manual and/or Compendium of Unofficial Methods for Rapid Screening of Pharmaceuticals by Thin Layer Chromatography that do not naturally quench fluorescence were studied and also found to be detectable by thermochemical activation on silica gel layers. The conditions of the thermochemical activation were studied, and in situ spectra of drug zones before and after heating were obtained. Heat activation of fluorescence quenching seems to be a widely applicable detection method that is safer and more convenient than the use of chemical spray, dip, or vapor phase reagents.

High‐Throughput Natural Products Discovery in Fungi Using FAC‐MS Technology

Ascomycete fungal genomes typically each contain >50 different secondary metabolite biosynthetic gene clusters (BGCs). With an estimated pool of >500,000 ascomycete fungal species on earth, the corresponding chemical potential that could be harnessed for human technological challenges such as disease and agriculture is truly massive. However, the vast majority of fungal-encoded chemical space is uncharted and difficult to access due to difficulties culturing and genetically manipulating most fungi. We report a high-throughput heterologous expression and metabolomic profiling technology using untargeted liquid chromatography-mass spectrometry (LC-MS) with ultrahigh mass accuracy to systematically identify the secondary metabolite (SM) products of heterologously expressed fungal BGCs. This platform uses fungal artificial chromosomes (FACs) to capture full-length BGCs derived from unbiased large-insert libraries of genomic DNA of Aspergillus terreus, A. aculeatus, and A. wenti (Fig. A). Host A. nidulans strains transformed with FACs (Fig. B) are then screened by LC-MS (Fig. C), and FAC-encoded products are identified by a robust scoring system to identify spectral features most likely associated with each FAC (Fig. D). Deletions of specific backbone and tailoring genes within FACs (achieved through facile E. coli genetics) then empirically validate assignment of metabolites to BGCs and facilitate analyses of biosynthesis (Fig. E). We use this “FAC-MS” platform to screen 56 FACs and report detection of 15 completely novel secondary metabolites from 56 FACs, a ~27% “hit rate”, including an A. terreus FAC encoding three distinct metabolites identified as a novel macrolactone, a sesterterpenoid, and the orphan benzodiazepine benzomalvin A, which is 300-fold overexpressed by the FAC-strain. Deletants of benzomalvin A backbone and tailoring genes allow us to propose and test the first biosynthetic model for this molecule, as well as demonstrate the readiness with which such studies can be carried out using the FAC-MS technology. Wide application of the FAC-MS pipeline should have a major impact on fungal natural products research in the mid-term future by greatly increasing the scale and rate at which fungal secondary metabolites can be systematically unearthed and introduced into pharmaceutical and agricultural screening campaigns. Support or Funding InformationThis work was supported in part by an SBIR award from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health under grant R44AI094885 to C.C.W. at Intact Genomics, Inc. and to J.W.B and N.L.K. N.L.K. also acknowledges AT009143 for partial support of this work. A) Fungal artificial chromosomes (FACs) capture full-length biosynthetic gene clusters (BGCs) derived from unbiased large-insert libraries of genomic DNA of Aspergillus terreus, A. aculeatus, and A. wenti. B and C) Host A. nidulans strains transformed with FACs are then screened by LC-MS. D) FAC-encoded products are identified by a robust scoring system. E) Deletions of backbone genes in FACs empirically validate assignment of metabolites to BGCs and dissect biosyntheses.

Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture.

Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications.

Pharmacists’ attitudes towards a pharmaceutical assessment screening tool to help prioritise pharmaceutical care in a UK hospital

ObjectiveTo establish the thoughts of pharmacists using the pharmaceutical assessment screening tool (PAST) when assigning a patient acuity level (PAL) and establish other decision factors. A PAL is a pharmaceutical...

Hepatic Spheroids for Long-Term Toxicity Studies

Abstract Drug-induced liver injury is a leading cause of drug failure and withdrawal from the market. Current in vitro screens rely on monolayers of primary hepatocytes to predict in vivo liver injury. Hepatocyte monolayers lose their phenotype and metabolic function within days of seeding, thereby creating a defined experimental window that limits the type, nature, and predictive capacity of the assay. Previous reports have shown that spheroids of primary hepatocytes possess structural polarity, functional bile canaliculi, and long-term viability. Common methods of spheroid formation are limited in pharmaceutical screening strategies due to inherent cell loss, cell stress, novel equipment, and assay requirements. The objective of this work is to develop a rat primary hepatocyte spheroid system that can maintain long-term cell viability, phenotype, and metabolic function, allowing for repeat compound exposure. In this article, we show that primary hepatocytes compact to form stable spheroids. At a seeding...

Human Umbilical Versus Coronary Cell Sources for Tissue-Engineered Blood Vessel Mimics

Along with the advancement of tissue engineering methods to produce implantable constructs has come the development of methods to create tissue-engineered constructs for in vitro preclinical testing of drugs, devices, and other therapeutics. Blood vessel mimics (BVMs) are examples of tissue-engineered in vitro models that have been created for the assessment of endothelial cell responses to implanted devices and intravascular imaging modalities. These BVMs could also serve as preclinical models for early-stage pharmaceutical screening. The current work aimed to build on previous BVM capabilities in three ways: by establishing and documenting a method to create dual-layer BVMs with smooth muscle and endothelial cells, by utilizing and comparing human umbilical and human coronary sources of cells, and by evaluating the ability of BVMs created with the new methods to withstand device implantation. In both umbilical and coronary BVMs, histology and scanning electron microscopy (SEM) confirmed the presence of a confluent cell layer within the lumen of the scaffold. After device deployment, SEM revealed that the cellular lining remained intact, and in umbilical vessels, some evidence of an early endothelial response was observed. The data presented here support the use of the documented methods to create dual-layer BVMs from a variety of cell sources.

Acute Methiopropamine Intoxication After “Synthacaine” Consumption

Use of methiopropamine (MPA), a synthetic metamfetamine analog, has been detected since 2011 in Europe, but there is limited information on its acute toxicity. A 30-year-old man was admitted to the emergency department in a confused state, with paranoid delusion, auditory and visual hallucinatory experiences, and incoherent speech following the use of "synthacaine" (a slang term derived from "synthetic" and "cocaine"). Toxicological screening for pharmaceuticals and drugs of abuse by liquid chromatography-diode-array detector, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS-MS) detected MPA, which was subsequently quantified by a specific LC-MS-MS method. Of note, 13 h after presentation to the emergency department, the plasma concentration of MPA was 14 ng/mL. This case report confirms the toxicity of MPA and the need for toxicological analysis to confirm the substance actually ingested by users of new psychoactive substances.

Mimicking the Kidney: A Key Role in Organ-on-Chip Development.

Pharmaceutical drug screening and research into diseases call for significant improvement in the effectiveness of current in vitro models. Better models would reduce the likelihood of costly failures at later drug development stages, while limiting or possibly even avoiding the use of animal models. In this regard, promising advances have recently been made by the so-called “organ-on-chip” (OOC) technology. By combining cell culture with microfluidics, biomedical researchers have started to develop microengineered models of the functional units of human organs. With the capacity to mimic physiological microenvironments and vascular perfusion, OOC devices allow the reproduction of tissue- and organ-level functions. When considering drug testing, nephrotoxicity is a major cause of attrition during pre-clinical, clinical, and post-approval stages. Renal toxicity accounts for 19% of total dropouts during phase III drug evaluation—more than half the drugs abandoned because of safety concerns. Mimicking the functional unit of the kidney, namely the nephron, is therefore a crucial objective. Here we provide an extensive review of the studies focused on the development of a nephron-on-chip device.