Quality control of high throughput screening

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Quality control of high throughput screening

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50 nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics.

A Rapid and Sensitive High-Throughput Screening Method to Identify Compounds Targeting Protein–Nucleic Acids Interactions

DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian highmobility- group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4,182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide and pyridazinone. Exploitation of analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

Design and Pilot Study for an Efficient High-Throughput Automated Computer-Vision Guided Intelligent De-Calyxing Machine for Post-Harvest Strawberry Processing

Strawberries harvested for processing as frozen fruits are currently de-calyxed manually in the field. This process requires the removal of the stem cap with green leaves (i.e. the calyx) and incurs many disadvantages when performed by hand. Not only does it necessitate the need to maintain cutting tool sanitation, but it also increases labor time and exposure of the de-capped strawberries before in-plant processing. This leads to labor inefficiency and decreased harvest yield. By moving the calyx removal process from the fields to the processing plants, this new practice would reduce field labor and improve management and logistics, while increasing annual yield. As labor prices continue to increase, the strawberry industry has shown great interest in the development and implementation of an automated calyx removal system. In response, this dissertation describes the design, operation, and performance of a full-scale automatic vision-guided intelligent de-calyxing (AVID) prototype machine. The AVID machine utilizes commercially available equipment to produce a relatively low cost automated de-calyxing system that can be retrofitted into existing food processing facilities. This dissertation is broken up into five sections. The first two sections include a machine overview and a 12-week processing plant pilot study. Results of the pilot study indicate the AVID machine is able to de-calyx grade-1-with-cap conical strawberries at roughly 66 percent output weight yield at a throughput of 10,000 pounds per hour. The remaining three sections describe in detail the three main components of the machine: a strawberry loading and orientation conveyor, a machine vision system for calyx identification, and a synchronized multi-waterjet knife calyx removal system. In short, the loading system utilizes rotational energy to orient conical strawberries. The machine vision system determines cut locations through RGB real-time feature extraction. The high-speed multi-waterjet knife system uses direct drive actuation to locate 30,000 psi cutting streams to precise coordinates for calyx removal. Based on the observations and studies performed within this dissertation, the AVID machine is seen to be a viable option for automated high-throughput strawberry calyx removal. A summary of future tasks and further improvements is discussed at the end.

Development of a novel platform for high-throughput gene design and artificial gene synthesis to produce large libraries of recombinant venom peptides for drug discovery

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas

Post-CMOS compatible high-throughput fabrication of AIN-based piezoelectric microcantilevers

A post-complementary metal oxide semiconductor (CMOS) compatible microfabrication process of piezoelectric cantilevers has been developed. The fabrication process is suitable for standard silicon technology and provides low-cost and high-throughput manufacturing. This work reports design, fabrication and characterization of piezoelectric cantilevers based on aluminum nitride (AlN) thin films synthesized at room temperature. The proposed microcantilever system is a sandwich structure composed of chromium (Cr) electrodes and a sputtered AlN film. The key issue for cantilever fabrication is the growth at room temperature of the AlN layer by reactive sputtering, making possible the innovative compatibility of piezoelectric MEMS devices with CMOS circuits already processed. AlN and Cr have been etched by inductively coupled plasma (ICP) dry etching using a BCl3–Cl2–Ar plasma chemistry. As part of the novelty of the post-CMOS micromachining process presented here, a silicon Si (1 0 0) wafer has been used as substrate as well as the sacrificial layer used to release the microcantilevers. In order to achieve this, the Si surface underneath the structure has been wet etched using an HNA (hydrofluoric acid + nitric acid + acetic acid) based solution. X-ray diffraction (XRD) characterization indicated the high crystalline quality of the AlN film. An atomic force microscope (AFM) has been used to determine the Cr electrode surface roughness. The morphology of the fabricated devices has been studied by scanning electron microscope (SEM). The cantilevers have been piezoelectrically actuated and their out-of-plane vibration modes were detected by vibrometry.

Design, construction, and analysis of cell line arrays and tissue microarrays for gene expression analysis

Cell line array (CMA) and tissue microarray (TMA) technologies are high-throughput methods for analysing both the abundance and distribution of gene expression in a panel of cell lines or multiple tissue specimens in an efficient and cost-effective manner. The process is based on Kononen's method of extracting a cylindrical core of paraffin-embedded donor tissue and inserting it into a recipient paraffin block. Donor tissue from surgically resected paraffin-embedded tissue blocks, frozen needle biopsies or cell line pellets can all be arrayed in the recipient block. The representative area of interest is identified and circled on a haematoxylin and eosin (H&E)-stained section of the donor block. Using a predesigned map showing a precise spacing pattern, a high density array of up to 1,000 cores of cell pellets and/or donor tissue can be embedded into the recipient block using a tissue arrayer from Beecher Instruments. Depending on the depth of the cell line/tissue removed from the donor block 100-300 consecutive sections can be cut from each CMA/TMA block. Sections can be stained for in situ detection of protein, DNA or RNA targets using immunohistochemistry (IHC), fluorescent in situ hybridisation (FISH) or mRNA in situ hybridisation (RNA-ISH), respectively. This chapter provides detailed methods for CMA/TMA design, construction and analysis with in-depth notes on all technical aspects including tips to deal with common pitfalls the user may encounter. © Springer Science+Business Media, LLC 2011.

Association study of MTHFD1 coding polymorphisms R134K and R653Q with migraine susceptibility

Objective There is evidence that folate metabolism has a role in migraine pathophysiology, particularly in the migraine with aura subtype. In this study we investigate whether two non-synonymous single nucleotide polymorphisms (SNPs), rs1950902 (C401T; R134K) and rs2236225 (G1958A; R653Q), in MTHDF1 are associated with migraine in an Australian case-control population. Background Increased plasma levels of homocysteine (HCy), one of the metabolites produced in the folate pathway, has been found to be a risk factor for migraine. There is also a genetic link, as a common polymorphism (C667T) that reduces the catalytic activity of MTHFR, the enzyme that catalyses the formation of HCy, is associated with an increase in risk of the migraine with aura (MA) subtype. MTHFD1 is a crucial multifunctional enzyme that catalyses three separate reactions of the folate pathway and therefore variants in MTHFD1 may also influence migraine susceptibility. Methods The R134K and R653Q variants in MTHFD1 were genotyped in an Australian cohort of 520 unrelated migraineurs (162 were diagnosed with migraine without aura [MO] and 358 with MA) and 520 matched controls. Data were analysed for association with migraine and for interaction with the MTHFR C667T polymorphism. Results We find no significant differences in genotype or allele frequencies for either SNP between migraineurs and controls, or when either MO or MA cases were compared to controls. In addition these MTHFD1 polymorphisms did not appear to influence the risk of MA conferred by the MTHFR 667T allele. Conclusions We find no evidence for association of the MTHFD1 R134K and R653Q polymorphisms with migraine in our Australian case-control population. However, as folate metabolism appears to be important in migraine, particularly with respect to the aura component, future studies using high throughput methods to expand the number of SNPs in folate-related genes genotyped and investigation of interactions between SNPs may be justified.

Cancer gene signatures in risk stratification: use in personalized medicine

Cancer is a complex disease which arises due to a series of genetic changes related to cell division and growth control. Cancer remains the second leading cause of death in humans next to heart diseases. As a testimony to our progress in understanding the biology of cancer and developments in cancer diagnosis and treatment methods, the overall median survival time of all cancers has increased six fold one year to six years during the last four decades. However, while the median survival time has increased dramatically for some cancers like breast and colon, there has been only little change for other cancers like pancreas and brain. Further, not all patients having a single type of tumour respond to the standard treatment. The differential response is due to genetic heterogeneity which exists not only between tumours, which is called intertumour heterogeneity, but also within individual tumours, which is called intratumoural heterogeneity. Thus it becomes essential to personalize the cancer treatment based on a specific genetic change in a given tumour. It is also possible to stratify cancer patients into low- and high-risk groups based on expression changes or alterations in a group of genes gene signatures and choose a more suitable mode of therapy. It is now possible that each tumour can be analysed using various high-throughput methods like gene expression profiling and next-generation sequencing to identify its unique fingerprint based on which a personalized or tailor-made therapy can be developed. Here, we review the important progress made in the recent years towards personalizing cancer treatment with the use of gene signatures.

Complex behavior and perception in Drosophila emerges from iterative feedback-regulated reflexes

For a hungry fruit fly, locating and landing on a fermenting fruit where it can feed, find mates, and lay eggs, is an essential and difficult task requiring the integration of both olfactory and visual cues. Understanding how flies accomplish this will help provide a comprehensive ethological context for the expanding knowledge of their neural circuits involved in processing olfaction and vision, as well as inspire novel engineering solutions for control and estimation in computationally limited robotic applications. In this thesis, I use novel high throughput methods to develop a detailed overview of how flies track odor plumes, land, and regulate flight speed. Finally, I provide an example of how these insights can be applied to robotic applications to simplify complicated estimation problems. To localize an odor source, flies exhibit three iterative, reflex-driven behaviors. Upon encountering an attractive plume, flies increase their flight speed and turn upwind using visual cues. After losing the plume, flies begin zigzagging crosswind, again using visual cues to control their heading. After sensing an attractive odor, flies become more attracted to small visual features, which increases their chances of finding the plume source. Their changes in heading are largely controlled by open-loop maneuvers called saccades, which they direct towards and away from visual features. If a fly decides to land on an object, it begins to decelerate so as to maintain a stereotypical ratio of expansion to retinal size. Once they reach a stereotypical distance from the target, flies extend their legs in preparation for touchdown. Although it is unclear what cues they use to trigger this behavior, previous studies have indicated that it is likely under visual control. In Chapter 3, I use a nonlinear control theoretic analysis and robotic testbed to propose a novel and putative mechanism for how a fly might visually estimate distance by actively decelerating according to a visual control law. Throughout these behaviors, a common theme is the visual control of flight speed. Using genetic tools I show that the neuromodulator octopamine plays an important role in regulating flight speed, and propose a neural circuit for how this controller might be implemented in the flies brain. Two general biological and engineering principles are evident across my experiments: (1) complex behaviors, such as foraging, can emerge from the interactions of simple independent sensory-motor modules; (2) flies control their behavior in such a way that simplifies complex estimation problems.