Project description:There are still numerous challenges to be overcome in microarray data analysis because advanced, state-of-the-art analyses are restricted to programming users. Here we present the Gene Expression Analysis Platform, a versatile, customizable, optimized, and portable software developed for microarray analysis. GEAP was developed in C# for the graphical user interface, data querying, storage, results filtering and dynamic plotting, and R for data processing, quality analysis, and differential expression. Through a new automated system that identifies microarray file formats, retrieves contents, detects file corruption, and solves dependencies, GEAP deals with datasets independently of platform. GEAP covers 32 statistical options, supports quality assessment, differential expression from single and dual-channel experiments, and gene ontology. Users can explore results by different plots and filtering options. Finally, the entire data can be saved and organized through storage features, optimized for memory and data retrieval, with faster performance than R. These features, along with other new options, are not yet present in any microarray analysis software. GEAP accomplishes data analysis in a faster, straightforward, and friendlier way than other similar software, while keeping the flexibility for sophisticated procedures. By developing optimizations, unique customizations and new features, GEAP is destined for both advanced and non-programming users.
Project description:Cryogels consisting of polyvinyl alcohol and iron (II, III) oxide magnetic nanoparticles coated with a model drug-acetaminophen, were developed as a tunable platform for thermally triggered drug release, based on shape-selective heat transfer. Two different shapes of cryogels; discs and spherical caps, were formed via adding polymer-nanoparticle-drug mixtures into 3D printed molds, followed by freeze-thawing five times. No additional chemical crosslinking agents were used for gel formation and the iron oxide nanoparticles were coated with acetaminophen using only citric acid as a hydrogen-bonding linker. The two gel shapes displayed varying levels of acetaminophen release within 42-50 °C, which are ideal temperatures for hyperthermia induced drug delivery. The amount and time of drug-release were shown to be tunable by changing the temperature of the medium and the shape of the gels, while keeping all other factors (ex. gel volume, surface area, polymer/nanoparticle concentrations and drug-loading) constant. The discs displayed higher drug release at all temperatures while being particularly effective at lower temperatures (42-46 °C), in contrast to the spherical caps, which were more effective at higher temperatures (48-50 °C). Magnetic hyperthermia-mediated thermal imaging and temperature profiling studies revealed starkly different heat transfer behavior from the two shapes of gels. The disc gels retained their structural integrity up to 51 °C, while the spherical caps were stable up to 59 °C, demonstrating shape-dependent robustness. The highly customizable physicochemical features, facile synthesis, biocompatibility and tunable drug release ability of these cryogels offer potential for their application as a low cost, safe and effective platform for hyperthermia-mediated drug delivery, for external applications such as wound care/muscle repair or internal applications such as melanoma treatment.
Project description:Employing polymer cantilevers has shown to outperform using their silicon or silicon nitride analogues concerning the imaging speed of atomic force microscopy (AFM) in tapping mode (intermittent contact mode with amplitude modulation) by up to one order of magnitude. However, tips of the cantilever made out of a polymer material do not meet the requirements for tip sharpness and durability. Combining the high imaging bandwidth of polymer cantilevers with making sharp and wear-resistant tips is essential for a future adoption of polymer cantilevers in routine AFM use. In this work, we have developed a batch fabrication process to integrate silicon nitride tips with an average tip radius of 9 ± 2 nm into high-speed SU8 cantilevers. Key aspects of the process are the mechanical anchoring of a moulded silicon nitride tip and a two-step release process. The fabrication recipe can be adjusted to any photo-processable polymer cantilever.
Project description:PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.
Project description:Hepatitis E virus is a small, nonenveloped RNA virus that is feco-orally transmitted and causes viral hepatitis in humans. A virus-like particle (VLP) expressed and purified from insect cells shares several properties with the virion but can be manipulated quite extensively through genetic engineering or chemical modification. This has exciting implications for exploiting the VLP as a nanocarrier for foreign epitopes or encapsulated deliverables. By exhaustively studying the structure of the virus, we have been successful in designing and synthesizing chimerized VLPs that either carry foreign epitopes, are capable of encapsulating foreign DNA, or both. Preliminary studies show that these particles provide specific and strong immune responses in mice when orally delivered. To appreciate the full potential of HEV VLPs, we have highlighted various properties of the virus with a strong focus on the VLP structure and the key features that make it suitable for oral delivery.
Project description:We describe the development of the practical manufacturing of Ensitrelvir, which was discovered as a SARS-CoV-2 antiviral candidate. Scalable synthetic methods of indazole, 1,2,4-triazole and 1,3,5-triazinone structures were established, and convergent couplings of these fragments enabled the development of a concise and efficient scale-up process to Ensitrelvir. In this process, introducing a meta-cresolyl moiety successfully enhanced the stability of intermediates. Compared to the initial route at the early research and development stage, the overall yield of the longest linear sequence (6 steps) was improved by approximately 7-fold. Furthermore, 9 out of the 12 isolated intermediates were crystallized directly from each reaction mixture without any extractive workup (direct isolation). This led to an efficient and environmentally friendly manufacturing process that minimizes waste of organic solvents, reagents, and processing time. This practical process for manufacturing Ensitrelvir should contribute to protection against COVID-19.
Project description:Bacterial pathogens are a major risk to human, animal, and plant health. To counteract the spread of antibiotic resistance, alternative antibacterial strategies are urgently needed. Here, we construct a proof-of-concept customizable, modular, and inducible antibacterial toxin delivery platform. By engineering a type VI secretion system (T6SS) that is controlled by an externally induced on/off switch, we transform the safe bacterium, Vibrio natriegens, into an effective antibacterial weapon. Furthermore, we demonstrate that the delivered effector repertoire, and thus the toxicity range of this platform, can be easily manipulated and tested. We believe that this platform can serve as a foundation for novel antibacterial bio-treatments, as well as a unique tool to study antibacterial toxins.
Project description:This paper presents a T-flask based screening platform for evaluating and identifying plant hydrolysates for cell culture processes. The development of this platform was driven by an urgent need of replacing a soy hydrolysate that was no longer available for the fed-batch process of recombinant Sp2/0 cell culture expressing a humanized antibody. Series of small-scale experiments in T-flasks and 3-l bioreactors were designed to gain an insight on how this soy hydrolysate benefits the culture. A comprehensive, function-oriented screening platform then was developed, consisting of three T-flask tests, namely the protection test, the growth promotion test, and the growth inhibition test. The cell growth in these three T-flask tests enabled a good prediction of the cell growth in the fed-batch bioreactor process. Fourteen plant hydrolysate candidates were quickly evaluated by this platform for their ability to exert strong protection, high cell growth promotion, and low cell growth inhibition to the culture. One soy hydrolysate was successfully identified to support the comparable cell growth as the discontinued soy hydrolysate. Because of the advantage of using small-scale batch culture to guide bioreactor fed-batch culture, this proposed platform approach has the potential for other applications, such as the medium and feeding optimization, and the mechanism study of plant hydrolysates, in a high throughput format.
Project description:BackgroundChromatin immunoprecipitation followed by next generation sequencing (ChIP-seq), enables unbiased and genome-wide mapping of protein-DNA interactions and epigenetic marks. The first step in ChIP-seq data analysis involves the identification of peaks (i.e., genomic locations with high density of mapped sequence reads). The next step consists of interpreting the biological meaning of the peaks through their association with known genes, pathways, regulatory elements, and integration with other experiments. Although several programs have been published for the analysis of ChIP-seq data, they often focus on the peak detection step and are usually not well suited for thorough, integrative analysis of the detected peaks.ResultsTo address the peak interpretation challenge, we have developed ChIPseeqer, an integrative, comprehensive, fast and user-friendly computational framework for in-depth analysis of ChIP-seq datasets. The novelty of our approach is the capability to combine several computational tools in order to create easily customized workflows that can be adapted to the user's needs and objectives. In this paper, we describe the main components of the ChIPseeqer framework, and also demonstrate the utility and diversity of the analyses offered, by analyzing a published ChIP-seq dataset.ConclusionsChIPseeqer facilitates ChIP-seq data analysis by offering a flexible and powerful set of computational tools that can be used in combination with one another. The framework is freely available as a user-friendly GUI application, but all programs are also executable from the command line, thus providing flexibility and automatability for advanced users.
Project description:Biofilms are communities of microbes that colonize surfaces. While several biofilm experimental models exist, they often have limited replications of spatiotemporal dynamics surrounding biofilms. For a better understanding dynamic and complex biofilm development, this manuscript presents a customizable platform compatible with off-the-shelf well plates that can monitor microbial adhesion, growth, and associated parameters under various relevant scenarios by taking advantage of 3D printing. The system i) holds any substrate in a stable, vertical position, ii) subjects samples to flow at different angles, iii) switches between static and dynamic modes during an experiment, and iv) allows multiplexing and real-time monitoring of biofilm parameters. Simulated fluid dynamics is employed to estimate flow patterns around discs and shear stresses at disc surfaces. A 3D printed peristaltic pump and a customized pH measurement system for real-time tracking of spent biofilm culture media are equipped with a graphical user interface that grants control over all experimental parameters. The system is tested under static and dynamic conditions with Streptococcus mutans using different carbon sources. By monitoring the effluent pH and characterizing biochemical, microbiological, and morphological properties of cultured biofilms, distinct properties are demonstrated. This novel platform liberates designing experimental strategies for investigations of biofilms under various conditions.