Fly Me to the Micron: Microtechnologies for Research.

Multicellular model organisms, such as (fruit fly), are frequently used in a myriad of biological research studies due to their biological significance and global standardization. However, traditional tools used in these studies generally require manual handling, subjective phenotyping, and bulk treatment of the organisms, resulting in laborious experimental protocols with limited accuracy. Advancements in microtechnology over the course of the last two decades have allowed researchers to develop automated, high-throughput, and multifunctional experimental tools that enable novel experimental paradigms that would not be possible otherwise.

One-pot method for preparing DNA, RNA, and protein for multiomics analysis.

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids.

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously.

action in the sperm produces developmentally deferred chromosome segregation defects during the mid-blastula transition.

, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved.

Microfluidics for understanding model organisms.

New microfluidic systems for whole organism analysis and experimentation are catalyzing biological breakthroughs across many fields, from human health to fundamental biology principles. This perspective discusses recent microfluidic tools to study intact model organisms to demonstrate the tremendous potential for these integrated approaches now and into the future. We describe these microsystems' technical features and highlight the unique advantages for precise manipulation in areas including immobilization, automated alignment, sorting, sensory, mechanical and chemical stimulation, and genetic and thermal perturbation.

LCVM infection generates tumor antigen-specific immunity and inhibits growth of nonviral tumors.

Antibodies and T cells specific for tumor-associated antigens (TAA) are found in individuals without cancer but with a history of infections and are associated with lowered cancer risk. We hypothesized that those immune responses were generated to transiently abnormally expressed self-antigens on infected cells (disease-associated antigens, DAA) and later on tumor cells as TAA. We tested this hypothesis in mice with a history of infection with lymphocytic choriomeningitis virus (LCMV) Armstrong strain (Arm) that causes acute infection when injected intraperitoneally or CL-13 strain that establishes chronic infection when injected intravenously.

Identification of Cell Surface Molecules That Determine the Macrophage Activation Threshold Associated With an Early Stage of Malignant Transformation.

The ability of immune cells to sense changes associated with malignant transformation as early as possible is likely to be important for the successful outcome of cancer immunosurveillance. In this process, the immune system faces a trade-off between elimination of cells harboring premalignant or malignant changes, and autoimmune pathologies. We hypothesized that the immune system has therefore evolved a threshold for the stage of transformation from normal to fully malignant cells that first provides a threat (danger) signal requiring a response.

Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides.

Inflammation-Induced Abnormal Expression of Self-molecules on Epithelial Cells: Targets for Tumor Immunoprevention.

Tumor-associated antigens (TAA) are self-molecules abnormally expressed on tumor cells, which elicit humoral and cellular immunity and are targets of immunosurveillance. Immunity to TAAs is found in some healthy individuals with no history of cancer and correlates positively with a history of acute inflammatory and infectious events and cancer risk reduction. This suggests a potential role in cancer immunosurveillance for the immune memory elicited against disease-associated antigens (DAA) expressed on infected and inflamed tissues that are later recognized on tumors as TAAs.

Mutant huntingtin disrupts mitochondrial proteostasis by interacting with TIM23.

Mutant huntingtin (mHTT), the causative protein in Huntington's disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space.

High-throughput mechanotransduction in Drosophila embryos with mesofluidics.

Developing embryos create complexity by expressing genes to coordinate movement which generates mechanical force. An emerging theory is that mechanical force can also serve as an input signal to regulate developmental gene expression. Experimental methods to apply mechanical stimulation to whole embryos have been limited, mainly to aspiration, indentation, or moving a coverslip; these approaches stimulate only a few embryos at a time and require manual alignment.

Molecular Sieving on the Surface of a Nano-Armored Protein.

The molecular sieving properties of protein surface-attached polymers are the central features in how polymers extend therapeutic protein lifetimes in vivo. Yet, even after 30 years of research, permeation rates of molecules through polymer-surrounded protein surfaces are largely unknown. As a result, the generation of protein-polymer conjugates remains a stochastic process, unfacilitated by knowledge of structure-function-polymer architecture relationships.

Two-Dimensional Difference Gel Electrophoresis.

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2D E) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios.

Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of β-catenin.

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization.

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases.

Proteomics technologies are often used for the identification of protein targets of the immune system. Here, we discuss the immunoproteomics technologies used for the discovery of autoantigens in autoimmune diseases where immune system dysregulation plays a central role in disease onset and progression. These autoantigens and associated autoantibodies can be used as potential biomarkers for disease diagnostics, prognostics and predicting/monitoring drug responsiveness (theranostics).

Immuno-proteomics: Development of a novel reagent for separating antibodies from their target proteins.

Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein.

In-gel equilibration for improved protein retention in 2DE-based proteomic workflows.

The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss.

High dynamic range proteome imaging with the structured illumination gel imager.

A current challenge for proteomics is detecting proteins over the large concentration ranges found in complex biological samples such as whole-cell extracts. Currently, no unbiased, whole-proteome analysis scheme is capable of detecting the full range of cellular proteins. This is due in part to the limited dynamic range of the detectors used to sense proteins or peptides.

Influenza virus infection elicits protective antibodies and T cells specific for host cell antigens also expressed as tumor-associated antigens: a new view of cancer immunosurveillance.

Most tumor-associated antigens (TAA) are self-molecules that are abnormally expressed in cancer cells and become targets of antitumor immune responses. Antibodies and T cells specific for some TAAs have been found in healthy individuals and are associated with lowered lifetime risk for developing cancer. Lower risk for cancer has also been associated with a history of febrile viral diseases.

Microfluidic system with integrated microinjector for automated Drosophila embryo injection.

Drosophila is one of the most important model organisms in biology. Knowledge derived from the recently sequenced 12 genomes of various Drosophila species can today be combined with the results of more than 100 years of research to systematically investigate Drosophila biology at the molecular level. In order to enable automated, high-throughput manipulation of Drosophila embryos, we have developed a microfluidic system based on a Pyrex-silicon-Pyrex sandwich structure with integrated, surface-micromachined silicon nitride injector for automated injection of reagents.

Assessing the critical period for Rho kinase activity during Drosophila ventral furrow formation.

Background: Drosophila ventral furrow formation (VFF), which is the first morphogenetic event during embryo development, serves as a model for epithelial sheet folding. VFF can be subdivided into five cell shape changes: apical membrane flattening, apicobasal nuclear migration, apicobasal cell shortening, random apical constriction, and concerted apical constriction. These processes are generally believed to be driven by Rho kinase (Rok) activation of myosin II to stimulate the constriction of the apical actomyosin network.

Two-dimensional difference gel electrophoresis.

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios.

DIGE: past and future.

This chapter provides a brief historical perspective of the development of difference gel electrophoresis, from its inception to commercialization and beyond.

Proteomic analysis reveals CCT is a target of Fragile X mental retardation protein regulation in Drosophila.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear.

pHMA, a pH-sensitive GFP reporter for cell engulfment, in Drosophila embryos, tissues, and cells.

Engulfment of apoptotic cells by phagocytosis ensures the removal of unwanted and defective cells. We developed a genetically encoded marker for cell engulfment, pHMA, which consists of the pH-Sensitive derivative of GFP, pHluorin, fused to the actin-binding domain of Moesin. In healthy cells of Drosophila embryos and cultured cells, pHMA resides at the cell cortex.