Adam T. Melvin

Micro total analysis systems: Fundamental advances and applications in the laboratory, clinic, and field

Applications of micro total analysis systems (μTAS) span basic-science research, clinical medicine, and field work. Assay devices designed for these applications offer improvements to existing methods or provide fundamentally new strategies. Both mature methods and novel techniques have benefited from the increased throughput, integration and miniaturization afforded by μTAS. Traditional assays such Western blots and binding assays are recapitulated in a μTAS format but with reduced reagent usage, decreased performance times and added capabilities. An increasingly vibrant area is the performance of drug screening and toxicology assays on-chip, enabling the efficient screening of very large numbers of molecules. Similarly, recent μTAS reactors demonstrate greater chemical synthetic yields and novel product synthesis compared to macro-systems, often as a result of accurate control over reaction conditions including precision reagent dispensing. These exciting systems are now enabling on-site production of short-lived radioactive compounds for medical applications. The greatest impact of μTAS may very well be the ability to perform massively parallel laboratory experiments, for example, the use millions of reaction vessels or the analysis of hundreds of thousands of single cells. Another strength of μTAS lies in the creation of multicellular communities, for example, the combination of many cell types into an interacting system to explore intercellular communication. Devices with multiple layers of co-cultured tissues benefit from precise placement of molecules, such as extracellular matrices or growth factors, in both space and time. Similarly, the complexity and variety of organ-on-chip and organism-on-chip technologies continues to escalate rapidly. Impressively, the types of organisms cultured on-chip now range from the simplest bacteria to complex animals such as fish. Automation, reliability, and integration must all increase as a device moves from the specialist environment of a lab to usage by non-expert personnel in the outside world, for example, at a clinical point-of-care or in environmental monitoring. Key innovations in recent months result in devices that operate with minimal external equipment, error-free operation, and unambiguous readouts, all critical for operation by untrained personnel. Lightweight, portable devices are increasingly used to identify chemical and biological toxins in water, air and soil with applications in public health, defense, and homeland security. Perhaps most exciting is the development of μTAS with sufficient robustness for operation in challenging environments, such as the ocean and outer space. A central component of these systems is the ability to withstand the unexpected. These systems push the boundaries of current integration principles and spur rapid growth of new design philosophies. This review focuses on advances in the area of μTAS or “lab-on-a-chip” systems over the time span of May 2011 through September 2012 with a focus on applications in basic research, clinical medicine and field usage. A range of journals with 2011 impact factors from 2.0 to 36.3 were screened to cover publications with highly specialized content as well as those directed at multidisciplinary audiences. These publications included discipline-specific journals such as Analytical Chemistry and Lab on a Chip as well as general scientific publications, e.g. Science and Nature. To identify material beyond the individually examined journals, extensive key word searches in databases such as PubMed, SciFinder, and Web of Science were performed. Recent reviews in the area of μTAS were also examined for appropriate references. Care was taken to identify impactful and exciting work from across the globe. Well over a thousand papers in the three target areas were identified and discussed. Due to space limitations, we were unable to include all papers but instead incorporated those most fitting into the review scheme and those reporting innovations in basic microdevice technology as well as in applications to biological, physical and engineering sciences. We apologize in advance for omitted papers and welcome feedback regarding any oversights on our part.

Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism

Migrating fibroblasts reorient directionality by PI3K-dependent branching and pivoting of protrusions, a mechanism that allows fibroblasts to align with an external chemotactic gradient.

Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts

During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3′-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3′-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts.

Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.

A Comparative Analysis of the Ubiquitination Kinetics of Multiple Degrons to Identify an Ideal Targeting Sequence for a Proteasome Reporter

The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif.

Identification of a p53-based portable degron based on the MDM2-p53 binding region

In recent years the ubiquitin proteasome system (UPS) has garnered increasing interest as a target for chemotherapeutics. Due to the success of the proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma, several new compounds have been developed to target E3 ubiquitin ligases and the proteasome in numerous human cancers. This has increased the need for new analytical methods to precisely measure intracellular enzyme activity in cells. A key component of a desired analytical method is a substrate that is capable of rapid intracellular ubiquitination yet easily incorporated into the next generation of more sophisticated UPS reporters. Portable degradation sequences, or degrons, have the ability to bind to E3 ligases and promote substrate ubiquitination when the sequence is presented in isolation or appended to other entities such as fluorescent peptide-based reporters. Previous work identified an E3 ligase (MDM2)-binding element at p53 amino acids 92-112, which was later demonstrated to be rapidly ubiquitinated in cytosolic lysates effectively functioning as a transportable degron. In this work, a shortened p53 sequence within amino acids 92-112 that displayed rapid ubiquitination kinetics was identified. A nine-member peptide library was synthesized using sequence elements of various sizes and lengths, all based on the initial 22 amino acid long sequence, containing a single ubiquitination site lysine. The ubiquitination kinetics were determined using a combination of gel electrophoresis and analytical high performance liquid chromatography (HPLC) to rank the members of the library and identify the optimal ubiquitination sequence. This analysis identified the five amino acid sequence, KGSYG, corresponding to residues 105-108 with an added N-terminal lysine, as a portable degron since this sequence demonstrated the most rapid ubiquitination kinetics.

Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

Regulation of the ubiquitin–proteasome system (UPS) to treat select types of cancer has become a popular area of drug discovery research. The FDA approval of proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma has led to an increased need for chemical reporters capable of detecting and quantifying protein ubiquitination and the activity of members of the UPS including E3 ubiquitin ligases and the proteasome in the tumor cells of the patients. One limitation of peptide-based reporters is their rapid degradation in the cellular environment by cytosolic peptidases. Conversely, β-hairpin “protectides” exhibit a pronounced secondary structure that significantly increases their lifetime under cellular conditions. The goal of this work was to develop a family of novel, ornithine-rich protectides that could act as primary degrons serving as substrates for in vitro ubiquitination. The fluorescent peptide-based reporters were demonstrated to be highly resistant to degradation in multiple myeloma cell lysates. The most stable β-hairpin primary degron, containing a single ornithine residue at the N-terminus, OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH2], demonstrated rapid ubiquitination kinetics and a 20-fold increase in stability when compared with an unstructured primary degron. A screen of E1 and E3 enzyme inhibitors in cell lysates showed that ubiquitination of OWRWR was significantly impaired by inhibitors of the SCF family of E3 ligases. Furthermore, this is the first report demonstrating the use of an ornithine residue on a primary degron as a ubiquitination site. This study serves as a strong foundation for the development of stable, fluorescent, peptide-based reporters capable of quantifying protein ubiquitination and the enzymatic activity of members of the UPS.

Microfluidic and Paper-Based Devices for Disease Detection and Diagnostic Research

Recent developments in microfluidic devices, nanoparticle chemistry, fluorescent microscopy, and biochemical techniques such as genetic identification and antibody capture have provided easier and more sensitive platforms for detecting and diagnosing diseases as well as providing new fundamental insight into disease progression. These advancements have led to the development of new technology and assays capable of easy and early detection of pathogenicity as well as the enhancement of the drug discovery and development pipeline. While some studies have focused on treatment, many of these technologies have found initial success in laboratories as a precursor for clinical applications. This review highlights the current and future progress of microfluidic techniques geared toward the timely and inexpensive diagnosis of disease including technologies aimed at high-throughput single cell analysis for drug development. It also summarizes novel microfluidic approaches to characterize fundamental cellular behavior and heterogeneity.

CPProtectides: Rapid uptake of well-folded β-hairpin peptides with enhanced resistance to intracellular degradation

Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors or drugs to intact cells. One limitation of CPPs is their rapid degradation by intracellular proteases. β‐hairpin “protectides” have previously been demonstrated to be long‐lived under cytosolic conditions due to their secondary structure. The goal of this work was to demonstrate that arginine‐rich β‐hairpin peptides function as both protectides and as CPPs. Peptides exhibiting a β‐hairpin motif were found to be rapidly internalized into cells with their uptake efficiency dependent on the number of arginine residues in the sequence. Cellular internalization of the β‐hairpin peptides was compared to unstructured, scrambled sequences and to commercially available, arginine‐rich CPPs. The unstructured peptides displayed greater uptake kinetics compared to the structured β‐hairpin sequences; however, intracellular stability studies revealed that the β‐hairpin peptides exhibited superior stability under cytosolic conditions with a 16‐fold increase in peptide half‐life. This study identifies a new class of long‐lived CPPs that can overcome the stability limitations of peptide‐based reporters or bioactive delivery mechanisms in intact cells.

Biophysical analysis of fluid shear stress induced cellular deformation in a microfluidic device

Even though the majority of breast cancers respond well to primary therapy, a large percentage of patients relapse with metastatic disease, for which there is no treatment. In metastasis, a tumor sheds a small number of cancerous cells, termed circulating tumor cells (CTCs), into the local vasculature, from where they spread throughout the body to form new tumors. As CTCs move through the circulatory system, they experience physiological forces not present in the initial tumor environment, namely, fluid shear stress (FSS). Evidence suggests that CTCs respond to FSS by adopting a more aggressive phenotype; however, to date single-cell morphological changes have not been quantified to support this observation. Furthermore, the methodology of previous studies involves inducing FSS by flowing cells through the tubing, which lacks a precise and tunable control of FSS. Here, a microfluidic approach is used for isolating and characterizing the biophysical response of single breast cancer cells to conditions experienced in the circulatory system during metastasis. To evaluate the single-cell response of multiple breast cancer types, two model circulating tumor cell lines, MDA-MB-231 and MCF7, were challenged with FSS at precise magnitudes and durations. As expected, both MDA-MB-231 and MCF7 cells exhibited greater deformability due to increasing duration and magnitudes of FSS. However, wide variations in single-cell responses were observed. MCF7 cells were found to rapidly deform but reach a threshold value after 5 min of FSS, while MDA-MB-231 cells were observed to deform at a slower rate but with a larger threshold of deformation. This behavioral diversity suggests the presence of distinct cell subpopulations with different phenotypes.