Academic Research Laboratory Research Articles

Enhanced Protein Production in Escherichia coli by Optimization of Cloning Scars at the Vector-Coding Sequence Junction.

Protein production in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research laboratories. Maximum production is usually sought, as this reduces costs and facilitates downstream purification steps. Frustratingly, many coding sequences are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction. These sequence variants modulated expression by up to 1000-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (ΔG) in this region were important, but not the only, determinants for high expression.

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.

Human Skin Models for Research Applications in Pharmacology and Toxicology: Introducing NativeSkin®, the “Missing Link” Bridging Cell Culture and/or Reconstructed Skin Models and Human Clinical Testing

The use of human skin models in pharmacology and toxicology has become a widely accepted approach for the evaluation of dermato-cosmetic products, both finished formulations and active ingredients. In particular, in vitro–reconstructed human skin models consisting on human keratinocytes and/or fibroblasts are used globally in both industrial and academic research laboratories, and regulatory bodies endorse the use of several of them as valid alternatives to animal testing requirements. Despite the recent progress that has been made in the reconstruction of more complex skin models containing other cell types, such models still have inherent limitations, as they are not an exact copy of human skin in vivo, and hence the predictability of the human in vivo response remains limited. Here we describe a new testing approach based on the use of NativeSkin®, a standardized ex vivo model of human skin, maintaining physiological skin biology, skin barrier function, and metabolism during 7 days of culture in tissue culture inserts. NativeSkin models are cultivated in quality-controlled and assured conditions, and demonstrate high reproducibility among donors. Where initial product testing is often performed on skin cell cultures and in vitro–reconstructed models, we propose the use of NativeSkin being the most complete skin model available, as highly predictive and cost-effective “last-line screen in laboratory conditions” prior to clinical evaluation in humans.

Effect of Haemophilus influenzae exposure on Staphylococcus aureus tympanostomy tube attachment and biofilm formation.

Tympanostomy tube (TT) biofilm formation may lead to sequelae. To determine whether the acute pathogen Haemophilus influenzae promotes TT attachment and biofilm formation by the chronic pathogen Staphylococcus aureus. Controlled, in vitro microbiological study at an academic research laboratory using TTs treated with 0 (untreated), 10, or 3000 µg/mL ciprofloxacin or ethylene oxide and TTs with and without prior H influenzae exposure. Fluoroplastic TTs (18 per treatment) were cultured with H influenzae. The TTs were gas-sterilized or exposed to 0, 10, or 3000 µg/mL ciprofloxacin. One-third of the TTs from each treatment group underwent H influenzae counts or scanning electron microscopy (SEM). Another one-third were used for an S aureus attachment assay. The remainder, as well as TTs not exposed to H influenzae, were cultured with S aureus and then treated with oxacillin to kill planktonic S aureus. S aureus counts and SEM were performed. Attachment and biofilm formation of S aureus on TTs assessed by quantitative bacterial counts and SEM. Mean (SD) H influenzae counts were lower on TTs treated with 3 mg/mL than with 10 µg/mL ciprofloxacin (2.06 × 103 [1.00 × 103] vs 4.21 × 105 [1.67 × 105]; P < .001). Mean (SD) S aureus attachment was higher on TTs with untreated preexisting H influenzae (8.88 × 105 [3.20 × 105]; P < .001) and lower on TTs with prior exposure to H influenzae treated with 10 (3.43 × 104 [2.10 × 104]; P = .006) or 3000 µg/mL ciprofloxacin (6.41 × 102 [3.59 × 102]; P < .001). S aureus biofilm formation was similar across groups, except TTs with prior exposure to H influenzae treated with ciprofloxacin 3 mg/mL, which had significantly less (9.30 × 101 [3.51 × 102]; P < .001). Exposure to live H influenzae may promote S aureus attachment on TTs. Treatment of H influenzae on TTs with ciprofloxacin, 3 mg/mL, as found in ototopical therapy, may reduce subsequent S aureus attachment and biofilm formation.

Mass Media as a Discursive Resource and the Construction of Engineering Selves

There have been different approaches to the study of the relations between mass media on the one hand and science and technological activities on the other. In this article, I summarize consumption approaches, point out some of their limitations, and then show how these limitations can be addressed by drawing on an ethnographic study I conducted of an academic engineering research laboratory. I analyze the discursive practices lab members use to interpret mass media. One practice treats media as reference points that mark a lab member’s growing alignment with the profession of electrical and computer engineering, and this constructs an engineering self by providing an opportunity for lab members to display knowledge of the prevalence and shapes of robots and align themselves with the firsthand knowledge prized in engineering culture. Another practice uses the discourse-in-practice of religion to construct an engineering self by aligning the speaker with things that are valued in engineering culture such as objectivity, science, evolution, and literature reviews. The final practice I analyze involves repurposing media technologies in ways that are helpful for both current and future lab research projects, which constructs selves by giving lab members an opportunity to show they know which features of media are relevant for engineering and how these features can be used to address engineering problems. I conclude with a discussion of how this contributes to studies of engineering identity and the relations between science, technology, and media.

High content screening in chemical biology: overview and main challenges

The last two decades have seen the development of high content screening (HCS) methodology and its adaptation for the evaluation of small molecules as drug candidates or their use as chemical tools for research purpose. HCS was initially set-up for the understanding of the mechanism of action of compounds by testing them on cell based-assays for pharmacological and toxicological studies. Since the last decade, the use of HCS has been extended to academic research laboratories and this technology has become the starting point for numerous projects aiming at the identification of molecular targets and cellular pathways for a given disease on which novel type of drugs could act. This screening approach relies on image capture of fluorescently labeled cells therefore generating a large amount of data that must be handled by appropriate automated image analysis methods and storage instrumentation. These latter in addition to the integration and data sharing are current challenges that HCS must still tackle.

Meeting the Google Expectation for Chemical Safety Information

Imagine a scenario in an academic research lab: you are working on a chemical system that has not yet been well characterized, perhaps looking at new catalysts, trying new reagent combinations, or scaling up a new reaction for more sample. How might you gauge the reactivity potential against the equipment and procedures in place to ensure control is maintained? How do you identify critical points in the system where subtle or even inadvertent changes in condition or operation could send things awry, possibly becoming dangerous? How do you assess the potential hazards of untried chemistry? Is there any helpful information readily available at your fingertips? Would a search engine as accessible as Google be able to turn up appropriate data for such scenarios?

A Pilot Study of Age-Related Changes in the Sphingolipid Composition of the Rat Sarcoplasmic Reticulum.

Muscle strength declines more rapidly than muscle size, manifesting as a loss of muscle quality. One putative contributor to this impairment of muscle quality is impaired sarcoplasmic reticulum (SR) function. The principal objective of this study was to characterize the sphingolipid composition of the SR in adult and aging rat muscles. A secondary, exploratory objective was to test for associations between SR sphingolipids and SR function (i.e., Ca2+ release). Using an animal model, the objectives were evaluated in a pre-clinical, cross-sectional study. Data were collected in an academic research laboratory. Medial gastrocnemius muscles of adult (n=8; 7-8 months) and aged (n=8; 24-25 months), male F344/BN hybrid rats were processed to extract SR. Sphingolipids in the SR were measured using tandem mass spectrometry. Fatty acid concentrations within the major sphingolipid classes were evaluated via Principal Component Analysis (PCA). In a subset of samples, SR Ca2+ release rates were determined using fluorometric methods, and associations with specific (based on results of PCA) fatty acid concentrations were evaluated. Aging SR showed an overall decline in the ratio of unsaturated to saturated fatty acids. Age-specific differences were observed for hexosylceramide and ceramide-1-phosphate. Within subset of samples with SR Ca2+ release data, a significant negative association between Ca2+ release and C1P18:0 and trends for positive associations with hexCER24:0 and 24:1 were observed. These preliminary, pre-clinical data suggest that changes in SR sphingolipids may play a role in age-related impairment of muscle function. Further work is needed to explore this hypothesis, as SR sphingolipids may prove a fruitful target for interventions, be they physical (i.e., exercise), nutritional or pharmacological.

Setting up an academic research laboratory in Canada in 2015.

Setting up an academic research laboratory in Canada in 2015.

Label-free biosensor assays in GPCR screening.

About one third of currently marketed drugs target G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins in the human proteome. GPCRs are ubiquitously expressed throughout the human body and play a pivotal role in a vast number of physiological and pathophysiological processes. Because of their intriguing complexity, their relevance, and yet unexploited potential in the treatment of diseases, GPCRs are studied intensively by both academic and industrial research labs.Classical biochemical and molecular biology techniques, including traditional second messenger assays, took biomedical research to the next level and represent the fascinating power of in vitro pharmacology. While extremely efficient in capturing one clearly defined cellular readout, those methods do not authentically portray the events taking place in living cells as a whole; hence the process of drug discovery runs the risk to lose sight of a wider context already in early stages. Label-free cell-based assays hold the promise to overcome these shortcomings by considering cellular processes holistically. If combined with diligent assay adjustments, dynamic mass redistribution (DMR) technology is an excellent tool to investigate GPCR signaling. In this article we aim to provide guidance for scientists seeking for information on how to set up and optimize DMR assays with the objective to establish a knowledge base on deciphering integrated cellular readouts. For this reason we focus on a basic DMR protocol for the investigation of the long-chain fatty acid FFA1 receptor as a model family A GPCR and complement it with information that allow a sophisticated approach to more specialized scientific questions with the use of this comparatively novel method.

Assessment of mitochondrial membrane potential in HEI-OC1 and LLC-PK1 cells treated with gentamicin and mitoquinone.

To determine the effects of concurrent treatment with gentamicin and the mitochondria-targeted antioxidant mitoquinone (MitoQ; which may prevent gentamicin ototoxicity) on change in the mitochondrial membrane potential (Δψ(m)), a precursor of apoptosis. Prospective and controlled. Academic research laboratory. LLC-PK1 (Lilly Laboratories Culture-Pig Kidney Type 1) and HEI-OC1 (House Ear Institute Organ of Corti 1) cells-renal and auditory cell lines, respectively-were used in this study. Δψ(m) was assessed by flow cytometry through the MitoProbe JC-1 Kit for Flow Cytometry in untreated LLC-PK1 and HEI-OC1 cells and cells exposed to low- (100µM) or high- (2000µM) dose gentamicin for 24 hours, with and without 0.5µM each of MitoQ or idebenone (IDB; an untargeted ubiquinone). Δψ(m) was not different in untreated LLC-PK1 cells and cells coincubated with low-dose gentamicin and MitoQ or IDB (P > .05). In HEI-OC1 cells, coincubation with low-dose gentamicin and MitoQ decreased Δψ(m) (P = .002). Coincubation of LLC-PK1 cells with high-dose gentamicin and DMSO, MitoQ, or IDB depolarized Δψ(m) (P < .0001), with MitoQ depolarizing the Δψ(m) to a greater extent than that of IDB (P = .03). In contrast, HEI-OC1 cells demonstrated a hyperpolarized Δψ(m) when coincubated with high-dose gentamicin and DMSO, MitoQ, or IDB (P < .001). The combination of gentamicin and MitoQ holds the potential to disrupt Δψ(m). This suggests a heightened need to monitor for toxicity in patients receiving both agents.

The novel immunotherapeutic oligodeoxynucleotide IMT504 protects neutropenic animals from fatal Pseudomonas aeruginosa bacteremia and sepsis.

IMT504 is a novel immunomodulatory oligonucleotide that has shown immunotherapeutic properties in early preclinical and clinical studies. IMT504 was tested in a neutropenic rat model of Pseudomonas aeruginosa bacteremia and sepsis. This animal system recapitulates many of the pathological processes found in neutropenic patients with Gram-negative, bacterial infections. The research was conducted in the setting of an academic research laboratory. The test subjects were Sprague-Dawley rats. Animals were rendered neutropenic by administration of cyclophosphamide, colonized with P. aeruginosa by oral feeding, and then randomized to receive IMT504 over a range of doses and treatment regimens representing early and late therapeutic interventions. IMT504 immunotherapy conferred a significant survival advantage over the 12-day study period compared with the results seen with placebo-treated animals when the therapy was administered at the onset of neutropenia and even in the absence of antibiotics and after the onset of fever and systemic infection. Notably, even late salvage IMT504 monotherapy was highly effective (13/14 surviving rats with IMT504 therapy versus 2/14 controls, P=<0.001). Moreover, late salvage IMT504 monotherapy was as effective as antibiotic therapy (13/14 surviving rats versus 21/21 rats, P=0.88). In addition, no antagonism or loss of therapeutic efficacy was noted with combination therapy of IMT504 plus antibiotics. IMT504 immunotherapy provides a remarkable survival advantage in bacteremia and sepsis in neutropenic animals and deserves further study as a new treatment option in patients with, or at risk for, severe Gram-negative bacterial infections and sepsis.

The outsourcing and commercialization of science: Is the lab CEO the future of academic research?

Once upon a time, there was a small academic research laboratory, in an old brick building, on a quaint college campus. Though small, the lab had everything it needed to do quality research and publish in high‐impact journals. There were a handful of keen undergraduate volunteers, a couple of energetic and optimistic grad students, and an old, silverback PI with a modest but sufficiently sized grant. If you peeked through the ivy‐covered windows of this small lab, you would likely spot some large pieces of equipment, like a fridge, fume hood, centrifuge, and computers, and a cluttering of microscopes, beakers, test tubes, and chemical jars. Occasionally, a student would need an instrument or a reagent that was not in the lab. When this happened, the PI grunted and guffawed and then bought the missing item or sent the student down the hall to borrow it from another team. But apart from that, the lab was self‐sufficient and produced a steady stream of papers. This depiction of autonomous research might sound foreign and outdated to many scientists reading this article, but it was once the norm across most universities—just ask that friendly professor emeritus who putters up and down your department hallways. Sure, contemporary research labs are still populated by ambitious students and grizzled PIs; they are still littered with high‐tech tools, flashy computers, and expensive chemicals; and they still try to publish in “big” journals. But in today's fast‐paced academic landscape, producing quality research is no longer a solely in‐house endeavor; rather, it is increasingly dependent on collaborations, alliances, and outsourcing [1]. The days of the lone‐wolf researcher are long gone, and nowhere is this more apparent than in the field of genomics. Anyone who has spent time sequencing or analyzing genomes in the past decade can attest …

Endonasal Skull Base Tumor Removal Using Concentric Tube Continuum Robots: A Phantom Study.

Objectives The purpose of this study is to experimentally evaluate the use of concentric tube continuum robots in endonasal skull base tumor removal. This new type of surgical robot offers many advantages over existing straight and rigid surgical tools including added dexterity, the ability to scale movements, and the ability to rotate the end effector while leaving the robot fixed in space. In this study, a concentric tube continuum robot was used to remove simulated pituitary tumors from a skull phantom. Design The robot was teleoperated by experienced skull base surgeons to remove a phantom pituitary tumor within a skull. Percentage resection was measured by weight. Resection duration was timed. Setting Academic research laboratory. Main Outcome Measures Percentage removal of tumor material and procedure duration. Results Average removal percentage of 79.8 ± 5.9% and average time to complete procedure of 12.5 ± 4.1 minutes (n = 20). Conclusions The robotic system presented here for use in endonasal skull base surgery shows promise in improving the dexterity, tool motion, and end effector capabilities currently available with straight and rigid tools while remaining an effective tool for resecting the tumor.

Abstract 4581: A novel modular polymer platform for the treatment of oral and head and neck carcinoma

Abstract Objective: Head and neck Squamous cell carcinoma (HNSCC) is difficult to resect completely by surgery due to complicated context and therefore exhibits high recurrence rate in the patient. To reduce tumor recurrence after surgery, we developed a biodegradable polymer to deliver CCL21 cytokine and cisplatin, and evaluate its efficacy in a partially resected xenograft animal model of HNSCC. Study Design: in vivo study. Setting: Academic research laboratory. Subjects and Methods: After xenograft HNSCC tumors were established by subcutaneous injection, C3H/HeJ mice underwent surgery to debulk their tumors to a size of 0.5x0.5x0.5 cm3 and received different polymer implantation. For cytokine studies, mice were grouped into: (1) no polymer; (2) plain polymer; (3) plain polymer with intratumoral injection of recombinant CCL21 twice a week; (4) polymer containing parental dendritic cells; (5) polymer containing dendritic cells secreting CCL21 (DC-CCL21). For cisplatin studies, mice were randomized to receive implantation of (1) no polymer; (2) plain polymer; (3) plain polymer with local cisplatin injection; (4) cisplatin polymer. The two groups of mice implanted with cisplatin polymer or no polymer were further randomized to receive (1) 4 Grays external beam radiation for 4 days; (2) no radiation. Tumor size was measured until the mice were euthanized. At necropsy, the tumors were excised and weighed. Results: Our results using this novel polymer platform demonstrate a significant reduction in tumor growth. The DC-CCL21 polymer reduced SCCVII/SF tumors in the C3H/HeJ mice by over 41% compared to the control groups (p&amp;lt;0.01). The cisplatin secreting polymer effectively reduced SCCVII/SF tumors in the C3H/HeJ mice by over 16-fold (P &amp;lt; 0.01) as compared to control, plain polymer group. We also observed a statistically significant lower tumor weight among mice treated with cisplatin polymer and concomitant CCL21 compared to the control group. Conclusion: Herein we demonstrate the efficacy of a novel polymer platform in delivering cisplatin and cytokines to a partially resected SCC in a murine model. We also demonstrate that we can effectively grow dendritic cells in the polymer that can actively secrete CCL21. Our results indicate that this polymer may represent a new therapeutic modality for patients with HNSCC. Once this polymer platform is optimized we will plan for validation in the context of a prospective trial in patients with unresectable advanced or recurrent HNSCC. Citation Format: Yuan Lin, Sherven Sharma, Steven M. Dubinett, Ben Wu, Maie St. John. A novel modular polymer platform for the treatment of oral and head and neck carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4581. doi:10.1158/1538-7445.AM2014-4581

An in silico model for rapid identification of multiple bacteria in resource limited laboratories.

We have developed an in silico model for simplified detection of five major food borne bacteria using common PCR primer based on conserved region flanking a variable region in the first rRNA operon sequences. The RFLP pattern exhibited on virtual SmaI and MboI digestion were unique to each genus. This design is based on the combination of comparative sequence analysis, conventional PCR and restriction digestion methods. We believe this approach as a better alternative to 16S rRNA sequencing based identification / detection of bacteria and suited for the resource limited academic research laboratories in which variety of bacteria are used in different students' projects.

Rapid and low-cost prototyping of medical devices using 3D printed molds for liquid injection molding.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

When will there be a cure for dementia?

There are now more than 5 million Americans suffering from Alzheimer’s dementia (AD) at an annual financial cost to the U.S. health care system of $200 billion a year but of course the real cost is the human tragedy that this disease causes as people lose their ability to function and as caregivers, often spouses and other family members, struggle to provide proper care for their loved ones. With the increasing baby boomer population, the number of Americans with Alzheimer’s dementia could skyrocket to 15 million by 2050 at an annual cost of $1.2 trillion.1 The extent of this rapidly developing crisis has not escaped the government, academic research laboratories and the pharmaceutical industry which are pouring significant dollars into research to find a cure for this menace. In order for a safe and effective drug to be developed research scientists must first define a disease on a molecular level. That has not yet been accomplished for AD however the understanding of the pathogenesis of AD has increased greatly since the early 1990s resulting in increasing optimism that better treatments can be developed.2 AD is currently perceived as a protein aggregation disorder and researchers now know that a protein called amyloid-beta (Ab) plays an important role in the pathogenesis of AD. Aggregates of this protein are present in the extracellular plaques that are found in the brains of those with AD and it is now thought to be a toxin that leads to the dysfunction of neurons eventually leading to the extensive neurological damage that is the hallmark of this disease.2 Briefly, Ab is produced when beta amyloid precursor protein is improperly cleaved.3 It is an important step inwhat is referred to as the amyloid cascade hypothesis which researchers are targeting in order to prevent, or at least significantly delay, the ultimate damage

Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society.

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology.