An automated centrifugal microfluidic assay for whole blood fractionation and isolation of multiple cell populations using an aqueous two-phase system.

Fractionating whole blood and separating its constituent components one from another is an essential step in many clinical applications. Currently blood sample handling and fractionation processes remain a predominantly manual task that require well-trained operators to produce reliable and reproducible results. Herein, we demonstrate an advanced on-chip whole human blood fractionation and cell isolation process combining (i) an aqueous two-phase system (ATPS) to create complex separation layers with (ii) a centrifugal microfluidic platform (PowerBlade) with active pneumatic pumping to control and automate the assay. We use a polyethylene glycol (PEG) and dextran (DEX) mixture as the two-phase density gradient media and our automated centrifugal microfluidic platform to fractionate blood samples. Different densities of precisely tuned PEG-DEX solutions were tested to match each of the cell types typically targeted during blood fractionation applications. By employing specially designed microfluidic devices, we demonstrate the automation of the following steps: loading of a whole blood sample on-chip, layering of the blood on the ATPS solution, blood fractionation, precise radial repositioning of the fractionated layers, and finally extraction of multiple, selected fractionated components. Fractionation of up to six distinct layers is shown: platelet-rich plasma, buffy coat, PEG, DEX with neutrophils, red blood cells (RBCs) and high density gradient media (HDGM). Furthermore, through controlled dispensing of HDGM to the fractionation chamber, we show that each of the fractionated layers can be repositioned radially, on-the-fly, without disturbing the interfaces, allowing precise transfer of target fractions and cell types into external vials via a chip-to-world interface. Cell counting analysis and cell viability studies showed equivalence to traditional, manual methods. An overall cell viability greater than 90% of extracted cells demonstrates that the proposed approach is suitable for cell isolation applications. This proof-of-principle demonstration highlights the utility of the proposed system for automated whole blood fractionation and isolation for blood cell applications. We anticipate that the proposed approach will be a useful tool for many clinical applications such as standard cell isolation procedures and other bioanalytical assays (e.g., circulating tumor cells, and cell and gene therapy).

Centrifugal microfluidic lab-on-a-chip system with automated sample lysis, DNA amplification and microarray hybridization for identification of enterohemorrhagic Escherichia coli culture isolates.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Buoyancy-driven step emulsification on pneumatic centrifugal microfluidic platforms.

We present here a new method for controlling the droplet size in step emulsification processes on a centrifugal microfluidic platform, which, in addition to the centrifugal force, uses pneumatic actuation for fluid displacement. We highlight the importance of the interplay between buoyancy effects and the flow rate at the step junction, and provide a simple analytical model relating these two quantities to the size of the droplets. Numerical models as well as experiments with water-in-oil emulsions are performed in support of the proposed model.

Polymer Micropillar Arrays for Colorimetric DNA Detection.

We describe the use of periodic micropillar arrays, produced from cyclic olefin copolymer using high-fidelity microfabrication, as templates for colorimetric DNA detection. The assay involves PCR-amplified gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) incorporating a detectable digoxigenin label, which is revealed through an immunoenzymatic process following hybridization with target-specific oligonucleotide capture probes. The capacity of micropillar arrays to induce wicking is used to distribute and confine capture probes with spatial control, making it possible to achieve a uniform signal while allowing multiple, independent probes to be arranged in close proximity on the same substrate. The kinetic profile of color pigment formation on the surface was followed using absorbance measurements, showing maximum signal increase between 20 and 60 min of reaction time. The relationship between microstructure and colorimetric signal was investigated through variation of geometric parameters, such as pitch (10-50 µm), pillar diameter (5-40 µm), and height (16-48 µm). Our findings suggest that signal intensity is largely influenced by the edges of the pillars and less by their height such that it deviates from a linear relationship when both aspect ratio and pillar density become very high. A theoretical model used to simulate the changes in surface composition at the molecular level suggests that differences in the temporal and spatial accumulation of assay components account for this observation.

Epigenetic subtyping of white blood cells using a thermoplastic elastomer-based microfluidic emulsification device for multiplexed, methylation-specific digital droplet PCR.

Epigenetic markers attract increasing attention for the study of phenotypic variations, which has led to the investigation of cell-lineage DNA methylation patterns that correlate with human leukocyte populations for obtaining counts of white blood cell (WBC) subsets. Current methods of DNA methylation analysis involve genome sequencing or loci-specific quantitative PCR (qPCR). Herein, a multiplexed digital droplet PCR (ddPCR) workflow for determining epigenetic-based WBC differential count is described for the first time. A microfluidic emulsification device fabricated from a commercially available thermoplastic elastomer (e.g., Mediprene) promotes customizability and cost-effectiveness of the methodology, which are prerequisites for translation into clinical and point-of-care diagnostics. Bisulfite-treated DNA from peripheral blood mononuclear cells and whole blood is encapsulated in droplets with ddPCR reagents containing primers and fluorescent hydrolysis probes specific for CpG loci correlated with WBC sub-population types. The method enables multiplexed detection of various methylation sites within a single droplet. Both qPCR and immunofluorescence staining (IF) were conducted to validate the capacity of the ddPCR methodology to accurately determine WBC sub-populations using epigenetic analysis of methylation sites. ddPCR results correlated closely to cell proportions obtained using IF, whereas qPCR significantly underestimated these values for both high and low copy number gene targets.

Extraction of nucleic acids from blood: unveiling the potential of active pneumatic pumping in centrifugal microfluidics for integration and automation of sample preparation processes.

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).

Two-level submicron high porosity membranes (2LHPM) for the capture and release of white blood cells (WBCs).

A method modifying a vacuum-assisted UV micro-molding (VAUM) process is proposed for the fabrication of polymer two-level submicron high porosity membranes (2LHPM). The modified process allows for the fabrication of robust, large-area membranes over 5 × 5 cm2 with a hierarchical architecture made from a 200 nm-thick layer having submicron level pores (as small as 500 nm) supported by a 20 µm-thick layer forming a microporous structure with 10-15 µm diameter pores. The fabricated freestanding membranes are flexible and mechanically robust enough for post manipulation and filtration of cell samples. Very high white blood cell (WBC) capture efficiencies (≈97%) from healthy blood samples after red blood cell (RBC) lysis are demonstrated using a 3D-printed filter cartridge incorporated within these 2LHPM. A high release efficiency of ≈95% is also proved using the same setup. Finally, on-filter multistep immunostaining of captured cells is also shown.

Elaboration of a finite element model of pancreatic islet dielectric response to gap junction expression and insulin release.

Dielectric spectroscopy could potentially be a powerful tool to monitor isolated human pancreatic islets for applications in diabetes therapy and research. Isolated intact human islets provide the most relevant means to understand the cellular and molecular mechanisms associated with diabetes. The advantages of dielectric spectroscopy for continuous islet monitoring are that it is a non-invasive, inexpensive and real-time technique. We have previously assessed the dielectric response of human islet samples during stimulation and differentiation. Because of the complex geometry of islets, analytical solutions are not sufficiently representative to provide a pertinent model of islet dielectric response. Here, we present a finite element dielectric model of a single intact islet that takes into account the tight packing of islet cells and intercellular junctions. The simulation yielded dielectric spectra characteristic of cell aggregates, similar to those produced with islets. In addition, the simulation showed that both exocytosis, such as what occurs during insulin secretion, and differential gap junction expression have significant effects on islet dielectric response. Since the progression of diabetes has some connections with dysfunctional islet gap junctions and insulin secretion, the ability to monitor these islet features with dielectric spectroscopy would benefit diabetes research.

Influence of Immunology Knowledge on Healthcare and Healthy Lifestyle.

Completing a course in Immunology is expected to improve health care knowledge (HCK), which in turn is anticipated to influence a healthy lifestyle (HLS), controlled use of health care services (HCS) and an awareness of emerging health care concerns (HCC). This cross-sectional study was designed to determine whether these interrelationships are empirically supported. Participants involved in this study were government servants from two ministries in Malaysia (n = 356) and university students from a local university (n = 147). Participants were selected using the non-random purposive sampling method. Data were collected using a self-developed questionnaire, which had been validated in a pilot study involving similar subjects. The questionnaire items were analyzed using Rasch analysis, SPSS version 21 and AMOS version 22. Results have shown that participants who followed a course in Immunology (CoI) had a higher primary HCK (Mean = 0.69 logit, SD = 1.29 logits) compared with those who had not (Mean = -0.27logit, SD = 1.26 logits). Overall, there were significant correlations among the HLS, the awareness of emerging HCC, and the controlled use of HCS (p <0.001). However, no significant correlations were observed between primary HCK and the other variables. However, significant positive correlation was observed between primary HCK and controlled use of HCS for the group without CoI. Path analysis showed that the awareness of emerging HCC exerted a positive influence on controlled use of HCS (ß = 0.156, p < .001) and on HLS (ß = 0.224, p < .001). These findings suggest that having CoI helps increase primary HCK which influences controlled use of HCS but does not necessarily influence HLS. Hence, introducing Immunology at various levels of education and increasing the public awareness of emerging HCC might help to improve population health en masse. In addition, further investigations on the factors affecting HLS is required to provide a better understanding on the relationship between primary HCK and HLS.

Investigation of the Viability, Adhesion, and Migration of Human Fibroblasts in a Hyaluronic Acid/Gelatin Microgel-Reinforced Composite Hydrogel for Vocal Fold Tissue Regeneration.

The potential use of a novel scaffold biomaterial consisting of cross-linked hyaluronic acid (HA)-gelatin (Ge) composite microgels is investigated for use in treating vocal fold injury and scarring. Cell adhesion integrins and kinematics of cell motion are investigated in 2D and 3D culture conditions, respectively. Human vocal fold fibroblast (hVFF) cells are seeded on HA-Ge microgels attached to a HA hydrogel thin film. The results show that hVFF cells establish effective adhesion to HA-Ge microgels through the ubiquitous expression of ß1 integrin in the cell membrane. The microgels are then encapsulated in a 3D HA hydrogel for the study of cell migration. The cells within the HA-Ge microgel-reinforced composite hydrogel (MRCH) scaffold have an average motility speed of 0.24 ± 0.08 µm min(-1) . The recorded microscopic images reveal features that are presumably associated with lobopodial and lamellipodial cell migration modes within the MRCH scaffold. Average cell speed during lobopodial migration is greater than that during lamellipodial migration. The cells move faster in the MRCH than in the HA-Ge gel without microgels. These findings support the hypothesis that HA-Ge MRCH promotes cell adhesion and migration; thereby they constitute a promising biomaterial for vocal fold repair.

SN-38 active loading in poly(lactic-co-glycolic acid) nanoparticles and assessment of their anticancer properties on COLO-205 human colon adenocarcinoma cells.

SN-38 is a highly effective drug against many cancers. The development of an optimal delivery system for SN-38 is extremely challenging due to its low solubility and labile lactone ring. Herein, SN-38 encapsulated in poly(D,L-lactide-co-glycolide) nanoparticles (NPs) is introduced to enhance its solubility, stability and cellular uptake. SN-38-loaded NPs prepared by spontaneous emulsification solvent diffusion (SESD) method had an average diameter of 310 nm, a zeta potential of -9.69 mV and a loading efficiency of 71%. They were able to protect the active lactone ring of SN-38 against inactivation under physiological condition. A colorectal adenocarcinoma cell line (COLO-205) was used to assess the NPs effects on cytotoxicity and cellular uptake. Result showed a significant decreased cell proliferation and cell apoptosis. These results suggest that these SN-38-loaded NPs can be an effective delivery system for the treatment of colon cancer and potentially for other types of cancers.

Annexin1 regulates DC efferocytosis and cross-presentation during Mycobacterium tuberculosis infection.

The phagocytosis of apoptotic cells and associated vesicles (efferocytosis) by DCs is an important mechanism for both self tolerance and host defense. Although some of the engulfment ligands involved in efferocytosis have been identified and studied in vitro, the contributions of these ligands in vivo remain ill defined. Here, we determined that during Mycobacterium tuberculosis (Mtb) infection, the engulfment ligand annexin1 is an important mediator in DC cross-presentation that increases efferocytosis in DCs and intrinsically enhances the capacity of the DC antigen-presenting machinery. Annexin1-deficient mice were highly susceptible to Mtb infection and showed an impaired Mtb antigen-specific CD8+ T cell response. Importantly, annexin1 expression was greatly downregulated in Mtb-infected human blood monocyte-derived DCs, indicating that reduction of annexin1 is a critical mechanism for immune evasion by Mtb. Collectively, these data indicate that annexin1 is essential in immunity to Mtb infection and mediates the power of DC efferocytosis and cross-presentation.

Covalent binding of nanoliposomes to the surface of magnetotactic bacteria for the synthesis of self-propelled therapeutic agents.

The targeted and effective delivery of therapeutic agents remains an unmet goal in the field of controlled release systems. Magnetococcus marinus MC-1 magnetotactic bacteria (MTB) are investigated as potential therapeutic carriers. By combining directional magnetotaxis-microaerophilic control of these self-propelled agents, a larger amount of therapeutics can be delivered surpassing the diffusion limits of large drug molecules toward hard-to-treat hypoxic regions in solid tumors. The potential benefits of these carriers emphasize the need to develop an adequate method to attach therapeutic cargos, such as drug-loaded nanoliposomes, without substantially affecting the cell's ability to act as delivery agents. In this study, we report on a strategy for the attachment of liposomes to MTB (MTB-LP) through carbodiimide chemistry. The attachment efficacy, motility, and magnetic response of the MTB-LP were investigated. Results confirm that a substantial number of nanoliposomes (∼70) are efficiently linked with MTB without compromising functionality and motility. Cytotoxicity assays using three different cell types (J774, NIH/3T3, and Colo205) reveal that liposomal attachments to MTB formulation improve the biocompatibility of MTB, whereas attachment does not interfere with liposomal uptake.

Polyelectrolyte multilayer coating of 3D scaffolds enhances tissue growth and gene delivery: non-invasive and label-free assessment.

Layer-by-layer (LbL) deposition is a versatile technique which is beginning to be be explored for inductive tissue engineering applications. Here, it is demonstrated that a polyelectrolyte multilayer film system composed of glycol-chitosan (Glyc-CHI) and hyaluronic acid (HA) can be used to coat 3D micro-fabricated polymeric tissue engineering scaffolds. In order to overcome many of the limitations associated with conventional techniques for assessing cell growth and viability within 3D scaffolds, two novel, real-time, label-free techniques are introduced: impedance monitoring and optical coherence phase microscopy. Using these methods, it is shown that LbL-coated scaffolds support in vitro cell growth and viability for a period of at least two weeks at levels higher than uncoated controls. These polyelectrolyte multilayer coatings are then further adapted for non-viral gene delivery applications via incorporation of DNA carrier lipoplexes. Scaffold-based delivery of the enhanced green fluorescent protein (EGFP) marker gene from these coatings is successfully demonstrated in vitro, achieving a two-fold increase in transfection efficiency compared with control scaffolds. These results show the great potential of Glyc-CHI/HA polyelectrolyte multilayer films for a variety of gene delivery and inductive tissue engineering applications.

Sub-femtomole detection of 16s rRNA from Legionella pneumophila using surface plasmon resonance imaging.

Legionellosis has been and continues to be a life-threatening disease worldwide, even in developed countries. Given the severity and unpredictability of Legionellosis outbreaks, developing a rapid, highly specific, and sensitive detection method is thus of great pertinence. In this paper, we demonstrate that sub-femtomole levels of 16s rRNA from pathogenic Legionella pneumophila can be timely and effectively detected using an appropriate designed capture, detector probes, and a QD SPRi signal amplification strategy. To achieve specific and sensitive detection, optimal hybridization conditions and parameters were implemented. Among these parameters, fragmentation of the 16s rRNA and further signal amplification by QDs were found to be the main parameters contributing to signal enhancement. The appropriate design of the detector probes also increased the sensitivity of the detection system, mainly due to secondary structure of 16s rRNA. The use of 16s rRNA from L. pneumophila allowed for the detection of metabolically active pathogens with high sensitivity. Detection of 16s rRNA in solutions as diluted as 1 pM at 450 µL (0.45 femtomole) was achieved in less than 3h, making our approach suitable for the direct, timely, and effective detection of L. pneumophila within man-made water systems.

Dielectric spectroscopy as a viable biosensing tool for cell and tissue characterization and analysis.

The use of dielectric spectroscopy to carry out real time observations of cells and to extract a wealth of information about their physiological properties has expanded in recent years. This popularity is due to the simple, easy to use, non-invasive and real time nature of dielectric spectroscopy. The ease of integrating dielectric spectroscopy with microfluidic devices has allowed the technology to further expand into biomedical research. Dielectric spectra are obtained by applying an electrical signal to cells, which is swept over a frequency range. This review covers the different methods of interpreting dielectric spectra and progress made in applications of impedance spectroscopy for cell observations. First, methods of obtaining specific electrical properties of cells (cell membrane capacitance and cytoplasm conductivity) are discussed. These electrical properties are obtained by fitting the dielectric spectra to different models and equations. Integrating models to reduce the effects of the electrical double layer are subsequently covered. Impedance platforms are then discussed including electrical cell substrate impedance sensing (ECIS). Categories of ECIS systems are divided into microelectrode arrays, interdigitated electrodes and those that allow differential ECIS measurements. Platforms that allow single cell and sub-single cell measurements are then discussed. Finally, applications of impedance spectroscopy in a range of cell observations are elaborated. These applications include observing cell differentiation, mitosis and the cell cycle and cytotoxicity/cell death. Future applications such as drug screening and in point of care applications are then covered.

Novel probiotic dissolvable carboxymethyl cellulose films as oral health biotherapeutics: in vitro preparation and characterization.

OBJECTIVES: Oral health is influenced by the mouth's resident microorganisms. Dental caries and periodontitis are oral disorders caused by imbalances in the oral microbiota. Probiotics have potential for the prevention and treatment of oral disorders. Current formulations, including supplements and foods, have limitations for oral delivery including short storage time, low residence time in the mouth, effects on food consistency, and low patient compliance. Oral thin films (OTFs) may be efficient in delivering probiotics to the mouth. This research aims to develop a novel carboxymethyl cellulose (CMC)-probiotic-OTF to deliver probiotics for the treatment/prevention of oral disorders. METHODS: CMC-OTFs were developed with varying CMC concentration (1.25 - 10 mg/mL), weight (5 - 40 g), thickness (16 - 262 µm), hygroscopicity (30.8 - 78.9 mg/cm(2) film), and dissolving time (135 - 600 s). The 10 g 5 mg/mL CMC-OTF was selected and used to incorporate Lactobacillus fermentum NCIMB 5221 (6.75 × 10(8) cells/film), a probiotic with anti-inflammatory potential for periodontitis treatment and capable of inhibiting microorganisms responsible for dental caries and oral candidiasis. RESULTS: The CMC-OTF maintained probiotic viability and antioxidant activity following 150 days of storage with a production of 549.52 ± 26.08 µM Trolox equivalents. CONCLUSION: This research shows the successful development and characterization of a novel probiotic-CMC-OTF with potential as an oral health biotherapeutic.

Rapid, guanosine 5'-diphosphate-induced, gelation of chitosan sponges as novel injectable scaffolds for soft tissue engineering and drug delivery applications.

Novel injectable chitosan sponges based on rapid ionic crosslinking using guanosine 5'-diphosphate are introduced. The rapid gelation, high water retention, desirable physicochemical properties, soft tissue-like mechanical properties, and excellent cytocompatibility make these injectable sponges promising candidates for tissue regeneration and drug delivery applications.

Mechanisms of action of islet neogenesis-associated protein: comparison of the full-length recombinant protein and a bioactive peptide.

Islet neogenesis-associated protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas as a factor inducing formation of new duct-associated islets. A bioactive portion of INGAP, INGAP(104-118) peptide (INGAP-P), has been shown to have neogenic and insulin-potentiating activity in numerous studies, including recent phase 2 clinical trials that demonstrated improved glucose homeostasis in both type 1 and type 2 diabetic patients. Aiming to improve INGAP-P efficacy and to understand its mechanism of action, we cloned the full-length protein (rINGAP) and compared the signaling events induced by the protein and the peptide in RIN-m5F cells that respond to INGAP with an increase in proliferation. Here, we show that, although both rINGAP and INGAP-P signal via the Ras/Raf/ERK pathway, rINGAP is at least 100 times more efficient on a molar basis than INGAP-P. For either ligand, ERK1/2 activation appears to be pertussis toxin sensitive, suggesting involvement of a G protein-coupled receptor(s). However, there are clear differences between the peptide and the protein in interactions with the cell surface and in the downstream signaling. We demonstrate that fluorescent-labeled rINGAP is characterized by clustering on the membrane and by slow internalization (≤5 h), whereas INGAP-P does not cluster and is internalized within minutes. Signaling by rINGAP appears to involve Src, in contrast to INGAP-P, which appears to activate Akt in addition to the Ras/Raf/ERK1/2 pathway. Thus our data suggest that interactions of INGAP with the cell surface are important to consider for further development of INGAP as a pharmacotherapy for diabetes.