Two-dimensional gallium nitride realized via graphene encapsulation.

The spectrum of two-dimensional (2D) and layered materials 'beyond graphene' offers a remarkable platform to study new phenomena in condensed matter physics. Among these materials, layered hexagonal boron nitride (hBN), with its wide bandgap energy (∼5.0-6.0 eV), has clearly established that 2D nitrides are key to advancing 2D devices. A gap, however, remains between the theoretical prediction of 2D nitrides 'beyond hBN' and experimental realization of such structures. Here we demonstrate the synthesis of 2D gallium nitride (GaN) via a migration-enhanced encapsulated growth (MEEG) technique utilizing epitaxial graphene. We theoretically predict and experimentally validate that the atomic structure of 2D GaN grown via MEEG is notably different from reported theory. Moreover, we establish that graphene plays a critical role in stabilizing the direct-bandgap (nearly 5.0 eV), 2D buckled structure. Our results provide a foundation for discovery and stabilization of 2D nitrides that are difficult to prepare via traditional synthesis.

Fabrication and Microscopic and Spectroscopic Characterization of Planar, Bimetallic, Micro- and Nanopatterned Surfaces.

Micropatterns and nanopatterns of gold embedded in silver and titanium embedded in gold have been prepared by combining either photolithography or electron-beam lithography with a glue-free template-stripping procedure. The obtained patterned surfaces have been topographically characterized using atomic force microscopy and scanning electron microscopy, showing a very low root-mean-square roughness (<0.5 nm), high coplanarity between the two metals (maximum height difference ≈ 2 nm), and topographical continuity at the bimetallic interface. Spectroscopic characterization using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF-SIMS), and Auger electron spectroscopy (AES) has shown a sharp chemical contrast between the two metals at the interface for titanium patterns embedded in gold, whereas diffusion of silver into gold was observed for gold patterns embedded in silver. Surface flatness combined with a high chemical contrast makes the obtained surfaces suitable for applications involving functionalization with molecules by orthogonal adsorption chemistries or for instrumental calibration. The latter possibility has been tested by determining the image sharpness and the analyzed area on circular patterns of different sizes for each of the spectroscopic techniques applied for characterization.This is the first study in which the analyzed area has been determined using XPS and AES on a flat surface, and the first example of a method for determining the analyzed area using ToF-SIMS.

Hydrocodone extended-release: pharmacodynamics, pharmacokinetics and behavioral pharmacology of a controversy.

Recently, the U.S. Food and Drug Administration (FDA) approved Zohydro(®), an extended release formulation of the opioid analgesic hydrocodone that contains no acetaminophen. This approval was against the recommendation of the FDA's Expert Panel. Subsequently, both chronic pain advocates and anti-drug abuse advocates have steadfastly expressed their support of, or astonishment at this decision. Here, we review the pharmacokinetics, pharmacodynamics, safety and abuse liability of this hydrocodone formulation and how it relates to the Expert Panel's opinion and the FDA decision. We discuss the important issues, risk mitigation, potential use of abuse deterrents, and how the different viewpoints of the Expert Panel and FDA decision makers resulted in the approval and subsequent controversy.

Ranolazine attenuates mechanical allodynia associated with demyelination injury.

OBJECTIVE: The aim of this study was to determine whether ranolazine, a new medication that targets sodium channels to improve cardiac ischemia and angina, could be an effective analgesic agent for pain associated with demyelination injury. BACKGROUND: Many agents have been used to treat neuropathic pain but not all neuropathic conditions respond similarly to treatment. We have demonstrated that ranolazine, an agent that blocks voltage-gated sodium channels Nav 1.4, 1.5, 1.7, and 1.8, is effective in attenuating mechanical hyperalgesia in both complete Freund's adjuvant and spared nerve injury preclinical models of inflammatory and neuropathic pain, respectively. Here we test the efficacy of this drug in a newly validated model of demyelination injury that responds uniquely to a number of treatment options. METHODS: After determination of baseline nerve conduction velocities (NCVs) and withdrawal responses from heat and mechanical stimulation in male Sprague-Dawley rats (300-350 g), 1 µg/30 µL of doxorubicin was injected into one sciatic nerve. The contralateral nerve provided a sham-injected control. Two weeks after doxorubicin injection, NCV and sensitivity to heat and mechanical stimulation were reassessed before and after treatment with ranolazine (10, 30, 50 mg/kg) administered intraperitoneally using an experimenter-blinded, randomized design. RESULTS: Doxorubicin injection produced a significant hyperalgesic effect in response to mechanical but not heat stimulation. Conduction velocities in the injected limbs were reduced when compared with controls. Ranolazine reduced mechanical allodynia with peak efficacy at 30 mg/kg. Fifty milligram/kilogram ranolazine restored NCVs by approximately 50%, but had no effect in the uninjected limb. CONCLUSIONS: Ranolazine exerts broad-spectrum actions to reduce mechanical allodynia that is associated with peripheral demyelination injury.

Spatially resolved correlation of active and total doping concentrations in VLS grown nanowires.

Controlling axial and radial dopant profiles in nanowires is of utmost importance for NW-based devices, as the formation of tightly controlled electrical junctions is crucial for optimization of device performance. Recently, inhomogeneous dopant profiles have been observed in vapor­liquid­solid grown nanowires, but the underlying mechanisms that produce these inhomogeneities have not been completely characterized. In this work, P-doping profiles of axially modulation-doped Si nanowires were studied using nanoprobe scanning Auger microscopy and Kelvin probe force microscopy in order to distinguish between vapor­liquid­solid doping and the vapor­solid doping. We find that both mechanisms result in radially inhomogeneous doping, specifically, a lightly doped core surrounded by a heavily doped shell structure. Careful design of dopant modulation enables the contributions of the two mechanisms to be distinguished, revealing a surprisingly strong reservoir effect that significantly broadens the axial doping junctions.

PDZK1 is a novel factor in breast cancer that is indirectly regulated by estrogen through IGF-1R and promotes estrogen-mediated growth.

Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels. PDZK1 exhibited an exclusive epithelial expression with mostly cytosolic subcellular localization. Additionally, 17ß-estradiol induced PDZK1 expression above its basal level more than 24 h after treatment in MCF-7 cells. PDZK1 expression was indirectly regulated by ER-α stimulation, requiring insulinlike growth factor 1 receptor (IGF-1R) expression and function. The molecular link between PDZK1 and IGF-1R was supported by a significant correlation between protein and mRNA levels (r = 0.591, p < 0.001, and r = 0.537, p < 0.001, respectively) of the two factors in two different cohorts of human breast cancer tissues. Interestingly, PDZK1 knockdown in MCF-7 cells blocked ER-dependent growth and reduced c-Myc expression, whereas ectopic expression of PDZK1 enhanced cell proliferation in the presence or absence of 17ß-estradiol potentially through an increase in c-Myc expression, suggesting that PDZK1 has oncogenic activity. PDKZ1 also appeared to interact with the Src/ER-α/epidermal growth factor receptor (EGFR) complex, but not with IGF-1R and enhanced EGFR-stimulated MEK/ERK1/2 signaling. Collectively, our results clarify the relationship between ER-α and PDZK1, propose a direct relationship between PDZK1 and IGF-1R, and identify a novel oncogenic activity for PDZK1 in breast cancer.

Targeted Osmotic Lysis: A Novel Approach to Targeted Cancer Therapies.

The conventional treatment of cancer has been based on the delivery of non-selective toxins and/or ionizing energy that affect both the cancer and normal tissues in the hope of destroying the offending disease before killing the patient. Unfortunately, resistance often develops to these treatments and patients experience severe, dose-limiting adverse effects that reduce treatment efficacy and compromise quality of life. Recent advances in our knowledge of the biology of tumor cells and their microenvironment, the recognition of surface proteins that are unique to specific cancers and essential to cell growth and survival and signaling pathways associate with invasion and metastasis have led to the development of targeted therapies that are able to identify specific cellular markers and more selectively deliver lethal treatment to the invading cancer thus improving efficacy and limiting adverse effects. In the context of targeted approaches to cancer therapy, we present targeted osmotic lysis as a novel and fundamentally different approach for treating advanced-stage carcinoma that exploits the conserved relationship between voltage-gated sodium channels and Na+, K+-ATPase and has the potential to increase survival without compromising quality of life in a broad spectrum of highly malignant forms of cancer.

Cancer as a Channelopathy-Appreciation of Complimentary Pathways Provides a Different Perspective for Developing Treatments.

Life depends upon the ability of cells to evaluate and adapt to a constantly changing environment and to maintain internal stability to allow essential biochemical reactions to occur. Ions and ion channels play a crucial role in this process and are essential for survival. Alterations in the expression of the transmembrane proteins responsible for maintaining ion balance that occur as a result of mutations in the genetic code or in response to iatrogenically induced changes in the extracellular environment is a characteristic feature of oncogenesis and identifies cancer as one of a constellation of diseases known as channelopathies. The classification of cancer as a channelopathy provides a different perspective for viewing the disease. Potentially, it may expand opportunities for developing novel ways to affect or reverse the deleterious changes that underlie establishing and sustaining disease and developing tolerance to therapeutic attempts at treatment. The role of ions and ion channels and their interactions in the cell's ability to maintain ionic balance, homeostasis, and survival are reviewed and possible approaches that mitigate gain or loss of ion channel function to contribute to new or enhance existing cancer therapies are discussed.

Effect of Cell Cycle on Cell Surface Expression of Voltage-Gated Sodium Channels and Na+,K+-ATPase.

Voltage-gated sodium channels (VGSCs) are the target for many therapies. Variation in membrane potential occurs throughout the cell cycle, yet little attention has been devoted to the role of VGSCs and Na+,K+-ATPases. We hypothesized that in addition to doubling DNA and cell membrane in anticipation of cell division, there should be a doubling of VGSCs and Na+,K+-ATPase compared to non-dividing cells. We tested this hypothesis in eight immortalized cell lines by correlating immunocytofluorescent labeling of VGSCs or Na+,K+-ATPase with propidium iodide or DAPI fluorescence using flow cytometry and imaging. Cell surface expression of VGSCs during phases S through M was double that seen during phases G0-G1. By contrast, Na+,K+-ATPase expression increased only 1.5-fold. The increases were independent of baseline expression of channels or pumps. The variation in VGSC and Na+,K+-ATPase expression has implications for both our understanding of sodium's role in controlling the cell cycle and variability of treatments targeted at these components of the Na+ handling system.

The Role of Targeted Osmotic Lysis in the Treatment of Advanced Carcinoma in Companion Animals: A Case Series.

Background: Targeted osmotic lysis (TOL) is a novel technology that involves concomitant stimulation of voltage-gated sodium channels (VGSCs) and the pharmacological blockade of Na+, K+-ATPase causing lysis of highly malignant cancer cells. Hypothesis/Objectives. TOL offers an option for treating advanced carcinomas in companion animals. Animals. Two cats and 2 dogs that presented to veterinary hospitals for evaluation and treatment of one of several forms of carcinoma. Methods: Digoxin was administered to achieve steady-state, therapeutic concentrations. The animals were then exposed to pulsed electric field stimulation. Pre- and posttreatment assessments of tumor size and quality of life were compared. The treatment frequency and survivability varied, based on the patient's premorbid functioning and response to treatment. Results: Regardless of cancer type, TOL consistently increased survival beyond expected, often improving, but without compromising of quality of life. Conclusions and Clinical Importance. TOL warrants consideration as an option for managing advanced carcinomas.

Measurement of CFA-induced hyperalgesia and morphine-induced analgesia in rats: dorsal vs plantar mechanical stimulation of the hindpaw.

OBJECTIVE: To compare the sensitivity of stimulating the plantar and dorsal hindpaw surfaces in the detection of mechanical allodynia and morphine analgesia. BACKGROUND: Several approaches are used to assess nociceptive reactivity to mechanical stimulation in animal models of pain. Although certain techniques seem to be favored for studying specific nociceptive conditions, the differences between techniques have not been directly compared and characterized. We chose to compare methods employing stimulation applied to the dorsum of the paw with stimulation of the plantar surface to demonstrate the utility of each approach in determining baseline nociceptive thresholds, changes in those thresholds after injury, and analgesic efficacy. METHODS: Withdrawal thresholds from mechanical stimulation applied to the dorsal and plantar surface of the hindpaw were measured in rats treated with morphine after receiving subcutaneous injections of complete Freund's adjuvant (CFA) using Semmes-Weinstein (S-W) monofilaments and electro von Frey (EVF) stimulation. RESULTS: In contrast to stimulation of the dorsal surface, plantar hindpaw stimulation seldom elicited an aversive withdrawal response. Differences in withdrawal response from baseline were only detectable within the first 5 hours post-CFA and only with EVF stimulation. No significant differences in stimulation techniques were observed after the initial 5-hour window. Effective dose 50 (ED(50)) for analgesic efficacy was consistently lower using dorsal stimulation. CONCLUSIONS: Stimulation of the plantar surface of the paw is superior for detecting small changes in paw sensitivity at very low stimulus intensities, whereas stimulation of the dorsal surface is superior for delineating baseline pain thresholds and for detecting robust analgesia. CLINICAL RELEVANCE: Reliable and sensitive assessment of animal pain behaviors is critical to translational pain research. This study demonstrates the importance of using proper test protocols in animal studies and its implication in preclinical screening of potential analgesics.

Emergency Use of Targeted Osmotic Lysis for the Treatment of a Patient with Aggressive Late-Stage Squamous Cell Carcinoma of the Cervix.

Upregulation of voltage-gated sodium channels (VGSCs) and Na+/K+-ATPase (sodium pumps) is common across most malignant carcinomas. Targeted osmotic lysis (TOL) is a developing technology in which the concomitant stimulation of VGSCs and pharmacological blockade of sodium pumps causes rapid selective osmotic lysis of carcinoma cells. This treatment of cervical carcinoma is evidence that TOL is a safe, well-tolerated and effective treatment for aggressive advanced carcinomas that has the potential to extend life without compromising its quality. TOL is likely to have broad application for the treatment of advanced-stage carcinomas.

Insulin is essential for the recovery from allodynia induced by complete Freund's adjuvant.

OBJECTIVE: To determine the effect of streptozotocin (STZ)-induced diabetes on the development and recovery of thermal and mechanical hyperalgesia associated with inflammation induced by subcutaneous injection of complete Freund's adjuvant (CFA). BACKGROUND: The response to nociceptive injury in diabetes differs from that seen in normal individuals in that diabetic patients have increased susceptibility to infections and recover slowly or incompletely from infections and tissue injury due to an abnormal inflammatory response. We have chosen to examine the effect of STZ-induced hypoinsulinemia on the hyperalgesia associated with the enhanced inflammatory state that is induced by the subcutaneous injection of CFA to delineate the potential role of insulin in the development of chronic pain. METHODS: STZ- and vehicle-treated Sprague-Dawley rats were tested using thermal and mechanical stimulation after subcutaneous injection of CFA. The behavioral response was compared with that similarly determined in non-diabetic controls and insulin-depleted rats that received insulin replacement. RESULTS: Recovery of the thermal hyperalgesic response to baseline levels occurred over a period of 9-14 days, but the allodynic response to mechanical stimulation persisted for the duration of the study in STZ-treated rats. Insulin replacement prevented the delay in recovery of mechanical allodynia, but had no obvious effect on nociception in uninflamed tissue. CONCLUSIONS: Normal insulin function is essential for recovery from mechanical allodynia associated with inflammation induced by CFA. Altered insulin metabolism may selectively influence fiber-type specific mechanisms related to mechanical allodynia associated with inflammation and wound healing.

Ranolazine attenuation of CFA-induced mechanical hyperalgesia.

OBJECTIVE: To determine whether ranolazine, a new anti-angina medication, could be an effective analgesic agent in complete Freund's adjuvant-induced inflammatory pain. BACKGROUND: Plantar injection of complete Freund's adjuvant (CFA) produces an extended period of hyperalgesia that is associated with a dramatic up-regulation of Na(v) 1.7 sodium channels in populations of large and small dorsal root ganglion neurons related to the injection site. Ranolazine appears to produce its anti-angina effect through blocking the late sodium current associated with the voltage-gated sodium channel, Na(v) 1.5. Because ranolazine also inhibits Na(v) 1.7, and 1.8, we sought to determine whether it could be an effective analgesic agent in CFA-induced inflammatory pain. METHODS: Baseline determinations of withdrawal from thermal and mechanical stimulation were made in Sprague-Dawley rats ( approximately 300-350 x g). Following determination of baseline, one hindpaw in each group was injected with 0.1 mL of CFA. The contralateral paw received saline. Thermal and mechanical stimulation was repeated on the third day post-injection. Vehicle (0.9% isotonic saline; pH 3.0) or ranolazine was then administered in randomized and blinded doses either by intraperitoneal (ip) injection (0, 10, 20, and 50 mg/kg) or by oral gavage (po; 0, 20, 50, 100, and 200 mg/kg). Animals were again tested 30 minutes (ip) and 1 hour (po) after drug administration. RESULTS: Ranolazine produced dose-dependant analgesia on mechanical allodynia induced by CFA injection, but had no effect on thermal hyperalgesia. CONCLUSIONS: Ranolazine's potential as a new option for managing both angina and chronic inflammatory pain warrants further study.

Current Understanding of Mineral and Bone Disorders of Chronic Kidney Disease and the Scientific Grounds on the Use of Exogenous Parathyroid Hormone in Its Management.

Chronic Kidney disease (CKD) disturbs mineral homeostasis leading to mineral and bone disorders (MBD). Serum calcium and phosphate (Pi) remain normal until the late stages of CKD at the expense of elevate fibroblast growth factor-23 (FGF-23), a phosphaturic hormone, followed by reduced 1,25-dihydroxy-vitamin D (1,25[OH]2D) and finally elevated parathyroid hormone (PTH). Pi retention is thought to be the initial cause of CKD-MBD. The management of MBD is a huge clinical challenge because the effectiveness of current therapeutic regimens to prevent and treat MBD is limited. An intermittent regimen of PTH, when administered at the early stages of CKD, through its phosphaturic action, could prevent FGF-23 increases, the drop of 1,25(OH)2D, and the development of renal osteodystrophy, including secondary hyperparathyroidism (HPT) and its catabolic effects on the skeleton. Even in more advanced stages of CKD that have not progressed to tertiary HPT, could be beneficial. Therapeutic effects could be achieved in vascular calcification as well. Limited experimental/clinical data support the effectiveness of PTH in CKD-MBD. Its safety, has been established only when it is used for the treatment of osteoporosis, including patients with CKD. The proposed intermittent PTH administration is biologically plausible but its effectiveness and safety has to be critically assessed in long term prospective studies in patients with CKD-MBD.

Targeted Osmotic Lysis of Highly Invasive Breast Carcinomas Using Pulsed Magnetic Field Stimulation of Voltage-Gated Sodium Channels and Pharmacological Blockade of Sodium Pumps.

Abstract: Concurrent activation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps causes a targeted osmotic lysis (TOL) of carcinomas that over-express the VGSCs. Unfortunately, electrical current bypasses tumors or tumor sections because of the variable resistance of the extracellular microenvironment. This study assesses pulsed magnetic fields (PMFs) as a potential source for activating VGSCs to initiate TOL in vitro and in vivo as PMFs are unaffected by nonconductive tissues. In vitro, PMFs (0-80 mT, 10 msec pulses, 15 pps for 10 min) combined with digoxin-lysed (500 nM) MDA-MB-231 breast cancer cells stimulus-dependently. Untreated, stimulation-only, and digoxin-only control cells did not lyse. MCF-10a normal breast cells were also unaffected. MDA-MB-231 cells did not lyse in a Na+-free buffer. In vivo, 30 min of PMF stimulation of MDA-MB-231 xenografts in J/Nu mice or 4T1 homografts in BALB/c mice, concurrently treated with 7 mg/kg digoxin reduced tumor size by 60-100%. Kidney, spleen, skin and muscle from these animals were unaffected. Stimulation-only and digoxin-only controls were similar to untreated tumors. BALB/C mice with 4T1 homografts survived significantly longer than mice in the three control groups. The data presented is evidence that the PMFs to activate VGSCs in TOL provide sufficient energy to lyse highly malignant cells in vitro and to reduce tumor growth of highly malignant grafts and improve host survival in vivo, thus supporting targeted osmotic lysis of cancer as a possible method for treating late-stage carcinomas without compromising noncancerous tissues.

A novel pipeline of 2-(benzenesulfonamide)-N-(4-hydroxyphenyl) acetamide analgesics that lack hepatotoxicity and retain antipyresis.

Although acetaminophen (ApAP) is one of the most commonly used medicines worldwide, hepatotoxicity is a risk with overdose or in patients with compromised liver function. ApAP overdose is the most common cause of acute fulminant hepatic failure. Oxidation of ApAP to N-acetyl-p-benzoquinone imine (NAPQI) is the mechanism for hepatotoxicity. 1 is a non-hepatotoxic, metabolically unstable lipophilic ApAP analog that is not antipyretic. The newly synthesized 3 is a non-hepatotoxic ApAP analog that is stable, lipophilic, and retains analgesia and antipyresis. Intraperitoneal or po administration of the new chemical entities (NCEs), 3b and 3r, in concentrations equal to a toxic dose of ApAP did not result in the formation of NAPQI. Unlike livers from NCE-treated mice, the livers from ApAP-treated mice demonstrated large amounts of nitrotyrosine, a marker of mitochondrial free radical formation, and loss of hepatic tight junction integrity. Given the widespread use of ApAP, hepatotoxicity risk with overuse, and the ongoing opioid epidemic, these NCEs represent a novel, non-narcotic therapeutic pipeline.

Ranolazine attenuates behavioral signs of neuropathic pain.

Ranolazine modulates the cardiac voltage-gated sodium channel (NaV 1.5) and is approved by the FDA in the treatment of ischemic heart disease. Ranolazine also targets neuronal (NaV 1.7, 1.8) isoforms that are implicated in neuropathic pain. Therefore, we determined the analgesic efficacy of ranolazine in a preclinical animal model of neuropathic pain. Both intraperitoneal and oral administration of ranolazine dose-dependently inhibited the mechanical and cold allodynia associated with spared nerve injury, without producing ataxia or other behavioral side effects. These data warrant clinical investigation of the potential use of ranolazine in the treatment of neuropathic pain.

Selective lysis of breast carcinomas by simultaneous stimulation of sodium channels and blockade of sodium pumps.

Sodium influx through voltage-gated sodium channels (VGSCs) coupled with balanced removal of sodium ions via Na+, K+-ATPase is a major determinant of cellular homeostasis and intracellular ionic concentration. Interestingly, many metastatic carcinomas express high levels of these channels. We hypothesized that if excess VGSCs are activated and Na+, K+-ATPase is simultaneously blocked, the intracellular Na+ concentration should increase, resulting in water movement into the cell, causing swelling and lytic cell death. MDA-MB-231 breast cancer cells over-express VGSCs by 7-fold. To test our hypothesis, we treated these cells in vitro with the Na+, K+-ATPase blocker, ouabain, and then stimulated with a sublethal electric current. For in vivo histologic and survival studies, MDA-MB-231 xenografts were established in Nu/J mice. Mice injected with saline or ouabain were electrically stimulated with trains of 10 msec 10V DC pulses. Within seconds to minutes, the cells swelled and lysed. MCF-10a cells, which express normal VGSCs levels, were unaffected by this treatment. Cells from the weakly-malignant cell line, MCF-7, which express 3-fold greater VGSCs than MCF-10a cells, displayed an intermediate time-to-lysis. The rate of lysis correlated directly with the degree of sodium channel expression and malignancy. We also demonstrated efficacy in cell lines from prostate, colon and lung carcinomas. Treated MDA-MB-231 xenografts showed 60-80% cell death. In survival studies, TOL-treated mice showed significantly slower tumor growth vs. controls. These results are evidence that this "targeted osmotic lysis" represents a novel method for selectively killing cancer cells and warrants further investigation as a potential treatment for advanced and end-stage breast cancer.

Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy.

Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intracluster distances (d). From these data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P < 0.001). For comparison, the prediction accuracy was only 61% (P = 0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Large clusters (N ≥ 3) with d > 50 nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d < 50 nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells.