Assessment of Punch Elevation Efficacy in Atrophic Scars with Unsatisfactory Results Following Prior Laser Treatment Using Three-Dimensional Image Analysis / 대한피부과학회지

Background@#Various treatments exist for addressing volume loss in atrophic scars. Although laser therapy has gained traction in treating atrophic scars, it is associated with side effects, such as post-inflammatory hyperpigmentation or erythema. Additionally, not all types of atrophic scars respond optimally to laser therapy, even after multiple sessions. @*Objective@#This study aimed to evaluate the efficacy and safety of punch elevation for atrophic scars that yielded unsatisfactory outcomes after repeated laser treatment sessions. @*Methods@#Seven patients with atrophic scars on their facial area underwent punch elevation, concurrently supplemented by fractional CO2 laser application to the scar margins. Improvement in volume restoration of atrophic scars was assessed via investigator evaluation and 3-dimensional (3D) image analysis. @*Results@#After 1 month, median volume (interquartile range) of depression improved from 4.39 mm3 (2.23∼9.90 mm3 ) to 1.97 mm3 (1.46∼7.50 mm3 ), indicating a statistically significant difference post-punch elevation (p=0.018). No serious adverse events were reported during follow-up. @*Conclusion@#The efficacy of the punch elevation was objectively evaluated. Punch elevation is a safe and effective therapeutic avenue for atrophic scars that exhibit resistance to laser or alternative interventions.

A Case of Cowden Syndrome Presenting with Diverse Cutaneous Manifestations

Cowden syndrome is caused by mutations in the phosphatase and tensin homolog (PTEN) gene and is part of the PTEN hamartoma tumor syndrome. Skin lesions including trichilemmomas, acral keratosis, mucocunateous neuromas and oral paillomas are the most prevalent feature found in patients with Cowden syndrome. It also possesses an increased risk of developing malignancies including breast, thyroid, endometrial, and colorectal cancers.Due to the increased risk of cancer, early diagnosis and regular surveillance are important for Cowden syndrome patients. Herein, we report a case of Cowden syndrome with diverse cutaneous manifestations and thyroid cancer.

Microcystic Adnexal Carcinoma on the Forehead Misdiagnosed as Cylindroma: A Case Report / 대한피부과학회지

Microcystic adnexal carcinoma (MAC) is a rare infiltrative cutaneous malignant tumor that presents as scar-like papules or plaques on sun-exposed skin. MAC can be misdiagnosed as a benign adnexal tumor, owing to its histopathologic similarity and slow growth rate. Nonetheless, MAC can show aggressive local invasion. Therefore, MAC should be treated with surgical excision or radiation therapy. Herein, we report the case of a 51-year-old woman with a flesh-colored plaque on her forehead. The patient was diagnosed with a benign adnexal tumor on the first punch biopsy. Notably, after a second incisional biopsy, the patient was finally diagnosed with MAC. This case demonstrates the difficulty of diagnosing MAC, suggesting that MAC should be considered for the differential diagnosis of slow-growing tumors in the head and neck and that multiple biopsies are needed when histological findings are ambiguous.

A Case of Paradoxical Vocal Fold Movement Due to Psychological Causes

Paradoxical vocal fold movement (PVFM) is a dystonic laryngeal disorder characterized by involuntary vocal fold adduction during inspiration and/or expiration. PVFM is uncommon and may aggravate airway obstruction. And patients with PVFM have a specific etiology; therefore, treatment must be individualized and given immediately. We present a case of 63-year-old male presenting with intermittent dyspnea. After multidisciplinary workup, we presumed psychogenic PVFM and evaluated with speech-language pathologist and psychologist. In this report, we describe a rare case of psychogenic PVFM patient.

Clinical Benefit of Early Laser Treatment of Scars in Children: A Single-Center Retrospective Study / 대한피부과학회지

Background@#Scars in children are highly concerning to most parents who usually seek prompt treatment for these lesions. However, compared with adults, children show a greater tendency to develop hypertrophic scars and a higher likelihood of scar widening with increasing age. @*Objective@#We investigated the role of laser treatment for scars in pediatric patients in view of the fact that this approach is challenging in this age group. @*Methods@#This retrospective study included patients aged ＜17 years who visited the Samsung Medical Center between 2013 and 2018 for scar treatment. Of the 28 children who visited the center during this time, 14 presented within 4 to 5 weeks from scar onset and received laser therapy and 14 presented after 4 to 5 weeks and received topical treatment. The Stony Brook Scar Evaluation Scale (SBSES) was used to assess scar formation. @*Results@#The mean initial SBSES scores were lower in the laser than in the topical group (1.93±0.92 vs. 2.71±0.83, p=0.0363). No intergroup difference was observed in SBSES scores upon treatment completion (4.50±0.94 vs. 4.21±1.19, p=0.4673). Multivariable analysis after adjustment for age, sex, and initial SBSES scores showed that the type (laser vs. topical) of treatment was not significantly associated with an SBSES score=5 or duration of treatment. @*Conclusion@#Laser intervention may be useful for scar therapy in children who present early and can receive prompt treatment before scar maturation; however, further studies are warranted to validate our results.

Clinical Benefit of Early Laser Treatment of Scars in Children: A Single-Center Retrospective Study / 대한피부과학회지

Background@#Scars in children are highly concerning to most parents who usually seek prompt treatment for these lesions. However, compared with adults, children show a greater tendency to develop hypertrophic scars and a higher likelihood of scar widening with increasing age. @*Objective@#We investigated the role of laser treatment for scars in pediatric patients in view of the fact that this approach is challenging in this age group. @*Methods@#This retrospective study included patients aged ＜17 years who visited the Samsung Medical Center between 2013 and 2018 for scar treatment. Of the 28 children who visited the center during this time, 14 presented within 4 to 5 weeks from scar onset and received laser therapy and 14 presented after 4 to 5 weeks and received topical treatment. The Stony Brook Scar Evaluation Scale (SBSES) was used to assess scar formation. @*Results@#The mean initial SBSES scores were lower in the laser than in the topical group (1.93±0.92 vs. 2.71±0.83, p=0.0363). No intergroup difference was observed in SBSES scores upon treatment completion (4.50±0.94 vs. 4.21±1.19, p=0.4673). Multivariable analysis after adjustment for age, sex, and initial SBSES scores showed that the type (laser vs. topical) of treatment was not significantly associated with an SBSES score=5 or duration of treatment. @*Conclusion@#Laser intervention may be useful for scar therapy in children who present early and can receive prompt treatment before scar maturation; however, further studies are warranted to validate our results.

Deficiencies of Homer2 and Homer3 accelerate aging-dependent bone loss in mice

Homer proteins are scaffold proteins that regulate calcium (Ca2+) signaling by modulating the activity of multiple Ca2+ signaling proteins. In our previous report, Homer2 and Homer3 regulated NFATc1 function through its interaction with calcineurin, which then acted to regulate receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone metabolism. However, to date, the role of Homers in osteoclastogenesis remains unknown. In this study, we investigated the roles of Homer2 and Homer3 in aging-dependent bone remodeling. Deletion of Homer2/Homer3 (Homer2/3 DKO) markedly decreased the bone density of the femur. The decrease in bone density was not seen in mice with Homer2 (Homer2−/−) and Homer3 (Homer3−/−) deletion. Moreover, RANKL treatment of bone marrow-derived monocytes/macrophages in Homer2/3 DKO mice significantly increased the formation of multinucleated cells and resorption areas. Finally, Homer2/3 DKO mice decreased bone density in an aging-dependent manner. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation during aging by Homer proteins, specifically Homer2 and Homer3.

Homer2 regulates amylase secretion via physiological calcium oscillations in mouse parotid gland acinar cells

The salivary glands secrete saliva, which plays a role in the maintenance of a healthy oral environment. Under physiological conditions, saliva secretion within the acinar cells of the gland is regulated by stimulation of specific calcium (Ca2+) signaling mechanisms such as increases in the intracellular Ca2+ concentration ([Ca2+]i) via storeoperated Ca2+ entry, which involves components such as Orai1, transient receptor potential (TRP) canonical 1, stromal interaction molecules, and inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs). Homer proteins are scaffold proteins that bind to G protein-coupled receptors, IP3Rs, ryanodine receptors, and TRP channels. However, their exact role in Ca2+ signaling in the salivary glands remains unknown. In the present study, we investigated the role of Homer2 in Ca2+ signaling and saliva secretion in parotid gland acinar cells under physiological conditions. Deletion of Homer2 (Homer2−/− markedly decreased the amplitude of [Ca2+]i oscillations via the stimulation of carbachol, which is physiologically concentrated in parotid acinar cells, whereas the frequency of [Ca2+]i oscillations showed no difference between wild-type and Homer2−/− mice. Homer2−/− mice also showed a significant decrease in amylase release by carbachol in the parotid gland in a dose-dependent manner. These results suggest that Homer2 plays a critical role in maintaining [Ca2+]i concentration and secretion of saliva in mouse parotid gland acinar cells.

Humanin suppresses receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation via AMP-activated protein kinase activation

Humanin (HN) is a mitochondrial peptide that exhibits cytoprotective actions against various stresses and diseases. HN has been shown to induce the phosphorylation of AMP-activated protein kinase (AMPK), which is a negative regulator of receptor activator of nuclear factor-κB ligand (RANKL). However, the role of HN in osteoclastogenesis or other skeletal disorders remains unknown. Here, we examined whether HN regulates osteoclastogenesis via AMPK activation using bone marrow-derived macrophage (BMM) cultures. Our results show that HN inhibited RANKL-induced osteoclast formation and reduced the expression of genes involved in osteoclastogenesis, including nuclear factor of activated T-cells cytoplasmic 1, osteoclast-associated receptor, cathepsin K, and tartrate-resistant acid phosphatase. Moreover, HN increased the levels of phosphorylated AMPK protein; compound C, an AMPK inhibitor, recovered HN-induced osteoclast differentiation. In addition, we found that HN significantly decreased the levels of RANKL-induced reactive oxygen species in BMMs. Therefore, these results indicate that HN plays an important role in osteoclastogenesis and may function as an inhibitor of bone disorders via AMPK activation.

Increased store-operated Ca²⁺ entry mediated by GNB5 and STIM1

Recent human genetic studies have shown that Gβ5 is related to various clinical symptoms, such as sinus bradycardia, cognitive disability, and attention deficit hyperactivity disorder. Although the calcium signaling cascade is closely associated with a heterotrimeric G-protein, the function of Gβ5 in calcium signaling and its relevance to clinical symptoms remain unknown. In this study, we investigated the in vitro changes of store-operated calcium entry (SOCE) with exogenous expression of Gβ5. The cells expressing Gβ5 had enhanced SOCE after depletion of calcium ion inside the endoplasmic reticulum. Gβ5 also augmented Stim1- and Orai1-dependent SOCE. An ORAI1 loss-of-function mutant did not show inhibition of Gβ5-induced SOCE, and a STIM1-ERM truncation mutant showed no enhancement of SOCE. These results suggested a novel role of GNB5 and Stim1, and provided insight into the regulatory mechanism of SOCE.

Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.

Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca2+ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca2+ concentration ([Ca2+]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca2+]i increase with nonspecific Ca2+ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca2+]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca2+ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca2+ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca2+ regulation of the WNK-OSR1 pathway in intact cells.

Bacterial PAMPs and Allergens Trigger Increase in Ca2+i-induced Cytokine Expression in Human PDL Fibroblasts

An oral environment is constantly exposed to environmental factors and microorganisms. The periodontal ligament (PDL) fibroblasts within this environment are subject to bacterial infection and allergic reaction. However, how these condition affect PDL fibroblasts has yet to be elucidated. PDL fibroblasts were isolated from healthy donors. We examined using reverse transcription-polymerase chain reaction and measuring the intracellular Ca2+ concentration ([Ca2+]i). This study investigated the receptors activated by exogenous bacterial pathogens (Lipopolysaccharide and peptidoglycan) and allergens (German cockroach extract and house dust mite) as well as these pathogenic mediators-induced effects on the intracellular Ca2+ signaling in human PDL fibroblasts. Moreover, we evaluated the expression of pro-inflammatory cytokines (interleukin (IL)-1beta, IL-6, and IL-8) and bone remodeling mediators (receptor activator of NF-kappaB ligand and osteoprotegerin) and intracellular Ca2+-involved effect. Bacterial pathogens and allergic mediators induced increased expression of pro-inflammatory cytokines, and these results are dependent on intracellular Ca2+. However, bacterial pathogens and allergic mediators did not lead to increased expression of bone remodeling mediators, except lipopolysaccharide-induced effect on receptor activator of NF-kappaB ligand expression. These experiments provide evidence that a pathogens and allergens-induced increase in [Ca2+]i affects the inflammatory response in human PDL fibroblasts.

Prevention and Decontamination of Chemical, Biological, Radiological, and Nuclear Contaminants for the Emergency Medical Personnel during Ambulance Services / 한양의대학술지

This paper will review proper protocol for Emergency Medical Technicians (EMTs) between the ambulance station, scene and hospital. EMTs must know how to protect themselves both inside and outside the ambulance from chemical, biological, radiological, and nuclear (CBRN) contaminants in order to provide the best quality care. EMTs should also know how to remove contamination after taking a patient to the hospital. A CBRN protocol at the scene of exposure is the best defense strategy for protecting both EMTs and the emergency ambulance vehicle from contamination. If EMTs and the emergency ambulance vehicle are exposed to CBRN, neither will be of great service as they both play a critical part in the quality of care given to a patient. In the event of possible exposure, EMTs should dress in high perceptibility personal protective equipment (PPE). Those who have been trained prior should arrive to the scene with an air purifying respirator. Before a patient with possible exposure is placed inside an emergency ambulance vehicle, all patients' compartments should be completely covered and sealed with plastic film. Once the emergency medical crew reaches the hospital and the patient is discharged from the emergency vehicle, all plastic film inside the ambulance, from the cockpit ceiling to the floor, should be removed and properly discarded. The patient's clothing and shoes should be placed into a double layered plastic bag. Adhering to proper protocols, within 10 minutes of arrival at hospital, a contaminated ambulance should undergo pre-washing, decontamination and rinsing operations.

House Dust Mite Extract Induces PLC/IP3-dependent Ca2+ Signaling and IL-8 Expression in Human Gingival Epithelial Cells

The gingival epithelium of the oral cavity is constantly exposed to exogenous stimuli such as bacterial toxins, allergens, and thermal changes. These exogenous stimuli are resisted by innate host defense in gingival epithelial cells. However, it is unclear exactly how the exogenous stimuli affect detrimentally on the human gingival epithelial cells. Here, we investigated whether the allergen, such as house dust mite (HDM) extract, is linked to Ca2+ signaling and proinflammatory cytokine expression in primary cultured human gingival epithelial cells. HDM extract induced an increase in intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. Extracellular Ca2+ depletion did not affected on the HDM extract-induced increase in [Ca2+]i. The HDM extract-induced increase in [Ca2+]i was abolished by the treatment with U73122 and 2-APB, which are inhibitors of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptor. Moreover, HDM extract induced the mRNA expression of pro-inflammatory cytokine, interleukin (IL)-8. These results suggest that HDM extract triggers PLC/IP3-dependent Ca2+ signaling and IL-8 mRNA expression in primary cultured human gingival epithelial cells.

DA-6034 Induces Ca2+i Increase in Epithelial Cells

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca2+ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca2+ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca2+-activated Cl- channels (CaCCs) and increased intracellular calcium concentrations ([Ca2+]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca2+]i in mouse salivary gland cells and human corneal epithelial cells. [Ca2+]i increase of DA-6034 was dependent on the Ca2+ entry from extracellular and Ca2+ release from internal Ca2+ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca2+ stores. These results suggest that DA-6034 induces Ca2+ signaling via extracellular Ca2+ entry and RyRs-sensitive Ca2+ release from internal Ca2+ stores in epithelial cells.

Xylitol Down-Regulates 1alpha,25-Dihydroxy Vitamin D3-induced Osteoclastogenesis via in Part the Inhibition of RANKL Expression in Osteoblasts

Xylitol is a sugar alcohol with a variety of functions including bactericidal and anticariogenic effects. However, the cellular mechanisms underlying the role of xylitol in bone metabolism are not yet clarified. In our present study, we exploited the physiological role of xylitol on osteoclast differentiation in a co-culture system of osteoblastic and RAW 264.7 cells. Xylitol treatment of these co-cultures reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells induced by 10 nM 1alpha,25(OH)2D3 in a dose-dependent manner. A cell viability test revealed no marked cellular damage by up to 100 mM of xylitol. Exposure of osteoblastic cells to xylitol decreased RANKL, but not OPG, mRNA expression in the presence of 10(-8) M 1alpha,25(OH)2D3 in a dose-dependent manner. Furthermore, bone resorption activity, assessed on bone slices in the co-culture system, was found to be dramatically decreased with increasing xylitol concentrations. RANKL and OPG proteins were assayed by ELISA and the soluble RANKL (sRANKL) concentration was decreased with an increased xylitol concentration. In contrast, OPG was unaltered by any xylitol concentration in this assay. These results indicate that xylitol inhibits 1alpha,25(OH)2D3-induced osteoclastogenesis by reducing the sRANKL/OPG expression ratio in osteoblastic cells.

Activation of G Proteins by Aluminum Fluoride Enhances RANKL-Mediated Osteoclastogenesis

Receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis is accompanied by intracellular Ca2+ mobilization in a form of oscillations, which plays essential roles by activating sequentially Ca2+/calmodulin-dependent protein kinase, calcineurin and NFATc1, necessary in the osteoclast differentiation. However, it is not known whether Ca2+ mobilization which is evoked in RANKL-independent way induces to differentiate into osteoclasts. In present study, we investigated Ca2+ mobilization induced by aluminum fluoride (AlF4-), a G-protein activator, with or without RANKL and the effects of AlF4- on the osteoclastogenesis in primary cultured mouse bone marrow-derived macrophages (BMMs). We show here that AlF4- induces intracellular Ca2+ concentration ([Ca2+]i) oscillations, which is dependent on extracellular Ca2+ influx. Notably, co-stimulation of AlF4- with RANKL resulted in enhanced NFATc1 expression and formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells. Additionally, we confirmed that mitogen-activated protein kinase (MAPK) is also activated by AlF4-. Taken together, these results demonstrate that G-protein would be a novel modulator responsible for [Ca2+]i oscillations and MAPK activation which lead to enhancement of RANKL-mediated osteoclastogenesis.

TRPM7 Is Essential for RANKL-Induced Osteoclastogenesis

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body Mg2+ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-kappaB ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of Mg2+. Knock-down of TRPM7 by siTRPM7 reduced intracellular Ca2+ concentration ([Ca2+]i) increases by 0 mM [Mg2+]e in HEK293 cells and inhibited the generation of RANKL-induced Ca2+ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced [Ca2+]i oscillations that triggers the late stages of osteoclastogenesis.

Fast temporal detection of intracellular hydrogen peroxide by HyPer

HyPer is the genetically encoded biosensor of intracellular hydrogen peroxide (H2O2), the most stable of the reactive oxygen species (ROS) generated by living cells. HyPer has a high sensitivity and specificity for detecting intracellular H2O2 by confocal laser microscopy. However, it was not known whether high speed ratiometric imaging of H2O2 by HyPer is possible. We thus investigated the sensitivity of HyPer in detecting changes to the intracellular H2O2 levels in HEK293 and PC12 cells using a microfluorometer imaging system. Increase in the HyPer ratio were clearly evident on stimulations of more than 100 microM H2O2 and fast changes in the HyPer ratio were observed on ratiometric fluorescent images after H2O2 treatment. These results suggest that HyPer is a potent biosensor of the fast temporal production of intracellular H2O2.