Embolization using both n-butyl cyanoacrylate and gelatin sponges in a patient with a posterior superior pancreaticoduodenal artery pseudoaneurysm that ruptured and bled into the drain tube.

Transcatheter arterial embolization is a useful treatment for postpancreatectomy hemorrhage, a severe complication of pancreatic surgery. N-butyl cyanoacrylate is a liquid and permanent embolic material that is widely used in transcatheter arterial embolization. However, its use can lead to the adherence of the catheter to the vessel wall and occlusion of the catheter lumen. This case report presents the case of a 63-year-old man with a postpancreatectomy posterior superior pancreaticoduodenal artery pseudoaneurysm, which ruptured and bled into a drain tube. The patient underwent transcatheter arterial embolization using N-butyl cyanoacrylate and a gelatin sponge without the incidence of adherence or occlusion of the drain tube. Gelatin sponge, which was used as a temporary embolic material, was effective in preventing the drain tube from adhering and occluding.

Creation of synthetic contrast-enhanced computed tomography images using deep neural networks to screen for renal cell carcinoma.

In this study, we elucidate if synthetic contrast enhanced computed tomography images created from plain computed tomography images using deep neural networks could be used for screening, clinical diagnosis, and postoperative follow-up of small-diameter renal tumors. This retrospective, multicenter study included 155 patients (artificial intelligence training cohort [n = 99], validation cohort [n = 56]) who underwent surgery for small-diameter (≤40 mm) renal tumors, with the pathological diagnosis of renal cell carcinoma, during 2010-2020. We created a learned deep neural networks using pix2pix. We examined the quality of the synthetic enhanced computed tomography images created using this deep neural networks and compared them with real enhanced computed tomography images using the zero-mean normalized cross-correlation parameter. We assessed concordance rates between real and synthetic images and diagnoses according to 10 urologists by creating a receiver operating characteristic curve and calculating the area under the curve. The synthetic computed tomography images were highly concordant with the real computed tomography images, regardless of the existence or morphology of the renal tumor. Regarding the concordance rate, a greater area under the curve was obtained with synthetic computed tomography (area under the curve = 0.892) than with only computed tomography (area under the curve = 0.720; p < 0.001). In conclusions, this study is the first to use deep neural networks to create a high-quality synthetic computed tomography image that was highly concordant with a real computed tomography image. Our synthetic computed tomography images could be used for urological diagnoses and clinical screening.

Tmax volume can predict clinical type in patients with acute ischemic stroke.

OBJECTIVE: Endovascular therapy (EVT) is performed for acute ischemic stroke (AIS) with large vessel occlusion (LVO), however, the treatment strategies and clinical outcomes differ between cardiac embolism (CE) and intracranial arteriosclerosis-derived LVO (ICAS-LVO). We analyzed whether the time-to-max (Tmax) volume derived from perfusion imaging predicted clinical classification in AIS patients before EVT. METHODS: Consecutive AIS patients with anterior circulation LVO evaluated by automated imaging software were retrospectively identified. Patients were classified into a CE group and an ICAS-LVO group, and parameters were compared between groups. RESULTS: Thirty-nine patients were included and Tmax volume and Tmax > 6 s volume/Tmax > 4 s volume were significantly greater in the CE group than in the ICAS-LVO group (Tmax > 4 s volume: 261 mL vs. 149 mL, p = .01, Tmax > 6 s volume: 143 mL vs. 59 mL, p = .001, Tmax > 6 s volume/Tmax > 4 s volume: 0.59 vs. 0.40, p < .001). Multiple logistic regression analysis indicated an association between clinical classification and high Tmax > 6 s volume/Tmax > 4 s volume (p = .04). CONCLUSION: The Tmax volume derived from perfusion imaging predicts the clinical classification of AIS patients before EVT.

Facial Spasm Caused by Compression of the Distal Portion of the Facial Nerve by the Anterior Inferior Cerebellar Artery, Resulting in Delayed Peripheral Facial Nerve Palsy: A Case Report.

Delayed onset of facial spasm following surgery for unilateral facial spasm after microvascular decompression (MVD) of the distal facial nerve is rare. We report a case of unilateral facial spasm caused by compression of the distal facial nerve successfully treated with MVD resulting in delayed peripheral facial nerve palsy. A 51-year-old male patient with a left facial spasm showed a gradual worsening of symptoms. Head imaging revealed that the left anterior inferior cerebellar artery (AICA) was in contact with the distal portion of the left facial nerve; hence, MVD was performed. The AICA was pressing on the distal facial nerve, so the AICA was transpositioned. Postoperatively, the facial spasm improved. On the eighth postoperative day, the patient showed left peripheral facial nerve palsy and was given conservative treatment. The patient was discharged home on the sixteenth postoperative day. One month after discharge, the facial palsy was in complete remission. Distal facial nerve compression may cause unilateral facial spasms. Although delayed facial nerve palsy may occur, the prognosis is good.

Severe Gastrointestinal Disorder Due to Capecitabine Associated with Dihydropyrimidine Dehydrogenase Deficiency: A Case Report and Literature Review.

Dihydropyrimidine dehydrogenase (DPD) deficiency induces severe adverse events in patients receiving fluoropyrimidines. We encountered a 64-year-old DPD-deficient man with a severe capecitabine-related gastrointestinal disorder. He received capecitabine-containing chemotherapy after rectal cancer resection. During the first course of chemotherapy, he developed severe diarrhea, a fever, and hematochezia. Endoscopy revealed mucosal shedding with bleeding throughout the gastrointestinal tract. DPD deficiency was suspected because he developed many severe adverse events of capecitabine early and was finally confirmed based on the finding of a low DPD activity level in peripheral blood mononuclear cells. After one month of intensive care, hemostasis and mucosal healing were noted, although his gastrointestinal function did not improve, and he had persistent nutritional management issues.

A convenient and reproducible method for the synthesis of astatinated 4-[211At]astato-l-phenylalanine via electrophilic desilylation.

The 211At-labeled compound, 4-[211At]astato-l-phenylalanine, is one of the most promising amino acid derivatives for use in targeted alpha therapy (TAT) for various cancers. Electrophilic demetallation of a stannyl precursor is the most widely used approach for labeling biomolecules with 211At. However, the low acid-resistance of the stannyl precursor necessitates the use of an N- and C-terminus-protected precursor, which results in a low overall radiochemical yield (RCY) due to the multiple synthetic steps involved. In this study, a deprotected organosilyl compound, 4-triethylsilyl-l-phenylalanine, was employed for the direct synthesis of astatinated phenylalanines. 211At was separately recovered from the irradiated 209Bi target using chloroform (CHCl3) and N-chlorosuccinimide-methanol (NCS-MeOH) solution. The RCYs of 4-[211At]astato-l-phenylalanine obtained from the triethylsilyl precursor with the use of 211At, isolated in CHCl3 and NCS-MeOH solution, were 75% and 64% respectively. In both cases, the retention time of the 4-[211At]astato-l-phenylalanine was found to be about 20 min, which showed reasonable correlation with the retention time of non-radioactive 4-halo-l-phenylalanines (4-chloro-, 4-bromo-, and 4-iodo-l-phenylalanine). The one-step reaction examined in this study involved mild reaction conditions (70 °C) and a short time (10 min) compared to the other currently reported procedures for astatination. Electrophilic desilylation was found to be very effective for the labeling of aromatic amino acids with 211At.

Mutant TP53 modulates metastasis of triple negative breast cancer through adenosine A2b receptor signaling.

PURPOSE: The identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis. EXPERIMENTAL DESIGN: TNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53. A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression. Functional assays were performed in vitro and in vivo to assess the role of ADORA2B in TNBC. RESULTS: Transcriptome profiling identified ADORA2B as up-regulated in basal-like TNBC cell lines with R248Q-mutated TP53, with shRNA-screening suggesting the potential for a synthetic-lethal interaction between these genes. In clinical samples, ADORA2B was highly expressed in 39.4% (26/66) of TNBC patients. ADORA2B-expression was significantly correlated with ER (P < 0.01), PgR (P = 0.027), EGFR (P < 0.01), and tumor size (P = 0.037), and was an independent prognostic factor for outcome (P = 0.036). In line with this, ADORA2B-transduced TNBC cells showed increased tumorigenesis, and ADORA2B knockdown, along with mutant p53 knockdown, decreased metastasis both in vitro and in vivo. Notably, the cytotoxic cyclic peptide SA-I suppressed ADORA2B expression and tumorigenesis in TNBC cell lines. CONCLUSIONS: ADORA2B expression increases the oncogenic potential of basal-like TNBC and is an independent factor for poor outcome. These data suggest that ADORA2B could serve as a prognostic biomarker and a potential therapeutic target for basal-like TNBC.

Lipoxygenase-mediated generation of lipid peroxides enhances ferroptosis induced by erastin and RSL3.

In cancer cells the small compounds erastin and RSL3 promote a novel type of cell death called ferroptosis, which requires iron-dependent accumulation of lipid reactive oxygen species. Here we assessed the contribution of lipid peroxidation activity of lipoxygenases (LOX) to ferroptosis in oncogenic Ras-expressing cancer cells. Several 12/15-LOX inhibitors prevented cell death induced by erastin and RSL3. Furthermore, siRNA-mediated silencing of ALOX15 significantly decreased both erastin-induced and RSL3-induced ferroptotic cell death, whereas exogenous overexpression of ALOX15 enhanced the effect of these compounds. Immunofluorescence analyses revealed that the ALOX15 protein consistently localizes to cell membrane during the course of ferroptosis. Importantly, treatments of cells with ALOX15-activating compounds accelerated cell death at low, but not high doses of erastin and RSL3. These observations suggest that tumor ferroptosis is promoted by LOX-catalyzed lipid hydroperoxide generation in cellular membranes.

Platelet × CRP Multiplier Value as an Indicator of Poor Prognosis in Patients With Resectable Pancreatic Cancer.

OBJECTIVE: Thrombocytosis in patients with various cancers has been considered a parameter for poor prognosis; however, its contribution to pancreatic cancer remains controversial. METHODS: Potential preoperative prognostic parameters (platelets, neutrophils, lymphocytes, the platelet-lymphocyte ratio, the neutrophil-lymphocyte ratio, the serum C-reactive protein [CRP], and carbohydrate antigen 19-9) were retrospectively analyzed in 95 patients with pancreatic cancer. Cutoff values were defined according to receiver operating characteristic curve analysis, and median survival times (MSTs) were compared. RESULTS: Median survival times (days) significantly differed according to platelet count (high [552] vs low [735], P = 0.017), CRP (high [471] vs low [750], P = 0.001), and carbohydrate antigen 19-9 level (high [639] vs low [765], P = 0.021), whereas there was no difference in the platelet-lymphocyte ratio and the neutrophil-lymphocyte ratio. Multivariate analysis identified thrombocytosis (hazard ratio, 2.015) and CRP level (hazard ratio, 1.771) as independent prognostic factors. The combinatory effects of platelets and the inflammatory response using a platelet × CRP multiplier value could effectively distinguished the MSTs (days) of patients with pancreatic cancer (high [482] vs low [812], P < 0.001). CONCLUSIONS: Thrombocytosis and CRP influenced pancreatic cancer patient prognosis. Platelet × CRP multiplier is assumed as a useful parameter that reflects the contribution of activated platelets to cancer progression.

Nasogastric tube syndrome induced by an indwelling long intestinal tube.

The nasogastric tube (NGT) has become a frequently used device to alleviate gastrointestinal symptoms. Nasogastric tube syndrome (NTS) is an uncommon but potentially life-threatening complication of an indwelling NGT. NTS is characterized by acute upper airway obstruction due to bilateral vocal cord paralysis. We report a case of a 76-year-old man with NTS, induced by an indwelling long intestinal tube. He was admitted to our hospital for treatment of sigmoid colon cancer. He underwent sigmoidectomy to release a bowel obstruction, and had a long intestinal tube inserted to decompress the intestinal tract. He presented acute dyspnea following prolonged intestinal intubation, and bronchoscopy showed bilateral vocal cord paralysis. The NGT was removed immediately, and tracheotomy was performed. The patient was finally discharged in a fully recovered state. NTS be considered in patients complaining of acute upper airway obstruction, not only with a NGT inserted but also with a long intestinal tube.

The fungicidal activity of amphotericin B requires autophagy-dependent targeting to the vacuole under a nutrient-starved condition in Saccharomyces cerevisiae.

In this study, we demonstrated that in distilled water, a nutrient-starved condition that elicits autophagy in Saccharomyces cerevisiae, an array of autophagy-deficient mutants are resistant to the fungicidal effects of amphotericin B. In addition, we found that a dansyl-labelled derivative of the antibiotic colocalized with disintegrated vacuoles throughout the cytoplasm in the amphotericin B-sensitive parental strain suspended in distilled water. In contrast, the dansyl-labelled derivative was not internalized in the Δatg18 strain, which is deficient in the formation of autophagosomes, a key early step in autophagy. However, the derivative accumulated without significant toxicity in structurally intact vacuoles in the Δvma1 mutant, which is deficient in the degradation of autophagic bodies, the final stage in autophagy. Our data support the idea that amphotericin B can utilize autophagy-dependent trafficking into the intra-vacuolar lumen, where it interacts with the luminal leaf of the membrane to cause structurally catastrophic effects.

Cetuximab delivery and antitumor effects are enhanced by mild hyperthermia in a xenograft mouse model of pancreatic cancer.

Even with current promising antitumor antibodies, their antitumor effects on stroma-rich solid cancers have been insufficient. We used mild hyperthermia with the intent of improving drug delivery by breaking the stromal barrier. Here, we provide preclinical evidence of cetuximab + mild hyperthermia therapy. We used four in vivo pancreatic cancer xenograft mouse models with different stroma amounts (scarce, MIAPaCa-2; moderate, BxPC-3; and abundant, Capan-1 and Ope-xeno). Cetuximab (1 mg/kg) was given systemically, and the mouse leg tumors were concurrently heated using a water bath method for 30 min at three different temperatures, 25°C (control), 37°C (intra-abdominal organ level), or 41°C (mild hyperthermia) (n = 4, each group). The evaluated variables were the antitumor effects, represented by tumor volume, and in vivo cetuximab accumulation, indirectly quantified by the immunohistochemical fluorescence intensity value/cell using antibodies against human IgG Fc. At 25°C, the antitumor effects were sufficient, with a cetuximab accumulation value (florescence intensity/cell) of 1632, in the MIAPaCa-2 model, moderate (1063) in the BxPC-3 model, and negative in the Capan-1 and Ope-xeno models (760, 461). By applying 37°C or 41°C heat, antitumor effects were enhanced shown in decreased tumor volumes. These enhanced effects were accompanied by boosted cetuximab accumulation, which increased by 2.8-fold (2980, 3015) in the BxPC-3 model, 2.5- or 4.8-fold (1881, 3615) in the Capan-1 model, and 3.2- or 4.2-fold (1469, 1922) in the Ope-xeno model, respectively. Cetuximab was effective in treating even stroma-rich and k-ras mutant pancreatic cancer mouse models when the drug delivery was improved by combination with mild hyperthermia.

An essential role for functional lysosomes in ferroptosis of cancer cells.

Pharmacological challenges to oncogenic Ras-expressing cancer cells have shown a novel type of cell death, ferroptosis, which requires intracellular iron. In the present study, we assessed ferroptosis following treatment of human fibrosarcoma HT1080 cells with several inhibitors of lysosomal activity and found that they prevented cell death induced by the ferroptosis-inducing compounds erastin and RSL3. Fluorescent analyses with a reactive oxygen species (ROS) sensor revealed constitutive generation of ROS in lysosomes, and treatment with lysosome inhibitors decreased both lysosomal ROS and a ferroptotic cell-death-associated ROS burst. These inhibitors partially prevented intracellular iron provision by attenuating intracellular transport of transferrin or autophagic degradation of ferritin. Furthermore, analyses with a fluorescent sensor that detects oxidative changes in cell membranes revealed that formation of lipid ROS in perinuclear compartments probably represented an early event in ferroptosis. These results suggest that lysosomal activity is involved in lipid ROS-mediated ferroptotic cell death through regulation of cellular iron equilibria and ROS generation.

Associations between responses to interferon therapy and genetic variation in interleukin-28B and the core region of hepatitis C virus genotype 3a.

The single-nucleotide polymorphism (SNP) of interleukin-28B (IL-28B) and mutations in the core region of hepatitis C virus (HCV) genotype 1b have been associated with response to interferon (IFN) therapy. However, whether this IL-28B SNP affects responses to INF therapy for HCV genotype 3a is not known. The aim of this study is to investigate whether this IL-28B SNP (rs8099917) and specific missense mutations in the HCV core region affect the response to IFN therapy for HCV genotype 3a. Patients (n = 19; median age 44.5) infected with HCV genotype 3a who received IFN therapy were studied. Of the 19 patients, 12 (63.1%) achieved sustained virological response. Of those 12 patients, 11 had the TT genotype (11/16; 68.7%), and one had the TG genotype (1/3; 33.3%). The difference in the sustained virological response rate between IL-28B genotype groups was not significant (P = 0.5232). HCV core region was well conserved; however, polymorphisms at position 72 were identified. Of the 19 HCV samples; 15 carried a glutamic acid at position 72, and these were defined as E type; the others (4/19) were defined as non-E type. Notably, there was a significant difference in the sustained virological response rate between E type and non-E-type; 12 of the 15 patients with E-type achieved sustained virological response, but none of the four patients with non-E-type achieved sustained virological response (P = 0.009). A glutamic acid at position 72 in the core region of HCV genotype 3a was associated with a good response to IFN therapy. J. Med. Virol. 87:1361-1367, 2015. © 2015 Wiley Periodicals, Inc.

Histological advantages of the tumor graft: a murine model involving transplantation of human pancreatic cancer tissue fragments.

OBJECTIVES: Experimental data based on cell line-derived xenograft models (cell xenograft) seldom reproduce the clinical situation, and therefore we demonstrated here the superiority of a murine model involving transplantation of human pancreatic cancer tissue fragments (tumor graft), focusing on the histological features and drug delivery characteristics. METHODS: Tumor pieces from 10 pancreatic cancer patients were transplanted into SCID (severe combined immunodeficient) mice. Histological characteristics of tumor grafts, including morphology, desmoplastic reaction, and vascularization, were compared with those of cell xenografts. Drug delivery was evaluated by quantifying the concentrations of injected drug, and the results were compared with its histological features. RESULTS: Eight of the 10 transplanted tumors successfully engrafted. Histological comparisons between tumor grafts and cell xenografts revealed the following: the amount of stroma was more (22.9% ± 11.8% vs 10.8% ± 5.4%; P < 0.05), vessel-cancer cell distance was longer (35.3 ± 39.0 vs 3.9 ± 3.1 µm; P < 0.001), and microvessel density was lower (6.8 ± 1.9 vs 10.8 ± 2.1 vessels/0.4 mm(2); P < 0.05) in tumor grafts. Drug concentrations in tumor grafts were lower than those in cell xenografts (3.3 ± 1.2 vs 6.0±0.2 µg/mL; P = 0.003), and the differences were correlated with the histological differences. CONCLUSIONS: Pancreatic tumor grafts better reproduce the histological nature of clinical cancer and thus provide a more realistic model that is applicable for pharmacokinetic studies.

Histological and prognostic importance of CD44(+) /CD24(+) /EpCAM(+) expression in clinical pancreatic cancer.

CD44(+) /CD24(+) /EpCAM(+) cells have been reported to be cancer stem cells in pancreatic cancer; however, the histological and clinical importance of these cells has not yet been investigated. Here we clarified the characteristics of CD44(+) /CD24(+) /EpCAM(+) cells in clinical specimens of pancreatic cancer using immunohistochemical assay. We used surgical specimens of pancreatic ductal adenocarcinoma from 101 patients. In view of tumor heterogeneity, we randomly selected 10 high-power fields per case, and triple-positive CD44(+) /CD24(+) /EpCAM(+) expression was identified using our scoring system. The distribution, histological characteristics, and prognostic importance of CD44(+) /CD24(+) /EpCAM(+) cells were then analyzed. As a result, the distribution of CD44(+) /CD24(+) /EpCAM(+) cells varied widely among the 101 cases examined, and CD44(+) /CD24(+) /EpCAM(+) expression was correlated with poor glandular differentiation and high proliferation. Survival analysis showed that CD44(+) /CD24(+) /EpCAM(+) expression was not correlated with patient outcome; however, CD44(+) /CD24(+) expression appeared to be correlated with poor prognosis. In conclusion, CD44(+) /CD24(+) /EpCAM(+) expression overlapped with poorly differentiated cells and possessed high proliferative potential in clinical pancreatic cancer. In particular, the presence of double-positive CD44(+) /CD24(+) expression seemed to have clinical relevance, associating with poor prognosis.

Effective delivery of chemotherapeutic nanoparticles by depleting host Kupffer cells.

Although chemotherapeutic nanoparticles would confer various advantages, the majority of administrated nanoparticles are known to be spoiled by the reticuloendothelial system (RES). Intending to more effectively deliver therapeutic nanoparticles to target regions in vivo, host RES, especially Kupffer cells in the liver, have been depleted ahead of drug administration. To demonstrate this hypothesis, clodronate liposomes were preinjected into BALB/c nude mice for depletion of Kupffer cells 2 days before, and pegylated liposomal doxorubicin (Doxil) at the doses of 1.25, 2.5 and 5.0 mg/kg was administered. As a result, doxorubicin accumulation in the liver was decreased from 36 to 26% injected dose/organ by the Kupffer cells depletion, and consequently, the plasma concentration of doxorubicin was significantly enhanced threefold (from 11 to 33 µg/mL) on day 1 at 1.25 mg/kg-dose group. Doxorubicin accumulation in the tumor was increased from 0.78 to 3.0 µg/g-tissue on day 3, and tumor growth inhibition by Doxil was significantly boosted (tumor volumes from 751 to 482 mm(3) on day 24) by the Kupffer cells depletion. In conclusion, Kupffer cells depletion by clodronate liposomes enhanced the plasma concentration and antitumor effects of Doxil, and would be widely applicable for various clinical cancer chemotherapies using nanoparticles.

Preparation of highly dispersible and tumor-accumulative, iron oxide nanoparticles Multi-point anchoring of PEG-b-poly(4-vinylbenzylphosphonate) improves performance significantly.

This paper describes the preparation of iron oxide nanoparticles, surface of which was coated with extremely high immobilization stability and relatively higher density of poly(ethylene glycol) (PEG), which are referred to as PEG protected iron oxide nanoparticles (PEG-PIONs). The PEG-PIONs were obtained through alkali coprecipitation of iron salts in the presence of the PEG-poly(4-vinylbenzylphosphonate) block copolymer (PEG-b-PVBP). In this system, PEG-b-PVBP served as a surface coating that was bound to the iron oxide surface via multipoint anchoring of the phosphonate groups in the PVBP segment of PEG-b-PVBP. The binding of PEG-b-PVBP onto the iron oxide nanoparticle surface and the subsequent formation of a PEG brush layer were proved by FT-IR, zeta potential, and thermogravimetric measurements. The surface PEG-chain density of the PEG-PIONs varied depending on the [PEG-b-PVBP]/[iron salts] feed-weight ratio in the coprecipitation reaction. PEG-PIONs prepared at an optimal feed-weight ratio in this study showed a high surface PEG-chain surface density (≈0.8 chainsnm(-2)) and small hydrodynamic diameter (<50 nm). Furthermore, these PEG-PIONs could be dispersed in phosphate-buffered saline (PBS) that contains 10% serum without any change in their hydrodynamic diameters over a period of one week, indicating that PEG-PIONs would provide high dispersion stability under in vivo physiological conditions as well as excellent anti-biofouling properties. In fact we have confirmed the prolong blood circulation time and facilitate tumor accumulation (more than 15% IDg(-1) tumor) of PEG-PIONs without the aid of any target ligand in mouse tumor models. The majority of the PEG-PIONs accumulated in the tumor by 96 h after administration, whereas those in normal tissues were smoothly eliminated by 96 h, proving the enhancement of tumor selectivity in the PEG-PION localization. The results obtained here strongly suggest that originally synthesized PEG-b-PVBP, having multipoint anchoring character by the phosphonate groups, is rational design for improvement in nanoparticle as in vivo application. Two major points, viz., extremely stable anchoring character and dense PEG chains tethered on the nanoparticle surface, worked simultaneously to become PEG-PIONs as an ideal biomedical devices intact for prolonged periods in harsh biological environments.

Identification of novel peptide agonists from a random peptide library for a 5-oxo-ETE receptor, a receptor for bioactive lipids.

A combinatorial peptide library contains an enormous combination of amino acid sequences and drug candidates, but an effective screening strategy to identify a variety of bioactive peptides has yet to be established. In this article, a random hexapeptide library was screened to identify novel peptide ligands for a 5-oxo-ETE receptor (OXER), which is a G-protein-coupled receptor for bioactive lipids, by using an OXER-Gi1alpha fusion protein. We successfully identified 2 hexapeptides-Ac-HMQLYF-NH2 and Ac-HMWLYF-NH(2)-that exhibited agonistic activity. Although the corresponding affinities were relatively low (EC50 values of 146 and 6.7 microM, respectively), the activities were confirmed by other independent cell-based assay methods, namely, intracellular calcium mobilization and cell chemotaxis. This study demonstrates that a combinatorial peptide library may be screened using a [35S]GTPgammaS binding assay with G-protein-coupled receptor (GPCR)-Galpha fusion proteins, in general, and that of peptide ligands can be obtained even for nonpeptide receptors.