Robotic radical distal gastrectomy for gastric cancer using the soft coagulation scissors technique.

We have developed a novel technique for safe and precise lymph-node dissection during robotic gastrectomy for gastric cancer using monopolar curved scissors with soft coagulation. This technique is called the soft coagulation scissors technique. The technical details of this technique are as follows: a long bipolar grasper in the first arm and monopolar curved scissors in the third arm are primarily used for lymph-node dissection, maximizing the use of the robotic arm articulation. The monopolar curved scissors were energized in the soft coagulation mode of the Valleylab™ FT10 energy platform or in the forced coagulation mode of the ERBE VIO®dV with an effect 1/power limit of 15 W. This limit was confirmed to be equivalent to the soft coagulation mode in preliminary experiments, and a long bipolar grasper applied adequate tension to the surgical site without strongly grasping the tissue or applying traction. The peak temperatures of our devices were more than 100 °C lower than those of the Harmonic ACE and the Maryland bipolar forceps with the forced coagulation mode. Overall, 80 patients with gastric cancer, including 36 (45.0%) with stage III or IV cancers, underwent robotic distal gastrectomy with this technique. The median estimated blood loss was 10 g. There were only four surgical complications (5.0%): two paralytic ileus, one intra-abdominal abscess, and one duodenal stump leakage. Robotic distal gastrectomy for gastric cancer is made possible by the soft coagulation scissors technique, which allows for safe and precise lymph-node dissection.

Synthesis of 2-Oxoquinoline Derivatives as Dual Pim and mTORC Protein Kinase Inhibitors.

Compound VBT-5445 was identified as an inhibitor to block the association of Pim and the protein Enhancer of Decapping 3 (EDC3), a Pim substrate, which normally functions to enhance the decapping of messenger RNA (mRNA). It was also shown to inhibit both the Pim and mTORC protein kinases. The activity of this compound class can be fine-tuned by structural modification. A series of VBT analogs were designed, synthesized, and evaluated. These compounds decrease the growth of multiple cancer types, including pancreas, prostate, breast, lung, and leukemia. Notably, 6-methyl (GRG-1-31, 6d), 4-Bromo (GRG-1-34, 6e), 4-Chloro (GRG-1-35, 6f), and phenylthio substituted (GRG-1-104, 6n) derivatives are highly potent at inhibiting tumor growth. The ability of these compounds to block cancer growth in vitro is highly correlated with their activity as mTORC inhibitors. The toxicity of GRG 1-34 is low in mice treated with twice-daily gavage for 30 days and did not induce weight loss. Pharmacokinetics of a single oral dose demonstrated a peak concentration at 0.5 hours after gavage. In summary, further development of this compound class has the potential to inhibit important signaling pathways and impact cancer treatment.

Modified side overlap esophagogastrostomy after laparoscopic proximal gastrectomy.

We report a new method of esophagogastrostomy after proximal gastrectomy, side overlap with fundoplication by Yamashita (SOFY) in 2017. Recently, even better treatment results can be obtained by modifying the SOFY method. We describe the technical details of the modified SOFY (mSOFY) after laparoscopic proximal gastrectomy. The stomach was dissected in the short axis direction and the esophagus was dissected in the left and right direction. After the proximal gastrectomy, the bilateral diaphragmatic crus were dissected to enhance gastric elevation. After confirming that the esophagus overlapped more than 5 cm at the center of the remnant stomach (we call it SOFY check), the remnant stomach was suture-fixed to the dissected diaphragmatic crus. The right wall of the esophageal stump and the remnant stomach were anastomosed using the full length of a 45 mm-linear stapler. The entry hole was closed in a direction that did not widen the anastomotic hole. Both sides of the esophagus, remnant stomach, and diaphragmatic crus were suture-fixed on the cranial side 1-2 cm away from the anastomosis. Moreover, the left wall and lower end of the esophagus was suture-fixed to the remnant stomach. The preserved dorsal esophageal wall is pressed and flattened by pressure from the pseudofornix, which is the reflux prevention mechanism. The mSOFY method had favorable treatment outcomes. In conclusion, mSOFY can be one of the safe and feasible reconstruction methods after laparoscopic proximal gastrectomy.

The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer.

Neuroendocrine (NE) prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (PCa) arising either de novo or from transdifferentiated prostate adenocarcinoma following androgen deprivation therapy (ADT). Extensive computational analysis has identified a high degree of association between the long noncoding RNA (lncRNA) H19 and NEPC, with the longest isoform highly expressed in NEPC. H19 regulates PCa lineage plasticity by driving a bidirectional cell identity of NE phenotype (H19 overexpression) or luminal phenotype (H19 knockdown). It contributes to treatment resistance, with the knockdown of H19 re-sensitizing PCa to ADT. It is also essential for the proliferation and invasion of NEPC. H19 levels are negatively regulated by androgen signaling via androgen receptor (AR). When androgen is absent SOX2 levels increase, driving H19 transcription and facilitating transdifferentiation. H19 facilitates the PRC2 complex in regulating methylation changes at H3K27me3/H3K4me3 histone sites of AR-driven and NEPC-related genes. Additionally, this lncRNA induces alterations in genome-wide DNA methylation on CpG sites, further regulating genes associated with the NEPC phenotype. Our clinical data identify H19 as a candidate diagnostic marker and predictive marker of NEPC with elevated H19 levels associated with an increased probability of biochemical recurrence and metastatic disease in patients receiving ADT. Here we report H19 as an early upstream regulator of cell fate, plasticity, and treatment resistance in NEPC that can reverse/transform cells to a treatable form of PCa once therapeutically deactivated.

Accuracy of Preoperative Endoscopy in Determining Tumor Location Required for Surgical Planning for Esophagogastric Junction Cancer.

PURPOSE: The surgical strategy for esophagogastric junction (EGJ) cancer depends on the tumor location as measured relative to the EGJ line. The purpose of this study was to clarify the accuracy of diagnostic endoscopy in different clinicopathological backgrounds. METHODS: Subjects were 74 consecutive patients with abdominal esophagus to upper gastric cancer who underwent surgical resection. Image-enhanced endoscopy with narrow-band imaging (NBI) was used to determine the EGJ line, prioritizing the presence of palisade vessels, followed by the upper end of gastric folds, as a landmark. The relative positional relationship between the tumor epicenter and the EGJ line was classified into six categories, and the agreement between endoscopic and pathologic diagnoses was examined to evaluate prediction accuracy. RESULTS: The concordance rate of 69 eligible cases was 87% with a kappa coefficient (K) of 0.81. The palisade vessels were observed in 62/69 patients (89.9%). Of the 37 pathological EGJ cancers centered within 2 cm above and below the EGJ line, Barrett's esophagus was found to be a significant risk factor for discordance (risk ratio, 4.40; p = 0.042); the concordance rate of 60% (K = 0.50) in the Barrett's esophagus group was lower than the rate of 91% (K = 0.84) in the non-Barrett's esophagus group. In five of six discordant cases, the EGJ line was estimated to be proximal to the actual line. CONCLUSION: Diagnostic endoscopy is beneficial for estimating the location of EGJ cancer, with a risk of underestimating esophageal invasion length in patients with Barrett's esophagus.

EDC3 phosphorylation regulates growth and invasion through controlling P-body formation and dynamics.

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P-bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P-body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P-body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P-bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin ß1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer-relevant functions and suggest that modulation of P-body activity may represent a new paradigm for cancer treatment.

PIM protein kinases regulate the level of the long noncoding RNA H19 to control stem cell gene transcription and modulate tumor growth.

The proviral integration site for Moloney murine leukemia virus (PIM) serine/threonine kinases have an oncogenic and prosurvival role in hematological and solid cancers. However, the mechanism by which these kinases drive tumor growth has not been completely elucidated. To determine the genes controlled by these protein kinases, we carried out a microarray analysis in T-cell acute lymphoblastic leukemia (T-ALL) comparing early progenitor (ETP-ALL) cell lines whose growth is driven by PIM kinases to more mature T-ALL cells that have low PIM levels. This analysis demonstrated that the long noncoding RNA (lncRNA) H19 was associated with increased PIM levels in ETP-ALL. Overexpression or knockdown of PIM in these T-ALL cell lines controlled the level of H19 and regulated the methylation of the H19 promoter, suggesting a mechanism by which PIM controls H19 transcription. In these T-ALL cells, the expression of PIM1 induced stem cell gene expression (SOX2, OCT-4, and NANOG) through H19. Identical results were found in prostate cancer (PCa) cell lines where PIM kinases drive cancer growth, and both H19 and stem cell gene levels. Small molecule pan-PIM inhibitors (PIM-i) currently in clinical trials reduced H19 expression in both of these tumor types. Importantly, the knockdown of H19 blocked the ability of PIM to induce stem cell genes in T-ALL cells, suggesting a novel signal transduction cascade. In PCa, increases in SOX2 levels have been shown to cause both resistance to the androgen deprivation therapy (ADT) and the induction of neuroendocrine PCa, a highly metastatic form of this disease. Treatment of PCa cells with a small molecule pan-PIM-i reduced stem cell gene transcription and enhanced ADT, while overexpression of H19 suppressed the ability of pan-PIM-i to regulate hormone blockade. Together, these results demonstrate that the PIM kinases control the level of lncRNA H19, which in turn modifies stem cell gene transcription regulating tumor growth.

Laparoscopic Suprapancreatic Lymph Node Dissection Using a Systematic Mesogastric Excision Concept for Gastric Cancer.

BACKGROUND: Gastrointestinal cancer surgery requires en bloc removal of the primary tumor and organ-specific mesentery1,2. However, this surgical concept for gastric cancer has not yet been applied because of the morphological complexity of the mesenteries of the stomach. Lymph node dissection in gastric cancer surgery can be roughly performed into three regions: lesser curvature, grater curvature, and suprapancreatic region. In this video, we introduced laparoscopic lymphadenectomy in the suprapancreatic region using a systematic mesogastric excision (SME), which has been reported as a concept to perform en bloc resection3. METHODS: This procedure was divided into three steps. First, mesenterization of the mesogastrium was performed by dissecting the embryological planes, and the mesogastrium was dissected from the retroperitoneal surface (Fig. 1a). Second, soft tissue, including the lymph node, was separated from the pancreas and the splenic artery by tracing the inner dissectable layer (Fig. 1b). Finally, the tumor-specific mesentery was transected according to the extent of the lymphadenectomy (Fig. 1c).Fig. 1Intraoperative findings during the stepwise procedure in dissecting the lymph node in the suprapancreatic region. The red broken line indicates the surgical outline. a The mesogastrium is dissected from the retroperitoneal tissue. b The mesogastrium is separated from the pancreas and splenic artery. c The mesogastric transection line is determined on the basis of the extent of the lymphadenectomy. Inf. phrenic a. inferior phrenic artery; PGA posterior gastric artery; Post. epiploic a. posterior epiploic artery; RV renal vein; SA splenic artery; SV splenic vein RESULTS: Between January 2017 and December 2017, six patients underwent laparoscopic distal gastrectomy with D2 lymphadenectomy using SME. The median time required to complete the suprapancreatic lymphadenectomy was 48 min. No patient underwent conversion to open surgery or experienced intraoperative complications. CONCLUSIONS: We believe that this laparoscopic suprapancreatic lymphadenectomy using SME takes advantage of the surgical anatomy and achieves en bloc removal of the primary tumor and gastric mesentery. This series is a proof of concept that this procedure can be performed in a timely manner and is feasible.

True esophagogastric junction adenocarcinoma: background of its definition and current surgical trends.

The definition of true esophagogastric junction (EGJ) adenocarcinoma and its surgical treatment are debatable. We review the basis for the current definition and the Japanese surgical strategy in managing true EGJ adenocarcinoma. The Siewert classification is a well-known anatomical classification system for EGJ adenocarcinomas: type II tumors in the region 1 cm above and 2 cm below the EGJ are described as "true carcinoma of the cardia". Coincidentally, this range matches gastric cardiac gland distribution. Conversely, Nishi's classification is generally used to describe EGJ carcinomas, defined as tumors with the center located within 2 cm above and 2 cm below the EGJ, regardless of their histological subtype. This range coincides with the extent of the lower esophageal sphincter combined with gastric cardiac gland distribution. The current Japanese surgical strategy focuses on the tumor range from the EGJ to the esophagus and stomach. According to previous studies, the strategy can be roughly classified into three types. The optimal surgical procedure for true EGJ adenocarcinoma is controversial. However, an ongoing Japanese nationwide prospective trial will help confirm the appropriate standard surgery, including the optimal extent of lymph node dissection.

Creation of Virtual Three-Dimensional Animation Using Computer Graphic Technology for Videoscopic Transcervical Upper Mediastinal Esophageal Dissection.

Background: Videoscopic transcervical mediastinal lymphadenectomy has been attempted to reduce thoracotomy-related complications of surgical treatment for esophageal cancer. However, many surgeons would hesitate to attempt this procedure because of the difficulty in understanding the anatomical orientation. In this study, we aimed to create a three-dimensional computer graphic (3D CG) animation and compare it with the real-life operation. Materials and Methods: LightWave 3D® version 7 was used as a rendering software to create the 3D CG. The 3D CG images were superimposed to generate an animation using AfterEffects CC®. Results: The 3D CG animation for videoscopic transcervical upper mediastinal esophageal dissection was successfully created; it dynamically shows the scene, especially the separation between the esophagus and trachea, and enables surgeons to easily understand the anatomical orientation when using transcervical approach. This 3D CG animation was of high quality and similar to the real-life operation. Conclusions: We created a virtual 3D CG animation for the transcervical approach, which will contribute to understanding this procedure for esophageal cancer preoperatively.

Phosphorylation of DEPDC5, a component of the GATOR1 complex, releases inhibition of mTORC1 and promotes tumor growth.

The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

Identification of lysine methylation in the core GTPase domain by GoMADScan.

RAS is the founding member of a superfamily of GTPases and regulates signaling pathways involved in cellular growth control. While recent studies have shown that the activation state of RAS can be controlled by lysine ubiquitylation and acetylation, the existence of lysine methylation of the RAS superfamily GTPases remains unexplored. In contrast to acetylation, methylation does not alter the side chain charge and it has been challenging to deduce its impact on protein structure by conventional amino acid substitutions. Herein, we investigate lysine methylation on RAS and RAS-related GTPases. We developed GoMADScan (Go language-based Modification Associated Database Scanner), a new user-friendly application that scans and extracts posttranslationally modified peptides from databases. The GoMADScan search on PhosphoSitePlus databases identified methylation of conserved lysine residues in the core GTPase domain of RAS superfamily GTPases, including residues corresponding to RAS Lys-5, Lys-16, and Lys-117. To follow up on these observations, we immunoprecipitated endogenous RAS from HEK293T cells, conducted mass spectrometric analysis and found that RAS residues, Lys-5 and Lys-147, undergo dimethylation and monomethylation, respectively. Since mutations of Lys-5 have been found in cancers and RASopathies, we set up molecular dynamics (MD) simulations to assess the putative impact of Lys-5 dimethylation on RAS structure. Results from our MD analyses predict that dimethylation of Lys-5 does not significantly alter RAS conformation, suggesting that Lys-5 methylation may alter existing protein interactions or create a docking site to foster new interactions. Taken together, our findings uncover the existence of lysine methylation as a novel posttranslational modification associated with RAS and the RAS superfamily GTPases, and putative impact of Lys-5 dimethylation on RAS structure.

Dynamic compartmentalization of purine nucleotide metabolic enzymes at leading edge in highly motile renal cell carcinoma.

Compartmentalization is vital for biological systems at multiple levels, including biochemical reactions in metabolism. Organelle-based compartments such as mitochondria and peroxisomes sequester the responsible enzymes and increase the efficiency of metabolism while simultaneously protecting the cell from dangerous intermediates, such as radical oxygen species. Recent studies show intracellular nucleotides, such as ATP and GTP, are heterogeneously distributed in cells with high concentrations at the lamellipodial and filopodial projections, or leading edge. However, the intracellular distribution of purine nucleotide enzymes remains unclear. Here, we report the enhanced localization of GTP-biosynthetic enzymes, including inosine monophosphate dehydrogenase (IMPDH isotype 1 and 2), GMP synthase (GMPS), guanylate kinase (GUK1) and nucleoside diphosphate kinase-A (NDPK-A) at the leading edge in renal cell carcinoma cells. They show significant co-localization at the membrane subdomain, and their co-localization pattern at the membrane is distinct from that of the cell body. While other purine nucleotide biosynthetic enzymes also show significant localization at the leading edge, their co-localization pattern with IMPDH is divergent. In contrast, a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), predominantly localized in the cytoplasm. Mechanistically, we found that plasma membrane localization of IMPDH isozymes requires active actin polymerization. Our results demonstrate the formation of a discrete metabolic compartment for localized purine biosynthesis at the leading edge, which may promote localized nucleotide metabolism for cell migration and metastasis in cancers.

Laparoscopic modified lymphadenectomy in gastric cancer surgery using systematic mesogastric excision: a novel technique based on a concept.

PURPOSE: Radical surgery for gastrointestinal cancer involves en bloc removal of the primary tumor and organ-specific mesenteries. However, the surgical concept and technique for lymphadenectomy during gastric cancer surgery remain unclear. We examined a novel technique for laparoscopic modified lymphadenectomy during gastric cancer surgery involving systematic mesogastric excision (SME) and focused on the topographic anatomy, surgical technique, and specimens. METHODS: Our surgical technique involved the following: mesenterization by dissociating embryological planes, separating fat tissue containing lymph nodes from the pancreas and its associated vessels by tracing the intramesenteric dissectable layers, and dissecting the lymph node that is dependent on the D1+ criteria. RESULTS: Between October 2011 and September 2016, 227 patients underwent laparoscopic D1+ gastrectomy using SME. Of these, total gastrectomy was performed in 47 cases and distal gastrectomy was performed in 180 cases. The median operative time was 303 min (range, 201-722 min), and estimated blood loss was 50 mL (range, 0-550 mL). The median number of harvested lymph nodes was 54 (range, 18-163). There was no conversion to open surgery. CONCLUSIONS: SME was adapted for modified gastrectomy and is considered safe. Modified lymphadenectomy during gastrectomy is determined by the resection margin of the mesogastrium.

The eCura system as a novel indicator for the necessity of salvage surgery after non-curative ESD for gastric cancer: A case-control study.

Endoscopic submucosal dissection (ESD) for early gastric cancer does not always lead to complete cancer resection. The aim of this study was to determine indicators for cancer residue (CR) status in cases of non-curative ESD. We analyzed 47 cases of non-curative ESD followed by salvage surgery and collected data regarding the rates of CR, which included both local CR and lymph node metastasis (LNM). To elucidate the risk factors for CR status, we compared the CR positive and the CR negative groups among surgical specimens according to the following variables obtained from ESD findings: tumor location, tumor size, depth of invasion, lympho-vascular invasion, histological margin, and histological diagnosis. The eCura system, which is an LNM risk scoring system, was also applied and scores were calculated in each case as follows: 3 points for lymphatic invasion and 1 point each for tumor size >30 mm, positive vertical margin, venous invasion, and submucosal invasion ≥500 µm. There were 9 (19%) CR positive cases, which included 6 cases of local CR and 4 cases of LNM; no cancer was detected in over 80% of the patients. The eCura scoring system was the only significant factor for CR status: the higher the eCura score, the greater the CR positivity (p = 0.0128). In particular, all patients in the low-risk group (score = 0-1 point) had no CR. Although no cancer recurrence was observed during a median follow-up of 4 years, 2 patients died of pneumonia. In conclusion, the eCura system might make it possible to select appropriate cases for salvage surgery.

Mechanisms Behind Resistance to PI3K Inhibitor Treatment Induced by the PIM Kinase.

Cancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity. PIM prevents cell death induced by PI3K-AKT-inhibitory drugs through a noncanonical mechanism of NRF2 ubiquitination and degradation and translational control of NRF2 protein levels through modulation of eIF4B and mTORC1 activity. Importantly, PIM also controls NAD(P)H production by increasing glucose flux through the pentose phosphate shunt decreasing ROS production, and thereby diminishing the cytotoxicity of PI3K-AKT inhibitors. Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.

Extracellular Bio-imaging of Acetylcholine-stimulated PC12 Cells Using a Calcium and Potassium Multi-ion Image Sensor.

In biochemistry, Ca2+ and K+ play essential roles to control signal transduction. Much interest has been focused on ion-imaging, which facilitates understanding of their ion flux dynamics. In this paper, we report a calcium and potassium multi-ion image sensor and its application to living cells (PC12). The multi-ion sensor had two selective plasticized poly(vinyl chloride) membranes containing ionophores. Each region on the sensor responded to only the corresponding ion. The multi-ion sensor has many advantages including not only label-free and real-time measurement but also simultaneous detection of Ca2+ and K+. Cultured PC12 cells treated with nerve growth factor were prepared, and a practical observation for the cells was conducted with the sensor. After the PC12 cells were stimulated by acetylcholine, only the extracellular Ca2+ concentration increased while there was no increase in the extracellular K+ concentration. Through the practical observation, we demonstrated that the sensor was helpful for analyzing the cell events with changing Ca2+ and/or K+ concentration.

A bio-image sensor for simultaneous detection of multi-neurotransmitters.

We report here a new bio-image sensor for simultaneous detection of spatial and temporal distribution of multi-neurotransmitters. It consists of multiple enzyme-immobilized membranes on a 128 × 128 pixel array with read-out circuit. Apyrase and acetylcholinesterase (AChE), as selective elements, are used to recognize adenosine 5'-triphosphate (ATP) and acetylcholine (ACh), respectively. To enhance the spatial resolution, hydrogen ion (H+) diffusion barrier layers are deposited on top of the bio-image sensor and demonstrated their prevention capability. The results are used to design the space among enzyme-immobilized pixels and the null H+ sensor to minimize the undesired signal overlap by H+ diffusion. Using this bio-image sensor, we can obtain H+ diffusion-independent imaging of concentration gradients of ATP and ACh in real-time. The sensing characteristics, such as sensitivity and detection of limit, are determined experimentally. With the proposed bio-image sensor the possibility exists for customizable monitoring of the activities of various neurochemicals by using different kinds of proton-consuming or generating enzymes.

Development of an ATP and hydrogen ion image sensor using a patterned apyrase-immobilized membrane.

A bio-image sensor using a patterned apyrase-immobilized membrane was developed to visualize the activities of adenosine triphosphate (ATP) and H+ ion in real-time. An enzymatic membrane patterning technique was suggested to immobilize apyrase on a specific sensing area of a charge coupled device (CCD)-type image sensor. It was able to observe the spatiotemporal information of ATP and H+ ion. The smallest size of a patterned membrane is 250×250µm2. The fabrication parameters of the patterned membrane, such as its thickness and the intensity of the incident light used for photolithography, were optimized experimentally. The sensing area under the patterned apyrase-immobilized membrane revealed a linear response up to 0.6mM of ATP concentration with a sensitivity of 37.8mV/mM. Meanwhile, another sensing area without the patterned membrane measured the diffused H+ ion from nearby membranes. This diffusion characteristics were analyzed to determine a measurement time that can minimize the undesirable impact of the diffused ions. In addition, the newly developed bio-image sensor successfully reconstructed ATP and H+ ion dynamics into sequential 2-dimensional images.