Search

KRAS activating mutations, which are present in more than 90% of pancreatic cancers, drive tumor dependency on the RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. Therefore, combined targeting of RAS/MAPK and PI3K/AKT signaling pathways may be required for optimal therapeutic effect in pancreatic cancer. However, the therapeutic efficacy of combined MAPK and PI3K/AKT signaling target inhibitors is unsatisfactory in pancreatic cancer treatment, because it is often accompanied by MAPK pathway reactivation by PI3K/AKT inhibitor. Therefore, we developed an inRas37 antibody, which directly targets the intra-cellularly activated GTP-bound form of oncogenic RAS mutation and investigated its synergistic effect in the presence of the PI3K inhibitor BEZ-235 in pancreatic cancer. In this study, inRas37 remarkably increased the drug response of BEZ-235 to pancreatic cancer cells by inhibiting MAPK reactivation. Moreover, the co-treatment synergistically inhibited cell proliferation, migration, and invasion and exhibited synergistic anticancer activity by inhibiting the MAPK and PI3K pathways. The combined administration of inRas37and BEZ-235 significantly inhibited tumor growth in mouse models. Our results demonstrated that inRas37 synergistically increased the antitumor activity of BEZ-235 by inhibiting MAPK reactivation, suggesting that inRas37 and BEZ-235 co-treatment could be a potential treatment approach for pancreatic cancer patients with KRAS mutations.

Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons of dorsal root ganglia in naive rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in the skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal glial fibrillary acidic protein-positive and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral nonneuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in dorsal root ganglia ipsilateral to plantar injection of complete Freund's adjuvant, and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to complete Freund's adjuvant injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate knee osteoarthritis pain model, compared with controls. We conclude that (1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch or proprioception mediated by large-sized low-threshold mechanosensitive primary sensory neurons; (2) Piezo2 may have functional roles involving sensory processing in the spinal cord, Schwann cells, and skin melanocytes; and (3) aberrant Piezo2 expression may contribute pain pathogenesis.

Here, we present evidence showing Piezo1 protein expression in the primary sensory neurons (PSNs) and non-neuronal cells of rat peripheral nervous system. Using a knockdown/knockout validated antibody, we detected Piezo1 immunoreactivity (IR) in ∼60% of PSNs of rat dorsal root ganglia (DRG) with higher IR density in the small- and medium-sized neurons. Piezo1-IR was clearly identified in DRG perineuronal glia, including satellite glial cells (SGCs) and Schwann cells; in sciatic nerve Schwann cells surrounding the axons and cutaneous afferent endings; and in skin epidermal Merkel cells and melanocytes. Neuronal and non-neuronal Piezo1 channels were functional since various cells (dissociated PSNs and SGCs from DRGs, isolated Schwann cells, and primary human melanocytes) exhibited a robust response to Piezo1 agonist Yoda1 by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses were abolished by non-specific Piezo1 antagonist GsMTx4. Immunoblots showed elevated Piezo1 protein in DRG proximal to peripheral nerve injury-induced painful neuropathy, while PSNs and SGCs from rats with neuropathic pain showed greater Yoda1-evoked elevation of [Ca2+]i and an increased frequency of cells responding to Yoda1, compared to controls. Sciatic nerve application of GsMTx4 alleviated mechanical hypersensitivity induced by Yoda1. Overall, our data show that Piezo1 is widely expressed by the neuronal and non-neuronal cells in the peripheral sensory pathways and that painful nerve injury appeared associated with activation of Piezo1 in PSNs and peripheral glial cells.

S-SCAM/MAGI-2 gene duplication is associated with schizophrenia (SCZ). S-SCAM overexpression in the forebrain induces SCZ-like phenotypes in a transgenic (Tg) mouse model. Interestingly, S-SCAM Tg mice show male-specific impairments in synaptic plasticity and working memory. However, mechanisms underlying the sex-specific deficits remain unknown. Here we report that S-SCAM Tg mice have male-specific deficits in synaptic GSK3β functions, as shown by reduced synaptic protein levels and increased inhibitory phosphorylation of GSK3β. This GSK3β hyper-phosphorylation was associated with increased CaMKII activities. Notably, synaptic levels of Axin1, to which GSK3β binds in competition with S-SCAM, were also reduced in male S-SCAM Tg mice. We demonstrated that Axin-binding is required for the S-SCAM overexpression-induced synaptic GSK3β reduction. Axin stabilization using XAV939 rescued the GSK3β deficits and restored the temporal activation of GSK3β during long-term depression in S-SCAM overexpressing neurons. Interestingly, synaptic Axin2 levels were increased in female S-SCAM Tg mice. Female sex hormone 17β-estradiol increased Axin2 expression and increased synaptic GSK3β levels in S-SCAM overexpressing neurons. These results reveal the role of S-SCAM in controlling Axin-dependent synaptic localization of GSK3β. Moreover, our studies point out the pathological relevance of GSK3β hypofunction found in humans and contribute to understanding the molecular underpinnings of sex differences in SCZ.

The monosodium iodoacetate knee osteoarthritis model has been widely used for the evaluation of osteoarthritis pain, but the pathogenesis of associated chronic pain is not fully understood. The T-type calcium channel 3.2 (CaV3.2) is abundantly expressed in the primary sensory neurons, in which it regulates neuronal excitability at both the somata and peripheral terminals and facilitates spontaneous neurotransmitter release at the spinal terminals. In this study, we investigated the involvement of primary sensory neuron-CaV3.2 activation in monosodium iodoacetate osteoarthritis pain. Knee joint osteoarthritis pain was induced by intra-articular injection of monosodium iodoacetate (2 mg) in rats, and sensory behavior was evaluated for 35 days. At that time, knee joint structural histology, primary sensory neuron injury, and inflammatory gliosis in lumbar dorsal root ganglia, and spinal dorsal horn were examined. Primary sensory neuron-T-type calcium channel current by patch-clamp recording and CaV3.2 expression by immunohistochemistry and immunoblots were determined. In a subset of animals, pain relief by CaV3.2 inhibition after delivery of CaV3.2 inhibitor TTA-P2 into sciatic nerve was investigated. Knee injection of monosodium iodoacetate resulted in osteoarthritis histopathology, weight-bearing asymmetry, sensory hypersensitivity of the ipsilateral hindpaw, and inflammatory gliosis in the ipsilateral dorsal root ganglia, sciatic nerve, and spinal dorsal horn. Neuronal injury marker ATF-3 was extensively upregulated in primary sensory neurons, suggesting that neuronal damage was beyond merely knee-innervating primary sensory neurons. T-type current in dissociated primary sensory neurons from lumbar dorsal root ganglia of monosodium iodoacetate rats was significantly increased, and CaV3.2 protein levels in the dorsal root ganglia and spinal dorsal horn ipsilateral to monosodium iodoacetate by immunoblots were significantly increased, compared to controls. Perineural application of TTA-P2 into the ipsilateral sciatic nerve alleviated mechanical hypersensitivity and weight-bearing asymmetry in monosodium iodoacetate osteoarthritis rats. Overall, our findings demonstrate an elevated CaV3.2 expression and enhanced function of primary sensory neuron-T channels in the monosodium iodoacetate osteoarthritis pain. Further study is needed to delineate the importance of dysfunctional primary sensory neuron-CaV3.2 in osteoarthritis pain.

The Sigma-1 receptor (σ(1)R) is highly expressed in the primary sensory neurons (PSNs) that are the critical site of initiation and maintenance of pain following peripheral nerve injury. By immunoblot and immunohistochemistry, we observed increased expression of both σ(1)R and σ1R-binding immunoglobulin protein (BiP) in the lumbar (L) dorsal root ganglia (DRG) ipsilateral to painful neuropathy induced by spared nerve injury (SNI). To evaluate the therapeutic potential of PSN-targeted σ(1)R inhibition at a selected segmental level, we designed a recombinant adeno-associated viral (AAV) vector expressing a small hairpin RNA (shRNA) against rat σ(1)R. Injection of this vector into the L4/L5 DRGs induced downregulation of σ(1)R in DRG neurons of all size groups, while expression of BiP was not affected. This was accompanied by attenuation of SNI-induced cutaneous mechanical and thermal hypersensitivity. Whole-cell current-clamp recordings of dissociated neurons showed that knockdown of σ(1)R suppressed neuronal excitability, suggesting that σ(1)R silencing attenuates pain by reversal of injury-induced neuronal hyperexcitability. These findings support a critical role of σ(1)R in modulating PSN nociceptive functions, and that the nerve injury-induced elevated σ(1)R activity in the PSNs can be a significant driver of neuropathic pain. Further understanding the role of PSN-σ(1)R in pain pathology may open routes to exploit this system for DRG-targeted pain therapy.

The duplication and deletion mutations of the S-SCAM/MAGI-2 gene are associated with schizophrenia and infantile spasms, respectively. S-SCAM is a unique synaptic scaffolding protein that localizes to both excitatory and GABAergic synapses. However, consequences of aberrant S-SCAM expression on GABAergic synapses is little studied. Here we report the effect of S-SCAM knockdown and overexpression on GABAergic synapses. S-SCAM knockdown in cultured hippocampal neurons caused a drastic loss of both pre- and post-synaptic components of GABAergic synapses, indicating its essential role in GABAergic synapse formation and maintenance. Surprisingly, S-SCAM overexpression also attenuated GABAergic synapses, but the effect is mediated by the loss of postsynaptic GABAA receptors, gephyrin, and neuroligin 2 and does not involve presynaptic component vesicular GABA transporters. Overexpression studies using S-SCAM mutants with various domain deletions indicated that GABAergic synapse loss correlates with their ability to increase excitatory synaptic function. Consistently, AMPA receptor antagonist CNQX or calcineurin inhibitor FK506 abolished the S-SCAM overexpression-induced loss of GABAA receptors, supporting that GABAergic synapse loss by S-SCAM overexpression is due to the activity-induced dispersal of synaptic GABAA receptors. These results suggest that abnormal S-SCAM protein levels disrupt excitation/inhibition balance in neurons, which may explain the pathogenic nature of S-SCAM copy number variations.

We report a potent pan-RAS–targeting antibody and the corresponding therapeutic strategy against RAS mutant tumors., Oncogenic RAS mutant (RASMUT) proteins have been considered undruggable via conventional antibody regimens owing to the intracellular location restricting conventional-antibody accessibility. Here, we report a pan-RAS–targeting IgG antibody, inRas37, which directly targets the intracellularly activated form of various RASMUT subtypes after tumor cell–specific internalization into the cytosol to block the interactions with effector proteins, thereby suppressing the downstream signaling. Systemic administration of inRas37 exerted a potent antitumor activity in a subset of RASMUT tumor xenografts in mice, but little efficacy in RASMUT tumors with concurrent downstream PI3K mutations, which were overcome by combination with a PI3K inhibitor. The YAP1 protein was up-regulated as an adaptive resistance-inducing response to inRas37 in RASMUT-dependent colorectal tumors; accordingly, a combination of inRas37 with a YAP1 inhibitor manifested synergistic antitumor effects in vitro and in vivo. Our study offers a promising pan-RAS–targeting antibody and the corresponding therapeutic strategy against RASMUT tumors.

This work was supported by the Industrial technology Innovation Program (No. 10063421, 'Development of the in-line welds quality estimation system and network-based quality control technology in arc and spot welds of ultra high strength steels for automotive parts assembly') funded By the Ministry of Trade, industry & Energy(MI, Korea).This research was respectfully supported by Engineering Development Research Center (EDRC) funded by the Ministry of Trade, Industry & Energy (MOTIE). (No. N0000990)

Oncogenic Ras mutants, and most frequently KRas mutants (86% of Ras-driven cancers), are found in approximately 25% of human cancers and are high-priority anticancer drug targets. Despite 30 years of effort to develop drugs that directly target oncogenic Ras mutants, no effective pharmacologic inhibitors for these mutants are clinically available, mainly because of the lack of suitable surface binding pockets for small molecules. More than 50 therapeutic antibodies have been clinically approved against many extracellular proteins. However, such antibodies do not have the capacity to localize in intracellular cytosolic regions after receptor-mediated endocytosis, restricting their therapeutic application for targeting cytosolic proteins. Our group recently developed a platform technology of cytosol-penetrating antibody, which in the IgG format can reach the cytosolic space of living cells owing to its endosomal escaping ability after receptor-mediated endocytosis. Exploiting the cytosol-penetrating antibody technology, we have engineered a human IgG1 format antibody, named iMab (internalizing and protein-protein interaction [PPI] interfering monoclonal antibody), which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of oncogenic Ras mutants. iMab specifically binds to the PPI interfaces of activated Ras with effector proteins to block the associations, thereby inhibiting the Ras downstream oncogenic signaling and exerting antiproliferation effects on oncogenic Ras mutant tumor cells. For in vivo antitumor efficacy assessment, we further engineer iMab to have tumor tissue-homing ability by fusion of tumor-associated integrin αvβ3/αvβ5 binding cyclic peptide to the N-terminus of light chain. When systemically administered, the iMab variant significantly inhibited the in vivo growth of oncogenic Ras-mutated tumor xenografts in mice, but not wild-type Ras-harboring tumors. Our results demonstrate the feasibility of developing antibody therapeutics that directly target cytosolic proteins involved in disease-associated PPIs, such as oncogenic Ras mutants, by systemic administration, similar to conventional therapeutic antibody regimens. Because the oncogenic Ras targeting antibody holds many desirable features of the conventional IgG antibody, it shows great potential for development as a first-in-class anticancer antibody. Citation Format: Seung-Min Shin, Ji-Sun Kim, Jin-Sun Hong, Seong-wook Park, Sei-Yong Jun, Hye-Jin Kweon, Dong-Ki Choi, Yong-Sung Kim. Direct targeting oncogenic Ras mutants by IgG-format cytosol-penetrating antibody [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B28.

Secreted Wnts play crucial roles in synaptogenesis and synapse maintenance, but endogenous factors promoting synapse elimination in central neurons remain unknown. Here we show that proline-rich 7 (PRR7) induces specific removal of excitatory synapses and acts as a Wnt inhibitor. Remarkably, transmembrane protein PRR7 is activity-dependently released by neurons via exosomes. Exosomal PRR7 is uptaken by neurons through membrane fusion and eliminates excitatory synapses in neighboring neurons. Conversely, PRR7 knockdown in sparse neurons greatly increases excitatory synapse numbers in all surrounding neurons. These non-cell autonomous effects of PRR7 are effectively negated by augmentation or blockade of Wnt signaling. PRR7 exerts its effect by blocking the exosomal secretion of Wnts, activation of GSK3β, and promoting proteasomal degradation of PSD proteins. These data uncover a proximity-dependent, reciprocal mechanism for the regulation of excitatory synapse numbers in local neurons and demonstrate the significance of exosomes in inter-neuronal signaling in the vertebrate brain., Wnts are important for synapse formation and maintenance. Here, the authors show that proline-rich 7 (PRR7) is a Wnt inhibitor that is secreted via exosomes to regulate excitatory synapse numbers.

This paper presents the results of an investigation into the effect of forced temperature change cycles on physical and mechanical properties of sand and weathered granite soil. The effect of forced temperature change cylecs on the particle arrangement and the thermal conductivity was first investigated. A series of triaxial compression tests on the soils were also performed to look into the effect of temperature change cycles on the stress-strain-strength behavior. The results indicated that the forced temperature change cycle does not significantly affect the particle arrangement and thermal conductivity. It is shown however that the heating duration showed some effect on the deviatoric stress at failure while no significant effect due to the number of heating-cooling cycle was observed.

In this study, the authors propose a signal detection technique of successive interference cancellation to reduce the effect of inter-symbol interference (ISI), which is caused by the time difference of arrivals among distributed nodes in an asynchronous wireless mesh network based on filtered multi-tone modulation. The proposed signal detection technique uses partial matrices of a transmission gain matrix to mitigate the ISI effect from the adjacent symbols. Under the assumption of perfect symbol time offset and channel estimation at each node, it is shown by simulation that the proposed technique can improve the bit error rate performance with lower complexity compared with the conventional technique.

This paper proposes a maximum efficiency operation strategy for three-level T-type inverter in entire operation areas. The three-level T-type inverter has higher and lower efficiency areas compared with two-level inverter. The proposed strategy aims to operate in the maximum efficiency point for the low-voltage and low-power home appliances. The three-level T-type inverter is analyzed in detail, and the two operation mode selection strategy is developed. The proposed algorithm is verified by theoretical analysis and experimental results.

Accumulating genetic evidence suggests that schizophrenia (SZ) is associated with individually rare copy number variations (CNVs) of diverse genes, often specific to single cases. However, the causality of these rare mutations remains unknown. One of the rare CNVs found in SZ cohorts is the duplication ofSynaptic Scaffolding Molecule(S-SCAM, also calledMAGI-2), which encodes a postsynaptic scaffolding protein controlling synaptic AMPA receptor levels, and thus the strength of excitatory synaptic transmission. Here we report that, in a transgenic mouse model simulating the duplication conditions, elevation of S-SCAM levels in excitatory neurons of the forebrain was sufficient to induce multiple SZ-related endophenotypes. S-SCAM transgenic mice showed an increased number of lateral ventricles and a reduced number of parvalbumin-stained neurons. In addition, the mice exhibited SZ-like behavioral abnormalities, including hyperlocomotor activity, deficits in prepulse inhibition, increased anxiety, impaired social interaction, and working memory deficit. Notably, the S-SCAM transgenic mice showed a unique sex difference in showing these behavioral symptoms, which is reminiscent of human conditions. These behavioral abnormalities were accompanied by hyperglutamatergic function associated with increased synaptic AMPA receptor levels and impaired long-term potentiation. Importantly, reducing glutamate release by the group 2 metabotropic glutamate receptor agonist LY379268 ameliorated the working memory deficits in the transgenic mice, suggesting that hyperglutamatergic function underlies the cognitive functional deficits. Together, these results contribute to validate a causal relationship of the rare S-SCAM CNV and provide supporting evidence for the rare CNV hypothesis in SZ pathogenesis. Furthermore, the S-SCAM transgenic mice provide a valuable new animal model for studying SZ pathogenesis.

Recombinant adeno-associated viral (AAV)-mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC-specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Muller glia-prone transduction. Titer-matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6-EGFP and AAVshH10-EGFP driven by a GFAP promoter (AAV6-GFAP-EGFP and AAVshH10-GFAP-EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6- and AAVshH10-GFAP-EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10-CMV-EGFP or AAVshH19-CMV-EGFP into DRG resulted in a similar in vivo transduction profile to AAV6-CMV-EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6-CMV-mCherry and AAV6-GFAP-EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.

Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology., Oncogenic RAS mutants are key anti-cancer targets as KRas mutations are very frequent in human cancers. Here, the authors engineer a cytosol-penetrating anti-Ras antibody and demonstrate its ability to block RAS-effector protein interactions inhibiting tumour growth of Ras mutant-driven cancers.

Purpose: To establish management protocol for mandibular angle fracture, we describe pertinent factors including cause, impacted third molar and recent treatment tendency. Methods: We examined the records of 62 patients who had unilateral mandibular angle fracture. Sixty patients who had open reduction surgery were examined at postoperative weeks 1, 4, 8, 12, and 28. Results: Left mandibular angle fracture is frequent in younger males. Presence of the mandibular third molar can increase fracture risk. Because of attached muscle, favorable fractures occurred primarily in the mandibular angle area. Conclusion: Extracting the mandibular third molar can prevent angle fractures, and open reduction with only one plate adaptation is generally the proper treatment method for mandibular angle fracture.

This paper proposes a compensation PWM technique for the extension of output voltage ranges in three-phase VSI applications using three shunt resistors. The proposed technique aims to solve the dead zone, which occurs in high modulation indexes. In the dead zone, two phase currents cannot be sampled correctly, so that the three-phase VSI cannot be operated up to the maximum output voltage. The dead zone is analyzed in detail, and the compensation PWM algorithm is developed. The proposed algorithm is verified by numerical analysis and experimental results.

Supplemental Digital Content is Available in the Text., Introduction: Tmem100 modulates interactions between TRPA1 and TRPV1. The cell specificity of Tmem100 expression in dorsal root ganglia (DRGs) is not well defined, nor is the effect of peripheral nerve injury on Tmem100 expression. Objective: This study was designed to determine the cell specificity of Tmem100 expression in DRG and its subcellular localization, and to examine how Tmem100 expression may be altered in painful conditions. Methods: Dorsal root ganglion Tmem100 expression was determined by immunohistochemistry, immunoblot, and quantitative real-time PCR, and compared between various experimental rat pain models and controls. Results: Tmem100 is expressed in both neurons and perineuronal glial cells in the rat DRG. The plasma membrane and intracellular localization of Tmem100 are identified in 83% ± 6% of IB4-positive and 48% ± 6% of calcitonin gene-related peptide–positive neurons, as well as in medium- and large-sized neurons, with its immunopositivity colocalized to TRPV1 (94% ± 5%) and TRPA1 (96% ± 3%). Tmem100 is also detected in the perineuronal satellite glial cells and in some microglia. Tmem100 protein is significantly increased in the lumbar DRGs in the complete Freund adjuvant inflammatory pain. By contrast, peripheral nerve injury by spinal nerve ligation diminishes Tmem100 expression in the injured DRG, with immunoblot and immunohistochemistry experiments showing reduced Tmem100 protein levels in both neurons and satellite glial cells of DRGs proximal to injury, whereas Tmem100 is unchanged in adjacent DRGs. The spared nerve injury model also reduces Tmem100 protein in the injured DRGs. Conclusion: Our data demonstrate a pain pathology–dependent alteration of DRG Tmem100 protein expression, upregulated during CFA inflammatory pain but downregulated during neuropathic pain.

How does chronic activity modulation lead to global remodeling of proteins at synapses and synaptic scaling? Here we report a role of guanylate-kinase-associated-protein (GKAP; also known as SAPAP), a scaffolding molecule linking NMDA receptor-PSD-95 to Shank-Homer complexes, in these processes. Over-excitation removes GKAP from synapses via ubiquitin-proteasome system, while inactivity induces synaptic accumulation of GKAP in rat hippocampal neurons. The bi-directional changes of synaptic GKAP levels are controlled by specific CaMKII isoforms coupled to different Ca2+ channels. α-CaMKII activated by NMDA receptor phosphorylates Serine-54 of GKAP to induce poly-ubiquitination of GKAP. In contrast, β-CaMKII activation via L-type voltage-dependent calcium channel promotes GKAP recruitment by phosphorylating Serine-340 and Serine-384 residues, which uncouples GKAP from MyoVa motor complex. Remarkably, overexpressing GKAP turnover mutants not only hampers activity-dependent remodeling of PSD-95 and Shank but also blocks bi-directional synaptic scaling. Therefore, activity-dependent turnover of PSD proteins orchestrated by GKAP is critical for homeostatic plasticity.

Synaptic plasticity, the cellular basis of learning and memory, involves the dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses. One of the remaining key unanswered aspects of AMPAR trafficking is the mechanism by which synaptic strength is preserved despite protein turnover. In particular, the identity of AMPAR scaffolding molecule(s) involved in the maintenance of GluA2-containing AMPARs is completely unknown. Here we report that the synaptic scaffolding molecule (S-SCAM; also called membrane-associated guanylate kinase inverted-2 and atrophin interacting protein-1) plays the critical role of maintaining synaptic strength. Increasing S-SCAM levels in rat hippocampal neurons led to specific increases in the surface AMPAR levels, enhanced AMPAR-mediated synaptic transmission, and enlargement of dendritic spines, without significantly effecting GluN levels or NMDA receptor (NMDAR) EPSC. Conversely, decreasing S-SCAM levels by RNA interference-mediated knockdown caused the loss of synaptic AMPARs, which was followed by a severe reduction in the dendritic spine density. Importantly, S-SCAM regulated synaptic AMPAR levels in a manner, dependent on GluA2 not GluA1, sensitive toN-ethylmaleimide-sensitive fusion protein interaction, and independent of activity. Further, S-SCAM increased surface AMPAR levels in the absence of PSD-95, while PSD-95 was dependent on S-SCAM to increase surface AMPAR levels. Finally, S-SCAM overexpression hampered NMDA-induced internalization of AMPARs and prevented the induction of long term-depression, while S-SCAM knockdown did not. Together, these results suggest that S-SCAM is an essential AMPAR scaffolding molecule for the GluA2-containing pool of AMPARs, which are involved in the constitutive pathway of maintaining synaptic strength.

This paper presents a design and topology selection of bi-direcional buck converter based on PV PCS for managing the electric power. Futhermore, Current Ringing on DCM bi-directional buck converter for soft switching is analyzed in detail. PSIM Simulation and Experiments at the various operating points show the propriety of this paper. Building on the result of simulation and experiment, a comparative analysis is performed with the approximate estimate. By use of a study, the selecion of switch and diode which improve efficiency of the overall system is appiled to DCM bi-directional buck converter based on PV PCS.

In this paper, an effective method has been proposed for selection of white Gaussian noise(WGN) band for radio background noise measurement system under broad band noise environment. MMN which comes from industrial devices and equipment mostly happens in the shape of broad band noise mostly like impulsive noise and this is the main reason for increasing level in the present radio noise measurements. The existing method based on singular value decomposition has weak point that it cannot give good performance for the broad band signal because it uses signal`s white property. The proposed method overcomes such a weakness of singular value decomposition based method by using signal`s Gaussian property based method in parallel. Moreover, this proposed method hires a modelling based method which uses parameter estimation algorithm like maximum likelihood estimation(MLE) and gives more accurate result than the method using amplitude probability distribution(APD) graph. Experiment results under the natural environment has done to verify feasibility of the proposed method.

Obese women have an increased risk for post-menopausal breast cancer. The physiological mechanism by which obesity contributes to breast tumourigenesis is not understood. We previously showed that HCCR-1 oncogene contributes to breast tumourigenesis as a negative regulator of p53 and detection of HCCR-1 serological level was useful for the diagnosis of breast cancer(.) In this study, we found that the HCCR-1 level is elevated in breast cancer tissues and cell lines compared to normal breast tissues. We identified apolipoprotein E (ApoE) interacting with HCCR-1. Our data show that HCCR-1 inhibits anti-proliferative effect of ApoE, which was mediated by diminishing ApoE secretion of breast cancer cells. Finally, HCCR-1 induced the severe obesity in transgenic mice. Those obese mice showed severe hyperlipidaemia. In conclusion, our results suggest that HCCR-1 might play a role in the breast tumourigenesis while the overexpression of HCCR-1 induces the obesity probably by inhibiting the cholesterol-lowering effect of ApoE. Therefore, HCCR-1 seems to provide the molecular link between the obesity and the breast cancer risk.

The human cervical cancer oncogene HCCR-1 is overexpressed in various human cancers, and might function as a negative regulator of the p53 tumor suppressor. To determine the regulatory pathway involved in the HCCR-1 gene expression, we searched the 5' flanking region of HCCR-1 and identified HCCR-1 promoter including putative homeodomain protein binding sites. The level of HCCR-1 expression was increased during the mouse embryogenesis. Expression of phosphatidylinositol 3-kinase (PI3K) in NIH/3T3 cells activated the HCCR-1 promoter. This promoter was also activated by wild type Akt but not by dominant negative Akt in K562 cells. In addition, the level of HCCR-1 was decreased by PI3K inhibitor, LY-294002, in a dose dependent manner. Northern blot analysis revealed that the HCCR-1 gene expression was down-regulated by LY-294002. These results suggest that the HCCR-1 oncogene expression was regulated by the PI3K/Akt signaling pathway.

Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira®) and bevacizumab (Avastin®), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.

Purpose: The purpose of our study was to identify an unique gene that shows cancer-associated expression, evaluates its potential usefulness in cancer diagnosis, and characterizes its function related to human carcinogenesis. Experimental Design: We used the differential display reverse transcription-PCR method with normal cervical, cervical cancer and metastatic tissues, and cervical cancer cell line to identify genes overexpressed in cancers. Results: We identified a minichromosome maintenance 3 (MCM3) gene that was overexpressed in various human cancers, including leukemia, lymphoma, and carcinomas of the uterine cervix, colon, lung, stomach, kidney and breast, and malignant melanoma. Western blot and immunohistochemical analyses also revealed that MCM3 protein was elevated in most of human cancer tissues tested. We compared the MCM3 protein expression levels in human cancers with conventional proliferation markers, Ki-67 and proliferating cell nuclear antigen. MCM3 antibody was the most specific for multiple human cancers, whereas proliferating cell nuclear antigen was relatively less effective in specificity, and Ki-67 failed to detect several human cancers. The down-regulation of MCM3 protein level was examined under serum starvation in both normal and cancer cells. Interestingly, MCM3 protein was stable in MCF-7 breast cancer cells even up to 96 hours after serum starvation, whereas it was gradually degraded in normal BJ fibroblast cells. Nude mice who received injections of HEK 293 cells stably transfected with MCM3 formed tumors in 6 weeks. Conclusions: Our study indicates that determination of MCM3 expression level will facilitate the assessment of many different human malignancies in tumor diagnosis, and MCM3 is involved in multiple types of human carcino-genesis.

Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of this study was to identify unique oncogenes that are differentially expressed in human cancers and characterize their functions in tumorigenesis. To discover new putative oncogenes, the differential display RT-PCR method was applied using normal cervical tissues, cervical cancer cell lines, cervical cancer tissues, and metastatic tissues. We identified a new human cervical cancer oncogene HCCR-2 that was overexpressed in various human tumors including leukemia, lymphoma, and carcinomas of the breast, kidney, ovary, stomach, colon, and uterine cervix. Ectopic expression of HCCR-2 resulted in direct tumorigenic conversions of NIH/3T3 and Rat1 fibroblasts. Nude mice injected with NIH/3T3 cells stably transfected with HCCR-2 formed tumors in 4 weeks. The resultant tumors display characteristics of an epithelial carcinoma. In HCCR-2 transfected NCI-H460 cells and RKO cells, stabilization of the p53 tumor suppressor occurred without genetic mutation and correlated with functional impairment, as indicated by the defective induction of p53-induced p21(WAF1), MDM2, and bax. These results indicate that HCCR-2 probably represents a new oncogene that is related to tumorigenesis, functioning as a negative regulator of the p53 tumor suppressor.

Mesothelioma is a rare aggressive tumor arising from the mesothelial cell and regarded as universally fatal disease with average survival around 1 year. The incidence rate is varied from one to forty per million in different countries and increasing by the year. The most common site of tumor origin is the pleura and only 20% to 33% of mesothelioma arise from the peritoneum. There are increasing reports of malignant mesothelioma with forty to fifty fatal cases per year in Korea. Histological studies with immunohistochemical stain is helpful for the diagnosis of peritoneal mesothelioma and imaging modality alone is not sufficient for diagnosis, so it is difficult to confirm diagnosis. A 64-year-old male patient was admitted to the hospital with a palpable mass on abdomen. The 6x6 cm sized huge mass was seen on the body of stomach adjacent to the peritoneum. We report a case of malignant peritoneal mesothelioma without evident exposure to asbestos, of which direct invasion to the gastric mucosa was confirmed by endoscopic biopsy and immunohistochemical stain.

Background Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules. Results We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell. Conclusions Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

Oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. HCCR transgenic mice developed breast cancers but it is unknown how HCCR-1 contributes to human tumorigenesis. This study identified a HCCR-1-binding protein 1 (HCCRBP-1) as an HCCR binding partner by performing yeast two hybrid screening. Their endogenous interaction was further confirmed by coimmunoprecipitation experiments. These two proteins colocalized in the mitochondria. HCCRBP-1 was overexpressed in various human tumors. In addition, HCCRBP-1 alone converted NIH/3T3 cells into tumor cells in combination with no other oncogenes. HCCRBP-1 induced tumorigenesis by markedly activating PKC activities but decreasing the pro-apoptotic PKC alpha and PKC delta isoform levels. We observed that p53 stabilization also occurred with functional impairment in HCCRBP-1-transfected 293 cells, as indicated by defective induction of p21, MDM2 and bax. Indeed, HCCRBP-1 decreased p21 promoter activity probably via p53 stabilization leading to the defective function. These results indicate that HCCRBP-1 oncogene induces p53 stabilization and thereby contributes to tumorigenesis.

Background The Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene. Results To investigate the biological function of HCCR-1 in the cell, we predicted biological features using bioinformatic tools, and have identified a LETM1 homologous domain at position 75 to 346 of HCCR-1. This domain contains proteins identified from diverse species predicted to be mitochondrial proteins. Fluorescence microscopy and fractionation experiments showed that HCCR-1 is located in mitochondria in the COS-7, MCF-7 and HEK/293 cell lines, and subcompartamentally at the outer membrane in the HEK/293 cell line. The topological structure was revealed as the NH2-terminus of HCCR-1 oriented toward the cytoplasm. We also observed that the D1-2 region, at position 1 to 110 of HCCR-1, was required and sufficient for posttranslational mitochondrial import. The function of HCCR-1 on mitochondrial membrane is to retard the intrinsic apoptosis induced by UVC and staurosporine, respectively. Conclusion Our experiments show the biological features of HCCR-1 in the cell, and suggest that uncontrolled expression of HCCR-1 may cause mitochondrial dysfunction that can result in resisting the UVC or staurosporine-induced apoptosis and progressing in the tumor formation.

Background Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of our study was to identify an unique gene that shows cancer-associated expression, and characterizes its function related to human carcinogenesis. Methods We used the differential display (DD) RT-PCR method using normal cervical, cervical cancer, metastatic cervical tissues, and cervical cancer cell lines to identify genes overexpressed in cervical cancers and identified gremlin 1 which was overexpressed in cervical cancers. We determined expression levels of gremlin 1 using Northern blot analysis and immunohistochemical study in various types of human normal and cancer tissues. To understand the tumorigenesis pathway of identified gremlin 1 protein, we performed a yeast two-hybrid screen, GST pull down assay, and immunoprecipitation to identify gremlin 1 interacting proteins. Results DDRT-PCR analysis revealed that gremlin 1 was overexpressed in uterine cervical cancer. We also identified a human gremlin 1 that was overexpressed in various human tumors including carcinomas of the lung, ovary, kidney, breast, colon, pancreas, and sarcoma. PIG-2-transfected HEK 293 cells exhibited growth stimulation and increased telomerase activity. Gremlin 1 interacted with homo sapiens tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide (14-3-3 eta; YWHAH). YWHAH protein binding site for gremlin 1 was located between residues 61–80 and gremlin 1 binding site for YWHAH was found to be located between residues 1 to 67. Conclusion Gremlin 1 may play an oncogenic role especially in carcinomas of the uterine cervix, lung, ovary, kidney, breast, colon, pancreas, and sarcoma. Over-expressed gremlin 1 functions by interaction with YWHAH. Therefore, Gremlin 1 and its binding protein YWHAH could be good targets for developing diagnostic and therapeutic strategies against human cancers.

Purpose: HCCR oncoprotein is reported to be related to tumorigenesis, including breast cancer, functioning as a negative regulator of p53. Mice transgenic for HCCR developed breast cancers. The objective of this study was to validate the HCCR oncoprotein as a candidate biomarker for breast cancer. Experimental Design: HCCR expression in breast cancer cells was analyzed by quantitative PCR, ELISA, immunohistochemistry, Western blotting, fluorescence-activated cell sorting, and confocal microscopy. Epitope areas were determined using mass spectrometry through the analysis of time-dependent tryptic fragment patterns of HCCR. HCCR expression profiles in breast cancer patient sera were analyzed, and correlations with clinicopathologic data and carbohydrate antigen 15-3 (CA15-3) levels were determined. Results: HCCR was up-regulated in breast cancer cells and tissues. The epitope regions of HCCR recognized by monoclonal antibody (BCS-1) were HFWTPK and QQTDFLDIYHAFR. According to fluorescence-activated cell sorting and confocal microscopic analysis, BCS-1 was bound to HCCR antigen on the cell surface. Serum HCCR concentrations were measured using ELISA from 299 subjects, including 129 patients with breast cancer, 24 patients with benign breast disease, and 158 normal volunteers, and comparisons were made to CA15-3. Serologic studies revealed an 86.8% sensitivity for HCCR in breast cancer, which was higher than 21.0% for CA15-3. Eighty-six of 98 (87.8%) patients with breast cancers that were negative for CA15-3 were positive for HCCR-1. A positive response rate of 83.3% was identified even at early stages for pathologic factors in breast cancer. Conclusions: The HCCR assay has an advantage over CA15-3 in diagnosing breast cancer and detecting early stages of the disease.

Transgenic mice containing novel oncogene HCCR-2 were generated to analyse the phenotype and to characterize the role of HCCR-2 in cellular events. Mice transgenic for HCCR-2 developed breast cancers and metastasis. The level of p53 in HCCR-2 transgenic mice was elevated in most tissues including breast, brain, heart, lung, liver, stomach, kidney, spleen, and lymph node. We examined whether stabilized p53 is functional in HCCR-2 transgenic mice. Defective induction of p53 responsive genes including p21WAF1, MDM2, and bax indicates that stabilized p53 in HCCR-2 transgenic mice exists in an inactive form. These results suggest that HCCR-2 represents an oncoprotein that is related to breast cancer development and regulation of the p53 tumor suppressor.

BACKGROUND/AIMS Few studies have assessed left ventricular (LV) dyssynchrony in cases of diastolic dysfunction that do not include overt symptoms of heart failure. We hypothesized that systolic or diastolic dyssynchrony involves unique features with respect to the degree of diastolic impairment in isolated diastolic dysfunction. METHODS We examined 105 subjects with no history of overt symptoms of heart failure and a left ventricular ejection fraction > 50% for mechanical dyssynchrony using tissue Doppler imaging. RESULTS In terms of longitudinal dyssynchrony, four cases showed (6.3%) LV intraventricular systolic dyssynchrony (SDS(LV)), whereas none had LV intraventricular diastolic dyssynchrony (DDS(LV)) or co-existing systolic dyssynchrony. Radial dyssynchrony (RD) was found in six cases (9.4%). After adjusting for age, SDS(LV) and DDS(LV) were found to be significantly related to increases in the E/E' ratio (r = 0.405 and p < 0.001 vs. r = 0.216 and p = 0.045, respectively). RD at the base and apex was also significantly related to increases in E/E' (r = 0.298 and p = 0.002 vs. r = 0.196 and p = 0.045, respectively). CONCLUSIONS Systolic and diastolic dyssynchrony in subjects with isolated diastolic dysfunction but without overt symptoms of heart failure was not as common as in patients with diastolic heart failure; however, the systolic and diastolic intraventricular time delay increased with increases in the E/E' ratio, an indicator of diastolic dysfunction.