Leucine 434 is essential for docosahexaenoic acid-induced augmentation of L-glutamate transporter current.

Astrocytic excitatory amino acid transporter 2 (EAAT2) plays a major role in removing the excitatory neurotransmitter L-glutamate (L-Glu) from synaptic clefts in the forebrain to prevent excitotoxicity. Polyunsaturated fatty acids such as docosahexaenoic acid (DHA, 22:6 n-3) enhance synaptic transmission, and their target molecules include EAATs. Here, we aimed to investigate the effect of DHA on EAAT2 and identify the key amino acid for DHA/EAAT2 interaction by electrophysiological recording of L-Glu-induced current in Xenopus oocytes transfected with EAATs, their chimeras, and single mutants. DHA transiently increased the amplitude of EAAT2 but tended to decrease that of excitatory amino acid transporter subtype 1 (EAAT1), another astrocytic EAAT. Single mutation of leucine (Leu) 434 to alanine (Ala) completely suppressed the augmentation by DHA, while mutation of EAAT1 Ala 435 (corresponding to EAAT2 Leu434) to Leu changed the effect from suppression to augmentation. Other polyunsaturated fatty acids (docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid, and α-linolenic acid) similarly augmented the EAAT2 current and suppressed the EAAT1 current. Finally, our docking analysis suggested the most stable docking site is the lipid crevice of EAAT2, in close proximity to the L-Glu and sodium binding sites, suggesting that the DHA/Leu434 interaction might affect the elevator-like slide and/or the shapes of the other binding sites. Collectively, our results highlight a key molecular detail in the DHA-induced regulation of synaptic transmission involving EAATs.

The postsynaptic density proteins Homer and Shank form a polymeric network structure.

The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of N-terminal EVH1 domains at each end of the coiled coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.

Postoperative Prognosis According to Pathologic Categorization of Desmoplastic Reaction in Patients with Extrahepatic Cholangiocarcinoma.

BACKGROUND: Recent studies have demonstrated the importance of desmoplastic reaction (DR) in predicting postoperative prognosis for patients with colorectal carcinoma. However, the impact of DR on the prognosis of extrahepatic cholangiocarcinomas (EHCCs) is not established. This study aimed to clarify the associations of pathologic DR categories with clinicopathologic factors and postoperative prognosis of perihilar cholangiocarcinoma (PHCC) and distal cholangiocarcinoma (DCC). METHODS: A pathologic review of 174 patients with PHCC and 109 patients with DCC who underwent surgical resection was performed. The patients were classified into three DR categories (immature, intermediate, and mature) based on the histologic features within the fibrotic stroma in the invasive front. The association between DR categories and the distribution of fibroblasts with anti-α-smooth muscle actin (SMA) expression, seeming to be tumor-promoting cancer-associated fibroblasts (CAFs), was evaluated in 191 tissue microarray specimens of EHCCs. RESULTS: Intermediate/immature DR categories were significantly associated with a more invasive nature, including higher pT and pN stages and more tumor buds than the mature category in both PHCC and DCC. The DR categories could stratify overall survival (OS) and relapse-free survival (RFS) in both PHCC and DCC patients. In the multivariate analysis, the DR category was an independent prognostic factor for OS and RFS in both PHCC and DCC (p < 0.001). The mature and immature DR categories were significantly associated respectively with the confined and pervasive distribution of fibroblasts with α-SMA expression. CONCLUSION: In patients with EHCCs, DR categorization was an independent prognostic factor reflecting the distribution of tumor-promoting CAFs in the invasive front.

Neurons Induce Tiled Astrocytes with Branches That Avoid Each Other.

Neurons induce astrocyte branches that approach synapses. Each astrocyte tiles by expanding branches in an exclusive territory, with limited entries for the neighboring astrocyte branches. However, how astrocytes form exclusive territories is not known. For example, the extensive branching of astrocytes may sterically interfere with the penetration of other astrocyte branches. Alternatively, astrocyte branches may actively avoid each other or remove overlapped branches to establish a territory. Here, we show time-lapse imaging of the multi-order branching process of GFP-labeled astrocytes. Astrocyte branches grow in the direction where other astrocyte branches do not exist. Neurons that had just started to grow dendrites were able to induce astrocyte branching and tiling. Upon neuronal loss by glutamate excitotoxicity, astrocytes' terminal processes retracted and more branches went over other branches. Our results indicate that neurons induce astrocyte branches and make them avoid each other.

Prenatal nicotine exposure affects cardiovascular function and growth of the developing fetus.

AIM: Effects of nicotine on fetal hemodynamics are not well known, especially in the first trimester fetus. We investigated the acute and chronic effects of nicotine on hemodynamics in pregnant mice and their fetuses using ultrasound. Postnatal health status including growth and hemodynamics was also examined. METHODS: To investigate the acute effects of nicotine on fetal hemodynamics, we injected nicotine 0.2 mg/kg subcutaneously into pregnant mice on gestational days (GD) 9.5, 11.5 and 13.5 and compared with saline-injected group. To determine the chronic effects of nicotine on fetal hemodynamics, we administered nicotine in drinking water (0.1 mg/mL) to pregnant mice from GD 6.5 until they gave birth and compared hemodynamics with water-administered mice. RESULTS: Regarding the acute effects of nicotine, we found no intergroup difference in maternal hemodynamics; however, fetal blood flow through the dorsal aorta, carotid artery and umbilical artery tended to decrease, particularly on GD 11.5. Regarding the chronic effects of nicotine, we observed no intergroup difference in maternal body weight changes and hemodynamics; however, blood flow to all fetal organs tended to be lower in the nicotine water group than in the water group with significant difference on GD 13.5. The offspring of the nicotine water group had significantly low birth weights and continued to have low body weight until 9 weeks of age. In addition, these offspring developed postnatal cardiac hypertrophy. CONCLUSION: Nicotine adversely affects fetal hemodynamics acutely and chronically in early pregnancy, potentially leading to fetal tissue hypoxia, intrauterine growth restriction and adverse postnatal health effects.

[Bronchial Stump Coverage Technique by da Vinci Xi].

da Vinci's greatest attractions are the fine images acquired with a high-resolution 3-dimensional( 3D) endoscope and the precise operation by forceps equipped with an articulated arm. We believed that complicated procedures can benefit from robot-assisted surgery. We have been actively performing collection and sewing of viable tissue using da Vinci Xi for the purpose of preventing bronchial stump fistulas. Of the 44 cases of lobectomy performed by the end of November 2019 using da Vinci Xi, 13 cases underwent bronchial stump covering. The covering procedure was intended for patients with conditions such as diabetes and a history of internal use of steroids. As the dressing, pedicled intercostal muscle was used in 4 cases, and free pericardial fat tissue was used in 9 cases. A good visual field was obtained by appropriately turning the oblique mirror upside down. The smoke emission effect of AirSeal was useful for securing the visual field. The dressing was sutured and fixed using 3-0 or 4-0 Prolene( SH) cut to a length of 10 to 12 cm. The postoperative course was good and bronchial stump fistula did not occur in all cases.

Hyaluronan synthesis supports glutamate transporter activity.

Hyaluronan is synthesized, secreted, and anchored by hyaluronan synthases (HAS) at the plasma membrane and comprises the backbone of perineuronal nets around neuronal soma and dendrites. However, the molecular targets of hyaluronan to regulate synaptic transmission in the central nervous system have not been fully identified. Here, we report that hyaluronan is a negative regulator of excitatory signals. At excitatory synapses, glutamate is removed by glutamate transporters to turn off the signal and prevent excitotoxicity. Hyaluronan synthesized by HAS supports the activity of glial glutamate transporter 1 (GLT1). GLT1 also retracted from cellular processes of cultured astrocytes after hyaluronidase treatment and hyaluronan synthesis inhibition. A serial knockout study showed that all three HAS subtypes recruit GLT1 to cellular processes. Furthermore, hyaluronidase treatment activated neurons in a dissociated rat hippocampal culture and caused neuronal damage due to excitotoxicity. Our findings reveal that hyaluronan helps to turn off excitatory signals by supporting glutamate clearance. Cover Image for this issue: doi: 10.1111/jnc.14516.

Plasma omentin levels are inversely associated with atherosclerosis in type 2 diabetes patients with increased plasma adiponectin levels: a cross-sectional study.

BACKGROUND: Omentin and adiponectin are among the anti-inflammatory and anti-atherogenic adipokines that have potentially beneficial effects on cardiovascular disorders. Recent studies indicate a paradoxical relationship between adiponectin and cardiovascular mortality across many clinical settings including type 2 diabetes. In this study, we characterized the clinical features of type 2 diabetes patients with increased adiponectin levels and examined the association between omentin and atherosclerosis in those patients. METHODS: The subjects were 413 patients with type 2 diabetes. Fasting plasma omentin and total adiponectin levels were measured by enzyme-linked immunosorbent assay. The intima-media thickness (IMT) of the common carotid artery was measured by ultrasonography. The subjects were stratified according to the median value of plasma adiponectin. RESULTS: In high-adiponectin group, omentin levels were higher, while IMT tended to be greater than those in low-adiponectin group. The high-adiponectin group also exhibited older age, higher systolic blood pressure, lower kidney function, body mass index, and insulin resistance index compared to the low-adiponectin group. Multivariate analysis revealed that omentin levels were independently and negatively associated with IMT in high-adiponectin group, but not in low-adiponectin group, after adjusting for adiponectin levels and traditional cardiovascular risk factors. On the other hand, adiponectin levels were not significantly associated with IMT in either group. CONCLUSIONS: Plasma omentin levels are inversely associated with IMT in type 2 diabetes patients with increased adiponectin levels and multiple cardiovascular risk factors. This study suggests a protective role of omentin against atherosclerosis in type 2 diabetes patients, which is potentially influenced by adiponectin level and cardiovascular risk status.

The role of CaMKII-Tiam1 complex on learning and memory.

A stimulation inducing long-term potentiation (LTP) of synaptic transmission induces a persistent expansion of dendritic spines, a phenomenon known as structural LTP (sLTP). We previously proposed that the formation of a reciprocally activating kinase-effector complex (RAKEC) between CaMKII and Tiam1, an activator of the small G-protein Rac1, locks CaMKII into an active conformation, which in turn maintains the phosphorylation status of Tiam1. This makes Rac1 persistently active, specifically in the stimulated spine. To understand the significance of the CaMKII-Tiam1 RAKEC in vivo, we generated a Tiam1 mutant knock-in mouse line in which critical residues for CaMKII binding were mutated into alanines. We confirmed the central role of this interaction on sLTP by observing that KI mice showed reduced Rac1 activity, had smaller spines and a diminished sLTP as compared to their wild-type littermates. Moreover, behavioral tests showed that the novel object recognition memory of these animals was impaired. We thus propose that the CaMKII-Tiam1 interaction regulates spine morphology in vivo and is required for memory storage.

Structure-Function Relationship of Transporters in the Glutamate-Glutamine Cycle of the Central Nervous System.

Many kinds of transporters contribute to glutamatergic excitatory synaptic transmission. Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters to be released from presynaptic terminals. After synaptic vesicle release, glutamate is taken up by neurons or astrocytes to terminate the signal and to prepare for the next signal. Glutamate transporters on the plasma membrane are responsible for transporting glutamate from extracellular fluid to cytoplasm. Glutamate taken up by astrocyte is converted to glutamine by glutamine synthetase and transported back to neurons through glutamine transporters on the plasma membranes of the astrocytes and then on neurons. Glutamine is converted back to glutamate by glutaminase in the neuronal cytoplasm and then loaded into synaptic vesicles again. Here, the structures of glutamate transporters and glutamine transporters, their conformational changes, and how they use electrochemical gradients of various ions for substrate transport are summarized. Pharmacological regulations of these transporters are also discussed.

Expression and localization of aquaporin-4 in sensory ganglia.

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4(-/-) mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4(-/-) mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system.

A general anaesthetic propofol inhibits aquaporin-4 in the presence of Zn²âº.

AQP4 (aquaporin-4), a water channel protein that is predominantly expressed in astrocyte end-feet, plays an important role in the brain oedema formation, and is thereby considered to be a potential therapeutic target. Using a stopped-flow analysis, we showed that propofol (2,6-diisopropylphenol), a general anaesthetic drug, profoundly inhibited the osmotic water permeability of AQP4 proteoliposomes in the presence of Zn²âº. This propofol inhibition was not observed in AQP1, suggesting the specificity for AQP4. In addition, the inhibitory effects of propofol could be reversed by the removal of Zn²âº. Other lipid membrane fluidizers also similarly inhibited AQP4, suggesting that the modulation of protein-lipid interactions plays an essential role in the propofol-induced inhibition of AQP4. Accordingly, we used Blue native PAGE and showed that the profound inhibition caused by propofol in the presence of Zn²âº is coupled with the reversible clustering of AQP4 tetramers. Site-directed mutagenesis identified that Cys²5³, located at the membrane interface connecting to the C-terminal tail, is responsible for Zn²âº-mediated propofol inhibition. Overall, we discovered that propofol specifically and reversibly inhibits AQP4 through the interaction between Zn²âº and Cys²5³. The findings provide new insight into the functional regulation of AQP4 and may facilitate the identification of novel AQP4-specific inhibitors.

Effect of orthodontic treatment on occlusal condition and masticatory function.

Change in occlusion and masticatory function after orthodontic treatment was investigated by a follow-up study in a group of 14 patients with malocclusion (3 men, 11 women, aged 12-46 years). All completed a food questionnaire and underwent occlusal force evaluation using a pressure-sensitive sheet at pre-treatment, post-treatment, and during the retention phase. The number of foods identified as difficult to eat decreased at post-treatment in all patients. The mean occlusal force was 646.6±223.5 N at pretreatment, 401.1±109.1 N at post-treatment, and 530.6±183.6 N during the retention phase. The mean occlusal contact area was 14.1±5.9 mm(2) at pre-treatment, 6.5±1.7 mm(2) at post-treatment, and 9.8±3.8 mm(2) during the retention phase. The mean average occlusal pressure was 47.6±6.6 MPa at pre-treatment, 62.2±5.7 MPa at post-treatment, and 55.4±6.7 MPa during the retention phase. A Dental Prescale film evaluation revealed that occlusal force and occlusal contact area increased over the 1-year retention phase. An improvement in level of satisfaction with mastication ability was reported at post-treatment and during the retention phase. Average occlusal pressure at post-treatment increased significantly (p <0.01), which may account for the observed increase in the ability of the patients to bite through food. These results suggest that an increase in average occlusal pressure improves level of satisfaction with mastication ability.

Perioperative chemotherapy with nivolumab for HER2-negative locally advanced gastric cancer: a case series.

BACKGROUND: Nivolumab with chemotherapy has been transformative for metastatic gastric cancer (GC). The potential of this regimen for local tumor control could be utilized for perioperative chemotherapy in locally advanced GC with bulky tumors or lymph node metastasis involving other organs. CASE PRESENTATION: Five patients with HER2-negative advanced GC were treated with nivolumab and oxaliplatin-based chemotherapy. All patients presented with clinical stage III or IVA GC with tumors in contact with either the pancreas or liver. Following chemotherapy, all tumors demonstrated shrinkage, allowing successful radical gastrectomies including four minimally invasive approach without postoperative complications. Four patients avoided combined resection of other organs. CONCLUSIONS: Perioperative chemotherapy with nivolumab was effective for local disease control in this case series. This regimen could be a promising treatment approach for locally advanced GC; however, its survival benefits should be evaluated in clinical trials.

CCR7 Mediates Cell Invasion and Migration in Extrahepatic Cholangiocarcinoma by Inducing Epithelial-Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC.

Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.

Pulmonary Artery Banding for Congenitally Corrected Transposition of the Great Arteries With Hydrops Fetalis: A Case Report.

At 32 weeks of gestation, a male fetus with congenitally corrected transposition of the great arteries developed hydrops fetalis caused by a combination of mitral valve regurgitation and tricuspid valve regurgitation (TR). We performed a pulmonary artery banding (PAB) at 108 days old for gradually progressing TR, after confirming that a balloon dilatation test in the main pulmonary artery reduced TR. As the patient grew, the PAB became tighter and systolic blood pressure in the morphological left ventricle increased. At present, the patient is waiting for a double switch operation.

Plasma omentin levels are associated with vascular endothelial function in patients with type 2 diabetes at elevated cardiovascular risk.

AIMS: Omentin is an adipokine that has protective effects against cardiovascular damage. Previous studies showed an inverse relationship between omentin and obesity, diabetes, and cardiovascular disease. This study aimed to investigate the association between omentin and vascular endothelial function in patients with type 2 diabetes (T2D). METHODS: The subjects were 425 patients with T2D and 223 non-diabetic controls. Fasting plasma omentin levels were measured by enzyme-linked immunosorbent assay, and the endothelium-dependent, flow-mediated dilatation (FMD) was measured by ultrasonography. RESULTS: Plasma omentin levels were higher, while FMD was lower in participants with T2D than in non-diabetic controls. No significant correlation was found between plasma omentin levels and FMD in either non-diabetic controls or participants with T2D on multivariate analysis. However, stratified analysis in T2D patients revealed that plasma omentin levels were independently and positively associated with FMD in high cardiovascular risk subgroups according to age (≥65 years), estimated glomerular filtration rate (<60 mL/min/1.73 m2), or preexisting cardiovascular diseases but not in low-risk subgroups. CONCLUSIONS: Plasma omentin levels are independently associated with endothelial function in subgroups of patients with T2D at elevated cardiovascular risk. This study suggests a protective role of omentin against endothelial dysfunction, particularly in high-risk patients.