Notch signaling genes and CD8+ T-cell dynamics: Their contribution to immune-checkpoint inhibitor therapy in oral squamous cell carcinoma: A retrospective study.

BACKGROUND: Aberrant Notch signaling pathway has been related with the tumorigenesis in head and neck region, involving oral cavity. Here, we report the correlation between mutations in the Notch signaling pathway and CD8+ T-cell infiltration via PD-L1, which lead to enhanced antitumor immunity and may target for immune-checkpoint inhibitors (ICIs) therapy. METHODS: This retrospective study analyzed the results of immunohistochemical staining for PD-L1 and CD8+ T-cell infiltration in 10 patients and whole-exome sequencing (WES) was conducted on five of these patients to identify frequently mutated genes. RESULTS: Four of 10 patients were positive for PD-L1 and CD8+ T. By analyzing WES in three of these four patients, we notably identified the mutations of NOTCH1, FBXW7, and noncoding RNA intronic mutation in NOTCH2NLR in two of these three patients. This study may enable better selection of ICI therapy with CD8+ T-cell infiltration via PD-L1 expression for oral squamous cell carcinoma patients with mutations in Notch signaling pathway.

Binuclear Complexes Supported by a Tetrapyridyl Ligand with a Bending Anthraquinodimethane Linker.

A tetrapyridyl ligand with a bending anthraquinodimethane linker has been synthesized, and its complexation with coinage metals has been examined. The treatment of the ligand with Ag(I) and Au(I) cations afforded binuclear complexes, wherein the two metal centers were in close proximity to the inside space of the ligand. X-ray analyses corroborated with theoretical calculations indicated that the ligand has reasonable flexibility toward a bending deformation of the linker moiety to provide a ligand pocket suitable for the proximal binuclear complexes, even though such deformations accompany a non-negligible amount of energetic cost. On the other hand, treatment with 2 equiv of Cu(I) salt afforded a binuclear complex, in which both copper atoms were coordinated at the periphery of the ligand.

One-pot Syntheses of Benzo- and Benzofuran-fused Iridaoxabenzenes via CH Bond Activations of Alkyl-bridged Diphenol Derivatives.

One-pot syntheses of new π-extended metallaaromatic compounds have been developed by utilizing Ir-mediated CH bond activation of ethylene- or ethylidene-bridged diphenol derivatives. Depending on the bridging alkyl groups, two types of iridaoxabenzenes, both of which are doubly fused with benzo and benzofuran units, have been obtained. Studies on their structures and electronic characters indicate that both complexes have an aromatic character on the iridaoxacycles, and their π-conjugated systems are fully delocalized over the whole molecular skeletons. These novel metallaaromatic complexes exhibited some reactivities which are distinct from those reported for the non-fused metallaaromatic compounds.

Perineural treatment with anti-TNF-α antibody ameliorates persistent allodynia and edema in novel mouse models with complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is an intractable chronic pain syndrome with various signs and symptoms including allodynia/hyperalgesia, edema, swelling, and skin abnormalities. However, a definitive therapeutic treatment for CRPS has not been established. In CRPS patients, inflammatory cytokines such as TNF-α and IL-1ß have been shown to increase in affected areas, suggesting that these molecules may be potential therapeutic targets for CRPS. Here, we first created a novel CRPS mouse model (CRPS-II-like) via sciatic nerve injury and cast immobilization, which was characterized by mechanical allodynia, local edema, and skin abnormalities, to evaluate the pathophysiology and pharmacotherapy of CRPS. When an anti-TNF-α antibody was consecutively administered near the injured sciatic nerve of CRPS model mice, persistent allodynia and CRPS-related signs in the ipsilateral hindpaw were markedly attenuated to control levels. Perineural administration of anti-TNF-α antibody also suppressed the upregulation of inflammatory cytokines as well as the activation of macrophages and Schwann cells in the injured sciatic nerve. These findings indicate that persistent allodynia and CRPS-related signs in CRPS models are primarily associated with TNF-α-mediated immune responses in injured peripheral nerves, suggesting that perineural treatment with anti-TNF-α antibody might be therapeutically useful.

A rare case report of diverticulum of the buccal mucosa.

We report a case of diverticulum of the buccal mucosa. A 56-year-old man had a small pouch-shaped lesion behind the parotid papilla that caused pain and food impaction. After resection, the lesion was histopathologically diagnosed as diverticulum without buccal muscle tear. There has been no recurrence during 1 year postoperatively.

Improvement of solubility of phospholipase D from Streptomyces antibioticus in recombinant Escherichia coli and its application for the enzymatic synthesis of a non-natural plasmalogen.

Plasmalogens are a subclass of glycerophospholipids that have a vinyl-ether bond at the sn-1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in the cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with ∼730 mU mg-1 protein of specific activity, and the yield was ∼420 mU l-1 culture, corresponding to 76 mU per gram of wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn-3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens.

Functional characteristics and therapeutic potential of SLC41 transporters.

Magnesium (Mg2+) plays an important role in various cellular functions such as protein synthesis, DNA stability, energy metabolism, enzyme and channel activities, and muscle contractility. Therefore, intracellular Mg2+ concentration is tightly regulated by multiple Mg2+ transporters and channels. So far, various candidate genes of Mg2+ transporters have been identified, and the research on their structure and function is currently in progress. The Solute Carrier 41 (SLC41) family, which is related to the bacterial Mg2+ transporter/channel MgtE, comprises three isoforms of SLC41A1, SLC41A2, and SLC41A3. Based on recent studies, SLC41A1 is thought to mediate Mg2+ influx or Na+-dependent Mg2+ efflux across the plasma membrane, whereas SLC41A2 and SLC41A3 may mediate Mg2+ fluxes across either the plasma membrane or organellar membranes. Intriguingly, SLC41A1 variants have been identified in patients with Parkinson's disease (PD) and nephronophthisis-related ciliopathies. Further genetic analyses reveal the association of SLC41A1 polymorphisms with PD risks. This review highlights the recent advances in the understanding of the molecular and functional characteristics of SLC41 family towards its therapeutic and diagnostic applications.

Inhibition of transient receptor potential cation channel 6 promotes capillary arterialization during post-ischaemic blood flow recovery.

BACKGROUND AND PURPOSE: Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischaemic blood flow. We previously demonstrated that the inhibition of transient receptor potential 6 subfamily C, member 6 (TRPC6) channels facilitate contractile differentiation of VSMCs under ischaemic stress. We here investigated whether TRPC6 inhibition promotes post-ischaemic blood flow recovery through capillary arterialization in vivo. EXPERIMENTAL APPROACH: Mice were subjected to hindlimb ischaemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischaemic left leg to non-ischaemic right one. The number and diameter of blood vessels were analysed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry. KEY RESULTS: Although the post-ischaemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production were inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by pyrazole-2 (Pyr2; BTP2; YM-58483) promoted post-ischaemic blood flow recovery in Apolipoprotein E-knockout mice. CONCLUSION AND IMPLICATIONS: Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischaemic peripheral blood circulation.

Acceptance of Murine Islet Allografts Without Immunosuppression in Inguinal Subcutaneous White Adipose Tissue Pretreated With bFGF.

Prevention of immune rejection without immunosuppression is the ultimate goal of transplant immunobiology. One way to achieve this in cellular transplantation, such as with islet transplantation, is to create a favorable local environment at the transplant site. In the current study, we found that C57BL/6 mice with streptozotocin-induced diabetes remained normoglycemic for >1 year after transplantation of BALB/c islets without immunosuppression when the inguinal subcutaneous white adipose tissue (ISWAT) was the site of transplantation and when the site was pretreated with basic fibroblast growth factor. Mechanistically, mesenchymal stem cells (MSCs) expanded in the ISWAT after the treatment was found to produce transforming growth factor-ß (TGF-ß), and prevention of islet allograft rejection could be achieved by cotransplantation with syngeneic MSCs isolated from the ISWAT after the treatment, which was abolished by anti-TGF-ß antibody treatment. Importantly, TGF-ß-producing cells remained present at the site of cotransplantation up to the end of observation period at 240 days after transplantation. These findings indicate that prevention of islet allograft rejection without immunosuppression is feasible with the use of syngeneic TGF-ß-producing MSCs expanded in the ISWAT after the treatment with bFGF, providing a novel strategy for prevention of islet allograft rejection without immunosuppression.

Lymphangiogenesis and angiogenesis rescue murine ischemic hindlimb via transient receptor potential vanilloid 4.

In this study, we assessed the regulation of transient receptor potential vanilloid 4 (TRPV4) promoting lymphangio/angiogenesis to improve the ischemic hindlimb animal model, and revealed that (1) a TRPV4 agonist improved the blood flow of ischemic hindlimbs by inducing both angiogenesis and lymphangiogenesis; (2) excessive TRPV4 expression was detected on lymphatic endothelial cells (LECs) in the ischemic hindlimb; and (3) hypoxic conditions promoted Ca2+ influx into LECs via TRPV4. It is considered that the upregulation of both lymphatic and blood vessels by activating TRPV4 would be a promising therapeutic strategy for peripheral artery disease.

Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms.

Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.

Genetic knockout and pharmacologic inhibition of NCX1 attenuate hypoxia-induced pulmonary arterial hypertension.

The Na+/Ca2+ exchanger type-1 (NCX1) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na+ and Ca2+. Vascular smooth muscle NCX1 plays an important role in intracellular Ca2+ homeostasis and Ca2+ signaling. We found that NCX1 was upregulated in the pulmonary arteries of mice exposed to chronic hypoxia (10% O2 for 4 weeks). Hence, we investigated the pathophysiological role of NCX1 in hypoxia-induced pulmonary arterial hypertension (PAH), using NCX1-heterozygous (NCX1+/-) mice, in which NCX1 expression is reduced by half, and SEA0400, a specific NCX1 inhibitor. NCX1+/- mice exhibited attenuation of hypoxia-induced PAH and right ventricular (RV) hypertrophy compared with wild-type mice. Furthermore, continuous administration of SEA0400 (0.5 mg/kg/day for 4 weeks) to wild-type mice by osmotic pumps significantly suppressed hypoxia-induced PAH and pulmonary vessel muscularization, with a slight reduction in RV hypertrophy. These findings indicate that the upregulation of NCX1 contributes to the development of hypoxia-induced PAH, suggesting that NCX1 inhibition might be a novel approach for the treatment of PAH.

Sodium-calcium exchanger 1 is the key molecule for urinary potassium excretion against acute hyperkalemia.

The sodium (Na+)-chloride cotransporter (NCC) expressed in the distal convoluted tubule (DCT) is a key molecule regulating urinary Na+ and potassium (K+) excretion. We previously reported that high-K+ load rapidly dephosphorylated NCC and promoted urinary K+ excretion in mouse kidneys. This effect was inhibited by calcineurin (CaN) and calmodulin inhibitors. However, the detailed mechanism through which high-K+ signal results in CaN activation remains unknown. We used Flp-In NCC HEK293 cells and mice to evaluate NCC phosphorylation. We analyzed intracellular Ca2+ concentration ([Ca2+]in) using live cell Ca2+ imaging in HEK293 cells. We confirmed that high-K+-induced NCC dephosphorylation was not observed without CaN using Flp-In NCC HEK29 cells. Extracellular Ca2+ reduction with a Ca2+ chelator inhibited high-K+-induced increase in [Ca2+]in and NCC dephosphorylation. We focused on Na+/Ca2+ exchanger (NCX) 1, a bidirectional regulator of cytosolic Ca2+ expressed in DCT. We identified that NCX1 suppression with a specific inhibitor (SEA0400) or siRNA knockdown inhibited K+-induced increase in [Ca2+]in and NCC dephosphorylation. In a mouse study, SEA0400 treatment inhibited K+-induced NCC dephosphorylation. SEA0400 reduced urinary K+ excretion and induced hyperkalemia. Here, we identified NCX1 as a key molecule in urinary K+ excretion promoted by CaN activation and NCC dephosphorylation in response to K+ load.

Endergonic addition of N-methylamines to aromatic ketones driven by photochemical offset of the entropic cost.

Intermolecular addition reactions are generally accompanied by an entropic penalty due to the decrease of molecular numbers during the reaction, which sometimes makes the reaction endergonic. Here we demonstrate that such an endergonic reaction can be promoted with light-energy as a driving force; N-methylamines were added to aromatic ketones to produce aminoalcohols under UV-light irradiation. The reaction represents an obvious example showing that the photochemical approach is effective to offset such an entropic cost, and thereby to drive thermodynamically uphill addition reactions. Moreover the present reactions are highly expedient from the synthetic view point, being transition-metal-catalyst-free, scalable, highly atom economical, and regioselective. The product amines can be converted in one step to functional multi-arylated enamines, which are potentially valuable compounds in electronic materials.

Iron-catalysed enantioselective Suzuki-Miyaura coupling of racemic alkyl bromides.

The first iron-catalysed enantioselective Suzuki-Miyaura coupling reaction has been developed. In the presence of catalytic amounts of FeCl2 and (R,R)-QuinoxP*, lithium arylborates are cross-coupled with tert-butyl α-bromopropionate in an enantioconvergent manner, enabling facile access to various optically active α-arylpropionic acids including several nonsteroidal anti-inflammatory drugs (NSAIDs) of commercial importance. (R,R)-QuinoxP* is specifically able to induce chirality when compared to analogous P-chiral ligands that give racemic products, highlighting the critical importance of transmetalation in the present asymmetric cross-coupling system.

Aberrant Amygdala-Dependent Cued Fear Memory in Na+/Ca2+ Exchanger 1 Heterozygous Mice.

Na+/Ca2+ exchangers (NCXs) are mainly expressed in the plasma membrane and exchange one Ca2+ for three Na+, depending on the electrochemical gradients across the plasma membrane. NCXs have three isoforms, NCX1-3, encoded by distinct genes in mammals. Here, we report that heterozygous mice lacking NCX1 (NCX1+/-) exhibit impaired amygdala-dependent cued fear memory. NCX1+/- mice showed significant impairment in fear-related behaviors measured with the elevated-plus maze, light-dark, open-field, and marble-burying tasks. In addition, NCX1+/- mice showed abnormality in cued fear memory but not in contextual fear memory in a fear-conditioning task. In immunohistochemical analyses, NCX1+/- mice had significantly increased number of c-Fos-positive cells in the lateral amygdala (LA) but not in the central amygdala following fear-related tone stimuli. c-Fos expression peaked at 1 h. In concordance with the aberrant fear-related behaviors in NCX1+/- mice, enhanced long-term potentiation was also observed in the LA of these mice. Furthermore, enhancement of CaMKII or CaMKIV activity in the LA was observed in NCX1+/- mice by immunoblot analyses. In contrast, CaMKII+/- but not CaMKIV-/- mice insufficiently exhibited tone-induced cued fear memory and there was no increase in the number of c-Fos-positive cells in the LA. Altogether, the increased CaMKII activity and consequent c-Fos expression likely account for the dysregulation of amygdala-dependent cued fear memory in NCX1+/- mice.

[Dysfunction of Na+/Ca2+ exchangers is associated with cognitive decline in Alzheimer's disease].

Na+/Ca2+ exchanger (NCX) is mainly expressed in the plasma membrane and mediates electrogenical exchange of one Ca2+ for three Na+, depending on the electrochemical gradients across the plasma membrane. NCX has three different isoforms (NCX1, NCX2, NCX3) encoded by distinct genes in mammals. Here, we report that NCX2 and NCX3 protein levels are relatively reduced in hippocampal CA1 of Alzheimer's disease model mice. Likewise, NCX2+/- or NCX3+/- mice exhibited impaired hippocampal LTP and memory-related behaviors. In immunoblot analyses, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in hippocampal CA1 of NCX2+/- mice compared to wild-type mice. By contrast, NCX2+/- mice was correlated with elevated calcineurin (CaN) activity and rescued by treatment with the calcineurin inhibitor FK506. Taken together, the imbalance of CaMKII and CaN activities with concomitant LTP impairment likely accounts for the learning disability observed in NCX2+/- mice.

Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na+/Ca2+ Exchanger.

BACKGROUND AND AIMS: Hydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question. METHODS: Isometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-ß-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function. RESULTS: Porcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-ß-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-ß-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp-induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice. CONCLUSIONS: Endogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.

Reduced CaM Kinase II and CaM Kinase IV Activities Underlie Cognitive Deficits in NCKX2 Heterozygous Mice.

Among five members of the K+-dependent Na+/Ca2+ exchanger (NCKX) family (NCKX1-5), only NCKX2 is highly expressed in mouse brain. NCKX2 in plasma membranes mediates cytosolic calcium excretion through electrogenic exchange of 4 Na+ for 1 Ca2+ and 1 K+. Here, we observed significantly decreased levels of NCKX2 protein and mRNA in the CA1 region of APP23 mice, a model of Alzheimer's disease. We also found that, like APP23 mice, heterozygous NCKX2-mutant mice exhibit mildly impaired hippocampal LTP and memory acquisition, the latter based on novel object recognition and passive avoidance tasks. When we addressed underlying mechanisms, we found that both CaMKII autophosphorylation and CaMKIV phosphorylation significantly decreased in CA1 regions of NCKX2+/- relative to control mice. Likewise, phosphorylation of GluA1 (Ser-831) and CREB (Ser-133), respective downstream targets of CaMKII and CaMKIV, also significantly decreased in the CA1 region. BDNF protein and mRNA levels significantly decreased in CA1 of NCKX2+/- relative to control mice. Finally, CaN activity increased in CA1 of NCKX2+/- mice. Our findings suggest that like APP23 mice, NCKX2+/- mice may exhibit impaired learning and hippocampal LTP due to decreased CaM kinase II and CaM kinase IV activities.