Soluble T-cadherin secretion from endothelial cells is regulated via insulin/PI3K/Akt signalling.

AIM AND OBJECTIVE: Our recent report showed that soluble T-cadherin promotes pancreatic beta-cell proliferation. However, how and where the secretion of soluble T-cadherin is regulated remain unclear. METHODS AND RESULTS: Soluble T-cadherin levels significantly increased in leptin receptor-deficient db/db mice with hypoinsulinaemia or in wild-type mice treated with insulin receptor blockade by S961. Similar results were observed in human subjects; Diabetic ketoacidosis patients at the time of hospitalization had increased plasma soluble T-cadherin levels, which decreased after insulin infusion therapy. Patients with recurrent ovarian cancer who were administered a phosphatidylinositol-3 kinase (PI3K)-alpha inhibitor (a new anticancer drug) had increased plasma soluble T-cadherin and plasma C-peptide levels. Endothelial cell-specific T-cadherin knockout mice, but not skeletal muscle- or cardiac muscle-specific T-cadherin knockout mice, showed a 26 % reduction in plasma soluble T-cadherin levels and a significant increase in blood glucose levels in streptozocin-induced diabetes. The secretion of soluble T-cadherin from human endothelial cells was approximately 20 % decreased by insulin and this decrease was canceled by blockade of insulin receptor/Akt signalling, not Erk signalling. CONCLUSION: We conclude that insulin regulates soluble T-cadherin levels and soluble T-cadherin secretion from endothelial cells is positively regulated by insulin/insulin receptor/Akt signalling.

Evaluation of functional group compatibility and development of reaction-accelerating additives in ammonium salt-accelerated hydrazinolysis of amides.

Functional group compatibility in an amide bond cleavage reaction with hydrazine was evaluated for 26 functional groups in the functional group evaluation (FGE) kit. Accurate and rapid evaluation of the compatibility of functional groups, such as nitrogen-containing heterocycles important in drug discovery research, will enhance the application of this reaction in drug discovery research. These data will be used for predictive studies of organic synthesis methods based on machine learning. In addition, these studies led to discoveries such as the unexpected positive additive effects of carboxylic acids, indicating that the FGE kit can propel serendipitous discoveries.

A case report of carcinoma of the papilla of Vater associated with a hyperplasia-dysplasia-carcinoma sequence by pancreaticobiliary maljunction.

BACKGROUND: Pancreaticobiliary maljunction (PBM) is a known risk factor for biliary tract cancer. However, its association with carcinoma of the papilla of Vater (PVca) remains unknown. We report a case with PVca that was thought to be caused by the hyperplasia-dysplasia-carcinoma sequence, which is considered a mechanism underlying PBM-induced biliary tract cancer. CASE PRESENTATION: A 70-year-old woman presented with white stool and had a history of cholecystectomy for the diagnosis of a non-dilated biliary tract with PBM. Esophagogastroduodenoscopy revealed a tumor in the papilla of Vater, and PVca was histologically proven by biopsy. We finally diagnosed her with PVca concurrent with non-biliary dilated PBM (cT1aN0M0, cStage IA, according to the Union for International Cancer Control, 8th edition), and subsequently performed subtotal stomach-preserving pancreaticoduodenectomy. Pathological findings of the resected specimen revealed no adenomas and dysplastic and hyperplastic mucosae in the common channel slightly upstream of the main tumor, suggesting a PBM related carcinogenic pathway with hyperplasia-dysplasia-carcinoma sequence. Immunostaining revealed positivity for CEA. CK7 positivity, CK20 negativity, and MUC2 negativity indicated that this PVca was of the pancreatobiliary type. Genetic mutations were exclusively detected in tumors and not in normal tissues, and bile ducts from formalin-fixed paraffin-embedded samples included mutated-ERBB2 (Mutant allele frequency, 81.95%). Moreover, of the cell-free deoxyribonucleic acid (cfDNA) extracted from liquid biopsy mutated-ERBB2 was considered the circulating-tumor deoxyribonucleic acid (ctDNA) of this tumor. CONCLUSIONS: Herein, we report the first case of PVca with PBM potentially caused by a "hyperplasia-dysplasia-carcinoma sequence" detected using immunostaining and next-generation sequencing. Careful follow-up is required if pancreaticobiliary reflux persists, considering the possible development of PVca.

Visible-Light-Induced Aminochlorination of Alkenes.

Photoinduced N-internal vicinal aminochlorination of styrene-type terminal alkenes was developed. The reaction proceeded without any catalyst, and the use of N-chloro(fluorenone imine) as both a photoactivatable aminating agent and a chlorinating agent was essential. The imine moiety, introduced at the internal position of the alkenes, could be hydrolyzed under mild conditions to provide versatile ß-chlorinated primary amines, the synthetic utility of which was demonstrated by several transformations.

One-Pot Catalytic Synthesis of α-Tetrasubstituted Amino Acid Derivatives via In Situ Generation of N-Unsubstituted Ketimines.

A one-pot catalytic synthesis of α-tetrasubstituted amino acid derivatives via in situ generation of N-unsubstituted ketimines is reported. Because of the irreversible formation of N-unsubstituted ketimines, the yields were higher than those generated under the conventional one-pot reaction conditions. This process prevents the need to isolate unstable N-unsubstituted ketimines with alkyl substituents and streamlines the synthesis of highly congested α-amino acid derivatives.

Evaluating the feasibility of a remote-based training program supported by information and communications technology in the older adults living at home.

BACKGROUND: Exercise has been one of the key strategies for preventing frailty. While training programs for preventing frailty have been mainly developed in person, which have now become difficult to perform due to the coronavirus disease pandemic. It would be worthwhile to explore a feasibility of methods for a remote-based training with information and communications technology (ICT) in the pre-frail/robust older adults living at home. METHODS: We assessed the feasibility of a remote-based training with ICT device in terms of 1) a measurement accuracy and 2) whether it could be used for remote-based training of different intensities. To evaluate a measurement accuracy of the ICT device, we evaluated an inter-rater reliability between a true score and scores obtaining from the ICT device in 20 participants aged 65 years and older. Intraclass correlation was calculated. To evaluate a feasibility of remote-based training interventions of different intensities, we did a parallel, randomized, active controlled trial. Participants aged 65 years or older were randomly allocated to the two 3-month intervention programs with different intensity of exercise with the ICT (i.e., an Exercise-Intensive program and a Light-load exercise program). The primary outcome was 3-month scores of the 30-s chair-stand test (CS-30), which was compared between two groups using mixed models for repeated measures to account for within-person correlations. RESULTS: The ICT device showed a high intraclass correlation of over 0.99 for all outcomes including CS-30. Between Aug and Oct 2020, 70 participants (36 and 34 in the Exercise-Intensive and Light-load exercise programs, respectively) were randomized. After 3 months of intervention, CS-30 scores and other physical function improved in both groups. Difference in the 3-month CS-30 scores between two programs was found to be 0.08 (95% confidence interval: - 2.64, 2.79; p = 0.955), which was not statistically significant. No harmful incidents, such as falls, occurred in either group. CONCLUSION: We showed a remote-based training with ICT device in the older adults living at home was feasible. Further studies are warranted to determine what kind of remote exercise intervention programs is more effective for maintaining a physical performance and, beyond that, preventing frailty. TRIAL REGISTRATION NUMBER: UMIN000041616 (05/09/2020) https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&recptno=R000047504&type=summary&language=E.

Catalytic Enantioselective Strecker Reaction of Isatin-Derived N-Unsubstituted Ketimines.

A catalytic enantioselective Strecker reaction of isatin-derived N-unsubstituted ketimines directly afforded the N-unprotected α-aminonitriles with a tetrasubstituted carbon stereocenter in up to 99% ee without requiring protection/deprotection steps. One-pot Strecker reactions from the parent carbonyl compounds were also realized with comparable yields and enantioselectivities. Direct transformations of the N-unprotected α-aminonitrile products streamlined the synthesis of unnatural amino acid derivatives and achieved the shortest one-pot stereoselective routes to a biologically active compound reported to date.

Scandium(III) Triflate Catalyzed Direct Synthesis of N-Unprotected Ketimines.

N-Unprotected ketimines are useful substrates and intermediates for synthesizing valuable nitrogen-containing compounds, but their potential applicability is limited by the available synthetic methods. To address this issue, we report a scandium(III) triflate catalyzed direct synthesis of N-unprotected ketimines. Using commercially available reagents and Lewis acid catalysts, ketones were directly transformed into the corresponding N-unprotected ketimines in high yields with broad functional group tolerance, even in multigram scales. The reactions were readily applicable for one-pot synthesis of important compounds such as a glycine Schiff base without isolation of N-unprotected ketimine intermediates. Preliminary mechanistic studies to clarify the reaction mechanism are also described.

Comparison of the gut microbiotas of laboratory and wild Asian house shrews (Suncus murinus) based on cloned 16S rRNA sequences.

The Asian house shrew, Suncus murinus, is an insectivore (Eulipotyphla, Mammalia) and an important laboratory animal for life-science studies. The gastrointestinal tract of Suncus is simple: the length of the entire intestine is very short relative to body size, the large intestine is quite short, and there are no fermentative chambers such as the forestomach or cecum. These features imply that Suncus has a different nutritional physiology from those of humans and mice, but little is known about whether Suncus utilizes microbial fermentation in the large (LI) or small (SI) intestine. In addition, domestication may affect the gastrointestinal microbial diversity of Suncus. Therefore, we compared the gastrointestinal microbial diversity of Suncus between laboratory and wild Suncus and between the SI and LI (i.e., four groups: Lab-LI, Lab-SI, Wild-LI, and Wild-SI) using bacterial 16S rRNA gene library sequencing analyses with a sub-cloning method. We obtained 759 cloned sequences (176, 174, 195, and 214 from the Lab-LI, Lab-SI, Wild-LI, and Wild-SI samples, respectively), which revealed that the gastrointestinal microbiota of Suncus is rich in Firmicutes (mostly lactic acid bacteria), with few Bacteroidetes. We observed different bacterial communities according to intestinal region in laboratory Suncus, but not in wild Suncus. Furthermore, the gastrointestinal microbial diversity estimates were lower in laboratory Suncus than in wild Suncus. These results imply that Suncus uses lactic acid fermentation in the gut, and that the domestication process altered the gastrointestinal bacterial diversity.

Effect of different knee flexion angles with a constant hip and knee torque on the muscle forces and neuromuscular activities of hamstrings and gluteus maximus muscles.

PURPOSE: This study examined the effect of different knee flexion angles with a constant hip and knee torque on the muscle force and neuromuscular activity of the hamstrings and gluteus maximus. METHODS: Twenty healthy males lay in prone position and held their lower limb with hip flexion at 45° and knee flexion at either 10° or 80°. At these angles, the hip and knee torques are identical. Under three load conditions: passive (referred to as Unloaded), active (Loaded), and active with 3-kg weight added to the shank (Loaded + 3 kg), the muscle stiffness (i.e., an indicator of muscle force) and neuromuscular activity of the hamstrings and gluteus maximus were measured using shear wave elastography and surface electromyography. RESULTS: The muscle stiffness and neuromuscular activity of the hamstrings and gluteus maximus increased significantly with the load. Muscle stiffness in the hamstrings was significantly lower at knee flexion of 80° than at 10° for Unloaded, but not for either Loaded or Loaded + 3 kg. The neuromuscular activity of the hamstrings was significantly greater at knee flexion of 80° than at 10° for both Loaded and Loaded + 3 kg. The muscle stiffness or neuromuscular activity of the gluteus maximus showed no significant differences between knee angles. CONCLUSIONS: When the passive force in the hamstrings decreases with knee flexion, sufficient muscle force to maintain the hip and knee torques against an external load is generated by preferentially increasing the neuromuscular activity of the hamstrings, rather than increasing the synergetic muscle force.

3-Mono-Substituted BINOL Phosphoric Acids as Effective Organocatalysts in Direct Enantioselective Friedel-Crafts-Type Alkylation of N-Unprotected α-Ketiminoester.

Although BINOL-derived phosphoric acids are among the most widely used chiral Brønsted acid organocatalysts, their structures are mostly limited to 3,3'-disubstituted ones and simple 3-mono-substituted ones without any polar functionalities on the 3-substituent have not been used in highly enantioselective reactions. This work reports such 3-mono-substituted analogues as effective organocatalysts in direct highly enantioselective Friedel-Crafts-type alkylation of N-unprotected α-ketiminoester. The origin of the observed high enantioselectivity with the 3-mono-substituted catalyst is also discussed.

Systemic administration of a TLR7 agonist attenuates regulatory T cells by dendritic cell modification and overcomes resistance to PD-L1 blockade therapy.

Research on immune checkpoint blockade therapy has made great progress in cancer immunotherapy, but the number of patients who benefit from this therapy remains limited. In this study, we examined the effects of monotherapy with systemic low-dose resiquimod, a synthesized TLR7 agonist, and examined its combined effects with PD-L1 blockade in two PD-L1 blockade-resistant tumor models (SCCVII and Colon 26). Resiquimod monotherapy in SCCVII tumors, representing impaired CD8+ T cell function and accelerated regulatory T cells (Tregs) within the tumors, efficiently reduced tumor growth with more recruitment of CD8+ T cells and a reduction of Treg. The results of resiquimod monotherapy in Colon 26, representing impaired Treg recruitment, were inferior to that in SCCVII. Combined resiquimod treatment with PD-L1 blockade exerted clear additional effects, as it was associated with reduced tumor size, attenuation of Tregs, and an increased ratio of CD8+ T cells/Tregs in both tumors. Systemic administration of low-dose resiquimod induced a transient and rapid activation of plasmacytoid and conventional dendritic cells, resulting in enhanced priming of T cells in regional lymph nodes. Experiments with more limited doses of resiquimod that did not yield beneficial effects after single treatment, showed additional effects to PD-L1 blockade and comparable antitumor effects when the frequency of anti-PD-L1 therapy was decreased. Our results suggest that systemic administration of low-dose resiquimod is useful as a companion drug to PD-1/PD-L1 blockade therapy.

The immune checkpoint molecule VISTA regulates allergen-specific Th2-mediated immune responses.

V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel immune checkpoint receptor and ligand that regulates T-cell activation. We investigated the functional involvement of VISTA in Th2 cell-mediated immune responses using an ovalbumin (OVA)-induced allergic asthma model. Treatment with an anti-VISTA monoclonal antibody (mAb) during allergen sensitization increased the production of antibodies, including total IgE, OVA-specific IgG1 and IgG2a and allergen-specific IL-5 and IL-13; it also increased the expression of IL-13 by splenic CD4+ T cells. However, treatment with the anti-VISTA mAb during sensitization did not accelerate asthmatic responses, including airway hyper-responsiveness (AHR) or the number of eosinophils in bronchoalveolar lavage (BAL) fluid. In contrast, treatment with the anti-VISTA mAb during allergen challenge significantly augmented AHR and BAL fluid eosinophilia. This treatment also increased the production of IL-5 and IL-13 in BAL fluid and the expression of IL-13 by CD4+ T cells in draining lymph nodes. These results suggest that VISTA is involved in the regulation of Th2 cell generation and Th2 cell-mediated antibody production and regulates asthmatic responses, especially in the effector phase.

Synthesis of 1-Tetrasubstituted 2,2,2-Trifluoroethylamine Derivatives via Palladium-Catalyzed Allylation of sp3 C-H Bonds.

This note describes the construction of tetrasubstituted carbon stereocenters via palladium-catalyzed allylation of sp3 C-H bonds of 2,2,2-trifluoroethylamine derivatives. The presence of 2-pyridyl group of the imines derived from 1-substituted-2,2,2-trifluoroethylamine was key to promoting the reaction efficiently, allowing an access to a variety of 1-allylated 2,2,2-trifluoroethylamine derivatives with tetrasubstituted carbon stereocenters.

Direct Access to N-Unprotected α- and/or ß-Tetrasubstituted Amino Acid Esters via Direct Catalytic Mannich-Type Reactions Using N-Unprotected Trifluoromethyl Ketimines.

Direct catalytic C-C bond-forming addition to N-unprotected ketimines is an efficient and straightforward method of synthesizing N-unprotected tetrasubstituted amines that eliminates prior protection/deprotection steps and allows facile transformation of the products. Despite its advantages, however, N-unprotected ketimines have difficulties in C-C bond-forming reactions, and only a limited number of reactions and substrates are reported compared with their N-protected counterparts. Herein we report that N-unprotected trifluoromethyl ketimines are effective for C-C bond-forming reactions using Mannich-type reactions as a model case. We demonstrate that Lewis acid catalysis was effective for promoting reactions with various N-unprotected trifluoromethyl ketimines, and thiourea organocatalysis was effective for promoting highly enantioselective reactions with various carbonyl nucleophiles, providing direct access to various N-unprotected α- and/or ß-tetrasubstituted amino acid esters. Furthermore, direct construction of vicinal tetrasubstituted chiral carbon stereocenters was achieved for the first time in a highly enantio- and diastereoselective manner. These results demonstrate the potential of N-unprotected ketimines as substrates applicable to many other addition reactions.

Secreted Metalloproteinase ADAMTS-3 Inactivates Reelin.

The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.

Differential contribution of three immune checkpoint (VISTA, CTLA-4, PD-1) pathways to antitumor responses against squamous cell carcinoma.

V domain-containing Ig suppressor of T-cell activation (VISTA)/PD-1H is a novel immune checkpoint molecule for regulating T-cell activation. We examined the effects of anti-VISTA mAb monotherapy and combination therapy with CTLA-4 or PD-1 blockade in a squamous cell carcinoma (SCCVII) model. VISTA monotherapy did not show clear tumor growth regression, but efficiently induced CD8(+) T cell activation by converting resting and exhausted cells into functional effector cells. VISTA monotherapy did not inhibit recruitment of regulatory T cells (Tregs) in the tumor microenvironment (TME). As an additional treatment to VISTA, CTLA-4 blockade, but not PD-1 blockade, elicited further tumor regression. The CTLA-4 and VISTA combination efficiently inhibited Treg recruitment and increased the ratios of both CD8 T/Treg and CD4 conventional T (Tcon)/Treg in the TME, whereas the PD-1 and VISTA combination dramatically increased tumor-recruiting CD8(+) T cells, but markedly reduced the Tcon/Treg ratio. Our results demonstrate that VISTA blockade efficiently converts CD8(+) T cells into functional effector T cells, but is not sufficient to regress tumor growth due to weak Treg suppression in the TME. Our results suggest that combined CTLA-4 and VISTA blockade is more efficacious than combined PD-1 and VISTA blockade for tumors like head and neck squamous cell carcinoma in which Treg-mediated immune regulation is dominant.

HPV16 E2 protein promotes innate immunity by modulating immunosuppressive status.

The balance between active immune responses against human papillomavirus (HPV) and HPV-induced immune escape regulates viral clearance and carcinogenesis. To understand the role of the early viral protein HPV16 E2 in host innate immune responses, the HPV16 E2-transfected murine squamous cell carcinoma cell line SCCVII (SCC/E2) was generated and anti-tumor responses in T-cell-depleted mice were evaluated. Tumor growth of SCC/E2 was markedly reduced. Cytotoxicity against the NK-sensitive targets YAC-1 and SCCVII was clearly enhanced in SCC/E2-inoculated mice. Despite the comparable ratio of NK cells, the proportion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) was significantly decreased in SCC/E2-inoculated mice. The transcription of MDSC-related mediators such as inducible nitric oxide synthase, indoleamine 2,3-dioxygenase, and heme oxygenase-1 was significantly impaired in the SCC/E2-inoculated tumor tissues on day 3. Our results suggest that HPV16 E2 promotes anti-tumor innate effector function by modulating immunoregulatory events mediated by MDSCs and their mediators. This report describes a new role for HPV16 E2 as a local immunomodulator at infected sites.