The roles of FHL2 as a mechanotransducer for cellular functions in the mechanical environment.

The cell has multiple mechanisms for sensing and responding to dynamic changes in the mechanical environment. In the process, intracellular signaling is activated to modulate gene expression. Recent studies have shown that multifunctional signaling molecules that link intracellular force and gene expression are important for understanding cellular functions in the mechanical environment. This review discusses recent studies on one of the mechanotransducers, Four-and-a-half LIM domains 2 (FHL2), which localizes to focal adhesions (FAs), actin cytoskeleton, and nucleus. FHL2 localizes to FAs and the actin cytoskeleton in the cell on stiff substrate. In this situation, intracellular tension of F-actin by Myosin II is critical for FHL2 localization to FAs and actin stress fibers. In the case, a conserved phenylalanine in each LIM domain is responsible for its localization to F-actin. On the other hand, lower tension of F-actin in the cell on a soft substrate causes FHL2 to be released into the cytoplasm, resulting in its localization in the nucleus. At the molecular level, phosphorylation of specific tyrosine in FHL2 by FAK, non-receptor tyrosine kinase, is critical to nuclear localization. Finally, by binding to transcription factors, FHL2 modulates gene expression for cell proliferation as a transcriptional co-factor. Thus, FHL2 is involved in mechano-sensing and -transduction in the cell in a mechanical environment.

General Factors and Dental-Related Risk Factors for Postoperative Pneumonia or Infectious Complications: A Retrospective Study.

Numerous studies report that perioperative oral care decreases the frequency of postoperative pneumonia or infection. However, no studies have analyzed the specific impact of oral infection sources on the postoperative course, and the criteria for preoperative dental care differ among institutions. This study aimed to analyze the factors and dental conditions present in patients with postoperative pneumonia and infection. Our results suggest that general factors related to postoperative pneumonia, including thoracic surgery, sex (male > female), the presence or absence of perioperative oral management, smoking history, and operation time, were identified, but there were no dental-related risk factors associated with it. However, the only general factor related to postoperative infectious complications was operation time, and the only dental-related risk factor was periodontal pocket (4 mm or higher). These results suggest that oral management immediately before surgery is sufficient to prevent postoperative pneumonia, but that moderate periodontal disease must be eliminated to prevent postoperative infectious complication, which requires periodontal treatment not only immediately before surgery, but also on a daily basis.

Chemical Synthesis and Molecular Interaction Analysis of α-Galactosyl Ceramide Derivatives as CD1d Ligands.

CD1d is a non-classical major histocompatibility complex (MHC) protein, responsible for lipid antigen presentation, which presents lipids to natural killer T (NKT) cells. Various CD1d lipid ligands have been reported, including microbial and endogenous glycolipids/phospholipids. Among them, an α-galactosylceramide (α-GalCer), a representative CD1d ligand, is one of the most potent ligands and its derivatives have been developed. In this chapter, the chemistry of α-GalCer and its derivatives are described with an emphasis on their chemical syntheses and molecular interaction analysis with CD1d are described.

Labial Vestibular Flap Closure of the Cleft Palate Is Advantageous for Maxillary Development.

Objective: Using labial vestibular flap was performed to close the primary alveolar and hard palate cleft at the second stage of early 2-stage closure surgery for unilateral cleft lip and palate for minimizing the damage to the maxillary periosteum. We analyzed maxillary development to clarify the influence of cleft palate surgery. Design: Retrospective longitudinal study in 5 years after primary palatal closure. Setting: Institutional study Patients: Study subjects included 214 patients with nonsyndromic complete unilateral cleft lip and palate who were consecutively treated in our clinic. Main Outcome: We used a 3D dental model scanner to assess maxillary development in patients aged 3 months to 5 years after using either the conventional pushback method (PB) (51 cases) or 2-stage closure (Local palatal flap closure: LF [67 cases] and Labial vestibular flap closure: VF [96 cases]). Results: Comparing the measurement results, the major axis of maxilla, width, intercanine distance, and intermolar distance was significantly larger in the LF group compared to the PB group. After the age of 3, the cleft side of VF group had grown significantly to compare with LF group in width. It was also confirmed that the inserted labial mucosal flap itself grew. Enlargement of the labial mucosal flap was observed at all sites except the canine. Conclusion: Good maxillary growth occurred in the following order: VF groups > LF group > PB group. Poor growth was correlated with the extent of periosteal damage during surgery and the degree of postoperative bone surface exposure.

Trehalose diesters containing a polar functional group-modified lipid moiety: Synthesis and evaluation of Mincle-mediated signaling activity.

Mincle, a C-type lectin receptor (CLR), activates the innate immune system by recognizing certain complex lipid compounds. In this study, we designed and synthesized trehalose disteate (TDS) and dibehenate (TDB), containing a polar-functional group in the middle of fatty acid moieties, based on a model of the Mincle-glycolipids interaction. The modified fatty acids were prepared using hydroxy fatty acids as common intermediates, and conjugated with an appropriate trehalose moiety to synthesize the desired trehalose diesters. TDE derivatives containing the modified fatty acid have different Mincle-mediated signaling activities depending on the position of the functional group and the length of the lipids. The newly developed TDE derivatives exhibit signaling activity comparable or superior to that of TDS or TDB, and the results suggest that Mincle tolerates polar functional groups at a certain position of the lipid chain of TDE. The introduction of the polar functional groups into the lipid moiety of the glycolipids also resulted in improved solubility in polar solvents, which would be advantageous for various analyses and applications.

Precise immunological evaluation rationalizes the design of a self-adjuvanting vaccine composed of glycan antigen, TLR1/2 ligand, and T-helper cell epitope.

Sialyl-Tn (STn), overexpressed on various tumors, has been investigated for its application in anti-cancer vaccine therapy. However, Theratope, an STn-based vaccine, failed in the phase III clinical trial due to poor immunogenicity and epitope suppression by the foreign carrier protein. We therefore developed a self-adjuvanting STn based-vaccine, a conjugate of clustered STn (triSTn) antigen, TLR1/2 ligand (Pam3CSK4), and T-helper (Th) cell epitope, and found that this three-component self-adjuvanting vaccine effectively resulted in the production of anti-triSTn IgG antibodies. We herein analyzed immune responses induced by this self-adjuvanting vaccine in detail. We newly synthesized two-component vaccines, i.e., Pam3CSK4- or Th epitope-conjugated triSTn, as references to evaluate the immune-stimulating functions of Pam3CSK4 and Th epitope. Immunological evaluation of the synthesized vaccine candidates revealed that Pam3CSK4 was essential for antibody production, indicating that the uptake of triSTn antigen by antigen-presenting cells (APCs) was promoted by the recognition of Pam3CSK4 by TLR1/2. The function of the Th epitope was also confirmed. Th cell activation was important for boosting antibody production and IgG subclass switching. Furthermore, flow cytometric analyses of immune cells, including T cells, B cells, dendritic cells, and other monocytes, were first employed in the evaluation of self-adjuvanting vaccines and revealed that the three-component vaccine was able to induce antigen-specific immune responses for efficient antibody production without excessive inflammatory responses. Importantly, the co-administration of Freund's adjuvants was suggested to cause excessive myeloid cell accumulation and decreased plasma cell differentiation. These results demonstrate that vaccines can be designed to achieve the desired immune responses via the bottom-up construction of each immune element.

Correction: Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity.

Correction for 'Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity' by Takanori Matsumaru et al., Chem. Commun., 2019, 55, 711-714, DOI: 10.1039/C8CC07322H.

Alkyne-Tagged Dopamines as Versatile Analogue Probes for Dopaminergic System Analysis.

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter's small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

Lipopolysaccharide Derived From the Lymphoid-Resident Commensal Bacteria Alcaligenes faecalis Functions as an Effective Nasal Adjuvant to Augment IgA Antibody and Th17 Cell Responses.

Alcaligenes spp., including A. faecalis, is a gram-negative facultative bacterium uniquely residing inside the Peyer's patches. We previously showed that A. faecalis-derived lipopolysaccharides (Alcaligenes LPS) acts as a weak agonist of toll-like receptor 4 to activate dendritic cells and shows adjuvant activity by enhancing IgG and Th17 responses to systemic vaccination. Here, we examined the efficacy of Alcaligenes LPS as a nasal vaccine adjuvant. Nasal immunization with ovalbumin (OVA) plus Alcaligenes LPS induced follicular T helper cells and germinal center formation in the nasopharynx-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs), and consequently enhanced OVA-specific IgA and IgG responses in the respiratory tract and serum. In addition, nasal immunization with OVA plus Alcaligenes LPS induced OVA-specific T cells producing IL-17 and/or IL-10, whereas nasal immunization with OVA plus cholera toxin (CT) induced OVA-specific T cells producing IFN-γ and IL-17, which are recognized as pathogenic type of Th17 cells. In addition, CT, but not Alcaligenes LPS, promoted the production of TNF-α and IL-5 by T cells. Nasal immunization with OVA plus CT, but not Alcaligenes LPS, led to increased numbers of neutrophils and eosinophils in the nasal cavity. Together, these findings indicate that the benign nature of Alcaligenes LPS is an effective nasal vaccine adjuvant that induces antigen-specific mucosal and systemic immune responses without activation of inflammatory cascade after nasal administration.

Validity of the combined use of two esthetic rating systems, the infant index and 5-point aesthetic index, for pre- and postsurgical evaluation of cleft lip repair.

OBJECTIVE: The present study was designed to investigate the usefulness of combining two different ordinal scaling indices, infant index (I-I) and 5-point aesthetic index (5-PAI), for the assessment and prediction of esthetic outcome of primary lip repair for patients with unilateral cleft lip. MATERIALS AND METHODS: The nasolabial appearance of the patients was evaluated before primary lip repair and at 5 years of age using cropped facial photographs with frontal and oblique views. The I-I and 5-PAI employ expanded reference photographs and objective esthetic variables for judgment. RESULTS: The inter- and intrarater Kappa values of both I-I and 5-PAI demonstrated good to very good agreement (range: 0.74-0.84 for I-I and 0.62-0.77 for 5-PAI). Furthermore, both the declination of the columella and the deformity of the alar cartilage in I-I showed a correlation with nasal rating score of 5-PAI and were identified as predictable independent parameters (declination of the columella: Rs = 0.37, P = 0.04; deformity of the alar cartilage: Rs = 0.35, P = 0.02). CONCLUSION: The combined use of I-I and 5-PAI with expanded reference photographs and objective variables could be useful for obtaining greater accuracy of the esthetic assessment and predicting postsurgical nasolabial esthetics at infancy.

Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid†A.

Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.

Polar functional group-containing glycolipid CD1d ligands modulate cytokine-biasing responses and prevent experimental colitis.

The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, and its ligands include glycolipids, such as α-GalCer. The complexes between CD1d and ligands activate natural killer T cells by T cell receptor recognition, leading to the secretion of various cytokines (IFN-γ, IL-4, IL-17A, etc.). Herein, we report structure-activity relationship studies of α-GalCer derivatives containing various functional groups in their lipid acyl chains. Several derivatives have been identified as potent CD1d ligands displaying higher cytokine induction levels and/or unique cytokine polarization. The studies also indicated that flexibility of the lipid moiety can affect the binding affinity, the total cytokine production level and/or cytokine biasing. Based on our immunological evaluation and investigation of physicochemical properties, we chose bisamide- and Bz amide-containing derivatives 2 and 3, and evaluated their in vivo efficacy in a DSS-induced model of ulcerative colitis. The derivative 3 that exhibits Th2- and Th17-biasing responses, demonstrated significant protective effects against intestinal inflammation in the DSS-induced model, after a single intraperitoneal injection.

The key entity of a DCAR agonist, phosphatidylinositol mannoside Ac1PIM1: its synthesis and immunomodulatory function.

Ac1PIM1 is a potential biosynthetic intermediate for phosphatidylinositol mannosides (PIMs) from Mycobacterium tuberculosis. We achieved the first synthesis of Ac1PIM1 by utilizing an allyl-type protecting group strategy and regioselective phosphorylation of inositol. A very potent agonist of an innate immune receptor DCAR, which is better than previously known agonists, is demonstrated.

Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system.

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and ß2-microglobulin (ß2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with ß2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 µg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

Design and Discovery of Covalent α-GalCer Derivatives as Potent CD1d Ligands.

CD1d is a nonpolymorphic antigen-presenting protein responsible for the regulation of natural killer T (NKT) cell activation. α-Galactosyl ceramide (α-GalCer, KRN7000) is the representative CD1d ligand that can bind to the CD1d protein. The resulting complex is recognized by the T cell receptors of the NKT cell, inducing various immune responses. Previous structure-activity relationship studies of α-GalCer have revealed that the ability of NKT cells to induce cytokines depends on the ligand structure, and in particular, ligands that form more stable complexes with CD1d display potent activity. We focused on the Cys residue of the large hydrophobic pockets of CD1d (A' pocket) and developed α-GalCer derivatives containing groups that can form covalent bonds. The assay results revealed that these ligands displayed higher levels of cytokine production and Th2 cell-type cytokine polarization response. Furthermore, the LC-MS/MS analysis indicated that the chloroacetylamide-containing ligand was covalently bound to Cys12 of CD1d, which suggests that the enhanced activities result from the formation of a stable CD1d-ligand complex. To our knowledge, this is the first ligand that allows covalent bond formation to CD1d under physiological conditions.

Occlusion at 5 Years of Age Following Hard Palate Closure With Vestibular Flap.

OBJECTIVE: This study aims to assess occlusal relationships and frequency of oronasal fistula at 5 years of age following 2 hard palate closure techniques and to compare results. DESIGN: Retrospective longitudinal study. SETTING: Institutional study. PATIENTS: Study patients included 57 patients with nonsyndromic complete unilateral cleft lip and palate who were consecutively treated. All patients underwent our early 2-stage protocol for palatoplasty, which consisted of soft palate plasty at 1 year of age and hard palate closure at 1.5 years of age. Twenty-nine patients underwent hard palate closure using vestibular flap (VF group) technique (2009-2011) and 28 patients underwent conventional hard palate closure with local palatal flap (LPF group) technique (2006-2008). MAIN OUTCOME MEASURES: Occlusal relationships were assessed with 5-year-olds' index, and frequency of oronasal fistula was investigated. RESULTS: Average 5-year-olds' index scores for VF and LPF groups were 3.11 and 3.57, respectively (P < .001). Oronasal fistula occurred in approximately 7% of patients in the VF group and in 18% of patients in the LPF group. CONCLUSION: Hard palate closure with VF technique may provide better occlusal relationships at 5 years of age than does conventional local closure with the LPF.

Monitoring respiratory rates with a wearable system using a stretchable strain sensor during moderate exercise.

Respiratory rate, a sensitive indicator of respiratory status, is rarely measured during the field walking test. Our objective was to develop and validate a non-invasive, wearable monitoring system using stretchable strain sensors and an accompanying algorithm capable of providing real-time measurements of respiration during exercise. Twenty-four healthy volunteers wore stretchable sensors during a walking test protocol that included standing, sitting, walking, and walking with a stick. Sensors were placed on the ribcage and abdomen. The Bland-Altman method was used to assess the accuracy and precision of breath counts; total respiration time and inspiration time ratio were determined by custom algorithms and compared with measurements obtained with the standard flow sensor. The output signal from the stretchable sensor was highly synchronized with flow signals. The limits of agreement were within 3 breaths/min throughout the test protocol. Differences between sensors for total respiration time and inspiration time ratio were less than 14% and 26%, respectively. The agreement was maintained regardless of respiratory rate or volume. The wearable respiratory monitoring system yielded accurate and precise breath counts and total duration of respiratory cycle during moderate exercise in healthy young individuals, suggesting that it might be useful in clinical practice. Graphical abstract.

Synthetic Studies on FNC-RED and Its Analogues Containing an All syn-Cyclopentanetetrol Moiety.

FNC-RED exhibits innate immune receptor Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD2) stimulatory activity. We have developed a divergent synthetic route to FNC-RED derivatives containing various alkyl side chains. Key features of the synthetic study include stepwise palladium catalyzed cross-coupling reactions and the construction of an all syn-cyclopentanetetrol moiety.

Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators.

CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.

Exercise-Induced Oxygen Desaturation as a Predictive Factor for Longitudinal Decline in 6-Minute Walk Distance in Subjects With COPD.

BACKGROUND: There are limited longitudinal studies reporting predictive factors for decline in 6-min walk distance (6MWD) in patients with COPD. While previous studies have confirmed the association between air-flow limitation and decline in 6MWD, other factors have not been clarified. The objective of this study was to investigate whether exercise-induced oxygen desaturation (EID) could be a predictive factor for decline in 6MWD in patients with COPD. The interactive effect of air-flow limitation on the association between EID and decline in 6MWD was also investigated. METHODS: A longitudinal observational study was conducted with 71 out-patients with COPD who were followed for 1 year. 6MWD, EID, spirometry, and clinical characteristics were assessed. The effect of EID on changes in 6MWD was examined using linear regression analyses. Furthermore, the subjects were categorized into 4 groups according to their EID and air-flow limitation status, and changes in 6MWD were compared among the groups. RESULTS: 51 subjects completed the follow-up assessments, and 29 (56.9%) experienced EID. Multiple linear regression model revealed that EID was the only predictive factor for changes in 6MWD after adjusting for confounders (ß = -38.9, P = .02). As results of multiple comparisons among the 4 groups based on EID and air-flow limitation status, changes in 6MWD in the EID and severe air-flow limitation group were the lowest. CONCLUSION: Our results revealed that EID was a predictive factor for decline in the functional capacity of subjects with COPD. The assessment of EID and air-flow limitation would thus be useful in estimating the prognosis of decline in the functional capacity of patients with COPD.