Investigation of Beckett–Casy model 3: Synthesis of novel naltrexone derivatives with contracted and expanded D-rings and their pharmacology

Novel naltrexone derivatives 7 and 8 with contracted and expanded D-rings were synthesized to investigate the importance of orientation of lone electron pair on the nitrogen for binding abilities to the opioid receptor. Compound 7 showed almost no binding affinity, whereas compound 8 was comparable to naltrexone (6) in binding affinity. Conformational analyses and NOE experiments in D₂O of compounds 6–8 suggested that the lone electron pairs of compounds 6 and 8 with respective six- and seven-membered D-rings would project in the pseudo-axial orientation, whereas compound 7 with five-membered D-ring would have the lone electron pair directing in pseudo-equatorial position. These results strongly supported the proposal that the axial orientation of the lone electron pair on nitrogen would provide sufficient binding abilities to the opioid receptor and that the 15–16 ethylene moiety in the morphine structure would play a role in fixation of the lone electron pair in the axial direction rather than interaction with the putative cavity in the Beckett–Casy model.     

Thorough Investigation of a Canine Autoinflammatory Disease (AID) Confirms One Main Risk Locus and Suggests a Modifier Locus for Amyloidosis

Autoinflammatory disease (AID) manifests from the dysregulation of the innate immune system and is characterised by systemic and persistent inflammation. Clinical heterogeneity leads to patients presenting with one or a spectrum of phenotypic signs, leading to difficult diagnoses in the absence of a clear genetic cause. We used separate genome-wide SNP analyses to investigate five signs of AID (recurrent fever, arthritis, breed specific secondary dermatitis, otitis and systemic reactive amyloidosis) in a canine comparative model, the pure bred Chinese Shar-Pei. Analysis of 255 DNA samples revealed a shared locus on chromosome 13 spanning two peaks of association. A three-marker haplotype based on the most significant SNP (p<2.6×10⁻⁸) from each analysis showed that one haplotypic pair (H13-11) was present in the majority of AID individuals, implicating this as a shared risk factor for all phenotypes. We also noted that a genetic signature (F _ST) distinguishing the phenotypic extremes of the breed specific Chinese Shar-Pei thick and wrinkled skin, flanked the chromosome 13 AID locus; suggesting that breed development and differentiation has played a parallel role in the genetics of breed fitness. Intriguingly, a potential modifier locus for amyloidosis was revealed on chromosome 14, and an investigation of candidate genes from both this and the chromosome 13 regions revealed significant (p<0.05) renal differential expression in four genes previously implicated in kidney or immune health (AOAH, ELMO1, HAS2 and IL6). These results illustrate that phenotypic heterogeneity need not be a reflection of genetic heterogeneity, and that genetic modifiers of disease could be masked if syndromes were not first considered as individual clinical signs and then as a sum of their component parts.     

Paradoxical Interventricular Septal Motion as a Major Determinant of Late Gadolinium Enhancement in Ventricular Insertion Points in Pulmonary Hypertension

Background

This study investigated the major clinical determinants of late gadolinium enhancement (LGE) at ventricular insertion points (VIPs) commonly seen in patients with pulmonary hypertension (PH).

Methods

Forty-six consecutive PH patients (mean pulmonary artery pressure ≥25 mmHg at rest) and 21 matched controls were examined. Right ventricular (RV) morphology, function and LGE mass volume at VIPs were assessed by cardiac magnetic resonance (CMR). Radial motion of the left ventricular (LV) wall and interventricular septum (IVS) was assessed by speckle-tracking echocardiography. Paradoxical IVS motion index was then calculated. Univariate and multivariate regression analysis were conducted to characterize the relationship between LGE volume at VIPs and PH-related clinical indices, including the paradoxical IVS motion index.

Results

Mean pulmonary arterial pressure (MPAP) of PH patients was 38±9 mmHg. LGE at VIPs was observed in 42 of 46 PH patients, and the LGE volume was 2.02 mL (0.47–2.99 mL). Significant correlations with LGE volume at VIPs were observed for MPAP (r = 0.50) and CMR-derived parameters [RV mass index (r = 0.53), RV end-diastolic volume index (r = 0.53), RV ejection fraction (r = −0.56), and paradoxical IVS motion index (r = 0.77)]. In multiple regression analysis, paradoxical IVS motion index alone significantly predicted LGE volume at VIPs (p<0.001).

Conclusions

LGE at VIPs seen in patients with PH appears to reflect altered IVS motion rather than elevated RV pressure or remodeling. Long-term studies would be of benefit to characterize the clinical relevance of LGE at VIPs.     

Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells.     

Neonatal Brain Metabolite Concentrations: An In Vivo Magnetic Resonance Spectroscopy Study with a Clinical MR System at 3 Tesla

Brain metabolite concentrations change dynamically throughout development, especially during early childhood. The purpose of this study was to investigate the brain metabolite concentrations of neonates (postconceptional age (PCA): 30 to 43 weeks) using single-voxel magnetic resonance spectroscopy (MRS) and to discuss the relationships between the changes in the concentrations of such metabolites and brain development during the neonatal period. A total of 83 neonatal subjects were included using the following criteria: the neonates had to be free of radiological abnormalities, organic illness, and neurological symptoms; the MR spectra had to have signal-to-noise ratios ≥ 4; and the estimated metabolite concentrations had to display Cramér-Rao lower bounds of ≤ 30%. MRS data (echo time/repetition time, 30/5000 ms; 3T) were acquired from the basal ganglia (BG), centrum semiovale (CS), and the cerebellum. The concentrations of five metabolites were measured: creatine, choline, N-acetylaspartate, myo-inositol, and glutamate/glutamine complex (Glx). One hundred and eighty-four MR spectra were obtained (83 BG, 77 CS, and 24 cerebellum spectra). Creatine, N-acetylaspartate, and Glx displayed increases in their concentrations with PCA. Choline was not correlated with PCA in any region. As for myo-inositol, its concentration decreased with PCA in the BG, whereas it increased with PCA in the cerebellum. Quantitative brain metabolite concentrations and their changes during the neonatal period were assessed. Although the observed changes were partly similar to those detected in previous reports, our results are with more subjects (n = 83), and higher magnetic field (3T). The metabolite concentrations examined in this study and their changes are clinically useful indices of neonatal brain development.     

AMP‐activated protein kinase counteracts brain‐derived neurotrophic factor‐induced mammalian target of rapamycin complex 1 signaling in neurons

Growth factors and nutrients, such as amino acids and glucose, regulate mammalian target of rapamycin complex 1 (mTORC1) signaling and subsequent translational control in a coordinated manner. Brain‐derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, activates mTORC1 and induces phosphorylation of its target, p70S6 kinase (p70S6K), at Thr389 in neurons. BDNF also increases mammalian target of rapamycin‐dependent novel protein synthesis in neurons. Here, we report that BDNF‐induced p70S6K activation is dependent on glucose, but not amino acids, sufficiency in cultured cortical neurons. AMP‐activated protein kinase (AMPK) is the molecular background to this specific nutrient dependency. Activation of AMPK, which is induced by glucose deprivation, treatment with pharmacological agents such as 2‐Deoxy‐d ‐glucose, metformin, and 5‐aminoimidazole‐4‐carboxamide ribonucleoside or forced expression of a constitutively active AMPKα subunit, counteracts BDNF‐induced phosphorylation of p70S6K and enhanced protein synthesis in cortical neurons. These results indicate that AMPK inhibits the effects of BDNF on mTORC1‐mediated translation in neurons.

     

Direct l-lysine production from cellobiose by Corynebacterium glutamicum displaying beta-glucosidase on its cell surface

We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of l-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l-lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum.     

Molecular epidemiology of Coxsackievirus A16 strains isolated from children in Yamagata,Japan between 1988 and 2011

To clarify the longitudinal molecular epidemiology of coxsackievirus A16, phylogenetic analysis based on the VP1 region of 220 isolates in Yamagata, Japan was performed. The resultant phylogenetic tree indicates that the Yamagata isolates and reference strains can be readily genotyped into three genogroups, and 0, 12 and 208 isolates belonged to the first, second, and third genogroups, respectively. The first genogroup includes only the prototype strain, the second strains that had disappeared by the end of the 20th century and the third comprises those that have been circulating since then in local communities, such as Yamagata.