The discovery of aminopyrazines as novel, potent Na(v)1.7 antagonists: hit-to-lead identification and SAR

Herein the discovery of a novel class of aminoheterocyclic Na(v)1.7 antagonists is reported. Hit compound 1 was potent but suffered from poor pharmacokinetics and selectivity. The compact structure of 1 offered a modular synthetic strategy towards a broad structure-activity relationship analysis. This analysis led to the identification of aminopyrazine 41, which had vastly improved hERG selectivity and pharmacokinetic properties.     

Characterisation of [¹¹C]PR04.MZ in Papio anubis baboon: a selective high-affinity radioligand for quantitative imaging of the dopamine transporter

N-(4-fluorobut-2-yn-1-yl)-2β-carbomethoxy-3β-(4'-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [(11)C]PR04.MZ ([(11)C]-1) has been developed using GMP compliant equipment. An adult female Papio anubis baboon was studied using a test-retest protocol with [(11)C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (f(P)), plasma input functions and metabolic degradation of the radiotracer [(11)C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (V(T)) and non-displaceable binding potentials (BP(ND)) for various brain regions and the blood were obtained from kinetic modelling. [(11)C]-1 shows promising results as a selective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man.     

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-β1 expression

The objective of this study was to investigate hormonal and TGF-β₁ characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n = 12, 301 ± 41.22 g) was significantly higher than that of AI-R (n = 10, 204.8 ± 24.89 g) (p < 0.05). There were significantly lower E2 (p < 0.05) or higher P4 (p < 0.01) and TGF-β₁ (p < 0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-β₁ protein level compared to that of AI-R (p < 0.01). Interestingly, the TGF-β₁ protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.     

MAPK mediates Hsp25 signaling in incisor development

Rodent incisors are continuously growing teeth that include all stages of amelogenesis. Understanding amelogenesis requires investigations of the genes and their gene products control the ameloblast phenotype. One of the mechanisms related to tooth differentiation is mitogen-activated protein kinase (MAPK) signaling. The extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) cascade is associated with mechanisms that control the cell cycle and cell survival. However, the roles of cascades in incisor development remain to be determined. In this study, we investigated incisor development and growth in the mouse based on MAPK signaling. Moreover, heat-shock protein (Hsp)-25 is well known to be a useful marker of odontoblast differentiation. We used anisomycin (a protein-synthesis inhibitor that activates MAPKs) and U0126 (a MAPK inhibitor that blocks ERK1/2 phosphorylation) to examine the role of MAPKs in Hsp25 signaling in the development of the mouse incisor. We performed immunohistochemistry and in vitro culture using incisor tooth germ, and found that phospho-ERK (pERK), pMEK, and Hsp25 localized in developing incisor ameloblasts and anisomycin failed to produce incisor development. In addition, Western blotting results showed that anisomycin stimulated the phosphorylation of ERK, MEK, and Hsp25, and that some of these proteins were blocked by the U0126. These findings suggest that MAPK signals play important roles in incisor formation, differentiation, and development by mediating Hsp25 signaling.     

Microscopic Observation Drug Susceptibility Assay (MODS) for Early Diagnosis of Tuberculosis in Children

MODS is a novel liquid culture based technique that has been shown to be effective and rapid for early diagnosis of tuberculosis (TB). We evaluated the MODS assay for diagnosis of TB in children in Viet Nam. 217 consecutive samples including sputum (n = 132), gastric fluid (n = 50), CSF (n = 32) and pleural fluid (n = 3) collected from 96 children with suspected TB, were tested by smear, MODS and MGIT. When test results were aggregated by patient, the sensitivity and specificity of smear, MGIT and MODS against “clinical diagnosis” (confirmed and probable groups) as the gold standard were 28.2% and 100%, 42.3% and 100%, 39.7% and 94.4%, respectively. The sensitivity of MGIT and MODS was not significantly different in this analysis (P = 0.5), but MGIT was more sensitive than MODS when analysed on the sample level using a marginal model (P = 0.03). The median time to detection of MODS and MGIT were 8 days and 13 days, respectively, and the time to detection was significantly shorter for MODS in samples where both tests were positive (P<0.001). An analysis of time-dependent sensitivity showed that the detection rates were significantly higher for MODS than for MGIT by day 7 or day 14 (P<0.001 and P = 0.04), respectively. MODS is a rapid and sensitive alternative method for the isolation of M.tuberculosis from children.     

Proteomic analysis of responses to osmotic stress in laboratory and sake-brewing strains of Saccharomyces cerevisiae

The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics.     

Conformational study of silk-like peptides containing the calcium-binding sequence from calbindin D9k using 13C CP/MAS NMR spectroscopy

The calcium-binding sites of calbindin D(9k) have a helix-loop-helix motif. In this study, the helix motifs were replaced by several Ala-Gly repeating regions designed on the basis of the primary sequences of several silk fibroins. The synthesized peptides were treated with several organic solvents to modify the secondary structure of the Ala-Gly repeating regions. The local structures of the Ala-Gly repeating regions, as well as the calcium-binding motif, D(9k)-loop (D(9k)L), were determined by (13)C CP/MAS NMR spectroscopy. In the four peptides containing D(9k)L synthesized, the poly(Ala) domains retain the ability to undergo a conformational transition from alpha-helical to beta-sheet in (A)(12)-D(9k)L despite the presence of the D(9k)L domain at the center of the peptide molecule, but the presence of this domain in the other model peptides synthesized has a marked effect on the conformation of the added silk-like domains. The results showed that the structures of the Ala-Gly repeating regions can be controlled by the choice of both the organic solvent and the amino acid sequence of the Ala-Gly repeating regions without disrupting the secondary structure of D(9k)L suggesting that it may retain its ability to bind calcium ions.     

Conformational study of silklike peptides modified by the addition of the calcium-binding sequence from the shell nacreous matrix protein MSI60 using 13C CP/MAS NMR spectroscopy

The calcium-binding site of the pearl oyster (Pinctada fucata) nacreous layer matrix protein MSI60 was introduced between different Ala-Gly repeating regions derived from the primary sequences of several silk fibroins. Several different organic solvents whose effect on the repetitive domains of silk peptides is well-understood were used to modify the secondary structure of the flanking Ala-Gly repeating regions. The local conformations of the flanking Ala-Gly repeating regions as well as the calcium-binding motif, MSI60, were determined by 13C CP/MAS NMR spectroscopy. The secondary structures of the polyalanine, poly(Ala), domains were modified by the solvent treatments in a predictable fashion, suggesting that only the solvent treatment and not the conformation of the MSI60 domain affected the conformation of poly(Ala) regions. Ala-Gly domains behaved differently, taking random coil conformation regardless of the choice of solvent, indicating that their secondary structure is affected by the central MSI60 domain. The conformation of the MSI60 domain is not altered by the solvent treatments, suggesting that it may retain its ability to bind calcium ions. This was confirmed using a calcium-binding assay. The assay further showed that the calcium-binding capability of MSI60 in the synthetic peptides was most effective when the flanking domain was in the beta-sheet structure.     

Microbial contamination of tattoo and permanent makeup inks marketed in the US: a follow-up study

In a 2018 survey, U.S. Food and Drug Administration (FDA) identified microbial contamination in 42 (49%) of 85 unopened tattoo and permanent makeup (PMU) inks purchased from 13 manufacturers in the US between November 2015 and April 2016. To confirm the results of our previous survey, we evaluated the level of microbial contamination in an additional 27 samples from 10 manufacturers from September 2017 to December 2017, including 21 unopened tattoo and PMU inks which were selected based on our previous survey results and 6 ink diluents that were not previously analysed. Aerobic plate count and enrichment culture methods from the FDA’s Bacteriological Analytical Manual revealed 11 (52%) out of 21 inks, from six manufacturers, were contaminated with micro-organisms, with contamination levels up to 3·6 × 10⁸ CFU per gram, consistent with our previous survey results. We identified 25 bacterial strains belonging to nine genera and 19 species. Strains of Bacillus sp. (11 strains, 44%) were dominant, followed by Paenibacillus sp. (5 strains, 20%). Clinically relevant strains, such as Kocuria rhizophila and Oligella ureolytica, were also identified, as similar to the findings in our previous survey. No microbial contamination was detected in any of the six ink diluents.