Expanding our Understanding of Sequence-Function Relationships of Type II Polyketide Biosynthetic Gene Clusters: Bioinformatics-Guided Identification of Frankiamicin A from Frankia sp. EAN1pec

A large and rapidly increasing number of unstudied “orphan” natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics. Here, we conduct global bioinformatic analysis of all available type II polyketide gene cluster sequences and identify a conserved set of gene clusters with unique ketosynthase α/β sequence characteristics in the genomes of Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia sp. EAN1pec as producing a compound with spectral characteristics consistent with the type II polyketide produced by this gene cluster. We isolated the compound, a pentangular polyketide which we named frankiamicin A, and elucidated its structure by NMR and labeled precursor feeding. We also propose biosynthetic and regulatory pathways for frankiamicin A based on comparative genomic analysis and literature precedent, and conduct bioactivity assays of the compound. Our findings provide new information linking this set of Frankia gene clusters with the compound they produce, and our approach has implications for accurate functional prediction of the many other type II polyketide clusters present in bacterial genomes.     

Cinnamtannin B-1 Promotes Migration of Mesenchymal Stem Cells and Accelerates Wound Healing in Mice

Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of Mallotus philippinensis bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with in vitro migratory activity against mesenchymal stem cells. However, the in vivo effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on in vivo migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration in vivo and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on mesenchymal stem cell migration.     

Crizotinib-Induced Abnormal Signal Processing in the Retina

Molecular target therapy for cancer is characterized by unique adverse effects that are not usually observed with cytotoxic chemotherapy. For example, the anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor crizotinib causes characteristic visual disturbances, whereas such effects are rare when another ALK-tyrosine kinase inhibitor, alectinib, is used. To elucidate the mechanism responsible for these visual disturbances, the responses to light exhibited by retinal ganglion cells treated with these agents were evaluated using a C57BL6 mouse ex vivo model. Both crizotinib and alectinib changed the firing rate of ON and OFF type retinal ganglion cells. However, the ratio of alectinib-affected cells (15.7%) was significantly lower than that of crizotinib-affected cells (38.6%). Furthermore, these drugs changed the response properties to light stimuli of retinal ganglion cells in some of the affected cells, i.e., OFF cells responded to both ON and OFF stimuli, etc. Finally, the expressions of ALK (a target receptor of both crizotinib and alectinib) and of MET and ROS1 (additional target receptors of crizotinib) were observed at the mRNA level in the retina. Our findings suggest that these drugs might target retinal ganglion cells and that the potency of the drug actions on the light responses of retinal ganglion cells might be responsible for the difference in the frequencies of visual disturbances observed between patients treated with crizotinib and those treated with alectinib. The present experimental system might be useful for screening new molecular target agents prior to their use in clinical trials.     

ABO Blood Type and Personality Traits in Healthy Japanese Subjects

There is no scientific consensus that a relationship exists between the ABO blood group and personality traits. However, a recent study hypothesized that the dopamine beta-hydroxylase (DBH) gene is in linkage with the ABO gene. The sample population consisted of 1,427 healthy Japanese subjects who completed the Temperament and Character Inventory (TCI). Each subject’s ABO blood type was determined by genotyping the rs8176719 and rs8176746 ABO gene single-nucleotide polymorphisms (SNPs) using a TaqMan genotyping assay. The relationships between the six ABO genotypes or four ABO phenotypes and personality traits were examined using a multivariate analysis of covariance (MANCOVA), controlling for age and sex. The MANCOVA data showed a significant difference in TCI scores among the ABO genotype groups (F [7, 1393] = 3.354, p = 0.001). A subsequent univariate analysis showed a significant difference in the mean scores for Persistence among the genotype groups (F = 2.680, partial η² = 0.010, p = 0.020). Similarly, dividing the ABO blood type into four phenotypes revealed a significant difference among the phenotype groups (F [7, 1397] = 2.529, p = 0.014). A subsequent univariate analysis showed a significant difference among the phenotype groups in the mean scores for Persistence (F = 2.952, partial η²= 0.006, p = 0.032). We observed a significant association between ABO blood group genotypes and personality traits in a large number of healthy Japanese subjects. However, these results should be regarded as preliminary and should be interpreted with caution because it is possible that the association between ABO blood group genotype and the Persistence trait is relatively weak.     

Gap Effect Abnormalities during a Visually Guided Pro-Saccade Task in Children with Attention Deficit Hyperactivity Disorder

Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder that starts in early childhood and has a comprehensive impact on psychosocial activity and education as well as general health across the lifespan. Despite its prevalence, the current diagnostic criteria for ADHD are debated. Saccadic eye movements are easy to quantify and may be a quantitative biomarker for a wide variety of neurological and psychiatric disorders, including ADHD. The goal of this study was to examine whether children with ADHD exhibit abnormalities during a visually guided pro-saccadic eye-movement and to clarify the neurophysiological mechanisms associated with their behavioral impairments. Thirty-seven children with ADHD (aged 5–11 years) and 88 typically developing (TD) children (aged 5–11 years) were asked to perform a simple saccadic eye-movement task in which step and gap conditions were randomly interleaved. We evaluated the gap effect, which is the difference in the reaction time between the two conditions. Children with ADHD had a significantly longer reaction time than TD children (p < 0.01) and the gap effect was markedly attenuated (p < 0.01). These results suggest that the measurement of saccadic eye movements may provide a novel method for evaluating the behavioral symptoms and clinical features of ADHD, and that the gap effect is a potential biomarker for the diagnosis of ADHD in early childhood.     

Tumor-Targeting Salmonella typhimurium A1-R Arrests a Chemo-Resistant Patient Soft-Tissue Sarcoma in Nude Mice

A patient-derived nude-mouse model of soft-tissue sarcoma has been established and treated in the following groups: (1) untreated controls; (2) gemcitabine (GEM) (80 mg/kg, ip, weekly, 3 weeks); (3) Pazopanib (100 mg/kg, orally, daily, 3 weeks) and (4) Salmonella typhimurium A1-R (5 × 10⁷ CFU/body, ip, weekly, 3 weeks). The sarcoma was resistant to GEM (p = 0.879). Pazopanib tended to reduce the tumor volume compared to the untreated mice, but there was no significant difference (p = 0.115). S. typhimurium A1-R significantly inhibited tumor growth compared to the untreated mice (p = 0.001). S. typhimurium A1-R was the only effective treatment for the soft-tissue sarcoma nude mouse model among all treatments including a newly approved multiple tyrosine kinase inhibitor; Pazopanib. These results suggest tumor-targeting S. typhimurium A1-R is a promising treatment for chemo-resistant soft-tissue sarcoma.     

Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5′ stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31.     

Noninvasive cross‐sectional imaging of proximal caries using swept‐source optical coherence tomography (SS‐OCT) in vivo

The aim of this study was to determine the diagnostic accuracy of swept‐source optical coherent tomography (SS‐OCT) in detecting and estimating the depth of proximal caries in posterior teeth in vivo. SS‐OCT images and bitewing radiographs were obtained from 86 proximal surfaces of 53 patients. Six examiners scored the locations according to a caries lesion depth scale (0–4) using SS‐OCT and the radiographs. The results were compared with clinical observations obtained after the treatment. SS‐OCT could detect the presence of proximal caries in tomograms that were synthesized based on the backscatter signal obtained from the proximal carious lesion through occlusal enamel. SS‐OCT showed significantly higher sensitivity and larger area under the receiver operating characteristic curve than radiographs for the detection of cavitated enamel lesions and dentin caries (Student's t ‐test, p < 0.05). SS‐OCT appears to be a more reliable and accurate method than bitewing radiographs for the detection and estimation of the depth of proximal lesions in the clinical environment. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Backbone 1H, 13C,and 15N assignments of yeast Ump1, an intrinsically disordered protein that functions as a proteasome assembly chaperone

Eukaryotic proteasome assembly is a highly organized process mediated by several proteasome-specific chaperones, which interact with proteasome assembly intermediates. In yeast, Ump1 and Pba1-4 have been identified as assembly chaperones that are dedicated to the formation of the proteasome 20S catalytic core complex. The crystal structures of Pba chaperones have been reported previously, but no detailed information has been provided for the structure of Ump1. Thus, to better understand the mechanisms underlying Ump1-mediated proteasome assembly, we characterized the conformation of Ump1 in solution using NMR. Backbone chemical shift data indicated that Ump1 is an intrinsically unstructured protein and largely devoid of secondary structural elements.