Demethylation and derepression of genomic retroelements in the skeletal muscles of aged mice

Changes in DNA methylation influence the aging process and contribute to aging phenotypes, but few studies have been conducted on DNA methylation changes in conjunction with skeletal muscle aging. We explored the DNA methylation changes in a variety of retroelement families throughout aging (at 2, 20, and 28 months of age) in murine skeletal muscles by methyl‐binding domain sequencing (MBD‐seq). The two following contrasting patterns were observed among the members of each repeat family in superaged mice: (a) hypermethylation in weakly methylated retroelement copies and (b) hypomethylation in copies with relatively stronger methylation levels, representing a pattern of “regression toward the mean” within a single retroelement family. Interestingly, these patterns depended on the sizes of the copies. While the majority of the elements showed a slight increase in methylation, the larger copies (>5 kb) displayed evident demethylation. All these changes were not observed in T cells. RNA sequencing revealed a global derepression of retroelements during the late phase of aging (between 20 and 28 months of age), which temporally coincided with retroelement demethylation. Following this methylation drift trend of “regression toward the mean,” aging tended to progressively lose the preexisting methylation differences and local patterns in the genomic regions that had been elaborately established during the early period of development.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the Japanese crayfish, Cambaroides japonicus, were fixed in buffered (pH 7.4) 4% formaldehyde followed by buffered (pH 7.4) 1% osmium tetroxide, and thin sections of the epoxy Epon resin-embedded tissue were studied with the electron microscope. Spermatozoa from vasa deferentia and spermatids from the testis were examined in smear preparations and thick sections by an ordinary light microscope, employing the Feulgen nuclear technique, fast green or periodic acid-Schiff reagent. On the other hand, testes fixed with buffered (pH 7.4) 4% formaldehyde were incubated in Novikoff and Goldfischer's medium or in Mölbert and coworkers' mixture for demonstrating thiamine pyrophosphatase (TPPase) or alkaline phosphatase, and observed in the electron microscope.The microtubules 220 Å to 310 Å in diameter appearing in the nuclear process seem to represent some unit structure of chromosomes in this species. The microtubules are composed of the tubular subunits which are disposed twisted along the peripheral part of major axis of the microtubules. Such tubular subunits are approximately 20 Å thick in wall and 10 Å wide in lumen. The acrosome in a helmet-like shape has been found to have a hornlike process at its proximal part, though the function of such process remains unsettled in the present study. With incubation in disodiumphenylphosphate, no final product is deposited in any part of the premature spermatozoa. The convoluted membrane as well as the invagination of nuclear envelope are revealed to be specific sites for TPPase activity, and such finding suggests that TPPase may act as an intermediary in formation of nuclear processes of the crayfish sperm.This study was supported by Grant GM-8327-05 from the United States Public Health Service.Scientist from the Laboratory of Electron Microscopy, Department of Biology, Kyung Pook National University, Taegu, Korea.     

Recognition and discrimination of target mRNAs by Sib RNAs,a cis-encoded sRNA family

Five Sib antitoxin RNAs, members of a family of cis-encoded small regulatory RNAs (sRNAs) in Escherichia coli, repress their target mRNAs, which encode Ibs toxins. This target repression occurs only between cognate sRNA–mRNA pairs with an exception of ibsA. We performed co-transformation assays to assess the ability of SibC derivatives to repress ibsC expression, thereby revealing the regions of SibC that are essential for ibsC mRNA recognition. SibC has two target recognition domains, TRD1 and TRD2, which function independently. The target site for TRD1 is located within the ORF of ibsC, whereas the target site for TRD2 is located in the translational initiation region. The TRD1 sequence is sufficient to repress ibsC expression. In contrast, TRD2 requires a specific structure in addition to the recognition sequence. An in vitro structural probing analysis showed that the initial interactions at these two recognition sites allowed base-pairing to progress into the flanking sequences. Displacement of the TRD1 and TRD2 domains of SibC by the corresponding domains of SibD changed the target specificity of SibC from ibsC to ibsD, suggesting that these two elements modulate the cognate target recognition of each Sib RNA by discriminating among non-cognate ibs mRNAs.     

Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon

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Expression, Purification, and Characterization of a Dentin Phosphoprotein Produced by Escherichia coli, and Its Odontoblastic Differentiation Effects on Human Dental Pulp Cells

To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p?<?0.05). Cell viability was also significantly increased by rhDPP at 5?days (p?<?0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.     

Mycobacterium massiliense is differentiated from Mycobacterium abscessus and Mycobacterium bolletii by erythromycin ribosome methyltransferase gene (erm) and clarithromycin susceptibility patterns

Erythromycin ribosome methyltransferase gene (erm) sequences of Mycobacterium massiliense and Mycobacterium bolletii isolates were newly investigated. Forty nine strains of M. massiliense that were analyzed in the present study had a deleted erm(41). Due to a frame‐shift mutation, large deletion, and truncated C‐terminal region, the Erm(41) of M. massiliense had only 81 amino acids encoded by 246 nucleotides. Corresponding to these findings, most of the M. massiliense isolates (89.8%) were markedly clarithromycin susceptible, but resistant strains invariably had a point mutation at the adenine (A₂₀₅₈ or A₂₀₅₉) in the peptidyltransferase region of the 23S rRNA gene, which is quite different from Mycobacterium abscessus and M. bolletii. In addition, erm(41) sequences of M. massiliense were more conserved than those of M. abscessus and M. bolletii. The results of species identification using erm(41) showed concordant results with those of multi‐locus sequence analysis (rpoB, hsp65, sodA and 16S‐23S ITS) where there were originally inconsistent results between rpoB and hsp65 sequence analysis in previous research. Therefore, erm(41) PCR that was used in the present study can be efficiently used to simply differentiate M. massiliense from M. abscessus and M. bolletii.     

Flow Visualization and Performance Measurements of a Flagellar Propeller

A new type of propeller that is optimized for low Reynolds numbers is required to propel a small object in a medium where the flow is dominated by viscous rather than inertial forces.A propeller in the shape of a bacterial flagellum seems an appropriate choice for driving a small object.Accordingly,in this study,we visualized the velocity field induced by a spring-like propeller inspired by the Escherichia coli flagellum,using a macroscopic model and applying stereoscopic particle image velocimetry.We also experimentally evaluated the effect of pitch and rotational speed on the performance of this flagellar propeller.Silicone oil,which has a kinematic viscosity 100,000 times that of water,was used as the working fluid to generate a low Reynolds number for the macroscopic model.Thrust,torque,and velocity were measured as functions of pitch and rotational speed,and the efficiency of the propeller was calculated from the measured results.We found that the flagellar propeller reached a maximum efficiency when the pitch angle was approximately 53°.Compared to pitch,rotational speed had a relatively small effect on the efficiency,and the pitch altered the flow pattern behind the rotating propeller.