BDNF Reduces eEF2 Phosphorylation and Enhances Novel Protein Synthesis in the Growth Cones of Dorsal Root Ganglia Neurons.

The local translation, which is regulated by extracellular stimuli such as guidance molecules, in growth cones of neurons provides a molecular mechanism for axonal development. In this study, we performed immunocytochemistry together with atomic force microscopy to investigate the localization of ribosomal proteins in the growth cones of rat dorsal root ganglion (DRG) neurons. The immunoreactivity of ribosomal protein P0/1/2 and S6, and novel protein synthesis were observed in the central, sterically bulky region of growth cones. Brain derived neurotrophic factor (BDNF) reduced the eEF2 phosphorylation, indicating its activation, and enhanced protein synthesis within 30 min. The effects of BDNF were completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). These results indicated that BDNF rapidly activates translation and enhances novel protein synthesis in growth cones of DRG though the mTOR signaling.

Dating igneous rocks using the Potassium-Argon Laser Experiment (KArLE) instrument: A case study for ~380 Ma basaltic rocks.

RATIONALE: We report new K-Ar isochron data for two ~380 Ma basaltic rocks, using an updated version of the Potassium-Argon Laser Experiment (KArLE), which is being developed for future in situ dating of planetary materials. These basalts have K contents comparable with those of lunar KREEP basalts or igneous lithologies found by Mars rovers, whereas previous proof-of-concept studies focused primarily on more K-rich rocks. We aim to measure these analogous samples to show the advancing capability of in situ K-Ar geochronology. METHODS: Combining laser-induced breakdown spectroscopy (LIBS), mass spectrometry (MS), and microscopic analyses, we measured the abundance of K and 40 Ar from 23 spots on the basalt samples. We then constructed K-Ar isochron plots from these rocks. The breadboard instrument consists of flight-equivalent devices including a 30-mJ Nd:YAG laser and a quadrupole mass spectrometer. RESULTS: Despite much lower K abundances than in previous studies, the isochron slopes yielded 380 ± 44 Ma and 398 ± 50 Ma for 380.7-Ma and 373.5-Ma rocks, respectively, indicating that accuracy better than 25 Ma (<7%) is achievable with our instrument. The isochron intercepts both yielded trapped 40 Ar approximately 1 × 10-6  cm3 STP/g. CONCLUSIONS: Our experimental results demonstrate that accurate and precise measurements are possible using the KArLE approach on basaltic rocks, which are ubiquitous on planetary surfaces, and are useful in addressing a wide range of questions in planetary science.

Preferential isolation of Megasphaera elsdenii from pig feces.

Lactic acid produced by intestinal bacteria is fermented by lactate-utilizing bacteria. In this study, we developed a selective culture medium (KMI medium) for Megasphaera elsdenii, a lactate-utilizing bacterium that is abundant in pig intestines. Supplementation of the medium with lactate and beef extract powder was necessary for the preferential growth of M. elsdenii. In addition, we designed a species-specific primer set to detect M. elsdenii. When pig fecal samples were plated on KMI agar medium, approximately 60-100% of the resulting colonies tested positive using the M. elsdenii-specific PCR primers. In fact, nearly all of the large, yellow-white colonies that grew on the KMI agar medium tested positive by PCR with this primer set. The 16S rRNA gene sequences of three representative PCR-positive strains showed strong similarities to that of M. elsdenii ATCC 25940T (98.9-99.2% identity). These three strains were approximately 1.5 µm sized cocci that were primarily arranged in pairs, as was observed for M. elsdenii JCM 1772T. The selective KMI medium and species-specific primer set developed in this study are useful for the isolation and detection of M. elsdenii and will be useful in research aimed at increasing our understanding of intestinal short-chain fatty acid metabolism in pigs.

Spectrally blue hydrated parent body of asteroid (162173) Ryugu.

Ryugu is a carbonaceous rubble-pile asteroid visited by the Hayabusa2 spacecraft. Small rubble pile asteroids record the thermal evolution of their much larger parent bodies. However, recent space weathering and/or solar heating create ambiguities between the uppermost layer observable by remote-sensing and the pristine material from the parent body. Hayabusa2 remote-sensing observations find that on the asteroid (162173) Ryugu both north and south pole regions preserve the material least processed by space weathering, which is spectrally blue carbonaceous chondritic material with a 0-3% deep 0.7-µm band absorption, indicative of Fe-bearing phyllosilicates. Here we report that spectrally blue Ryugu's parent body experienced intensive aqueous alteration and subsequent thermal metamorphism at 570-670 K (300-400 °C), suggesting that Ryugu's parent body was heated by radioactive decay of short-lived radionuclides possibly because of its early formation 2-2.5 Ma. The samples being brought to Earth by Hayabusa2 will give us our first insights into this epoch in solar system history.

In vitro Neo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System.

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

In Situ Geochronology on Mars and the Development of Future Instrumentation.

We review the in situ geochronology experiments conducted by the Mars Science Laboratory mission's Curiosity rover to understand when the Gale Crater rocks formed, underwent alteration, and became exposed to cosmogenic radiation. These experiments determined that the detrital minerals in the sedimentary rocks of Gale are ∼4 Ga, consistent with their origin in the basalts surrounding the crater. The sedimentary rocks underwent fluid-moderated alteration 2 Gyr later, which may mark the closure of aqueous activity at Gale Crater. Over the past several million years, wind-driven processes have dominated, denuding the surfaces by scarp retreat. The Curiosity measurements validate radiometric dating techniques on Mars and guide the way for future instrumentation to make more precise measurements that will further our understanding of the geological and astrobiological history of the planet.

The quantitative analysis of the human papillomavirus DNA load in submandibular gland lesions with droplet digital polymerase chain reaction.

BACKGROUND: The relationship between infection with human papillomavirus (HPV) and tumorigenesis of salivary gland remains controversial. OBJECTIVES: This study explored the relationship between HPV and salivary gland lesions as well as that of the HPV infection status and p16INK4A immunoreactivity. The HPV DNA loads were also quantitatively evaluated. MATERIALS AND METHODS: Tissue samples from 31 submandibular gland lesions were evaluated. p16INK4A immunohistochemical (IHC) staining, nested polymerase chain reaction (PCR), DNA sequencing, and droplet digital PCR (ddPCR) were performed. RESULTS: Non-neoplastic lesion, benign tumors, and malignant tumors were noted in 9, 16, 6 cases, respectively. p16INK4A immunoreactivity was higher in malignant tumors than in benign tumors (50.0% vs. 6.3%). Single PCR with MY09/11 found that all samples were negative. Nevertheless, nested PCR revealed a high HPV-DNA positivity rate of 96.8%. No relationship between the HPV status and p16INK4A immunoreactivity was shown. HPV-18 was the only subtype identified in this study. ddPCR showed significantly lower HPV-18 DNA loads in submandibular gland lesions than in oropharyngeal cancers. CONCLUSIONS: HPV-DNA positivity and p16INK4A-immunoreactivity were not correlated in submandibular gland lesions. The loads of HPV DNA detected in this study were small. HPV positivity therefore may not be associated with tumorigenesis of the submandibular gland.

Morphological analysis of the external anal sphincter motor nerve and its motoneurons in the cat.

To investigate the spinal neural circuitry that controls the tonus of the external anal sphincter (EAS) in the cat, the size distribution of EAS motor fibers and their motoneurons (MN) was examined, and the presence of muscle spindles in EAS musculature was also tested for. The size distribution of EAS motor fibers was examined after degeneration of afferent fibers and that of their MN was measured, after labeling the cells with horseradish peroxidase. Both distributions were unimodal, thereby demonstrating the difficulty of distinguishing between alpha and potential gamma MN; but muscle spindles were not found in the musculature. Mechanisms underlying the spinally controlled tonus of the EAS remain unclear, including the nature and role of spinal reflexes. It is argued that non-spindle sensory receptors in the anal canal may provide the sensory component of a reflex circuit that contributes to this tonus.

[The effect of air massage on serum constituents].

This study investigated the effect of air massage of the legs on serum constituents. Five volunteers (aged 48.8+/-12.98 years, n=5) served as subjects. The study consisted of three periods as follows: control period for one month(no-mas), massage period receiving the massage for 15 minutes per day for one month(mas), and another massage period receiving the massage for 15 minutes per day along with 40% oxygen uptake for one month (O2-mas). Venous blood samples were drawn every week during the study periods. Serum constituents were examined using an auto analyzer(Hitachi Ltd., JEOL Ltd.). Albumin concentration significantly increased under the O2-mas condition, whereas free-cholesterol concentration was significantly decreased under the same condition. In addition, significant increases in concentrations of creatinine and calcium were seen in men under the O2-mas condition. Furthermore, there was a significant decrease in the activity of alkaline phosphatase in women under both the mas and O2-mas conditions. These findings suggested that massage has positive effects on nutritional recovery and metabolism of lipids, bone and muscle. Thus, it may be argued that massage could potentially be effective as a form of exercise.

[Use of a computer-assisted simulation system in undergraduate education of laboratory technologists].

In the undergraduate education of laboratory technologists, practical training courses in a hospital are extremely important to gain knowledge, skills and attitudes. In present laboratories, however, senior technologists have less time for teaching their juniors because of increased demands of hospital tasks. To supplement practical training of students, we therefore decided to employ clinical simulation teaching materials using a computer assisted education system. First, a series of e learning coursewares on laboratory tests for blood transfusion were created using pictures taken during our daily work. The pictures were arranged with questions, answers and commentaries, uploaded to the server in our university, and offered to the students. They were received with high rates of student satisfaction. Since e-learning seemed to be an effective and enjoyable strategy to achieve good learning outcomes, it is desirable to establish an organization to collect peer reviewed high quality e learning materials that can be shared with nationwide educators teaching tomorrow's laboratory technologists.

Quantitative Potassium Measurements with Laser-Induced Breakdown Spectroscopy Using Low-Energy Lasers: Application to In Situ K-Ar Geochronology for Planetary Exploration.

In situ radiogenic isotope measurements to obtain the absolute age of geologic events on planets are of great scientific value. In particular, K-Ar isochrons are useful because of their relatively high technical readiness and high accuracy. Because this isochron method involves spot-by-spot K measurements using laser-induced breakdown spectroscopy (LIBS) and simultaneous Ar measurements with mass spectrometry, LIBS measurements are conducted under a high vacuum condition in which emission intensity decreases significantly. Furthermore, using a laser power used in previous planetary missions is preferable to examine the technical feasibility of this approach. However, there have been few LIBS measurements for K under such conditions. In this study, we measured K contents in rock samples using 30 mJ and 15 mJ energy lasers under a vacuum condition (10-3 Pa) to assess the feasibility of in situ K-Ar dating with lasers comparable to those used in NASA's Curiosity and Mars 2020 missions. We obtained various calibration curves for K using internal normalization with the oxygen line at 777 nm and continuum emission from the laser-induced plasma. Experimental results indicate that when K2O < 1.1 wt%, a calibration curve using the intensity of the K emission line at 769 nm normalized with that of the oxygen line yields the best results for the 30 mJ laser energy, with a detection limit of 88 ppm and 20% of error at 2400 ppm of K2O. Futhermore, the calibration curve based on the K 769 nm line intensity normalized with continuum emission yielded the best result for the 15 mJ laser, giving a detection limit of 140 ppm and 20% error at 3400 ppm K2O. Error assessments using obtained calibration models indicate that a 4 Ga rock with 3000 ppm K2O would be measured with 8% (30 mJ) and 10% (15 mJ) of precision in age when combined with mass spectrometry of 40Ar with 10% of uncertainty. These results strongly suggest that high precision in situ isochron K-Ar dating is feasible with a laser used in previous and upcoming Mars rover missions.

The expression and localization of RNase and RNase inhibitor in blood cells and vascular endothelial cells in homeostasis of the vascular system.

RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may accelerate pathologic thrombus formation.

Ultrasonic anisotropy measured in 2-dimensional echocardiograms in vitro and verified by histology.

This study aims to validate ultrasonic anisotropy in 2-dimensional echocardiograms by one-to-one histological correlation. Thirteen echograms were obtained in vitro from 9 specimens of 2 left ventricles of healthy adult beagle dogs and were correlated with corresponding histology in regard to ultrasound orientation relative to the local fiber direction. Median value of pixel echogenicity (255-rank) was lower in areas with parallel (n = 9, 18 ± 13) than in those with oblique (n = 7, 41 ± 14, P < 0.05) and perpendicular (n = 13, 57 ± 21, P < 0.01) ultrasound orientations. Echogenicity in the proximal part within 0.5 cm from an edge was relatively high (n = 9, 33 ± 15; n = 5, 66 ± 27; n = 8, 65 ± 32, respectively) and was different between parallel and perpendicular orientations (P < 0.05). The ratio of the distal adjacent 0.5-cm part to the proximal part echogenicity was lower for either parallel or oblique orientation (0.35 ± 0.25, 0.51 ± 0.16) than for perpendicular orientation (0.95 ± 0.16, P < 0.01, respectively). Acoustic dropouts appeared beyond myocardial areas with parallel or oblique ultrasound orientation. These results disclosed precise acoustic anisotropy in the 2-dimensional echocardiograms.

Development of a new measurement method for serum calcium with chlorophosphonazo-III.

BACKGROUND: Although serum calcium has been measured using the o-cresolphthalein complexone (oCPC) method in the clinical laboratory, this method still has some problems regarding linearity and reagent stability. We developed a new measurement procedure using chlorophosphonazo-III (CPZ-III: 2,7-bis (4-chloro-2-phosphonophenylazo) -1,8- dihydroxy-3, 6-naphthalenedisulphonic acid, disodium salt) as a chelator with an acid medium for serum calcium measurement. The present method showed better linearity and reagent stability compared with the oCPC method. METHODS: Characteristics were studied in optimized conditions measuring wavelength by absorption spectra analysis, and interference of protein and metals with Mg(2+), Fe(2+), Cu(2+) and Zn(2+). The method was applied to an automated analyser (7170; Hitachi High Technologies Corp). The measurement performance was evaluated for accuracy, precision, recovery rate, linearity and reagent stability with a comparison study against atomic absorption spectrophotometry (AAS). RESULTS: The within-run and between-run variations (coefficient of variation [CV]) were 0.92-1.01% and 0.75-1.43%, respectively. The linearity was 0-7.0 mmol/L. The comparison study obtained y = 1.002x (AAS) - 0.10, Sy/x = 0.18 mmol/L, n = 50. Reagent stability was at least 20 d at 4 degrees C without daily calibration. CONCLUSION: The new calcium measurement method in serum was demonstrated to have reliable and acceptable performances as a routine test in clinical laboratory.