Characterization of C-4-methylated sterols by pyridine-induced solvent shifts in proton magnetic resonance spectroscopy

The proton magnetic resonance (PMR) spectra were measured in deuterochloroform (CDCl₃) and pyridine solutions for some 4-desmethyl, 4α-methy1, 4β-methyl, and 4,4-dimethyl sterols related to 5α-cholestane series as well as for their C-3-oxo derivatives. The influence of pyridine, relative to CDCl₃, on methyl group chemical shifts was discussed. The technique utilizing pyridine-induced solvent shifts in PMR spectroscopy was found useful in characterizing the individual classes of sterols.     

Plant regeneration via somatic embryogenesis from protoplasts of six plant species related to Citrus

Protoplasts isolated from embryogenic callus of Fortunella polyandra (Ridl.), Atalantia bilocularis (Pieree ex Guill.), Hesperethusa crenulata (Roxb.), Glycosmis pentaphylla (Retz.) Corr., Triphasia trifolia (Burm. f.) P. Wils. and Murraya koenigii (L.) Spreng. were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l^–1 BA and 0.6 M sorbitol. The highest plating efficiencies for all species were obtained on MT basal medium containing 5% sucrose supplemented with 0.001 mg l^–1 BA. F. polyandra produced higher percentages of globular somatic embryo development, while A. bilocularis consistently showed a lower percentage of globular somatic embryo development in all 5 concentrations of BA. MT basal medium containing 5% sucrose and supplemented with 0.001 mg l^–1 BA was found to be a suitable medium for development of globular somatic embryos derived from protoplasts to form heart-shaped somatic embryos with cotyledon-like structures. The highest percentages of shoot formation for all 6 species were obtained using 0.1 mg l^–1 GA₃. A complete protoplast-to-plant system was developed for F. polyandra, A. bilocularis and T. trifolia, which could facilitate the transfer of nuclear and cytoplasmic genes from these species into cultivated Citrus through protoplast fusion.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellin A₃     

Chlorine sensitivity of feline calicivirus, a norovirus surrogate

The sensitivity to free chlorine of feline calicivirus (FCV), a norovirus surrogate, was examined relative to chlorine demand. When a crude suspension of FCV was treated with a sodium hypochlorite solution containing 10 microg/ml free chlorine, the extent of the decrease of viral infectivity clearly depended on the volume of the reaction mixture. The apparent sensitivity of FCV to free chlorine increased with the reduction of host cell debris, indicating that chlorine demand must be minimized to know the true sensitivity of the virus. We therefore partially purified the viruses from the host cell components and found that the infectivity of FCV was reduced by more than log 4.6 by 5 min of treatment with 300 ng/ml free chlorine.     

A set of external reference controls/probes that enable quality assurance between different microarray platforms

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration–response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT–PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT–PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.     

Diminished medial prefrontal activity behind autistic social judgments of incongruent information

Individuals with autism spectrum disorders (ASD) tend to make inadequate social judgments, particularly when the nonverbal and verbal emotional expressions of other people are incongruent. Although previous behavioral studies have suggested that ASD individuals have difficulty in using nonverbal cues when presented with incongruent verbal-nonverbal information, the neural mechanisms underlying this symptom of ASD remain unclear. In the present functional magnetic resonance imaging study, we compared brain activity in 15 non-medicated adult males with high-functioning ASD to that of 17 age-, parental-background-, socioeconomic-, and intelligence-quotient-matched typically-developed (TD) male participants. Brain activity was measured while each participant made friend or foe judgments of realistic movies in which professional actors spoke with conflicting nonverbal facial expressions and voice prosody. We found that the ASD group made significantly less judgments primarily based on the nonverbal information than the TD group, and they exhibited significantly less brain activity in the right inferior frontal gyrus, bilateral anterior insula, anterior cingulate cortex/ventral medial prefrontal cortex (ACC/vmPFC), and dorsal medial prefrontal cortex (dmPFC) than the TD group. Among these five regions, the ACC/vmPFC and dmPFC were most involved in nonverbal-information-biased judgments in the TD group. Furthermore, the degree of decrease of the brain activity in these two brain regions predicted the severity of autistic communication deficits. The findings indicate that diminished activity in the ACC/vmPFC and dmPFC underlies the impaired abilities of individuals with ASD to use nonverbal content when making judgments regarding other people based on incongruent social information.     

Crystallization and amino acid composition of a serine protease from rat skeletal muscle

A serine protease from rat skeletal muscle was crystallized in good yield, and the homogeneity of the preparation was proved by ultracentrifugical analysis and polyacrylamide disc electrophoresis. The S_{20, w} value of the enzyme was 2.2 S and the molecular weight was calculated to be 22,000–24,000 from the results of sedimentation equilibrium analysis. Analysis showed 87% coincidence in the amino acid composition with that of a serine protease from the small intestine. The apparent Km and k_cat(sec^?1) values for N-acetyl-L-tyrosine ethyl ester were 1.1 × 10^?3 M and 9.0, respectively.     

Altered Network Topologies and Hub Organization in Adults with Autism: A Resting-State fMRI Study

Recent functional magnetic resonance imaging (fMRI) studies on autism spectrum condition (ASC) have identified dysfunctions in specific brain networks involved in social and non-social cognition that persist into adulthood. Although increasing numbers of fMRI studies have revealed atypical functional connectivity in the adult ASC brain, such functional alterations at the network level have not yet been fully characterized within the recently developed graph-theoretical framework. Here, we applied a graph-theoretical analysis to resting-state fMRI data acquired from 46 adults with ASC and 46 age- and gender-matched controls, to investigate the topological properties and organization of autistic brain network. Analyses of global metrics revealed that, relative to the controls, participants with ASC exhibited significant decreases in clustering coefficient and characteristic path length, indicating a shift towards randomized organization. Furthermore, analyses of local metrics revealed a significantly altered organization of the hub nodes in ASC, as shown by analyses of hub disruption indices using multiple local metrics and by a loss of “hubness” in several nodes (e.g., the bilateral superior temporal sulcus, right dorsolateral prefrontal cortex, and precuneus) that are critical for social and non-social cognitive functions. In particular, local metrics of the anterior cingulate cortex consistently showed significant negative correlations with the Autism-Spectrum Quotient score. Our results demonstrate altered patterns of global and local topological properties that may underlie impaired social and non-social cognition in ASC.     

Comparison of the quick Gram stain method to the B&M modified and favor methods

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.     

Nicotinamide phosphoribosyltransferase/visfatin does not catalyze nicotinamide mononucleotide formation in blood plasma

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined K(m) values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases K(m) values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of "NAMPT-mediated systemic NAD biosynthesis." Our study would advance current understanding of visfatin physiology.