Complete Mitochondrial Genomes of Carcinoscorpius rotundicauda and Tachypleus tridentatus (Xiphosura,Arthropoda) and Implications for Chelicerate Phylogenetic Studies

Horseshoe crabs (order Xiphosura) are often referred to as an ancient order of marine chelicerates and have been considered as keystone taxa for the understanding of chelicerate evolution. However, the mitochondrial genome of this order is only available from a single species, Limulus polyphemus. In the present study, we analyzed the complete mitochondrial genomes from two Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus tridentatus to offer novel data for the evolutionary relationship within Xiphosura and their position in the chelicerate phylogeny. The mitochondrial genomes of C. rotundicauda (15,033 bp) and T. tridentatus (15,006 bp) encode 13 protein-coding genes, two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. Overall sequences and genome structure of two Asian species were highly similar to that of Limulus polyphemus, though clear differences among three were found in the stem-loop structure of the putative control region. In the phylogenetic analysis with complete mitochondrial genomes of 43 chelicerate species, C. rotundicauda and T. tridentatus were recovered as a monophyly, while L. polyphemus solely formed an independent clade. Xiphosuran species were placed at the basal root of the tree, and major other chelicerate taxa were clustered in a single monophyly, clearly confirming that horseshoe crabs composed an ancestral taxon among chelicerates. By contrast, the phylogenetic tree without the information of Asian horseshoe crabs did not support monophyletic clustering of other chelicerates. In conclusion, our analyses may provide more robust and reliable perspective on the study of evolutionary history for chelicerates than earlier analyses with a single Atlantic species.     

Poly-lysine peptidomimetics having potent antimicrobial activity without hemolytic activity

Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure–activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.     

Protein NMR Structures Refined without NOE Data

The refinement of low-quality structures is an important challenge in protein structure prediction. Many studies have been conducted on protein structure refinement; the refinement of structures derived from NMR spectroscopy has been especially intensively studied. In this study, we generated flat-bottom distance potential instead of NOE data because NOE data have ambiguity and uncertainty. The potential was derived from distance information from given structures and prevented structural dislocation during the refinement process. A simulated annealing protocol was used to minimize the potential energy of the structure. The protocol was tested on 134 NMR structures in the Protein Data Bank (PDB) that also have X-ray structures. Among them, 50 structures were used as a training set to find the optimal “width” parameter in the flat-bottom distance potential functions. In the validation set (the other 84 structures), most of the 12 quality assessment scores of the refined structures were significantly improved (total score increased from 1.215 to 2.044). Moreover, the secondary structure similarity of the refined structure was improved over that of the original structure. Finally, we demonstrate that the combination of two energy potentials, statistical torsion angle potential (STAP) and the flat-bottom distance potential, can drive the refinement of NMR structures.     

Mg2+ Effect on Argonaute and RNA Duplex by Molecular Dynamics and Bioinformatics Implications

RNA interference (RNAi), mediated by small non-coding RNAs (e.g., miRNAs, siRNAs), influences diverse cellular functions. Highly complementary miRNA-target RNA (or siRNA-target RNA) duplexes are recognized by an Argonaute family protein (Ago2), and recent observations indicate that the concentration of Mg²⁺ ions influences miRNA targeting of specific mRNAs, thereby modulating miRNA-mRNA networks. In the present report, we studied the thermodynamic effects of differential [Mg²⁺] on slicing (RNA silencing cycle) through molecular dynamics simulation analysis, and its subsequent statistical analysis. Those analyses revealed different structural conformations of the RNA duplex in Ago2, depending on Mg²⁺ concentration. We also demonstrate that cation effects on Ago2 structural flexibility are critical to its catalytic/functional activity, with low [Mg²⁺] favoring greater Ago2 flexibility (e.g., greater entropy) and less miRNA/mRNA duplex stability, thus favoring slicing. The latter finding was supported by a negative correlation between expression of an Mg²⁺ influx channel, TRPM7, and one miRNA’s (miR-378) ability to downregulate its mRNA target, TMEM245. These results imply that thermodynamics could be applied to siRNA-based therapeutic strategies, using highly complementary binding targets, because Ago2 is also involved in RNAi slicing by exogenous siRNAs. However, the efficacy of a siRNA-based approach will differ, to some extent, based on the Mg²⁺ concentration even within the same disease type; therefore, different siRNA-based approaches might be considered for patient-to-patient needs.     

Xanthene Derivatives Increase Glucose Utilization through Activation of LKB1-Dependent AMP-Activated Protein Kinase

5′ AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.     

Glioma: Application of Whole-Tumor Texture Analysis of Diffusion-Weighted Imaging for the Evaluation of Tumor Heterogeneity

Background and Purpose

To apply a texture analysis of apparent diffusion coefficient (ADC) maps to evaluate glioma heterogeneity, which was correlated with tumor grade.

Materials and Methods

Forty patients with glioma (WHO grade II (n = 8), grade III (n = 10) and grade IV (n = 22)) underwent diffusion-weighted imaging (DWI), and the corresponding ADC maps were obtained. Regions of interest containing the lesions were drawn on every section of the ADC map containing the tumor, and volume-based data of the entire tumor were constructed. Texture and first order features including entropy, skewness and kurtosis were derived from the ADC map using in-house software. A histogram analysis of the ADC map was also performed. The texture and histogram parameters were compared between low-grade and high-grade gliomas using an unpaired student’s t-test. Additionally, a one-way analysis of variance analysis with a post-hoc test was performed to compare the parameters of each grade.

Results

Entropy was observed to be significantly higher in high-grade gliomas than low-grade tumors (6.861±0.539 vs. 6.261±0.412, P  = 0.006). The fifth percentiles of the ADC cumulative histogram also showed a significant difference between high and low grade gliomas (836±235 vs. 1030±185, P = 0.037). Only entropy proved to be significantly different between grades III and IV (6.295±0.4963 vs. 7.119±0.3165, P<0.001). The diagnostic accuracy of ADC entropy was significantly higher than that of the fifth percentile of the ADC histogram (P = 0.0034) in distinguishing high- from low-grade glioma.

Conclusion

A texture analysis of the ADC map based on the entire tumor volume can be useful for evaluating glioma grade, which provides tumor heterogeneity.     

Increased Frequency of Aneuploidy in Long-Lived Spermatozoa

Aneuploidy commonly causes spontaneous abortions, stillbirths, and aneuploid births in humans. Notably, the majority of sex chromosome aneuploidies in live births have a paternal origin. An increased frequency of aneuploidy is also associated with male infertility. However, the dynamics and behavior of aneuploid spermatozoa during fertilization in humans have not been studied in detail. Therefore, we compared the frequency of aneuploidy and euploidy in live spermatozoa from normozoospermic men over a 3-day period. To assess the dynamics and behavior of aneuploid spermatozoa, we simultaneously evaluated sperm viability using the hypo-osmotic swelling test and sperm aneuploidy using fluorescence in situ hybridization. Whereas the frequency of viable euploid spermatozoa significantly decreased over 3 days, the frequency of viable spermatozoa with aneuploidy interestingly showed a time-dependent increase. In addition, spermatozoa with abnormal sex chromosomes survived longer. To compared with spermatozoa with other swelling patterns, those with tail-tip swelling patterns had a lower frequency of aneuploidy at all time points. This study revealed the novel finding that the frequency of aneuploid spermatozoa with fertilization capability significantly increased compared to that of euploid spermatozoa over 3 days, suggesting that aneuploid spermatozoa can survive longer than euploid spermatozoa and have a greater chance of fertilizing oocytes.     

Stigmasterol isolated from marine microalgae Navicula incerta induces apoptosis in human hepatoma HepG2 cells

Plant sterols have shown potent anti-proliferative effects and apoptosis induction against breast and prostate cancers. However, the effect of sterols against hepatic cancer has not been investigated. In the present study, we assessed whether the stigmasterol isolated from Navicula incerta possesses apoptosis inductive effect in hepatocarcimona (HepG2) cells. According to the results, Stigmasterol has up-regulated the expression of pro-apoptotic gene expressions (Bax, p53) while down-regulating the anti-apoptotic genes (Bcl-2). Probably via mitochondrial apoptosis signaling pathway. With the induction of apoptosis caspase-8, 9 were activated. The DNA damage and increase in apoptotic cell numbers were observed through Hoechst staining, annexin V staining and cell cycle analysis. According to these results, we can suggest that the stigmasterol shows potent apoptosis inductive effects and has the potential to be tested as an anti-cancer therapeutic against liver cancer. [BMB Reports 2014; 47(8): 433-438]