Germline selection of PTPN11 (HGNC:9644) variants make a major contribution to both Noonan syndrome's high birth rate and the transmission of sporadic cancer variants resulting in fetal abnormality.

Some spontaneous germline gain-of-function mutations promote spermatogonial stem cell clonal expansion and disproportionate variant sperm production leading to unexpectedly high transmission rates for some human genetic conditions. To measure the frequency and spatial distribution of de novo mutations we divided three testes into 192 pieces each and used error-corrected deep-sequencing on each piece. We focused on PTPN11 (HGNC:9644) Exon 3 that contains 30 different PTPN11 Noonan syndrome (NS) mutation sites. We found 14 of these variants formed clusters among the testes; one testis had 11 different variant clusters. The mutation frequencies of these different clusters were not correlated with their case-recurrence rates nor were case recurrence rates of PTPN11 variants correlated with their tyrosine phosphatase levels thereby confusing PTPN11's role in germline clonal expansion. Six of the PTPN11 exon 3 de novo variants associated with somatic mutation-induced sporadic cancers (but not NS) also formed testis clusters. Further, three of these six variants were observed among fetuses that underwent prenatal ultrasound screening for NS-like features. Mathematical modeling showed that germline selection can explain both the mutation clusters and the high incidence of NS (1/1000-1/2500).

Driving Simulator Brake Reaction Parameters After Total Hip Arthroplasty According to Different Surgical Approaches.

BACKGROUND: The aim of this study is to validate the point of normalization of braking following total hip arthroplasty (THA) and to determine the relevance of the surgical approach. METHODS: Brake reaction parameters (BRPs), including brake reaction time, total brake time, and brake pedal depression force were measured in 90 patients who underwent primary arthroplasty of the right hip (42 with direct anterior approach and 48 with posterolateral approach) using a modern driving simulator. The driving parameters were measured preoperatively and every 2 weeks postoperatively until the eighth week. BRPs were measured in 40 subjects without hip problems, and the results were used as a control. Statistical assessment was performed to analyze when the patients' reaction to braking recovered to that of the control group with respect to different surgical approaches and also according to the pain. RESULTS: Preoperative BRPs of the patients undergoing THA were prolonged compared to the control group and were normalized at the sixth week following the operation. Although BRPs of the direct anterior approach group showed significantly better improvement compared to the posterolateral approach group (total brake time at week 2, brake reaction time and brake pedal depression at week 4), both groups reached baseline value at week 6. In addition, we found no correlation between the pain score and BRPs. CONCLUSION: The results of the current study indicate that the response to braking events normalizes at 6 weeks following THA in young active patients and is irrelevant to the surgical approach.

Positive selection for new disease mutations in the human germline: evidence from the heritable cancer syndrome multiple endocrine neoplasia type 2B.

Multiple endocrine neoplasia type 2B (MEN2B) is a highly aggressive thyroid cancer syndrome. Since almost all sporadic cases are caused by the same nucleotide substitution in the RET proto-oncogene, the calculated disease incidence is 100-200 times greater than would be expected based on the genome average mutation frequency. In order to determine whether this increased incidence is due to an elevated mutation rate at this position (true mutation hot spot) or a selective advantage conferred on mutated spermatogonial stem cells, we studied the spatial distribution of the mutation in 14 human testes. In donors aged 36-68, mutations were clustered with small regions of each testis having mutation frequencies several orders of magnitude greater than the rest of the testis. In donors aged 19-23 mutations were almost non-existent, demonstrating that clusters in middle-aged donors grew during adulthood. Computational analysis showed that germline selection is the only plausible explanation. Testes of men aged 75-80 were heterogeneous with some like middle-aged and others like younger testes. Incorporating data on age-dependent death of spermatogonial stem cells explains the results from all age groups. Germline selection also explains MEN2B's male mutation bias and paternal age effect. Our discovery focuses attention on MEN2B as a model for understanding the genetic and biochemical basis of germline selection. Since RET function in mouse spermatogonial stem cells has been extensively studied, we are able to suggest that the MEN2B mutation provides a selective advantage by altering the PI3K/AKT and SFK signaling pathways. Mutations that are preferred in the germline but reduce the fitness of offspring increase the population's mutational load. Our approach is useful for studying other disease mutations with similar characteristics and could uncover additional germline selection pathways or identify true mutation hot spots.

A germ-line-selective advantage rather than an increased mutation rate can explain some unexpectedly common human disease mutations.

Two nucleotide substitutions in the human FGFR2 gene (C755G or C758G) are responsible for virtually all sporadic cases of Apert syndrome. This condition is 100-1,000 times more common than genomic mutation frequency data predict. Here, we report on the C758G de novo Apert syndrome mutation. Using data on older donors, we show that spontaneous mutations are not uniformly distributed throughout normal testes. Instead, we find foci where C758G mutation frequencies are 3-4 orders of magnitude greater than the remaining tissue. We conclude this nucleotide site is not a mutation hot spot even after accounting for possible Luria-Delbruck "mutation jackpots." An alternative explanation for such foci involving positive selection acting on adult self-renewing Ap spermatogonia experiencing the rare mutation could not be rejected. Further, the two youngest individuals studied (19 and 23 years old) had lower mutation frequencies and smaller foci at both mutation sites compared with the older individuals. This implies that the mutation frequency of foci increases as adults age, and thus selection could explain the paternal age effect for Apert syndrome and other genetic conditions. Our results, now including the analysis of two mutations in the same set of testes, suggest that positive selection can increase the relative frequency of premeiotic germ cells carrying such mutations, although individuals who inherit them have reduced fitness. In addition, we compared the anatomical distribution of C758G mutation foci with both new and old data on the C755G mutation in the same testis and found their positions were not correlated with one another.

Flame retardant polymeric foams based on poly(ethylene vinylacetate)/clay nanocomposites.

Poly(ethylene vinylacetate)/clay nanocomposites were prepared by a "one pot" method, using Cloisite-Na+ with a compatibilizer at different ratios. The nano-composite prepared was used to prepare a flame retarding polymeric foams. Flame retarding properties were examined by the limiting oxygen index and cone calorimetry. CO and CO2 production were reduced remarkably by using the nanocomposite in EVA blends.

Flame retardant polyethylene/organo-nanocomposite foams cross-linked by electron beam irradiation.

Cross-linking of low-density polyethylene (PE)/organo-nanocomposite blends by electron beam irradiation was studied with the aim of flame retarding foam production by thermal expansion. Flammability and thermal properties of the obtained foams were studied and it was found that relatively low doses up to 42.5 kGy could be used leading to a product with excellent surface appearance and flame retardancy.

Augmented sphingosylphosphorylcholine-induced Ca2+-sensitization of mesenteric artery contraction in spontaneously hypertensive rat.

Sphingosylphosphorylcholine (SPC) is a vasoconstricting lysosphingolipid, and the RhoA/Rho-kinase pathway plays an important role in SPC-induced contraction. Since RhoA/Rho-kinase-mediated signaling is involved in the generation and/or maintenance of hypertension, we compared the effect of SPC on the contractility of endothelium-denuded small mesenteric arteries in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Fura-2 Ca2+ signals, contractile responses, and phosphorylation of 20-kDa myosin light chains (MLC20) were measured. Ten microM SPC induced a gradual and sustained vasoconstriction, which was greater in arteries of the SHR (82.5 +/- 4.3%, n=9) than in those of the WKY (26.7 +/- 4.5%, n=10). In Ca2+-free media, SPC gradually increased vascular tone in the SHR, but caused little vasoconstriction in the WKY. In the SHR and WKY, SPC evoked a greater vasoconstriction than did high K+ depolarization at a given Ca2+ ratio, and the Ca2+ ratio-tension curve induced by SPC was significantly shifted to the left compared with that induced by high K+ depolarization. However, the magnitude of shift to the left was greater in the SHR than in the WKY. The Rho-kinase inhibitor Y-27632 significantly inhibited SPC-induced contractions, but neither the protein kinase C inhibitor calphostin-C nor PD98059, which inhibits activation of some mitogen-activated protein kinases, had any effect on the SHR or the WKY. SPC significantly increased the phosphorylation of MLC20 in both the SHR and the WKY, and Y-27632 inhibited the SPC-induced increase in MLC(20) phosphorylation in the SHR. Our results suggest that SPC induces greater vascular tone in the SHR than in the WKY. Furthermore, our results indicate that activation of the Rho-kinase pathway plays an important role in the SPC-induced Ca2+ sensitization in the SHR.

[Effects of a cardiocerebrovascular disease prevention education program for postmenopausal middle-aged women].

PURPOSE: This study was conducted to examine effects of a cardiocerebrovascular disease (CVD) prevention education program on knowledge, self-efficacy and health behavior among postmenopausal middle-aged women. METHODS: A non-equivalent control group pretest-posttest design was used. Participants were 53 postmenopausal middle-aged women who registered in two community culture centers in G metropolitan city. Experimental group (n=26) received a CVD prevention education program 8 times over 8 weeks. Knowledge, self-efficacy and health behavior of the participants were examined with self-report structured questionaries. Data were collected between October 15 and December 11, 2013, and were analyzed using chi-square test, Fisher's exact test, independent t-test, and analysis of covariance with SPSS/PC version 21.0. RESULTS: After the intervention the experimental group showed significant increases in the knowledge of CVD symptoms (p<.001) and CVD risk factors (p<.001), level of self-efficacy (p=.028) and health behavior (p<.001) compared to the control group. However, no significant difference was found between groups for knowledge of CVD prevention (p<.133). CONCLUSION: Results suggest that a CVD prevention education program can be an effective strategy to improve knowledge on CVD symptoms and risk factors, self-efficacy and health behavior for postmenopausal middle-aged women.

Anticancer effect of gene/peptide co-delivery system using transferrin-grafted LMWSC.

A series of ternary complex was designed to deliver psiRNA-bcl2 and (KLA)4 peptide into cancer cells for cancer therapy. The delivered psiRNA-bcl2 induced gene-silencing in a nucleus of cancer cells, while (KLA)4 peptide inhibited cancer growth via mitochondrial apoptosis, indicating that the ternary complexes exerted very strong synergistic effects on cancer growth suppression by acting on psiRNA-bcl2 and (KLA)4 peptide simultaneously. The ternary complexes having a targeting-ligand, transferrin (TfP), were found to be especially effective at binding to the TfP receptor rich cancer cells, HCT119. The plasmid DNA (pDNA) in ternary complexes was completely condensed at various content of LMWSC-PEG-TfP (32-64 times more than pDNA) and released into cells. pDNA in the complexes was protected from DNase present on the exterior of cells. The size (165-248 nm) of ternary complexes with LMWSC-PEG-TfP was increased, but surface charges (3-4.5 mV) were decreased. These results likely occurred because the free amine-group of LMWSC decreased in response to conjugated transferrin. Moreover, transfected ternary complexes with LMWSC-PEG-TfP were not expressed in the normal cells (HEK293), but were over expressed in HCT119 cells. These findings indicate that the ternary complexes can be specifically targeted to HCT119 cancer cells. The useful complexes for gene and peptide delivery had high anticancer activities via a synergistic effect due to co-operative action of psiRNA and (KLA)4 peptide in HCT119 cells.

Degradation of fucoidans from Sargassum fulvellum and their biological activities.

A fucoidan extracted from Sargassum fulvellum, was degraded by ultrasound (US) or electron beam (EB) irradiation in the presence of hydrogen peroxide aqueous solution. From the energetic point of view, the most effective method for the fucoidan degradation was found to be EB radiation method in H2O2 with a yield of scission Gs 3.310 × 10(-8)mol/J at the reaction condition of 1.5% hydrogen peroxide and irradiation 2.5 kGy. The degradation took place by the formation of a reactive hydroxyl radical due to the dissociation of H2O2 in the presence of US or EB. The low molecular weight fucoidans (LMWFs) prevented P-selectin binding to Sialyl Lewis X with an IC50 (inhibitory concentration 50) of 20 nM as compared to 400 nM for heparin and 25,000 nM for dextran sulfate. The LMWFs showed no hemolytic activity at concentrations up to 950 µg/ml.

3-aminopropyl functionalized magnesium phyllosilicate as an organoclay based drug carrier for improving the bioavailability of flurbiprofen.

This study aimed to develop an oral delivery system using clay-based organic-inorganic hybrid materials to improve the bioavailability of the drug, flurbiprofen, which is poorly soluble in water. 3-aminopropyl functionalized magnesium phyllosilicate (AMP clay) was synthesized by a one-pot direct sol-gel method, and then flurbiprofen (FB) was incorporated into AMP clay (FB-AMP) at different drug/clay ratios. The structural characteristics of AMP and FB-AMP formulation were confirmed by X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy. Among tested formulations, FB-AMP(3), dramatically increased the dissolution of FB and achieved rapid and complete drug release within 2 hours. More than 60% of FB was released from FB-AMP(3) after 30 minutes; the drug was completely dissolved in the water within 2 hours. Under the acidic condition (pH 1.2), FB-AMP(3) also increased the dissolution of FB by up to 47.1% within 1 hour, which was three-fold higher than that of untreated FB. Furthermore, following an oral administration of FB-AMP(3) to Sprague-Dawley rats, the peak plasma concentration and area under the plasma concentration-time curve of FB increased two-fold, and the time to reach the peak plasma concentration was shortened compared with that in the untreated FB. This result suggests that the oral drug delivery system using clay-based organic-inorganic hybrid material might be useful to improve the bioavailability of FB.

Genome-wide enrichment screening reveals multiple targets and resistance genes for triclosan in Escherichia coli.

Triclosan is a widely used biocide effective against different microorganisms. At bactericidal concentrations, triclosan appears to affect multiple targets, while at bacteriostatic concentrations, triclosan targets FabI. The site-specific antibiotic-like mode-of-action and a widespread use of triclosan in household products claimed to possibly induce cross-resistance to other antibiotics. Thus, we set out to define more systematically the genes conferring resistance to triclosan; A genomic library of Escherichia coli strain W3110 was constructed and enriched in a selective medium containing a lethal concentration of triclosan. The genes enabling growth in the presence of triclosan were identified by using a DNA microarray and confirmed consequently by ASKA clones overexpressing the selected 62 candidate genes. Among these, forty-seven genes were further confirmed to enhance the resistance to triclosan; these genes, including the FabI target, were involved in inner or outer membrane synthesis, cell-surface material synthesis, transcriptional activation, sugar phosphotransferase (PTS) systems, various transporter systems, cell division, and ATPase and reductase/dehydrogenase reactions. In particular, overexpression of pgsA, rcsA, or gapC conferred to E. coli cells a similar level of triclosan resistance induced by fabI overexpression. These results indicate that triclosan may have multiple targets other than well-known FabI and that there are several undefined novel mechanisms for the resistance development to triclosan, thus probably inducing cross antibiotic resistance.

Synthesis and anti-cancer efficacy of rapid hydrolysed water-soluble paclitaxel pro-drugs.

A new series of poly(ethylene glycol)(PEG)-paclitaxel conjugates that increases water solubility of paclitaxel was synthesized. We developed well-designed self-immolating linkers between a drug and a water-soluble polymer moiety which gave an extremely rapid hydrolysis rate to convert a pro-drug into a parent drug without any reduction in drug efficacy. The self-immolating spacer groups were introduced between the solubilizing PEG and C7-OH of paclitaxel in order to control the rate of enzymatic hydrolysis. All these pro-drugs had a water-solubility of 400 mg/ml or more compared with a solubility of about 0.01 mg/ml. The rate of hydrolysis for the pro-drugs in rat plasma showed considerable variation of t((1/2)) ranging from 0.94 min to 42.7 min. To evaluate the anti-tumor efficacy of the pro-drug which had the fastest enzymatic hydrolysis rate, the growth inhibitory effect (IC(50)), the anti-tumor activity and the anti-metastatic potential of the pro-drug were examined. The pro-drug was potent to inhibit the growth of various cancer cell lines, such as human lung, ovarian, colon and melanoma cancer cells. On the development of melanoma lung colonies in C57B/6 mice following intravenous administration of metastatic murine B16/F10 melanoma cells, the pro-drug seems to be more efficacious than paclitaxel. The reduction of the number of melanoma lung colonies was 46.9% (dose: 5 mg/kg) with pure paclitaxel, and 24.5%, and 40.0% with the pro-drug in the dose of 0.71 mg paclitaxel equivalent/kg and 1.42 mg paclitaxel equivalent/kg, respectively.