Circulating Methylated Septin 9 Nucleic Acid in the Plasma of Patients with Gastrointestinal Cancer in the Stomach and Colon

BACKGROUND: Methylated Septin 9 (mSEPT9) in plasma has recently been suggested as a screening marker for colorectal cancer (CRC) with variable sensitivity. We aimed to determine the usefulness of plasma mSEPT9 for screening CRC and gastric cancer (GC) and its diagnostic role in postoperative CRC patients. METHODS: A total of 350 peripheral blood samples from 101 CRC patients, 153 GC patients, and 96 healthy persons were collected. In addition, we obtained 35 follow-up blood samples from 27 CRC patients after curative radical surgery. Plasma mSEPT9, serum carcinoembryonic antigen (CEA), and serum CA19-9 were evaluated with clinicopathologic features. RESULTS: The sensitivity of plasma mSEPT9 was 36.6% for detecting CRC and 17.7% for detecting GC, and the specificity was 90.6%. During follow-up periods, mSEPT9 showed negative conversion in eight of nine CRC patients (88.9%) whose plasma mSEPT9 had been positive before radical surgery. The patients with plasma mSEPT9 had a tendency of presence of distant metastasis and lower disease-free survival in both CRC and GC. In GC patients, plasma mSEPT9 was more frequently observed in intestinal (23.5%) and mixed type (40.0%) than diffuse type (7.3%; P =.009). Combined analysis of mSEPT9, CEA, and CA19-9 increased the sensitivity for diagnosing GC to 32.7% (P = .002). CONCLUSION: Considering the high incidence of plasma mSEPT9 in intestinal or mixed type GCs similar to CRCs, GC should be examined through the plasma mSEPT9 screening test. In addition, plasma mSEPT9 is proposed as a follow-up marker in CRC patients, but further validation is required.     

Non-Invasive Prenatal Testing of Trisomy 18 by an Epigenetic Marker in First Trimester Maternal Plasma

Background

Quantification of cell-free fetal DNA by methylation-based DNA discrimination has been used in non-invasive prenatal testing of fetal chromosomal aneuploidy. The maspin (Serpin peptidase inhibitor, clade B (ovalbumin), member 5; SERPINB5) gene, located on chromosome 18q21.33, is hypomethylated in the placenta and completely methylated in maternal blood cells. The objective of this study was to evaluate the accuracy of non-invasive detection of fetal trisomy 18 using the unmethylated-maspin (U-maspin) gene as a cell-free fetal DNA marker and the methylated-maspin (M-maspin) gene as a cell-free total DNA marker in the first trimester of pregnancy.

Methodology/Principal Findings

A nested case-control study was conducted using maternal plasma collected from 66 pregnant women, 11 carrying fetuses with trisomy 18 and 55 carrying normal fetuses. Median U-maspin concentrations were significantly elevated in women with trisomy 18 fetuses compared with controls (27.2 vs. 6.7 copies/mL; P<0.001). Median M-maspin concentrations were also significantly higher in women with trisomy 18 fetuses than in controls (96.9 vs. 19.5 copies/mL, P<0.001). The specificities of U-maspin and M-maspin concentrations for non-invasive fetal trisomy 18 detection were 96.4% and 74.5%, respectively, with a sensitivity of 90.9%.

Conclusions

Our results suggest that U-maspin and M-maspin concentrations may be useful as potential biomarkers for non-invasive detection of fetal trisomy 18 in the first trimester of pregnancy, irrespective of the sex and genetic variations of the fetus.     

Molecular Identification of Armillaria gallica from the Niobrara Valley Preserve in Nebraska

Armillaria isolates were collected from a unique forest ecosystem in the Niobrara Valley Preserve in Nebraska, USA, which comprises a glacial and early postglacial refugium in the central plains of North America. The isolates were collected from diverse forest trees representing a unique mixture of forest types. Combined methods of rDNA sequencing and flow cytometric measurements of nuclear DNA content determined that all Armillaria isolates collected from the site were A. gallica.     

Chlorpromazine exacerbates hepatic insulin sensitivity via attenuating insulin and leptin signaling pathway, while exercise partially reverses the adverse effects

Investigated in this study are the effects and mechanisms of exercise and chlorpromazine (CPZ), a widely used conventional antipsychotic drug, on the hepatic insulin sensitivity of 90% pancreatectomized (Px) male Sprague–Dawley rats. The Px diabetic rats were provided with 0, 5, or 50 mg CPZ per kg of body weight (No-CPZ, LCPZ, or HCPZ) for 8 weeks, and half of each group had regular exercise. LCPZ did not exacerbate hepatic insulin sensitivity through insulin and leptin signaling in diabetic rats. However, HCPZ decreased whole-body glucose infusion rates in hyperinsulinemic clamped states, but not whole-body glucose uptake. This was due to the elevated hepatic glucose output in hyperinsulinemic states. The decreased hepatic insulin sensitivity was associated with insulin receptor substrate-2 (IRS2) protein levels in the liver. Decreased IRS2 levels attenuated hepatic insulin and leptin signaling pathways in hyperinsulinemic states, which elevated glucose production by inducing phosphoenolpyruvate carboxykinase expression. Long-term exercise recovered hepatic insulin sensitivity attenuated by HCPZ to reduce the hepatic glucose output in hyperinsulinemic clamped states. This recovery was related to enhanced insulin and leptin signaling via increased IRS2 gene and protein levels by activating the cAMP responding element-binding protein, but exercise improved only insulin signaling. In conclusion, HCPZ exacerbates hepatic insulin action by attenuating insulin and leptin signaling in type 2 diabetic rats, while regular exercise partially reverses the attenuation of hepatic insulin sensitivity by improving insulin signaling. Enhancement of insulin and leptin signaling through an induction of IRS2 may play an important role in improving hepatic glucose homeostasis.     

The light switch effect upon the association of [Ru(1,10-phenanthroline)2dipyrido[3,2-a:2',3'-c]phenazine]2+ with single stranded poly(dA) and poly(dT)

Large enhancement in the luminescence intensity of the Delta- and Lambda-Ru(phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) complexes upon their association with single stranded poly(dA) and poly(dT) is reported in this work. As the mixing ratio ([[Ru(phen)(2)DPPZ](2+)]/[DNA base]) increases, the luminescence intensity increase in a sigmoidal manner, indicating that the enhancement involves some cooperativity. At a high mixing ratio, the luminescence properties are affected by the nature of the DNA bases and not by the absolute configuration of the [Ru(phen)(2)DPPZ](2+) complex, indicating that the single stranded poly(dA) and poly(dT) do not recognize the configuration of the metal complex. In the case of the Lambda-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex, the manner of the enhancement is somewhat different from the other Ru(II) complex-polynucelotide combinations: the luminescence intensity reached a maximum at an intermediate mixing ratio of 0.32, and gradually decreased as the mixing ratio increased. In contrast to other complexes at high mixing ratios, an upward bending curve was found in the Stern-Volmer plot, which indicates that the micro-environment of the Lambda-[Ru(phen)(2)DPPZ](2+) is heterogeneous. In the Delta-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex case, formation of this highly luminescent species at an intermediate mixing ratio is far less effective.     

Property of the M-DNA probed by a minor groove binding dye 4',6-diamidino-2-phenylindole

Spectral properties including circular and linear dichroism (CD and LD) of M-DNA, a molecular electric wire, formed at a high Zn(2+) concentration have been studied using a minor groove binding drug 4',6-diamidino-2-phenylindole (DAPI) as a probe. As the Zn(2+) concentration increased, the magnitude of LD in the DNA absorption region decreased at pH 8.5, implying the aggregation of DNA, which is in contrast with the retained LD magnitude at pH 7.0. As the M-DNA formed, change in the secondary structure of DNA was observed by CD spectrum, which resembles that of the C-form DNA, although overall structure seemed to remain as a right handed double helix. The DAPI-DNA complex in the presence of high concentration of Zn(2+) ions at pH 7.0 exhibited the similar CD spectrum with that in the absence of Zn(2+) ion, consisting of type I, II and III. In contrast, at pH 8.5 at a high Zn(2+) concentration in which DNA is in its M-form, DNA bound DAPI produced only the type III CD, suggesting that DAPI binds at the surface of M-DNA: the presence of Zn(2+) ions prevents the minor groove binding of DAPI.     

Hepatic copper-transporting ATPase ATP7B: function and inactivation at the molecular and cellular level

Copper-transporting ATPase ATP7B (Wilson disease protein) is a member of the P-type ATPase family with characteristic domain structure and distinct ATP-binding site. ATP7B plays a central role in the regulation of copper homeostasis in the liver by delivering copper to the secretory pathway and mediating export of excess copper into the bile. The dual function of ATP7B in hepatocytes is coupled with copper-dependent intracellular relocalization of the transporter. The final destination of ATP7B in hepatocytes during the copper-induced trafficking process is still under debate. We show the results of immunocytochemistry experiments in polarized HepG2 cells that support the model in which elevated copper induces trafficking of ATP7B to sub-apical vesicles, and transiently to the canalicular membrane. In Atp7b ^-/- mice, an animal model of Wilson disease, both copper delivery to the trans-Golgi network and copper export into the bile are disrupted despite large accumulation of copper in the cytosol. We review the biochemical and physiological changes associated with Atp7b inactivation in mouse liver and discuss the pleiotropic consequences of the common Wilson disease mutation, His1069Gln.     

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.     

Deleterious impact of feto-maternal MHC compatibility on the success of pregnancy in a macaque model

The impact of feto-maternal histocompatibility on reproduction has inspired long-lasting debates. However, after the review of numerous articles, the impact of HLA allele sharing within couples on fecundity remains questionable. We decided to explore the impact of major histocompatibility complex (MHC) feto-maternal compatibility on reproduction in a cynomolgus macaque facility composed of animals of Mauritian descent. The Mauritian-derived macaque population presents a very restricted MHC polymorphism (only seven founding haplotypes) due to a strong founding bottleneck effect. The MHC polymorphism was investigated in 237 trios (male, female and offspring) using 17 microsatellite markers distributed across the MHC. Haplotypes were confirmed by segregation analysis. We evaluated the relative frequencies of MHC-compatible and MHC-semi-compatible offspring with the mothers. Among the 237 trios, we selected 42 trios for which the identity of the father is certain and for which the theoretical probabilities of fully compatible and semi-compatible offspring were equal. We found 11 offspring fully compatible and 31 offspring semi-compatible with their respective mother. The observed proportions were clearly outside the interval of confidence of 99 % and therefore most probably resulted from a selection of the semi-compatible offspring during pregnancy. We concluded that MHC fully compatible cynomolgus macaque offspring have a selective survival disadvantage in comparison with offspring inheriting a paternal MHC haplotype differing from maternal haplotypes.