Epimedium-derived phytoestrogen exert beneficial effect on preventing steroid-associated osteonecrosis in rabbits with inhibition of both thrombosis and lipid-deposition

OBJECTIVES: This study tested the effect of Epimedium-derived phytoestrogen (PE) on preventing steroid-associated osteonecrosis (ON) in rabbit model. METHODS: Thirty 28-week-old male New-Zealand white rabbits were divided into control group (CON; n=14) and PE group (PE; n=16; 5 mg/kg body weight/day) after receiving an established inductive protocol for inducing steroid-associated ON. Before and after inductive protocol, Dynamic-MRI was employed on bilateral femora for local intra-osseous perfusion, blood samples were examined for coagulation, fibrinolysis and lipid-transportation, and marrow samples were quantified for adipogenesis-gene mRNA expression. Six weeks later, bilateral femora were dissected for Micro-CT-based micro-angiography, and then ON lesion, intravascular thrombosis and extravascular fat-cell-size were examined histopathologically. RESULTS: The incidence of ON in the PE group (31%) was significantly lower than that in the CON group (93%). Compared to the CON group, local intra-osseous perfusion was maintained in the PE group. Blocked trunk vessels were seldom found in micro-angiography of the PE-treated rabbits. Thrombosis incidence and fat-cell-size were both significantly lower in the PE group than those in the CON group. During the early period after induction, indicator of coagulation, fibrinolysis, lipid-transportation and adipogenesis-gene expression were found with significantly changing pattern in the PE group compared to the CON group. CONCLUSION: PE was able to exert beneficial effect on preventing steroid-associated ON in rabbits with inhibition of both thrombosis and lipid deposition.     

Biochemical Responses and Accumulation Properties of Long-Chain Perfluorinated Compounds (PFOS/PFDA/PFOA) in Juvenile Chickens (<Emphasis Type="Italic">Gallus gallus</Emphasis>)

One-day-old male chickens were exposed via oral gavage to mixtures of perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorodecanoate (PFDA) at either a low dose (0.1 mg/kg body weight [b.w.]) or a high dose (1.0 mg/kg b.w.), or a saline/ethanol vehicle control, three times a week for 3 weeks. After 3 weeks of exposure, half of the chicks were sacrificed and the other half were allowed to depurate for a further 3 weeks. No dose-dependent statistically significant differences in body/organ weights were observed among treatment and control groups after 3 weeks of exposure or after three 3 of depuration. Neither 15 histological nor 14 measured plasma biochemical parameters were significantly different in chicks from the exposed groups and vehicle controls. PFOS, PFDA, and PFOA concentrations in blood/liver/kidney samples were measured throughout the exposure and depuration periods at different time intervals. PFOS and PFDA accumulated at much higher concentrations than PFOA during the experimental periods. Interestingly, PFOS and PFDA accumulation patterns in the blood were similar during the exposure and depuration periods. The half-lives for each PFC at the 0.1 and 1.0 mg/kg doses were, respectively, approximately 15 and 17 days for PFOS, 11 and 16 days for PFDA, and 3.9 and 3.9 days for PFOA. PFDA accumulation in organs was greater than or similar to that of PFOS: the liver was the main target during exposure and the blood was the main reservoir during depuration. These results indicate that exposure to a 1.0-mg mixture of PFOS/PFDA/PFOA/kg b.w. has no adverse effect on juvenile chickens.     

KCNQ-encoded channels regulate Na+ transport across H441 lung epithelial cells

H441 cells are a model of absorptive airway epithelia that are characterised by a pronounced apical Na⁺ flux through amiloride-sensitive Na⁺ channels. The flux of Na⁺ is intimately linked to Na⁺ handling by the cell as well as the membrane potential across the apical membrane. As KCNQ-encoded K⁺ channels influence chloride secretion in gastrointestinal epithelia, the goal of the present study was to ascertain the expression of KCNQ genes in H441 cells and determine the functional role of the expression products. Message for KCNQ3 and KCNQ5 was detected by RT-polymerase chain reaction and the translated proteins were observed by immunocytochemistry. Ussing experiments showed that the pan-KCNQ channel blocker XE991, but not KCNQ1 selective blockers, reduced the short circuit current and the amiloride-sensitive component. These data show for the first time that potassium channels encoded by KCNQ3 or KCNQ5 are crucial determinants of epithelial Na⁺ flux.     

Effect of soft segment crystallization and hard segment physical crosslink on shape memory function in antibacterial segmented polyurethane ionomers

Shape memory polyurethane (SMPU) ionomers containing constant 75 wt.% soft segment content were synthesized using poly(ε-caprolactone)diol, 4,4′-diphenylmethane diisocyanate, 1,4-butanediol and/or N,N-bis(2-hydroxyethyl)-isonicotinamide. To introduce substrate bonding antibacterial activity, pyridinium was prepared through a neutralization reaction using 1-iodooctane as neutralization agent. For the SMPU ionomer film obtained, tensile testing at 70 °C and dynamic mechanical analysis suggests that, at temperatures > T_ms (the melting point of soft segments), 6.72 and 29.55 mol.% pyridinium within hard segments significantly decreased the mechanical properties such as the stress at 100% elongation (70 °C), the initial modulus (70 °C) and the elastic modulus (75–110 °C). Cyclic tensile investigation demonstrated that the two factors, soft segment crystallization and hard segment physical crosslink, play a very important role in shape memory function in SMPU ionomers. For the each individual specimen, the fixity ratio increased, and the recovery ratio decreased with the extension of cooling time. After sufficient cooling time, the fixity ratio of all specimens can reach a high value (95%). Owing to the disrupted physical crosslink in the sample containing 29.55 mol.% pyridinium, the crystallization rate of soft segments has less effect on shape fixity. Therefore, a high fixity ratio (93.8%) can be achieved in a short cooling time (30 s). In the control sample, the fixity ratio is only 73.7% after 30 s cooling. In addition, the admirable substrate bonding antibacterial activity of prepared SMPU ionomers was verified using standards AACTT 147 and ASTM E2149 in comparison with the control sample. The antibacterial activity of SMPU ionomers on Gram-positive bacteria (Staphylococcus aureus) is significant, and the rate of reduction of bacteria is 100%; the antibacterial activity on Gram-negative bacteria (Klebsiella pneumoniae) increases from 83.6% to 90.7% with increase in pyridinium content from 6.72 to 29.55 mol.%.     

Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): antiparasitic activity of a PKG inhibitor

Cyclic GMP-dependent protein kinase (PKG) has been biochemically and genetically validated in Toxoplasma gondii as a primary target responsible for the antiparasitic activity of the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (Compound 1) [Biftu T, Feng D, Ponpipom M, et al. Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents. Bioorg Med Chem Lett 2005;15:3296-301; Gurnett AM, Liberator PA, Dulski PM, et al. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J Biol Chem 2002;277:15913-22; Donald RGK, Allocco J, Singh SB, et al. Toxoplasma gondii cyclic GMP-dependent kinase: Chemotherapeutic targeting of an essential parasite protein kinase. Eukaryotic Cell 2002;1:317-28; Nare B, Allocco J, Liberator PA, Donald RGK. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine Toxoplasmosis: Gamma interferon is required for efficacy. Antimicrob Agents Chemother 2002;46:300-7]. Compound 1 inhibits the growth of several related protozoan parasites of the subphylum Apicomplexa. Native PKG activity has been partially purified by cGMP-affinity and MonoQ ion exchange chromatography from Plasmodium falciparum (PfPKG). Biochemical fractions enriched for a 98kDa protein detected using anti-PKG antisera, contain cGMP-induced protein kinase activity that is sensitive to inhibition by Compound 1. To enable a more thorough characterization of PfPKG we expressed a synthetic cDNA incorporating T. gondii codon preference (Pf(Tg)PKG) in T. gondii parasites. The protein kinase activity of purified recombinant Pf(Tg)PKG is stimulated by cGMP, with significant cooperativity as demonstrated by a Hill coefficient of 2. Both substrate preference and inhibition of Pf(Tg)PKG kinase activity by Compound 1 are similar to that seen with native PfPKG, as well as PKG enzymes from Eimeria spp. and T. gondii. We conclude that PfPKG has biochemical and pharmacological properties that are similar to previously characterized apicomplexan PKG enzymes. Compound 1 is active against blood cell stages of P. falciparum cultured in vitro. In a Plasmodium berghei mouse model of infection, Compound 1 delays the onset of parasitemia but does not cure the parasite infection.     

The role of endometrial and subendometrial vascularity measured by three-dimensional power Doppler ultrasound in the prediction of pregnancy during frozen-thawed embryo transfer cycles

BACKGROUND: A good blood supply to the endometrium is usually considered as an essential requirement for implantation. OBJECTIVE: The aim of this study was to evaluate the role of endometrial and subendometrial vascularity in the prediction of pregnancy during frozen-thawed embryo transfer (FET) cycles. METHODS: Women undergoing FET in natural or clomiphene-induced cycles after the first stimulated IVF treatment were recruited. A three-dimensional (3D) ultrasound examination with power Doppler was performed 1 day after the LH surge to determine endometrial thickness, endometrial pattern, pulsatility index (PI) and resistance index (RI) of uterine vessels, endometrial volume, vascularization index, flow index and vascularization flow index of endometrial and subendometrial regions. RESULTS: Women in the pregnant group were significantly younger and used less gonadotrophins in their stimulated cycle. Endometrial thickness, endometrial volume, endometrial pattern, uterine PI, uterine RI, endometrial and subendometrial 3D power Doppler flow indices were similar between the nonpregnant and the pregnant groups. The age of women was the only predictive factor for pregnancy. Receiver operating characteristic curve analysis revealed that the area under the curve was around 0.5 for all ultrasound parameters for endometrial receptivity. CONCLUSION: Vascularity of endometrial and subendometrial layers measured by 3D power Doppler ultrasound is not a good predictor of pregnancy in FET cycles if measured at one time point only.     

Interference and blood sample preparation for a pyruvate enzymatic assay

BACKGROUND: To assess the severity of circulatory failure, a pyruvate enzymatic assay was performed on whole blood using lactate dehydrogenase to catalyze the conversion of pyruvate to lactate. We investigated factors related to blood sample collection and preparation that might influence the results, including the timing of blood deproteinization, temperature of sample storage, and hemolysis. METHOD: A total of 25 whole blood specimens were collected for this study. Each sample was divided into 2 parts: one stored at room temperature (RT) and another kept on ice. The samples were deproteinizied by using 8% perchloric acid (PCA) at varying times after collection; the first deproteinization was immediately after the blood was drawn (0 h), then at 1 h intervals for 6 h and also in samples kept overnight. The supernatant samples were analyzed soon after deproteinization using a COBAS Centrifugal Analyzer. In another set of samples, the blood was immediately deproteinized, and the supernatants were stored at RT and 4 degrees C and assayed for pyruvate at varying times, as above. Finally, the effect of hemolysis on the blood pyruvate enzymatic assay was also evaluated. RESULTS: When samples were stored at RT, pyruvate levels remained constant until the third h after deproteinization, when there was an approximately 13.3% increase in pyruvate concentration. When whole blood samples were kept at 4 degrees C before deproteinization, pyruvate levels were significantly reduced over time, ranging from 37.8% to 62.2% (paired t test showed a significant mean difference, P < 0.001). No significant differences in pyruvate concentration were observed in supernatant stored at either RT or 4 degrees C. Hemolysis caused a 33.7% increase in the pyruvate concentration, equivalent to 0.18 mg pyruvate per gram per deciliter of hemoglobin. CONCLUSIONS: For a pyruvate enzymatic assay, keeping a whole blood sample at RT will not cause a significant difference in the pyruvate level as long as the sample is immediately deproteinized. Whole blood samples should not be stored in an ice bath for transport, nor should hemolyzed samples be used for a blood pyruvate enzymatic assay.     

Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis

BACKGROUND: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma. METHODS: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection. RESULTS: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses. CONCLUSIONS: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.     

Association of Interferon-Gamma Gene Polymorphisms in Taiwanese Children with Biliary Atresia

Background

Biliary atresia (BA) is a devastating neonatal hepatobiliary disease characterized by bile duct inflammation and fibrosis. The pathogenesis remains unclear, but immunologically mediated injury to bile ducts following an infectious insult is likely to play a critical role. Interferon-gamma (IFN-γ) is a key cytokine that affects immune-mediated inflammatory responses.

Objective

This study aims to investigate whether polymorphisms of the IFN-γ (IFNG) gene were associated with susceptibility to BA.

Methods

The IFNG ?1615 C/T, ?183 G/T, +874 A/T, and +2197 A/G polymorphisms were genotyped using the TaqMan assay, and CA repeat microsatellite was analyzed using capillary electrophoresis in 50 children with BA and 788 ethnically matched healthy controls.

Results

The distribution of genotype, allele, and haplotype frequencies of these IFNG gene variants did not differ significantly between children with BA and controls.

Conclusion

Polymorphisms of the IFNG gene do not appear to play a major role in the genetic predisposition to BA in Taiwanese children.