同型半胱氨酸、子宫动脉血流参数与反复妊娠丢失患者胰岛素抵抗和妊娠结局的关系

摘要 目的：探讨同型半胱氨酸（Hcy）、子宫动脉血流参数与反复妊娠丢失（RPL）患者胰岛素抵抗和妊娠结局的关系。方法：选择2020年6月至2022年10月昆明市妇幼保健院收治的162例RPL患者作为RPL组和同期82例规律产检的健康孕妇作为对照组。按照妊娠结局将RPL患者分为活产组（85例）和流产组（77例）。检测血清Hcy水平,胰岛素抵抗指数（HOMA-IR）,超声检查子宫动脉血流参数,包括子宫动脉收缩期峰值/舒张末期流速（S/D）、搏动指数（PI）、血流阻力指数（RI）。Pearson相关性分析Hcy、子宫动脉血流参数与HOMA-IR的相关性。采用多因素Logistic回归分析RPL 患者妊娠结局的影响因素。采用受试者工作特征（ROC）曲线分析Hcy、子宫动脉血流参数对RPL 患者妊娠结局的预测价值。结果：RPL组血清Hcy水平,S/D、PI、RI以及HOMA-IR均高于对照组（P＜0.05）。RPL组血清Hcy,S/D、PI、RI与HOMA-IR均呈正相关（P＜0.05）。流产组血清Hcy水平,S/D、PI、RI以及HOMA-IR均高于活产组（P＜0.05）,多因素Logistic回归分析显示高HOMA-IR、高Hcy、高S/D、高RI、染色体异常是RPL患者流产的危险因素（P＜0.05）。ROC曲线分析显示联合Hcy、S/D、RI预测RPL患者流产的曲线下面积（AUC）为0.849,高于单独预测。结论：RPL患者子宫动脉血流参数S/D、RI、PI和血清Hcy水平均增高,高S/D、RI和Hcy与RPL患者胰岛素抵抗以及流产风险增加有关。联合S/D、RI和Hcy可提高RPL流产风险评估效能。     

Effect of serinate ligation at each of the iron sites of the [Fe4S4] cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties.

Pyrococcus furiosus ferredoxin (Fd) contains a single [Fe(4)S(4)] cluster coordinated by three cysteine (at positions 11, 17, and 56) and one aspartate ligand (at position 14). In this study, the spectroscopic, redox, and functional consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutations have been investigated. The four serine variants each contain a potential cluster coordination sphere of one serine and three cysteine residues, with serine ligation at each of the four Fe sites of the [Fe(4)S(4)] cluster. All five variants were expressed in Escherichia coli, and each contained a [Fe(4)S(4)](2+,+) cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein and EPR and variable-temperature magnetic circular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced protein. Changes in both the absorption and resonance Raman spectra are consistent with changing from complete cysteinyl cluster ligation in the D14C variant to three cysteines and one oxygenic ligand in each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited state properties for the paramagnetic [Fe(4)S(4)](+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = (1)/(2) ground states and the D14S, D14C/C17S, and D14C/C56S variants having mixed-spin, S = (1)/(2) and (3)/(2) ground states. The midpoint potentials (pH 7.0, 23 degrees C) of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (E(m) = -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were more negative by 49 and 78 mV, respectively. Since the VTMCD spectra indicate the presence of a valence-delocalized Fe(2. 5+)Fe(2.5+) pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibited a pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of the serinate ligand to the reduced cluster (pK(a) = 4.75). All five variants had similar K(m) and V(m) values in a coupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized by ferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of ligation at each Fe atom in the [Fe(4)S(4)] cluster appears to have little effect on the interaction and the electron transfer between Fd and FNOR.     

A lipid transfer protein that transfers lipid

Very few lipid transfer proteins (LTPs) have been caught in the act of transferring lipids in vivo from a donor membrane to an acceptor membrane. Now, two studies (Halter, D., S. Neumann, S.M. van Dijk, J. Wolthoorn, A.M. de Maziere, O.V. Vieira, P. Mattjus, J. Klumperman, G. van Meer, and H. Sprong. 2007. J. Cell Biol. 179:101-115; D'Angelo, G., E. Polishchuk, G.D. Tullio, M. Santoro, A.D. Campli, A. Godi, G. West, J. Bielawski, C.C. Chuang, A.C. van der Spoel, et al. 2007. Nature. 449:62-67) agree that four-phosphate adaptor protein 2 (FAPP2) transfers glucosylceramide (GlcCer), a lipid that takes an unexpectedly circuitous route.     

Bioelectrical impedance estimation of fat-free body mass in children and youth: a cross-validation study.

The purposes of this study were to develop and cross-validate the "best" prediction equations for estimating fat-free body mass (FFB) from bioelectrical impedance in children and youth. Predictor variables included height2/resistance (RI) and RI with anthropometric data. FFB was determined from body density (underwater weighing) and body water (deuterium dilution) (FFB-DW) and from age-corrected density equations, which account for variations in FFB water and bone content. Prediction equations were developed using multiple regression analyses in the validation sample (n = 94) and cross-validated in three other samples (n = 131). R2 and standard error of the estimate (SEE) values ranged from 0.80 to 0.95 and 1.3 to 3.7 kg, respectively. The four samples were then combined to develop a recommended equation for estimating FFB from three regression models. R2 and SEE values and coefficients of variation from these regression equations ranged from 0.91 to 0.95, 2.1 to 2.9 kg, and 5.1 to 7.0%, respectively. As a result of all cross-validation analyses, we recommend the equation FFB-DW = 0.61 RI + 0.25 body weight + 1.31, with a SEE of 2.1 kg and adjusted R2 of 0.95. This study demonstrated that RI with body weight can predict FFB with good accuracy in Whites 10-19 yr old.     

Replication of Bacteriophage Ribonucleic Acid: Some Properties of Native and Denatured Replicative Intermediate

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli cells infected with the ribonucleic acid (RNA) bacteriophage R17 were denatured with dimethyl sulfoxide at 37 C or in aqueous solvents of low ionic strength at 97 C. Denaturation was demonstrated for RF and RI by an increase in specific infectivity and a striking change in the hyperchromicity curves after treatment. RI denaturation was also demonstrated by a shift in the buoyant density in Cs(2)SO(4) from 1.619 to the buoyant density of single-stranded R17 RNA (1.627). Analysis of the denatured RI hyperchromicity curves and the equilibrium distributions of denatured RI in Cs(2)SO(4) gradients revealed, however, a residual double-stranded component. Velocity sedimentation of denatured RI was performed, and the weight distribution of S values was calculated. From the known relation between molecular weight and S values, it was possible to transform the weight distribution into a number distribution of chain lengths. This distribution was compared with that predicted from the steady-state hypothesis for RI. Deviations from the predicted distribution may be due to the residual double-stranded component.     

Availability of applying diaphragm matching with the breath-holding technique in stereotactic body radiation therapy for liver tumors

PurposeImage-guided radiotherapy (IGRT) based on bone matching can produce large target-positioning errors because of expiration breath-hold reproducibility during stereotactic body radiation therapy (SBRT) for liver tumors. Therefore, the feasibility of diaphragm-based 3D image matching between planning computed tomography (CT) and pretreatment cone-beam CT was investigated.MethodsIn 59 liver SBRT cases, Lipiodol uptake after transarterial chemoembolization was defined as a tumor marker. Further, the relative isocenter coordinate that was obtained by Lipiodol matching was defined as the reference coordinate. The distance between the relative isocenter coordinate and reference coordinate, which was obtained from diaphragm matching and bone matching techniques, was defined as the target positioning error. Furthermore, the target positioning error between liver matching and Lipiodol matching was evaluated.ResultsThe positioning errors in all directions by the diaphragm matching were significantly smaller than those obtained by using by the bone matching technique (p < 0.05). Further, the positioning errors in the A-P and C-C directions that were obtained by using liver matching were significantly smaller than those obtained by using bone matching (p < 0.05). The estimated PTV margins calculated by the formula proposed by van Herk for diaphragm matching, liver matching, and bone matching were 5.0 mm, 5.0 mm, and 11.6 mm in the C-C direction; 3.6 mm, 2.4 mm, and 6.9 mm in the A-P direction; and 2.6 mm, 4.1 mm, and 4.6 mm in the L-R direction, respectively.ConclusionsDiaphragm matching-based IGRT may be an alternative image matching technique for determining liver tumor positions in patients.     

The effect of APOA5 and APOC3 variants on lipid parameters in European Whites, Indian Asians and Afro-Caribbeans with type 2 diabetes

Common variants in APOA5 and APOC3 have been associated with differences in plasma triglyceride (TG) levels in healthy individuals. The aim of this study was to examine the association of APOA5 (-1131T>C, S19W) and APOC3 (-482C>T, 1100C>T) polymorphisms in patients with type 2 diabetes (T2D) of European White (EW) (n=931), Indian Asian (IA) (n=610) and Afro-Caribbean (AC) (n=167) origin, with lipid and T2D parameters. Rare allele frequencies and linkage disequilibrium differed significantly amongst ethnic groups. Compared to APOA5 -1131T and 19S homozygotes, -1131C and 19W carriers had higher TGs in all groups, but this effect was only statistically significant for the -1131C in the EWs (P=0.04) and 19W in the IAs (P<0.001). APOC3 SNPs showed no significant association with lipid levels in any ethnic group. While haplotypes carrying -1131C allele showed significant TG-raising in the EWs only, the 19W defined haplotype showed significant TG-raising in both IAs and EWs. Comparing all four SNPs in EW T2D subjects with healthy EWs (n=2579), the APOC3 1100C>T frequency was significantly higher in T2D [0.26 (0.24, 0.28)] vs. healthy EWs [0.22 (0.20, 0.23)], P=0.001. While the variable size effects of the two APOA5 SNPs on TG levels may result from ethnically different gene-gene or gene-environment interactions, APOA5 and APOC3 variants did not affect parameters of T2D. However, comparison between EWs with T2D and healthy EWs suggest APOC3 1100C>T is associated with increased risk of diabetes probably through mechanisms other than direct effects on TG.     

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a light-scattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI; (b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted.     

Human polymorphic variants of the NEIL1 DNA glycosylase

In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864-1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type beta,delta-elimination reaction but primarily yielded the beta-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and gamma-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome.     

Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A

Onconase (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI.RNase A complex having a K(d) value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI.RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the K(d) value of the pRI.RNase A complex by 20 x 10(6)-fold (to 1.4 microM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 microM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.     

EcoRI variant N199H has enhanced specific activity

The Asn¹⁹⁹ residue of the EcoRI restriction endonuclease has been replaced with other amino acids to investigate whether it mediates nucleotide recognition or catalytic activity. Cassette mutagenesis gave variants of EcoRI: N199D, N199H, N199L, N199R, N199S and N199V. Their relative cleavage rates were found to be in the following order: N199H>EcoRI (wild type; wt)>N199L>N199V>N199S>N199R>N199D. In particular, EcoRI variant N199H showed about a twofold higher specific activity than that of the wt enzyme.     

Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions

The role of solvent composition and temperature on equilibrium electron transfer in seven rubredoxin variants [ Clostridium pasteurianum ( Cp), V8D, V8R, V8A, V44A Cp, Pyrococcus furiosus ( Pf), and A44V Pf] were investigated to examine the role of both monopolar and dipolar interactions. The reduction potentials of all variants decreased as the polarity of the solvent decreased. The enthalpy and entropy associated with electron transfer were determined from temperature-controlled voltammetric studies. The entropic contribution [delta( Tdelta S degrees )] to the change in the reduction potential was larger for charged variants (V8D and V8R), while the enthalpic contribution [delta(-delta H degrees )] was larger for the other mutants. The large entropy change observed for monopolar variants is likely due to solvent reorganization that occurs between oxidation states. Entropic-enthalpic compensation phenomena, an observation that most proteins have an entropic term [delta( Tdelta S degrees )] and enthalpic term [delta(-delta H degrees )] with opposite signs, was observed. A correlation of the size of the amino acid side chain with delta E degrees ', delta(-delta H degrees ), and delta( Tdelta S degrees ) is also discussed.     

A novel cardiopulmonary exercise test protocol and criterion to determine maximal oxygen uptake in chronic heart failure

Cardiopulmonary exercise testing for peak oxygen uptake (Vo(2peak)) can evaluate prognosis in chronic heart failure (CHF) patients, with the peak respiratory exchange ratio (RER(peak)) commonly used to confirm maximal effort and maximal oxygen uptake (Vo(2max)). We determined the precision of RER(peak) in confirming Vo(2max), and whether a novel ramp-incremental (RI) step-exercise (SE) (RISE) test could better determine Vo(2max) in CHF. Male CHF patients (n = 24; NYHA class I-III) performed a symptom-limited RISE-95 cycle ergometer test in the format: RI (4-18 W/min; ～10 min); 5 min recovery (10 W); SE (95% peak RI work rate). Patients (n = 18) then performed RISE-95 tests using slow (3-8 W/min; ～15 min) and fast (10-30 W/min; ～6 min) ramp rates. Pulmonary gas exchange was measured breath-by-breath. Vo(2peak) was compared within patients by unpaired t-test of the highest 12 breaths during RI and SE phases to confirm Vo(2max) and its 95% confidence limits (CI(95)). RER(peak) was significantly influenced by ramp rate (fast, medium, slow: 1.21 ± 0.1 vs. 1.15 ± 0.1 vs. 1.09 ± 0.1; P = 0.001), unlike Vo(2peak) (mean n = 18; 14.4 ± 2.6 ml·kg(-1)·min(-1); P = 0.476). Group Vo(2peak) was similar between RI and SE (n = 24; 14.5 ± 3.0 vs. 14.7 ± 3.1 ml·kg(-1)·min(-1); P = 0.407); however, within-subject comparisons confirmed Vo(2max) in only 14 of 24 patients (CI(95) for Vo(2max) estimation averaged 1.4 ± 0.8 ml·kg(-1)·min(-1)). The RER(peak) in CHF was significantly influenced by ramp rate, suggesting its use to determine maximal effort and Vo(2max) be abandoned. In contrast, the RISE-95 test had high precision for Vo(2max) confirmation with patient-specific CI(95) (without secondary criteria), and showed that Vo(2max) is commonly underestimated in CHF. The RISE-95 test was well tolerated by CHF patients, supporting its use for Vo(2max) confirmation.     

Synthesis of [10S(19)-3H]-dihydrotachysterol2 from ergocalciferol and preliminary investigations into its metabolic fate in rats

Dihydrotachysterol2 (DHT2), a 5,6-trans derivative of vitamin D2, is very successfully used in the treatment of hypoparathyroidism and renal osteodystrophy. However, the metabolism and the action of DHT2 are poorly understood. Investigations into metabolism of DHT2 start at the synthesis of the radioactively labelled compound. This paper deals with a two-step synthesis of [10S(19)-3H]-dihydrotachysterol2 from vitamin D2. Vitamin D2 can be converted into 5,6-trans vitamin D2 by iodination under irradiation and by triplet-sensitized isomerization. The first method led to unwanted side-reaction products which were difficult to separate from 5,6-trans vitamin D2. Triplet-sensitized isomerization yielded merely 5,6-trans vitamin D2 which could be separated from the residual starting material by chromatography on silica gel and recrystallization. [10S](19)-3H]-Dihydrotachysterol2 with a specific radioactivity of 56 kCi/mol was prepared from 5,6-trans vitamin D2 via partial, homogeneous catalytic reduction of the 10S(19) double bond with tritium gas. It was purified by high-performance liquid chromatography (h.p.l.c.) and characterized by chromatography and ultra-violet absorption spectrophotometry. The biological usefulness of the material was demonstrated in rats following intragastric administration. Blood was collected after 24 and 48 h and fractionation of serum lipids on h.p.l.c. showed 5 peaks of radioactivity.     

Direct stereoselective assay of fluoxetine and norfluoxetine enantiomers in human plasma or serum by two-dimensional gas-liquid chromatography with nitrogen-phosphorus selective detection

A method was developed and validated for the direct enantioselective assay of fluoxetine and norfluoxetine in human plasma or serum by two-dimensional capillary gas-liquid chromatography (GC). A Rtx-1 fused-silica capillary (15 mx0.25 mm I.D., 1.0 micrometer film thickness) and a hydrodex-beta-6-TBDM fused-silica capillary (25 mx0.25 mm I.D., 0.25 micrometer film thickness) were used. A three-step liquid-liquid extraction was used for sample preparation with fluvoxamine and nisoxetine as internal standards. The method provided linear calibration between about 5 and 250 ng/ml for (R)- and (S)-fluoxetine as well as 15 and 250 ng/ml for (R)- and (S)-norfluoxetine. The limits of detection were about 1.5 and 6 ng/ml, respectively. Intra-day precision (coefficient of variation) was estimated as being between 5.4 and 12.7% at plasma levels of 25, 100 and 200 ng/ml for the four enantiomers. Inter-day precision was between 5.3 and 9.1% at 100 ng/ml. The enantioselective separation of some racemic psychopharmaceuticals was tested with various cyclodextrin GC-capillaries. Advantages and disadvantages of direct enantioselective GC are discussed for the assay of racemic psychopharmaceuticals. Samples from a patient who was treated with racemic fluoxetine were measured. In agreement with literature, plasma levels of the (R)-enantiomers of fluoxetine and norfluoxetine were considerably decreased in comparison to the (S)-enantiomers.     

The genetic structure of recombinant inbred mice: High-resolution consensus maps for complex trait analysis

Background

Recombinant inbred (RI) strains of mice are an important resource used to map and analyze complex traits. They have proved particularly effective in multidisciplinary genetic studies. Widespread use of RI strains has been hampered by their modest numbers and by the difficulty of combining results derived from different RI sets.

Results

We have increased the density of typed microsatellite markers 2- to 5-fold in each of several major RI sets that share C57BL/6 as a parental strain (AXB, BXA, BXD, BXH, and CXB). A common set of 490 markers was genotyped in just over 100 RI strains. Genotypes of another ~1100 microsatellites were generated, collected, and error checked in one or more RI sets. Consensus RI maps that integrate genotypes of ~1600 microsatellite loci were assembled. The genomes of individual strains typically incorporate 45-55 recombination breakpoints. The collected RI set - termed the BXN set - contains approximately 5000 breakpoints. The distribution of recombinations approximates a Poisson distribution and distances between breakpoints average about 0.5 cM. Locations of most breakpoints have been defined with a precision of < 2 cM. Genotypes deviate from Hardy-Weinberg equilibrium in only a small number of intervals.

Conclusions

Consensus maps derived from RI strains conform almost precisely with theoretical expectation and are close to the length predicted by the Haldane-Waddington equation (X3.6 for a 2-3 cM interval between markers). Non-syntenic associations among different chromosomes introduce predictable distortions in QTL data sets that can be partly corrected using two-locus correlation matrices.     

Accuracy of Image Guidance Using Free-Breathing Cone-Beam Computed Tomography for Stereotactic Lung Radiotherapy

Movement of the target object during cone-beam computed tomography (CBCT) leads to motion blurring artifacts. The accuracy of manual image matching in image-guided radiotherapy depends on the image quality. We aimed to assess the accuracy of target position localization using free-breathing CBCT during stereotactic lung radiotherapy. The Vero4DRT linear accelerator device was used for the examinations. Reference point discrepancies between the MV X-ray beam and the CBCT system were calculated using a phantom device with a centrally mounted steel ball. The precision of manual image matching between the CBCT and the averaged intensity (AI) images restructured from four-dimensional CT (4DCT) was estimated with a respiratory motion phantom, as determined in evaluations by five independent operators. Reference point discrepancies between the MV X-ray beam and the CBCT image-guidance systems, categorized as left-right (LR), anterior-posterior (AP), and superior-inferior (SI), were 0.33 ± 0.09, 0.16 ± 0.07, and 0.05 ± 0.04 mm, respectively. The LR, AP, and SI values for residual errors from manual image matching were -0.03 ± 0.22, 0.07 ± 0.25, and -0.79 ± 0.68 mm, respectively. The accuracy of target position localization using the Vero4DRT system in our center was 1.07 ± 1.23 mm (2 SD). This study experimentally demonstrated the sufficient level of geometric accuracy using the free-breathing CBCT and the image-guidance system mounted on the Vero4DRT. However, the inter-observer variation and systematic localization error of image matching substantially affected the overall geometric accuracy. Therefore, when using the free-breathing CBCT images, careful consideration of image matching is especially important.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.     

Rapid determination of the synthetic pyrethroid insecticide, deltamethrin, in rat plasma and tissues by HPLC

Deltamethrin (DLM), [(S)-alpha-cyano-d-phenoxybenzyl-(1R,3R)-e-(2,2 dibromovinyl)-2,2-dimethylcyclo-propane-1-carboxylate], is a pyrethroid insecticide widely used in agriculture and households. There are several methods for analysis of DLM in biological fluids and tissues, but these methods are time consuming. They generally involve the extraction of DLM with lipid-soluble solvents such as n-pentane, n-hexane, diethylether or acetone, and subsequent evaporation of the solvent. A more rapid and sensitive high-performance liquid chromatography (HPLC) method to analyze DLM in plasma and tissues (liver, kidney, and brain) was developed and validated according to U.S. Food and Drug Administration (U.S. FDA) and International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. The limit of detection (S/N of 3/1) for DLM was 0.01 microg/ml for plasma, liver, kidney and brain. The method performances were shown to be selective for DLM and linear over the concentration range 0.01-20.0 microg/ml. For five replications of samples at 0.05, 0.1, 0.2, 1.5 and 4.0 microg/ml, intraday precision and accuracy values were in the range of 0.7-13.1% relative standard deviation (%R.S.D.) and 1.8-14.1%Error, respectively. Interday (n = 15) precision and accuracy values at 0.05, 0.1, 0.2, 1.5, and 4.0 microg/ml were in the range of 3.2-15.2% (%R.S.D.) and 3.7-14.8%Error, respectively. The absolute recoveries of DLM ranged from 93 to 103% for plasma, 95 to 114% for liver, 97 to 108% for kidney, and 95 to 108% for brain. This method can be quite useful for DLM pharmacokinetic and tissue distribution studies, for which multiple plasma and tissue samples have to be analyzed quickly with high reproducibility.