Identifying a novel mutation of CYP17A1 gene from five Chinese 17α-hydroxylase/17, 20-lyase deficiency patients

Mutations of CYP17A1 gene could cause complete or partial, combined or isolated 17α-hydroxylase/17,20-lyase enzyme deficiencies (17OHD). We intended to investigate the CYP17A1 mutation in five unrelated patients and analyze its possible influence on phenotype of an atypical 17OHD patient presented with micropenis, hypertension and intermittent hypokalemia. Steroid hormones were assayed in these patients. A novel missense mutation (c.1169C>G, p. Thr390Arg) located in exon 7 was detected in one of the patients. Homozygous c. 985_987delinsAA, p. Tyr329fs mutation was found in two patients, while compound heterozygous mutations (c. 985_987delinsAA, p. Tyr329fs/c. 932–939 del, p. Val311fs and c. 287G>A, p. Arg96Gln/c. 985_987delinsAA, p. Tyr329fs) were found in two other patients, respectively. Then, steric model analysis of CYP17A1 showed that the novel mutation T390R changed the local structure as well as the electrostatic potential of the nearby beta sheet. Finally, site-directed mutagenesis and in vitro expression were used to analyze the activity of novel mutant CYP17A1. It indicated the T390R mutant retained part of enzyme activity, which was consistent to the clinical features. In conclusion, we identified a novel missense mutation of CYP17A1 gene from a patient with micropenis, hypertension and intermittent hypokalemia, which varied from other four patients. It also expanded our understanding of genotype–phenotype correlation of the disease.     

Site-specifically C-labeled Sel-tagged annexin A5 and a size-matched control for dynamic in vivo PET imaging of protein distribution in tissues prior to and after induced cell death

Background

Radiolabeled annexin A5 (AnxA5) is widely used for detecting phosphatidylserine exposed on cell surfaces during apoptosis. We describe here a new method for labeling AnxA5 and a size-matched control protein with short-lived carbon-11, for probing the specificity of in vivo cell death monitoring using positron emission tomography (PET) imaging.

Methods

AnxA5 and the control protein were recombinantly expressed with a C-terminal “Sel-tag”, the tetrapeptide –Gly-Cys-Sec-Gly–COOH. The proteins were then labeled either fluorescently for in vitro corroborations of binding behaviors or with ¹¹C for dynamic in vivo PET studies.

Results

AnxA5 demonstrated retained calcium-dependent binding to apoptotic cells after the C-terminus modification. The control protein showed no functional binding. The ¹¹C-ligands demonstrated similar in vivo pharmacokinetic behavior in healthy mice except for higher uptake in kidney and higher intact elimination to urine of AnxA5. After inducing hepatic apoptosis, however, the uptake of labeled AnxA5 in the targeted tissue increased compared to baseline levels while that of the control protein tended to decrease.

Conclusions

These data suggest that the combined use of these two tracers can facilitate differentiating specific AnxA5 binding and its changes caused by induced cell death from uptake due to non-specific permeability and retention effects at baseline or after therapy.

General significance

The Sel-tag enables rapid and mild reactions with electrophilic agents giving site-specifically labeled proteins for multi-probe analyses. The combined use of ¹¹C-labeled AnxA5 and a size-matched control protein with dynamic PET can be useful for evaluating drug effects on target as well as off-target tissues.     

Biomarkers in Parkinson’s disease (recent update)

Parkinson’s disease (PD) is the second most common neurodegenerative disorder mostly affecting the aging population over sixty. Cardinal symptoms including, tremors, muscle rigidity, drooping posture, drooling, walking difficulty, and autonomic symptoms appear when a significant number of nigrostriatal dopaminergic neurons are already destroyed. Hence we need early, sensitive, specific, and economical peripheral and/or central biomarker(s) for the differential diagnosis, prognosis, and treatment of PD. These can be classified as clinical, biochemical, genetic, proteomic, and neuroimaging biomarkers. Novel discoveries of genetic as well as nongenetic biomarkers may be utilized for the personalized treatment of PD during preclinical (premotor) and clinical (motor) stages. Premotor biomarkers including hyper-echogenicity of substantia nigra, olfactory and autonomic dysfunction, depression, hyposmia, deafness, REM sleep disorder, and impulsive behavior may be noticed during preclinical stage. Neuroimaging biomarkers (PET, SPECT, MRI), and neuropsychological deficits can facilitate differential diagnosis. Single-cell profiling of dopaminergic neurons has identified pyridoxal kinase and lysosomal ATPase as biomarker genes for PD prognosis. Promising biomarkers include: fluid biomarkers, neuromelanin antibodies, pathological forms of α-Syn, DJ-1, amyloid β and tau in the CSF, patterns of gene expression, metabolomics, urate, as well as protein profiling in the blood and CSF samples. Reduced brain regional N-acetyl-aspartate is a biomarker for the in vivo assessment of neuronal loss using magnetic resonance spectroscopy and T₂ relaxation time with MRI. To confirm PD diagnosis, the PET biomarkers include [¹⁸F]-DOPA for estimating dopaminergic neurotransmission, [¹⁸F]dG for mitochondrial bioenergetics, [¹⁸F]BMS for mitochondrial complex-1, [¹¹C](R)-PK11195 for microglial activation, SPECT imaging with ¹²³Iflupane and βCIT for dopamine transporter, and urinary salsolinol and 8-hydroxy, 2-deoxyguanosine for neuronal loss. This brief review describes the merits and limitations of recently discovered biomarkers and proposes coenzyme Q₁₀, mitochondrial ubiquinone-NADH oxidoreductase, melatonin, α-synculein index, Charnoly body, and metallothioneins as novel biomarkers to confirm PD diagnosis for early and effective treatment of PD.     

Analyse texturale en TEP au FDG des cancers pulmonaires non à petites cellules de stade localement avancé : valeur pronostique de l’entropie

IntroductionIn the current context of personalized medicine, textural analysis promises to be an accurate approach of cancer prognosis. The lack of standardization and the multitude of textural indices limited radiomics studies reproducibility as an obstacle of introduction of textual analysis into clinical practice. Our study assessed the prognostic value of entropy in 18F-FDG PET/CT in locally advanced non-small cell lung cancer (NSCLC).MethodPatients who performed 18F-FDG PET/CT for lung cancer staging between September 2015 and April 2017 in 2 hospitals were included for conventional and textural PET parameters extraction. A retrospective analysis of patient was performed over 24 months to determine the progression-free survival and overall survival.ResultsForty-two patients were included. Progression-free survival was significantly correlated with entropy on multivariate regression (cut-off at 8.4) with a hazard ratio of 3.04 (95 % CI 1.13–8.16) (P = 0.03), as MTV (P < 0.001). Neither conventional PET parameters nor entropy was a significant association with overall survival.ConclusionThese results confirmed the external validity and robustness of FDG PET entropy as an independent prognostic factor of progression-free survival in patients with locally advanced NSCLC, in addition to Conventional PET.     

Identifying the pathways for foliar water uptake in beech (Fagus sylvatica L.): a major role for trichomes

Foliar water uptake (FWU), the direct uptake of water into leaves, is a global phenomenon, having been observed in an increasing number of plant species. Despite the growing recognition of its functional relevance, our understanding of how FWU occurs and which foliar surface structures are implicated, is limited. In the present study, fluorescent and ionic tracers, as well as microcomputed tomography, were used to assess potential pathways for water entry in leaves of beech, a widely distributed tree species from European temperate regions. Although none of the tracers entered the leaf through the stomatal pores, small amounts of silver precipitation were observed in some epidermal cells, indicating moderate cuticular uptake. Trichomes, however, were shown to absorb and redistribute considerable amounts of ionic and fluorescent tracers. Moreover, microcomputed tomography indicated that 72% of empty trichomes refilled during leaf surface wetting and microscopic investigations revealed that trichomes do not have a cuticle but are covered with a pectin‐rich cell wall layer. Taken together, our findings demonstrate that foliar trichomes, which exhibit strong hygroscopic properties as a result of their structural and chemical design, constitute a major FWU pathway in beech.     

Production of 64Cu-labeled monobody for imaging of human EphA2-expressing tumors

We previously reported on the monobody E1, which specifically targets the tumor marker hEphA2. In this study, we labeled NOTA-conjugated E1 with ⁶⁴Cu (⁶⁴Cu-NOTA-E1) and evaluated biologic characteristics. The uptake of ⁶⁴Cu-NOTA-E1 in PC3 cells (a human prostate cancer cell line) with high expression of hEphA2 increased in a time-dependent manner. In PC3 xenograft mice, ⁶⁴Cu-NOTA-E1 injected via the tail vein allowed visualization of tumors on positron emission tomography after 1 h and the highest uptake measured at 24 h post-injection. By contrast, the radioactivity of other tissues either did not increase or decreased over 24 h. This indicates that ⁶⁴Cu-NOTA-E1 has high tumor uptake and retention, with rapid clearance, and low background values in other tissues. Therefore, ⁶⁴Cu-NOTA-E1 should be suitable as a novel PET imaging agent for hEphA2-expressing tumors.     

Micro-computed tomography and alizarin red evaluations of boric acid–induced fetal skeletal changes in Sprague-Dawley rats

BACKGROUND: Assessment of developmental toxicity has historically included assessment of fetal skeletal morphology after alizarin red staining. X-ray micro-computed tomography (micro-CT) produces high-resolution images of skeletal structures and was investigated as an alternative method. METHODS: Groups of 5 mated Crl:CD (SD) female rats each were administered vehicle or boric acid (40 to 500 mg/kg/day) from GD 6 through 11. On GD 21, all live fetuses were weighed, euthanized, and viscera removed. Each litter was placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 sec (∼20 litters per hour) and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 min later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. RESULTS: Micro-CT evaluation of fetal rat skeletons detected essentially the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. The specific skeletal abnormalities that did not match exactly involved the smallest skeletal elements with minimal degrees of ossification (i.e., cervical ribs, hypoplastic 13^th ribs, supernumerary ribs, the 5^th sternebra, and numbers of caudal vertebrae), but the differences did not impact the overall conclusions. Additional measures such as femur length were easily measured by micro-CT. CONCLUSIONS: These results indicate that micro-CT imaging can effectively assess rat fetal skeletal structures, and for those laboratories with this resource, it may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining. Birth Defects Res (Part B) 33:214–219, 2009 © 2009 Wiley-Liss, Inc.     

构建基于患者个体化的Stanford B型主动脉夹层计算流体力学数值模拟分析模型

目的:探索简便、高效、精确的构建基于真实人体解剖形态结构的Stanford B型主动脉夹层计算流体力学数值模拟分析模型的方法.方法:利用Siemens Sensation Cardiac 64层螺旋CT薄层扫描技术,基于1mm层厚获取6例Stanford B型主动脉夹层连续断层DICOM格式图像,导入Materialise MIMICS v12.11软件,界定目标区域后生成三维动脉模型,经网格优化处理去除低质量及相交面网格,保存结果输出,导入TGrid 5.0软件,对主动脉面网格模型进行几何修复,使面网格扭曲率<0.75,采用自由分网方式生成Stanford B型主动脉夹层计算流体力学分析体网格模型,并对所构建模型进行血流属性、流场边界等界定,初步验证模型的有效性.结果:通过初步计算求解,确定所构建的6例Stanford B型主动脉夹层计算流体力学分析模型分别包含1857030,1820501,1844181,1849651,1858246及1814914个四面体单元.结论:利用64层螺旋CT薄层扫描技术获取DICOM格式连续断层CT图像可快速、准确地构建Stanford B型主动脉夹层计算流体力学数值模拟分析模型,为进一步的计算流体力学分析奠定了良好的基础.     

PET imaging of the basal ganglia

The present chapter reviews PET imaging in basal ganglia disorders; Parkinson's disease is used as a model of these disorders because the neurochemical pathobiology of this disease is well known and great advances in the imaging area have been achieved. Other basal ganglia disorders including Tourette's syndrome, dystonia, Huntington's chorea and Wilson's disease are also dealt with. With PET and SPECT techniques, the whole integrative dopaminergic network of neurons can be studied, which plays an important role in differential diagnostics. Furthermore, pharmacological effects of medication can be visualized and the role of stereotaxic neurosurgery can be evaluated. Finally, functional imaging gives clues about the prognosis and rehabilitation aspects of the basal ganglia disorders.