Computer Simulations Suggest a Key Role of Membranous Nanodomains in Biliary Lipid Secretion

The bile fluid contains various lipids that are secreted at the canalicular membrane of hepatocytes. As the secretion mechanism is still a matter of debate and a direct experimental observation of the secretion process is not possible so far, we used a mathematical model to simulate the extraction of the major bile lipids cholesterol, phosphatidylcholine and sphingomyelin from the outer leaflet of the canalicular membrane. Lipid diffusion was modeled as random movement on a triangular lattice governed by next-neighbor interaction energies. Phase separation in liquid-ordered and liquid-disordered domains was modeled by assigning two alternative ordering states to each lipid species and minimization of next-neighbor ordering energies. Parameterization of the model was performed such that experimentally determined diffusion rates and phases in ternary lipid mixtures of model membranes were correctly recapitulated. The model describes the spontaneous formation of nanodomains in the external leaflet of the canalicular membrane in a time window between 0.1 ms to 10 ms at varying lipid proportions. The extraction of lipid patches from the bile salt soluble nanodomain into the bile reproduced observed biliary phospholipid compositions for a physiologi-cal membrane composition. Comparing the outcome of model simulations with available experi-mental observations clearly favors the extraction of tiny membrane patches composed of about 100–400 lipids as the likely mechanism of biliary lipid secretion

mRNA-Expression of KRT5 and KRT20 Defines Distinct Prognostic Subgroups of Muscle-Invasive Urothelial Bladder Cancer Correlating with Histological Variants

Recently, muscle-invasive bladder cancer (MIBC) has been subclassified by gene expression profiling, with a substantial impact on therapy response and patient outcome. We tested whether these complex molecular subtypes of MIBC can be determined by mRNA detection of keratin 5 (KRT5) and keratin 20 (KRT20). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was applied to quantify gene expression of KRT5 and KRT20 using TaqMan (R)-based assays in 122 curatively treated MIBC patients (median age 68.0 years). Furthermore, in silico analysis of the MD Anderson Cancer Center (MDACC) cohort (GSE48277 + GSE47993) was performed. High expression of KRT5 and low expression of KRT20 were associated with significantly improved recurrence-free survival (RFS) and disease-specific survival disease specific survival (DSS: 5-year DSS for KRT5 high: 58%; 5-year DSS for KRT20 high: 29%). KRT5 and KRT20 were associated with rates of lymphovascular invasion and lymphonodal metastasis. The combination of KRT5 and KRT20 allowed identification of patients with a very poor prognosis (KRT20(+)/KRT5(-), 5-year DSS 0%, p < 0.0001). In silico analysis of the independent MDACC cohorts revealed congruent results (5-year DSS for KRT20 low vs. high: 84% vs. 40%, p = 0.042). High KRT20-expressing tumors as well as KRT20(+)/KRT- tumors were significantly enriched with aggressive urothelial carcinoma variants (micropapillary, plasmacytoid, nested)

Metabolic heterogeneity of human hepatocellular carcinoma: implications for personalized pharmacological treatment

Metabolic reprogramming is a characteristic feature of cancer cells, but there is no unique metabolic program for all tumors. Genetic and gene expression studies have revealed heterogeneous inter- and intratumor patterns of metabolic enzymes and membrane transporters. The functional implications of this heterogeneity remain often elusive. Here, we applied a systems biology approach to gain a comprehensive and quantitative picture of metabolic changes in individual hepatocellular carcinoma (HCC). We used protein intensity profiles determined by mass spectrometry in samples of 10 human HCCs and the adjacent noncancerous tissue to calibrate Hepatokin1, a complex mathematical model of liver metabolism. We computed the 24-h profile of 18 metabolic functions related to carbohydrate, lipid, and nitrogen metabolism. There was a general tendency among the tumors toward downregulated glucose uptake and glucose release albeit with large intertumor variability. This finding calls into question that the Warburg effect dictates the metabolic phenotype of HCC. All tumors comprised elevated β-oxidation rates. Urea synthesis was found to be consistently downregulated but without compromising the tumor's capacity for ammonia detoxification owing to increased glutamine synthesis. The largest intertumor heterogeneity was found for the uptake and release of lactate and the size of the cellular glycogen content. In line with the observed metabolic heterogeneity, the individual HCCs differed largely in their vulnerability against pharmacological treatment with metformin. Taken together, our approach provided a comprehensive and quantitative characterization of HCC metabolism that may pave the way for a computational a priori assessment of pharmacological therapies targeting metabolic processes of HCC

Functional Consequences of Metabolic Zonation in Murine Livers: Insights for an Old Story

Background and Aims: Zone-dependent differences in expression of metabolic enzymes along the portocentral axis of the acinus are a long-known feature of liver metabolism. A prominent example is the preferential localization of the enzyme, glutamine synthetase, in pericentral hepatocytes, where it converts potentially toxic ammonia to the valuable amino acid, glutamine. However, with the exception of a few key regulatory enzymes, a comprehensive and quantitative assessment of zonal differences in the abundance of metabolic enzymes and, much more important, an estimation of the associated functional differences between portal and central hepatocytes is missing thus far. Approach and Results: We addressed this problem by establishing a method for the separation of periportal and pericentral hepatocytes that yields sufficiently pure fractions of both cell populations. Quantitative shotgun proteomics identified hundreds of differentially expressed enzymes in the two cell populations. We used zone-specific proteomics data for scaling of the maximal activities to generate portal and central instantiations of a comprehensive kinetic model of central hepatic metabolism (Hepatokin1). Conclusions: The model simulations revealed significant portal-to-central differences in almost all metabolic pathways involving carbohydrates, fatty acids, amino acids, and detoxification

Laser Speckle Simulation in Rotationally Symmetric Triangulation Sensor

散斑是激光三角传感器测量不确定度极限的根本影响因素。提出了一种用于旋转对称激光三角传感器的激光散斑的仿真方法,获得了仿真散斑图像。在旋转对称的三角传感器中,投射的激光点在检测器上被成像为一个环,从而散斑也相应是圆弧形。研究了散斑的特性,该散斑在环的半径方向上服从主观散斑的特性,其尺寸由光学系统的数值孔径决定。而在环的切线方向上,其本质上是客观散斑,由于光学系统存在折返光路,其尺寸由物体到检测器的光程、投射的激光光斑尺寸和成像圆环的半径决定。实验结果表明,仿真结果与散斑理论一致。基于仿真给出了对旋转对称三角传感器位移测量不确定度极限的分析,结果表明,使用旋转对称形式的传感器光学布局,在相同的光学系统数值孔径和使用同样的灰度质心算法的情况下,可达到传统激光三角测量不确定度的1/5。Speckle is the fundamental uncertainty factor in laser triangulation.A method to simulate the speckle in rotationally symmetric triangulation was presented and the simulated image was obtained.In this kind of triangulation sensors,the incident laser point will be imaged to a ring on the detector and the speckle is accordingly arc shaped.Properties of this kind of speckle were studied.The speckle size in radius direction of the ring obeyed the subjective speckle,and is determined by the number aperture of the optical system.In tangent direction of the ring,the speckle is essentially an objective speckle,its size is determined by the optical path length from the object to the detector,the area of the incident laser spot,as well as the radius of the imaged ring because of optical path was folded.Experiments showed that the simulation result was coincident with speckle theory.Based on the simulation,an analysis of the uncertainty limits of rotationally symmetric triangulation sensor was given.It shows that using the optical layout in our sensor,an uncertainty about 1/5 of traditional triangulation was estimated with same optical system numerical aperture and grey centroid algorithm.国家自然基金国际合作项目(60416312);; 国家自然基金项目(60375011,60575028);; 安徽省自然基金项目(04042044);; 2006年度中德合作PPP项目;; 新世纪优秀人才支持计划资助项目(NCET-04-0560

Technical assistance to assess the potential of renewable liquid and gaseous transport fuels of non-biological origin (RFNBOs) as well as recycled carbon fuels (RCFs), to establish a methodology to determine the share of renewable energy from RFNBOs as well as to develop a framework on additionality in the transport sector

This report is a summary of the work conducted in Task 1 of the technical assistance to assess the potential of renewable fuels of non-biological origin (RFNBOs) and recycled carbon fuels (RCFs) to establish a methodology to determine the share of renewable energy from RFNBOs as well as to develop a framework on additionality in the transport sector. The goal of Task 1 within the entire project was the assessment of the deployment potential of RFNBOs and RCFs over the period from 2020 to 2050 in the EU transport sector. All relevant transport sub-sectors and modalities are considered: road transport, maritime and inland shipping, aviation, and railway. Furthermore, the competition for RFNBOs and RCFs between the transport sectors and other sectors and applications of RFNBOs is considered. A central result is the potential gross final consumption of RFNBOs and RCFs that would count towards the RES target in the transport sector. In addition, the needed resources and the arising costs for this deployment as well as the impacts on greenhouse gas emissions and local environments are analyzed. Finally, barriers to the deployment and options to overcome these are outlined

Tumor budding correlates with tumor invasiveness and predicts worse survival in pT1 non-muscle-invasive bladder cancer

Tumor budding is defined as a single cell or a cluster of up to 5 tumor cells at the invasion front. Due to the difficulty of identifying patients at high risk for pT1 non-muscle-invasive bladder cancer (NMIBC) and the difficulties in T1 substaging, tumor budding was evaluated as a potential alternative and prognostic parameter in these patients. Tumor budding as well as growth pattern, invasion pattern and lamina propria infiltration were retrospectively evaluated in transurethral resection of the bladder (TURB) specimens from 92 patients with stage pT1 NMIBC. The presence of tumor budding correlated with multifocal tumors (p = 0.003), discontinuous invasion pattern (p = 0.039), discohesive growth pattern (p < 0.001) and extensive lamina propria invasion (p < 0.001). In Kaplan–Meier analysis, tumor budding was associated with significantly worse RFS (p = 0.005), PFS (p = 0.017) and CSS (p = 0.002). In patients who received BCG instillation therapy (n = 65), the absence of tumor budding was associated with improved RFS (p = 0.012), PFS (p = 0.011) and CSS (p = 0.022), with none of the patients suffering from progression or dying from the disease. Tumor budding is associated with a more aggressive and invasive stage of pT1 NMIBC and a worse outcome. This easy-to-assess parameter could help stratify patients into BCG therapy or early cystectomy treatment groups

Methods for L-ribooligonucleotide sequence determination using LCMS

The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation. Spiegelmer sequencing is effected by affixing a label or hapten to the 5′-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control