Three Dimensional Culture of Human Renal Cell Carcinoma Organoids

Renal cell carcinomas arise from the nephron but are heterogeneous in disease biology, clinical behavior, prognosis, and response to systemic therapy. Development of patient-specific in vitro models that efficiently and faithfully reproduce the in vivo phenotype may provide a means to develop personalized therapies for this diverse carcinoma. Studies to maintain and model tumor phenotypes in vitro were conducted with emerging three-dimensional culture techniques and natural scaffolding materials. Human renal cell carcinomas were individually characterized by histology, immunohistochemistry, and quantitative PCR to establish the characteristics of each tumor. Isolated cells were cultured on renal extracellular matrix and compared to a novel polysaccharide scaffold to assess cell-scaffold interactions, development of organoids, and maintenance of gene expression signatures over time in culture. Renal cell carcinomas cultured on renal extracellular matrix repopulated tubules or vessel lumens in renal pyramids and medullary rays, but cells were not observed in glomeruli or outer cortical regions of the scaffold. In the polysaccharide scaffold, renal cell carcinomas formed aggregates that were loosely attached to the scaffold or free-floating within the matrix. Molecular analysis of cell-scaffold constructs including immunohistochemistry and quantitative PCR demonstrated that individual tumor phenotypes could be sustained for up to 21 days in culture on both scaffolds, and in comparison to outcomes in two-dimensional monolayer cultures. The use of three-dimensional scaffolds to engineer a personalized in vitro renal cell carcinoma model provides opportunities to advance understanding of this disease.     

An In Vivo Experimental Validation of a Computational Model of Human Foot

Reliable computational foot models offer an alternative means to enhance knowledge on the biomechanics of human foot.Model validation is one of the most critical aspects of the entire foot modeling and analysis process.This paper presents an invivo experiment combining motion capture system and plantar pressure measure platform to validate a three-dimensional finiteelement model of human foot.The Magnetic Resonance Imaging(MRI)slices for the foot modeling and the experimental datafor validation were both collected from the same volunteer subject.The validated components included the comparison of staticmodel predictions of plantar force,plantar pressure and foot surface deformation during six loading conditions,to equivalentmeasured data.During the whole experiment,foot surface deformation,plantar force and plantar pressure were recorded simultaneouslyduring six different loaded standing conditions.The predictions of the current FE model were in good agreementwith these experimental results.     

A Three Dimensional Model of Human Thiopurine Methyltransferase; Ligand Interactions and Structural Consequences of Naturally Occurring Mutations

The three-dimensional model of human thiopurine methyltransferase (hTPMT) was constructed by molecular modeling. A multiple alignment of AdoMet dependent methyltransferases based on a structural superposition of the AdoMet binding domain of Hhai, TaqI and rCOMT was used in the modeling procedure. The reliability of the model was examined by comparing its conformation and packing properties with those of Hhai, TaqI and rCOMT and structures in the PDB-database. The examined criteria indicated a reliable model structure. The model gave insight into the structural effects of naturally occurring mutations of the hTPMT allele, and was used to characterize the ligand interactions of the protein. The residues Gln42 and Glu91 were predicted to participate in AdoMet binding through H-bond interactions whereas Phe146 participates through Van der Waal interaction. The cationic methyl-sulphonium group of AdoMet was located close to the aromatic residue Phe40. The model also indicated that substrates interact with hTPMT situated in a pocket consisting of the hydrophobic residues Phe40, Met148, Val184, Val220 and the charged residues Lys145, Glu218, Lys219. These residues were also included in a predictive explanation for the inhibitor/substrate preference of the enzyme. The most frequent of naturally occurring mutations was predicted to cause alterations on the surface of the protein with minor/none structural consequences. The mutation Ala80-Pro seemed directly to cause an inactive enzyme by disrupting the structure of the binding site of AdoMet.Electronic Supplementary Material available.     

Capturing the Dynamics of a Hybrid Multiscale Cancer Model with a Continuum Model

Cancer is a complex disease involving processes at spatial scales from subcellular, like cell signalling, to tissue scale, such as vascular network formation. A number of multiscale models have been developed to study the dynamics that emerge from the coupling between the intracellular, cellular and tissue scales. Here, we develop a continuum partial differential equation model to capture the dynamics of a particular multiscale model (a hybrid cellular automaton with discrete cells, diffusible factors and an explicit vascular network). The purpose is to test under which circumstances such a continuum model gives equivalent predictions to the original multiscale model, in the knowledge that the system details are known, and differences in model results can be explained in terms of model features (rather than unknown experimental confounding factors). The continuum model qualitatively replicates the dynamics from the multiscale model, with certain discrepancies observed owing to the differences in the modelling of certain processes. The continuum model admits travelling wave solutions for normal tissue growth and tumour invasion, with similar behaviour observed in the multiscale model. However, the continuum model enables us to analyse the spatially homogeneous steady states of the system, and hence to analyse these waves in more detail. We show that the tumour microenvironmental effects from the multiscale model mean that tumour invasion exhibits a so-called pushed wave when the carrying capacity for tumour cell proliferation is less than the total cell density at the tumour wave front. These pushed waves of tumour invasion propagate by triggering apoptosis of normal cells at the wave front. Otherwise, numerical evidence suggests that the wave speed can be predicted from linear analysis about the normal tissue steady state.     

上颌第一前磨牙三维模型的建立与研究

目的：建立上颌第一前磨牙三维模型,以辅助牙体解剖学数字化教学和指导临床根管治疗术。方法：对人离体上颌第一前磨牙通过ConebeamCT扫描,获得DICOM格式影像,将获得的影像定位后利用Mimicsl0．0三维重建软件采集牙釉质、牙本质及髓腔的点数据,然后将采集到的点数据导入到MagicslO．0软件进行面网格化,将网格化后的图像标本进行光滑处理后保存,再次利用MimicslO．0三维重建软件进行数据处理,最终获得清晰的牙体及根管系统三维立体图像。结果：准确的建立了包含牙釉质、牙本质、牙髓腔的三维立体模型。结论：本实验方法建立的上颌第一前磨牙的三维模型,具有极高的真实性和精确性,对辅助教学、指导临床根管治疗都具有重要意义。为牙体解剖教学和口腔临床应用提供了一种简捷而精确的建模方法。     

Computational Modelling of Cancer Development and Growth: Modelling at Multiple Scales and Multiscale Modelling

In this paper, we present two mathematical models related to different aspects and scales of cancer growth. The first model is a stochastic spatiotemporal model of both a synthetic gene regulatory network (the example of a three-gene repressilator is given) and an actual gene regulatory network, the NF-\(\upkappa \)B pathway. The second model is a force-based individual-based model of the development of a solid avascular tumour with specific application to tumour cords, i.e. a mass of cancer cells growing around a central blood vessel. In each case, we compare our computational simulation results with experimental data. In the final discussion section, we outline how to take the work forward through the development of a multiscale model focussed at the cell level. This would incorporate key intracellular signalling pathways associated with cancer within each cell (e.g. p53–Mdm2, NF-\(\upkappa \)B) and through the use of high-performance computing be capable of simulating up to \(10^9\) cells, i.e. the tissue scale. In this way, mathematical models at multiple scales would be combined to formulate a multiscale computational model.     

人类YAC库PCR三维筛选体系的建立及质量考核

为了在知道某个区域、位点、基因或ＤＮＡ片段的部分信息后,能从ＣＥＰＨＹＡＣ库中筛选出与其对应的ＹＡＣ克隆,为进一步研究奠定基础,需要建立一个筛选体系。本文概述了这一筛选体系的建立过程。随后,用两对与已知基因对应的引物进行了筛选验证工作,证明了这一体系的可用性,同时提出了以后筛选的途径,即首先筛选ＹＡＣ库的ＭｅｇａＹＡＣ部分,并以５块板为一组进行筛选。另外,运用荧光原位杂交技术（ＦＢＨ）,对ＣＥＰＨＹＡＣ库的质量及其第一代人类基因组物理图谱进行了考察。我们取２６个ＹＡＣ克隆进行ＦＩＳＨ定位,结果其中嵌合体１３个,占５０％。定位错误的克隆有６个,占２３％。非嵌合体且定位正确的共９个,占３５％。     

Electron Microscope Observations of the Melanocyte of the Human Epidermis

Using standard osmium fixation and methacrylate embedding techniques, a study has been made of the melanocyte of human biopsy skin removed under general and local anaesthesia. Melanogenesis was easily observable in the melanocytes, but immature pigment granules were rarely seen in the Malpighian cells. The passage of melanin from melanocyte to Malpighian cell—cytocrine secretion—is thought to have been observed. Phagocytes near the dermal-epidermal junction seem to have their pigment granules in vacuoles, rather than surrounded directly by the cytoplasmic matrix as in the melanocytes. This, together with the failure to observe "effete" melanocytes, prompts the suggestion that the phagocytes are melanocytes which have migrated from the epidermis into the dermis. A melanin granule is shown with alternating dark and lighter transverse striations, concerning which structure little can at present be said.     

How to Build a Multiscale Model in Biology

Biological processes span several scales in space, from the single molecules to organisms and ecosystems. Multiscale modelling approaches in biology are useful to take into account the complex interactions between different organisation levels in those systems. We review several single- and multiscale models, from the most simple to the complex ones, and discuss their properties from a multiscale point of view. Approaches based on master equations for stochastic processes, individual-based models, hybrid continuous-discrete models and structured PDE models are presented.     

泽米铁科三种植物的叶表皮特征

在光镜下观察了泽米铁科分属于3个属的3种植物叶表皮特征,结果表明：3种植物叶表皮细胞及气孔器差异显著,一定程度上证实了3属是自然的分类群。在鳞秕泽米铁(Zamia furfuracea)中发现了气孔簇。本文还对3种植物之间的系统演化关系及它们对环境的适应性进行了讨论。     

Cell Communication in the Basal Cells of the Human Epidermis

Electrotonic spread can be measured in the basal cells of the human epidermis. The communication between neighboring cells is high, whereas no leak to the intercellular spaces could be detected. The specific resistance of the membranes between the cells is about 10 Ωcm². This finding suggests that for those particles that are able to pass the cell membrane the intracellular path through the epidermis is at least as suitable as the path through the intercellular spaces.     

Computational Modeling Deduced Three Dimensional Structure of Cry1Ab16 Toxin from Bacillus thuringiensis AC11

The first theoretical structural model of newly reported Cry1Ab16 δ-endotoxin produced by Bacillus thuringiensis AC11 was predicted using homology modeling technique. Cry1Ab16 resembles the Cry1Aa protein structure by sharing a common three domains structure responsible in pore forming and specificity determination along with few structural deviations. The main differences between the two is in the length of loops, absence of α7b, α9a, α10b, α11a and presence of additional β12b, α13 components while α10a is spatially located at downstream position in Cry1Ab16. A better understanding of the 3D structure shall be helpful in the design of domain swapping and mutagenesis experiments aimed at improving toxicity.     

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

Background

Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data.

Methods

FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging.

Results

The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections.

Conclusion

We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.     

A Computational Model Predicting Disruption of Blood Vessel Development

Vascular development is a complex process regulated by dynamic biological networks that vary in topology and state across different tissues and developmental stages. Signals regulating de novo blood vessel formation (vasculogenesis) and remodeling (angiogenesis) come from a variety of biological pathways linked to endothelial cell (EC) behavior, extracellular matrix (ECM) remodeling and the local generation of chemokines and growth factors. Simulating these interactions at a systems level requires sufficient biological detail about the relevant molecular pathways and associated cellular behaviors, and tractable computational models that offset mathematical and biological complexity. Here, we describe a novel multicellular agent-based model of vasculogenesis using the CompuCell3D (http://www.compucell3d.org/) modeling environment supplemented with semi-automatic knowledgebase creation. The model incorporates vascular endothelial growth factor signals, pro- and anti-angiogenic inflammatory chemokine signals, and the plasminogen activating system of enzymes and proteases linked to ECM interactions, to simulate nascent EC organization, growth and remodeling. The model was shown to recapitulate stereotypical capillary plexus formation and structural emergence of non-coded cellular behaviors, such as a heterologous bridging phenomenon linking endothelial tip cells together during formation of polygonal endothelial cords. Molecular targets in the computational model were mapped to signatures of vascular disruption derived from in vitro chemical profiling using the EPA''s ToxCast high-throughput screening (HTS) dataset. Simulating the HTS data with the cell-agent based model of vascular development predicted adverse effects of a reference anti-angiogenic thalidomide analog, 5HPP-33, on in vitro angiogenesis with respect to both concentration-response and morphological consequences. These findings support the utility of cell agent-based models for simulating a morphogenetic series of events and for the first time demonstrate the applicability of these models for predictive toxicology.     

The Emergence of Three Human Development Clubs

We examine the joint distribution of levels of income per capita, life expectancy, and years of schooling across countries in 1960 and in 2000. In 1960 countries were clustered in two groups; a rich, highly educated, high longevity “developed” group and a poor, less educated, high mortality, “underdeveloped” group. By 2000 however we see the emergence of three groups; one underdeveloped group remaining near 1960 levels, a developed group with higher levels of education, income, and health than in 1960, and an intermediate group lying between these two. This finding is consistent with both the ideas of a new “middle income trap” that countries face even if they escape the “low income trap”, as well as the notion that countries which escaped the poverty trap form a temporary “transition regime” along their path to the “developed” group.     

Cx26 Affects the in Vitro Reconstruction of Human Epidermis

To study the function of connexins in human keratinocytes, we have used a three-dimensional culture system, in which a tissue is reconstructed using cells from the outer root sheet of hair follicles. This tissue reproduces in vitro the histological organisation of human epidermis in situ and the normal distribution of several keratinocyte markers. Furthermore, it shows characteristics of a differentiating epidermis, including the expression of connexin26. Connexin26 protein expression is increased under physiological and pathological conditions resulting in increased keratinocyte turnover. Loss of this protein in keratinocytes, obtained from patients carrying a stop mutation, resulted in a reduced stratification of the in vitro reconstructed tissue, probably due to a lower proliferation and migration capacity of the keratinocytes, although dye coupling and persistence of other gap junctions is maintained. No changes were seen in tissues reconstructed with keratinocytes from patients carrying a non stop mutation of connexin30. The data indicate that, at least in vitro, connexin26 affects the function of human keratinocytes, independently of obvious changes in coupling.     

Development of Epidermis on Banana Fruits

Fruit epidermis ofMusa (AAB) cv. Poovan (S) remains single-layered throughout its development. There is no change in stomatal number but its frequency and index decrease due to slight increase in epidermal cell number and size. The external wall of the epidermal cells shows stratification of wall layers that is characteristic of normal epidermal cell with cuticle and epicuticular wax deposits. Surface wax deposits show qualitative and quantitative variations during fruit development and ripening.     

激光扫描共聚焦显微镜对人类染色体三维结构的观察

为了获得染色体内部结构的多种信息,以及对染色体形态构建提供有益的尝试,本试验利用荧光染料的特异性标记及激光扫描共聚焦显微镜的连续断层扫描和三维重建的特点,对人类染色体的形态结构进行观测,结果显示：本方法不仅能显示染色体的荧光带纹的分布状况,而且能作染色体内部的一系列光学切片和染色体三维结构的观测。     

Three Dimensional Structure of Haemoglobin Rainier

The difference electron density map of deoxyhaemoglobin Rainier (α₂β₂^145Tyr→Cys) shows that the cysteine introduced by the mutation forms a disulphide bridge with the reactive cysteine, F9(93)β, of the same β-chain. The resulting structural distortions affect the C-terminus of the β-chain, thereby inhibiting part of the alkaline Bohr effect and haem–haem interaction.