Sex- and estrous-cycle dependent dorsal hippocampal phosphoproteomic changes induced by low-dose ketamine.

Numerous emotional and cognitive processes mediated by the hippocampus present differences between sexes and can be markedly influenced by hormonal status in males and females of several species. In rodents, the dorsal hippocampus (dHPC) is known to contribute to the rapid antidepressant actions of the NMDA receptor antagonist ketamine. We and others have demonstrated a greater sensitivity to the fast-acting antidepressant ketamine in female versus male rats that is estrogen- and progesterone-dependent.

3D and 4D Tumorigenesis Model for the Quantitative Analysis of Cancer Cell Behavior and Screening for Anticancer Drugs.

Cancer cells from cell lines and tumor biopsy tissue undergo aggregation and aggregate coalescence when dispersed in a 3D Matrigel™ matrix. Coalescence is a dynamic process mediated by a subset of cells within the population of cancer cells. In contrast, non-tumorigenic cells from normal cell lines and normal tissues do not aggregate or coalesce, nor do they possess the motile cell types that orchestrate coalescence of cancer cells.

Directed movement toward, translocation along, penetration into and exit from vascular networks by breast cancer cells in 3D.

We developed a computer-assisted platform using laser scanning confocal microscopy to 3D reconstruct in real-time interactions between metastatic breast cancer cells and human umbilical vein endothelial cells (HUVECs). We demonstrate that MB-231 cancer cells migrate toward HUVEC networks, facilitated by filopodia, migrate along the network surfaces, penetrate into and migrate within the HUVEC networks, exit and continue migrating along network surfaces. The system is highly amenable to 3D reconstruction and computational analyses, and assessments of the effects of potential anti-metastasis monoclonal antibodies and other drugs.

Reciprocal signaling and direct physical interactions between fibroblasts and breast cancer cells in a 3D environment.

Tumorigenic cells undergo cell aggregation and aggregate coalescence in a 3D Matrigel environment. Here, we expanded this 3D platform to assess the interactions of normal human dermal fibroblasts (NHDFs) and human primary mammary fibroblasts (HPMFs) with breast cancer-derived, tumorigenic cells (MDA-MB-231). Medium conditioned by MDA-MB-231 cells activates both types of fibroblasts, imbuing them with the capacity to accelerate the rate of aggregation and coalescence of MDA-MB-231 cells more than four fold.

Quantification of Urinary Protein Biomarkers of Autosomal Dominant Polycystic Kidney Disease by Parallel Reaction Monitoring.

Purpose: Autosomal dominant polycystic kidney disease (ADPKD) is a life-long disease in which the genes responsible are known, but the pathogenesis of cyst formation and cyst growth are not understood. Cyst growth ultimately leads to end-stage renal failure in most patients. Analysis of the urinary proteome offers the potential to identify proteins that indicate the presence of cysts (and thus provides diagnosis) as well as the rates of cyst growth (providing prognostic information).

A multiregional proteomic survey of the postnatal human brain.

Detailed observations of transcriptional, translational and post-translational events in the human brain are essential to improving our understanding of its development, function and vulnerability to disease. Here, we exploited label-free quantitative tandem mass-spectrometry to create an in-depth proteomic survey of regions of the postnatal human brain, ranging in age from early infancy to adulthood. Integration of protein data with existing matched whole-transcriptome sequencing (RNA-seq) from the BrainSpan project revealed varied patterns of protein-RNA relationships, with generally increased magnitudes of protein abundance differences between brain regions compared to RNA.

Melanoma cells undergo aggressive coalescence in a 3D Matrigel model that is repressed by anti-CD44.

Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid. Non-tumorigenic cells did not undergo coalescence. Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model.

4D Tumorigenesis Model for Quantitating Coalescence, Directed Cell Motility and Chemotaxis, Identifying Unique Cell Behaviors, and Testing Anticancer Drugs.

A 4D high-resolution computer-assisted reconstruction and motion analysis system has been developed and applied to the long-term (14-30 days) analysis of cancer cells migrating and aggregating within a 3D matrix. 4D tumorigenesis models more closely approximate the tumor microenvironment than 2D substrates and, therefore, are improved tools for elucidating the interactions within the tumor microenvironment that promote growth and metastasis. The model we describe here can be used to analyze the growth of tumor cells, aggregate coalescence, directed cell motility and chemotaxis, matrix degradation, the effects of anticancer drugs, and the behavior of immune and endothelial cells mixed with cancer cells.

Mediated coalescence: a possible mechanism for tumor cellular heterogeneity.

Recently, we demonstrated that tumorigenic cell lines and fresh tumor cells seeded in a 3D Matrigel model, first grow as clonal islands (primary aggregates), then coalesce through the formation and contraction of cellular cables. Non-tumorigenic cell lines and cells from normal tissue form clonal islands, but do not form cables or coalesce. Here we show that as little as 5% tumorigenic cells will actively mediate coalescence between primary aggregates of majority non-tumorigenic or non-cancerous cells, by forming cellular cables between them.

Quantitative Motion Analysis in Two and Three Dimensions.

This chapter describes 2D quantitative methods for motion analysis as well as 3D motion analysis and reconstruction methods. Emphasis is placed on the analysis of dynamic cell shape changes that occur through extension and retraction of force generating structures such as pseudopodia and lamellipodia. Quantitative analysis of these structures is an underutilized tool in the field of cell migration.

A computer-assisted 3D model for analyzing the aggregation of tumorigenic cells reveals specialized behaviors and unique cell types that facilitate aggregate coalescence.

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction.

Computer-assisted analysis of filopod formation and the role of myosin II heavy chain phosphorylation in Dictyostelium.

To investigate the role played by filopodia in the motility and chemotaxis of amoeboid cells, a computer-assisted 3D reconstruction and motion analysis system, DIAS 4.0, has been developed. Reconstruction at short time intervals of Dictyostelium amoebae migrating in buffer or in response to chemotactic signals, revealed that the great majority of filopodia form on pseudopodia, not on the cell body; that filopodia on the cell body originate primarily on pseudopodia and relocate; and that filopodia on the uropod are longer and more stable than those located on other portions of the cell.

Three-dimensional structures of idiotypically related Fabs with intermediate and high affinity for fluorescein.

Multi-disciplinary studies of fluorescein-protein conjugates have led to the generation of a family of antibodies with common idiotypes and affinities for fluorescein ranging over five orders of magnitude. The high affinity 4-4-20 prototype traps the ligand in a highly complementary binding slot, which is lined by multiple aromatic side-chains. An antibody (9-40) of intermediate affinity belongs to the same idiotypic family as 4-4-20 and shares substantial amino acid identities within the VL and VH domains.

Computer-assisted reconstruction and motion analysis of the three-dimensional cell.

Even though several microscopic techniques provide three-dimensional (3D) information on fixed and living cells, the perception persists that cells are two-dimensional (2D). Cells are, in fact, 3D and their behavior, including the extension of pseudopods, includes an important 3D component. Although treating the cell as a 2D entity has proven effective in understanding how cells locomote, and in identifying defects in a variety of mutant and abnormal cells, there are cases in which 3D reconstruction and analysis are essential.

Discrete bathochromic shifts exhibited by fluorescein ligand bound to rabbit polyclonal anti-fluorescein Fab fragments.

Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e.

3D-DIASemb: a computer-assisted system for reconstructing and motion analyzing in 4D every cell and nucleus in a developing embryo.

A computer-assisted three-dimensional (3D) system, 3D-DIASemb, has been developed that allows reconstruction and motion analysis of cells and nuclei in a developing embryo. In the system, 75 optical sections through a live embryo are collected in the z axis by using differential interference contrast microscopy. Optical sections for one reconstruction are collected in a 2.