Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects

Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors.We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions.HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations

Mycoplasma hominis deep wound infection after neuromuscular scoliosis surgery: the use of real-time polymerase chain reaction (PCR)

Mycoplasma hominis is a commensal of the genitourinary tract. It mostly causes infections to associated structures of this system; however, occasionally it is a pathogen in nongenitourinary tract infections. Since, M. hominis strains require special growth conditions and cannot be Gram stained, they may be missed or delay diagnosis. This report describes a deep wound infection caused by M. hominis after neuromuscular scoliosis surgery; M. hominis was recovered by real-time polymerase chain reaction (PCR). An awareness of the role of M. hominis as an extragenital pathogen in musculoskeletal infections, especially in neuromuscular scoliosis, being a high-risk group for postoperative wound infection, it is necessary to identify this pathogen. Real-time PCR for postoperative deep wound infection, in patients with a history of genitourinary infections, decreases the delay in diagnosis and treatment. In these cases rapid real-time PCR on deep cultures should be considered

Implementation of a program for type 2 diabetes based on the Chronic Care Model in a hospital-centered health care system: "the Belgian experience"

Background: Most research publications on Chronic Care Model (CCM) implementation originate from organizations or countries with a well-structured primary health care system. Information about efforts made in countries with a less well-organized primary health care system is scarce. In 2003, the Belgian National Institute for Health and Disability Insurance commissioned a pilot study to explore how care for type 2 diabetes patients could be organized in a more efficient way in the Belgian healthcare setting, a setting where the organisational framework for chronic care is mainly hospital-centered. Methods: Process evaluation of an action research project (2003-2007) guided by the CCM in a well-defined geographical area with 76,826 inhabitants and an estimated number of 2,300 type 2 diabetes patients. In consultation with the region a program for type 2 diabetes patients was developed. The degree of implementation of the CCM in the region was assessed using the Assessment of Chronic Illness Care survey (ACIC). A multimethod approach was used to evaluate the implementation process. The resulting data were triangulated in order to identify the main facilitators and barriers encountered during the implementation process. Results: The overall ACIC score improved from 1.45 (limited support) at the start of the study to 5.5 (basic support) at the end of the study. The establishment of a local steering group and the appointment of a program manager were crucial steps in strengthening primary care. The willingness of a group of well-trained and motivated care providers to invest in quality improvement was an important facilitator. Important barriers were the complexity of the intervention, the lack of quality data, inadequate information technology support, the lack of commitment procedures and the uncertainty about sustainable funding. Conclusion: Guided by the CCM, this study highlights the opportunities and the bottlenecks for adapting chronic care delivery in a primary care system with limited structure. The study succeeded in achieving a considerable improvement of the overall support for diabetes patients but further improvement requires a shift towards system thinking among policy makers. Currently primary care providers lack the opportunities to take up full responsibility for chronic care

Systems microscopy approaches to understand cancer cell migration and metastasis

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration

An ultrasoft X-ray multi-microbeam irradiation system for studies of DNA damage responses by fixed- and live-cell fluorescence microscopy

Localized induction of DNA damage is a valuable tool for studying cellular DNA damage responses. In recent decades, methods have been developed to generate DNA damage using radiation of various types, including photons and charged particles. Here we describe a simple ultrasoft X-ray multi-microbeam system for high dose-rate, localized induction of DNA strand breaks in cells at spatially and geometrically adjustable sites. Our system can be combined with fixed- and live-cell microscopy to study responses of cells to DNA damage

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field

A comprehensive overview of radioguided surgery using gamma detection probe technology

The concept of radioguided surgery, which was first developed some 60 years ago, involves the use of a radiation detection probe system for the intraoperative detection of radionuclides. The use of gamma detection probe technology in radioguided surgery has tremendously expanded and has evolved into what is now considered an established discipline within the practice of surgery, revolutionizing the surgical management of many malignancies, including breast cancer, melanoma, and colorectal cancer, as well as the surgical management of parathyroid disease. The impact of radioguided surgery on the surgical management of cancer patients includes providing vital and real-time information to the surgeon regarding the location and extent of disease, as well as regarding the assessment of surgical resection margins. Additionally, it has allowed the surgeon to minimize the surgical invasiveness of many diagnostic and therapeutic procedures, while still maintaining maximum benefit to the cancer patient. In the current review, we have attempted to comprehensively evaluate the history, technical aspects, and clinical applications of radioguided surgery using gamma detection probe technology