Profiling of toxicity and identification of distinct apoptosis profiles using a 384-well high-throughput flow cytometry screening platform.

Methods and techniques used to detect apoptosis have benefited from advances in technologies such as flow cytometry. With a large arsenal of lasers, fluorescent labels, and readily accessible biological targets, it is possible to detect multiple targets with unique combinations of fluorescent spectral signatures from a single sample. Traditional flow cytometry has been limited as a screening tool as the sample throughput has been low, whereas the data analysis and generation of screening relevant results have been complex. The HTFC Screening System running ForeCyt software is an instrument platform designed to perform high-throughput, multiplexed screening with seamless transformation of flow cytometry data into screening hits. We report the results of a screen that simultaneously quantified caspase 3/7 activation, annexin V binding, cell viability, and mitochondrial integrity. Assay performance over 5 days demonstrated robustness, reliability, and performance of the assay. This system is high throughput in that a 384-well plate can be read and fully analyzed within 30 min and is sensitive with an assay window of at least 10-fold for all parameters and a Z' factor of ≥0.75 for all endpoints and time points. From a screen of 231 compounds, 11 representative toxicity profiles highlighting differential activation of apoptotic pathways were identified.

Low-level vibrations retain bone marrow's osteogenic potential and augment recovery of trabecular bone during reambulation.

Mechanical disuse will bias bone marrow stromal cells towards adipogenesis, ultimately compromising the regenerative capacity of the stem cell pool and impeding the rapid and full recovery of bone morphology. Here, it was tested whether brief daily exposure to high-frequency, low-magnitude vibrations can preserve the marrow environment during disuse and enhance the initiation of tissue recovery upon reambulation. Male C57BL/6J mice were subjected to hindlimb unloading (HU, n = 24), HU interrupted by weight-bearing for 15 min/d (HU+SHAM, n = 24), HU interrupted by low-level whole body vibrations (0.2 g, 90 Hz) for 15 min/d (HU+VIB, n = 24), or served as age-matched controls (AC, n = 24). Following 3 w of disuse, half of the mice in each group were released for 3 w of reambulation (RA), while the others were sacrificed. RA+VIB mice continued to receive vibrations for 15 min/d while RA+SHAM continued to receive sham loading. After disuse, HU+VIB mice had a 30% greater osteogenic marrow stromal cell population, 30% smaller osteoclast surface, 76% greater osteoblast surface but similar trabecular bone volume fraction compared to HU. After 3 w of reambulation, trabecular bone of RA+VIB mice had a 30% greater bone volume fraction, 51% greater marrow osteoprogenitor population, 83% greater osteoblast surfaces, 59% greater bone formation rates, and a 235% greater ratio of bone lining osteoblasts to marrow adipocytes than RA mice. A subsequent experiment indicated that receiving the mechanical intervention only during disuse, rather than only during reambulation, was more effective in altering trabecular morphology. These data indicate that the osteogenic potential of bone marrow cells is retained by low-magnitude vibrations during disuse, an attribute which may have contributed to an enhanced recovery of bone morphology during reambulation.

Autophagy regulates adipose mass and differentiation in mice.

The relative balance between the quantity of white and brown adipose tissue can profoundly affect lipid storage and whole-body energy homeostasis. However, the mechanisms regulating the formation, expansion, and interconversion of these 2 distinct types of fat remain unknown. Recently, the lysosomal degradative pathway of macroautophagy has been identified as a regulator of cellular differentiation, suggesting that autophagy may modulate this process in adipocytes. The function of autophagy in adipose differentiation was therefore examined in the current study by genetic inhibition of the critical macroautophagy gene autophagy-related 7 (Atg7). Knockdown of Atg7 in 3T3-L1 preadipocytes inhibited lipid accumulation and decreased protein levels of adipocyte differentiation factors. Knockdown of Atg5 or pharmacological inhibition of autophagy or lysosome function also had similar effects. An adipocyte-specific mouse knockout of Atg7 generated lean mice with decreased white adipose mass and enhanced insulin sensitivity. White adipose tissue in knockout mice had increased features of brown adipocytes, which, along with an increase in normal brown adipose tissue, led to an elevated rate of fatty acid, beta-oxidation, and a lean body mass. Autophagy therefore functions to regulate body lipid accumulation by controlling adipocyte differentiation and determining the balance between white and brown fat.

Automated separation of visceral and subcutaneous adiposity in in vivo microcomputed tomographies of mice.

Reflecting its high resolution and contrast capabilities, microcomputed tomography (microCT) can provide an in vivo assessment of adiposity with excellent spatial specificity in the mouse. Herein, an automated algorithm that separates the total abdominal adiposity into visceral and subcutaneous compartments is detailed. This algorithm relies on Canny edge detection and mathematical morphological operations to automate the manual contouring process that is otherwise required to spatially delineate the different adipose deposits. The algorithm was tested and verified with microCT scans from 74 C57BL/6J mice that had a broad range of body weights and adiposity. Despite the heterogeneity within this sample of mice, the algorithm demonstrated a high degree of stability and robustness that did not necessitate changing of any of the initially set input variables. Comparisons of data between the automated and manual methods were in complete agreement (R (2) = 0.99). Compared to manual contouring, the increase in precision and accuracy, while decreasing processing time by at least an order of magnitude, suggests that this algorithm can be used effectively to separately assess the development of total, visceral, and subcutaneous adiposity. As an application of this method, preliminary data from adult mice suggest that a relative increase in either subcutaneous, visceral, or total fat negatively influences skeletal quantity and that fat infiltration in the liver is greatly increased by a high-fat diet.

Mechanical Signals As a Non-Invasive Means to Influence Mesenchymal Stem Cell Fate, Promoting Bone and Suppressing the Fat Phenotype.

Pluripotent mesenchymal stem cells (MSCs) are considered ideal therapeutic targets in regenerative medicine, as they hold the capacity to differentiate into higher order connective tissues. The potential to harness MSCs for disease treatment and acceleration of repair will ultimately depend on an improved understanding of how physical and/or chemical signals regulate their activity, and the ability of exogenous stimuli to enhance MSC proliferation and define MSC fate. Recent appreciation that bone marrow osteoprogenitors are inversely proportional to adipocyte precursors suggests that their shared progenitor, the MSC, will commit to one lineage at the cost of the other. This interrelationship may contribute to the phenotype of sedentary subjects who have more fat and less bone, while conversely, to the outcome of exercise being less fat and more bone. Mechanical biasing of MSC lineage selection suggests that physical signals may influence the quantity of both fat and bone through developmental, as well as metabolic or adaptive pathways. Considered with the recent finding that low magnitude mechanical signals (LMMS) suppress the development of subcutaneous and visceral fat without elevating energy expenditure, this indicates that MSCs are ideally positioned as mechanosensitive elements central to musculoskeletal adaptation, but that the signals needn't be large to be influential. The biasing of MSC differentiation by mechanical signals represents a unique means by which adiposity can be inhibited while simultaneously promoting a better skeleton, and may provide the basis for a safe, non-invasive, non-pharmacologic strategy to prevent both obesity and osteoporosis, yet uniquely - without targeting the resident fat or bone cell.

In vitro non-viral gene delivery with nanofibrous scaffolds.

Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (beta-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1x TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L8E78L8), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro.

Incorporation and controlled release of a hydrophilic antibiotic using poly(lactide-co-glycolide)-based electrospun nanofibrous scaffolds.

The successful incorporation and sustained release of a hydrophilic antibiotic drug (Mefoxin, cefoxitin sodium) from electrospun poly(lactide-co-glycolide) (PLGA)-based nanofibrous scaffolds without the loss of structure and bioactivity was demonstrated. The morphology and density of the electrospun scaffold was found to be dependent on the drug concentration, which could be attributed to the effect of ionic salt on the electrospinning process. The drug release behavior from the electrospun scaffolds and its antimicrobial effects on Staphylococcus aureus cultures were also investigated. In all tested scaffolds, the maximum dosage of drug was released after 1 h of incubation in water at 37 degrees C. The usage of the amphiphilic block copolymer (PEG-b-PLA) reduced the cumulative amount of the released drug at earlier time points and prolonged the drug release rate at longer times (up to a 1-week period). The antibiotic drug released from these electrospun scaffolds was effective in their ability to inhibit Staphylococcus aureus growth (>90%). The combination of mechanical barriers based on non-woven nanofibrous biodegradable scaffolds and their capability for local delivery of antibiotics increases their desired utility in biomedical applications, particularly in the prevention of post-surgical adhesions and infections.