Suppression of Ca2+ signaling enhances melanoma progression.

The role of store-operated Ca2+ entry (SOCE) in melanoma metastasis is highly controversial. To address this, we here examined UV-dependent metastasis, revealing a critical role for SOCE suppression in melanoma progression. UV-induced cholesterol biosynthesis was critical for UV-induced SOCE suppression and subsequent metastasis, although SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression was responsible for UV-dependent differences in gene expression associated with both increased invasion and reduced glucose metabolism. Functional analyses further established that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed cholesterol biosynthesis-dependent reduced SOCE. Invasiveness could be reversed with either cholesterol biosynthesis inhibitors or pharmacological SOCE potentiation. Collectively, we provide evidence that, contrary to current thinking, Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis.

Infrared Spectroscopy-Determined Bone Compositional Changes Associated with Anti-Resorptive Treatment of the oim/oim Mouse Model of Osteogenesis Imperfecta.

Applications of vibrational spectroscopy to assess bone disease and therapeutic interventions are continually advancing, with tissue mineral and protein composition frequently investigated. Here, we used two spectroscopic approaches for determining bone composition in a mouse model (oim) of the brittle bone disease osteogenesis imperfecta (OI) with and without antiresorptive agent treatment (alendronate, or ALN, and RANK-Fc). Near-infrared (NIR) spectral analysis using a fiber optic probe and attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) mode were applied to investigate bone composition, including water, mineral, and protein content. Spectral parameters revealed differences among the control wildtype (WT) and OIM groups. NIR spectral analysis of protein and water showed that OIM mouse humerii had ∼50% lower protein and ∼50% higher overall water content compared to WT bone. Moreover, some OIM-treated groups showed a reduction in bone water compared to OIM controls, approximating values observed in WT bone. Differences in bone quality based on increased mineral content and reduced carbonate content were also found between some groups of treated OIM and WT bone, but crystallinity did not differ among all groups. The spectroscopically determined parameters were evaluated for correlations with gold-standard mechanical testing values to gain insight into how composition influenced bone strength. As expected, bone mechanical strength parameters were consistently up to threefold greater in WT mice compared to OIM groups, except for stiffness in the ALN-treated OIM groups. Furthermore, bone stiffness, maximum load, and post-yield displacement showed the strongest correlations with NIR-determined protein content (positive correlations) and bound-water content (negative correlations). These results demonstrate that in this study, NIR spectral parameters were more sensitive to bone composition differences than ATR parameters, highlighting the potential of this nondestructive approach for screening of bone diseases and therapeutic efficacy in pre-clinical models.

Glucose consumption of vascular cell types in culture: toward optimization of experimental conditions.

In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and adventitial fibroblasts (AFs) with or without angiotensin II (AngII) stimulation. In a subconfluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-h incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at ∼275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AFs and VSMCs but not in ECs. Enhanced extracellular acidification rate by AngII was also observed in AFs. In AFs, AngII induction of target proteins at 48 h varied depending on the glucose concentration used. In low glucose media, induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggests essential roles of angiotensin II type-1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, this study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

Clinical application of near infrared fiber optic spectroscopy for noninvasive bone assessment.

Approaches for noninvasive bone quality assessment are of great clinical need, particularly in individuals that require close monitoring of disease progression. X-ray measurements are standard approaches to assess bone quality; however, they have several disadvantages. Here, a nonionizing approach for noninvasive assessment of the second metacarpal bone based on near infrared (NIR) spectroscopy was investigated. Transcutaneous bone signal detection was experimentally confirmed with cadaveric hand data, and Monte Carlo modeling further indicated that 50% of the measured signals arise from bone. Spectral data were collected via a NIR fiber optic from the bone of individuals with osteogenesis imperfecta, a disease marked by frequent bone fractures and fragility. Multiple significant correlations were found between spectral parameters related to water, protein and fat, and standard bone quality parameters obtained by X-ray measurements. The results from this preliminary study highlight the potential application of NIR spectroscopy for the noninvasive assessment of bone quality.

Fourier transform infrared spectroscopy of developing bone mineral: from amorphous precursor to mature crystal.

Bone mineral development has been described to proceed through an amorphous precursor prior to apatite crystallization. However, further analytical approaches are necessary to identify specific markers of amorphous mineral components in bone. Here, we establish an original Fourier transform infrared (FTIR) spectroscopy approach to allow the specific identification of the amorphous and/or crystalline nature of bone mineral. Using a series of standards, our results demonstrate that obtaining the second derivative of the FTIR spectra could reveal a peak specifically corresponding to amorphous calcium phosphate (ACP) at ∼992 cm-1. The intensity of this peak was strongly correlated to ACP content in standard mixtures. The analysis of a variety of bones showed that a clear ACP peak could be identified as a specific marker of the existence of an amorphous mineral component in developing bones. In contrast, the ACP peak was not detected in the mature bones. Moreover, subjecting developing bones to ex vivo crystallization conditions led to a clear reduction of the ACP peak, further substantiating the conversion of amorphous mineral precursor into mature apatite crystals. Analysis of mineralization in osteogenic cell cultures corroborated our observations, showing the presence of ACP as a major transient component in early mineralization, but not in the mature matrix. Additionally, FTIR imaging revealed that ACP was present in areas of matrix development, distributed around the edges of mineralizing nodules. Using an original analytical approach, this work provides strong evidence to support that bone mineral development is initiated by an amorphous precursor prior to apatite crystallization.