An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells.

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.

Raman biophysical markers in skin cancer diagnosis.

Raman spectroscopy (RS) has demonstrated great potential for in vivo cancer screening; however, the biophysical changes that occur for specific diagnoses remain unclear. We recently developed an inverse biophysical skin cancer model to address this issue. Here, we presented the first demonstration of in vivo melanoma and nonmelanoma skin cancer (NMSC) detection based on this model. We fit the model to our previous clinical dataset and extracted the concentration of eight Raman active components in 100 lesions in 65 patients diagnosed with malignant melanoma (MM), dysplastic nevi (DN), basal cell carcinoma, squamous cell carcinoma, and actinic keratosis. We then used logistic regression and leave-one-lesion-out cross validation to determine the diagnostically relevant model components. Our results showed that the biophysical model captures the diagnostic power of the previously used statistical classification model while also providing the skin's biophysical composition. In addition, collagen and triolein were the most relevant biomarkers to represent the spectral variances between MM and DN, and between NMSC and normal tissue. Our work demonstrates the ability of RS to reveal the biophysical basis for accurate diagnosis of different skin cancers, which may eventually lead to a reduction in the number of unnecessary excisional skin biopsies performed.

Capillary collagen as the physical transport barrier in drug delivery to tumor microenvironment.

The capillary wall is among the most important barriers that controls mass exchange between tumor microenvironment and systemic circulation. There are numerous studies on endothelial cells role in this mass exchange, but the role of capillary collagen of Type-IV in transport of small molecules and nanotherapeutics is less known. Our recent study revealed that the capillary wall collagen modulates the drug transport across the wall, and that it can be taken as a biophysical marker for drug transport. In our in vivo investigations with the 3LL and 4T1 tumors we noticed the differences in the collagen content in capillary walls. The imaging analysis and transport computational model of the capillary microenvironment showed that the penetration of doxorubicin (DOX) and pegylated liposomal doxorubicin (PLD) is substantially reduced by larger collagen content in the capillaries of the 3LL tumors. The results pointed to the importance of transport oncophysics, which opens a new avenue with respect to classical biology in understanding and improving drug delivery by nanotherapeutics, and aims to better explain the therapeutic resistance.

Sperm volumetric dynamics during in vitro capacitation process in bovine spermatozoa.

Previous studies have demonstrated that sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in bovine spermatozoa. In this study, sperm head morphometry was used to investigate its value as a biophysical marker for detecting volumetric changes in bovine spermatozoa under in vitro capacitating and non-capacitating incubation conditions. To further test this hypotesis, aliquots of pooled, washed bovine sperm were incubated in either Tyrode's complete medium with heparin (TCMH; a capacitating medium containing Ca2+, NaHCO3 and heparin), Tyrode's complete medium heparin-free (TCM; a medium containing just Ca2+ and NaHCO3) or Tyrode's basal medium (TBM; a non-capacitating medium free of Ca2+, NaHCO3 and heparin, used as control). Aliquots of sperm were processed for morphometric analysis at different incubation-time intervals (0, 3 and 6 h at 38°C), and the chlortetracycline assay was used simultaneously to confirm the ability of the sperm to undergo capacitation (B pattern) and the acrosome reaction (AR pattern) status in each medium. After 3 h of incubation under TCMH conditions, a significant increase was observed in the percentage of B and AR patterns and a significant decrease was found in all sperm morphometric parameters (P<0.01). Interestingly, after 6 h of incubation in TCMH, the percentage of B and AR patterns increased drastically over time and marked differences were found in the dimensional and shape parameters, which were significantly smaller compared with TBM or TCM media (P<0.001). Significant correlations were observed between sperm size and AR pattern (r=-0.875, P<0.01). In conclusion, sperm head morphometry can be used as a potential biophysical marker for detecting volumetric changes during capacitation process in bovine spermatozoa.