Quantitative histochemical analysis of human artery using Raman spectroscopy.

We have developed a method for using near infrared Raman spectroscopy to quantitatively analyze the histochemical composition of human artery. The main contributors to bands observed in the Raman spectra of normal and atherosclerotic aorta are the proteins collagen and elastin, cholesterol lipids, and calcium hydroxyapatite. The Raman scattering cross-sections of different bands for these components have been determined in order to understand their relative contributions to the Raman spectra of biological tissue. The Raman signal is observed to behave linearly with the concentration of the components, even in a highly scattering medium such as a powder. Using these data, we have developed a linear model that can be used to extract the quantitative contribution of an individual component to the spectrum of a mixture. The model has been applied to several mixtures of known composition of tissue constituents in order to evaluate its precision and accuracy. The calculated fit coefficients from the spectra are in agreement with the measured values within experimental uncertainties. The spectra of different types of atherosclerotic aorta have also been modeled, and we have extracted quantitative information regarding the relative concentration of biological constituents in atherosclerotic aorta.     

Breast Cancer Stem Cell Potency of Nickel(II)-Polypyridyl Complexes Containing Non-steroidal Anti-inflammatory Drugs

We report the breast cancer stem cell (CSC) potency of two nickel(II)-3,4,7,8-tetramethyl-1,10-phenanthroline complexes, 1 and 3 , containing the non-steroidal anti-inflammatory drugs (NSAIDs), naproxen and indomethacin, respectively. The nickel(II) complexes, 1 and 3 kill breast CSCs and bulk breast cancer cells in the micromolar range. Notably, 1 and 3 display comparable or better potency towards breast CSCs than salinomycin, an established CSC-active agent. The complexes, 1 and 3 also display significantly lower toxicity towards non-cancerous epithelial breast cells than breast CSCs or bulk breast cancer cells (up to 4.6-fold). Mechanistic studies suggest that 1 and 3 downregulate cyclooxygenase-2 (COX-2) in breast CSCs and kill breast CSCs in a COX-2 dependent manner. Furthermore, the potency of 1 and 3 towards breast CSCs decreased upon co-treatment with necroptosis inhibitors (necrostatin-1 and dabrafenib), implying that 1 and 3 induce necroptosis, an ordered form of necrosis, in breast CSCs. As apoptosis resistance is a hallmark of CSCs, compounds like 1 and 3 , which potentially provide access to alternative (non-apoptotic) cell death pathways could hold the key to overcoming hard-to-kill CSCs. To the best of our knowledge, 1 and 3 are the first compounds to be associated to COX-2 inhibition and necroptosis induction in CSCs.     

Metal-sensitive and thermostable trypsin from the crevalle jack (<Emphasis Type="Italic">Caranx hippos</Emphasis>) pyloric caeca: purification and characterization

Background

Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.

Results

A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-_DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. K _m , k_cat, and k _cat /K _m values using BApNA as substrate were 0.689 mM, 6.9 s^-1, and 10 s^-1 mM^-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd²⁺, Al³⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Hg²⁺ at 1 mM, revealing high sensitivity of the enzyme to metal ions.

Conclusions

Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.

     

Mucin-electrolyte interactions at the solid-liquid interface probed by QCM-D

The interaction between mucin and ions has been investigated by employing the quartz crystal microbalance technique with measurement of energy dissipation. The study was partially aimed at understanding the adsorption of mucin on surfaces with different chemistry, and for this purpose, surfaces exposing COOH, OH, and CH(3) groups were prepared. Mucin adsorbed to all three types of functionalized gold surfaces. Adsorption to the hydrophobic surface and to the charged hydrophilic surface (COOH) occured with high affinity despite the fact that in the latter case both mucin and the surface were negatively charged. On the uncharged hydrophilic surface exposing OH groups, the adsorption of mucin was very low. Another aim was to elucidate conformational changes induced by electrolytes on mucin layers adsorbed on hydrophobic surfaces from 30 mM NaNO(3). To this end, we investigated the effect of three electrolytes with increasing cation valance: NaCl, CaCl(2) and LaCl(3). At low NaCl concentrations, the preadsorbed layer expands, whereas at higher concentrations of NaCl the layer becomes more compact. This swelling/compacting of the mucin layer is fully reversible for NaCl. When the mucin layer instead is exposed to CaCl(2) or LaCl(3), compaction is observed at 1 mM. For CaCl(2), this process is only partially reversible, and for LaCl(3), the changes are irreversible within the time frame of the experiment. Finally, mucin interaction with the DTAB cationic surfactant in an aqueous solution of different electrolytes was evaluated with turbidimetry measurements. It is concluded that the electrolytes used in this work screen the association between mucin and DTAB and that the effect increases with increasing cation valency.     

Imaging voltage-dependent cell motions with heterodyne Mach-Zehnder phase microscopy

We describe a heterodyne Mach-Zehnder interferometric microscope capable of quantitative phase imaging of biological samples with subnanometer sensitivity and frame rates up to 10 kHz. We use the microscope to image cultured neurons and measure nanometer-scale voltage-dependent motions in cells expressing the membrane motor protein prestin.