Pathological alterations in the expression status of rotator cuff tendon matrix components in hyperlipidemia.

Hyperlipidemia is an important risk factor in the development and progression of tendon pathology, however its role in aggravating rotator cuff tendon injury (RCTI) is largely unknown. We aimed to assess the expression status of key extracellular matrix (ECM) components in the tendon tissues and tenocytes under hyperlipidemia. Shoulder rotator cuff (RC) tendon tissues harvested from the swine model of hyperlipidemia displayed alterations in histomorphometry and the expression status of major ECM component proteins including COL-I, COL-III, COL-IV, COL-V, COL-VI, MMP2, and MMP9. Similarly, the LDL- and oxLDL-challenged tenocytes displayed altered expression of the same proteins at both transcriptional and translational levels. In addition, the lipid uptake and cellular reactive oxygen radicals predominated in the lipid-challenged tenocytes compared to the control. Overall, the LDL-treated cells displayed predominant pathological alterations compared to the ox-LDL-treated cells. Further understanding regarding the underlying molecular mechanisms driving the tendon matrisome alteration and subsequent aggravated RCTI pathology in hyperlipidemia could open novel translational avenues in the management of RCTI.

A microfluidic device for the automated derivatization of free fatty acids to fatty acid methyl esters.

Free fatty acid (FFA) compositions are examined in feedstock for biodiesel production, as source-specific markers in soil, and because of their role in cellular signaling. However, sample preparation of FFAs for gas chromatography-mass spectrometry (GC-MS) analysis can be time and labor intensive. Therefore, to increase sample preparation throughput, a glass microfluidic device was developed to automate derivatization of FFAs to fatty acid methyl esters (FAMEs). FFAs were delivered to one input of the device and methanolic-HCl was delivered to a second input. FAME products were produced as the reagents traversed a 29 µL reaction channel held at 55 °C. A Design of Experiment protocol was used to determine the combination of derivatization time (T(der)) and ratio of methanolic-HCl:FFA (R(der)) that maximized the derivatization efficiencies of tridecanoic acid and stearic acid to their methyl ester forms. The combination of T(der) = 0.8 min and R(der) = 4.9 that produced optimal derivatization conditions for both FFAs within a 5 min total sample preparation time was determined. This combination of T(der) and R(der) was used to derivatize 12 FFAs with a range of derivatization efficiencies from 18% to 93% with efficiencies of 61% for tridecanoic acid and 84% for stearic acid. As compared to a conventional macroscale derivatization of FFA to FAME, the microfluidic device decreased the volume of methanolic-HCl and FFA by 20- and 1300-fold, respectively. The developed microfluidic device can be used for automated preparation of FAMEs to analyze the FFA compositions of volume-limited samples.

A new model isolates glioblastoma clonal interactions and reveals unexpected modes for regulating motility, proliferation, and drug resistance.

Tumor clonal heterogeneity drives treatment resistance. But robust models are lacking that permit eavesdropping on the basic interaction network of tumor clones. We developed an in vitro, functional model of clonal cooperation using U87MG glioblastoma cells, which isolates fundamental clonal interactions. In this model pre-labeled clones are co-cultured to track changes in their individual motility, growth, and drug resistance behavior while mixed. This highly reproducible system allowed us to address a new class of fundamental questions about clonal interactions. We demonstrate that (i) a single clone can switch off the motility of the entire multiclonal U87MG cell line in 3D culture, (ii) maintenance of clonal heterogeneity is an intrinsic and influential cancer cell property, where clones coordinate growth rates to protect slow growing clones, and (iii) two drug sensitive clones can develop resistance de novo when cooperating. Furthermore, clonal communication for these specific types of interaction did not require diffusible factors, but appears to depend on cell-cell contact. This model constitutes a straightforward but highly reliable tool for isolating the complex clonal interactions that make up the fundamental "hive mind" of the tumor. It uniquely exposes clonal interactions for future pharmacological and biochemical studies.

Establishment of a reference standard database for use in the qualitative and semi-quantitative analysis of pharmaceutical contact materials within an extractables survey by GC-MS.

The analysis of reference standards may be performed to enhance the qualitative and quantitative data generated by non-specific screening methods utilized in extractables studies performed on pharmaceutical contact materials. However, the establishment of a database containing relative response factor and retention index values obtained from these standards has not been published. In this study, the establishment of such a database for GC-MS, a methodology commonly included in extractables studies, on an intra-lab basis was investigated. A set of 154 organic compounds representing a diverse range of chemical functionalities and properties was analyzed at eight time points on four GC-MS instruments that represent the diversity of age and model at our laboratory. The results of this study have shown that any variance in relative response factor between instruments was not significant from a practical perspective as supported by the coefficient of variation values (n = 32), which were ≤15% and ≤10% for 75% and 45% of the compounds tested, respectively. Furthermore, the retention index of the compounds, as expressed by retention time and relative retention time, did not have more than a 2% coefficient of variation between instruments or columns in most cases. It was concluded that a database of these values could be established for future use in extractables studies on an intra-laboratory basis.

Recent advances in microfluidic detection systems.

There are numerous detection methods available for microfluidic analyses. Both conventional and novel detection methods are being put to use for detection on these miniaturized systems, with the analyte of interest driving the choice of detection method. In this article, we summarize microfluidic-based detection strategies from the last 2 years. More focus is given to unconventional approaches to detection routes and novel strategies for performing high-sensitivity detection.

Mitochondrial proton leak rates in the slow, oxidative myotomal muscle and liver of the endothermic shortfin mako shark (Isurus oxyrinchus) and the ectothermic blue shark (Prionace glauca) and leopard shark (Triakis semifasciata).

Mitochondrial proton leak was assessed as a potential heat source in the slow, oxidative (red) locomotor muscle and liver of the shortfin mako shark (Isurus oxyrinchus), a regional endotherm that maintains the temperature of both tissues elevated above ambient seawater temperature. We hypothesized that basal proton leak rates in red muscle and liver mitochondria of the endothermic shortfin mako shark would be greater than those of the ectothermic blue shark (Prionace glauca) and leopard shark (Triakis semifasciata). Respiration rate and membrane potential in isolated mitochondria were measured simultaneously at 20 degrees C using a Clark-type oxygen electrode and a lipophilic probe (triphenylmethylphosphonium, TPMP(+)). Succinate-stimulated respiration was titrated with inhibitors of the electron transport chain, and the non-linear relationship between respiration rate and membrane potential was quantified. Mitochondrial densities of both tissues were measured by applying the point-contact method to electron micrographs so that proton leak activity of the entire tissue could be assessed. In all three shark species, proton leak occurred at a higher rate in red muscle mitochondria than in liver mitochondria. For each tissue, the proton leak curves of the three species overlapped and, at a membrane potential of 160 mV, mitochondrial proton leak rate (nmol H(+) min(-1) mg(-1) protein) did not differ significantly between the endothermic and ectothermic sharks. This finding indicates that red muscle and liver mitochondria of the shortfin mako shark are not specialized for thermogenesis by having a higher proton conductance. However, mako mitochondria did have higher succinate-stimulated respiration rates and membrane potentials than those of the two ectothermic sharks. This means that under in vivo conditions mitochondrial proton leak rates may be higher in the mako than in the ectothermic species, due to greater electron transport activity and a larger proton gradient driving proton leak. We also estimated each tissue's total proton leak by combining mitochondrial proton leak rates at 160 mV and tissue mitochondrial density data with published values of relative liver or red muscle mass for each of the three species. In red muscle, total proton leak was not elevated in the mako shark relative to the two ectothermic species. In the liver, total proton leak would be higher in the mako shark than in both ectothermic species, due to a lower proton conductance in the blue shark and a lower liver mitochondrial content in the leopard shark, and thus may contribute to endothermy.