Two Subpopulations of Human Monocytes That Differ by Mitochondrial Membrane Potential

Atherosclerosis is associated with a chronic local inflammatory process in the arterial wall. Our previous studies have demonstrated the altered proinflammatory activity of circulating monocytes in patients with atherosclerosis. Moreover, atherosclerosis progression and monocyte proinflammatory activity were associated with mitochondrial DNA (mtDNA) mutations in circulating monocytes. The role of mitochondria in the immune system cells is currently well recognized. They can act as immunomodulators by releasing molecules associated with bacterial infection. We hypothesized that atherosclerosis can be associated with changes in the mitochondrial function of circulating monocytes. To test this hypothesis, we performed live staining of the mitochondria of CD14+ monocytes from healthy donors and atherosclerosis patients with MitoTracker Orange CMTMRos dye, which is sensitive to mitochondrial membrane potential. The intensity of such staining reflects mitochondrial functional activity. We found that parts of monocytes in the primary culture were characterized by low MitoTracker staining (MitoTracker-low monocytes). Such cells were morphologically similar to cells with normal staining and able to metabolize 5-aminolevulinic acid and accumulate the heme precursor protoporphyrin IX (PplX), indicative of partially preserved mitochondrial function. We assessed the proportion of MitoTracker-low monocytes in the primary culture for each study subject and compared the results with other parameters, such as monocyte ability to lipopolysaccharide (LPS)-induced proinflammatory activation and the intima-media thickness of carotid arteries. We found that the proportion of MitoTracker-low monocytes was associated with the presence of atherosclerotic plaques. An increased number of such monocytes in the primary culture was associated with a reduced proinflammatory activation ability of cells. The obtained results indicate the presence of circulating monocytes with mitochondrial dysfunction and the association of such cells with chronic inflammation and atherosclerosis development

Novel Fluoride Matrix for Dual-Range Optical Sensors and Visualization

Novel tetragonal matrix Ba0.5−xLn0.5NaxF2.5−x with x = 0.08, doped by Yb3+, Ho3+, Er3+, was synthesized by molten salt synthesis (MSS) from nitrate flux. XRD data show that the tetragonal phase with a = 4.122(1) Å, c = 17.672(1) Å is stable in an argon atmosphere up to 960 °C. Luminescence spectra recorded in 500–900 nm and 1050–1700 nm upon 974 nm pumping demonstrated the characteristic luminescence at 1550 nm (4I13/2 → 4I15/2) for Er3+ and 1150 nm (5I6 → 5I8) for Ho3+. The relative thermal sensitivity (Sr) at 296–316 K were 0.3%×K−1 and 5.5%×K−1 in shortwave infrared (SWIR) and visible range, respectively. Synthesized luminophores can be used as dual-range optical temperature sensors, which simultaneously operate in visible and SWIR ranges

FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems