Native mass spectrometry analysis of conjugated HSA and BSA complexes with various flavonoids.

Mass spectrometry was used to study the binding interaction between serum albumin proteins (BSA and HSA) and flavone dyes, which is known to induce large fluorescence signals for protein detection. By electrospray ionization mass spectrometry (ESI-MS), multiple charged species/states could be produced in ammonium acetate buffer, while preserving the native structures of the proteins. Subsequent introduction of a flavone dye into the buffered solution resulted in an immediate interaction, forming the respective protein-dye conjugates associated by non-covalent interactions. Formation of protein-dye conjugates induced a notable response in the ESI-MS spectra, including changes in both the charge states and molecular mass of the protein species. The resulting data pointed out that the protein-flavone dye maintained a 1 : 1 ratio in the conjugate, although multiple binding sites for drug molecules are present in albumin proteins.

Synthesis, characterization, in vitro SAR study, and preliminary in vivo toxicity evaluation of naphthylmethyl substituted bis-imidazolium salts.

A series of novel bis-imidazolium salts was synthesized, characterized, and evaluated in vitro against a panel of non-small cell lung cancer (NSCLC) cells. Two imidazolium cores were connected with alkyl chains of varying lengths to develop a structure activity relationship (SAR). Increasing the length of the connecting alkyl chain was shown to correlate to an increase in the anti-proliferative activity. The National Cancer Institute's NCI-60 human tumor cell line screen confirmed this trend. The compound containing a decyl linker chain, 10, was chosen for further in vivo toxicity studies with C578BL/6 mice. The compound was well tolerated by the mice and all of the animals survived and gained weight over the course of the study.

Synthesis of highly selective lysosomal markers by coupling 2-(2'-hydroxyphenyl)benzothiazole (HBT) with benzothiazolium cyanine (Cy): the impact of substituents on selectivity and optical properties.

HBT-Cy 1 has been previously reported as a highly selective fluorescent probe for lysosome visualization in live cells. To further investigate the role of the structural components of HBT-Cy in lysosome selectivity, cyanine based fluorescent probe series (2-5) have been synthesized in good yields by connecting benzothiazolium cyanine (Cy) with 2-hydroxyphenylbenzothiazole (HBT) via a meta phenylene ring. Probes 2-5 exhibited exceptional photophysical properties including bright red-emission (λem≈ 630-650 nm), a large Stokes shift (Δλ > 130 nm) and high fluorescence quantum yields (φfl≈ 0.1-0.5). Probes 2, 3, and 5 exhibited exceptional selectivity towards cellular lysosomes in NHLF and MO3.13 cells. Our further study revealed that the phenyl benzothiazolium cyanine component (6) was the lysosome directing group in the HBT-Cy probe structure. The attachment of the hydroxyphenyl benzothiazole (HBT) component to the HBT-Cy probe structure has significantly improved its photophysical properties. Lysosome probes 2, 3 and 5 exhibited excellent biocompatibility, quick staining, bright red fluorescence, and wash-free application for live cell imaging. These probes further exhibited excellent characteristics for bioimaging experiments including a non-alkalinizing nature, high biocompatibility, high photostability and long-term imaging ability (>4 hours).

NIR-emitting benzothiazolium cyanines with an enhanced stokes shift for mitochondria imaging in live cells.

A series of benzothiazolium-based hemicyanines (3a-3f) have been synthesized. Evaluation of their photophysical properties shows that they exhibit improved photophysical characteristics. In comparison with the available commercial MitoTrackers, the new probes revealed an enhanced Stokes shift (Δλ ∼ 80 nm) and minimized aggregation for increased sensitivity. The synthesized probes are found to exhibit excellent selectivity for mitochondrial staining in an oligodendrocyte cell line. Probes show almost no fluorescence in aqueous environments, while the fluorescence is increased by ∼10-fold in organic solvents, making it possible for mitochondrial imaging without the need for post-staining washing. Since the absorption peaks of probes are close to the laser wavelengths of 561 and 640 nm on a commercial confocal microscope, e.g.3a exhibits λabs ∼ 620 nm and λem ∼ 702 nm, they could be useful probes for mitochondrial tracking in live cells.

A fluorescent flavonoid for lysosome detection in live cells under "wash free" conditions.

Lysosomes are vital organelles in living cells, which have acidic environments (pH 4.0-5.0) where macrobiomolecules and malfunctioning organelles are broken down into monomers by hydrolase activity. The majority of the currently reported fluorescent probes for detecting lysosomes suffer from small Stokes shifts (Δλ < 20 nm) and higher cytotoxicity due to an "alkalinizing effect". An interesting flavonoid-based lysosome probe is synthesized by introducing a morpholine moiety onto the flavonoid skeleton. This new probe has shown excellent selectivity to detect lysosomes in MO3.13 oligodendrocytes and normal human lung fibroblast cell lines. Probes 1a and 1b have shown excellent fluorescence quantum yield (φfl up to 0.43 in non-aqueous solvents) and large Stokes shifts (120-150 nm). These new fluorescent probes also exhibit a large quantum yield difference from an aqueous to organic environment, making them potentially useful as "wash-free" stains for visualizing lysosomes. Cell viability evaluation of these probes shows excellent biocompatibility with the median lethal concentration being LC50 ≈ 50 µM.

Varus and valgus flexion laxity of total knee alignment methods in loaded cadaveric knees.

Both total knee alignment methods, the anatomic and classic, seek to achieve stability in flexion and extension. However, posterior femoral condyle referencing (anatomic alignment) combined with perpendicular tibial resection (classic alignment) results in a 3 degree relative internal rotation of the femoral component with lateral joint opening. The current cadaver study investigated the influence of total knee alignment methods and femoral component malrotation (3 degrees and 6 degrees internal and external malrotation) on femorotibial laxity. Varus and valgus excursion tests were done at 0 degrees, 30 degrees, 60 degrees, and 90 degrees knee flexion under vertical loading conditions of 150 N. None of the alignments produced increased laxity in extension. The largest laxity was found on the varus test at 60 degrees flexion with the femoral component at 6 degrees internal rotation. A 3 degree internal rotation of the femoral component showed increased varus laxity only for the combined alignment method. This finding shows that the femoral component position of the combined alignment method is a 3 degree relative internal malrotation and that an additional internal malrotation may compromise varus stability. Posterior femoral condyle referencing did not provide proper femoral component rotation. A ligament tensor may be helpful in determining femoral component rotation after soft tissue release in extension is performed.