Dynamic evaluation of blood flow microcirculation by combined use of the laser Doppler flowmetry and high‐speed videocapillaroscopy methods

The dynamic light scattering methods are widely used in biomedical diagnostics involving evaluation of blood flow. However, there exist some difficulties in quantitative interpretation of backscattered light signals from the viewpoint of diagnostic information. This study considers the application of the high‐speed videocapillaroscopy (VCS) method that provides the direct measurement of the red blood cells (RBCs) velocity into a capillary. The VCS signal presents true oscillation nature of backscattered light caused by moving RBCs. Thus, the VCS signal can be assigned as a reference one with respect to more complicated signals like in laser Doppler flowmetry (LDF). An essential correlation between blood flow velocity oscillations in a separate human capillary and the integral perfusion estimate obtained by the LDF method has been found. The observation of blood flow by the VCS method during upper arm occlusion has shown emergence of the reverse blood flow effect in capillaries that corresponds to the biological zero signal in the LDF. The reverse blood flow effect has to be taken into account in interpretation of LDF signals.      

Axial resolution enhancement of light‐sheet microscopy by double scanning of Bessel beam and its complementary beam

The side lobes of Bessel beam will create significant out‐of‐focus background when scanned in light‐sheet fluorescence microscopy (LSFM), limiting the axial resolution of the imaging system. Here, we propose to overcome this issue by scanning the sample twice with zeroth‐order Bessel beam and another type of propagation‐invariant beam, complementary to the zeroth‐order Bessel beam, which greatly reduces the out‐of‐focus background created in the first scan. The axial resolution can be improved from 1.68 μm of the Bessel light‐sheet to 1.07 μm by subtraction of the two scanned images across a whole field‐of‐view of up to 300 μm × 200 μm × 200 μm. The optimization procedure to create the complementary beam is described in detail and it is experimentally generated with a spatial light modulator. The imaging performance is validated experimentally with fluorescent beads as well as eGFP‐labeled mouse brain neurons.      

In situ diode laser fenestration: An ex‐vivo evaluation of irradiation effects on human aortic tissue

The in situ laser fenestration is an interesting option for the endovascular treatment of short‐necked aneurysms with an intraoperative modification of a standard endograft. According to literature evidence, diode laser emitting in the near‐infrared wavelength (810 nm) can be successfully used to fenestrate the endograft fabric. This paper describes a three‐dimensional navigation system for the accurate targeting of the fenestration site, then reports results of an ex vivo study to assess whether the laser operative conditions, which ensure the fabric fenestration, are harmless for the biological tissue surrounding the endoprosthesis. Two hundred twenty‐five samples of human aorta, including healthy specimens and abdominal aortic aneurysm samples, were irradiated ex vivo using a 810 nm diode laser. Energy and pulse duration were varied. Irradiated tissues were fixed in formaldehyde, sectioned and subjected to histological examination. Only 7.5% of the irradiated samples exhibited a thermal damage, which was always confined to the contact point between the laser fiber tip and the aortic wall. These experiments suggest that the diode laser can be safely used for the proposed surgical application.     

Protective effect of low-intensity laser irradiation under acute toxic stress

We studied how short-term preexposure of the thymus zone in male outbred NMRI mice to helium-neon laser light (632.8 nm, 0.2 mW/cm²) affects the activity of cells of the immune system under acute toxic stress. The stress was modeled by introducing a bacterial lipopolysaccharide that significantly enhanced the production of a number of cytokines in macrophages: interleukins 1α, 1β, 6, and 10, and tumor necrosis factor TNF-α. Single exposure of healthy mice to laser light did not cause any significant change in the production of cytokines and nitric oxide in cells but increased the production of the heat shock proteins HSP25, HSP70, and HSP90. Nonetheless, if mice were exposed to red light before inducing toxic stress, then the production of almost all the cytokines studied and nitric oxide was noticeably normalized. Moreover, the production of the heat shock proteins studied was also normalized. Thus, preexposure of a small region of the animal skin surface to laser light markedly decreased the toxic effect of lipopolysaccharide.     

Fluorescence characteristics and photophysical parameters of light-harvesting chlorophyll <Emphasis Type="Italic">a/b</Emphasis> complex aggregates

Fluorescence characteristics and molecular photophysical parameters of light-harvesting chlorophyll a/b complexes isolated from pea were studied in relation to their aggregation state. The aggregate size was varied by changing the Triton X-100 concentration from 0 to 0.23 mM at a chlorophyll concentration of 2.45 μg/ml. Molecular photophysical parameters were determined with laser fluorimetry. Dispersion of large aggregates into smaller ones drastically decreased the intensity of low-temperature (77 K) fluorescence at 700 nm, reduced the singlet-singlet annihilation rate by more than two orders of magnitude, and prolonged the fluorescence lifetime from 0.16 to 3.2 ns.     

Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.     

Entamoeba histolytica: ADP-ribosylation of secreted glyceraldehyde-3-phosphate dehydrogenase

In addition to its classic glycolytic role, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in many activities unrelated to glycolysis, such as membrane fusion, binding to host proteins and signal transduction. GAPDH can be the target of several modifications that allow incorporation to membranes and possible regulation of its activity; among these modifications is mono-ADP-ribosylation. This post-translational modification is important for the regulation of many cellular processes and is the mechanism of action of several bacterial toxins. In a previous study, we observed the extracellular ADP-ribosylation of a 37-kDa ameba protein. We report here that GAPDH and cysteine synthase A are the main ADP-ribosylated proteins in Entamoeba histolytica extracellular medium, GAPDH is secreted from ameba at 37 degrees C in a time-dependent manner, and its enzymatic activity is not inhibited by ADP-ribosylation. Extracellular GAPDH from ameba may play an important role in the survival of this human pathogen or in interaction with host molecules, as occurs in other organisms.     

Glycomics-driven discoveries in schistosome research

Schistosome glycans and glycoconjugates play a prominent role in the parasite's biology, in particular in the interaction with the human host. A large amount of structural data regarding glycosylation of different schistosome life stages and glycoconjugate subsets has been collected in the last decade, but many significant gaps in our knowledge of the schistosomal glycome remain. Here we will present a concise review of the already available data guided by a selection of recently generated stage-specific glycan profiles, and discuss implications and prospects of glycomics studies of schistosomes.     

Clusterin is associated with spontaneous breast cancer in TA2 mice

Two-dimensional electrophoresis and Matrix-assisted laser desorption ionization-time of flight-time of mass spectrometry were used to detect the differentially expressed proteins in serum of tientsin albinao 2 mice with spontaneous breast cancer, normal tientsin albinao 2 mice and tientsin albinao 1 mice. Only nuclear clusterin (n-CLU) was expressed in tientsin albinao 1. Immunohistochemistry and western blot validated that n-CLU was present in normal tientsin albinao 2 and tientsin albinao 1 mammary epithelium, and secretory clusterin expressed in the cytoplasm of normal tientsin albinao 2 mammary epithelium and spontaneous breast cancer. n-CLU may play an important role in tientsin albinao 2 spontaneous breast cancer initiation and development.