Dmitry A. Voronov

Transcription Factors Runx1 to 3 Are Expressed in the Lacrimal Gland Epithelium and Are Involved in Regulation of Gland Morphogenesis and Regeneration

PURPOSE Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration. METHODS Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT(2)Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration. RESULTS We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation. CONCLUSIONS Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration.

Lacrimal Gland Repair Using Progenitor Cells

In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous‐deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild‐type mouse LGs by transplanting them into the LGs of TSP ‐1−/− mice, which represent a novel mouse model for ADDE. TSP‐1−/− mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c‐kit‐positive epithelial cell adhesion molecule (EpCAM+) populations sorted from mouse LGs, the c‐kit+dim/EpCAM+/Sca1 − /CD34 − /CD45 − cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three‐dimensional cultures. Moreover, when transplanted into injured or “diseased” LGs, they engraft into acinar and ductal compartments. EPCP‐injected TSP‐1−/− LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88–98

Cryptomonad alga Rhodomonas sp. ( Cryptophyta, Pyrenomonadaceae ) bloom in the redox zone of the basins separating from the White Sea

Bloom of a cryptomonad alga Rhodomonas sp. (Cryptophyta, Pyrenomonadaceae) was observed in the chemocline of saline basins separating from the White Sea, resulting in red coloration of the relevant water layer. According to the sequence of the 18S nuclear rRNA gene, this species was identical to Rhodomonas sp. RCC2020 (GenBank accession no. JN934672) from the Beaufort Sea. The presence of the red layer formed by mass development of Rhodomonas sp. is considered an indicator of a certain stage of separation of a basin from the sea.

The study of coastal meromictic water basins in the Kandalaksha Gulf of the White Sea by spectral and physicochemical methods

Research is initiated to study water samples from stratified water basins in the Kandalaksha Gulf of the White Sea at different stages of their separation from the sea. The objects of research are lakes Elovoe and Nizhnee Ershovskoe located close to the Nikolai Pertsov White Sea Biological Station. Depth profiles of physico-chemical characteristics such as temperature, salinity, pH and dissolved oxygen were measured. Brightly colored green water layers were found in both lakes. Concentrations of photosynthetic organisms were estimated using absorption and fluorescence spectra of water samples from various depths.

Depth profiles of spectral and hydrological characteristics of water and their relation to abundances of green sulfur bacteria in the stratified lakes of the White Sea

We analyze the results received from two expeditions performed in August-September 2013, August-September 2014 and February 2015 in the Kandalaksha Bay of the White Sea. Depth profiles of hydrological characteristics and optical properties of water were recorded for five marine lakes being on different stages of isolation from the White Sea. Those relic lakes demonstrate a tendency to meromixis and are characterized by apparent stratification of the water bodies from the brackish top layer to the bottom salt water. Maximal concentrations of anoxygenic phototrophs (green sulfur bacteria) were found at depths close to the redox interface in all the studied lakes. To discriminate differently pigmented groups of microorganisms the fluorescence emission spectra of bacteriochlorophylls from the living cells were used. We puzzle out the data on light spectrum propagation through the water body in each lake using optical properties of water (attenuation spectra) in the UV, visible and NIR ranges, as well as direct measurements of the total irradiances at various depths. The changes in optical characteristics of water in the stratified reservoirs due to cromophoric dissolved organic matter (CDOM) and microbial pigments affect the light intensity and its spectral distribution at each water layer thus influencing the living conditions for differently pigmented phototrophic microorganisms and determining the composition of microbial community.

Electrophysiology of the rhythmic defecation program in nematode Heterorhabditis megidis

The nervous system controls most rhythmic behaviors, with a remarkable exception. In Caenorhabditis elegans periodic defecation rhythm does not appear to involve the nervous system. Such oscillations are studied in detail with genetic and molecular biology tools. The small size of C. elegans cells impairs the use of standard electrophysiological methods. We studied a similar rhythmic pacemaker in the noticeably larger gut cells of Heterorhabditis megidis nematode. H. megidis defecation cycle is driven by a central pattern generator (CPG) associated with unusual all-or-none hyper-polarization “action potential”. The CPG cycle period depends on the membrane potential and CPG cycling also persisted in experiments where the membrane potential of gut cells was continuously clamped at steady voltage levels. The usual excitable tissue description does not include the endoderm or imply the generation of hyper-polarization spikes. The nematode gut cells activity calls for a reevaluation of the excitable cells definition.

Spectroscopic Study of Green Sulfur Bacteria in Stratified Water Bodies of the Kandalaksha Gulf of the White Sea

Optical characteristics of water in stratified lakes of the White Sea are of particular interest in connection with the observation of thin colored layers resulting from massive development of anoxygenic phototrophic bacteria around the chemocline. While optical properties of chlorophyll are widely used in remote sensing, spectral characteristics of bacteriochlorophylls (BChl) for natural microbial communities have been little studied. In this work, spectral study of green sulfur bacteria of four water bodies of the Kandalaksha Gulf of the White Sea was carried out. Absorption and fluorescence spectra were measured for water samples taken in March 2017 from different depths and compared with spectra of monocultures isolated from the same water bodies earlier. It has been shown that the BChl fluorescence in the living cells of green sulfur bacteria has two overlapping emission bands: in the region of 740–770 nm (BChl d and e) and at 815 nm (BChl a). The wavelength of the maximum of the first band depends on the ratio of the concentrations of green- and brown-colored forms of bacteria containing different types of BChl. A new method for separating the contributions of two types of bacteria is proposed, based on the deconvolution of the BChl fluorescence spectrum into three bands whose parameters are determined from the spectra of monocultures. The BChl content at various water depths has been estimated and the percentage ratio of different types of phototrophic bacteria has been determined.

Spectroscopic study of the microbial community in chemocline zones of relic meromictic lakes separating from the White Sea

As a result of a recent years study on the Karelia shore of the White Sea more than ten relict lakes in different stages of separation from the sea have been discovered. Five of them are located close to the Nikolai Pertsov White Sea Biological Station of Moscow State University. Such separated lakes are interesting to explore for their firm vertical stratification. Water layers differ not only by temperature, salinity and other physic and chemical characteristics and optical properties, but also by ibhabiting microorganisms and by the quality of dissolved organic matter. To study phototropic organisms in water sampled from different depths we used spectroscopic techniques. Identification of the main bands in the absorption and fluorescence spectra showed that there are two main groups of photosynthetic organisms in the redox zone (chemocline): unicellular algae containing chlorophyll a and green sulfur bacteria with bacteriochlorophylls c, d, e. Spectral data were compared with physical and chemical characteristics of the water layer (temperature, salinity, pH, dissolved oxygen and sunlight illumination at certain depth). It gave an opportunity to compare vertical profiles of oxygen and hydrogen sulphide concentration with the number and distribution of oxygenic and anoxygenic phototrophic microorganisms. Maximum abundance of both algae and green sulfur bacteria were achieved within the redox zone. Typical thickness of the layer with the highest concentration of microorganisms did not exceed 10-20 cm.