Materno-fetal nutrient transfer across primary human trophoblast layer.

MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionallyon cultured primary trophoblasts, or bidirectionally on the Transwell® system using BeWo cells treated with forskolin. As forskolin can induce various gene alterations (e.g. cAMP response element genes), we aimed to establish a physiological primary trophoblast model for materno-fetal nutrient exchange studies without forskolin application. Methods: Human term cytotrophoblasts were isolated by enzymatic digestion and Percoll® gradient separation. The purity of the primary cells was assessed by flow cytometry using the trophoblast-specific marker cytokeratin-7. After screening different coating matrices, we optimized the growth conditions for the primary cytotrophoblasts on Transwell/ inserts. The morphology of 5 days cultured trophoblasts was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally transport studies were performed on the polarized trophoblasts in the Transwell® system. Results: During 5 days culture, the trophoblasts (>90% purity) developed a modest trans-epithelial electrical resistance (TEER) and a sizedependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ~400-70’000D). SEM analyses confirmed a confluent trophoblast layer with numerous microvilli at day six, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein ZO-1, and the membrane proteins ABCA1 and Na+/K+-ATPase. Vectorial glucose and cholesterol transport studies confirmed functionality of the cultured trophoblast barrier. Conclusion: Evidence from cell morphology, biophysical parameters and cell marker expressions indicate the successful and reproducible establishment of a primary trophoblast monolayer model suitable for transport studies. Application of this model to pathological trophoblasts will help to better understand the mechanism underlying gestational diseases, and to define the consequences of placental pathology on materno-fetal nutrient transport.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.

Expression of Imprinted Genes Is Aberrant in Deceased Newborn Cloned Calves and Relatively Normal in Surviving Adult Clones

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r, and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen, and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle, and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level of those three imprinted genes.

Molecular interactions and signal transfer between sensory rhodopsin-I and its cognate transducer

SRI is unique among known photoreceptors in that it produces opposite signals depending on the color of light stimuli. Absorption of orange light (587 nm) triggers an attractant response by the cell, whereas absorption of orange light followed by near-UV light (373 run) triggers a repellent response. Using behavioral mutants that exhibit aberrant color-sensing ability, we tested a two-conformation equilibrium model, using FRET and EPR spectroscopy. The essence of the model applied to SRI-HtrI is that the complex exists in a metastable two-conformer equilibrium which is shifted in one direction by orange light absorption (producing an attractant signal) and in the opposite direction by a second UV-violet photon (producing a repellent signal). First, by FRET we found that the E-F cytoplasmic loop of SRI moves toward the RAMP domain of the HtrI transducer during the formation of the orange-light activated signaling state of the complex. This is the first localization of a change in the physical relationship between the receptor and transducer subunits of the complex and provides a structural property of the two proposed conformers that we can monitor. Second, EPR spectra of a spin label probe at this cytoplasmic position showed shifts in the dark in the mutants toward shorter or longer EF loop-RAMP distances, explaining their behavior in terms of their mutations causing pre-stimulus shifts into one or the other conformer. ^ Next, we applied a novel electrophysiological method for monitoring the directionality of proton movement during photoactivation of SRI, to investigate the process of proton transfer in the photoactive site from the chromophore to proton acceptors on both the wildtype and aberrant color-response mutants. We observed an unexpected and critical difference in the two signaling conformations of the SRI-HtrI complex. The finding is that the vectoriality (i.e. movement away or toward the cytoplasm) of the light-induced proton transfer from the chromophore to the protein is opposite in formation of the two conformations. Retinylidene proton transfer is a common critical process in rhodopsins and these results are the first to show differences in vectoriality in a rhodopsin receptor, and to demonstrate functional importance of the direction of proton transfer. ^

Water, bottom deposits, and macrobenthos in the south part of the East Siberian Sea in September 2003

Macrobenthos biomass and bottom biocoenoses were studied in the sublittoral zone of the southern East Siberian Sea. The macrobenthos is characterized by relatively high abundance (from 30 to 2680 #/m**2), biomass (from 0.25 to 578.8 g/m**2), and diversity (83 species in total). Lateral distribution of macrobenthos biomass correlates with a substrate type and salinity and is substantially higher in areas washed by the Arctic water mass than in estuaries with mixed fresh and Arctic waters and shows a tendency to decreasing in the convergence zone of different water masses. The highest macrobenthos biomass is observed in cores of water masses in the Long Strait area and in the eastern part of the sea.

234Th and particulate fluxes in the Kara Sea in October 1993

The mean residence time of 234Th associated with suspended matter in the Kara Sea was calculated from distributions of dissolved and suspended 234Th. Integral particulate fluxes at different levels were estimated for two stations. The flux increases only in the pycnocline; below it changes insignificantly. Two maxima of differential fluxes are noted in vertical profiles: in the surface layer where primary production is maximal, and in the interface layer where zooplankton realizing active transport of suspended matter is usually concentrated. Differential fluxes were determined at 10 stations; their space distribution is controlled by primary production, which depends usually on turbidity of river water in estuaries.

Multi-year high-frequency current and water quality in-situ observations at the Guadiana Estuary

High-frequency data collected continuously over a multiyear time frame are required for investigating the various agents that drive ecological and hydrodynamic processes in estuaries. Here, we present water quality and current in-situ observations from a fixed monitoring station operating from 2008 to 2014 in the lower Guadiana Estuary, southern Portugal (37°11.30' N, 7°24.67' W). The data were recorded by a multi-parametric probe providing hourly records (temperature, salinity, chlorophyll, dissolved oxygen, turbidity, and pH) at a water depth of ~1 m, and by a bottom-mounted acoustic Doppler current profiler measuring the pressure, near-bottom temperature, and flow velocity through the water column every 15 min. The time-series data, in particular the probe ones, present substantial gaps arising from equipment failure and maintenance, which are ineluctable with this type of observations in harsh environments. However, prolonged (months-long) periods of multi-parametric observations during contrasted external forcing conditions are available. The raw data are reported together with flags indicating the quality status of each record. River discharge data from two hydrographic stations located near the estuary head are also provided to support data analysis and interpretation.

(Table 1) Productivity characteristics of phytoplankton and some concomitant factors in the Kara Sea in September 1993

Primary production in water column (P_p) varied from 107 to 312 mg C/m**2/day in Yenisey Bay: from 25 to 63 mg C/m**2/day in Obskaya Guba: and from 20 to 359 mg C/m**2/day in the open sea, that is: in the western Kara Sea and Ob-Yenisey shoals. The average concentration of chlorophyll a in the photosynthesis layer (C_ph) ranged from 0.2 to 1.8 mg/m**3 in these two regions, lower than in the estuaries of Ob (1.6-21.7 mg/m**3) and Yenisey (2.0-5.2 mg/m**3) Rivers. An inverse relation between surface salinity (S) and chlorophyll concentration (C_s) and chlorophyll concentration in the photosynthesis layer was found for all of the regions. The highest values of C_s and C_ph (0.8-22 mg/m**3) were measured at S<10 ppt, and the lowest values (0.2-0.8 mg/m**3) at S>22 ppt. A similar correlation of S with values of Pp occurred only in the Yenisey Bay and offshore regions. Obtained results agree well with the "outwelling" hypothesis. It states that large part of organic matter produced in estuaries is not used in estuarine trophic chains but is transported into adjacent sea areas and increases their productivity. Low values of Pp in the study regions may be attributed to such unfavorable factors as deficiency in nutrients, low temperature and turbidity, and lack of solar radiation.

Suspended matter in waters of the Kem' River estuary (white Sea) and its composition in July-August 2003 and 2002

On the base of detailed studies in the Keret' and Kem' estuaries (Karelian coast of the White Sea) in 2000-2003 a comparative analysis has been carried out. It includes: salinity and freshening of the water column, variations of suspended matter concentration and its chemical composition, current velocity and zooplankton species composition during flood- and ebb tides.