deCLUTTER²⁺ – a pipeline to analyze calcium traces in a stem cell model for ventral midbrain patterned astrocytes

Astrocytes are the most populous cell type of the human central nervous system and are essential for physiological brain function. Increasing evidence suggests multiple roles for astrocytes in Parkinson’s disease, nudging a shift in the research focus, which historically pivoted around ventral midbrain dopaminergic neurons (vmDANs). Studying human astrocytes and other cell types in vivo remains challenging. However, in vitro-reprogrammed human stem cell-based models provide a promising alternative. Here, we describe a novel protocol for astrocyte differentiation from human stem cell-derived vmDAN-generating progenitors. This protocol simulates the regionalization, gliogenic switch, radial migration and final differentiation that occur in the developing human brain. We characterized the morphological, molecular and functional features of these ventral midbrain patterned astrocytes with a broad palette of techniques and identified novel candidate midbrain-astrocyte specific markers. In addition, we developed a new pipeline for calcium imaging data analysis called deCLUTTER2+ (deconvolution of Ca2+ fluorescent patterns) that can be used to discover spontaneous or cue-dependent patterns of Ca2+ transients. Altogether, our protocol enables the characterization of the functional properties of human ventral midbrain patterned astrocytes under physiological conditions and in disease.</p

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.Genetics of disease, diagnosis and treatmen

Hydrochemical parameters and trophic state of an urban lake used for recreation

Every lake located in an urban area or in the vicinity of a town is a unique element of the landscape. However, lakes situated in cities are particularly endangered as they have a role of receiving water for municipal, industrial and precipitation wastes. The load of nutrients introduced with sewage is so high that it disturbs the biological balance and disrupts biogeochemical processes in an ecosystem. The object of the study was an urban Lake Sajmino (21.6 ha, max. depth 7.8 m) used for recreation. Its catchment is smaller than the topographic catchment area because some of the precipitation water is redirected outside by a storm water drainage system. The lake is fed by 3 permanent inflows (I-III). The goal of the study was to find correlations between the hydrochemical and biological parameters of the lake water and the quality of water supplied by the inflows. Simultaneously, an attempt was made to identify the hazards, in order to undertake actions in the near future preventing deterioration of the lake’s trophic state. The amounts of pollutants flowing into the lake from various sources exceeded the acceptable levels, therefore leading to an increase in its trophy, exhibited by algal blooms and limitation in the transparency of the lake water expressed as Secchi disc visibility. A significant (p ≤ 0.05) inverse correlation between Chl a and SD (r = –0.755) was found for the lake water, and between the Chl a of the lake and the pollutants of the inflows: NNH4 (I), r = 0.786, NNO2 (I), r = 0.731, NNO2 (II), r = 0.881, suspension (III), r = –0.719. TSI indicates that the eutrophic character of the lake is still unsettled. TSI (TP) > TSI (TN) > TSI (SD) > TSI (Chl a). The SD value (2.2÷3.55 m) may be caused by dissolved organic matter, the suspension or the water’s colour. Phytoplankton (Chl a ≤ 4.840 μg dm-3) could be limited by nitrogen, zooplankton feeding or toxic compounds

Monitoring vertical displacements of an engineering object with masonry walls

The paper presents a method for conducting measurements and processing the results that makes it possible to determine the vertical displacements of measurement and control network points stabilized outside and inside a building in which changes in the form of scratches and cracks on the external and internal walls were noticed. These changes were so disturbing that a number of technical opinions were issued on the technical condition of the building, the ground and water conditions as well as the location of other buildings in relation to that object. The measurement methodology is generally known, but the problem of correctly defining a reference system and estimating the values of vertical displacements still remains to be solved. Moreover, the paper presents the characteristics of the object subjected to research, its technical condition in 2006 and in 2015 as well as an analysis of the impact of a heating duct on the behaviour of the building