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A method for display of 3'-end restriction fragments of cDNAs is proposed, extending the idea reported recently. First, representative pools of such fragments are selectively amplified using PCR suppression effect. Then, simplified subsets of these fragments suitable for comparison by PAGE are amplified by adapter-specific primers extended by two randomly picked bases at their 3'ends. By testing all possible combinations of extended primers the whole mRNA pool may be systematically investigated. The method was applied to search for molecular regional markers of freshwater planarian Dugesia tigrina .

BackgroundMesenchymal stromal/stem cells derived from human umbilical cord (UC-MSCs) uniquely combine properties of embryonic and postnatal MSCs and may be the most acceptable, safe, and effective source for allogeneic cell therapy e.g. for therapeutic angiogenesis. In this report we describe pro-angiogenic properties of UC-MSCs as manifested in vitro.MethodsUC-MSCs were isolated from human Wharton’s jelly by enzymatic digestion. Presence of soluble forms of VEGF-A in UC-MSC-conditioned media was measured by ELISA. Effects of the conditioned media on human umbilical vein-derived endothelial EA.hy926 cells proliferation were measured by MTT-assay; changes in cell motility and directed migration were assessed by scratch wound healing and transwell chamber migration assays. Angiogenesis was modeled in vitro as tube formation on basement membrane matrix. Progressive differentiation of MSCs to endothelioid progeny was assessed by CD31 immunostaining.ResultsAlthough no detectable quantities of soluble VEGF-A were produced by UC-MSCs, the culture medium, conditioned by the UC-MSCs, effectively stimulated proliferation, motility, and directed migration of EA.hy926 cells. In 2D culture, UC-MSCs were able to acquire CD31+ endothelial cell-like phenotype when stimulated by EA.hy926-conditioned media supplemented with VEGF-A165. UC-MSCs were capable of forming unstable 2D tubular networks either by themselves or in combinations with EA.hy926 cells. Active spontaneous sprouting from cell clusters, resulting from disassembling of such networks, was observed only in the mixed cultures, not in pure UC-MSC cultures. In 3D mode of sprouting experimentation, structural support of newly formed capillary-like structures was provided by UC-MSCs that acquired the CD31+ phenotype in the absence of exogenous VEGF-A.ConclusionThese data suggest that a VEGF-A-independent paracrine mechanism and at least partially VEGF-A-independent differentiation mechanism are involved in the pro-angiogenic activity of UC-MSCs.

The WHO Classification of Tumors of Soft Tissue and Bone subdivides rhabdomyosarcomas (RMS) into alveolar, embryonal, pleomorphic, and spindle cell RMS. Advances in molecular genetic diagnostics have made it possible to identify new RMS subgroups within traditional morphological entities. One of these subgroups comprises rare tumors characterized by epithelioid and spindle cell morphology, highly aggressive clinical course with pronounced tendency to intraosseous growth, and the presence of pathognomonic recurring genetic aberrations- chimeric genes/transcripts EWSR1::TFCP2, FUS::TFCP2, or MEIS1::NCOA2. Starting from 2018, only 26 reported cases of RMS have been assigned to this subgroup. The rarity of such tumors hampers their correct diagnostics for both anatomic pathologists and molecular oncologists. Here we describe a clinical case of intraosseous spindle cell RMS expressing EWSR1::TFCP2 fusion gene, encountered for the first time in our practice, in a 16-year-old female patient presenting with mandibular lesion. The diagnostic process took considerable time and involved RNA sequencing; a high-throughput method of molecular genetic research. The tumor was extremely aggressive, showing resistance to polychemotherapy, radiation therapy, and crizotinib targeted therapy, with the fatal outcome.

The cerebral cortex is composed of a large variety of different neuron types. All cortical neurons, except some interneurons, are born in two proliferative zones, the cortical ventricular (VZ) and subventricular (SVZ) zones. The relative contribution of both proliferative zones to the generation of the diversity of the cortical neurons is not well understood. To further dissect the underlying mechanism, molecular markers specific for the SVZ are required. Towards this end we performed a subtraction of cDNA libraries, generated from E15.5 and E18.5 mouse cerebral cortex. A novel cDNA, Svet1, was cloned which was specifically expressed in the proliferating cells of the SVZ but not the VZ. The VZ is marked by the expression of the Otx1 gene. Later in development, Svet1 and Otx1 were expressed in subsets of cells of upper (II-IV) and deep (V-VI) layers, respectively. In the reeler cortex, where the layers are inverted, Svet1 and Otx1 label precursors of the upper and deeper layers, respectively, in their new location. Interestingly, in the Pax6/small eye mutant, Svet1 activity was abolished in the SVZ and in the upper part of the cortical plate while the Otx1 expression domain remained unchanged. Therefore, using Svet1 and Otx1 as cell-type-specific molecular markers for the upper and deep cortical layers we conclude that the Sey mutation affects predominantly the differentiation of the SVZ cells that fail to migrate into the cortical plate. The abnormality of the SVZ coincides with the absence of upper layer cells in the cortex. Taken together our data suggest that while the specification of deep cortical layers occurs in the ventricular zone, the SVZ is important for the proper specification of upper layers.

Neurons can communicate with each other either via exchange of specific molecules at synapses or by direct electrical connections between the cytoplasm of either cell [for review see Bruzzone et al. (1996) Eur. J. Biochem., 238, 1-27]. Although electrical connections are abundant in many nervous systems, little is known about the mechanisms which govern the specificity of their formation. Recent cloning of the innexins--gap junction proteins responsible for electrical coupling in invertebrates (Phelan et al. (1998) Trends Genet., 14, 348-349], has made it possible to study the molecular mechanisms of patterning of the electrical connections between individual neurons in model systems. Here we demonstrate that intracellular injection of mRNA encoding the molluscan innexin Panx1 (Panchin et al. 2000 Curr. Biol., 10, R473-R474) drastically alters the specificity of electrical coupling between identified neurons of the pteropod mollusc Clione limacina.

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