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Neuroblastomas, the most prevalent malignant solid neoplasms of childhood, originate from progenitor cells of the sympathetic nervous system. Their genetic causation is diverse and involves multiple molecular mechanisms. This review highlights multiple roles of microRNA in neuroblastoma pathogenesis and discusses the prospects of harnessing these important natural regulator molecules as biomarkers, therapeutic targets and pharmaceuticals in neuroblastoma.

Background Choroid plexus carcinomas (CPCs) are rare aggressive pediatric tumors of the brain with no treatment standards. Genetic profiling of CPCs is often confined to possible association with Li–Fraumeni syndrome, though only about a half of CPCs develop from syndromic predispositions. Whole-chromosome gains and losses typical of CPCs reflect genomic instability of these tumors, but only partially explain the aggressive clinical course. Methods This retrospective study enrolled 25 pediatric patients with CPC, receiving treatment between January 2009 and June 2022. Molecular-genetic testing was performed for 20 cases with available tumor tissue and encompassed mutational status, chromosomal aberrations, and gene expression profiles. We analyzed several factors presumably influencing the outcomes, including molecular profiles and clinical parameters. The median follow-up constituted 5.2 years (absolute range 2.8–12.6 years). Results All studied CPCs had smooth mutational profiles with the only recurrent event being TP53 variants, either germline or somatic, encountered in 13 cases. Unbalanced whole-chromosome aberrations, notably multiple monosomies, were highly typical. In 7 tumors, chromosome losses were combined with complex genomic rearrangements: segmental gains and losses or signs of chromothripsis. This phenomenon was associated with extremely low 5-year survival: 20.0 ± 17.9% vs 85.7 ± 13.2%; P = .009. Transcriptomically, the cohort split into 2 polar clusters Ped_CPC1 and Ped_CPC2 differing by survival: 31.3 ± 17.8% vs 100%; P = .012. Conclusion CPCs split into at least 2 molecular subtypes distinguished both genomically and transcriptomically. Clusterization of the tumors into Ped_CPC1 and Ped_CPC2 significantly correlates with survival. The distinction may prove relevant in clinical trials for dedicated and patient-oriented optimization of clinical protocols for these rare tumors.

Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adopt a protocol for sensitive detection of the

At the normal physiological conditions, hepatocytes predominantly reside in G0 phase of cell cycle; they actively proceed to G1 phase upon damage to the organ. As it was shown in experiments with restoration of liver mass in rats after subtotal hepatectomy (resection of 80% of the organ mass may be considered as a model of the 'small for size' liver syndrome), the growth inhibition is due to prolonged arrest of hepatocyte proliferation, molecular mechanisms of which remain understudied. In a rat model of liver regeneration after surgical removal of 80% of its mass, we observe a delayed onset of hepatocyte proliferation: Ki67+ hepatocytes begin to appear as late as at 30 h after liver subtotal resection. Their appearance coincides with the beginning of transcription of genes for cyclins A2, B1, D 1 , and E 1 at 24-30 h after surgery. The corresponding increase in concentrations of cyclin D 1 and E proteins is further delayed till 48 h after liver resection. We have also observed a prolonged decrease in the expression of proto-oncogene c-met (the hepatocyte growth factor receptor-encoding gene Met), an increase in expression of the transforming growth factor β1 (TGFβ 1 ) receptor-encoding gene Tgfbr2. At the same time, irreversible block of hepatocyte proliferation is prevented by expression of certain factors, notably of the TWEAK/Fn14 signaling pathway: concentrations of the corresponding proteins in remnant livers have peaked from 24 to 48 h after liver subtotal resection.

The objective of this study was to evaluate physical, mechanical, and biological properties of the polydioxanone (PDO) monofilament meshes and polyglycolide (PGA) polyfilament meshes in comparison with Permacol® implants. In rat experimental model, a 1.5 × 2.0 cm defect in abdominal wall was reconstructed by using the Permacol surgical implant or knitted meshes produced from either PDO monofilament, or PGA multifilament. The implant sites were assessed for the tensile strength and the extents of material resorption, host inflammatory response and host tissue replacement on days 3, 10, 30, or 60 after the surgery. The PDO and PGA meshes were rapidly pervaded by the host connective tissue with elements of skeletal muscle histogenesis. The degree of adhesions was significantly higher in the Permacol group. All of the prostheses underwent resorption, which correlated with gradual decreases in the overall tensile strength of the site and the Col1a1 gene expression level. Elevated expression of Fgf2 gene maintained longer in the PDO group, and the Mmp9 gene expression level in this group was higher than in the other groups. Gene expression levels of inflammatory cytokines were higher in the Permacol group. The foreign body giant cell numbers were lower in the PDO and Permacol groups than in the PGA group. Minimal macrophage infiltration with predominance of M2 cells was observed in the PDO group. Overall, the PDO prosthesis turned out to be significantly better than the PGA or Permacol prostheses by a number of indicators of biocompatibility and efficacy. © 2018 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 652-663, 2019.

AIM To investigate the influence of the umbilical cord-derived multipotent stromal cells (MSCs) on recovery of the liver after the subtotal resection, that is, removal of 80% of the organ mass, a renowned model of the small-for-size liver remnant syndrome. METHODS The MSCs were obtained from the intervascular tissue of umbilical cords, dissected from rat fetuses, by the explant culture technique. The vital labeling of MSCs with РКН26 was carried out on the 3rd passage. The subtotal resection was performed on male Sprague-Dawley rats. The experimental group animals received a transplant 106 MSCs infused into the spleen. Hepatocyte proliferation was assessed by counting of either mitotic figures or Ki67-positive cells in microscopic images. MSC differentiation was assessed with antibodies to hepatocyte-specific marker cytokeratin 18 (CK18), cholangiocyte-specific protein CK19, smooth muscle cell-specific protein α-SMA, the endothelial cell marker CD31, or the active fibroblast marker FAPα. Total macrophages of the liver were selectively stained in cryosections incubated with anti-CD68 antibodies (1:100, Abcam), while the M2a and M2c macrophage populations were selectively stained with anti-CD206 antibodies. Expression of interleukin and growth factor genes was evaluated with PCR-RT. RESULTS Intrasplenic allogeneic transplantation of the umbilical cord-derived multipotent stromal cells stimulates reparative processes within the residual liver tissue after subtotal resection (removal of 80% of the organ mass), as indicated by increased rates of hepatocyte proliferation and accelerated organ mass recovery. These effects may result from paracrine influence of the transplanted cells on the resident macrophage population of the liver. The transplantation favors polarization of macrophages to M2 phenotype (the M2-polarized macrophages specifically express CD206; they are known to suppress inflammation and support tissue repair). No differentiation of the transplanted cells into any of the liver cell types have been observed in the study. CONCLUSION We found no direct evidence for the paracrine effect of MSCs on liver regeneration after the subtotal liver resection in rats. However, the paracrine mechanism of the therapeutic activity of transplanted MSC is indirectly indicated by a decrease in the total number of CD68 + macrophages and an increase in the proportion of M2 pro-repair macrophages in the regenerating liver as compared to animals in which the transplantation was only mimicked.

This study delves into possible mechanisms underlying the stimulating influence of UC-MSCs transplantation on functional and structural recovery of ischemic skeletal muscles. Limb ischemia was created in Sprague-Dawley rats by excision of femoral and popliteal arteries. Allogeneic rat PKH26-labeled UC-MSCs were administered by direct intramuscular injection. Animals of experimental group responded to the transplantation by improvement in their locomotor function as assessed by the rotarod performance test on day 9 and 29 after transplantation. Histomorphometric analysis showed that relative area of the lesions in the experimental group was significantly smaller than in the control group at all time points during the observation. Calculated densities of microcirculation vessels within the lesions were significantly higher in the experimental group than in the control group on day 10 after transplantation. Only a part of the transplanted allogeneic UC-MSCs survived within the ischemic muscle tissue, and a considerable portion of these surviving cells were found alongside the VEGF-producing preserved muscle fibers. The PKH26 label was not found within the walls of capillaries or larger blood vessels. The administration of allogeneic UC-MSCs significantly increased the proportion of M2 macrophages, exhibiting proangiogenic and anti-inflammatory properties, for at least 10 days following the transplantation.

Background In many clinical cases of extensive liver resection (e.g. due to malignancy), the residual portion is too small to maintain the body homeostasis. The resulting acute liver failure is associated with the compensatory growth inhibition, which is a typical manifestation of the ‘small for size’ liver syndrome. The study investigates possible causes of the delayed onset of hepatocyte proliferation after subtotal hepatectomy (80% liver resection) in rats. Results The data indicate that the growth inhibition correlates with delayed upregulation of the Tnf gene expression and low content of the corresponding Tnfα protein within the residual hepatic tissue. Considering the involvement of Tnf/Tnfα, the observed growth inhibition may be related to particular properties of liver macrophages – the resident Kupffer cells with CD68+CX1CR3−CD11b− phenotype. Conclusions The delayed onset of hepatocyte proliferation correlates with low levels of Tnfα in the residual hepatic tissue. The observed growth inhibition possibly reflects specific composition of macrophage population of the liver. It is entirely composed of embryonically-derived Kupffer cells, which express the ‘proregeneratory’ M2 macrophage-specific marker CD206 in the course of regeneration. Electronic supplementary material The online version of this article (10.1186/s12865-018-0260-1) contains supplementary material, which is available to authorized users.

Background Mesenchymal 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. Methods UC-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. Results Although 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. Conclusion These 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.

Background: Poor ovarian response represents a major negative contribution to the efficacy of In vitro fertilization programs. It has been shown that specific nucleotide sequence variants of BMP15 gene, which encodes the growth factor specifically expressed by oocytes to regulate follicle development, are related to certain forms of ovarian dysfunction. The aim of the study was to search for novel variants related to poor ovarian response phenotypes by means of exon sequencing, and also to check possible relations of already known BMP15 variants to this particular form of ovarian insufficiency. Methods: A total of 150 patients (65 women with poor ovarian response and 85 women with normal ovarian response as a control group) participated in this retrospective case-control study. All patients received ovarian stimulation according to the protocol with follicle-stimulating hormone and gonadotropin-releasing hormone antagonist. Genotyping was carried out by polymerase chain reaction with consequent readout of melting curves by means of modified kissing probes assay. Statistical tests were two-sided, with percent values compared by χ2 test and associations measured by odds ratio. Results: Two novel single nucleotide polymorphisms of BMP15 were revealed by exon sequencing. Of these, the c.607 C>T substitution was found only in the control group. In contrast, the novel single nucleotide deletion c.-8 delC in the 5’ non-coding region of BMP15 mRNA was significantly more common in the poor ovarian response group. For the previously known sequence variants, the statistical analysis revealed associations of poor ovarian response with two single nucleotide substitutions, the exonic c.308 A>G and the intronic c.328+905 A>G. Conclusion: Two novel variants of BMP15, both of plausible clinical relevance, were found in this study. Examination of larger patient cohorts is required to further elucidate their connection with the phenomenon of poor ovarian response in In vitro fertilization programs.

We have cloned the genes PANX1, PANX2 and PANX3, encoding putative gap junction proteins homologous to invertebrate innexins, which constitute a new family of mammalian proteins called pannexins. Phylogenetic analysis revealed that pannexins are highly conserved in worms, mollusks, insects and mammals, pointing to their important function. Both innexins and pannexins are predicted to have four transmembrane regions, two extracellular loops, one intracellular loop and intracellular N and C termini. Both the human and mouse genomes contain three pannexin-encoding genes. Mammalian pannexins PANX1 and PANX3 are closely related, with PANX2 more distant. The human and mouse pannexin-1 mRNAs are ubiquitously, although disproportionately, expressed in normal tissues. Human PANX2 is a brain-specific gene; its mouse orthologue, Panx2, is also expressed in certain cell types in developing brain. In silico evaluation of Panx3 expression predicts gene expression in osteoblasts and synovial fibroblasts. The apparent conservation of pannexins between species merits further investigation.

Proliferation of hepatocytes is known to be the main process in the hepatectomy-induced liver regrowth; however, in cases of extensive loss it may be insufficient for complete recovery unless supported by some additional sources e.g. mobilization of undifferentiated progenitors. The study was conducted on rat model of 80% subtotal hepatectomy; the objective was to evaluate contributions of hepatocytes and resident progenitor cells to the hepatic tissue recovery via monitoring specific mRNA and/or protein expression levels for a panel of genes implicated in growth, cell differentiation, angiogenesis, and inflammation. Some of the genes showed distinctive temporal expression patterns, which were loosely associated with two waves of hepatocyte proliferation observed at 2 and 7 days after the surgery. Focusing on genes implicated in regulation of the progenitor cell activity, we came across slight increases in expression levels for Sox9 and two genes encoding tumor necrosis factor-like cytokine TWEAK (Tnfsf12) and its receptor Fn14 (Tnfrsf12a). At the same time, no increase in numbers of cytokeratin 19-positive (CK19+) cells was observed in periportal areas, and no CK19+ cells were found in hepatic plates. Since CK19 is thought to be a specific marker of both cholangiocytes and the hepatic progenitor cells, the data indicate a lack of activation of the resident progenitor cells during recovery of hepatic tissue after 80% subtotal hepatectomy. Thus, proliferation of hepatocytes invariably makes the major contribution to the hepatic tissue recovery, although in the cases of subtotal loss this contribution is distinctively modulated. In particular, induction of Sox9 and TWEAK/Fn14 regulatory pathways, conventionally attributed to progenitor cell activation, may incidentally stimulate mitotic activity of hepatocytes.

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.

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.

For a wide range of research purposes it is necessary to perform genotyping, i.e. to test which alleles, each corresponding to a particular locus, are present in the individual genome. Here we suggest a protocol of genotyping for mice with scrambler (scm) mutation. This mutation results in the aberrant splicing of the corresponding mRNA and affects the expression of mdab1 protein. Traditional approaches using genomic Southern hybridization or PCR with specific primers are not suitable for the genotyping of scm because of the lack of comprehensive information on the organization of the gene and on the presence of repetitive sequences in the known region. Here we propose a quick and highly reproducible method for genotyping scm mutant mice. The protocol consists of the following steps: isolation of genomic DNA, digestion with the restriction endonuclease, anchoring of resulting fragments with the adapter, and PCR amplification using adapter-specific primers. The final product of PCR amplification has a characteristic length which is different for the wt (wild type) and scm alleles. Thus, the characteristic pattern of bands obtained for each individual mouse specimen serves as criteria for the presence of wild type and/or scm allele. We believe that this approach could have wider application. The protocol can be easily modified and used as a convenient tool for identification of other genomic defects including those artificially introduced into genome by knockout or gene-trap techniques.

Subtractive hybridization was used to search for the early difference in gene expression between anteriorly and posteriorly regenerating tissues of the same region of the planarian body. A sequence (named scarf) specific for posteriorly regenerating tissue was isolated, encoding a soluble C-type lectin consisting of two slightly different carbohydrate-recognition domains. Such an unusual bivalent structure allows attribution of the protein to a novel subfamily of C-type lectins. scarf expression in intact planarian is rather abundant and follows a characteristic pattern not linked to any known morphological structure. We performed a series of experiments using scarf as a molecular marker. Its expression was monitored during different types of regeneration by whole-mount in situ hybridization and reverse-transcription polymerase chain reaction. The obtained data suggest that scarf expression is positively regulated by anterior tissues closely adjacent to the scarf-expressing region, so that their surgical removal results in rapid scarf switch-off. In turn, tissues expressing scarf seem to inhibit its activation anteriorly. This indicates that at least some of the body patterning events in planarians are based on a system of reciprocal inductive interactions rather than on a global morphogen gradient. q 1998 Academic Press

Here we describe a method for preparing high-quality cDNA libraries from total RNA. By this method, double-stranded (ds) cDNA ligated with a specially designed ds adaptor is amplified by PCR using a modified T-primer and another primer corresponding to the outer part of the adaptor. The suppression PCR effect strongly inhibits the amplification of poly(A)− RNA, thereby reducing background. This method leads to amplification of high-quality cDNA, facilitating the construction of representative cDNA libraries from as little as 10-100 ng of total RNA.

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.

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 .