Coupled mutation finder: A new entropy-based method quantifying phylogenetic noise for the detection of compensatory mutations

Background: The detection of significant compensatory mutation signals in multiple sequence alignments (MSAs) is often complicated by noise. A challenging problem in bioinformatics is remains the separation of significant signals between two or more non-conserved residue sites from the phylogenetic noise and unrelated pair signals. Determination of these non-conserved residue sites is as important as the recognition of strictly conserved positions for understanding of the structural basis of protein functions and identification of functionally important residue regions. In this study, we developed a new method, the Coupled Mutation Finder (CMF) quantifying the phylogenetic noise for the detection of compensatory mutations.Results: To demonstrate the effectiveness of this method, we analyzed essential sites of two human proteins: epidermal growth factor receptor (EGFR) and glucokinase (GCK). Our results suggest that the CMF is able to separate significant compensatory mutation signals from the phylogenetic noise and unrelated pair signals. The vast majority of compensatory mutation sites found by the CMF are related to essential sites of both proteins and they are likely to affect protein stability or functionality.Conclusions: The CMF is a new method, which includes an MSA-specific statistical model based on multiple testing procedures that quantify the error made in terms of the false discovery rate and a novel entropy-based metric to upscale BLOSUM62 dissimilar compensatory mutations. Therefore, it is a helpful tool to predict and investigate compensatory mutation sites of structural or functional importance in proteins. We suggest that the CMF could be used as a novel automated function prediction tool that is required for a better understanding of the structural basis of proteins. The CMF server is freely accessible at http://cmf.bioinf.med.uni-goettingen.de

Когнітивні структури репрезентації хрематонімійних знань

В запропонованій статті розглядаються основні структури репрезентації хрематонімів в ментальному лексиконі індивіда. Питання побудови моделей та схем організації ментального лексикону привертає велику увагу лінгвістів сьогодні.В предложенной статье рассматриваются основные структуры репрезентации хрематонимов в ментальном лексиконе индивида. Вопрос построения моделей и схем организации ментального лексикона привлекает огромное внимание лингвистов.The article deals with the main representation structures of chrematonyms in the mental lexicon of an individual. The task of model construction and scheme organization of mental lexicon attracts much attention of linguists today

Long-term expansion, enhanced chondrogenic potential, and suppression of endochondral ossification of adult human MSCs via WNT signaling mo

Mesenchymal stem cells (MSCs) are a potential source of chondrogenic cells for the treatment of cartilage disorders, but loss of chondrogenic potential during in vitro expansion and the propensity of cartilage to undergo hypertrophic maturation impede their therapeutic application. Here we report that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion of human bone marrow-derived MSCs. The cells retained their chondrogenic potential and other phenotypic and functional properties of multipotent MSCs, which were gradually lost in the absence of WNT3A. Moreover, we discovered that endogenous WNT signals are the main drivers of the hypertrophic maturation that follows chondrogenic differentiation. Inhibition of WNT signals during differentiation prevented calcification and maintained cartilage properties following implantation in a mouse model. By maintaining potency during expansion and preventing hypertrophic maturation following differentiation, the modulation of WNT signaling removes two major obstacles that impede the clinical application of MSCs in cartilage repair

Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures

Abstract Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with LinCD34+ CD38lowCD45RACD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC

Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells

Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein

Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells

Therapeutic application of human embryonic stem cells (hESCs) requires precise control over their differentiation. However, spontaneous differentiation is prevalent, and growth factors induce multiple cell types; e.g., the mesoderm inducer BMP4 generates both mesoderm and trophoblast. Here we identify endogenous WNT signals as BMP targets that are required and sufficient for mesoderm induction, while trophoblast induction is WNT independent, enabling the exclusive differentiation toward either lineage. Furthermore, endogenous WNT signals induce loss of pluripotency in hESCs and their murine counterparts, epiblast stem cells (EpiSCs). WNT inhibition obviates the need to manually remove differentiated cells to maintain cultures and improves the efficiency of directed differentiation. In EpiSCs, WNT inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras. Our findings show that endogenous WNT signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs

Wnt3a inhibits SR1-enhanced CD34⁺ cell expansion.

<p>UCB-derived CD34⁺ HSPC were cultured for 14 days in SFT or SFTSR1 medium with or without the addition of Wnt3a. Flowcytometric analysis was performed at day 14. Depicted is the CD34⁺ cell expansion after 14 days of culture (n = 5). <i>* p<0</i>.<i>05</i>, <i>** p<0</i>.<i>01</i></p

Exogenous Wnt3a reduces growth factor-driven expansion of CD34⁺ cells.

<p>UCB-derived CD34⁺ cells were cultured in serum free SFT medium with or without the addition of Wnt3a. Cells were analyzed using flow cytometry at 7 and 14 days of culture. Shown are (A) the total nucleated cell expansion compared to input (n = 8), (B) the frequency of CD34⁺ cells within the TNC population during culture (n = 8), (C) expression of CD45 and CD34 after 14 days of culture in SFT with (lower panel) or without (upper panel) Wnt3a (representative experiment out of 8), (D) the expansion of CD34⁺ cells compared to input (n = 8) and (E) the frequency of cells expressing lineage markers after 7 and 14 days of culture (n = 6). (F) Frequency of CFU-GEMM, BFU-E and CFU-GM in 250 CD34⁺ cells cultured for 2 weeks in SFT or SFT+Wnt3a (n = 2, 3 dishes per experiment). (G) CD34⁺ cell expansion compared to SFT medium after 14 days of culture with different dosages of Wnt3a (n = 2). (H) Levels of human chimerism at several time points after transplantation with the progeny of 10⁵ CD34⁺ cells cultured for 7 days in SFT or SFT+Wnt3a medium (n = 5 mice per group). (I) Levels of human chimerism in bone marrow 17 weeks after transplantation with the progeny of 10⁵ CD34⁺ cells cultured for 7 days in SFT or SFT+Wnt3a medium (n = 5 mice per group). <i>* p<0</i>.<i>05</i>, <i>** p<0</i>.<i>01</i>, <i>*** p<0</i>.<i>001</i></p

Wnt3a-mediated inhibition of HSPC expansion is due to activation of the canonical Wnt3a pathway.

<p>(A) Expansion of CD34⁺ cells after 7 and 14 days of culture in SFT, SFT+Wnt3a, SFT+Fr8CRD and SFT+Wnt3a+Fr8CRD (n = 2). (B) CD34⁺ cell frequency within the TNC population during these cultures (n = 2). (C) CD34⁺ cell expansion in SFT and SFT+CHIR99021 after 7 and 14 days of culture (n = 2).</p