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Overexpression of the myeloid Src-family kinases Fgr and Hck has been linked to the development of acute myeloid leukemia (AML). Here we characterized the contribution of active forms of these kinases to AML cell cytokine dependence, inhibitor sensitivity, and AML cell engraftment in vivo. The human TF-1 erythroleukemia cell line was used as a model system as it does not express endogenous Hck or Fgr. To induce constitutive kinase activity, Hck and Fgr were fused to the coiled-coil (CC) oligomerization domain of the breakpoint cluster region protein associated with the Bcr-Abl tyrosine kinase in chronic myeloid leukemia. Expression of CC-Hck or CC-Fgr transformed TF-1 cells to a granulocyte–macrophage colony-stimulating factor (GM-CSF)-independent phenotype that correlated with enhanced phosphorylation of the kinase domain activation loop. Both CC-Hck and CC-Fgr cell populations became sensitized to growth arrest by Src-family kinase inhibitors previously shown to suppress the growth of bone marrow cells from AML patients in vitro and decrease AML cell engraftment in immunocompromised mice. Methionine substitution of the ‘gatekeeper’ residue (Thr338) also stimulated Hck and Fgr kinase activity and transformed TF-1 cells to GM-CSF independence without CC fusion. TF-1 cells expressing either active form of Hck or Fgr engrafted immunocompromised mice faster and developed more extensive tumors compared to mice engrafted with the parent cell line, resulting in shorter survival. Expression of wild-type Hck also significantly enhanced bone marrow engraftment without an activating mutation. Reverse phase protein array analysis linked active Hck and Fgr to the mammalian target of rapamycin complex-1/p70 S6 ribosomal protein (mTORC-1/S6) kinase and focal adhesion kinase (Fak) signaling pathways. Combining Hck and Fgr inhibitors with existing mTORC-1/S6 kinase or Fak inhibitors may improve clinical responses and reduce the potential for acquired resistance.

Development of acute myeloid leukemia (AML) is often associated with overexpression of non-receptor tyrosine kinases, including the three Src-family kinase (SFK) members Hck, Lyn and Fgr. SFK inhibitors with significant anti-AML efficacy include the pyrrolopyrimidine A-419259 and the N-phenylbenzamide TL02-59. Previously solved x-ray crystal structures of near-full-length Fgr with both inhibitors revealed that Fgr assumed dramatically different conformations, despite both inhibitors binding the ATP-binding pocket. When bound to A-419259, the regulatory SH3 and SH2 domains packed against the back of the kinase domain, resulting in a closed conformation. Conversely, TL02-59 induced allosteric displacement of the SH3 and SH2 domains from their regulatory positions, resulting in an open conformation.In this study, we aimed to determine if the allosteric displacement of the SH3 and SH2 domains from the back of the kinase domain of Fgr is an important feature for ATP-site inhibitor binding potency of Type-II inhibitors like TL02-59. We investigated the TL02-59 binding mechanism with an emphasis on the role of allosteric changes to the overall Fgr kinase conformation on inhibitor action. Fgr mutants were generated with enhanced SH3 domain interaction with the SH2-kinase linker (high affinity linker or ‘HAL’ mutants), to shift the conformational preference of Fgr to a closed arrangement. X-ray crystal structures of all three Fgr SH3-SH2-HAL proteins were determined at high resolution (≤ 1.6 Å), revealing a global domain arrangement and key residue orientations compatible with enhanced SH3-linker interaction. Fluorescence polarization and surface plasmon resonance experiments confirmed enhanced intramolecular SH3:linker interactions in Fgr HAL mutants. Interestingly, restriction of Fgr regulatory domain dynamics increased the Km for ATP and enhanced the potency of TL02-59 and A-419259 in vitro, and in AML cells expressing an active version of Fgr, suggesting that both ATP-site inhibitors prefer the closed Fgr conformation. TF-1 cells expressing the HAL variant of Fgr showed significantly decreased bone marrow engraftment in immunocompromised mice compared to the wild-type, suggesting that survival of these cells in the bone marrow requires SH3-dependent Fgr signaling activity. This work supports the development of small molecule SH3-antagonists, with the potential to enhance ATP-site inhibitor potency and interfere with AML cell survival.

Despite the fact that AML is the most common acute leukemia in adults, patient outcomes are poor necessitating the development of novel therapies. We identified that inhibition of Thioredoxin Reductase (TrxR) is a promising strategy for AML and report a highly potent and specific inhibitor of TrxR, S-250. Both pharmacologic and genetic inhibition of TrxR impairs the growth of human AML in mouse models. We found that TrxR inhibition leads to a rapid and marked impairment of metabolism in leukemic cells subsequently leading to cell death. TrxR was found to be a major and direct regulator of metabolism in AML cells through impacts on both glycolysis and the TCA cycle. Studies revealed that TrxR directly regulates GAPDH leading to a disruption of glycolysis and an increase in flux through the pentose phosphate pathway (PPP). The combined inhibition of TrxR and the PPP led to enhanced leukemia growth inhibition. Overall, TrxR abrogation, particularly with S-250, was identified as a promising strategy to disrupt AML metabolism.

From business transactions to the obtaining of degrees online in the University of Puerto Rico, to the procedures that every citizen carries out daily in agencies like the Department of Transportation and in public corporations like the electric power and aqueducts authorities. Not only has this allowed for multi-million dollar savings, it has resulted in a significant improvement in citizen services at a lower cost.

La presente investigación busca demostrar cómo, en nuestro país, las normas laborales sobre conciliación de la vida familiar y personal, al no contar con una perspectiva de género ni que considere a la niñez, generan una triple discriminación dentro de la familia formando lo que denominamos el “triángulo de la vulnerabilidad”, en que se encasilla a la madre en un rol de cuidado; se despoja al padre de su derecho sin valorar la importancia del respeto y fomento al vínculo paterno-filial; y se impide que el menor pueda gozar de la atención, cuidados y crianza de ambos padres. Para demostrarlo, la tesis se divide en tres capítulos. El primero busca resaltar la importancia y necesidad de la deconstrucción normativa a través de la perspectiva de género, y presenta diferentes conceptos esenciales, tales como la discriminación estructural o sistémica, que serán herramientas necesarias para la fiel comprensión del tema que abordamos. El segundo capítulo plantea que el derecho del trabajo, al responder con indiferencia normativa, actualmente enfrenta una “nueva cuestión social” que, en realidad, se puede resolver recordando sus bases fundamentales y su esencia. Además, se exploran los conceptos de conciliación y corresponsabilidad para arribar, luego, a la conciliación con corresponsabilidad, que ―considerando siempre el interés superior del niño e incorporando el mainstreaming de género―, desde la normativa internacional, consolida una serie de deberes y derechos. Finalmente, el tercer capítulo revisa la normativa nacional al respecto y nos permite afirmar que, aunque de forma inadvertida, nuestra actual Constitución reconoce este derecho a la conciliación con corresponsabilidad como autónomo y jurídicamente exigible. De esta forma, la deconstrucción de la división sexual del trabajo se transforma en un deber constitucional, y se deja de percibir la conciliación entre esfera laboral y familiar como un reto exclusivo de las mujeres. Para su consecución, es imperativo motivar la participación activa de los hombres en el ámbito familiar, ya que, sin su intervención consciente, no existirá cambio verdadero.

While ATP-site inhibitors for protein-tyrosine kinases are often effective drugs, their clinical utility can be limited by off-target activity and acquired resistance mutations due to the conserved nature of the ATP-binding site. However, combining ATP-site and allosteric kinase inhibitors can overcome these shortcomings in a double-drugging framework. Here we explored the allosteric effects of two pyrimidine diamines, PDA1 and PDA2, on the conformational dynamics and activity of the Src-family tyrosine kinase Hck, a promising drug target for acute myeloid leukemia. Using 1H-15N HSQC NMR, we mapped the binding site for both analogs to the SH3 domain. Despite the shared binding site, PDA1 and PDA2 had opposing effects on near-full-length Hck dynamics by hydrogen-deuterium exchange mass spectrometry, with PDA1 stabilizing and PDA2 disrupting the overall kinase conformation. Kinase activity assays were consistent with these observations, with PDA2 enhancing kinase activity while PDA1 was without effect. Molecular dynamics simulations predicted selective bridging of the kinase domain N-lobe and SH3 domain by PDA1, a mechanism of allosteric stabilization supported by site-directed mutagenesis of N-lobe contact sites. Cellular thermal shift assays confirmed SH3 domain-dependent interaction of PDA1 with wild-type Hck in myeloid leukemia cells and with a kinase domain gatekeeper mutant (T338M). These results identify PDA1 as a starting point for Src-family kinase allosteric inhibitor development that may work in concert with ATP-site inhibitors to suppress the evolution of resistance.Competing Interest StatementThe authors have declared no competing interest.

Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO 2 by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity of acetate kinase and N- acetyl-γ-glutamyl phosphate reductase, we demonstrate this phosphate (P i )-based route in vitro and in vivo. We further engineer a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism and interface the P i route with a recently developed formaldehyde assimilation pathway to enable C2 compound formation from formate as the sole carbon source in Escherichia coli . The P i route therefore offers a potent tool in expanding the landscape of synthetic formate assimilation. A key step in the assimilation of formate is its reduction into formaldehyde. Here, the authors develop a two-enzyme route in which formate is activated into formyl phosphate and reduced by NAD(P)H into formaldehyde and confirm its functionality in vitro and in vivo.

Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to population-averaged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships) is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation). Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity.

The aim of this study is to analyze the influence of Confucianism on health behaviors, health outcomes and medical decisions. The research was conducted using the following databases: PubMed, Scopus, CINHAL, PsycINFO and Web of Science, without restrictions of language and year of publication. The search process identified 833 publications matching the search criteria, and after the review process, 40 articles were included. Family is a central aspect of Confucianism, and it seems to affect participation in medical decisions, taking care of relatives, ethical dilemmas and mental health problems. Although most Confucianist influence seems to be positive, some ways of thinking could increase suffering, burnout and a delay in healthcare seeking. Understanding these values could help health professionals to deal with the growing contingent of patients with different cultures and religious beliefs.