Quantification of the interfacial and bulk contributions to the longitudinal spin Seebeck effect

This article appeared in P. Jiménez-Cavero et al. Applied Physics Letters 118, 092404 (2021) and may be found at https://aip.scitation.org/doi/10.1063/5.0038192We report the disentanglement of bulk and interfacial contributions to the thermally excited magnon spin current in the spin Seebeck effect under static heating. For this purpose, we have studied the dependence of the inverse spin Hall voltage and the thermal conductivity on the magnetic layer thickness. Knowledge of these quantities allows us to take into account the influence of both sources of thermal spin current in the analysis of the voltage dependence. The magnetic layer thickness modulates the relative magnitude of the involved thermal drops for a fixed total thermal difference throughout the sample. In the end, we attain the separate contributions of both sources of thermal spin current—bulk and interfacial—and obtain the value of the thermal magnon accumulation length scale in maghemite, which we find to be 29(1) nm. According to our results, bulk magnon accumulation dominates the spin Seebeck effect in our studied range of thicknesses, but the interfacial component is by no means negligibleThis work was supported by the Spanish Ministry of Science [through Project Nos. MAT2014-51982-C2-R, MAT2016-80762-R, MAT2017-82970-C2-R, and PID2019-104150RB-I00 (including FEDER funding) and the Aragón Regional government (Project No. E26)]. P.J.-C. acknowledges Spanish MECD for support through FPU program (Reference No. FPU014/02546). D.B. acknowledges support from MINECO (Spain) through an FPI program (No. BES-2017-079688). R.R. also acknowledges support from the European Commission through the Project No. 734187-SPICOLOST (H2020-MSCA-RISE-2016), the European Union's Horizon 2020 research and innovation program through the Marie Sklodowska-Curie Actions Grant Agreement SPEC No. 894006, and the Spanish Ministry of Science (No. RYC 2019-026915-I). Authors acknowledge the Advanced Microscopy Laboratory-INA University of Zaragoza for offering access to their instruments. C. L-B. acknowledges Xunta de Galicia and ESF for a PhD Grant (ED481A-2018/013)S

A 17-residue sequence from the matrix metalloproteinase-9 (MMP-9) hemopexin domain binds α4β1 integrin and inhibits MMP-9-induced functions in chronic lymphocytic leukemia B cells

13 páginas, 7 figuras, 2 tablas -- PAGS nros. 27601-27613We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4β1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4β1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC50 values of 138 and 279 μm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesisThis work was supported by Grants SAF2009–07035 and RTICC RD06/0020/0011 (to A. G.-P.) and RTICC RD06/0020/0080 (to M. J. T.) from the Ministerio de Ciencia e Innovación, Spain, and by a grant from the Fundación Puerta de Hierro (to J. A. G. M.)Peer reviewe

Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

Investigación joven con perspectiva de género II

Actas del II Congreso de jóvenes investigadorxs con perspectiva de género (Getafe, 26 y 27 de junio de 2017) organizado por el Instituto Universitario de Estudios de Género de la Universidad Carlos III de Madrid

Papel del microambiente en la respuesta de células de leucemia linfocítica crónica a trióxido de arsénico: mecanismos de resistencia

182 p.-56 fig.-12 tab.Introduction Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant CD5+ B lymphocytes (CLL cells) in the peripheral blood. The progression of the disease is determined by CLL cell infiltration in lymphoid organs, mainly lymph nodes and bone marrow, where they receive survival signals from common components of these tissue microenvironments, such as MMP-9 and stromal cells. These survival signals impair therapy, indeed, despite the efforts made, CLL remains an incurable disease, becoming crucial to continue searching for new therapeutic options based on the contribution of the microenvironment to the disease progression. Arsenic trioxide (ATO) is successfully employed for the treatment of acute promyelocytic leukemia and is being trialed for other hematological malignancies, including CLL. Indeed, ATO induces apoptosis in all CLL cases and could constitute an efficient therapy for this disease. Objectives In this context, the work that makes up this Doctoral Thesis is organized around three main objectives: - Molecular characterization of the ATO effect in CLL cells. - Determination of the role of MMP-9 in the CLL cell response to ATO. - Analysis of the influence of other molecules and signaling pathways induced by the microenvironment in the CLL cell response to ATO. Methodology We used B lymphocytes from the peripheral blood of 47 CLL patients (CLL cells) and MEC-1 cells (CLL-derived), both the parental cell line as MMP-9 stable transfectants. We used also co-cultures of CLL cells and bone marrow stromal cells (BMSCs), as well as BMSC-conditioned medium. The effect of drugs (ATO and Fludarabine) was determined by flow cytometry, using Annexin V-FITC/propidium iodide, and by the MTT assay. The global gene expression profile of MEC-1 cells in response to ATO was analyzed by RNA microarrays. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression and association was analyzed by Western blotting and immunoprecipitation, respectively. Gene silencing was performed by nucleofection of specific siRNAs. Statistical analyses were performed using the two-tailed Student's t-test and a p value of ≤ 0.05 was considered significant. Results The gene expression profile induced by ATO in CLL cells reflects an antioxidant and defensive response. Indeed, among the most significantly enriched Gene Ontology terms in the group of upregulated genes were found regulation of apoptosis, response to wounding and response to oxidative stress. Since the main effect of ATO in CLL cells is the induction of apoptosis, we were interested in the genes related to regulation of this process (HMOX1, HSPA1B, CLU, TNFRSF9, GCLM, NQO1, SQSTM1, CTSB and MMP9), particularly in those encoding HMOX-1 and MMP-9 due to their roles on oxidative stress regulation and CLL cell survival, respectively. We have studied the role of HMOX-1 and MMP-9 in the CLL cell response to ATO and found that this agent upregulates HMOX-1 (mRNA and protein) in CLL cells and this seem to favor its cytotoxic effect, thus suggesting a proapoptotic role for HMOX-1 in response to ATO. On the other hand, ATO also upregulates MMP-9 (mRNA and protein) and its localization at the surface of early apoptotic cells, being both processes dependent on apoptosis onset. These results suggest a role for MMP-9 in the CLL cell response to these drugs. Indeed, isolated or stromal MMP-9 induce CLL cell resistance to ATO and fludarabine, which is also achieved by overexpressing MMP-9 in MEC-1 cells and is reversed by MMP9 gene silencing. The molecular mechanism underlying MMP-9-induced drug resistance involves modulation of the balance between the anti- and pro-apoptotic Bcl-2 family members.Culturing CLL cells on BMSCs also protects CLL cells from the ATO-induced apoptosis and this protection is overcome by blocking MMP-9 or α4β1 and/or αLβ2 integrins. The BMSC-conditioned medium also partially protects CLL cells against ATO-induced apoptosis, indicating that both cell-cell interactions and soluble factors are contributing to BMSC-induced resistance to ATO in CLL cells. We have studied the molecular mechanism involves in this protection and found that BMSCs induce PI3K/Akt, PKC, Lyn and ERK activation, as well as NF-κB and STAT3, and also upregulates Mcl-1 and Bcl-xL in CLL cells. Using inhibitors of these pathways, we demonstrated that PI3K and PKC are involved in the stroma-induced resistance to ATO. Further, blocking PI3Kδ or PKCβ isoforms with Idelalisib or Sotrastaurin, respectively, inhibits Akt phosphorylation, NF-κB/STAT3 activation and Mcl-1 upregulation and restores CLL cell sensibility to ATO. Likewise, MCL1 gene silencing also overcomes the protecting effect of stroma and confirms the key role of this protein in the mechanism of stroma-induced resistance to ATO in CLL cells. Conclusions In CLL cells, ATO induces expression of genes related with an antioxidant and defensive response. HMOX-1 seems to favor the cytotoxic effect of ATO, while MMP-9 promotes cell survival via modulation of the balance between the pro- and anti-apoptotic members of the Bcl-2 family. BMSCs also induce resistance to ATO in CLL cells and this protection is overcame by blocking MMP-9 or the integrins α4β1 or αLβ2. The molecular mechanism underlying this resistance involves activation of the PI3Kδ/PKCβ→Akt→NF-κB/STAT3→Mcl-1, and blocking PI3Kδ and PKCβ restores sensibility to ATO. Taken together, our results indicate that combination of ATO with specific inhibitors for MMP-9 and/or PI3Kδ and PKCβ may constitute an efficient alternative/complementary therapy for CLL.Proyectos SAF2009-07035, SAF2012-31613 (AGP) Y RTICC (Red Temática de Investigación Cooperativa en Cáncer) RD06/0020/0011 (AGP) y RD12/0036/0061 (AGP) del Ministerio de Economía y Competividad (MINECO) Proyecto S2010/BMD-2314-Neoplasbim (AGP) DE LA Comunidad de Madrid/Unión Europea Beca (JAEPre2010-00607) del CSICPeer reviewe

Papel de microambiente en la respuesta de células de leucemia linfocítica crónica a trióxido de arsénico: mecanismos de resistencia

La leucemia linfocítica crónica, LLC, se caracteriza por acumulación en sangre periférica de linfocitos B aberrantes CD5 positivos, en adelante células LLC. La enfermedad progresa cuando estas células infiltran órganos linfoides, donde reciben señales de supervivencia que dificultan la terapia. A pesar de los avances, la LLC continúa siendo una enfermedad incurable, siendo necesario desarrollar nuevas estrategias terapéuticas sobre la base de la contribución del microambiente a la patogénesis de la enfermedad. El trióxido de arsénico, en lo sucesivo ATO, induce eficientemente apoptosis en todos los casos de LLC, presentándose como una potencial terapia. El perfil de expresión génica inducido por ATO en células LLC es consistente con una respuesta antioxidante y defensiva. De hecho, ATO induce la expresión de genes como los codificantes de HMOX1 y MMP9, con funciones en la regulación del estrés oxidativo y la supervivencia de las células LLC, respectivamente. HMOX1 parece favorecer el efecto del ATO y, por tanto, media señales proapoptóticas, mientras que MMP9 promueve la supervivencia celular por un mecanismo que implica la modulación del balance entre los miembros pro y antiapoptóticos de la familia BCL2. El cocultivo con estroma de médula ósea, en adelante BMS, también protege a las células LLC de la apoptosis inducida por ATO, y esta protección es revertida por el bloqueo de MMP9 o de las integrinas alfa4beta1 o alfaLbeta2, indicando que tanto factores solubles como interacciones directas célula-célula están implicados en la misma. Además, el cocultivo con BMS induce activación en las células LLC de AKT dependiente de PI3Kdelta y PKCbeta. Esto conduce, a su vez, a la activación de los factores de transcripción NFkappaB y STAT3 que, finalmente, median un incremento en la expresión de la proteína antiapoptótica MCL1. Esta inducción de MCL1 en las células LLC por el mecanismo anterior es esencial en la resistencia a ATO inducida por estroma. De hecho, el bloqueo de PI3Kdelta y PKCbeta o la disminución de los niveles de MCL1 revierten esta protección. Considerados conjuntamente, estos resultados sugieren que la combinación de ATO con inhibidores específicos de MMP9, PI3Kdelta y,o PKCbeta podría constituir una eficiente terapia alternativa o complementaria para la LLC

Gene expression profile induced by arsenic trioxide in chronic lymphocytic leukemia cells reveals a central role for heme oxygenase-1 in apoptosis and regulation of matrix metalloproteinase-9

19 p.-9 fig.-1 tab.CLL remains an incurable disease in spite of the many new compounds being tested. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the gene expression profile induced by ATO in CLL cells. ATO modulated many genes, largely involved in oxidative stress, being HMOX1 the most upregulated gene, also induced at the protein level. ATO also increased MMP-9, as we previously observed, both at the mRNA and protein level. Using specific inhibitors, qPCR analyses, and gene silencing approaches we demonstrate that upregulation of MMP-9 by ATO involved activation of the p38 MAPK/AP-1 signaling pathway. Moreover, gene silencing HMOX1 or inhibiting HMOX1 activity enhanced p38 MAPK phosphorylation and c-jun expression/activation, resulting in transcriptional upregulation of MMP-9. Overexpression of HMOX1 or enhancement of its activity, had the opposite effect. Cell viability analyses upon modulation of HMOX1 expression or activity demonstrated that HMOX1 had a proapoptotic role and enhanced the cytotoxic effect of ATO in CLL cells. We have therefore identified a new mechanism in which HMOX1 plays a central role in the response of CLL cells to ATO and in the regulation of the anti-apoptotic protein MMP-9. Thus, HMOX1 arises as a new therapeutic target in CLL and the combination of HMOX1 modulators with ATO may constitute an efficient therapeutic strategy in CLL.This work was supported by grants SAF2012-31613 and SAF2015-69180R (AGP) and RTICC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0061 (AGP), from the Ministry of Economy and Competitiveness (MINECO), Spain; S2010/BMD-2314-Neoplasbim (AGP) from the Comunidad de Madrid/European Union; and a grant from the Fundación para la Investigación Biomédica Hospital Universitario Puerta de Hierro, Madrid (JAGM).Peer reviewe

Overexpression of progelatinase B/proMMP-9 affects migration regulatory pathways and impairs chronic lymphocytic leukemia cell homing to bone marrow and spleen

This study addresses the role of (pro)MMP-9 overexpression in CLL cell migration. We have used primary CLL cells and CLL-derived MEC-1 cells transfected with empty (mock cells) or proMMP-9-encoding (MMP-9 cells) lentiviral vectors. The constitutive (pro)MMP-9 expression in mock cells and primary CLL cells was similar, whereas in MMP-9 cells, expression resembled that of CLL cells incubated with proMMP-9. In xenograft models, in NOD/SCID mice, MMP-9-MEC-1 transfectants showed significantly reduced homing to bone marrow and spleen compared with mock cells. Likewise, incubation of primary CLL cells with proMMP-9, before injection into mice, inhibited their homing to these organs. This inhibition was specific, dose-dependent, and observed in all CLL tested, independently of prognostic markers or disease stage. Additionally, the MMP-9 catalytic activity was only partially involved, as the inactive mutant proMMP-9MutE had a partial effect. MMP-9 cells also showed impaired migration in vitro, which was reverted by reducing (pro)MMP-9 expression with siRNAs. CLL migration thus requires optimal (pro)MMP-9 expression levels, below or above which migration is hampered. Biochemical analysis of the (pro)MMP-9 effect indicated that MMP-9 cells or primary CLL cells incubated with proMMP-9 had reduced activation of migration regulatory molecules, including RhoAGTPase, Akt, ERK, and FAK. In contrast, p190RhoGAP (RhoA inhibitor) and PTEN (Akt/ERK/FAK inhibitor) were up-regulated in MMP-9 cells. Reduction of (pro)MMP-9 expression by siRNAs restored RhoA activity and diminished PTEN levels. Our results reveal a novel function for (pro)MMP-9 in modulating signaling pathways leading to CLL cell arrest. Therefore, local high (pro)MMP-9 expression may contribute to malignant cell retention in lymphoid organs and disease progression.status: publishe

MMP-9 expression in MEC-1 cells prevents downregulation of anti-apoptotic Bcl-2 family proteins in response to ATO.

<p>(A) 5×10⁶ Mock- or MMP-9-MEC-1 cells were treated or not with 5 µM ATO. After 24 h cells were lysed and the expression of the indicated proteins was analyzed by Western blotting, using vinculin as an internal control. A representative experiment is shown for each case and numbers indicate the average values from all experiments performed, after normalizing Mock control values to 1. (B) The indicated ratios of anti-apoptotic/pro-apoptotic proteins are shown. (C) 3×10⁷ Mock- or MMP-9-cells were treated or not with 5 µM ATO for 24 h. Cells were lysed and lysates immunoprecipitated with anti-Mcl-1 or control Abs and analyzed by Western blotting. Values indicate the amount of Bim found in the Mcl-1 immunoprecipitates in both types of MEC-1 transfectants. * or ^#P≤0.05; ** or ^##P≤0.01; *** or ^###P≤0.001. Symbols are: *, Mock- vs MMP-9-cells; ^#, Mock- or MMP-9-cells treated with ATO compared to their respective untreated counterparts.</p

MMP-9 localizes to the CLL cell surface in response to ATO and in correlation with induction of apoptosis.

<p>(A) 5×10⁶ CLL cells in RPMI/0.1% FBS were treated or not with 3 µM ATO for 24 h. The conditioned media was collected, concentrated 20× and analyzed by gelatin zymography. The results from four representative samples and the average normalized values (arbitrary units, AU) from all six samples studied are shown. (B) 1.5×10⁵ CLL cells were incubated with or without 3 µM ATO for 24 h. MMP-9 surface expression was analyzed by flow cytometry using an anti-MMP-9 pAb or a control pAb. Histograms from six representative cases are shown, where white areas correspond to control/untreated cells and grey areas to ATO treated cells. Arrows indicate specific fluorescence (SF). Average normalized values from all ten samples analyzed are also shown. (C) 30×10⁶ CLL cells in RPMI/0.1%FBS were incubated with or without 3 µM ATO for 24 h. Membrane (Mb) and cytosolic (Cyt) fractions were separated and analyzed by gelatin zymography. RhoGDI and CD45 detected by Western blotting in the same lysates were used as controls for the procedure. The conditioned media (CM) of these cells was also analyzed by gelatin zymography and the normalized average values of the quantitated bands are shown (D) 1.5×10⁵ CLL cells in RPMI/0.1%FBS were incubated for 1 h with or without the indicated antibodies. 3 µM ATO was added and after 24 h, surface-bound MMP-9 was determined by flow cytometry using an anti-MMP-9 pAb or a control pAb. Histograms for two representative cases are shown. White areas correspond to untreated cells (−ATO) and grey areas (both light and dark) to ATO-treated cells (+ATO). Average normalized SF values are also shown. **P≤0.01; ***P≤0.001.</p