cAMP controls cytosolic Ca(2+ )levels in Dictyostelium discoideum

BACKGROUND: Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca(2+ )concentration ([Ca(2+)](i)) that is composed of liberation of stored Ca(2+ )and extracellular Ca(2+)-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca(2+)](i). RESULTS: We analyzed Ca(2+)-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca(2+ )was reduced as compared to wild type. Yet, single cell [Ca(2+)](i)-imaging of regA(- )amoebae revealed a cAMP-induced [Ca(2+)](i )increase even in the absence of extracellular Ca(2+). The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca(2+)](i )in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca(2+)](i)-transients also in nominally Ca(2+)-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced. CONCLUSION: We conclude that there is a link between the second messengers cAMP and Ca(2+). cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca(2+)](i )of regA(- )cells. Intracellular cAMP levels control [Ca(2+)](i )by regulating Ca(2+)-fluxes of stores which in turn affect Ca(2+)-influx, light scattering oscillations and chemotactic performance

Dysregulation of H/ACA ribonucleoprotein components in chronic lymphocytic leukemia

Telomeres are protective repeats of TTAGGG sequences located at the end of human chromosomes. They are essential to maintain chromosomal integrity and genome stability. Telomerase is a ribonucleoprotein complex containing an internal RNA template (hTR) and a catalytic subunit (hTERT). The human hTR gene consists of three major domains; among them the H/ACA domain is essential for telomere biogenesis. H/ACA ribonucleoprotein (RNP) complex is composed of four evolutionary conserved proteins, including dyskerin (encoded by DKC1 gene), NOP10, NHP2 and GAR1. In this study, we have evaluated the expression profile of the H/ACA RNP complex genes: DKC1, NOP10, NHP2 and GAR1, as well as hTERT and hTR mRNA levels, in patients with chronic lymphocytic leukemia (CLL). Results were correlated with the number and type of genetic alteration detected by conventional cytogenetics and FISH (fluorescence in situ hybridization), IGHV (immunoglobulin heavy chain variable region) mutational status, telomere length (TL) and clinico pathological characteristics of patients. Our results showed significant decreased expression of GAR1, NOP10, DKC1 and hTR, as well as increased mRNA levels of hTERT in patients compared to controls (p=0.04). A positive correlation between the expression of GAR1-NHP2, GAR1-NOP10, and NOP10-NHP2 (p=0.0001), were observed. The analysis taking into account prognostic factors showed a significant increased expression of hTERT gene in unmutated-IGHV cases compared to mutated-CLL patients (p = 0.0185). The comparisons among FISH groups exhibited increased expression of DKC1 in cases with two or more alterations with respect to no abnormalities, trisomy 12 and del13q14, and of NHP2 and NOP10 compared to those with del13q14 (p = 0.03). The analysis according to TL showed a significant increased expression of hTERT (p = 0.0074) and DKC1 (p = 0.0036) in patients with short telomeres compared to those with long TL. No association between gene expression and clinical parameters was found. Our results suggest a role for these telomere associated genes in genomic instability and telomere dysfunction in CLL.Fil: Dos Santos, Patricia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Palau Nagore, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Stella, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Ibrutinib impairs the phagocytosis of rituximab-coated leukemic cells from chronic lymphocytic leukemia patients by human macrophages

We have read with great interest the recent article of Kohrt, H.E. et al1 showing that Ibrutinib prevented NK cell mediated cytotoxicity of antibody-coated CLL cells in vitro. They also found that the concurrent treatment with Ibrutinib and rituximab or trastuzumab reduces the therapeutic efficacy of both anti-CD20 antibodies in a mouse model, while the sequential treatment with Ibrutinib and rituximab restored its anti-lymphoma activity. Since macrophages are the most important effector cells in CD20-directed cytotoxicity in murine models2,3 and they probably play a key role in human anti-CD20 therapy4,5, we determined whether Ibrutinib interferes the capacity of human macrophages to mediate phagocytosis of rituximab-coated CLL cells. To address this issue, macrophages differentiated from healthy peripheral blood monocytes were treated with or without Ibrutinib for 30 minutes and then cultured for 1, 2 or 3 hours with CFSE-labeled CLL cells or rituximab-coated CFSE-labeled CLL cells. Then, cells were tripsinized and the proportion of macrophages that have taken up CFSE-labeled CLL cells (CFSE+ macrophages) were scored by flow cytometry and verified using confocal microscopy, as previously described6. As expected, we found that the cultures with rituximab-coated CLL cells showed the highest percentage of CFSE+ macrophages, which increase in a time dependent manner (open circles in Figure 1A). Ibrutinib was able to reduce these values in all the times evaluated (solid circles in Figure 1A). Low percentages of CFSE+ macrophages were obtained in cultures with uncoated CLL cells, which were not modified by Ibrutinib (open and solid squares in Figure 1A). In addition, we found that Ibrutinib diminishes the percentage of CFSE+ macrophages in the cultures with rituximab-coated cells in a dose dependent manner (Figure 1B), which was not associated to a decreased viability of the macrophages (not shown). Moreover, the inhibitory effect of Ibrutinib was not limited to rituximab since comparable results were obtained when campath-coated CFSE-labeled CLL cells were employed (Figure 1C). Similar results were found when macrophages from CLL patients were used: mean±SE of the % of CFSE+ macrophages: 26.8 ± 2.1 vs, 17.3 ± 2.7 vs 10.8 ± 0.7 for rituximab-coated CFSE-labeled CLL cells alone, with 0.5μM or 5μM of Ibrutinib (n= 6). Representative dot plots are shown in Figure 1D. The results obtained by flow cytometry analysis were validated by confocal microscopy quantifying the number of macrophages that engulfed at least one tumor target cell (Figure 1E). A representative experiment is shown in Figure 1F. In addition, by performing a binding assay at 4oC, we confirmed that Ibrutinib did not reduce the binding of rituximab-coated CFSE-labeled CLL cells to macrophages (Figure 1G). Interestingly, while the presence of Ibrutinib during the assay impairs the phagocytosis of rituximab-coated CLL cells, when Ibrutinib was washed out, macrophages recovered their phagocytic capacity in a time-dependent manner (Figure 1H). In conclusion we found that the presence of Ibrutinib impairs the phagocytosis of rituximab-opsonized CLL cells by human macrophages, which was restored when the inhibitor was removed from the cultures. Our results, and those obtained by Kohrt et al1 suggest that the sequential administration of Ibrutinib followed by rituximab, and not the concurrent treatment of the patients with these agents, might enhance their anti-tumor activity in vivo.Fil: Borge, Mercedes. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Almejún, María Belén. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología e Inmunología; ArgentinaFil: Podaza, Enrique Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Colado, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fernández Grecco, Horacio. Sanatorio Municipal Dr. Julio Méndez; ArgentinaFil: Cabrejo, María. Sanatorio Municipal Dr. Julio Méndez; ArgentinaFil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos ; ArgentinaFil: Giordano, Mirta Nilda. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin

Extraordinary linear dynamic range in laser-defined functionalized graphene photodetectors

This is the author accepted manuscript. The final version is available from AAAS via the DOI in this record.Graphene-based photodetectors have demonstrated mechanical flexibility, large operating bandwidth, and broadband spectral response. However, their linear dynamic range (LDR) is limited by graphene’s intrinsic hot-carrier dynamics, which causes deviation from a linear photoresponse at low incident powers. At the same time, multiplication of hot carriers causes the photoactive region to be smeared over distances of a few micrometers, limiting the use of graphene in high-resolution applications. We present a novel method for engineering photoactive junctions in FeCl3-intercalated graphene using laser irradiation. Photocurrent measured at these planar junctions shows an extraordinary linear response with an LDR value at least 4500 times larger than that of other graphene devices (44 dB) while maintaining high stability against environmental contamination without the need for encapsulation. The observed photoresponse is purely photovoltaic, demonstrating complete quenching of hot-carrier effects. These results pave the way toward the design of ultrathin photodetectors with unprecedented LDR for high-definition imaging and sensing.S.R. and M.F.C. acknowledge financial support from the Engineering and Physical Sciences Research Council (grant nos. EP/J000396/1, EP/K017160/1, EP/K010050/1, EP/G036101/1, EP/M001024/1, and EP/M002438/1), from the Royal Society’s International Exchanges Scheme 2012/R3 and 2013/R2, and from the European Commission (FP7-ICT-2013-613024-GRASP)

SEPT10 expression in chronic lymphocytic leukemia. Correlation with clinical and biological prognostic factors

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. Microarray studies allowed highlight genes differentially expressed in this pathology. In this study, we have evaluated the prognostic significance of SEPT10 expression in CLL patients. Results were correlated with immunoglobulin heavy-chain variable (IGHV) genes mutational status, genomic rearrangements and clinical parameters. SEPT10 mRNA levels were determined by quantitative real-time PCR in 70 newly diagnosed CLL patients consecutively referred to our Institution. A wide heterogeneity for SEPT10 expression was found. Gene upregulation was observed in 18.5% of cases. The univariate analysis showed a positive association between gen expression and platelet count (p < 0.0001) and a negative correlation with hemoglobin levels (p = 0.0094). Although no significant differences were observed, mean treatment free survival was shorter in patients with high expression (31 months) with respect to those with low mRNA levels (72 months). Cases with abnormal karyotypes had increased expression compared to those with normal karyotypes and no association between gene expression and FISH (fluorescence in situ hybridization) risk groups and IGHV mutational status was found. Cases using IGHV3-23 gene rearrangement had low SEPT10 expression. Our results showed an association between SEPT10 expression and features of adverse outcome but without independent prognostic value. The study of SEPT10 expression may be important for a better understanding of disease heterogeneity, adding further information to those provided by established prognostic factors.Fil: Travella, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentin

Ca(2+ )regulation in the absence of the iplA gene product in Dictyostelium discoideum

BACKGROUND: Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca(2+ )concentration ([Ca(2+)](i)). The [Ca(2+)](i)-change is composed of liberation of stored Ca(2+ )and extracellular Ca(2+)-entry. The significance of the [Ca(2+)](i)-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca(2+)-buffers in the cytosol indicates that a [Ca(2+)](i)-increase is required for chemotaxis. Yet, the iplA(- )mutant disrupted in a gene bearing similarity to IP(3)-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca(2+)](i)-transient which favours the view that [Ca(2+)](i)-changes are insignificant for chemotaxis. RESULTS: We investigated Ca(2+)-fluxes and the effect of their disturbance on chemotaxis and development of iplA(- )cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca(2+). Chemotaxis was impaired when [Ca(2+)](i)-transients were suppressed by the presence of a Ca(2+)-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca(2+)-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca(2+)-influx and liberation of stored Ca(2+), respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca(2+)-uptake and Ca(2+)-release activated by fatty acids or Ca(2+)-ATPase inhibitors were similar to wild type. Mn(2+)-quenching of fura2 fluorescence allows to study Ca(2+)-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn(2+ )was necessary to observe agonist-induced Mn(2+)-influx. cAMP evoked a [Ca(2+)](i)-elevation when stores were strongly loaded with Ca(2+), again with a similar shift in sensitivity in the mutant. In addition, basal [Ca(2+)](i )was significantly lower in iplA(- )than in wild type amoebae. CONCLUSION: These results support the view that [Ca(2+)](i)-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca(2+)-release from stores to Ca(2+)-entry in Dictyostelium. The iplA(- )strain retains the capacitative Ca(2+)-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure

Extraordinary linear dynamic range in laser-defined functionalized graphene photodetectors

Graphene-based photodetectors have demonstrated mechanical flexibility, large operating bandwidth, and broadband spectral response. However, their linear dynamic range (LDR) is limited by graphene's intrinsichot-carrier dynamics, which causes deviation from a linear photoresponse at low incident powers. At the same time, multiplication of hot carriers causes the photoactive region to be smeared over distances of a few micro-meters, limiting the use of graphene in high-resolution applications. We present a novel method for engineer-ing photoactive junctions in FeCl3-intercalated graphene using laser irradiation. Photocurrent measured at these planar junctions shows an extraordinary linear response with an LDR value at least 4500 times larger than that of other graphene devices (44 dB) while maintaining high stability against environmental contamination without the need for encapsulation. The observed photoresponse is purely photovoltaic, demonstrating complete quenching of hot-carrier effects. These results pave the way toward the design of ultrathin photode-tectors with unprecedented LDR for high-definition imaging and sensing.Comment: 44 pages, includes supplementar

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo, Determined by Immunolabelling

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.AJOM was financed by a PhD Grant of the FPI program from the Spanish Ministerio de Ciencia e Innovación; TCB's PhD Grant belonged to the FPU Program of the same Ministry. AJOM and AGC were supported by Projects CGL2010-20748-C02-01 and CGL2013-48247-P of the mentioned Ministry, and RNM6433 of the Consejería de Economía, Innovación y Ciencia of the Junta de Andalucía. The European COST Action TD0903 contributed via two Short Term Scientific Missions to AJOM in FM's lab in Dijon

Anomalías cromosómicas estructurales nuevas en leucemia linfocítica crónica. Su valor pronóstico

El análisis citogenético permite la detección de anomalías estructurales (AE) que pasan desapercibidas con la técnica de FISH (fluorescence in situ hybridization). En este trabajo se analizaron 34 pacientes con leucemia linfocítica crónica (LLC) portadores de anomalías estructurales (AE) clonales y un grupo control de 78 pacientes sin alteraciones. Se realizó estudio cromosómico por bandeo G complementado con FISH, y análisis molecular del estado mutacional de IGVH y de la expresión de los genes LPL y ADAM-29. Se detectaron 16 casos (47%) con AE nuevas. El cromosoma 8 fue el más implicado con 7 alteraciones, seguido por los pares 13 (6), 12 (5) y 15 (4). Se observó una similar distribución de AE entre los pacientes con IGVH mutado y no mutado. Los casos con AE mostraron una tendencia a mayor expresión de LPL y menor de ADAM-29. Se encontraron diferencias significativas en el recuento de blancos (p=0,019), plaquetas (p=0,002), LDH (p=0,029) y sobrevida libre de tratamiento (13 meses) en los pacientes con AE nuevas respecto de controles (69 meses) (p=0,087). Los resultados obtenidos confirman el pronóstico adverso de las AE en pacientes con LLC reforzando la importancia del análisis citogenético en esta patología.Fil: Travella, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; ArgentinaFil: Rodriguez, Andrea. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Harmonized Soil Database of Ecuador (HESD): data from 2009 to 2015

One of the largest challenges with soil information around the world is how to harmonize archived soil data from different sources and how to make it accessible to soil scientist. In Ecuador, there have been two major projects that have provided soil information, but the methodology of these projects, although comparable, did not coincide, especially with respect to how information was reported. Here, we present a new soil database for Ecuador, comprising 13 542 soil profiles with 51 713 measured soil horizons, including 92 different edaphic variables. The original data were in a non-editable format (i.e., PDF), which made it difficult to access and process the information. Our study provides an integrated framework that combines multiple analytic tools for automatically converting legacy soil information from an analog format into usable digital soil mapping inputs across Ecuador. This framework allowed us to incorporate quantitative information on a broad set of soil properties and retrieve qualitative information on soil morphological properties collected in the profile description phase, which is rarely included in soil databases. We present a new harmonized national soil database using a specific methodology to preserve relevant information. The national representativeness of soil information has been enhanced compared with other international databases, and this new database contributes to filling the gaps in publicly available soil information across the country. The database is freely available at https://doi.org/10.6073/pasta/1560e803953c839e7aedef78ff7d3f6c (Armas et al., 2022).</p