Transcriptional and post-transcriptional study of calcium-dependent protein kinase, StCDPK1 in Solanum tuberosum and analysis of its potential targets

La papa, Solanum tuberosum L., es el tercer cultivo alimenticio luego del arroz y del trigo (FAOSTAT, 2009). En el proceso de tuberización, un tallo subterráneo especializado, el estolón, sediferencia en un órgano de reserva, el tubérculo. Con el fin de comprender los mecanismos quesubyacen a este proceso de desarrollo y para mejorar la calidad y el rendimiento del cultivo se hanrealizado numerosas investigaciones. Entre los diversos factores que regulan la tuberización, loscambios en los niveles intracelulares de calcio y en el estado de fosforilación de las proteínas soneventos primarios que coordinan en tiempo y forma la respuesta de la planta. El calcio es unsegundo mensajero esencial en plantas y las quinasas de proteínas dependientes de calcio, CDPKs,son sensores/transductores del catión que se inducen/activan en respuesta a estímulos bióticos yabióticos (Boudsocq & Sheen 2013). Estas enzimas monoméricas poseen un dominio quinasa deproteínas (DK) unido a través de una región bisagra o dominio autoinhibitorio (DAI) a un dominioregulador homólogo a la calmodulina (CLD) con 4 sitios de unión al calcio (EF-hands). Componenfamilias multigénicas compuestas por aproximadamente 30 miembros que presentan granhomología de secuencia entre sí a excepción del dominio amino terminal variable (NTV) queregula la localización subcelular de la enzima. En Solanum tuberosum, nuestro grupo identificó tresisoformas de CDPK, StCDPK1, 2 y 3, que se expresan durante el proceso de tuberización (Gargantini et al. 2009; Giammaria et al. 2011; Grandellis et al. 2012; Raices et al. 2001; Raices etal. 2003a; Raices et al. 2003b; Raices et al. 2003c). Con el fin de estudiar la regulación transcripcional de la isoforma StCDPK1 durante el ciclo de vidade la planta de papa, se clonó y analizó su secuencia promotora, se obtuvieron y caracterizaronplantas transgénicas de papa que expresan el gen reportero β-glucuronidasa (GUS) bajo dichopromotor (plantas StCDPK1pro::GUS) y se realizaron ensayos de qRT-PCR en diferentes tejidos dela planta y estadios de tuberización. Se comparó la actividad GUS observada en las plantas StCDPK1pro::GUS con la observada en plantas CDPK3pro::GUS desarrolladas previamente (Grandellis et al. 2012) y con la de plantas CDPK2pro::GUS obtenidas también durante eltranscurso de esta tesis. Los ensayos histoquímicos indican que la isoforma StCDPK1 estáfuertemente asociada al sistema vascular de la planta y confirman que presenta una expresiónespacio temporal definida durante el proceso de tuberización. Asimismo se analizó si la expresión de StCDPK1 podía ser regulada post transcripcionalmente pormicroARNs. A través de los programas online psRNATarget y TargetAlign se identificó el miR390que tendría como blanco el transcripto de StCDPK1 provocando el clivaje del ARNm ointerrumpiendo su traducción. Los perfiles de expresión opuestos del miR390 y de StCDPK1 enestadíos de tuberización indicaron que este miARN podría regular la expresión de StCDPK1. Posteriormente, en ensayos de co-agroinfiltración en Nicotiana benthamiana se determinó que lostranscriptos de StCDPK1 son blanco de acción del miR390, demostrando, por primera vez, que una CDPK es regulada a este nivel (manuscrito enviado a Plant Mol. Biol). Además se obtuvo la proteína recombinante StCDPK1 etiquetada con 6xHis para proceder a sucaracterización bioquímica y determinar sus parámetros cinéticos. Se demostró que la proteína 6xHis-StCDPK1 es una quinasa activa dependiente de calcio, capaz de autofosforilarse en presenciadel catión y que fosforila diversos sustratos, entre los cuales se encuentra AtDi19, un factor detranscripción involucrado en la respuesta a estrés salino y sequía en Arabidopsis thaliana. Recientemente obtuvimos las proteínas recombinantes del homólogo de AtDi19 en papa y otrasdos proteínas que han sido implicadas en la inducción de la tuberización, la proteína StSP6A,homólogo en papa de flowering locus T (FT) y StPIN4, un transportador de auxinas. Próximamentese ensayará la capacidad de la enzima de fosforilar in vitro a estos sustratos. Finalmente, seprodujeron plantas que sobrexpresan la proteína StCDPK1 como una primera aproximaciónfuncional para determinar si esta isoforma interviene en la respuesta de la planta a estresesabióticos.Potato (Solanum tuberosum L.) is the world's most important non-grain food crop. Tuberdevelopment (Tuberization) in potato has been extensively studied in order to understand themechanism as well as to improve tuber yield and quality. Among the many factors that regulatetuberization, calcium signaling plays an important role. Calcium is the key second messenger inplants and Calcium Dependent Protein Kinases (CDPKs) transduce calcium signatures into specificresponses including tuberization. These enzymes are encoded by multigene families, whosemembers share a unique structure consisting of an N-terminal variable domain (NTV), aserine/threonine kinase catalytic domain (KD) fused through a junction region or autoinhibitorydomain to a carboxyterminal calmodulin-like domain (CLD) with four EF hands Ca2+ binding sites. Previous reports from our group showed that CDPK activity increases at the onset of tuberformation and that StCDPK1 is strongly induced in swollen stolons (Gargantini et al. 2009; Giammaria et al. 2011; Grandellis et al. 2012; Raices et al. 2001; Raices et al. 2003a; Raices et al. 2003b; Raices et al. 2003c). To elucidate the transcription pattern and post transcriptional regulation of StCDPK1, we analyzedits expression in different tissues and stages of potato life cycle and characterized potato plantsharboring GUS under the control of StCDPK1 promoter (StCDPK1pro::GUS). Histochemical analysisindicates that StCDPK1 is strongly associated to the vascular system of stems, roots and duringstolon to tuber transition correlating with the cis-acting elements present in its promotersequence. qRT-PCR data demonstrated that StCDPK1 is ubiquitously expressed having highexpression in stolons, swollen stolons, leaves, and stems. On the contrary, miR390 expressionexhibited an inverse pattern in stolons, swollen stolons and tubers suggesting a possible regulationof StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays,demonstrating that StCDPK1 is targeted by miR390. Overall, our results indicate that StCDPK1expression varied in tissue specific manner having significant expression in vasculature, and itcould be modulated by miR390 during potato development. The recombinant 6xHis-StCDPK1 protein was produced and biochemically characterized. StCDPK1kinetic parameters differed from those exhibited by other isoforms, consistent with the fact that CDPKs are involved in various signal transduction pathways and are differentially expressed amongtissues. In addition, the kinase was autophosphorylated in the presence of calcium and 2D analysisrevealed multiple phosphorylation states as predicted by in silico analysis. This isoform was able tophosphorylate some targets in vitro, among which, AtDi19 was reported to be involved in salt anddrought stress in Arabidopsis thaliana. Recently, we produced the recombinant proteins of thepotato homolog of AtDi19 (StDi19) as well as two other proteins implicated in tuber induction, StSP6A and StPIN4. Shortly, the capability of StCDPK1 to phosphorylate these proteins will beassayed in vitro. Finally, 35S::StCDPK1 over-expressing plants were produced as a first functionalapproach to study the role of this isoform in the abiotic stress response.Fil: Santin, Franco. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A fluorometric method for the assay of protein kinase activity

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD+, which is quantified by its fluorescent properties after alkaline treatment (linear range 0–10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.Fil: Rojas, Bruno Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Santin, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

In vitro response of different biotypes and explants of Passiflora caerulea L

Passiflora caerulea L., al igual que otras especies de la familia Passifloraceae, es utilizada en la medicina popular por sus propiedades antiespasmódicas y para el tratamiento de la ansiedad, el insomnio y el nerviosismo. La belleza de sus flores les otorga valor ornamental, mientras que sus frutos son apreciados por su importancia alimenticia. Se evaluó la respuesta in vitro de diferentes explantos y tres biotipos de P. caerulea: Corral de Bustos (provincia de Córdoba), Zavalla (provincia de Santa Fe) y Neuquén (provincia de Neuquén). Se utilizaron dos tipos de explantos: entrenudos y segmentos nodales, y como medio de cultivo Murashige y Skoog (1962) (MS), suplementado con vitaminas de Gamborg (1976) y 1 mg/L-1 de benciladenina (BA). Las respuestas fueron diferentes según el genotipo y el explanto. Los entrenudos ubicados tanto horizontal como verticalmente en medio de cultivo generaron callos como única respuesta. El biotipo de Neuquén mostró los mayores porcentajes de segmentos nodales con brotes. A través de estudios histológicos se determinó que en medio de cultivo MS con 1 mg/L-1 de BA, los segmentos nodales de P. caerulea originan brotes a partir de las yemas axilares preformadas y raíces que parten de callos en la base de los mismos. En iguales condiciones, los entrenudos originan callo como única respuesta.As other species of the Passifloraceae family, Passiflora caerulea L. is used in popular medicine for its antispasmodic properties and as a remedy for anxiety, insomnia and nervousness. It is also highly prized for the ornamental value of its beautiful flowers, as well as for the nutritional importance of its fruits. The in vitro response of different explants and three biotypes of P. caerulea: the Corral de Bustos (Province of Córdoba), the Zavalla (Province of Santa Fe) and the Neuquén (Province of Neuquén) genotypes, was evaluated using two types of explants: internodes and nodal segments on Murashige and Skoog (1962) (MS) culture medium supplemented with Gamborg’s vitamins (1976) and 1 mg.L-1 of benzyladenine (BA). There were different responses depending on the genotype and the explant. The internodes placed both horizontally and vertically in the culture medium produced callus as sole response. The Neuquén biotype showed the highest percentages of nodal segments with shoots. Histological tests allowed to establish that in MS culture medium with 1 mg.L-1 of BA, the nodal segments of P. caerulea produce shoots from the preformed axillary buds and roots that develop from the callus situated on its base. Under similar conditions, the internodes produce callus as sole response.Fil: Severin, Cecilia. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Bueno, Mirian. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Santin, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Giubileo, María Graciela. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; Argentin

20-ps resolution Clock Distribution Network for a fast-timing single photon detector

The time resolution of active pixel sensors whose timestamp mechanism is based on Time-to-Digital Converters is critically linked to the accuracy in the distribution of the master clock signal that latches the timestamp values across the detector. The Clock Distribution Network that delivers the master clock signal must compensate process-voltage-temperature variations to reduce static time errors (skew), and minimize the power supply bounce to prevent dynamic time errors (jitter). To achieve sub-100ps time resolution within pixel detectors and thus enable a step forward in multiple imaging applications, the network latencies must be adjusted in steps well below that value. Power consumption must be kept as low as possible. In this work, a self-regulated Clock Distribution Network that fulfills these requirements is presented for the FastICpix single photon detector ¿ aiming at a 65nm process. A 40 MHz master clock is distributed to 64x64 pixels over an area of 2.4x2.4 cm2 using digital Delay-Locked Loops, achieving clock leaf skew below 20 ps with a power consumption of 26 mW. Guidelines are provided to adapt the system to arbitrary chip area and pixel pitch values, yielding a versatile design with very fine time resolution

Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery

Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients

Claudin3 is localized outside the tight junctions in human carcinomas

Claudin3 is an integral component of the tight junction proteins in polarized epithelia. The expression of claudin3 was assessed in epithelial-derived tumors using Oncomine database. To determine the gene alteration during carcinogenesis, copy number alterations and mutations of claudin3 were evaluated using cBioPortal database. Claudin3 is overexpressed in several tumors including gynecological, bladder, breast and prostate carcinomas. 38% of the 163 evaluated studies show mutations and/or amplification of claudin3. 3D reconstruction of tissue samples following immunofluorescence analysis clearly demonstrated that, unlike in healthy tissues, claudin3 is mislocalized and unengaged in the formation of tight junction in tumor samples. These data strongly support the evaluation of unengaged claudin3 as a target for the development of novel diagnostic probes, optical approaches for real time detection of tumoral tissues during surgery, and target therapeutic drugs

Mammaglobin B is an independent prognostic marker in epithelial ovarian cancer and its expression is associated with reduced risk of disease recurrence

<p>Abstract</p> <p>Background</p> <p>Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients.</p> <p>Methods</p> <p>MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome.</p> <p>Results</p> <p>MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08–0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17–0.99, p = 0.048) had an independent prognostic value for disease-free survival.</p> <p>Conclusion</p> <p>This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.</p

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London