STAT3 in cancer: A double edged sword

The transcription factor signal transducer and activator of transcription (STAT) 3 is activated downstream of cytokines, growth factors and oncogenes to mediate their functions under both physiological and pathological conditions. In particular, aberrant/unrestrained STAT3 activity is detected in a wide variety of tumors, driving multiple pro-oncogenic functions. For that, STAT3 is widely considered as an oncogene and is the object of intense translational studies. One of the distinctive features of this factor is however, its ability to elicit different and sometimes contrasting effects under different conditions. In particular, STAT3 activities have been shown to be either pro-oncogenic or tumor-suppressive according to the tumor aetiology/mutational landscape, suggesting that the molecular bases underlining its functions are still incompletely understood. Here we discuss some of the properties that may provide the bases to explain STAT3 heterogeneous functions, and in particular how post-translational modifications contribute shaping its sub-cellular localization and activities, the cross talk between these activities and cell metabolic conditions, and finally how its functions can control the behaviour of both tumor and tumor microenvironment cell populations

SOCS3 is a novel bi-functional regulator of muscle growth and wasting

poster abstractDisease states such as cancer and other inflammatory conditions often show elevated IL-6 levels that correlate with muscle wasting and mortality. Previously we reported that STAT3, a transcription factor downstream of IL-6 binding to its receptor, plays a causative role in cancer cachexia, and that STAT3 inhibition prevents muscle wasting. Others have also shown that STAT3 blockade rescues cachexia in a murine model of kidney failure. Altogether these results established STAT3 as a regulator of muscle mass. One of STAT3 downstream target genes is the Suppressor of cytokine signaling-3 (SOCS-3). Interestingly, SOCS3 has been reported to inhibit the IL-6/STAT3 signaling by means of a feedback mechanism. In particular, SOCS3 can prevent further STAT3 activation by inhibiting the activation of JAK kinases, competing for receptor binding motifs and targeting the receptor for proteasomal degradation. We thus sought to determine the role of SOCS3 in muscle growth regulation and whether SOCS3 can improve muscle wasting in conditions of high IL-6. Adenoviral-mediated SOCS3 overexpression in C2C12 myotubes caused hypertrophy and rescued IL-6-induced myofiber shrinkage. Similarly, SOCS3 gene transfer in the tibialis muscle of tumor hosts and burn-injured mice prevented muscle atrophy due to elevated IL-6. We then generated MLC-SOCS3 transgenic mice overexpressing SOCS3 from a muscle-specific promoter. Interestingly, these animals exhibit a complex sexually dimorphic phenotype. Indeed, female mice showed higher SOCS3 protein levels in skeletal muscle compared to the males, consistently with decreased pSTAT3 expression. Despite reduced or unchanged body weights, the MLC-SOCS3 transgenics generally showed larger skeletal muscles compared to their wild-type littermates. 1-weekold and adult MLC-SOCS3 mice were also characterized by significantly larger muscle cross-sectional area. However, only adult male mice showed reduced number of muscle fibers and increased number of central nuclei, thus suggesting that SOCS3 could affect myogenesis and differentiation. On this line and consistent with previous reports, primary myoblasts isolated from MLC-SOCS3 mice were shown to proliferate at a lower rate and formed hypertrophic fibers upon differentiation. Furthermore, MLC-SOCS3 myotubes as well as C2C12 expressing SOCS3 were refractory to both catabolic (IL-6) and anabolic (IGF-1 and GH) stimuli. These data suggest that SOCS3 could act as a bi-functional regulator of muscle growth, possibly by affecting differentiation and limiting both IL-6/STAT3- induced wasting as well as IGF-1/GH-associated signaling. Further investigation is needed to define whether SOCS3 may play a role in the activation of muscle satellite cells and to support the use of SOCS3 as a therapeutic approach in cachexia and sarcopenia

Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification

The interest in therapeutic monoclonal antibodies (mAbs) has significantly grown in the pharmaceutical industry, exceeding 100 FDA mAbs approved. Although the upstream processing of their industrial production has been significantly improved in the last years, the downstream processing still depends on immobilized protein A affinity chromatography. The high cost, low capacity and short half-life of immobilized protein A chromatography matrices, encouraged the design of alternative short-peptide ligands for mAb purification. Most of these peptides have been obtained by screening combinatorial peptide libraries. These low-cost ligands can be easily produced by solid-phase peptide synthesis and can be immobilized on chromatographic supports, thus obtaining matrices with high capacity and selectivity. Furthermore, matrices with immobilized peptide ligands have longer half-life than those with protein A due to the higher stability of the peptides. In this review the design and synthesis of peptide ligands, their immobilization on chromatographic supports and the evaluation of the affinity supports for their application in mAb purification is described.Fil: Barredo, Gabriela Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Giudicessi, Silvana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Martínez Ceron, María Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Camperi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

Temporins: An Approach of Potential Pharmaceutic Candidates

Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), are small and mostlypolycationic molecules that form part of the innate immune response. There are currently more than3000 experimentally reported AMPs. Particularly, in frogs, the temporin family, have beendiscovered as potential AMPs. The aim of this work is to review the latest publications about thisclass of peptides, discuss their properties and make an update of the last studies and new discoveriesin the field.More than 130 temporins have been identified in this family. The most studied temporins aretemporin A (TA), temporin B (TB) and temporin L (TL). These peptides showed antimicrobialactivity against Gram-negative, Gram-positive bacteria and fungi. Since the discovery of temporinsin 1996, several groups of research isolated different peptides from various species of frogs thatwere included as members of this family. Although antimicrobial activity of many temporins has not been analyzed yet, most of them showed antimicrobial and antifungal activities. Combination ofnanotechnology and AMPs as temporins in different antimicrobial treatments could be a promisingalternative for resistance pathogens. These studies demonstrate that, even with the advancement inscientific research on the composition and antimicrobial activity of temporins, further studies arenecessary to wholly understand their components and mechanisms of action.Fil: Romero, Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Cardillo, Alejandra Beatriz. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Martínez Ceron, María Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Camperi, Silvia Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Giudicessi, Silvana Laura. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

Fully automated screening of a combinatorial library to avoid false positives: application to tetanus toxoid ligand identification

Peptide ligands are widely used in protein purification by affinity chromatography. Here, we applied a fully automated two-stage library screening method that avoids false positive peptidyl-bead selection and applied it to tetanus toxoid purification. The first library screening was performed using only sulforhodamine (a fluorescent dye), and fluorescent beads were isolated automatically by flow cytometry and discarded. A second screening was then performed with the rest of the library, using the target protein (tetanus toxoid)-rhodamine conjugate. This time, fluorescent beads were isolated, and peptide sequences were identified by matrix-assisted laser desorption/ionization tandem mass spectrometry. Those appearing with greater frequency were synthesized and immobilized on agarose to evaluate a range of chromatographic purification conditions. The affinity matrix PTx1-agarose (Ac-Leu-Arg-Val-Tyr-His-Gly-Gly-Ala-Gly-Lys-agarose) showed the best performance when 20 mM sodium phosphate, 0.05% Tween 20, pH 5.9 as adsorption buffer and 100 mM Tris-HCl, 100 mM NaCl, pH 8.0 as elution buffer were used. A pure tetanus toxoid (Ttx) was loaded on a chromatographic column filled with the PTx1 matrix, and 96% adsorption was achieved, with a Kd of 9.18 ± 0.07 nmol/L and a qm of 1.31 ± 0.029 μmol Ttx/mL matrix. Next, a Clostridium tetani culture supernatant treated with formaldehyde (to obtain the toxoid) was applied as a sample. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a band, identified by electrospray ionization mass spectrometry as the Ttx, that appeared only in the elution fraction, where an S-layer protein was also detected.Fil: Martínez Ceron, María Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Avila, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Giudicessi, Silvana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Minoia, Juan Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Fingermann, Matias. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Camperi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Albericio, Fernando. Universidad de Barcelona; EspañaFil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

Friendly Strategy to Prepare Encoded One Bead−One Compound Cyclic Peptide Library

One bead−one peptide libraries allow the screening of suitable ligands for any target protein. Short cyclic peptides are ideal ligands for affinity chromatography because of their high affinity and selectivity for the target protein and stability against proteases. We designed a library synthesis strategy to facilitate the identification of cyclic peptides by MS consisting of (a) sequential incorporation of a mixture of Fmoc-Ala-OH and Fmoc-Asp[2-phenylisopropyl (OPp)]-OH (15:85) to Gly-oxymethylbenzamide-ChemMatrix (Gly-HMBA-CM) resin, (b) synthesis of the combinatorial library on the resin by the divide−couple−recombine method, (c) removal of OPp with 4% TFA, (d) peptide cyclization on solid phase through side-chain Asp and amino terminus, and (e) removal of side chain protecting groups with a 95% TFA cocktail. Peptides were cleaved from the beads with ammonia and the linear code was sequenced by MALDI-TOF MS/MS. The high capacity of ChemMatrix resin together with the sensitivity of MS allows code sequencing from a single bead.Fil: Giudicessi, Silvana Laura. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Gurevich Messina, Juan Manuel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina;Fil: Martínez Ceron, María Camila. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Erra Balsells, Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Albericio, F.. Instituto de Investigación Biomédica; España; Universidad de Barcelona; España; Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina; España;Fil: Cascone, Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Camperi, Silvia Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Vitamin D and VDR in cancer cachexia and muscle regeneration

Low circulating levels of vitamin D were associated with decreased muscle strength and physical performance. Along this line, the present study was aimed to investigate: i) the therapeutic potential of vitamin D in cancer-induced muscle wasting; ii) the mechanisms by which vitamin D affects muscle phenotype in tumor-bearing animals.Rats bearing the AH130 hepatoma showed decreased circulating vitamin D compared to control rats, while muscle vitamin D receptor (VDR) mRNA was up-regulated. Both circulating vitamin D and muscle VDR expression increased after vitamin D administration, without exerting appreciable effects on body weight and muscle mass.The effects of vitamin D on muscle cells were studied in C2C12 myocytes. Vitamin D-treated myoblasts did not differentiate properly, fusing only partially and forming multinucleated structures with aberrant shape and low myosin heavy chain content. Vitamin D treatment resulted in VDR overexpression and myogenin down-regulation. Silencing VDR expression in C2C12 cultures abrogated the inhibition of differentiation exerted by vitamin D treatment.These data suggest that VDR overexpression in tumor-bearing animals contributes to muscle wasting by impairing muscle regenerative program. In this regard, attention should be paid when considering vitamin D supplementation to patients affected by chronic pathologies where muscle regeneration may be involved

Immobilized short peptides chromatography applied to therapeutic antibodies purification

Los anticuerpos monoclonales (mAbs) tienen un enorme y creciente interés en la industria farmacéutica. Actualmente hay más de 70 mAbs terapéuticos aprobados por la Food and Drug Administration (FDA). Su proceso de producción industrial consiste en una etapa de crecimiento de las células productoras del mAb en un biorreactor y en una etapa de recuperación y purificación del mAb a partir del caldo de cultivo celular. La cromatografía de afinidad (AC) con proteína A inmovilizada es el método de elección para su purificación. Ésta se basa en un reconocimiento molecular entre la proteína de interés y la proteína A inmovilizada sobre un soporte. Sin embargo, las matrices con proteína A inmovilizada son costosas, de baja capacidad, lábiles y de corta vida útil. Las desventajas de la proteína A han incentivado el desarrollo de métodos de purificación alternativos con péptidos cortos sintéticos (entre 5 y12 aminoácidos) como ligandos de afinidad. Éstos son ideales para separaciones industriales por afinidad ya que pueden ser producidos a un costo muy inferior a los ligandos proteicos y en forma aséptica bajo normas GMP. A su vez, son mucho más estables porque no requieren de una estructura terciaria específica para mantener su actividad biológica y las interacciones entre los péptidos y las proteínas son generalmente moderadas, lo que resulta en condiciones suaves de elución. La presente revisión describe:1. El desarrollo de ligandos peptídicos con afinidad por anticuerpos; 2. La síntesis y evaluación de soportes de afinidad con ligandos peptídicos inmovilizados; y 3. Las aplicaciones de las matrices de afinidad con péptidos inmovilizados en la purificación de anticuerpos.Monoclonal antibodies (mAbs) have an enormous and growing interest in the pharmaceutical industry. Currently, there are more than 70 therapeutic mAbs approved by the Food and Drug Administration (FDA). Its industrial production process consists of a first step of growing the mAb-producing cells in a bioreactor and a step of recovering and purifying the mAb from the cell culture broth. Affinity chromatography (AC) with immobilized protein A is the method of choice for their purification. Itis based on a molecular recognition between the antibody and protein A immobilized on a support. However, matrices with immobilized protein A are expensive, with low capacity, labile and of shorthalf-life. The disadvantages of protein A have encouraged the development of alternative purification methods with synthetic short peptides (from 5 to 12 amino acids) as affinity ligands. Peptides are ideal for affinity industrial separations since they can be synthesized at lower cost than proteins and in an aseptic way under GMP standards. Furthermore, they are much more stable because they do not require a specific tertiary structure to maintain their biological activity. Also, interactions between peptides and proteins are generally moderate, resulting in mild elution conditions. The present review describes the development of peptide ligands with affinity for antibodies, the synthesis and evaluation of affinity supports with immobilized peptide ligands and the applications of affinity matrices with immobilized peptides to antibodies purification.Fil: Barredo, Gabriela Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Giudicessi, Silvana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Saavedra, Soledad Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Martínez Ceron, María Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Cascone, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Camperi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin