Antibodies on demand: a fast method for the production of human scFvs with minimal amounts of antigen

<p>Abstract</p> <p>Background</p> <p>Antibodies constitute a powerful tool to study protein function, protein localization and protein-protein interactions, as well as for diagnostic and therapeutic purposes. High-throughput antibody development requires faster methodologies with lower antigen consumption.</p> <p>Results</p> <p>Here, we describe a novel methodology to select human monoclonal recombinant antibodies by combining <it>in vitro </it>protein expression, phage display antibody libraries and antibody microarrays. The application of this combination of methodologies permitted us to generate human single-chain variable fragments (scFvs) against two proteins: green fluorescent protein (GFP) and thioredoxin (Trx) in a short time, using as low as 5 μg of purified protein. These scFvs showed specific reactivity against their respective targets and worked well by ELISA and western blot. The scFvs were able to recognise as low as 31 ng of protein of their respective targets by western blot.</p> <p>Conclusion</p> <p>This work describes a novel and miniaturized methodology to obtain human monoclonal recombinant antibodies against any target in a shorter time than other methodologies using only 5 μg of protein. The protocol could be easily adapted to a high-throughput procedure for antibody production.</p

Sistema de presentación de antígenos basado en el virus de la sharka

Referencia OEPM: P9800623.-- Fecha de solicitud: 24/03/1998.-- Titulares: Inmunología y Genética Aplicada, S.A., Consejo Superior de Investigaciones Científicas (CSIC).El sistema de presentación de antígenos heterólogos basado en el virus de la sharka (PPV) comprende una partícula de PPV quimérica formada por ensamblaje de la proteína de la cápsida (CP) de PPV modificada que contiene, al menos, un antígeno heterólogo en dicha CP modificada, estando dicho antígeno dispuesto en la superficie exterior de dicha partícula viral de PPV. Preferentemente, dicho antígeno heterólogo se encuentra en el extremo amino terminal de la CP modificada de PPV. En una realización concreta se describe la construcción de un sistema de presentación de un epítopo inmunológicamente activo derivado de la proteína VP2 del parvovirus canino (CPV). Estos sistemas de presentación de antígenos tienen utilidad en diagnóstico y vacunas.Peer reviewe

Snail1 induces IL-17 expression to inhibit adipogenesis

44 p.-7 fig.-1 tab. Peláez-García, Alberto et al.Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.This research was supported by a grant to established research groups (AECC), grant BIO2012-31023 from the Ministry of Economy and Competitiveness, grant S2011/BMD-2344/ (Colomics2) from Comunidad de Madrid and ProteoRed-ISCIII support. Work at AGH’s lab was supported by a grant from the Ministry of Economy and Competitiveness (SAF2010-16089)Peer reviewe

VE-cadherin RGD motifs promote metastasis and constitute a potential therapeutic target in melanoma and breast cancers

13 p.-6 fig.We have investigated the role of vascular-endothelial (VE)-cadherin in melanoma and breast cancer metastasis. We found that VE-cadherin is expressed in highly aggressive melanoma and breast cancer cell lines. Remarkably, inactivation of VEcadherin triggered a significant loss of malignant traits (proliferation, adhesion, invasion and transendothelial migration) in melanoma and breast cancer cells. These effects, except transendothelial migration, were induced by the VE-cadherin RGD motifs. Co-immunoprecipitation experiments demonstrated an interaction between VE-cadherin and α2β1 integrin, with the RGD motifs found to directly affect β1 integrin activation. VE-cadherin-mediated integrin signaling occurred through specific activation of SRC, ERK and JNK, including AKT in melanoma. Knocking down VEcadherin suppressed lung colonization capacity of melanoma or breast cancer cells inoculated in mice, while pre-incubation with VE-cadherin RGD peptides promoted lung metastasis for both cancer types. Finally, an in silico study revealed the association of high VE-cadherin expression with poor survival in a subset of melanoma patients and breast cancer patients showing low CD34 expression. These findings support a general role for VE-cadherin and other RGD cadherins as critical regulators of lung and liver metastasis in multiple solid tumours. These results pave the way for cadherin-specific RGD targeted therapies to control disseminated metastasis in multiple cancers.BEP was an FPI fellow from Ministry of Economy and Competitiveness (MINECO). This research was supported by grants BIO2012-31023 and BIO2015-66849 from MINECO and PRB2 (IPT13/0001-ISCIII-SGEFI/FEDER) to JIC.Peer reviewe

Model to Track Wild Birds for Avian Influenza by Means of Population Dynamics and Surveillance Information

Design, sampling and data interpretation constitute an important challenge for wildlife surveillance of avian influenza viruses (AIV). The aim of this study was to construct a model to improve and enhance identification in both different periods and locations of avian species likely at high risk of contact with AIV in a specific wetland. This study presents an individualbased stochastic model for the Ebre Delta as an example of this appliance. Based on the Monte-Carlo method, the model simulates the dynamics of the spread of AIV among wild birds in a natural park following introduction of an infected bird. Data on wild bird species population, apparent AIV prevalence recorded in wild birds during the period of study, and ecological information on factors such as behaviour, contact rates or patterns of movements of waterfowl were incorporated as inputs of the model. From these inputs, the model predicted those species that would introduce most of AIV in different periods and those species and areas that would be at high risk as a consequence of the spread of these AIV incursions. This method can serve as a complementary tool to previous studies to optimize the allocation of the limited AI surveillance resources in a local complex ecosystem. However, this study indicates that in order to predict the evolution of the spread of AIV at the local scale, there is a need for further research on the identification of host factors involved in the interspecies transmission of AI

Heat dissipation mechanisms in hybrid superconductor-semiconductor devices revealed by Joule spectroscopy

Understanding heating and cooling mechanisms in mesoscopic superconductor-semiconductor hybrid devices is crucial for their application in quantum technologies. Owing to the poor thermal conductivity of typical devices, heating effects can drive superconducting-to-normal phase transitions even at low applied bias, observed as sharp conductance dips through the loss of Andreev excess currents. Tracking such dips across magnetic field, cryostat temperature, and applied microwave power, which constitutes Joule spectroscopy, allows to uncover the underlying cooling bottlenecks in different parts of a device. By applying this technique, we analyze heat dissipation in devices based on full-shell InAs-Al nanowires and reveal that superconducting islands are strongly susceptible to heating as their cooling is limited by the rather inefficient electron-phonon coupling, as opposed to grounded superconductors that primarily cool by quasiparticle diffusion. Our measurements indicate that powers as low as 50-150 pW are able to fully suprpress the superconductivity of an island. Finally, we show that applied microwaves lead to similar heating effects as DC signals, and explore the interplay of the microwave frequency and the effective electron-phonon relaxation time.Comment: 9 pages, 4 figure

A survey of biosecurity measures and serological status for bovine viral diarrhoea virus and bovine herpesvirus 1 on dairy cattle farms in north-west and north-east Spain

Biosecurity is a key measure to reduce and prevent the introduction of diseases to farms and minimise spread of diseases within a herd. The aim of the study was to characterise the current application of biosecurity measures on dairy cattle farms in Spain along with their bovine viral diarrhoea and infectious bovine rhinotracheitis status. Data on biosecurity measures for 124 dairy herds were collected using a questionnaire. The sanitary status of these farms for bovine viral diarrhoea and infectious bovine rhinotracheitis was also assessed using antibody ELISA. Data were analysed using multiple correspondence analysis and a two-step cluster analysis. Three main clusters of farms were identified: clusters 1 and 2 included herds of small and intermediate sizes. These, particularly cluster 1, showed the most deficiencies in the control of vehicles and visitors. However, laboratory tests were always performed on purchased animals. Cluster 3 had the largest herd sizes, with somewhat better biosecurity control of vehicles and visitors. However, farms in this cluster also purchased the most animals, sometimes without testing, and hired external workers more often. The study indicated that, in the study population, there are serious shortcomings in the application of biosecurity measures on dairy farms, exposing them to disease transmission. This survey also highlights regional and herd size-related differences in the implementation of biosecurity. Collecting data is an important first step to identification of specific weaknesses in different farm typologies, and an adequate follow-up is needed to ensure that measures are implemented correctly on farms

Schnurri-3 drives tumor growth and invasion in cancer cells expressing interleukin-13 receptor alpha 2

Interleukin 13 receptor alpha 2 (IL13Rα2) is a relevant therapeutic target in glioblastoma (GBM) and other tumors associated with tumor growth and invasion. In a previous study, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) is a key mediator of the IL-13/IL13Rα2 signaling pathway. PTP1B regulates cancer cell invasion through Src activation. However, PTP1B/Src downstream signaling mechanisms that modulate the invasion process remain unclear. In the present research, we have characterized the PTP1B interactome and the PTP1B-associated phosphoproteome after IL-13 treatment, in different cellular contexts, using proteomic strategies. PTP1B was associated with proteins involved in signal transduction, vesicle transport, and with multiple proteins from the NF-κB signaling pathway, including Tenascin-C (TNC). PTP1B participated with NF-κB in TNC-mediated proliferation and invasion. Analysis of the phosphorylation patterns obtained after PTP1B activation with IL-13 showed increased phosphorylation of the transcription factor Schnurri-3 (SHN3), a reported competitor of NF-κB. SHN3 silencing caused a potent inhibition in cell invasion and proliferation, associated with a down-regulation of the Wnt/β-catenin pathway, an extensive decline of MMP9 expression and the subsequent inhibition of tumor growth and metastasis in mouse models. Regarding clinical value, high expression of SHN3 was associated with poor survival in GBM, showing a significant correlation with the classical and mesenchymal subtypes. In CRC, SHN3 expression showed a preferential association with the mesenchymal subtypes CMS4 and CRIS-B. Moreover, SHN3 expression strongly correlated with IL13Rα2 and MMP9-associated poor prognosis in different cancers. In conclusion, we have uncovered the participation of SNH3 in the IL-13/IL13Rα2/PTP1B pathway to promote tumor growth and invasion. These findings support a potential therapeutic value for SHN3.Angela Martín-Regalado was supported by an FPU fellowship (FPU18/05766-MEFP). Laura Pintado-Berninches was supported by a Margarita Salas contract (CA1/RSUE/2021-00208) from the Ministry of Universities (Spain). Javier Robles and Issam Boukich were supported by IND2019/BMD-17153 and IND2022/BMD-23554 fellow ships of the Comunidad de Madrid. This project was supported by grants RTI2018-095055-B-I00, PID2021-122227OB-I00 and CPP2021-008337 from the MCIN/AEI/10.13039/501100011033 using Next Generation EU/PRTR funds to JIC, and PI21CIII/00002 from the MCIN and FEDER funds to PSG and NINDS R01 NS122395 to IVB.S