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This research was funded by the FUR (Fondo Unico della Ricerca—University of Verona) of M. Perduca. C.J.-L. acknowledges funding from projects CGL2016-76723 from the Ministerio de Economía y Competitividad from Spain and Fondo Europeo de Desarrollo Regional (FEDER) and Programa Operativo FEDER 2014–2020 (A-BIO-376-UGR18) Junta de Andalucia. M.P.C.-J. acknowledges funding from projects PID2019-109294RB-100 from the Ministerio de Ciencia e Innovación from Spain., We are grateful to the “Centro Piattaforme Tecnologiche” of the University of Verona for giving access to DLS equipment. CJL acknowledges. the Unidad Cientıfica de Excelencia UCE PP 2016.05 (U. Granada) and Instituto de Biotecnología. Y.J. wants to acknowledge a FPU2016 grant (ref. FPU16_04580) from the Ministerio de Educación, Ciencia y Deporte y Competitividad (Spain). AS-L is funded by the Spanish Ministry of Science, Innovation and Universities: Formación de Doctores 2018 (ref. PRE2018-0854409). Thanks go to the Scientific Instrumentation Center (CIC) personnel of the University of Granada for technical assistance with the TEM.We also thank Salvatore Calogero Gaglio for his help in preparing Figure S4., Magnetococcus marinus magnetosome-associated protein MamC, expressed as recombinant, has been proven to mediate the formation of novel biomimetic magnetic nanoparticles (BMNPs) that are successful drug nanocarriers for targeted chemotherapy and hyperthermia agents. These BMNPs present several advantages over inorganic magnetic nanoparticles, such as larger sizes that allow the former to have larger magnetic moment per particle, and an isoelectric point at acidic pH values, which allows both the stable functionalization of BMNPs at physiological pH value and the molecule release at acidic (tumor) environments, simply based on electrostatic interactions. However, difficulties for BMNPs cell internalization still hold back the efficiency of these nanoparticles as drug nanocarriers and hyperthermia agents. In the present study we explore the enhanced BMNPs internalization following upon their encapsulation by poly (lactic-co-glycolic) acid (PLGA), a Food and Drug Administration (FDA) approved molecule. Internalization is further optimized by the functionalization of the nanoformulation with the cell-penetrating TAT peptide (TATp). Our results evidence that cells treated with the nanoformulation [TAT-PLGA(BMNPs)] show up to 80% more iron internalized (after 72 h) compared to that of cells treated with BMNPs (40%), without any significant decrease in cell viability. This nanoformulation showing optimal internalization is further characterized. In particular, the present manuscript demonstrates that neither its magnetic properties nor its performance as a hyperthermia agent are significantly altered due to the encapsulation. In vitro experiments demonstrate that, following upon the application of an alternating magnetic field on U87MG cells treated with BMNPs and TAT-PLGA(BMNPs), the cytotoxic effect of BMNPs was not affected by the TAT-PLGA enveloping. Based on that, difficulties shown in previous studies related to poor cell uptake of BMNPs can be overcome by the novel nanoassembly described here., FUR (Fondo Unico della Ricerca-University of Verona), Ministerio de Economia y Competitividad from Spain CGL2016-76723, European Commission CGL2016-76723, Junta de Andalucia A-BIO-376-UGR18, Spanish Government PID2019-109294RB-100

This research work was supported by the Ministerio de Economia y Competitividad (CGL2016-76723), the Ministerio de Ciencia e Innovacion (PID2019-109294RB-100) projects, Ramon y Cajal program (RYC-2014-16901), the Junta de Andalucia Programa Operativo FEDER 2014-2020 (A1-FQM-341-UGR18, C-FQM-497-UGR18, A-BIO-376-UGR18), and the Proyectos de I + D + I, del Plan Andaluz de Investigacion, Desarrollo e Innovacion (P20_00208 and P20_00346). This research was also aided by the Andalusian regional government (CTS-236) and by the FUR (Fondo Unico della Ricerca, University of Verona)of Dr. M. Perduca. Alberto Sola-Leyva holds a Formacion de Doctores 2018 grant (ref. PRE2018-085440) from the Ministerio de Ciencia, Innovacion, y Universidades (Spain). Ylenia Jabalera wants to acknowledge an FPU2016 grant (ref. FPU16_04580) from the Ministerio de Educacion, Ciencia, y Deporte y Competitividad (Spain)., The synergy between directed chemotherapy and thermal therapy (both magnetic hyperthermia and photothermia) mediated by a nanoassembly composed of functionalized biomimetic magnetic nanoparticles (BMNPs) with the chemotherapeutic drug doxorubicin (DOXO) covered by the polymer poly(lactic-co-glycolic acid) (PLGA), decorated with TAT peptide (here referred to as TAT–PLGA(DOXO-BMNPs)) is explored in the present study. The rationale behind this nanoassembly lies in an optimization of the nanoformulation DOXO-BMNPs, already demonstrated to be more efficient against tumor cells, both in vitro and in vivo, than systemic traditional therapies. By embedding DOXO-BMNPs into PLGA, which is further functionalized with the cell-penetrating TAT peptide, the resulting nanoassembly is able to mediate drug transport (using DOXO as a drug model) and behaves as a hyperthermic agent (induced by an alternating magnetic field (AMF) or by laser irradiation with a laser power density of 2 W/cm2). Our results obtained using the HepG2 cell line show that there is a synergy between chemotherapy and thermal therapy that results in a stronger cytotoxic effect when compared to that caused by the soluble DOXO. This is probably due to the enhanced DOXO release occurring upon the application of the thermal therapy, as well as the induced local temperature rise mediated by BMNPs in the nanoassembly following exposition to AMF or to near-infrared (NIR) laser irradiation. These results represent a proof of concept demonstrating that TAT–PLGA(DOXO-BMNPs) can be used to efficiently combine therapies against tumor cells, which is a step forward in the transition from systemic to local treatments., Spanish Government CGL2016-76723, Instituto de Salud Carlos III Spanish Government, European Commission PID2019-109294RB-100, Spanish Government RYC-2014-16901, Junta de Andalucia A1-FQM-341-UGR18 C-FQM-497-UGR18 A-BIO-376-UGR18, Proyectos de I + D + I, del Plan Andaluz de Investigacion, Desarrollo e Innovacion P20_00208 P20_00346, Ministerio de Ciencia, Innovacion, y Universidades (Spain) PRE2018-085440, Ministerio de Educacion, Ciencia, y Deporte y Competitividad (Spain) FPU16_04580, Andalusian regional government CTS-236

16 p.-6 fig., Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy., Work at MJT and MIM laboratory was supported by Instituto de Salud Carlos III (ISCIII) of MICINN (grants cofunded by ERDF: “Una manera de hacer Europa” (PI17/01237, PI18/00095, RETIC ARADYAL RD16/0006/0001 and Euronanomed Program AC19/00082), Miguel Servet I program (CP15/00103) and Sara Borrell program (CD17/00146)), Andalusian Regional Ministry of Health (PI-0179-2014, PE-0172-2018). Work at EP-I laboratory was supported by the Spanish Ministerio de Economía, Industria y Competitividad (CTQ 2016-75870-P), Ministerio de Ciencia y Educación (PID 2019-104293GB-I00), Ministerio de Ciencia e Innovación [Proyectos de I+D+I Programación Conjunta Internacional, EuroNanoMed 2019 (PCI 2019-111825-2)], ISCIII RETIC ARADYAL RD16/0006/0012 and Junta de Andalucía (UMA18-FEDERJA-007). Work at DP-S laboratory was supported by Grants from Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (MICINN, Spain) and European Regional Development Fund, SAF 2015-68590-R and RTI 2018-097624-B-I00, ISCIII RETIC ARADyAL RD16/0006/0021.

CD300a receptor is found on different CD8+ T cell subsets and its expression has been associated to a more cytotoxic molecular signature. CD300a has an important role in some viral infections and its expression levels are known to be modulated by human immunodeficiency virus (HIV)−1 infection on several cell types. The main objective of this work was to investigate CD300a expression and its regulation during HIV-1 specific CD8+ T cell responses. CD300a receptor expression was analysed by multiparametric flow cytometry on CD8+ T lymphocytes from HIV negative donors, naive HIV-1+ individuals and HIV-1+ subjects under suppressive combined antiretroviral therapy (cART). HIV-1 specific CD8+ T cell response was studied by stimulating cells with HIV-1 derived peptides or with a Gag HIV-1 peptide. Our results showed that HIV-1 specific CD8+ T cells expressing higher levels of CD300a were more polyfunctional showing an increased degranulation and cytokine production. Moreover, we observed an up-regulation of CD300a expression after Gag HIV-1 peptide stimulation. Finally, our results demonstrated an inverse correlation between CD300a expression on CD8+ T lymphocytes and HIV disease progression markers. In conclusion, CD300a expression is associated to a better and more polyfunctional HIV-1 specific CD8+ T cell response., This study was supported by a grant from “Plan Estatal de I+ D+ I 2013–2016, ISCIII-Subdirección de Evaluación y Fomento de la Investigación-Fondo Europeo de Desarrollo Regional (FEDER) (Grant PI13/00889)” and Marie Curie Actions, Career Integration Grant, European Commission (Grant CIG 631674). Joana Vitallé and Iñigo Terrén are recipients of a predoctoral contract funded by the Department of Education, Basque Government (PRE_2017_2_0242 and PRE_2018_1_0032). Joana Vitallé and Iñigo Terrén are recipients of a fellowship from the Jesús de Gangoiti Barrera Foundation (FJGB15/008 and FJGB17/003). Laura Tarancón-Díez was supported by Instituto de Salud Carlos III, PFIS (FI00/00431). Olatz Zenarruzabeitia is recipient of a postdoctoral contract funded by “Instituto de Salud Carlos III-Contratos Sara Borrell 2017 (CD17/0128)” and the European Social Fund (ESF)-The ESF invests in your future. Ezequiel Ruiz-Mateos is supported by Programa Nicolás Monardes, C0032-2017, Consejería de Salud y Bienestar Social, Junta de Andalucía. Francisco Borrego is an Ikerbasque Research Professor, Ikerbasque, Basque Foundation for Science.

The authors thank I. Linares, V. Sanz, A.B. Rodriguez, A.I. Rodriguez and E. Arias for technical advice and R. Davies for editorial assistance., The capacity of cytotoxic-T lymphocytes to recognize and destroy tumor cells depends on the surface expression by tumor cells of MHC class I molecules loaded with tumor antigen peptides. Loss of MHC-I expression is the most frequent mechanism by which tumor cells evade the immune response. The restoration of MHC-I expression in cancer cells is crucial to enhance their immune destruction, especially in response to cancer immunotherapy. Using mouse models, we recovered MHC-I expression in the MHC-I negative tumor cell lines and analyzed their oncological and immunological profile. Fhit gene transfection induces the restoration of MHC-I expression in highly oncogenic MHC-I-negative murine tumor cell lines and genes of the IFN-γ transduction signal pathway are involved. Fhit-transfected tumor cells proved highly immunogenic, being rejected by a T lymphocyte-mediated immune response. Strikingly, this immune rejection was more frequent in females than in males. The immune response generated protected hosts against the tumor growth of non-transfected cells and against other tumor cells in our murine tumor model. Finally, we also observed a direct correlation between FHIT expression and HLA-I surface expression in human breast tumors. Recovery of Fhit expression on MHC class I negative tumor cells may be a useful immunotherapeutic strategy and may even act as an individualized immunotherapeutic vaccine., FEDER funds (EU) from the Instituto de Salud Carlos III PI12/02031 PI14/01978 PI15/00528 PI17/00197 PI19/01179 PT13/0010/0039 PT17/0015/0041, Worldwide Cancer Research 15-1166, Junta de Andalucia CTS-143 CTS-3952 CVI-4740, Instituto de Salud Carlos III, Rio-Hortega Contract from ISCIII CM12/00033, ibs.Granada Fellowship 496

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License.-- et al., [Objectives]: Treatment for celiac disease (CD) is a lifelong strict gluten-free diet (GFD). Patients should be followed-up with dietary interviews and serology as CD markers to ensure adherence to the diet. However, none of these methods offer an accurate measure of dietary compliance. Our aim was to evaluate the measurement of gluten immunogenic peptides (GIP) in stools as a marker of GFD adherence in CD patients and compare it with traditional methods of GFD monitoring. [Methods]: We performed a prospective, nonrandomized, multicenter study including 188 CD patients on GFD and 84 healthy controls. Subjects were given a dietary questionnaire and fecal GIP quantified by enzyme-linked immunosorbent assay (ELISA). Serological anti-tissue transglutaminase (anti-tTG) IgA and anti-deamidated gliadin peptide (anti-DGP) IgA antibodies were measured simultaneously. [Results]: Of the 188 celiac patients, 56 (29.8%) had detectable GIP levels in stools. There was significant association between age and GIP in stools that revealed increasing dietary transgressions with advancing age (39.2% in subjects ≥13 years old) and with gender in certain age groups (60% in men ≥13 years old). No association was found between fecal GIP and dietary questionnaire or anti-tTG antibodies. However, association was detected between GIP and anti-DGP antibodies, although 46 of the 53 GIP stool-positive patients were negative for anti-DGP. [Conclusions]: Detection of gluten peptides in stools reveals limitations of traditional methods for monitoring GFD in celiac patients. The GIP ELISA enables direct and quantitative assessment of gluten exposure early after ingestion and could aid in the diagnosis and clinical management of nonresponsive CD and refractory CD. Trial registration number NCT02711397., This work was supported by grants from Ministerio de Ciencia e Innovación and FEDER funds (DELIAC, IPT-2011-0952-900000), and Corporación Tecnológica de Andalucía (SINGLUCHECK, 1737/0118).