Orchestration of lymphocyte chemotaxis by mitochondrial dynamics

Lymphocyte traffic is required to maintain homeostasis and perform appropriate immunological reactions. To migrate into inflamed tissues, lymphocytes must acquire spatial and functional asymmetries. Mitochondria are highly dynamic organelles that distribute in the cytoplasm to meet specific cellular needs, but whether this is essential to lymphocyte functions is unknown. We show that mitochondria specifically concentrate at the uropod during lymphocyte migration by a process involving rearrangements of their shape. Mitochondrial fission facilitates relocation of the organelles and promotes lymphocyte chemotaxis, whereas mitochondrial fusion inhibits both processes. Our data substantiate a new role for mitochondrial dynamics and suggest that mitochondria redistribution is required to regulate the motor of migrating cells

Metal substrate catalysis in the confined space for platinum drug delivery

[EN] Catalysis-based approaches for the activation of anticancer agents hold considerable promise. These principally rely on the use of metal catalysts capable of deprotecting inactive precursors of organic drugs or transforming key biomolecules available in the cellular environment. Nevertheless, the efficiency of most of the schemes described so far is rather low, limiting the benefits of catalytic amplification as strategy for controlling the therapeutic effects of anticancer compounds. In the work presented here, we show that flavin reactivity within a hydrogel matrix provides a viable solution for the efficient catalytic activation and delivery of cisplatin, a worldwide clinically-approved inorganic chemotherapy agent. This is achieved by ionically adsorbing a flavin catalyst and a Pt(iv) prodrug as substrate into porous amino-functionalized agarose beads. The hydrogel chassis supplies high local concentrations of electron donating groups/molecules in the surrounding of the catalyst, ultimately boosting substrate conversion rates (TOF >200 min(-1)) and enabling controlled liberation of the drug by light or chemical stimuli. Overall, this approach can afford platforms for the efficient delivery of platinum drugs as demonstrated herein by using a transdermal diffusion model simulating the human skin.We acknowledge financial support from the Spanish State Research Agency (grants CTQ2016-80844-R, PID2019-109111RBI00, RTI2018-094398-B-I00, BIO2014-61838-EXP) and the Basque Government (Eusko Jaurlaritza, grant PIBA_2021_1_0034). S. V. L. thanks the Mexican Council of Science and Technology (CONACyT) for the postdoctoral fellowship she received (ref. CVU-267390). C. S. C. thanks Gipuzkoa Foru Aldundia (Gipuzkoa Fellows program; grant number 2019-FELL-000018-01/62/2019) for.nancial support. L. S. thanks the Spanish MultiMetDrugs network (RED2018-102471-T) for fruitful discussion. FLG thanks the Spanish Biocatalysis network (RED2018-102403T) and the European Research Council (ERC-Co-2018 818089). This work was performed under the Maria de Maeztu and Severo Ochoa Centres of Excellence Programme run by the Spanish State Research Agency, Grant No. MDM-2017-0720 (CIC biomaGUNE) and CEX2018-000867-S (DIPC)

A review of nature-based solutions for greywater treatment: Applications, hydraulic design, and environmental benefits

Abstract Recognizing greywater as a relevant secondary source of water and nutrients represents an important chance for the sustainable management of water resource. In the last two decades, many studies analysed the environmental, economic, and energetic benefits of the reuse of greywater treated by nature-based solutions (NBS). This work reviews existing case studies of traditional constructed wetlands and new integrated technologies (e.g., green roofs and green walls) for greywater treatment and reuse, with a specific focus on their treatment performance as a function of hydraulic operating parameters. The aim of this work is to understand if the application of NBS can represent a valid alternative to conventional treatment technologies, providing quantitative indications for their design. Specifically, indications concerning threshold values of hydraulic design parameters to guarantee high removal performance are suggested. Finally, the existing literature on life cycle analysis of NBS for greywater treatment has been examined, confirming the provided environmental benefits

Mice with reduced expression of the telomere-associated protein Ft1 develop p53-sensitive progeroid traits

Human AKTIP and mouse Ft1 are orthologous ubiquitin E2 variant proteins involved in telomere maintenance and DNA replication. AKTIP also interacts with A- and B-type lamins. These features suggest that Ft1 may be implicated in aging regulatory pathways. Here, we show that cells derived from hypomorph Ft1 mutant (Ft1kof/kof ) mice exhibit telomeric defects and that Ft1kof/kof animals develop progeroid traits, including impaired growth, skeletal and skin defects, abnormal heart tissue, and sterility. We also demonstrate a genetic interaction between Ft1 and p53. The analysis of mice carrying mutations in both Ft1 and p53 (Ft1kof/kof ; p53ko/ko and Ft1kof/kof ; p53+/ko ) showed that reduction in p53 rescues the progeroid traits of Ft1 mutants, suggesting that they are at least in part caused by a p53-dependent DNA damage response. Conversely, Ft1 reduction alters lymphomagenesis in p53 mutant mice. These results identify Ft1 as a new player in the aging process and open the way to the analysis of its interactions with other progeria genes using the mouse model

Synthesis and Biological Evaluation of RGD and isoDGR-Monomethyl Auristatin Conjugates Targeting Integrin (V3)

This work reports the synthesis of a series of small molecule-drug conjugates containing the \u3b1V\u3b23-integrin ligand cyclo[DKP-RGD] or cyclo[DKP-isoDGR], a lysosomally cleavable Val-Ala (VA) linker or an "uncleavable" version devoid of this sequence, and monomethyl Auristatin E (MMAE) or F (MMAF) as cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide-alkyne cycloaddition as key-step. The conjugates were tested for their binding affinity to the isolated \u3b1v\u3b23 receptor, and shown to retain nanomolar IC50 values, in the same range of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with \u3b1v\u3b23 integrin over-expressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess a markedly lower cytotoxic activity compared to the free drugs, which is consistent with an inefficient integrin-mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, cyclo[DKP-isoDGR]-VA-MMAE conjugate has low nanomolar IC50 values in cell viability assays with both cancer cell lines tested (U87: 11.50 \ub1 0.13 nM; M21: 6.94 \ub1 0.09 nM) and is therefore a promising candidate for in vivo experiments.This work reports the synthesis of a series of small-molecule\u2013drug conjugates containing the \u3b1 V \u3b2 3 -integrin ligand cyclo[DKP-RGD] or cyclo[DKP-isoDGR], a lysosomally cleavable Val-Ala (VA) linker or an \u201cuncleavable\u201d version devoid of this sequence, and monomethyl auristatin E (MMAE) or F (MMAF) as the cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide\u2013alkyne cycloaddition as the key step. The conjugates were tested for their binding affinity for the isolated \u3b1 v \u3b2 3 receptor and were shown to retain nanomolar IC 50 values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with \u3b1 v \u3b2 3 integrin overexpressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess markedly lower cytotoxic activity than the free drugs, which is consistent with inefficient integrin-mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, the cyclo[DKP-isoDGR]-VA-MMAE conjugate has low nanomolar IC 50 values in cell viability assays with both cancer cell lines tested (U87: 11.50\ub10.13 nm; M21: 6.94\ub10.09 nm) and is therefore a promising candidate for in vivo experiments

Soluble galectin-3 as a microenvironment-relevant immunoregulator with prognostic and predictive value in lung adenocarcinoma

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer (NSCLC) patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS, which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve (AUC) of 0.801 (p=0.011). Moreover, high levels might predict decreased progression-free survival and overall survival to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD

Preclinical and clinical characterization of fibroblast-derived neuregulin-1 on trastuzumab and pertuzumab activity in HER2-positive breast cancer

[Purpose]: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab.[Experimental Design]: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2–based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial.[Results]: NRG1 was expressed in HER2-positive breast cancer–derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group.[Conclusions]: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab.This work is supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219 and by Generalitat de Catalunya (2017 SGR 507). S. Menendez is supported by Department de Salut, Generalitat de Catalunya (PERIS SLT006/17/00040). MARBiobanc is supported by ISCiii/FEDER (PT17/0015/0011) and by “Xarxa de Bancs de tumors” sponsored by Pla Director d’ Oncologia de Catalunya (XBTC) and Fundacion Jimenez Díaz Biobanks Platform by PT13/0010/0012 grant. Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R) and the CRIS Cancer Foundation provides support to A. Pandiella. Work carried out in our laboratories receive support from the European Community through the Regional Development Funding Program (FEDER). J.C. Montero is funded by the ISCIII through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI18/00796). J. Albanell is supported by Breast Cancer Research Foundation (BCRF20-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociacion Espanola Contra el Cáncer (AECC)

Interlaboratory performance of a Real-Time PCR method for detection of Ceratocystis platani, the agent of canker stain of Platanus spp

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters-diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility-were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes

Inhibitory activity of the liquid fase of Copahue Thermal mud (Neuquén, Argentina) on strains of Staphylococcus aureus

El Complejo Termal de Copahue se sitúa a unos 1980 m sobre el nivel del mar, a 18 Km del volcán Copahue, sobre la cordillera de los Andes, Neuquén, Argentina. En él se halla la laguna Madre cuya temperatura oscila entre 70° y 90º C. En el fondo de la laguna se encuentra el fango gris, compuesto principalmente por azufre, silicio, oxígeno y aluminio. Empíricamente se conocen propiedades antiflogísticas y antibacterianas de los fangos minerales. Actualmente son utilizados en tratamientos de enfermedades de piel, reumáticas, infecciones de heridas quirúrgicas y también con fines estéticos. Staphylococcus aureus es un microorganismo integrante de la microbiota humana y como colonizante de piel y mucosas está implicado en diferentes procesos infecciosos. Su presencia en la microbiota del hombre aumenta el riesgo de padecer infecciones invasivas. La resistencia a antimicrobianos constituye un problema emergente en el tratamiento de este tipo de infecciones. El objetivo de éste trabajo fue analizar las propiedades antimicrobianas de la fase líquida (FL) del fango termal de Copahue, mediante ensayos in vitro, sobre cepas de S. aureus colonizantes de piel y mucosas, aisladas de pacientes provenientes de la comunidad y hospitales. El fango se obtuvo a 70 cm de profundidad del borde perimetral de la laguna. Se realizaron pruebas de difusión en agar y velocidad bactericida. Con la prueba de difusión en agar se observó halo inhibitorio frente a seis cepas de S. aureus ensayadas. La estimación del efecto inhibitorio frente a todas las cepas evidenció una declinación rápida del recuento de colonias viables antes de las 4 h (en el orden de ≤ 3 log), visualizándose un recrecimiento microbiano a las 24 h. La técnica de Killing Curve permitió establecer el tiempo óptimo de contacto para lograr inhibición del crecimiento bacteriano entre los microorganismos y la FL del fango. Este trabajo demuestra la actividad inhibitoria de la FL del fango del volcán Copahue sobre cepas de S. aureus colonizantes aisladas de piel y mucosas.The Copahue Thermal Complex is located at 1,980 m above sea level, at 18 km from the Copahue Volcano, on the Andes mountain range, in Neuquén, Argentina, where Lake Madre, whose temperature ranges from 70°C to 90° C is found. Grey fango composed of sulfur, silicon, oxygen and aluminum is found at the bottom of this lake. Empirically, mineral fango is known to have antiflogistic and antibacterial properties, which are currently used for treating skin conditions, rheumatic diseases and surgical wound infections and for cosmetic purposes. Staphylococcus aureus is a microorganism which forms part of the human microbiota, and as a skin and mucous membranes colonizer, it is involved in several infectious processes. Its presence in the human microbiota increases the risk of invasive infections. Antimicrobial resistance is a consequence arising from the treatment of this type of infections. The purpose of this work was to analyze the antimicrobial properties of the liquid phase of Copahue thermal fango by means of in vitro assays using skin and mucous membranes colonizer strains of S. aureus, isolated from community and hospital patients. The fango was obtained from the perimeter edge of the lake at a depth of 70 cm. Agar diffusion and bactericidal effect tests were conducted. The agar diffusion test showed an inhibition zones for six assayed strains of S. aureus. The estimated inhibitory effect for all the strains showed a fast decline in the viable colony count before 4 hs. (≤ 3 log order), visualizing a microbial regrowth at 24 hs. The Killing Curve technique allowed to establish the optimum contact time to achieve bacterial growth inhibition among microorganisms and the fango liquid phase. This work shows the antimicrobial properties of the liquid phase of fango from the Copahue Volcano against colonizer strains of S. aureus.Facultad de Ciencias Médica