Ultrasonication processing for the production of plant-based nanoemulsions

Plant-derived proteins have been emerging and growing in interest over the past few years, due to their interesting properties and the trend to replace animal-derived proteins [1]. Ultrasonication processing can be used to develop nanoemulsions based on plant proteins that are kinetically stabilized by their small dimension, unlike classic emulsions [2]. In this work, oil-in-water nanoemulsions were produced through high-speed homogenization, followed by ultrasonic homogenization (US), using different plant-derived proteins, including potato (Solanum tuberosum), lupin (Lupinus angustifolius), pea (Pisum sativum), chickpea (Cicer arietinum) and faba bean (Vicia faba) protein as emulsifiers. A central composite rotatable experimental design was used to evaluate the influence of three independent variables: water/oil ratio (65-75% of water), protein content (1-6%) and US time (1-7 min) on the size average (by intensity) and polydispersity index (PDI) of the nanoemulsions. A total of 17 experiments were performed with 14 three-level experimental points, and 3 replicates at the central point. The effect of the US time (0, 3, 4.5 and 6 min) in the potato and lupin proteins primary and secondary structures were analysed through SDS-PAGE electrophoresis and circular dichroism, respectively. Results showed that the use of potato, lupin and pea proteins lead to the formation of stable nanoemulsions, while chickpea and faba bean proteins resulted in non-stable nanoemulsions, with phase separation. The smallest mean droplet size for potato protein was 439.9 nm and PDI value 0.464 [21:73 (w/w) oil/water ratio, 6% of protein and 6 min of US]. The smallest mean droplet size for lupin protein was 505.5 nm and PDI value 0.434, and for pea protein the droplet size was 551.3 nm and PDI value 0.249 [23.6:73 (w/w) oil/water ratio, 3.4% of protein and 6 min of US]. Electrophoresis results show that for native potato and lupin samples the ultrasonication did not induce significant changes in the protein pattern, indicating that the US treatment did not modify the primary structure. Regarding the second structure, US did not change the secondary structure of potato protein but induced a slight increase of -helix for all US treatments for lupin protein. Stable nanoemulsions can be developed using plant-derived proteins and ultrasonication, foreseeing different applications in the food industry.This study was supported by the project cLabel+ (POCI-01-0247-FEDER-046080) cofinanced by Compete 2020, Lisbon 2020, Portugal 2020 and the European Union, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Targeting of Aberrant αvβ6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells.

Expression of the αvβ6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvβ6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αβ) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvβ6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvβ6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvβ6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin

Accuracy of the ADNEX MR scoring system based on a simplified MRI protocol for the assessment of adnexal masses

PURPOSE:We aimed to evaluate the ADNEX MR scoring system for the prediction of adnexal mass malignancy, using a simplified magnetic resonance imaging (MRI) protocol.METHODS:In this prospective study, 200 patients with 237 adnexal masses underwent MRI between February 2014 and February 2016 and were followed until February 2017. Two radiologists calculated ADNEX MR scores using an MRI protocol with a simplified dynamic study, not a high temporal resolution study, as originally proposed. Sensitivity, specificity, positive and negative predictive values, likelihood ratios, and the area under the receiver operating characteristic curve were calculated (cutoff for malignancy, score ≥ 4). The reference standard was histopathologic diagnosis or imaging findings during >12 months of follow-up.RESULTS:Of 237 lesions, 79 (33.3%) were malignant. The ADNEX MR scoring system, using a simplified MRI protocol, showed 94.9% (95% confidence interval [CI], 87.5%–98.6%) sensitivity and 97.5% (95% CI, 93.6%–99.3%) specificity in malignancy prediction; it was thus highly accurate, like the original system. The level of interobserver agreement on simplified scoring was high (κ = 0.91).CONCLUSION:In a tertiary cancer center, the ADNEX MR scoring system, even based on a simplified MRI protocol, performed well in the prediction of malignant adnexal masses. This scoring system may enable the standardization of MRI reporting on adnexal masses, thereby improving communication between radiologists and gynecologists

In vitro potency, in vitro and in vivo efficacy of liposomal alendronate in combination with γδ T cell immunotherapy in mice

Nitrogen-containing bisphosphonate (N-BP), including zoledronic acid (ZOL) and alendronate (ALD), have been proposed as sensitisers in γδ T cell immunotherapy in pre-clinical and clinical studies. Therapeutic efficacy of N-BPs is hampered by their rapid renal excretion and high affinity for bone. Liposomal formulations of N-BP have been proposed to improve accumulation in solid tumours. Liposomal alendronate (L-ALD) has been suggested as a suitable alternative to liposomal ZOL (L-ZOL), due to unexpected mice death experienced in pre-clinical studies with the latter. Only one study so far has proven the therapeutic efficacy of L-ALD, in combination with γδ T cell immunotherapy, after intraperitoneal administration of γδ T cell resulting in delayed growth of ovarian cancer in mice. This study aims to assess the in vitro efficacy of L-ALD, in combination with γδ T cell immunotherapy, in a range of cancerous cell lines, using L-ZOL as a comparator. The therapeutic efficacy was tested in a pseudo-metastatic lung mouse model, following intravenous injection of γδ T cell, L-ALD or the combination. In vivo biocompatibility and organ biodistribution studies of L-BPs were undertaken simultaneously. Higher concentrations of L-ALD (40–60 μM) than L-ZOL (3–10 μM) were required to produce a comparative reduction in cell viability in vitro, when used in combination with γδ T cells. Significant inhibition of tumour growth was observed after treatment with both L-ALD and γδ T cells in pseudo-metastatic lung melanoma tumour-bearing mice after tail vein injection of both treatments, suggesting that therapeutically relevant concentrations of L-ALD and γδ T cell could be achieved in the tumour sites, resulting in significant delay in tumour growth

Sentimientos de adolescentes con Diabetes Mellitus delante del proceso de vivir con la enfermedad

Qualitative research conducted in a diabetes service in the countryside of the state of Ceará, Brazil, with 11 teenagers with diabetes mellitus. The study aimed to understand the experience of adolescents facing the process of living with diabetes. Data were collected in 2007 May and June, through semi-structured interviews. It was observed that the teenager faces difficulties since the moment of diagnosis, especially because their food habits and lifestyles need to change, triggering feelings like fear, insecurity and anger. Over time, they incorporate the necessary changes to treatment and care, and begin to see the disease as normal. One concludes that it is necessary to understand teenagers, their behaviors, fears and desires and support them in the different areas of this experience.Investigación cualitativa, llevada a cabo en un servicio de diabetes del interior del estado de Ceará, Brasil, con 11 adolescentes con diabetes mellitus. El objetivo fue comprender la experiencia del adolescente delante del proceso de vivir con diabetes. Los datos fueron recolectados entre mayo y junio de 2007 a través de entrevistas semi estructuradas. Se observó que el adolescente enfrenta dificultades desde el momento del diagnóstico, sobre todo en el plan alimentar y cambios en el estilo de vida, desencadenando sentimientos como miedo, inseguridad e ira. Con el tiempo, incorporan los cambios necesarios al tratamiento y atención, y llegan a ver la enfermedad como algo normal. En conclusión, que es necesario entender los adolescentes, sus comportamientos, miedos y deseos, y apoyarlos en las diferentes áreas de esta experiencia.Pesquisa de natureza qualitativa, realizada em um serviço de diabetes do interior do Ceará com 11 adolescentes portadores de diabetes mellitus. O estudo objetivou compreender a experiência do adolescente frente ao processo de viver com o diabetes. Os dados foram coletados nos meses de maio e junho de 2007 por meio de entrevista semiestruturada. Constatou-se que o adolescente enfrenta dificuldades desde o momento do diagnóstico, principalmente no plano alimentar e nas mudanças no estilo de vida, desencadeando sentimentos como medo, insegurança e revolta. Com o passar do tempo, incorporam as mudanças necessárias ao tratamento e cuidados; e passam a ver a doença de forma normal. Conclui-se que é necessário compreender os adolescentes, seus comportamentos, medos e anseios e apoiá-los nos diversos âmbitos dessa experiência.Secretaria Municipal de Saúde de Pio IX-PI Programa de Saúde da FamíliaUniversidade Federal do Ceará Faculdade de Farmácia Odontologia e Enfermagem Departamento de EnfermagemUniversidade Federal de São Paulo (UNIFESP) Programa de Pós-Graduação em Saúde ColetivaUNIFESP, Programa de Pós-Graduação em Saúde ColetivaSciEL

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study

Background: Locally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based chimeric antigen receptor T-cell (CAR-T) approach named T4 immunotherapy. Patient-derived T-cells are engineered by retroviral transduction to coexpress a panErbB-specific CAR called T1E28ζ and an IL-4-responsive chimeric cytokine receptor, 4αβ, which allows IL-4-mediated enrichment of transduced cells during manufacture. These cells elicit preclinical antitumor activity against HNSCC and other carcinomas. In this trial, we used intratumoral delivery to mitigate significant clinical risk of on-target off-tumor toxicity owing to low-level ErbB expression in healthy tissues. // Methods: We undertook a phase 1 dose-escalation 3+3 trial of intratumoral T4 immunotherapy in HNSCC (NCT01818323). CAR T-cell batches were manufactured from 40 to 130 mL of whole blood using a 2-week semiclosed process. A single CAR T-cell treatment, formulated as a fresh product in 1–4 mL of medium, was injected into one or more target lesions. Dose of CAR T-cells was escalated in 5 cohorts from 1×107−1×109 T4+ T-cells, administered without prior lymphodepletion. // Results: Despite baseline lymphopenia in most enrolled subjects, the target cell dose was successfully manufactured in all cases, yielding up to 7.5 billion T-cells (67.5±11.8% transduced), without any batch failures. Treatment-related adverse events were all grade 2 or less, with no dose-limiting toxicities (Common Terminology Criteria for Adverse Events V.4.0). Frequent treatment-related adverse events were tumor swelling, pain, pyrexias, chills, and fatigue. There was no evidence of leakage of T4+ T-cells into the circulation following intratumoral delivery, and injection of radiolabeled cells demonstrated intratumoral persistence. Despite rapid progression at trial entry, stabilization of disease (Response Evaluation Criteria in Solid Tumors V.1.1) was observed in 9 of 15 subjects (60%) at 6 weeks post-CAR T-cell administration. Subsequent treatment with pembrolizumab and T-VEC oncolytic virus achieved a rapid complete clinical response in one subject, which was durable for over 3 years. Median overall survival was greater than for historical controls. Disease stabilization was associated with the administration of an immunophenotypically fitter, less exhausted, T4 CAR T-cell product. // Conclusions: These data demonstrate the safe intratumoral administration of T4 immunotherapy in advanced HNSCC

Proteomic Analysis Reveals That Iron Availability Alters the Metabolic Status of the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways

A search for ultra-high-energy photons at the Pierre Auger Observatory exploiting air-shower universality

The Pierre Auger Observatory is the most sensitive detector to primary photons with energies above ∼0.2 EeV. It measures extensive air showers using a hybrid technique that combines a fluorescence detector (FD) with a ground array of particle detectors (SD). The signatures of a photon-induced air shower are a larger atmospheric depth at the shower maximum (X$_{max}$) and a steeper lateral distribution function, along with a lower number of muons with respect to the bulk of hadron-induced background. Using observables measured by the FD and SD, three photon searches in different energy bands are performed. In particular, between threshold energies of 1-10 EeV, a new analysis technique has been developed by combining the FD-based measurement of X$_{max}$ with the SD signal through a parameter related to its muon content, derived from the universality of the air showers. This technique has led to a better photon/hadron separation and, consequently, to a higher search sensitivity, resulting in a tighter upper limit than before. The outcome of this new analysis is presented here, along with previous results in the energy ranges below 1 EeV and above 10 EeV. From the data collected by the Pierre Auger Observatory in about 15 years of operation, the most stringent constraints on the fraction of photons in the cosmic flux are set over almost three decades in energy

Study on multi-ELVES in the Pierre Auger Observatory

Since 2013, the four sites of the Fluorescence Detector (FD) of the Pierre Auger Observatory record ELVES with a dedicated trigger. These UV light emissions are correlated to distant lightning strikes. The length of recorded traces has been increased from 100 μs (2013), to 300 μs (2014-16), to 900 μs (2017-present), to progressively extend the observation of the light emission towards the vertical of the causative lightning and beyond. A large fraction of the observed events shows double ELVES within the time window, and, in some cases, even more complex structures are observed. The nature of the multi-ELVES is not completely understood but may be related to the different types of lightning in which they are originated. For example, it is known that Narrow Bipolar Events can produce double ELVES, and Energetic In-cloud Pulses, occurring between the main negative and upper positive charge layer of clouds, can induce double and even quadruple ELVES in the ionosphere. This report shows the seasonal and daily dependence of the time gap, amplitude ratio, and correlation between the pulse widths of the peaks in a sample of 1000+ multi-ELVES events recorded during the period 2014-20. The events have been compared with data from other satellite and ground-based sensing devices to study the correlation of their properties with lightning observables such as altitude and polarity