Spin Label Studies of the Hemoglobin–Membrane Interaction During Sickle Hemoglobin Polymerization

An enhanced hemoglobin–membrane association has been previously documented in sickle cell anemia. However, it is not known how this interaction is modified during the hemoglobin S polymerization process. In this work, we use a model of reconstituted erythrocytes from ghost membranes whose cytoskeleton proteins had been previously labeled with the 4-maleimido Tempo spin label, and that were subsequently resealed with hemoglobin S or A solutions. Using electron paramagnetic resonance spectroscopy, we studied the time dependence of the spectral W/S parameter, indicative of the conformational state of cytoskeleton proteins (mainly spectrin) under spontaneous deoxygenation, with the aim of detecting the eventual effects due to hemoglobin S polymerization. The differences observed in the temporal behavior of W/S in erythrocytes reconstituted with both hemoglobins were considered as experimental evidence of an increment in hemoglobin S–membrane interaction as a result of the polymerization process of hemoglobin S under spontaneous deoxygenation.Fil: Falcón Dieguez, Jose. Universidad de Oriente; CubaFil: Rodi, Pablo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Lores Guevara, Manuel. Universidad de Oriente; CubaFil: Gennaro, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

Negative thermal expansion of nanoporous anodic aluminum oxide membranes

We have measured the thermal expansion of Ni nanowires (NWs) electrodeposited into self-organized nanoporous amorphous aluminum oxide (AAO) membranes without an Al substrate using X-ray diffraction between 110 K and 350 K. The results indicate an average thermal expansion of the Ni NWs - along the wire axis - of α ¯ NiNW = − 1.6 ± 1.5 × 10 − 6 K − 1. Assuming a bulk-like thermal expansion of the isolated Ni NWs, this result indicates that AAO also has a negative thermal expansion. We estimate the thermal expansion of nanoporous AAO to be α AAO = − 5 ± 1 × 10 − 6 K − 1. We show that data obtained previously on the thermal expansion of metallic NWs grown in the nanoporous AAO may be interpreted as originating from a negative thermal expansion of the matrix.Fil: Forzani, Luisina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Física del Litoral. Universidad Nacional del Litoral. Instituto de Física del Litoral; ArgentinaFil: Ramos, C. A.. Centro Atómico Bariloche (cab); ArgentinaFil: Vassallo Brigneti, Ettore Ciro. Instituto Tecnológico y de Estudios Superiores de Occid; MéxicoFil: Gennaro, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Física del Litoral. Universidad Nacional del Litoral. Instituto de Física del Litoral; ArgentinaFil: Koropecki, Roberto Roman. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Física del Litoral. Universidad Nacional del Litoral. Instituto de Física del Litoral; Argentin

Estudio de la microviscosidad de la Hemoglobina A (HbA) y la Hemoglobina S (HbS) en condiciones de desoxigenación espontánea

La microviscosidad de la hemoglobina A y la hemoglobina S es analizada en muestras con concentración intracelular, durante el proceso de desoxigenación espontánea y a 36 ºC, empleando glutatión y carbonmonoxihemoglobina, marcados con 4-maleimido-tempo. El glutatión marcado muestra un comportamiento constante de la microviscosidad de la hemoglobina S durante el estudio. La carbonmonoxihemoglobina marcada muestra un incremento de la microviscosidad durante el proceso de polimerización de la hemoglobina S, lo cual pudiera explicar el comportamiento de los tiempos de relajación magnética protónica determinados en otros artículos para las mismas muestras. Tanto el glutatión como la carbonmonoxihemoglobina marcados, reportan un comportamiento constante de la microviscosidad en la hemoglobina A.The Microviscosity of hemoglobin A and hemoglobin S are analyzed in samples of intracellular concentration, during the free deoxygenation process and at 36 ºC, using Glutathione and Carbonmonoxihemoglobin labeled with 4-maleimido-tempo. Labeled Glutathione shows a constant behaviour of the hemoglobin S microviscosity during the study. Labeled Carbonmonoxihemoglobin shows an increase of the microviscosity during the hemoglobin S polymerization process, which could explain the proton relaxation times behaviour determined in other articles for the same samples. Labeled Glutathione and Carbonmoxihemoglobing report constant behaviour of microviscosity in hemoglobin A.Fil: Lores, M.. Universidad de Oriente; CubaFil: Cabal, C.. Universidad de Oriente; CubaFil: Nascimento, Otaciro R.. Universidade de Sao Paulo; BrasilFil: Gennaro, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin

The new world of RNA diagnostics and therapeutics

The 5th Workshop IRE on Translational Oncology was held in Rome (Italy) on 27–28 March at the IRCCS Regina Elena National Cancer Institute. This meeting entitled “The New World of RNA diagnostics and therapeutics” highlightes the significant progress in the RNA field made over the last years. Research moved from pure discovery towards the development of diagnostic biomarkers or RNA-base targeted therapies seeking validation in several clinical trials. Non-coding RNAs in particular have been the focus of this workshop due to their unique properties that make them attractive tools for the diagnosis and therapy of cancer. This report collected the presentations of many scientists from different institutions that discussed recent oncology research providing an excellent overview and representative examples for each possible application of RNA as biomarker, for therapy or to increase the number of patients that can benefit from precision oncology treatment. In particular, the meeting specifically emphasized two key features of RNA applications: RNA diagnostic (Blandino, Palcau, Sestito, Díaz Méndez, Cappelletto, Pulito, Monteonofrio, Calin, Sozzi, Cheong) and RNA therapeutics (Dinami, Marcia, Anastasiadou, Ryan, Fattore, Regazzo, Loria, Aharonov)

Phosphorylation of FAM134C by CK2 controls starvation-induced ER-phagy.

Selective degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is initiated by ER-phagy receptors, which facilitate the incorporation of ER fragments into autophagosomes. FAM134 reticulon family proteins (FAM134A, FAM134B, and FAM134C) are ER-phagy receptors with structural similarities and nonredundant functions. Whether they respond differentially to the stimulation of ER-phagy is unknown. Here, we describe an activation mechanism unique to FAM134C during starvation. In fed conditions, FAM134C is phosphorylated by casein kinase 2 (CK2) at critical residues flanking the LIR domain. Phosphorylation of these residues negatively affects binding affinity to the autophagy proteins LC3. During starvation, mTORC1 inhibition limits FAM134C phosphorylation by CK2, hence promoting receptor activation and ER-phagy. Using a novel tool to study ER-phagy in vivo and FAM134C knockout mice, we demonstrated the physiological relevance of FAM134C phosphorylation during starvation-induced ER-phagy in liver lipid metabolism. These data provide a mechanistic insight into ER-phagy regulation and an example of autophagy selectivity during starvation.We thank G. Diez Roux and P. Ashley-Norman for critical reading of the manuscript. We thank the microscopy, MS, advanced histopathology, and FACS facilities at TIGEM Institute. We thank E. Nusco for helping us with AAV injections. Funding: This work was supported by European Research Council (ERC) (714551), Telethon intramural grants, and Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG 2015 Id 17717) (to C.S.) and Telethon Foundation (TMPGCBX16TT), AFM Telethon (Trampoline Grant), and AIRC (MFAG-2020-24856) (to P.G.). G.D.L. is a recipient of AIRC fellowship “Francesco Alicino” (25407). V.L. acknowledges funding from the ERC (101001784), the Italian MIUR-PRIN 2017 (2017FJZZRC), and the Swiss National Supercomputing Center (CSCS) (project ID u8). The work of A.S. was supported by the German Research Foundation DFG (SFB1177/2 and WO210/20-2) and the Dr. Rolf M. Schwiete Stiftung (13/2017). A.E. is supported by the RETOS projects Programme of Spanish Ministry of Science, Innovation and Universities, Spanish State Research Agency (grants SAF2015-67538-R and PID2019-104012RB-I00), and the ERC (638891). A.B.P.-G. is a recipient of Ph.D. fellowship from MICIU/AEI (BES-2017-081381). A.R. is a recipient of Umberto Veronesi Foundation postdoctoral fellowship. Author contributions: G.D.L. and F.I. performed most of the experiments. F.I. and A.B.P.-G. performed in vivo experiments. M.M. performed mutagenesis experiments. S.A. and V.L. performed LC3-FAM134C binding analysis. C.P.Q.M. performed in vitro phosphorylation assays. L.C. analyzed CK2 substrate phosphorylation. F.S., A.P., C.C., and A.S. analyzed proteomic data. G.N. provided critical suggestions. A.R. performed proteomic experiments. A.E. supervised in vivo experiments. M.R., L.A.P., and O.M. supervised CK2 experiments. C.S. designed the study. P.G. and C.S. conceived and supervised the experiments. C.S., P.G., V.L., and M.R. wrote the paper. G.D.L. and F.I. prepared the figures. All the authors read the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.S

Assessment of genetically modified maize DP4114 × MON 89034 × MON 87411 × DAS‐40278‐9 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA GMO‐NL‐2020‐171)

Genetically modified maize DP4114 × MON 89034 × MON 87411 × DAS-40278-9 was developed by crossing to combine four single events: DP4114, MON 89034, MON 87411 and DAS-40278-9. The GMO Panel previously assessed the four single maize events and two of the subcombinations and did not identify safety concerns. No new data on the single maize events or the assessed subcombinations were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the four-event stack maize does not give rise to food and feed safety and nutritional concerns. Therefore, no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable four-event stack maize grains into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in eight of the maize subcombinations not previously assessed and concludes that these are expected to be as safe as the single events, the previously assessed subcombinations and the four-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize DP4114 × MON 89034 × MON 87411 × DAS-40278-9. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the four-event stack maize and its subcombinations are as safe as its non-GM comparator and the tested non-GM maize varieties with respect to potential effects on human and animal health and the environment

Assessment of genetically modified maize MON 95379 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2020‐170)

Genetically modified maize MON 95379 was developed to confer insect protection against certain lepidopteran species. These properties were achieved by introducing the cry1B.868 and cry1Da_7 expression cassettes. The molecular characterisation data and bioinformatic analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between maize MON 95379 and its conventional counterpart needs further assessment. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the Cry1B.868 and Cry1Da_7 proteins as expressed in maize MON 95379. The GMO Panel finds no evidence that the genetic modification impacts the overall safety of maize MON 95379. In the context of this application, the consumption of food and feed from maize MON 95379 does not represent a nutritional concern in humans and animals. Therefore, no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable maize MON 95379 grains into the environment, this would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize MON 95379. The GMO Panel concludes that maize MON 95379 is as safe as its conventional counterpart and the tested non-GM maize varieties with respect to potential effects on human and animal health and the environment

CARCINOMA MAMÁRIO DUCTAL INVASOR: CORRELAÇÃO DA EXPRESSÃO IMUNOISTOQUÍMICA DE AXL E Β-CATENINA COM A AGRESSIVIDADE TUMORAL

Introduction: Invasive ductal carcinoma corresponds to the most common histological type of the breast, coexisting with different forms of clinical evolution, histological grading, expression of certain tissue markers and genomic profiles that seek a better understanding of the disease. Objectives: To analyze the correlation of β-catenin and AXL markers with tumor aggressiveness, with reference to overall survival, tumor progression and histopathological prognostic factors. Methods: A study of 101 samples of invasive ductal mammary carcinoma was performed. Those with a diagnosis of ductal type, initially submitted to biopsy or definitive surgical treatment, were included. For control purposes, 20 samples of intraductal carcinoma, 35 of breast fibroadenoma and 10 of breast tissue without any alteration were included. Those undergoing neoadjuvant chemotherapy, those without a tumor sample prior to chemotherapy, those lost to follow-up, and those with incomplete data, were excluded. Results: When the β-catenin expression was analyzed, it was negative. As for AXL, different degrees of expression were observed without statistical significance between them. Conclusion: When analyzing invasive ductal breast adenocarcinoma in TMA, there was no correlation in the expression of ß-Catenin and AXL when compared to overall survival, tumor progression and histological grade.Introdução: O carcinoma ductal invasor corresponde ao tipo histológico mais comum da mama coexistindo com formas diferentes de evolução clínica, graduação histológica, expressão de determinados marcadores teciduais e perfis genômicos que procuram melhor entendimento da doença. Objetivos: Analisar a correlação dos marcadores β-catenina e AXL com a agressividade tumoral, tendo como referência a sobrevida global, progressão tumoral e fatores prognósticos histopatológicos. Métodos: Foi realizado estudo de 101 amostras de carcinoma mamário ductal invasor. Foram incluídas aquelas com diagnóstico do tipo ductal, submetidas inicialmente à biópsia ou tratamento cirúrgico definitivo. Incluiu-se para fins de controle 20 amostras de carcinoma intraductal, 35 de fibroadenoma mamário e 10 de tecido mamário sem qualquer alteração. Foram excluídos os submetidos à quimioterapia neoadjuvante, que não tivessem amostra tumoral prévia ao tratamento quimioterápico, que perderam o seguimento, e com dados incompletos. Resultados: Quando analisada a expressão da β-catenina, foi negativa. Quanto ao AXL foram observados diferentes graus de expressão sem significância estatística entre eles. Conclusão: Quando analisados adenocarcinoma mamário do tipo ductal invasor em TMA não houve correlação na expressão de ß-catenina e AXL quando comparados a sobrevida global, progressão tumoral e grau histológico

Assessment of genetically modified maize GA21 × T25 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐DE‐2016‐137)

Genetically modified maize GA21 x T25 was developed by crossing to combine two single events: GA21 and T25. The GMO Panel previously assessed the two single maize events and did not identify safety concerns. No new data on the single maize events were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in maize GA21 x T25 does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that maize GA21 x T25, as described in this application, is as safe as its conventional counterpart and the non-GM reference varieties tested, and no post-market monitoring of food and feed is considered necessary. In the case of accidental release of viable maize GA21 x T25 grains into the environment, this would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize GA21 x T25. Post-market monitoring of food and feed is not considered necessary. The GMO Panel concludes that maize GA21 x T25 is as safe as its conventional counterpart and the non-GM reference varieties tested, with respect to potential effects on human and animal health and the environment

Assessment of genetically modified Maize MON 87429 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2019‐161)

Maize MON 87429 was developed to confer tolerance to dicamba, glufosinate, quizalofop and 2,4-D herbicides. The molecular characterisation data and bioinformatic analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between maize MON 87429 and its conventional counterpart needs further assessment, except for the levels of phytic acid in grains, which do not raise nutritional and safety concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the DMO, PAT, FT_T and CP4 EPSPS proteins as expressed in maize MON 87429. The GMO Panel finds no evidence that the genetic modification impacts the overall safety of maize MON 87429. In the context of this application, the consumption of food and feed from maize MON 87429 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that maize MON 87429 is as safe as the conventional counterpart and non-GM maize reference varieties tested, and no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable maize MON 87429 grains into the environment, this would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize MON 87429. The GMO Panel concludes that maize MON 87429, as described in this application, is as safe as its conventional counterpart and the tested non-GM maize reference varieties with respect to potential effects on human and animal health and the environment