Tannin-rich extracts improve the performance of amidated pectin as an alternative microencapsulation matrix to alginate

This work was funded by Teagasc, the Irish Agricultural and Food Development Authority (Fermoy, Ireland), by the “Plan propio de Investigaci ́on y Transferencia” of the University of Granada under both programs “Intensificaci ́on de la Investigaci ́on, modalidad B′′ and by the European Research Commission (Research Executive Agency) under the research project Stance4Health (Grant Contract N 816303) granted to Jos ́e A. Rufi ́an-Henares.Microencapsulation of tannin extracts through extrusion-gelation method was performed comparing two alter- native encapsulation matrices: alginate and amidated pectin. The microstructure of the generated microbeads was studied, as well as their microencapsulation efficiency and release properties. Overall, pectin-based beads performed better than their alginate-based counterparts. This, combined with a greater incorporation of tannins in the feed formulations led to a higher tannin load in the final beads. The best microencapsulation efficiency was given by pectin microbeads loaded with 10% tannin extract (w/w), but the final tannin content could be further increased by adding a 20% (w/w) concentration of the extracts. During a 14-days storage, only a marginal loss of tannins was recorded for pectin-based microbeads. The results reveal that great potential exists in producing pectin-based microbeads in presence of tannins, which allow better loading capacities and improving structural properties, thanks to the interactions between the tannins and the amidated polysaccharide.Teagasc, the Irish Agricultural and Food Development Authority (Fermoy, Ireland)Plan propio de Investigación y Transferencia” of the University of Granada under both programs “Intensificación de la Investigación, modalidad BEuropean Research Commission (Research Executive Agency) under the research project Stance4Health (Grant Contract N 816303

Tannin-rich extracts improve the performance of amidated pectin as an alternative microencapsulation matrix to alginate

peer-reviewedMicroencapsulation of tannin extracts through extrusion-gelation method was performed comparing two alternative encapsulation matrices: alginate and amidated pectin. The microstructure of the generated microbeads was studied, as well as their microencapsulation efficiency and release properties. Overall, pectin-based beads performed better than their alginate-based counterparts. This, combined with a greater incorporation of tannins in the feed formulations led to a higher tannin load in the final beads. The best microencapsulation efficiency was given by pectin microbeads loaded with 10% tannin extract (w/w), but the final tannin content could be further increased by adding a 20% (w/w) concentration of the extracts. During a 14-days storage, only a marginal loss of tannins was recorded for pectin-based microbeads. The results reveal that great potential exists in producing pectin-based microbeads in presence of tannins, which allow better loading capacities and improving structural properties, thanks to the interactions between the tannins and the amidated polysaccharide

Heterogeneidad fenotípica en el Sistema de Secreción tipo III de Pseudomonas syringae durante la interacción con la planta

La heterogeneidad fenotípica es un fenómeno que se ha descrito en poblaciones bacterianas de diversas especies. Un patrón de expresión génica heterogéneo puede llegar a volverse bimodal en ambientes homogéneos, proceso conocido como bistabilidad. El desarrollo de métodos de análisis de células individuales, como la microscopía confocal, la citometría o la microfluídica, ha llevado a la identificación de nuevos ejemplos de variación fenotípica y de biestabilidad. La relevancia de estos procesos se ha demostrado en patógenos humanos y de animales. No obstante, se conoce muy poco sobre la relevancia de este tipo de procesos en el proceso de adaptación a huéspedes no animales. P. syringae es una bacteria patógena de plantas con un amplio rango de hospedador, existiendo más de 50 patovares. La virulencia de Pseudomonas syringae dependiende del Sistema de Secreción Tipo III (T3SS) y de los efectores tipo III (T3E). Mediante fusiones transcripcionales a proteínas fluorescentes generadas en el genoma de P. syringae pv. phaseolicola, y el uso de microscopía de fluorescencia y citometría de flujo, nuestro laboratorio ha demostrado que la expresión del T3SS y de T3E es heterogénea en el interior de la planta y biestable en medio mínimo. En este trabajo presentamos los resultados obtenidos en nuestro análisis del impacto de la expresión heterogénea del T3SS para la adaptación a la planta, que incluyen la evaluación de la viabilidad de las variantes (T3SSON/T3SSOFF) en el apoplasto de la hoja, y de la dinámica de expresión en la población en diferentes escenarios de activación de defensa en la planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Ratones knock-out del receptor lpa1 de ácido lisofosfatídico presentan un acusado déficit de la isoenzima glutaminasa KGA (GLS) y una morfología alterada en las espinas dendríticas de hipocampo y corteza

Objectives: The objective of the present study was to utilize mice with knocked-down lysophosphatidic acid 1 (LPA1) receptor to ascertain changes in glutamatergic transmission that may help to explain part of the cognitive and memory deficits shown by these KO-LPA1 mice. Material & methods: A well characterized KO-LPA1 mouse strain was used as animal model and compared with wild-type (WT) and heterozygous animals. Expression studies were implemented by immunohistochemistry and Western analysis of mouse brain regions, real-time quantitative RT-PCR of GA isoforms, enzymatic analysis of regional GA activity and Golgi staining to assess dendritic spine morphology and density. Results: A strong reduction of KGA immunoreactivity was mostly revealed in cerebral cortex and hippocampus of KO-LPA1 mice versus WT and heterozygous animals. In contrast, neither mRNA levels nor enzyme activity were significantly altered in KO mice suggesting compensatory mechanisms for neurotransmitter Glu synthesis. Interestingly, Golgi staining of hippocampal and cortical neurons revealed a clear morphology change toward a less-mature undifferentiated spine phenotype, without changes in the total number of spines. Conclusions: The molecular mechanisms underlying KGA downregulation in null LPA1 mutant mice are unknown. However, LPA increases neuronal differentiation, arborization and neurite outgrowth of developing neurons, while Gln-derived Glu, through GA reaction, has been also involved in neuronal growth and differentiation. It is tempting to speculate that downregulation of KGA protein in KO-LPA1 mice induce morphological changes in dendritic spines of cortical and hippocampal neurons which, in turn, may account for memory and cognitive deficits shown by KO-LPA1 mice.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Acknowledgements: Red de Trastornos Adictivos, RTA, (RD12/0028/0013/) RETICS, ISCIII, y Consejería Innovación, Ciencia y Empresa, Junta de Andalucía (Proyecto de Excelencia CVI-6656)

Glutaminase and MMP-9 downregulation in cortex and hippocampus of LPA1 receptor null mice correlate with altered dendritic spine plasticity

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions acting through G protein-coupled receptors (GPCRs). Here we explore the crosstalk between LPA1 receptor and glutamatergic transmission by examining expression of glutaminase (GA) isoforms in different brain areas isolated from wild-type (WT) and KOLPA1 mice. Silencing of LPA1 receptor induced a severe down-regulation of Gls-encoded long glutaminase protein variant (KGA) (glutaminase gene encoding the kidney-type isoforms, GLS) protein expression in several brain regions, particularly in brain cortex and hippocampus. Immunohistochemical assessment of protein levels for the second type of glutaminase (GA) isoform, glutaminase gene encoding the liver-type isoforms (GLS2), did not detect substantial differences with regard to WT animals. The regional mRNA levels of GLS were determined by real time RT-PCR and did not show significant variations, except for prefrontal and motor cortex values which clearly diminished in KO mice. Total GA activity was also significantly reduced in prefrontal and motor cortex, but remained essentially unchanged in the hippocampus and rest of brain regions examined, suggesting activation of genetic compensatory mechanisms and/or post-translational modifications to compensate for KGA protein deficit. Remarkably, Golgi staining of hippocampal regions showed an altered morphology of glutamatergic pyramidal cells dendritic spines towards a less mature filopodia-like phenotype, as compared with WT littermates. This structural change correlated with a strong decrease of active matrix-metalloproteinase (MMP) 9 in cerebral cortex and hippocampus of KOLPA1 mice. Taken together, these results demonstrate that LPA signaling through LPA1 influence expression of the main isoenzyme of glutamate biosynthesis with strong repercussions on dendritic spines maturation, which may partially explain the cognitive and learning defects previously reported for this colony of KOLPA1 mice

Phenotypically heterogeneous loci in the plant pathogen Pseudomonas syringae.

Phenotypic heterogeneity usually refers to the co-existance of different phenotypes within a population. Phenotypic differences may arise through genetic variation (genomic rearrangements or mutations) or through the response to differences in the stimuli as encountered within the microenvironment. But also, sometimes, the sources of variation may not be deterministic, i.e. directly related to stimuli, but a consequence of molecular noise in gene expression and/or a programmed event under genetic or epigenetic control. A particular example of phenotypic heterogeneity is bistability. Bistability occurs when a bacterial clonal population splits into two subpopulations showing distinct phenotypes. Phenotypic heterogeneity can be beneficial in fluctuating environments by allowing some individuals within the clonal population to survive sudden changes (risk-spreading). It can also benefit the entire population through cooperation between individuals displaying phenotypic differences (division of labour). These processes and their biological relevance have been described in some animal pathogens, but little is known about them in plant-pathosystems. Pseudomonas syringae is a plant-pathogenic bacterium whose virulence depends on the expression of a type III secretion system (T3SS). Our team has previously reported that T3SS expression is bistable under inducing conditions, generating two subpopulations (T3SSON/T3SSOFF) that show differences in virulence. We have identified other loci including genes related to motility, biofilm formation and DNA methylation that also display phenotypic heterogeneity to very different degrees and with different dynamics and are at different stages on their molecular and biological characterization. Our latest advances on this front will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Real-life disease monitoring in follicular lymphoma patients using liquid biopsy ultra-deep sequencing and PET/CT

In the present study, we screened 84 Follicular Lymphoma patients for somatic mutations suitable as liquid biopsy MRD biomarkers using a targeted next-generation sequencing (NGS) panel. We found trackable mutations in 95% of the lymph node samples and 80% of the liquid biopsy baseline samples. Then, we used an ultra-deep sequencing approach with 2 · 10−4 sensitivity (LiqBio-MRD) to track those mutations on 151 follow-up liquid biopsy samples from 54 treated patients. Positive LiqBio-MRD at first-line therapy correlated with a higher risk of progression both at the interim evaluation (HRINT 11.0, 95% CI 2.10–57.7, p = 0.005) and at the end of treatment (HREOT, HR 19.1, 95% CI 4.10–89.4, p < 0.001). Similar results were observed by PET/CT Deauville score, with a median PFS of 19 months vs. NR (p < 0.001) at the interim and 13 months vs. NR (p < 0.001) at EOT. LiqBio-MRD and PET/CT combined identified the patients that progressed in less than two years with 88% sensitivity and 100% specificity. Our results demonstrate that LiqBio-MRD is a robust and non-invasive approach, complementary to metabolic imaging, for identifying FL patients at high risk of failure during the treatment and should be considered in future response-adapted clinical trials.This study has been funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union through the projects PI21/00314, PI19/01430, PI19/01518 and PI18/00295, PTQ2020-011372, CP19/00140, CP22/00082, Doctorado industrial CAM IND2020/TIC-17402 and CRIS cancer foundation

Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase

Personalized monitoring of circulating tumor DNA with a specific signature of trackable mutations after chimeric antigen receptor T-cell therapy in follicular lymphoma patients

BackgroundCART therapy has produced a paradigm shift in the treatment of relapsing FL patients. Strategies to optimize disease surveillance after these therapies are increasingly necessary. This study explores the potential value of ctDNA monitoring with an innovative signature of personalized trackable mutations.MethodEleven FL patients treated with anti-CD19 CAR T-cell therapy were included. One did not respond and was excluded. Genomic profiling was performed before starting lymphodepleting chemotherapy to identify somatic mutations suitable for LiqBio-MRD monitoring. The dynamics of the baseline mutations (4.5 per patient) were further analyzed on 59 cfDNA follow-up samples. PET/CT examinations were performed on days +90, +180, +365, and every six months until disease progression or death.ResultsAfter a median follow-up of 36 months, all patients achieved a CR as the best response. Two patients progressed. The most frequently mutated genes were CREBBP, KMT2D and EP300. Simultaneous analysis of ctDNA and PET/CT was available for 18 time-points. When PET/CT was positive, two out of four ctDNA samples were LiqBio-MRD negative. These two negative samples corresponded to women with a unique mesenteric mass in two evaluations and never relapsed. Meanwhile, 14 PET/CT negative images were mutation-free based on our LiqBio-MRD analysis (100%). None of the patients had a negative LiqBio-MRD test by day +7. Interestingly, all durably responding patients had undetectable ctDNA at or around three months after infusion. Two patients presented discordant results by PET/CT and ctDNA levels. No progression was confirmed in these cases. All the progressing patients were LiqBio-MRD positive before progression.ConclusionThis is a proof-of-principle for using ctDNA to monitor response to CAR T-cell therapy in FL. Our results confirm that a non-invasive liquid biopsy MRD analysis may correlate with response and could be used to monitor response. Harmonized definitions of ctDNA molecular response and pinpointing the optimal timing for assessing ctDNA responses are necessary for this setting. If using ctDNA analysis, we suggest restricting follow-up PET/CT in CR patients to a clinical suspicion of relapse, to avoid false-positive results