RESUMEN
Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.
Asunto(s)
Dihidroxifenilalanina , Mytilus edulis , Animales , Dihidroxifenilalanina/química , Dihidroxifenilalanina/metabolismo , Mytilus edulis/genética , Mytilus edulis/química , Mytilus edulis/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Verrucomicrobia/genética , Verrucomicrobia/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Monofenol Monooxigenasa/química , Proteínas/genética , Proteínas/química , Proteínas/aislamiento & purificación , Hidroxilación , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Increased signs of DNA damage have been associated to aging and neurodegenerative diseases. DNA damage repair mechanisms are tightly regulated and involve different pathways depending on cell types and proliferative vs. postmitotic states. Amongst them, fused in sarcoma (FUS) was reported to be involved in different pathways of single- and double-strand break repair, including an early recruitment to DNA damage. FUS is a ubiquitously expressed protein, but if mutated, leads to a more or less selective motor neurodegeneration, causing amyotrophic lateral sclerosis (ALS). Of note, ALS-causing mutation leads to impaired DNA damage repair. We thus asked whether FUS recruitment dynamics differ across different cell types putatively contributing to such cell-type-specific vulnerability. For this, we generated engineered human induced pluripotent stem cells carrying wild-type FUS-eGFP and analyzed different derivatives from these, combining a laser micro-irradiation technique and a workflow to analyze the real-time process of FUS at DNA damage sites. All cells showed FUS recruitment to DNA damage sites except for hiPSC, with only 70% of cells recruiting FUS. In-depth analysis of the kinetics of FUS recruitment at DNA damage sites revealed differences among cellular types in response to laser-irradiation-induced DNA damage. Our work suggests a cell-type-dependent recruitment behavior of FUS during the DNA damage response and repair procedure. The presented workflow might be a valuable tool for studying the proteins recruited at the DNA damage site in a real-time course.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Daño del ADN , MutaciónRESUMEN
The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein-Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail.
Asunto(s)
Retrovirus Endógenos , Infecciones por Virus de Epstein-Barr , Esclerosis Múltiple , Animales , Retrovirus Endógenos/genética , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Melfalán , Ratones , gammaglobulinasRESUMEN
Low phosphorus (P) availability is a major limiting factor for potatoes. P fertilizer is applied to enhance P availability; however, it may become toxic when plants accumulate at high concentrations. Therefore, it is necessary to gain more knowledge of the morphological and biochemical processes associated with P deficiency and toxicity for potatoes, as well as to explore an alternative approach to ameliorate the P deficiency condition. A comprehensive study was conducted (I) to assess plant morphology, mineral allocation, and metabolites of potatoes in response to P deficiency and toxicity; and (II) to evaluate the potency of plant growth-promoting rhizobacteria (PGPR) in improving plant biomass, P uptake, and metabolites at low P levels. The results revealed a reduction in plant height and biomass by 60-80% under P deficiency compared to P optimum. P deficiency and toxicity conditions also altered the mineral concentration and allocation in plants due to nutrient imbalance. The stress induced by both P deficiency and toxicity was evident from an accumulation of proline and total free amino acids in young leaves and roots. Furthermore, root metabolite profiling revealed that P deficiency reduced sugars by 50-80% and organic acids by 20-90%, but increased amino acids by 1.5-14.8 times. However, the effect of P toxicity on metabolic changes in roots was less pronounced. Under P deficiency, PGPR significantly improved the root and shoot biomass, total root length, and root surface area by 32-45%. This finding suggests the potency of PGPR inoculation to increase potato plant tolerance under P deficiency.
Asunto(s)
Fósforo/metabolismo , Desarrollo de la Planta , Rhizobiaceae/fisiología , Solanum tuberosum/anatomía & histología , Solanum tuberosum/metabolismo , Estrés Fisiológico , Hojas de la Planta/anatomía & histología , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Raíces de Plantas/anatomía & histología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/microbiologíaRESUMEN
Amyotropic lateral sclerosis (ALS) is a lethally progressive and irreversible neurodegenerative disease marked by apparent death of motor neurons present in the spinal cord, brain stem and motor cortex. While more and more gene mutants being established for genetic ALS, the vast majority suffer from sporadic ALS (>90%). It has been challenging, thus, to model sporadic ALS which is one reason why the underlying pathophysiology remains elusive and has stalled the development of therapeutic strategies of this progressive motor neuron disease. To further unravel these pathological signaling pathways, human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs) from FUS- and SOD1 ALS patients and healthy controls were systematically compared to independent published datasets. Here through this study we created a gene profile of ALS by analyzing the DEGs, the Kyoto encyclopedia of Genes and Genomes (KEGG) pathways, the interactome and the transcription factor profiles (TF) that would identify altered molecular/functional signatures and their interactions at both transcriptional (mRNAs) and translational levels (hub proteins and TFs). Our findings suggest that FUS and SOD1 may develop from dysregulation in several unique pathways and herpes simplex virus (HSV) infection was among the topmost predominant cellular pathways connected to FUS and not to SOD1. In contrast, SOD1 is mainly characterized by alterations in the metabolic pathways and alterations in the neuroactive-ligand-receptor interactions. This suggests that different genetic ALS forms are singular diseases rather than part of a common spectrum. This is important for patient stratification clearly pointing towards the need for individualized medicine approaches in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína FUS de Unión a ARN , Superóxido Dismutasa-1 , Anciano , Esclerosis Amiotrófica Lateral/clasificación , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Femenino , Estudio de Asociación del Genoma Completo , Herpes Simple/genética , Herpes Simple/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Simplexvirus/genética , Simplexvirus/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , TranscriptomaRESUMEN
The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.
Asunto(s)
Retrovirus Endógenos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Superantígenos/genética , Superantígenos/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Células A549 , Animales , Células COS , Línea Celular , Sistema Libre de Células , Chlorocebus aethiops , Retrovirus Endógenos/química , Retrovirus Endógenos/genética , Chaperón BiP del Retículo Endoplásmico , Regulación de la Expresión Génica , Glicosilación , Células HEK293 , Humanos , Proteínas de la Membrana/química , Conformación Molecular , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/química , Superantígenos/química , Transcripción Genética , Proteínas del Envoltorio Viral/químicaRESUMEN
BACKGROUND: Potato disorders lead to a significant reduction in the yield and quality of marketable tubers. Thumbnail cracks are physiological tuber skin disorders that can significantly affect the tuber's appearance and, hence, the overall quality. The aim of this study was to characterize fracture properties of the tuber skin. Knowledge of the physiological reasons that influence the resistance of potato tubers to mechanical impacts and thus to cracking and fracturing is limited. Tuber dry matter (DM) and starch content were found to correlate with the rheological properties of tubers, which, in turn, could affect the resistance of the tubers to cracking and fracturing. Moreover, divalent cations, such as calcium (Ca) and magnesium (Mg), and their distribution in the tuber can affect the tuber's resistance to mechanical impacts via cell-wall stabilizing properties. RESULTS: Tubers with higher DM, starch, and Ca concentrations exhibited the highest resistance to mechanical impacts. CONCLUSIONS: The reason for the increased resistance of tubers with higher DM and starch concentrations to mechanical impacts is assumed to be related to a certain cell structure of these tubers, which is why considerable force is needed to damage the cell structures. The relation between higher Ca concentrations and an improved resistance of tubers to mechanical impacts is assumed to be connected with the role of Ca in linking cell-wall polymers and thus stabilizing the cell wall. © 2018 Society of Chemical Industry.
Asunto(s)
Minerales/análisis , Tubérculos de la Planta/química , Solanum tuberosum/química , Almidón/análisis , Calcio/análisisRESUMEN
BACKGROUND: The market for cocktail tomatoes is growing continuously, mainly because of their good taste. Titratable acids (TA), total soluble solids (TSS) and dry matter (DM) contents correlate positively with good taste. So far, the impact of different potassium (K) applications on yield and consumer-oriented quality traits has not been comprehensively described for cultivars of smaller fruits. To fill this gap, we tested the effect of different K fertilization regimes on three cultivars of small-sized or cocktail tomatoes. RESULTS: A positive impact on quality parameters (TSS, TA, DM, color and firmness) was detected by raising the K fertilizer application for the cocktail tomato cultivars Primavera and Yellow Submarine. The cultivar Resi showed no response to fertilization, except for TSS and TA. Yield increased significantly by higher K application only in Primavera, the most productive cultivar. The K concentration in tomato rose with increasing K application. CONCLUSION: Because TSS and TA increased in all cultivars, whereas only one cultivar showed an increase in yield, the results of the present study emphasize the importance of the application of cultivar-specific mineral fertilizers on yield- and consumer-oriented quality traits. © 2018 Society of Chemical Industry.
Asunto(s)
Potasio/metabolismo , Solanum lycopersicum/química , Color , Comportamiento del Consumidor , Fertilizantes/análisis , Frutas/química , Frutas/metabolismo , Humanos , Solanum lycopersicum/metabolismo , Minerales/análisis , GustoRESUMEN
The (1,3)-ß-glucan callose is a major component of cell wall thickenings in response to pathogen attack in plants. GTPases have been suggested to regulate pathogen-induced callose biosynthesis. To elucidate the regulation of callose biosynthesis in Arabidopsis thaliana, we screened microarray data and identified transcriptional upregulation of the GTPase RabA4c after biotic stress. We studied the function of RabA4c in its native and dominant negative (dn) isoform in RabA4c overexpression lines. RabA4c overexpression caused complete penetration resistance to the virulent powdery mildew Golovinomyces cichoracearum due to enhanced callose deposition at early time points of infection, which prevented fungal ingress into epidermal cells. By contrast, RabA4c(dn) overexpression did not increase callose deposition or penetration resistance. A cross of the resistant line with the pmr4 disruption mutant lacking the stress-induced callose synthase PMR4 revealed that enhanced callose deposition and penetration resistance were PMR4-dependent. In live-cell imaging, tagged RabA4c was shown to localize at the plasma membrane prior to infection, which was broken in the pmr4 disruption mutant background, with callose deposits at the site of attempted fungal penetration. Together with our interactions studies including yeast two-hybrid, pull-down, and in planta fluorescence resonance energy transfer assays, we concluded that RabA4c directly interacts with PMR4, which can be seen as an effector of this GTPase.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Glucanos/metabolismo , Glucosiltransferasas/metabolismo , Enfermedades de las Plantas/inmunología , Proteínas de Unión al GTP rab/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Ascomicetos/fisiología , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Glucosiltransferasas/genética , Fenotipo , Enfermedades de las Plantas/microbiología , Epidermis de la Planta/genética , Epidermis de la Planta/inmunología , Epidermis de la Planta/fisiología , Epidermis de la Planta/ultraestructura , Inmunidad de la Planta , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/fisiología , Hojas de la Planta/ultraestructura , Plantas Modificadas Genéticamente , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rab/genéticaRESUMEN
Despite decades of research on amyotrophic lateral sclerosis (ALS), there is only one approved drug, which minimally extends patient survival. Here, we investigated pathophysiological mechanisms underlying ALS using motor neurons (MNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying mutations in FUS or SOD1. Patient-derived MNs were less active and excitable compared to healthy controls, due to reduced Na(+) /K(+) ratios in both ALS groups accompanied by elevated potassium channel (FUS) and attenuated sodium channel expression levels (FUS, SOD1). ALS iPSC-derived MNs showed elevated endoplasmic reticulum stress (ER) levels and increased caspase activation. Treatment with the FDA approved drug 4-Aminopyridine (4AP) restored ion-channel imbalances, increased neuronal activity levels and decreased ER stress and caspase activation. This study provides novel pathophysiological data, including a mechanistic explanation for the observed hypoexcitability in patient-derived MNs and a new therapeutic strategy to provide neuroprotection in MNs affected by ALS. Stem Cells 2016;34:1563-1575.
Asunto(s)
4-Aminopiridina/farmacología , Esclerosis Amiotrófica Lateral/patología , Células Madre Pluripotentes Inducidas/patología , Neuronas Motoras/patología , Esclerosis Amiotrófica Lateral/genética , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Canales Iónicos/metabolismo , Masculino , Persona de Mediana Edad , Mutación/genética , Neuroprotección/efectos de los fármacos , Fenotipo , Proteína FUS de Unión a ARN/genética , Superóxido Dismutasa/genética , Sinapsis/efectos de los fármacos , Sinapsis/metabolismoRESUMEN
Autosomal-dominant mutations within the gene FUS (fused in sarcoma) are responsible for 5% of familial cases of amyotrophic lateral sclerosis (ALS). The FUS protein is physiologically mainly located in the nucleus, while cytoplasmic FUS aggregates are pathological hallmarks of FUS-ALS. Data from non-neuronal cell models and/or models using heterologous expression of FUS mutants suggest cytoplasmic FUS translocation as a pivotal initial event which leads to neurodegeneration depending on a second hit. Here we present the first human model of FUS-ALS using patient-derived neurons carrying endogenous FUS mutations leading to a benign (R521C) or a more severe clinical phenotype (frameshift mutation R495QfsX527). We thereby showed that the severity of the underlying FUS mutation determines the amount of cytoplasmic FUS accumulation and cellular vulnerability to exogenous stress. Cytoplasmic FUS inclusions formed spontaneously depending on both, severity of FUS mutation and neuronal aging. These aggregates showed typical characteristics of FUS-ALS including methylated FUS. Finally, neurodegeneration was not specific to layer V cortical neurons perfectly in line with the current model of disease spreading in ALS. Our study highlights the value and usefulness of patient-derived cell models in FUS-ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Células Madre Pluripotentes Inducidas/patología , Neuronas/patología , Proteína FUS de Unión a ARN/genética , Adulto , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Progresión de la Enfermedad , Femenino , Humanos , Cuerpos de Inclusión/patología , Cuerpos de Inclusión/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Masculino , Persona de Mediana Edad , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Mutación , Neuronas/fisiología , Fenotipo , Proteína FUS de Unión a ARN/metabolismo , Índice de Severidad de la Enfermedad , Médula Espinal/patología , Médula Espinal/fisiopatologíaRESUMEN
The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.
Asunto(s)
Pared Celular/metabolismo , Genes de Plantas , Familia de Multigenes , Populus/genética , Regiones Promotoras Genéticas , Madera/metabolismo , Xilanos/biosíntesis , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Vectores Genéticos/metabolismo , Glucuronidasa/metabolismo , Plantas Modificadas Genéticamente , Activación Transcripcional/genética , Madera/genéticaRESUMEN
A common response by plants to fungal attack is deposition of callose, a (1,3)-ß-glucan polymer, in the form of cell wall thickenings called papillae, at site of wall penetration. While it has been generally believed that the papillae provide a structural barrier to slow fungal penetration, this idea has been challenged in recent studies of Arabidopsis (Arabidopsis thaliana), where fungal resistance was found to be independent of callose deposition. To the contrary, we show that callose can strongly support penetration resistance when deposited in elevated amounts at early time points of infection. We generated transgenic Arabidopsis lines that express POWDERY MILDEW RESISTANT4 (PMR4), which encodes a stress-induced callose synthase, under the control of the constitutive 35S promoter. In these lines, we detected callose synthase activity that was four times higher than that in wild-type plants 6 h post inoculation with the virulent powdery mildew Golovinomyces cichoracearum. The callose synthase activity was correlated with enlarged callose deposits and the focal accumulation of green fluorescent protein-tagged PMR4 at sites of attempted fungal penetration. We observed similar results from infection studies with the nonadapted powdery mildew Blumeria graminis f. sp. hordei. Haustoria formation was prevented in resistant transgenic lines during both types of powdery mildew infection, and neither the salicylic acid-dependent nor jasmonate-dependent pathways were induced. We present a schematic model that highlights the differences in callose deposition between the resistant transgenic lines and the susceptible wild-type plants during compatible and incompatible interactions between Arabidopsis and powdery mildew.
Asunto(s)
Arabidopsis/inmunología , Arabidopsis/microbiología , Ascomicetos/fisiología , Resistencia a la Enfermedad/inmunología , Glucanos/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Adaptación Fisiológica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Oxilipinas/metabolismo , Fenotipo , Plantas Modificadas Genéticamente , Ácido Salicílico/metabolismo , Factores de Tiempo , Transcripción GenéticaAsunto(s)
Magnesio/metabolismo , Tubérculos de la Planta/química , Potasio/metabolismo , Solanum tuberosum/metabolismo , Biomasa , Dióxido de Carbono/metabolismo , Hojas de la Planta/metabolismo , Tubérculos de la Planta/crecimiento & desarrollo , Solanum tuberosum/crecimiento & desarrollo , Almidón/análisis , Azúcares/análisis , Simportadores/metabolismoRESUMEN
Trichoderma afroharzianum, a ubiquitous soil-borne fungus found on plant roots and decaying residues, displays competitive traits and mycoparasitic behavior against diverse microorganisms. Selected strains of this fungus are known in agriculture for their beneficial effects on plant growth and as bio-fungicides. However, recent findings have pinpointed Trichoderma afroharzianum as the causal agent behind maize ear rot disease in Europe since 2018, notably impacting maize cobs in Germany, France, and Italy. This study aims to evaluate the severity of Trichoderma ear rot disease on maize fresh matter content and specific quality parameters under semi-field conditions. Two distinct maize varieties were artificially inoculated with a pathogenic Trichoderma isolate at the flowering stage using needle pin or silk channel methods. Disease severity was assessed visually at the time of harvest based on the percentage of infected kernels according to EPPO Guidelines (PP 1/285). Fresh matter content and quality parameters such as alpha-amylase activity, C/N ratio, water, and sugar content were analyzed. Results showed that needle pin inoculation led to higher disease severity (60%) compared to silk channel inoculation (39%). Cob weight decreased significantly at the highest disease severity level by up to 50% compared to control plants. In both varieties, alpha-amylase activity increased significantly with higher Trichoderma disease severity, resulting in starch degradation and increased glucose release. The germination rate was severely affected by the infection, with only 22% of grains germinating, and the seedlings showed shortened and deformed growth. This is the first report on Trichoderma ear rot infection and its effect on fresh matter content and quality parameters in maize after artificial inoculation under field conditions. The results address an important knowledge gap and provide valuable insights into the infection pathway and impact on maize quality.
RESUMEN
High-temperature (HT) geothermal wells can provide green power 24 hours a day, 7 days a week. Under harsh environmental and operational conditions, the long-term durability requirements of such wells require special cementitious composites for well construction. This paper reports a comprehensive assessment of geothermal cement composites in cyclic pressure function laboratory tests and field exposures in an HT geothermal well (300-350 °C), as well as a numerical model to complement the experimental results. Performances of calcium-aluminate cement (CAC)-based composites and calcium-free cement were compared against the reference ordinary Portland cement (OPC)/silica blend. The stability and degradation of the tested materials were characterized by crystalline composition, thermo-gravimetric and elemental analyses, morphological studies, water-fillable porosity, and mechanical property measurements. All CAC-based formulations outperformed the reference blend both in the function and exposure tests. The reference OPC/silica lost its mechanical properties during the 9-month well exposure through extensive HT carbonation, while the properties of the CAC-based blends improved over that period. The Modified Cam-Clay (MCC) plasticity parameters of several HT cement formulations were extracted from triaxial and Brazilian tests and verified against the experimental results of function cyclic tests. These parameters can be used in well integrity models to predict the field-scale behavior of the cement sheath under geothermal well conditions.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder causing progressive loss of motor neurons. Mutations in Fused in sarcoma (FUS) leading to its cytoplasmic mislocalization cause a subset of ALS. Under stress, mutant FUS localizes to stress granules (SGs)-cytoplasmic condensates composed of RNA and various proteins. Aberrant dynamics of SGs is linked to the pathology of ALS. Here, using motor neurons (MNs) derived from human induced pluripotent stem cells, we show that, in mutant FUS, MN dynamics of SGs is disturbed. Additionally, heat-shock response (HSR) and integrated stress response (ISR) involved in the regulation of SGs are upregulated in mutant MNs. HSR activation correlates with the amount of cytoplasmic FUS mislocalization. While inhibition of SG formation, translation, or ISR does not influence survival of FUS ALS neurons, proteotoxicity that cannot be compensated with the activation of stress pathways is the main driver of neurodegeneration in early FUS ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Humanos , Esclerosis Amiotrófica Lateral/patología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Mutación , Citoplasma/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons, resulting in progressive weakness of all voluntary muscles and eventual respiratory failure. Non-motor symptoms, such as cognitive and behavioral changes, frequently occur over the course of the disease. Considering its poor prognosis with a median survival time of 2 to 4 years and limited causal treatment options, an early diagnosis of ALS plays an essential role. In the past, diagnosis has primarily been determined by clinical findings supported by electrophysiological and laboratory measurements. To increase diagnostic accuracy, reduce diagnostic delay, optimize stratification in clinical trials and provide quantitative monitoring of disease progression and treatment responsivity, research on disease-specific and feasible fluid biomarkers, such as neurofilaments, has been intensely pursued. Advances in imaging techniques have additionally yielded diagnostic benefits. Growing perception and greater availability of genetic testing facilitate early identification of pathogenic ALS-related gene mutations, predictive testing and access to novel therapeutic agents in clinical trials addressing disease-modified therapies before the advent of the first clinical symptoms. Lately, personalized survival prediction models have been proposed to offer a more detailed disclosure of the prognosis for the patient. In this review, the established procedures and future directions in the diagnostics of ALS are summarized to serve as a practical guideline and to improve the diagnostic pathway of this burdensome disease.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/patología , Diagnóstico Tardío , Neuronas Motoras/patología , BiomarcadoresRESUMEN
Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuEPO) towards a thermoresistant variant microbial transglutaminase (mTGase), TG16 with the aim of developing novel rHuEPO conjugates that may potentially enhance its biological efficacy. As a model bioconjugation, we studied the reactivity of rHuEPO towards TG16 with a low molar mass amine group containing substrate, monodansyl cadaverine (MDC). The reactions were carried out at a Tm of 54.3 °C, the transition temperature of rHuEPO. Characterization by SDS-PAGE and mass spectrometry confirmed the conjugates formation. Then, we examined the conjugation of rHuEPO with a biodegradable and biocompatible polyester, poly(D-sorbitol adipate) (PDSA). To achieve this, PDSA was enzymatically synthesized using lipase B from Candida antartica (CAL-B), chemically modified with side chains having free primary amine (NH2) groups that can be acyl acceptor substrate of TG16, thoroughly characterized by 1H NMR spectroscopy, and then applied for the TG16-mediated conjugation reaction with rHuEPO. rHuEPO conjugates generated by this approach were identified by SDS-PAGE proving that the amine-grafted PDSA is accepted as a substrate for TG16. The successful conjugation was further verified by the detection of high molar mass fluorescent bands after labelling of amine-grafted PDSA with rhodamine B-isothiocyanate. Overall, this enzymatic procedure is considered as an effective approach to prepare biodegradable rHuEPO-polymer conjugates even in the presence of N- and O-glycans.
Asunto(s)
Anemia , Eritropoyetina , Anemia/tratamiento farmacológico , Humanos , Poliésteres , Proteínas Recombinantes/uso terapéutico , TransglutaminasasRESUMEN
Potatoes are an important staple food with high yield potential and great nutritional value. Potassium (K) fertilisation can increase both tuber yield and quality, but its effects differ depending on the K fertilisation form. Potatoes are known to be chloride sensitive, since chloride ions can influence, for example, the starch content. Therefore, fertilisations shortly before planting using potassium sulphate (K2SO4) are often recommended instead of potassium chloride (KCl). However, the use of different fertilisation forms is contradictory, and the chloride sensitivity of potatoes remains unclear. To examine this issue in more detail, a 2-year field experiment using two cultivars, "Laura" and "Marabel," was conducted. K fertilisation with 240 kg K2O as K2SO4 and KCl was applied, and the control remained unfertilised. Quality traits, including internal and external parameters, were analysed after harvest and after 5 months of storage at 6°C. The results revealed minor effects on yield, but the starch content and ascorbic acid concentration were reduced due to the KCl supply. Furthermore, the reducing sugar concentration in tubers increased during storage more after KCl compared to K2SO4 fertilisation. Moreover, volatile compounds were affected by the K fertilisation form, with higher levels of lipid-derived off-flavour compounds after KCl application. However, the effects of cultivation year, cultivar, and storage interacted with the influence of the fertilisation form. In summary, KCl fertilisation can disadvantageously influence several quality traits, but the use of potato cultivars should also be considered when recommending fertilisers.