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The integrin CD49a marks highly cytotoxic epidermal-tissue-resident memory (TRM) cells, but their differentiation from circulating populations remains poorly defined. We demonstrate enrichment of RUNT family transcription-factor-binding motifs in human epidermal CD8+CD103+CD49a+ TRM cells, paralleled by high RUNX2 and RUNX3 protein expression. Sequencing of paired skin and blood samples revealed clonal overlap between epidermal CD8+CD103+CD49a+ TRM cells and circulating memory CD8+CD45RA-CD62L+ T cells. In vitro stimulation of circulating CD8+CD45RA-CD62L+ T cells with IL-15 and TGF-ß induced CD49a expression and cytotoxic transcriptional profiles in a RUNX2- and RUNX3-dependent manner. We therefore identified a reservoir of circulating cells with cytotoxic TRM potential. In melanoma patients, high RUNX2, but not RUNX3, transcription correlated with a cytotoxic CD8+CD103+CD49a+ TRM cell signature and improved patient survival. Together, our results indicate that combined RUNX2 and RUNX3 activity promotes the differentiation of cytotoxic CD8+CD103+CD49a+ TRM cells, providing immunosurveillance of infected and malignant cells.
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Linfocitos T CD8-positivos , Melanoma , Humanos , Linfocitos T CD8-positivos/metabolismo , Integrina alfa1/metabolismo , Integrinas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Memoria Inmunológica , Antígenos Comunes de Leucocito/metabolismo , Melanoma/metabolismoRESUMEN
Microsatellite mutations involving the expansion of tri-, tetra-, penta-, or hexanucleotide repeats cause more than 40 different neurological disorders. Although, traditionally, the position of the repeat within or outside of an open reading frame has been used to focus research on disease mechanisms involving protein loss of function, protein gain of function, or RNA gain of function, the discoveries of bidirectional transcription and repeat-associated non-ATG (RAN) have blurred these distinctions. Here we review what is known about RAN proteins in disease, the mechanisms by which they are produced, and the novel therapeutic opportunities they provide.
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Expansión de las Repeticiones de ADN/genética , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/genética , Biosíntesis de Proteínas , Codón Iniciador/genética , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/fisiología , Mutación con Ganancia de Función , Código Genético , Humanos , Mutación con Pérdida de Función , Repeticiones de Microsatélite/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Transcripción GenéticaRESUMEN
PURPOSE: The provision and funding of long-term care (LTC) for older people varies between European countries. Despite differences, there is limited information about the comparative performance of LTC systems in Europe. In this study, we compared quality of life (QoL) of informal carers of home care service users in Austria, England and Finland. METHODS: Informal carers were surveyed in Austria, England and Finland. The study data (n = 835) contained information on social care-related quality of life (SCRQoL) associated with the ASCOT-Carer measure, and characteristics of carers and care recipients from each country. We applied risk-adjustment methods using a fractional regression model to produce risk-adjusted SCRQoL scores for the comparative analysis. In a sensitivity analysis, we applied multiple imputation to missing data to validate our findings. RESULTS: We found that the mean values of the risk-adjusted SCRQoL of informal carers in England were 1.4-2.9% and 0.3-0.5% higher than in Finland and Austria, and the mean values of the risk-adjusted SCRQoL of carers in Austria were 0.8-2.7% higher than in Finland. Differences in the mean values of the country-specific risk-adjusted SCRQoL scores were small and statistically non-significant. English informal carers were less healthy and co-resided with care resipients more often than carers in Austria or Finland. CONCLUSION: Small differences between the risk-adjusted SCRQoL scores between Austria, England and Finland are consistent with the observation that the countries provide different types of support for informal carers. Our results help local and national decision-makers in these countries to benchmark their informal care support systems.
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BACKGROUND: WAS gene mutational analysis is crucial to establish a definite diagnosis of Wiskott-Aldrich syndrome (WAS). Data on the genetic background of WAS in Vietnamese patients have not been reported. METHODS: We recruited 97 male, unrelated patients with WAS and analyzed WAS gene mutation using Sanger sequencing technology. RESULTS: We identified 36 distinct hemizygous pathogenic mutations, with 17 novel variants, from 38 patients in the entire cohort (39.2%). The mutational spectrum included 14 missense, 12 indel, five nonsense, four splicing, and one non-stop mutations. Most mutations appear only once, with the exception of c.37C>T (p.R13X) and c.374G>A (p.G125E) each of which occurs twice in unrelated patients. CONCLUSION: Our data enrich the mutational spectrum of the WAS gene and are crucial for understanding the genetic background of WAS and for supporting genetic counseling.
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Síndrome de Wiskott-Aldrich , Humanos , Masculino , Análisis Mutacional de ADN , Mutación , Vietnam , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
The Polar Qualification System (PQS) was applied on hue spectra fingerprinting to describe color changes in tomato during storage. The cultivar 'Pitenza' was harvested at six different maturity stages, and half of the samples were subjected to gaseous 1-methylcyclopropene (1-MCP) treatment. Reference color parameters were recorded with a vision system colorimeter instrument, and the fruit pigment concentration was assessed with the DA-index®. Additionally, acoustic firmness (Stiffness) was measured. All acquired reference parameters were used to grade fruit in the supply chain. The applied 1-MCP treatments were used to control the ripening of climacteric horticultural produce. Both the DA-index® and stiffness values, presented as chlorophyll concentration and acoustic firmness, showed significant differences among maturity stages and treated and control samples and in their kinetics during storage. The machine vision parameter PQS-X was significantly affected by 1-MCP treatment (F = 10.18, p < 0.01), while PQS-Y was primarily affected by storage time (F = 18.18, p < 0.01) and maturity stage (F = 11.15, p < 0.01). A significant correlation was achieved for acoustic firmness with normalized color (r > 0.78) and PQS-Y (r > 0.80), as well as for the DA-index® (r > 0.9). The observed color changes agreed with the reference measurements. The significant statistical effect on the PQS coordinates suggests that hue spectra fingerprinting with this data compression technique is suitable for quality assessment based on color.
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Color , Ciclopropanos , Solanum lycopersicum , Solanum lycopersicum/efectos de los fármacos , Solanum lycopersicum/química , Solanum lycopersicum/crecimiento & desarrollo , Ciclopropanos/farmacología , Frutas/química , Frutas/efectos de los fármacos , Pigmentación/efectos de los fármacos , Pigmentación/fisiología , Clorofila/análisis , Clorofila/metabolismo , Pigmentos Biológicos/análisisRESUMEN
Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)-/- and PKR-like endoplasmic reticulum kinase (PERK)-/- cells, with more substantial reductions of CAGG-encoded QAGR in PKR-/- cells. Experiments using mutant eIF2α-S51A HEK293T cells show that p-eIF2α is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNAs. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNAs that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2α is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.
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Factor 2 Eucariótico de Iniciación/genética , Distrofia Miotónica/genética , eIF-2 Quinasa/genética , Sistemas CRISPR-Cas/genética , Expansión de las Repeticiones de ADN/genética , Células HEK293 , Humanos , Repeticiones de Microsatélite/genética , Distrofia Miotónica/fisiopatología , Biosíntesis de Proteínas/genéticaRESUMEN
C9orf72 ALS/FTD patients show remarkable clinical heterogeneity, but the complex biology of the repeat expansion mutation has limited our understanding of the disease. BAC transgenic mice were used to better understand the molecular mechanisms and repeat length effects of C9orf72 ALS/FTD. Genetic analyses of these mice demonstrate that the BAC transgene and not integration site effects cause ALS/FTD phenotypes. Transcriptomic changes in cell proliferation, inflammation and neuronal pathways are found late in disease and alternative splicing changes provide early molecular markers that worsen with disease progression. Isogenic sublines of mice with 800, 500 or 50 G4C2 repeats generated from the single-copy C9-500 line show longer repeats result in earlier onset, increased disease penetrance and increased levels of RNA foci and dipeptide RAN protein aggregates. These data demonstrate G4C2 repeat length is an important driver of disease and identify alternative splicing changes as early biomarkers of C9orf72 ALS/FTD.
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Empalme Alternativo , Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN , Modelos Animales de Enfermedad , Demencia Frontotemporal/patología , Penetrancia , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteína C9orf72/genética , Demencia Frontotemporal/etiología , Demencia Frontotemporal/metabolismo , Humanos , Ratones , Ratones Transgénicos , Mutación , FenotipoRESUMEN
Septic tanks in low- and middle-income countries are often not emptied for a long time, potentially resulting in poor pollutant removal efficiency and increased greenhouse gas emissions, including methane (CH4). We examined the impact of long emptying intervals (4.0-23 years) on the biochemical oxygen demand (BOD) removal efficiency of 15 blackwater septic tanks and the CH4 emission rates of 23 blackwater septic tanks in Hanoi. The average BOD removal efficiency was 37% (-2-65%), and the average CH4 emission rate was 10.9 (2.2-26.8) g/(cap·d). The emptying intervals were strongly negatively correlated with BOD removal efficiency (R = -0.676, p = 0.006) and positively correlated with CH4 emission rates (R = 0.614, p = 0.001). CH4 emission rates were positively correlated with sludge depth (R = 0.596, p = 0.002), but against expectation, negatively correlated with BOD removal efficiency (R = -0.219, p = 0.451). These results suggest that shortening the emptying interval improves the BOD removal efficiency and reduces the CH4 emission rate. Moreover, the CH4 emission estimation of the Intergovernmental Panel on Climate Change, which is a positive conversion of BOD removal, might be inaccurate for septic tanks with long emptying intervals. Our findings suggest that emptying intervals, sludge depth, and per-capita emission factors reflecting long emptying intervals are potential parameters for accurately estimating CH4 emissions from septic tanks.
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Gases de Efecto Invernadero , Metano , Metano/análisis , Aguas del Alcantarillado , Cambio ClimáticoRESUMEN
Wolfram syndrome is a rare multisystem disease characterized by childhood-onset diabetes mellitus and progressive neurodegeneration. Most cases are attributed to pathogenic variants in a single gene, Wolfram syndrome 1 (WFS1). There currently is no disease-modifying treatment for Wolfram syndrome, as the molecular consequences of the loss of WFS1 remain elusive. Because diabetes mellitus is the first diagnosed symptom of Wolfram syndrome, we aimed to further examine the functions of WFS1 in pancreatic ß cells in the context of hyperglycemia. Knockout (KO) of WFS1 in rat insulinoma (INS1) cells impaired calcium homeostasis and protein kinase B/Akt signaling and, subsequently, decreased cell viability and glucose-stimulated insulin secretion. Targeting calcium homeostasis with reexpression of WFS1, overexpression of WFS1's interacting partner neuronal calcium sensor-1 (NCS1), or treatment with calpain inhibitor and ibudilast reversed deficits observed in WFS1-KO cells. Collectively, our findings provide insight into the disease mechanism of Wolfram syndrome and highlight new targets and drug candidates to facilitate the development of a treatment for this disorder and similar diseases.
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Antineoplásicos/farmacología , Proteínas de Unión a Calmodulina/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Piridinas/farmacología , Síndrome de Wolfram/tratamiento farmacológico , Animales , Calcio/metabolismo , Proteínas de Unión a Calmodulina/genética , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Glicoproteínas , Homeostasis , Hiperglucemia , Secreción de Insulina , Proteínas de la Membrana/genética , Receptor de Insulina , Transcriptoma , Síndrome de Wolfram/genéticaRESUMEN
Repeat associated non-AUG (RAN) translation is found in a growing number of microsatellite expansion diseases, but the mechanisms remain unclear. We show that RAN translation is highly regulated by the double-stranded RNA-dependent protein kinase (PKR). In cells, structured CAG, CCUG, CAGG, and G4C2 expansion RNAs activate PKR, which leads to increased levels of multiple RAN proteins. Blocking PKR using PKR-K296R, the TAR RNA binding protein or PKR-KO cells, reduces RAN protein levels. p-PKR is elevated in C9orf72 ALS/FTD human and mouse brains, and inhibiting PKR in C9orf72 BAC transgenic mice using AAV-PKR-K296R or the Food and Drug Administration (FDA)-approved drug metformin, decreases RAN proteins, and improves behavior and pathology. In summary, targeting PKR, including by use of metformin, is a promising therapeutic approach for C9orf72 ALS/FTD and other expansion diseases.
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Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72 , Metformina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , eIF-2 Quinasa , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Modelos Animales de Enfermedad , Demencia Frontotemporal/metabolismo , Humanos , Ratones , Ratones Transgénicos , Repeticiones de Microsatélite/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Determining and applying 'good' postharvest and quality control practices for otherwise highly sensitive fruits, such as sour cherry, is critical, as they serve as excellent media for a wide variety of microbial contaminants. The objective of this research was to report two series of experiments on the modified atmosphere storage (MAP) of sour cherries (Prunus cerasus L. var. Kántorjánosi, Újfehértói fürtös). Firstly, the significant effect of different washing pre-treatments on various quality indices was examined (i.e., headspace gas composition, weight loss, decay rate, color, firmness, soluble solid content, total plate count) in MAP-packed fruits. Subsequently, the applicability of near infrared (NIR) spectroscopy combined with chemometrics was investigated to detect the effect of various storage conditions (packed as control or MAP, stored at 3 or 5 °C) on sour cherries of different perceived ripeness. Significant differences were found for oxygen concentration when two perforations were applied on the packages of 'Kántorjánosi' (p < 0.01); weight loss when 'Kánorjánosi' (p < 0.001) and 'Újfehértói fürtös' (p < 0.01) were packed in MAP; SSC when 'Újfehértói fürtös' samples were ozone-treated (p < 0.05); and total plate count when 'Kántorjánosi' samples were ozone-treated (p < 0.01). The difference spectra reflected the high variability in the samples, and the detectable effects of different packaging. Based on the investigations with the soft independent modelling of class analogies (SIMCA), different packaging and storage resulted in significant differences in most of the cases even on the first storage day, which in many cases increased by the end of storage. The soft independent modelling of class analogies proved to be suitable for classification with apparent error rates between 0 and 0.5 during prediction regardless of ripeness. The research findings suggest the further correlation of NIR spectroscopic and reference parameters to support postharvest handling and fast quality control.
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Ozono , Prunus avium , Prunus avium/química , Espectroscopía Infrarroja Corta , Frutas/química , Ozono/análisis , AtmósferaRESUMEN
Calcium (Ca2+ ) signaling is critical for neuronal functioning and requires the concerted interplay of numerous Ca2+ -binding proteins, including neuronal calcium sensor 1 (NCS1). Although an important role of NCS1 in neuronal processes and in neurodevelopmental and neurodegenerative diseases has been established, the underlying mechanisms remain enigmatic. Here, we systematically investigated the functions of NCS1 in the brain. Using Golgi-Cox staining, we observed a reduction in dendritic complexity and spine density in the prefrontal cortex and the dorsal hippocampus of Ncs1-/- mice, which may underlie concomitantly observed deficits in memory acquisition. Subsequent RNA sequencing of Ncs1-/- and Ncs1+/+ mouse brain tissues revealed that NCS1 modulates gene expression related to neuronal morphology and development. Investigation of developmental databases further supported a molecular role of NCS1 during brain development by identifying temporal gene expression patterns. Collectively, this study provides insights into NCS1-dependent signaling and lays the foundation for a better understanding of NCS1-associated diseases.
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Calcio/metabolismo , Hipocampo/patología , Enfermedades del Sistema Nervioso/patología , Proteínas Sensoras del Calcio Neuronal/fisiología , Neuronas/patología , Neuropéptidos/fisiología , Animales , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Neuronas/metabolismo , RNA-Seq , Transducción de Señal , TranscriptomaRESUMEN
In this study, sonication with mild heat treatment was used to reduce the E. coli count in inoculated liquid whole egg, egg yolk and albumen. Ultrasonic equipment (20/40 kHz, 180/300 W) has been used for 30/60 min with a 55 °C water bath. The combination of sonication and low-heat treatment was able to reduce the concentration of E. coli from 5-log CFU × mL-1 below 10 CFU × mL-1 at 300 W, 40 kHz and 60 min of sonication in liquid egg products. The 60 min treatment was able to reduce the E. coli concentration below 10 CFU × mL-1 in the case of egg yolk regardless of the applied frequency, absorbed power or applied energy dose. The 30 min treatment of sonication and heating was able to reduce significantly the number of E. coli in the egg products, as well. Our results showed that sonication with mild heat treatment can be a useful technique to decrease the number of microorganisms in liquid egg products to a very low level. Near-infrared spectroscopy was used to investigate structural changes in the samples, induced by the combined treatment. Principal component analysis showed that this method can alter the C-H, C-N, -OH and -NH bonds in these egg products.
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Escherichia coli , Espectroscopía Infrarroja Corta , Calor , Recuento de Colonia Microbiana , Huevos/análisisRESUMEN
The activities of social insect colonies are supported by exocrine glands and the tremendous functional diversity of the compounds that they secrete. Many social wasps in the subfamilies Vespinae and Polistinae have two sternal glands-the van der Vecht and Richards' glands-that vary in their features and function across the species in which they are found. Field observations suggest that giant hornets use secretions from the van der Vecht gland to chemically mark targeted nests when workers initiate group attacks on social insect prey. However, descriptions of giant hornets' sternal glands and details about their recruitment behavior are lacking. We describe the morphology of the sternal glands of the giant hornet Vespa soror du Buysson and consider their potential to contribute to a marking pheromone. We also assess the gastral rubbing behavior of workers as they attacked Apis cerana F. (Hymenoptera: Apidae) colonies. V. soror workers have well-developed van der Vecht and Richards' glands on their terminal gastral sternites, with morphologies that robustly support the synthesis, storage, and dissemination of their secretory products. Observations confirm that the van der Vecht gland is exposed during gastral rubbing, but that the Richards' gland and glands associated with the sting apparatus may also contribute to a marking pheromone. Workers briefly but repeatedly rubbed their gasters around hive entrances and on overhead vegetation. Colonies were heavily marked over consecutive attacks. Our findings provide insight into the use of exocrine secretions by giant hornets as they recruit nestmates to prey colonies for group attacks.
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PURPOSE: To examine the associations of excessive internet use with depression, anxiety, and sleep quality among high school students in northern Vietnam, a country experiencing rapid economic growth. DESIGN AND METHODS: A cross-sectional study was conducted in a high school in northern Vietnam from July to September 2019. Symptoms of depression and anxiety were respectively assessed using the Center for Epidemiological Studies Depression Scale and the Vietnamese Anxiety Scale. Sleep quality was measured using the Pittsburgh Sleep Quality Index. Internet use and demographic characteristics were obtained using structured questionnaires. A multiple logistic regression was performed. RESULTS: In total, 678 participants with an average age of 16.1 (standard deviation 0.9) years were included. Nearly one-third of the adolescents (30.7%) exhibited excessive internet use (> 4 h/day), 19.6% experienced depressive symptoms, 14.5% presented anxiety symptoms, and 58.8% reported poor sleep quality. Compared to non-excessive internet users, excessive internet users (> 4 h/day) experienced significantly higher levels of depressive symptoms (p = .001), anxiety (p = .008), and poorer sleep quality (p < .001). Students who were female and with fair/poor self-rated health experienced higher depression, anxiety, and poor sleep quality (p < .05). After adjusting for demographic and health-related factors, students with excessive internet use were 58% more likely to experience poor sleep quality (odds ratio, 1.58, 95% confidence interval [1.06, 2.35]). CONCLUSIONS: Excessive internet use in Vietnamese high school students was significantly associated with poor sleep quality, but not with depression or anxiety.
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Depresión , Calidad del Sueño , Adolescente , Ansiedad/diagnóstico , Ansiedad/epidemiología , Estudios Transversales , Depresión/diagnóstico , Depresión/epidemiología , Femenino , Humanos , Internet , Uso de Internet , Instituciones Académicas , Sueño , Estudiantes , Encuestas y Cuestionarios , Vietnam/epidemiologíaRESUMEN
Septic systems are potentially a significant source of greenhouse gases (GHGs). The present study investigated GHGs from the blackwater septic systems that are widely used especially in low- and middle-income countries. Ten blackwater septic tanks in Hanoi, Vietnam, were investigated using the floating chamber method. The average methane and carbon dioxide emission rates measured at the first compartment (65% of total capacity) of the septic tanks were 11.92 and 20.24 g/cap/day, respectively, whereas nitrous oxide emission was negligible. Methane emission rate was significantly correlated with septage oxidation-reduction potential (ORP) (R = -0.67, p = 0.034), chemical oxygen demand mass (R = 0.78, p = 0.007), and biochemical oxygen demand mass (R = 0.78, p = 0.008), whereas it was not significantly correlated with water temperature (R = 0.26, p = 0.47) and dissolved oxygen (R = -0.59, p = 0.075) within the limited range: 30.6-31.7 °C and 0.03-0.34 mg-O2/L. The methane emission rates from septic tanks accumulating septage for >5 years were significantly higher than those at 0-5 years (p = 0.016). These results suggest that lower ORP and higher biodegradable carbon mass, in association with longer septage storage periods are key conditions for methane emissions. To the best of our knowledge, this is the first study to characterize GHG emissions from septic systems.
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Gases de Efecto Invernadero , Dióxido de Carbono/análisis , Efecto Invernadero , Metano/análisis , Óxido Nitroso/análisis , VietnamRESUMEN
Ultrasonic testing is an emerging non-destructive testing technology with high repeatability and precision. Milk is a very complex liquid and the change of its viscosity is a highly relevant property throughout conversion into other dairy products. In the following paper, we propose a novel method for the monitoring of viscosity during enzymatic milk clotting by ultrasonic spectroscopy. An ultrasonic transducer-receiver couple with a 250 kHz nominal frequency was submerged in the samples and an enveloped sweep ("chirp") signal was applied in a through-transmission mode. Simultaneously, the change in viscosity was measured with a rotational viscometer at a constant shearing speed. The data were analyzed with an algorithm developed by the authors for spectral ultrasonic testing. Estimations yielded a high adjusted R2 (0.963-0.998) and low cross-validated estimation error (RPD: 4.38-14.22), suggesting that the method is suitable for industrial use given the right instrumentation.
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Algoritmos , Leche/química , Reología , Espectrofotometría/métodos , Ultrasonido/métodos , Animales , Bovinos , Análisis de los Alimentos , ViscosidadRESUMEN
Inositol 1,4,5-trisphosphate receptors (InsP3Rs) are endoplasmic reticulum-localized channels that mediate Ca2+ release from the endoplasmic reticulum into the cytoplasm. We previously reported that an EF-hand Ca2+-binding protein, neuronal calcium sensor 1 (NCS1), binds to the InsP3R and thereby increases channel open probability, an event associated with chemotherapy-induced peripheral neuropathy. However, the exact NCS1-binding site on InsP3R remains unknown. Using protein docking, co-immunoprecipitation, and blocking peptides, we mapped the NCS1-binding site to residues 66-110 on the suppressor domain of InsP3R type 1 (InsP3R1). We also identified Leu-89, a residue in the hydrophobic pocket of NCS1, as being critical for facilitating the NCS1-InsP3R1 interaction. Overexpression of WT NCS1 in MDA-MB231 breast cancer cells increased Ca2+ signaling and survival, whereas overexpression of Leu-89 NCS1 variants decreased Ca2+ signaling and survival, further suggesting the importance of this residue in the NCS1-InsP3R1 interaction. In conclusion, we show that NCS1-InsP3R1 interaction enhances intracellular Ca2+ signaling in cells and can be modulated by altering or occluding the hydrophobic pocket of NCS1. This improved understanding of the NCS1-InsP3R1 interaction may facilitate the development of management strategies for diseases resulting from aberrant NCS1 expression.
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Neoplasias de la Mama/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Sitios de Unión , Femenino , Humanos , Unión ProteicaRESUMEN
Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Femenino , Humanos , Ratones , Ratones Desnudos , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Exercise is an effective therapeutic intervention for cancer survivors. Concerns about the completeness of reporting of exercise interventions have been raised in the literature, but without any formal analysis. This study aimed to evaluate the completeness of reporting of exercise interventions for cancer survivors in a large sample of randomized clinical trials (RCTs). METHODS: We developed a pre-defined protocol. We searched MEDLINE, EMBASE, and CENTRAL for exercise trials in oncology between 2010 and 2017. Pairs of independent researchers screened the records, extracted study characteristics, and assessed 16 items on the TIDieR checklist (i.e., the 12 items, with item 5 divided into two and item 8 divided into four). For each of these items, the percentage of interventions in the included studies that reported the item was calculated. RESULTS: We included 131 RCTs reporting 138 interventions in the analysis. Breast cancer was the most common type of cancer (69, 50%), and aerobic exercise was the most studied exercise modality (43, 30%) followed by combined aerobic and resistance training (40, 28%). Completeness of reporting ranged from 42 to 96% among the TIDieR items; none of the items was fully reported. 'Intervention length' was the most reported item across interventions (133, 96%), followed by 'rationale' (131, 95%), whereas 'provider' (58, 42%) and 'how well (planned)' (63, 46%) were the two least reported items. Half of the TIDieR items were completely reported in 50 to 70% of the interventions, and only four items were reported in more than 80% of the interventions (Items 2 and 8a to c). The seven items deemed to be core for replication (Items 3 to 9) exhibited a mean reporting of 71%, ranging from 42 to 96%. CONCLUSION: Exercise training interventions for cancer survivors are incompletely reported across RCTs published between 2010 and 2017. The reporting of information about the provider, materials, and modifications require urgent improvements. Stronger reporting will enhance usability of trial reports by both healthcare providers and survivors, and will help to reduce research waste.