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Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca2+ levels, and of the heart rate.
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Técnicas Biossensoriais , Cálcio , Larva , Contração Miocárdica , Peixe-Zebra , Animais , Cálcio/metabolismo , Contração Miocárdica/fisiologia , Coração/fisiologia , Troponina T/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Troponina C/metabolismoRESUMO
AIM: Bradyarrhythmias result from inhibition of automaticity, prolonged repolarization, or slow conduction in the heart. The ERG channels mediate the repolarizing current IKr in the cardiac action potential, whereas T-type calcium channels (TTCC) are involved in the sinoatrial pacemaker and atrioventricular conduction in mammals. Zebrafish have become a valuable research model for human cardiac electrophysiology and disease. Here, we investigate the contribution of ERG channels and TTCCs to the pacemaker and atrioventricular conduction in zebrafish larvae and determine the mechanisms causing atrioventricular block. METHODS: Zebrafish larvae expressing ratiometric fluorescent Ca2+ biosensors in the heart were used to measure Ca2+ levels and rhythm in beating hearts in vivo, concurrently with contraction and hemodynamics. The atrioventricular delay (the time between the start of atrial and ventricular Ca2+ transients) was used to measure impulse conduction velocity and distinguished between slow conduction and prolonged refractoriness as the cause of the conduction block. RESULTS: ERG blockers caused bradycardia and atrioventricular block by prolonging the refractory period in the atrioventricular canal and in working ventricular myocytes. In contrast, inhibition of TTCCs caused bradycardia and second-degree block (Mobitz type I) by slowing atrioventricular conduction. TTCC block did not affect ventricular contractility, despite being highly expressed in cardiomyocytes. Concomitant measurement of Ca2+ levels and ventricular size showed mechano-mechanical coupling: increased preload resulted in a stronger heart contraction in vivo. CONCLUSION: ERG channels and TTCCs influence the heart rate and atrioventricular conduction in zebrafish larvae. The zebrafish lines expressing Ca2+ biosensors in the heart allow us to investigate physiological feedback mechanisms and complex arrhythmias.
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Bloqueio Atrioventricular , Canais de Cálcio Tipo T , Marca-Passo Artificial , Humanos , Animais , Peixe-Zebra , Frequência Cardíaca/fisiologia , Bradicardia , Canais de Cálcio Tipo T/fisiologia , Canais de Potássio Éter-A-Go-Go , Miócitos Cardíacos , Mamíferos , Regulador Transcricional ERGRESUMO
Glycans cover the surfaces of all mammalian cells through a process called glycosylation. Nearly all proteins and receptors that integrate the intricate series of co-stimulatory/inhibitory pathways of the immune system are glycosylated. Growing evidence indicates that the development of the immune system at the origins of T and B cell development is tightly regulated by glycosylation. In this opinion, we hypothesize that the glycome composition of developing T and B cells is developmentally regulated. We discuss how glycans play fundamental roles in lymphocyte development and how glycans early define T and B cell functionality in multiple aspects of adaptive immunity. These advances can provide opportunities for the discovery of novel disease factors and more effective candidate treatments for various conditions.
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Imunidade Adaptativa , Ativação Linfocitária , Animais , Humanos , Glicosilação , Polissacarídeos , MamíferosRESUMO
T-cell development ensures the formation of diverse repertoires of T-cell receptors (TCRs) that recognize a variety of antigens. Glycosylation is a major posttranslational modification present in virtually all cells, including T-lymphocytes, that regulates activity/functions. Although these structures are known to be involved in TCR-selection in DP thymocytes, it is unclear how glycans regulate other thymic development processes and how they influence susceptibility to disease. Here, we discovered stage-specific glycome compositions during T-cell development in human and murine thymocytes, as well as dynamic alterations. After restricting the N-glycosylation profile of thymocytes to high-mannose structures, using specific glycoengineered mice (Rag1CreMgat1fl/fl), we showed remarkable defects in key developmental checkpoints, including ß-selection, regulatory T-cell generation and γδT-cell development, associated with increased susceptibility to colon and kidney inflammation and infection. We further demonstrated that a single N-glycan antenna (modeled in Rag1CreMgat2fl/fl mice) is the sine-qua-non condition to ensure normal development. In conclusion, we revealed that mannosylated thymocytes lead to a dysregulation in T-cell development that is associated with inflammation susceptibility.
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Timócitos , Timo , Camundongos , Animais , Humanos , Glicosilação , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas de Homeodomínio/genética , PolissacarídeosRESUMO
To explore the processes of soil erosion at the plot scale, Digital surface model of Differences (DoD) maps (Unmanned Aerial Vehicle - Structure from Motion (UAV-SfM) method) and data from Radio Frequency Identification (RFID) tags were analysed. The comparison of differences in accuracy of UAV-SfM and 3D terrestrial laser scanner (TLS) measurements, and the integration of the UAV-SfM method and soil particle tracing with RFID tag locations were conducted to assess sediment transport in a plot in Fukushima prefecture, Japan. The Universal Soil Loss Equation (USLE) plot was installed and kept with no vegetation and no cultivation. Water and sediment discharges were measured at the outlet of the plot, and the topographic index of runoff and sediment connectivity (IC) -focused on surface roughness- was also estimated. Based on field surveys, four periods were defined. Locations of RFID tags were firstly determined by using orthoimages derived from the UAV-SfM method and then compared with those locations measured with a laser total station. The mean and standard deviation of difference amounts of UAV-SfM were of 1 and 3.3 mm, respectively. On average, the RFID tags were located with an accuracy of 3.1 cm (RMSE). Although data of tags tracing showed short transport distances with rill erosion, the results of the UAV-SfM surveys showed an increase of sediment connectivity (SC) over the study period that may explain the largest sediment discharge, especially of fine soil particles. The concurrence of higher values of SC as well as the development of new and longer rills demonstrated the important activity of net soil loss in our study site. The combination of distinct methods and techniques, all providing accurate measurements, shed light on the sediment transport process at short distances, which affects the net water and sediment discharge at larger scales.
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A new low-cost material with a polymeric base formed from sodium silicate was developed. The material presents a nanostructured, highly rich iron surface with a large phosphorus retention capacity and potential reuse as a crop fertilizer. In the present study, we demonstrate that iron is the element that acts as an adsorbent for phosphate, while the polymeric base functions exclusively as a support for iron. The iron is uniformly adsorbed on the surface of the material, forming nanostructures, which ensure that iron works similarly to nanoparticles in solution but avoid other problems, such as particle agglomeration or the difficulty of separating them after the removal process. Materials were characterised by SEM, EDS, N2 sorption, and image processing, and the effect of pH, ionic strength, and temperature was studied. Sorption kinetics were analysed using Boyd's diffusion model, and adsorption equilibria were studied using several adsorption models. A maximum iron adsorption on the polymeric base of 23.9 ± 0.3 mg Feâg-1 was found, while maximum phosphorus adsorption was 366 ± 21 mg Pâg-1 Fe. Thus, phosphorus is recovered from the aqueous medium with an inexpensive material that has the potential to be used directly as a fertilizer.
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PURPOSE: Common clinical application of auditory brainstem response (ABR) testing is limited to 0.25-4 kHz. Prior research has demonstrated associations between ABR and behavioral thresholds for tone burst stimuli > 4 kHz in adults, but there are no comparable data for children. The ability to predict behavioral thresholds > 4 kHz clinically based on the ABR would provide valuable audiologic information for individuals who are unable to provide behavioral thresholds. This study included children with hearing loss and children with normal hearing to determine the association between ABR and behavioral thresholds at 6 and 8 kHz. METHOD: ABR and behavioral thresholds were obtained for children ages 4.7-16.7 years (M = 10.5, SD = 3.4) with sensorineural hearing loss (n = 24) or normal hearing sensitivity (n = 16) and for adults ages 18.4-54.4 years (M = 32.7, SD = 10.4) with sensorineural hearing loss (n = 13) or normal hearing sensitivity (n = 11). Thresholds obtained for 6 and 8 kHz using ABR and conventional audiometry were compared. RESULTS: Differences between ABR and behavioral thresholds averaged 5-6 dB for both children and adults for both test frequencies, with differences of ≤ 20 dB in all instances. Linear mixed modeling for data from participants with hearing loss suggested that ABR threshold is a good predictor of behavioral threshold at 6 and 8 kHz for both children and adults. Test specificity was 100%; no participants with behavioral thresholds ≤ 20 dB HL had ABR thresholds > 25 dB nHL. CONCLUSIONS: Initial evidence suggests that ABR testing at 6 and 8 kHz is reliable for estimating behavioral threshold in listeners with hearing loss and accurately identifies normal hearing sensitivity. The results of this study contribute to efforts to improve outcomes for vulnerable populations by reducing barriers to clinical implementation of ABR testing at > 4 kHz.
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Surdez , Perda Auditiva Neurossensorial , Perda Auditiva , Adulto , Criança , Humanos , Audiometria/métodos , Audiometria de Tons Puros/métodos , Limiar Auditivo/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Perda Auditiva Neurossensorial/diagnóstico , Pré-Escolar , Adolescente , Adulto Jovem , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Individuals with Down syndrome are known to have high rates of hearing loss, but it is unclear how this impacts their ability to communicate and function in real-world environments. METHODS: Sixteen English-speaking and Spanish-speaking mothers of individuals with Down syndrome ages 6-40 years participated in individual, semi-structured interviews using a videoconferencing platform. Session transcripts were analysed using applied thematic analysis. RESULTS: Mothers described listening environments, the impact of hearing on daily life, barriers to successful listening, and strategies to overcome communication barriers for their children with Down syndrome. CONCLUSIONS: Hearing was largely discussed in terms of challenges and detriments, suggesting that hearing experiences are predominately considered to negatively impact the functional abilities of individuals with Down syndrome. Background noise and hearing loss were sources of communication difficulties. Parent-reported barriers and strategies can inform ecologically valid research priorities aimed at improving outcomes for individuals with Down syndrome.
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Síndrome de Down , Perda Auditiva , Deficiência Intelectual , Criança , Feminino , Humanos , Adolescente , Adulto Jovem , Adulto , Mães , AudiçãoRESUMO
Essentially all cells are covered with a dense coat of different glycan structures/sugar chains, giving rise to the so-called glycocalyx. Changes in cellular glycosylation are a hallmark of cancer, affecting most of the pathophysiological processes associated with malignant transformation, including tumour immune responses. Glycans are chief macromolecules that define T-cell development, differentiation, fate, activation and signalling. Thus, the diversity of glycans expressed at the surface of T cells constitutes a fundamental molecular interface with the microenvironment by regulating the bilateral interactions between T-cells and cancer cells, fine-tuning the anti-tumour immune response. In this review, we will introduce the power of glycans as orchestrators of T-cell-mediated immune response in physiological conditions and in cancer. We discuss how glycans modulate the glyco-metabolic landscape in the tumour microenvironment, and whether glycans can synergize with immunotherapy as a way of rewiring T-cell effector functions against cancer cells.
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Neoplasias , Humanos , Polissacarídeos , Linfócitos T , Glicosilação , Imunidade , Microambiente TumoralRESUMO
PURPOSE: Normative auditory brainstem response (ABR) data for infants and young children are available for 0.25-4 kHz, limiting clinical assessment to this range. As such, the high-frequency hearing sensitivity of infants and young children remains unknown until behavioral testing can be completed, often not until late preschool or early school ages. The purpose of this study was to obtain normative ABR data at 6 and 8 kHz in young infants. METHOD: Participants were 173 full-term infants seen clinically for ABR testing at 0.4-6.7 months chronological age (M = 1.4 months, SD = 1.0), 97% of whom were ≤ 12 weeks chronological age. Stimuli included 6 and 8 kHz tone bursts presented at a rate of 27.7/s or 30.7/s using Blackman window gating with six cycles (6 kHz) or eight cycles (8 kHz) rise/fall time and no plateau. Presentation levels included 20, 40, and 60 dB nHL. The ABR threshold was estimated in 5- to 10-dB steps. RESULTS: As previously observed with lower frequency stimuli, ABR waveforms obtained in response to 6 and 8 kHz tone bursts decreased in latency with increasing intensity and increasing age. Latency was shorter for 8-kHz tone bursts than 6-kHz tone bursts. Data tables are presented for clinical reference for infants ≤ 4 weeks, 4.1-8 weeks, and 8.1-12 weeks chronological age including median ABR latency for Waves I, III, and V and the upper and lower boundaries of the 90% prediction interval. Interpeak Latencies I-III, III-V, and I-V are also reported. CONCLUSION: The results from this study demonstrate that ABR assessment at 6 and 8 kHz is feasible for young infants within a standard clinical appointment and provide reference data for clinical interpretation of ABR waveforms for frequencies above 4 kHz.
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Potenciais Evocados Auditivos do Tronco Encefálico , Audição , Lactente , Criança , Humanos , Pré-Escolar , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Audiometria de Tons Puros/métodos , Limiar Auditivo/fisiologia , Audição/fisiologia , Estimulação Acústica/métodosRESUMO
The phospholamban mutation Arg 9 to Cys (R9C) has been found to cause a dilated cardiomyopathy in humans and in transgenic mice, with ventricular dilation and premature death. Emerging evidence suggests that phospholamban R9C is a loss-of-function mutation with dominant negative effect on SERCA2a activity. We imaged calcium and cardiac contraction simultaneously in 3 and 9 days-post-fertilization (dpf) zebrafish larvae expressing plnbR9C in the heart to unveil the early pathological pathway that triggers the disease. We generated transgenic zebrafish lines expressing phospholamban wild-type (Tg(myl7:plnbwt)) and phospholamban R9C (Tg(myl7:plnbR9C)) in the heart of zebrafish. To measure calcium and cardiac contraction in 3 and 9 dpf larvae, Tg(myl7:plnbwt) and Tg(myl7:plnbR9C) fish were outcrossed with a transgenic line expressing the ratiometric fluorescent calcium biosensor mCyRFP1-GCaMP6f. We found that PlnbR9C raised calcium transient amplitude, induced positive inotropy and lusitropy, and blunted the ß-adrenergic response to isoproterenol in 3 dpf larvae. These effects can be attributed to enhanced SERCA2a activity induced by the PlnbR9C mutation. In contrast, Tg(myl7:plnbR9C) larvae at 9 dpf exhibited ventricular dilation, systolic dysfunction and negative lusitropy, hallmarks of a dilated cardiomyopathy in humans. Importantly, N-acetyl-L-cysteine rescued this deleterious phenotype, suggesting that reactive oxygen species contribute to the pathological pathway. These results also imply that dysregulation of calcium homeostasis during embryo development contributes to the disease progression at later stages. Our in vivo model in zebrafish allows characterization of pathophysiological mechanisms leading to heart disease, and can be used for screening of potential therapeutical agents.
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Proteínas de Ligação ao Cálcio , Cálcio , Contração Miocárdica , Peixe-Zebra , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cardiomegalia , Cardiomiopatia Dilatada/patologia , Mutação , Peixe-Zebra/genéticaRESUMO
We introduce how to image calcium ion levels in the heart of zebrafish embryos and larvae up to 5 days post-fertilization with the photoprotein green fluorescent protein (GFP)-aequorin (GA) in the transgenic line Tg(myl7:GA). Incubation of the embryos with CTZ to obtain the functional photoprotein yields few emission counts, suggesting that, when the heart is beating, the rate of aequorin consumption is faster than that of the reconstitution with CTZ. In this chapter, we present an improved aequorin reconstitution protocol. We further describe the experimental procedure as well as the bioluminescence data analysis and processing.
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Equorina , Peixe-Zebra , Equorina/genética , Equorina/metabolismo , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Íons/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Peixe-Zebra/metabolismoRESUMO
Shelled walnuts are considered a microbiologically low-risk food but have been linked to some outbreaks, and a treatment aiming to decrease this risk is desirable. Pulsed light (PL) may be an alternative, providing it does not seriously impair their quality. This work assessed the impact of PL on some quality attributes of walnuts. To do this, measurements of rancidity, volatiles, total phenols, antioxidant activity, and descriptive sensory analysis were carried out on untreated and PL (43 J/cm2)-treated kernels. PL had no statistically significant (p > 0.05) effects on TBARS, peroxide value, total phenols, and antioxidant activity but significantly increased the concentration of volatiles related to green/herbaceous odors and decreased compounds related to fruity and citrus odors. The descriptors nut overall, walnut odor and flavor, and aftertaste were given statistically significantly (p < 0.05) higher scores, while descriptors woody odor and sweet received lower scores; 16 other traits such as all those related to color, texture, and rancidity were unaffected. No significant (p > 0.05) effects on total phenols and antioxidant activity in general were observed during the course of PL treatment. It can be concluded that PL technology may be used in shelled walnuts with only mild effects on their quality; a storage study must be carried out in order to determine the effect of PL treatment on its shelf-life.
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The nature of the immune responses associated with COVID-19 pathogenesis and disease severity, as well as the breadth of vaccine coverage and duration of immunity, is still unclear. Given the unpredictability for developing a severe/complicated disease, there is an urgent need in the field for predictive biomarkers of COVID-19. We have analyzed IgG Fc N-glycan traits of 82 SARS-CoV-2+ unvaccinated patients, at diagnosis, by nano-LC-ESI-MS. We determined the impact of IgG Fc glyco-variations in the induction of NK cells activation, further evaluating the association between IgG Fc N-glycans and disease severity/prognosis. We found that SARS-CoV-2+ individuals display, at diagnosis, variations in the glycans composition of circulating IgGs. Importantly, levels of galactose and sialic acid structures on IgGs are able to predict the development of a poor COVID-19 disease. Mechanistically, we demonstrated that a deficiency on galactose structures on IgG Fc in COVID-19 patients appears to induce NK cells activation associated with increased release of IFN-γ and TNF-α, which indicates the presence of pro-inflammatory immunoglobulins and higher immune activation, associated with a poor disease course. This study brings to light a novel blood biomarker based on IgG Fc glycome composition with capacity to stratify patients at diagnosis.
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COVID-19 , Biomarcadores , COVID-19/diagnóstico , Teste para COVID-19 , Galactose , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Polissacarídeos , SARS-CoV-2 , Índice de Gravidade de DoençaRESUMO
In vivo models of cardiac function maintain the complex relationship of cardiomyocytes with other heart cells, as well as the paracrine and mechanoelectrical feedback mechanisms. We aimed at imaging calcium transients simultaneously with heart contraction in zebrafish larvae. Methods: To image calcium in beating hearts, we generated a zebrafish transgenic line expressing the FRET-based ratiometric biosensor Twitch-4. Since emission ratioing canceled out the motion artifacts, we did not use myosin inhibitors or tnnt2a morpholinos to uncouple contraction from changes in calcium levels. We wrote an analysis program to automatically calculate kinetic parameters of the calcium transients. In addition, the ventricular diameter was determined in the fluorescence images providing a real-time measurement of contraction correlated with calcium. Results: Expression of Twitch-4 did not affect the force of contraction, the size of the heart nor the heart rate in 3- and 5-days post-fertilization (dpf) larvae. Comparison of 3 and 5 dpf larvae showed that calcium levels and transient amplitude were larger at 5 dpf, but the fractional shortening did not change. To validate the model, we evaluated the effect of drugs with known effects on cardiomyocytes. Calcium levels and the force of contraction decreased by the L-type calcium channel blocker nifedipine, whereas they increased with the activator Bay-K 8644. Caffeine induced bradycardia, markedly decreased ventricular diastolic calcium levels, increased the size of the calcium transients, and caused an escape rhythm in some larvae. Conclusions: The Tg(myl7:Twitch-4) line provides a physiological approach to image systolic and diastolic calcium levels in the heart of zebrafish larvae. Since the heart is beating, calcium levels and contraction can be correlated. This line will be a useful tool to address pathophysiological mechanisms in diseases like heart failure and arrhythmia, in cardiotoxicity studies and for drug screening.
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Cálcio , Peixe-Zebra , Animais , Cálcio/metabolismo , Coração/diagnóstico por imagem , Larva/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Peixe-Zebra/metabolismoRESUMO
Even during the continuing world pandemic of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), consumers remain exposed to the risk of getting infected by existing, emerging, or re-emerging foodborne and waterborne viruses. SARS-CoV-2 is different in that it is transmitted directly via the airborne route (droplets and aerosols) or indirect contact (surfaces contaminated with SARS-CoV-2). International food and health organizations and national regulatory bodies have provided guidance to protect individuals active in food premises from potential occupational exposure to SARS-CoV-2, and have recommended chemicals effective in controlling the virus. Additionally, to exclude transmission of foodborne and waterborne viruses, hygiene practices to remove viral contaminants from surfaces are applied in different stages of the food chain (e.g., food plants, food distribution, storage, retail sector, etc.), while new and enhanced measures effective in the control of all types of viruses are under development. This comprehensive review aims to analyze and compare efficacies of existing cleaning practices currently used in the food industry to remove pathogenic viruses from air, nonfood, and food contact surfaces, as well as from food surfaces. In addition, the classification, modes of transmission, and survival of food and waterborne viruses, as well as SARS-CoV-2 will be presented. The international guidelines and national regulations are summarized in terms of virucidal chemical agents and their applications.
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COVID-19 , Cadeia Alimentar , Aerossóis , COVID-19/prevenção & controle , Descontaminação , Humanos , SARS-CoV-2RESUMO
In arid and semi-arid areas, unsuitable afforestation programs may cause soil water deficit and even, in some cases, deep-soil desiccation and the subsequent death of part of the vegetation. However, the benefits of preferential flow through decayed root channels for supplying water in the dried soil layers are rarely evaluated. This study examines the effects of root channels on soil water content (SWC) and infiltration (initial and steady-state) in two planted species with high drought tolerance, namely: Korshinsk peashrub (Caragana korshinskii Kom.), and red plum apricot (Armeniaca vulgaris Lam.), using bare land as control site and published data from alfalfa (Medicago sativa L.) plots to refine results analysis. The infiltration rates were measured using a double-ring infiltrometer in areas with alive and decayed roots; and methylene blue was used to trace the pathways of water flow. The highest SWC appeared in the Korshinsk peashrub land, whereas the alfalfa grassland had the lowest SWC; and the differences of SWC among treatments were significant at all soil layers. Korshinsk peashrub had the highest steady infiltration rates, which were about 23% and 83% higher than those rates measured in the fruit tree plantation and alfalfa grasslands, respectively. The steady infiltration rates were significantly and positively correlated with the average root diameter and area. Within the same species, the decayed root plots significantly increased the soil water infiltration capacity compared to the alive root plots. Considering the water scarcity conditions of the study area, the preferential flow formed through decomposed roots was conducive to soil water supplement, appearing as a natural compensation mechanism caused by dead vegetation. Our findings contribute to a better understanding of soil water infiltration during root decay of the plants in water-limited ecosystems, and how this dynamic replenishes soil moisture and alleviates soil desiccation.
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Caragana , Solo , China , Ecossistema , Árvores , Água/análiseRESUMO
The repurposing of the CRISPR/Cas bacterial defense system against bacteriophages as simple and flexible molecular tools has revolutionized the field of gene editing. These tools are now widely used in basic research and clinical trials involving human somatic cells. However, a global moratorium on all clinical uses of human germline editing has been proposed because the technology still lacks the required efficacy and safety. Here we focus on the approaches developed since 2013 to decrease the frequency of unwanted mutations (the off-targets) during CRISPR-based gene editing.
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Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetylh-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The ß-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.
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The diversity of glycan presentation in a cell, tissue and organism is enormous, which reflects the huge amount of important biological information encoded by the glycome which has not been fully understood. A compelling body of evidence has been highlighting the fundamental role of glycans in immunity, such as in development, and in major inflammatory processes such as inflammatory bowel disease, systemic lupus erythematosus and other autoimmune disorders. Glycans play an instrumental role in the immune response, integrating the canonical circuits that regulate innate and adaptive immune responses. The relevance of glycosylation in immunity is demonstrated by the role of glycans as important danger-associated molecular patterns and pathogen-associated molecular patterns associated with the discrimination between self and non-self; also as important regulators of the threshold of T cell activation, modulating receptors signalling and the activity of both T and other immune cells. In addition, glycans are important determinants that regulate the dynamic crosstalk between the microbiome and immune response. In this chapter, the essential role of glycans in the immunopathogenesis of inflammatory disorders will be presented and its potential clinical applications (diagnosis, prognosis and therapeutics) will be highlighted.