RESUMEN
Dave Garbers' work significantly contributed to our understanding of sperm's regulated motility, capacitation, and the acrosome reaction. These key sperm functions involve complex multistep signaling pathways engaging numerous finely orchestrated elements. Despite significant progress, many parameters and interactions among these elements remain elusive. Mathematical modeling emerges as a potent tool to study sperm physiology, providing a framework to integrate experimental results and capture functional dynamics considering biochemical, biophysical, and cellular elements. Depending on research objectives, different modeling strategies, broadly categorized into continuous and discrete approaches, reveal valuable insights into cell function. These models allow the exploration of hypotheses regarding molecules, conditions, and pathways, whenever they become challenging to evaluate experimentally. This review presents an overview of current theoretical and experimental efforts to understand sperm motility regulation, capacitation, and the acrosome reaction. We discuss the strengths and weaknesses of different modeling strategies and highlight key findings and unresolved questions. Notable discoveries include the importance of specific ion channels, the role of intracellular molecular heterogeneity in capacitation and the acrosome reaction, and the impact of pH changes on acrosomal exocytosis. Ultimately, this review underscores the crucial importance of mathematical frameworks in advancing our understanding of sperm physiology and guiding future experimental investigations.
Asunto(s)
Reacción Acrosómica , Transducción de Señal , Capacitación Espermática , Motilidad Espermática , Espermatozoides , Masculino , Espermatozoides/metabolismo , Espermatozoides/fisiología , Humanos , Reacción Acrosómica/fisiología , Capacitación Espermática/fisiología , Transducción de Señal/fisiología , Animales , Motilidad Espermática/fisiología , Modelos Biológicos , Modelos TeóricosRESUMEN
The combination of signals from the T-cell receptor (TCR) and co-stimulatory molecules triggers transcriptional programs that lead to proliferation, cytokine secretion, and effector functions. We compared the impact of engaging the TCR with CD28 and/or CD43 at different time points relative to TCR engagement on T-cell function. TCR and CD43 simultaneous engagement resulted in higher CD69 and PD-1 expression levels than in TCR and CD28-stimulated cells, with a cytokine signature of mostly effector, inflammatory, and regulatory cytokines, while TCR and CD28-activated cells secreted all categories of cytokines, including stimulatory cytokines. Furthermore, the timing of CD43 engagement relative to TCR ligation, and to a lesser degree that of CD28, resulted in distinct patterns of expression of cytokines, chemokines, and growth factors. Complete cell activation was observed when CD28 or CD43 were engaged simultaneously with or before the TCR, but ligating the TCR before CD43 or CD28 failed to complete a cell activation program regarding cytokine secretion. As the order in which CD43 or CD28 and the TCR were engaged resulted in different combinations of cytokines that shape distinct T-cell immune programs, we analyzed their upstream sequences to assess whether the combinations of cytokines were associated with different sets of regulatory elements. We found that the order in which the TCR and CD28 or CD43 are engaged predicts the recruitment of specific sets of chromatin remodelers and TFSS, which ultimately regulate T-cell polarization and plasticity. Our data underscore that the combination of co-stimulatory molecules and the time when they are engaged relative to the TCR can change the cell differentiation program.
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Antígenos CD28 , Receptores de Antígenos de Linfocitos T , Antígenos CD28/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T , Activación de Linfocitos , Diferenciación Celular , Citocinas/metabolismoRESUMEN
Defect detection is a key element of quality control in today's industries, and the process requires the incorporation of automated methods, including image sensors, to detect any potential defects that may occur during the manufacturing process. While there are various methods that can be used for inspecting surfaces, such as those of metal and building materials, there are only a limited number of techniques that are specifically designed to analyze specialized surfaces, such as ceramics, which can potentially reveal distinctive anomalies or characteristics that require a more precise and focused approach. This article describes a study and proposes an extended solution for defect detection on ceramic pieces within an industrial environment, utilizing a computer vision system with deep learning models. The solution includes an image acquisition process and a labeling platform to create training datasets, as well as an image preprocessing technique, to feed a machine learning algorithm based on convolutional neural networks (CNNs) capable of running in real time within a manufacturing environment. The developed solution was implemented and evaluated at a leading Portuguese company that specializes in the manufacturing of tableware and fine stoneware. The collaboration between the research team and the company resulted in the development of an automated and effective system for detecting defects in ceramic pieces, achieving an accuracy of 98.00% and an F1-Score of 97.29%.
RESUMEN
Mammalian sperm capacitation is a prerequisite for successful fertilization. Capacitation involves biochemical and physiological modifications of sperm as they travel through the female reproductive tract. These modifications prepare the sperm to undergo the acrosome reaction (AR), an acrosome vesicle exocytosis that is necessary for gamete fusion. Capacitation requires an increase in both intracellular calcium ([Ca2+]i) and pH (pHi). Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the acrosome pH (pHa) is sufficient to trigger the AR in mouse and human sperm, but it is unknown if pHa increases naturally during human sperm capacitation. We used single-cell imaging and image-based flow cytometry to evaluate pHa during capacitation and its regulation. We found that pHa progressively increases during capacitation. The V-ATPase, which immunolocalized to the acrosome and equatorial segment, is mainly responsible for the acidity of the acrosome. It is likely that the regulation of V-ATPase is at least in part responsible for the progressive increase in pHa during capacitation. Acrosome alkalinization was dependent on extracellular HCO3- and Ca2+. Inhibition of the HCO3--dependent adenylyl cyclase and protein kinase A induced significant pHa changes. Overall, alkalinization of the acrosome may be a key step in the path toward the AR.
Asunto(s)
Reacción Acrosómica , Calcio , Capacitación Espermática , Acrosoma/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Calcio/metabolismo , Humanos , Masculino , Mamíferos , Ratones , Capacitación Espermática/fisiología , Espermatozoides/metabolismoRESUMEN
Plant-microbe symbiosis is pervasive in the Earth's ecosystems and dates back to the early land colonisation by plants. Mutualistic partnership with rhizobia bacteria and mycorrhizal fungi promotes plant nutrition, growth and diversity, impacting important ecosystem functions. However, how the global behaviour and dynamical properties of an ecosystem are modified by plant-microbe symbiosis is still unclear. To tackle this theoretical question, we resorted to the Daisyworld as a toy model of the global ecosystem. We redesigned the original model to allow accounting for seed production, spreading, germination, and seedling development to mature seed-producing plants to describe how symbiotic and non-symbiotic daisy species differ in these key processes. Using the steady-state and bifurcation analysis of this model, we demonstrate that symbiosis with microbes broadens the habitability range of the Daisyworld by enhancing plant growth and/or facilitating plant access to otherwise uninhabitable nutrient-poor regions.
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Micorrizas , Simbiosis , Ecosistema , Desarrollo de la Planta , Plantas/microbiologíaRESUMEN
How cells control the numbers of subcellular components is a fundamental question in biology. Given that biosynthetic processes are fundamentally stochastic it is utterly puzzling that some structures display no copy number variation within a cell population. Centriole biogenesis, with each centriole being duplicated once and only once per cell cycle, stands out due to its remarkable fidelity. This is a highly controlled process, which depends on low-abundance rate-limiting factors. How can exactly one centriole copy be produced given the variation in the concentration of these key factors? Hitherto, tentative explanations of this control evoked lateral inhibition- or phase separation-like mechanisms emerging from the dynamics of these rate-limiting factors but how strict centriole number is regulated remains unsolved. Here, a novel solution to centriole copy number control is proposed based on the assembly of a centriolar scaffold, the cartwheel. We assume that cartwheel building blocks accumulate around the mother centriole at supercritical concentrations, sufficient to assemble one or more cartwheels. Our key postulate is that once the first cartwheel is formed it continues to elongate by stacking the intermediate building blocks that would otherwise form supernumerary cartwheels. Using stochastic models and simulations, we show that this mechanism may ensure formation of one and only one cartwheel robustly over a wide range of parameter values. By comparison to alternative models, we conclude that the distinctive signatures of this novel mechanism are an increasing assembly time with cartwheel numbers and the translation of stochasticity in building block concentrations into variation in cartwheel numbers or length.
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Centriolos/metabolismo , Centriolos/ultraestructura , Modelos Biológicos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Centriolos/química , Biología Computacional , Simulación por Computador , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Cuaternaria de Proteína , Procesos EstocásticosRESUMEN
Phenotypic variation in the copy number of gene products expressed by cells or tissues has been the focus of intense investigation. To what extent the observed differences in cellular expression levels are persistent or transient is an intriguing question. Here, we develop a quantitative framework that resolves the expression variation into stable and unstable components. The difference between the expression means in two cohorts isolated from any cell population is shown to converge to an asymptotic value, with a characteristic time, τT, that measures the timescale of the unstable dynamics. The asymptotic difference in the means, relative to the initial value, measures the stable proportion of the original population variance [Formula: see text]. Empowered by this insight, we analysed the T-cell receptor (TCR) expression variation in CD4 T cells. About 70% of TCR expression variance is stable in a diverse polyclonal population, while over 80% of the variance in an isogenic TCR transgenic population is volatile. In both populations the TCR levels fluctuate with a characteristic time of 32 hours. This systematic characterisation of the expression variation dynamics, relying on time series of cohorts' means, can be combined with technologies that measure gene or protein expression in single cells or in bulk.
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Linfocitos T CD4-Positivos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Separación Celular , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.
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Canales de Calcio/metabolismo , Erizos de Mar/metabolismo , Espermatozoides/fisiología , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Quimiotaxis/fisiología , Biología Computacional/métodos , Iones/metabolismo , Masculino , Potenciales de la Membrana/fisiología , Modelos Teóricos , Transducción de Señal , Motilidad Espermática/fisiologíaRESUMEN
BACKGROUND: Kidney transplantation is the gold standard treatment for children with end-stage chronic kidney disease. Graft thrombosis is an important cause of graft failure, with high morbidity, mortality, and impact on quality of life and to the health system. The role of thromboprophylaxis in this setting is still uncertain. We describe the demographic characteristics and thrombotic risk factors in pediatric renal transplant recipients, determining the rate of renal graft thrombosis, and discuss the role of thromboprophylaxis. METHODS: This retrospective study reviewed 96 pediatric renal transplantations between 2008 and 2017 in a single hospital. Patients were assigned to one of two groups: children who did not receive thromboprophylaxis after transplantation and those who did. We reported their characteristics, comparing the incidence of graft thrombosis and hemorrhagic complications between the groups. RESULTS: Forty-nine patients (51%) received thromboprophylaxis. Thrombosis occurred in 5 patients who did not receive thromboprophylaxis (5.2%) compared with none in the group that did (p = .025). In all patients, renal graft thrombosis resulted in early graft loss. Thirteen patients had hemorrhagic complications. Seven were unrelated to pharmacological thromboprophylaxis (2 major, 1 moderate, and 4 minor bleeding, which either did not receive thromboprophylaxis or had bleeding prior to thromboprophylaxis), while six occurred during heparinization (2 major, 1 moderate, and 3 minor bleeding). There was no significant difference in the rate of hemorrhagic complications between the groups (p = .105). CONCLUSIONS: The rate of renal graft thrombosis was 5.2%. Thrombosis remains an important cause of early graft loss. Thromboprophylaxis was associated with a reduction in graft thrombosis without increased risk of bleeding.
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Fallo Renal Crónico/cirugía , Trasplante de Riñón , Complicaciones Posoperatorias/prevención & control , Tromboembolia/prevención & control , Adolescente , Anticoagulantes/uso terapéutico , Brasil , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Estudios Retrospectivos , Factores de Riesgo , Receptores de TrasplantesRESUMEN
OBJECTIVE: To report a single-center experience with thrombolytic therapy using recombinant tissue plasminogen activator (rt-PA) in preterm neonates with severe thrombotic events, in terms of thrombus resolution and bleeding complications. STUDY DESIGN: This retrospective study included 21 preterm neonates with severe venous thrombotic events admitted to the neonatal intensive care unit, identified in our pharmacy database from January 2001 to December 2016, and treated with rt-PA until complete or partial clot lysis, no-response or bleeding complications. Our primary outcome was thrombus resolution. RESULTS: Twenty-one preterm neonates were treated with rt-PA for an average of 2.9 cycles. Seventeen patients (80.9%) had superior vena cava thrombosis and superior vena cava syndrome. All patients had a central venous catheter, parenteral nutrition, mechanical ventilation, and sepsis. Fifteen patients (71.4%) were extremely preterm, 11 (52.4%) were extremely low birth weight, and seven (33.3%) were very low birth weight. The patency rate was 85.7%, complete lysis occurred in 11 (52.4%) patients, and partial lysis in seven (33.3%). Minor bleeding occurred in five (23.8%) patients, three patients (14.2%) had clinically relevant nonmajor bleeding events, and major bleeding occurred in six (28%) patients. CONCLUSION: In this study, the rate of thrombus resolution in preterm neonates treated with rt-PA were similar to the percentages reported in children and adolescents, with a high rate of bleeding. Therefore, rt-PA thrombolytic therapy should only be considered as a treatment option for severe life-threatening thrombosis in premature neonates for whom the benefits of the thrombolytic treatment outweigh the risks of bleeding.
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Hemorragia/prevención & control , Recien Nacido Prematuro , Síndrome de la Vena Cava Superior/tratamiento farmacológico , Terapia Trombolítica/métodos , Trombosis/tratamiento farmacológico , Activador de Tejido Plasminógeno/administración & dosificación , Vena Cava Inferior/efectos de los fármacos , Femenino , Estudios de Seguimiento , Humanos , Recién Nacido , Masculino , Pronóstico , Estudios Retrospectivos , Síndrome de la Vena Cava Superior/patología , Trombosis/patología , Vena Cava Inferior/patologíaRESUMEN
Autoantibodies are biomarkers for autoimmune disease diagnosis, monitoring, and prediction. Therefore, this study established the frequency of latent and overt polyautoimmunity in children and adolescents with >6 months of diagnosis of immune thrombocytopenia (ITP). Forty-seven patients with chronic or persistent disease had non-organ-specific and organ-specific autoantibodies assessed. Frequency of latent polyautoimmunity was 36.2%, and, of overt polyautoimmunity, it was 4.3%. Of ITP patients with latent polyautoimmunity, 52.9% were positive for antinuclear antibody (ANA), 47.1% for autoantibodies other than ANA, and 64.7% for multiple autoantibodies. In addition, patients with latent polyautoimmunity and those positive for ANA were significantly older at disease onset. Both ITP patients positive and negative for autoantibodies reported family members with autoimmune diseases. The autoantibodies observed were as follows: ANA, anti-dsDNA, anti-SSA/Ro, IgM aCL, anti-GAD, anti-IA2, anti-IAA, anti-TG, anti-TPO, anti-LKM1, and SMA. Of ITP patients with overt polyautoimmunity, 1 was diagnosed with type 1 diabetes mellitus and the other with thyroiditis. In conclusion, children and adolescents with ITP present high frequency of latent and overt polyautoimmunity even for autoantibodies other than ANA. Therefore, ANA and other non-organ-specific and organ-specific autoantibodies should be considered for assessment during ITP patients' follow-up.
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Autoanticuerpos/inmunología , Púrpura Trombocitopénica Idiopática/inmunología , Adolescente , Autoantígenos/inmunología , Niño , Estudios Transversales , Femenino , Humanos , MasculinoRESUMEN
Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in γ-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix γ-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patient's GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo.
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Ligasas de Carbono-Carbono/genética , Mutación , Sangrado por Deficiencia de Vitamina K/genética , Anomalías Múltiples/genética , Secuencia de Aminoácidos , Secuencia de Bases , Pruebas de Coagulación Sanguínea , Sistemas CRISPR-Cas , Calcinosis/genética , Proteínas de Unión al Calcio/genética , Enfermedades de los Cartílagos/genética , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular/genética , Femenino , Genes Reporteros , Estudios de Asociación Genética , Genotipo , Células HEK293 , Deformidades Congénitas de la Mano/genética , Hemorragia , Humanos , Lactante , Recién Nacido , Datos de Secuencia Molecular , Fenotipo , Estenosis de la Válvula Pulmonar/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteína Gla de la MatrizRESUMEN
Due to their unique electronic and optical features, gold nanoparticles (AuNP) have received a great deal of attention for application in different fields such as catalysis, electronics, and biomedicine. The large-volume manufacturing predicted for future decades and the inevitable release of these substances into the environment necessitated an assessment of potential adverse human and ecological risks due to exposure to AuNP. Accordingly, this study aimed to examine the acute and developmental toxicity attributed to a commercial suspension of Au nanorods stabilized with cetyltrimethylammonium bromide (CTAB-AuNR) using early embryonic stages of zebrafish (Danio rerio), a well-established model in ecotoxicology. Zebrafish embryos were exposed to CTAB-AuNR (0-150 µg/L) to determine for developmental assessment until 96 hr post fertilization (hpf) and lethality. Uptake of CTAB-AuNR by embryos and nanoparticles potential to induce DNA damage was also measured at 48 and 96 hpf. Analysis of the concentration-response curves with cumulative mortality at 96 hpf revealed a median lethal concentration (LC50,96h) of 110.2 µg/L. At sublethal concentrations, CTAB-AuNR suspensions were found to produce developmental abnormalities such as tail deformities, pericardial edema, decreased body length, and delayed eye, head, and tail elongation development. Further, less than 1% of the initial concentration of CTAB-AuNR present in the exposure media was internalized by zebrafish embryos prior to (48 hpf) and after hatching (96 hpf). In addition, no marked DNA damage was detected in embryos after exposure to CTAB-AuNR. Overall, CTAB-AuNR suspensions produced lethal and sublethal effects on zebrafish embryos with possible repercussions in fitness of adult stages. However, these results foresee a low risk for fish since the observed effects occurred at concentrations above the levels expected to find in the aquatic environment.
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Desarrollo Embrionario/efectos de los fármacos , Oro/toxicidad , Nanotubos/toxicidad , Pez Cebra/embriología , Animales , Ensayo Cometa , Embrión no Mamífero/efectos de los fármacos , Microscopía Electrónica de Transmisión , Nanotubos/ultraestructura , Pez Cebra/crecimiento & desarrolloAsunto(s)
ADN Circular/genética , Púrpura Trombocitopénica Idiopática/genética , Receptores de Antígenos de Linfocitos T/genética , Adolescente , Niño , Preescolar , ADN Circular/inmunología , Femenino , Humanos , Masculino , Púrpura Trombocitopénica Idiopática/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/inmunología , Timo/metabolismoRESUMEN
In many broadcast-spawning marine organisms, oocytes release chemicals that guide conspecific spermatozoa towards them through chemotaxis. In the sea urchin Lytechinus pictus, the chemoattractant peptide speract triggers a train of fluctuations of intracellular Ca(2+) concentration in the sperm flagella. Each transient Ca(2+) elevation leads to a momentary increase in flagellar bending asymmetry, known as a chemotactic turn. Furthermore, chemotaxis requires a precise spatiotemporal coordination between the Ca(2+)-dependent turns and the form of chemoattractant gradient. Spermatozoa that perform Ca(2+)-dependent turns while swimming down the chemoattractant gradient, and conversely suppress turning events while swimming up the gradient, successfully approach the center of the gradient. Previous experiments in Strongylocentrotus purpuratus sea urchin spermatozoa showed that niflumic acid (NFA), an inhibitor of several ion channels, drastically altered the speract-induced Ca(2+) fluctuations and swimming patterns. In this study, mathematical modeling of the speract-dependent Ca(2+) signaling pathway suggests that NFA, by potentially affecting hyperpolarization-activated and cyclic nucleotide-gated channels, Ca(2+)-regulated Cl(-) channels and/or Ca(2+)-regulated K(+) channels, may alter the temporal organization of Ca(2+) fluctuations, and therefore disrupt chemotaxis. We used a novel automated method for analyzing sperm behavior and we identified that NFA does indeed disrupt chemotactic responses of L. pictus spermatozoa, although the temporal coordination between the Ca(2+)-dependent turns and the form of chemoattractant gradient is unaltered. Instead, NFA disrupts sperm chemotaxis by altering the arc length traveled during each chemotactic turning event. This alteration in the chemotactic turn trajectory disorientates spermatozoa at the termination of the turning event. We conclude that NFA disrupts chemotaxis without affecting how the spermatozoa decode environmental cues.
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Flagelos/efectos de los fármacos , Canales Iónicos/antagonistas & inhibidores , Ácido Niflúmico/farmacología , Transporte Espermático/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Flagelos/metabolismo , Masculino , Oligopéptidos/farmacología , Erizos de Mar , Transducción de Señal , Espermatozoides/fisiologíaRESUMEN
A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases G1, S, G2, and M, and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However, whereas mean lengths are precisely estimated for all phases, the respective variances remain uncertain. To overcome this limitation, a redesigned experimental protocol is derived and validated in silico. The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases. The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase-specific biosensor-based fluorescent imaging.
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Ciclo Celular/fisiología , Células Eucariotas/citología , Células Eucariotas/fisiología , Modelos Biológicos , Nucleósidos/metabolismo , Muerte Celular , Proliferación Celular , Técnicas Citológicas , Cinética , Procesos EstocásticosRESUMEN
Two highly similar RNA polymerase sigma subunits, σ(F) and σ(G), govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ(F) drives synthesis of σ(G) but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σ(G) while discriminating between σ(F) and σ(G) in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σ(G) (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ(G) in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ(G). CsfB is normally produced in the forespore, under σ(F) control, but sigGN45E mutant cells also expressed csfB and did so in a σ(G)-dependent manner, autonomously from σ(F). Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ(G) activity. N45 is invariant in the homologous position of σ(G) orthologues, whereas its functional equivalent in σ(F) proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ(F), a E39N substitution in σ(F) resulted in efficient binding of CsfB to σ(F). Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ(F) in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis.
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Bacillus subtilis/genética , Bacillus subtilis/fisiología , ARN Polimerasas Dirigidas por ADN/genética , Factor sigma/genética , Factor sigma/metabolismo , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Asparagina/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Retroalimentación Fisiológica , Regulación Bacteriana de la Expresión Génica , Ácido Glutámico/genética , Datos de Secuencia Molecular , Mutación , Esporas Bacterianas/genética , Esporas Bacterianas/fisiologíaRESUMEN
This study aimed to evaluate the impact of body condition score (BCS) at calving, parity, and the calving season on the performance of dairy cows and their offspring. Data from 521 Holstein cows that calved a female calf and had their BCS evaluated at calving from a single commercial farm located in Southern Brazil were used. Cows were categorized into five BCS classes: class 1: <3.0 (n = 19), class 2: 3.0-3.25 (n = 134), class 3: 3.5-3.75 (n = 160), class 4: 4.0-4.25 (n = 142), and class 5: >4.25 (n = 66). Data were also categorized by calving order (primiparous and multiparous dams) and by calving season. The study was designed as a prospective cohort study. Variables with normal distribution were analyzed by the MIXED procedure of SAS, while binary outcomes were analyzed by the GLIMMIX procedure of SAS. Daughters from primiparous dams were born lighter (39.1 ± 0.42 vs. 41.4 ± 0.29 kg, p < 0.01), but they had the same weights as the daughters from multiparous cows at weaning (121.5 ± 1.67 vs. 120.4 ± 1.58 kg, p = 0.20). As expected, primiparous cows showed lower (p < 0.01) 305-day milk yields than multiparous ones: 8633 ± 363 vs. 10,761 ± 249 kg, respectively. Regarding the calving season, cows that calved in the winter were the most productive ones, and those that calved in the fall had lower milk yields (p = 0.01). Calves born in the winter were heavier at birth (p < 0.01), calved younger (p = 0.04), and produced more milk at first lactation (p = 0.03). The BCS class had an impact (p < 0.01) on calf birth weights; daughters from Class 1 cows (BCS < 3.0) were lighter (38.0 ± 1.0 kg) than the calves from Class 5 cows with a BCS > 4.25 (41.9 ± 0.57 kg). Calves from dams with a BCS < 3 (Class 1) had a 31.8% culling rate until weaning, while calves from cows with a BCS of 3.0-3.25 (Class 2) had a 9.6% culling rate (p = 0.12). These results suggest that maternal and environmental factors, such as calving season and parity, in addition to the dams' body condition score at calving, are associated with different offspring performances.
RESUMEN
INTRODUCTION: Although still rare, pulmonary embolism (PE) in children has been increasing over the years. Data regarding this group of patients are still sparse, which contributes to the lack of standardized prophylaxis protocols and the misdiagnosis. This study aimed to determine the incidence of pediatric PE at a Brazilian tertiary hospital, describe clinical characteristics and identify possible risk factors. We also analyzed the diagnosis and management of PE. METHODS: This was a retrospective review of tertiary Brazilian single-center data of all pediatric patients (0 - 18 years) with acute PE, diagnosed radiologically, from September 2009 to May 2019. RESULTS: The incidence of PE was 3.3 cases per 10,000 hospitalized children. All the twenty-three cases had some risk factor identified and sixteen of them (69.5%) had more than one risk factor. The most important were central venous catheter (39.1%), malignancy (34.8%) and recent surgery (34.8%). Among the children with identifiable symptoms (69.5%), the most common was dyspnea (56.2%). Only one patient did not receive antithrombotic therapy because of the high bleeding risk and most patients (70.6%) were treated for 3 to 6 months. Among the nineteen patients alive at the end of the six-month follow-up, ten (52.6%) repeated the PE image control. Seven of them (70.0%) had complete or partial resolution of the thrombosis and none had worsening images. CONCLUSION: Our lower incidence than that of the current literature may reflect underdiagnosis due to low suspicion of PE. At least one risk factor was identified in all patients, which emphasizes the importance of increasing awareness of high-risk children.
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Surface defect detection with machine learning has become an important tool in industries and a large field of study for researchers or workers in recent years. It is necessary to have a simplified source of information that helps us to better focus on one type of surface. In this systematic review, we present a classification for surface defect detection based on convolutional neural networks (CNNs) focused on surface types. Findings: Out of 253 records identified, 59 primary studies were eligible. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we analyzed the structures of each study and the concepts related to defects and their types on surfaces. The presented review is mainly focused on finding a classification for the types of surfaces most used in industry (metal, building, ceramic, wood, and special). We delve into the specifics of each surface category, offering illustrative examples of their applications within both industrial and laboratory settings. Furthermore, we propose a new taxonomy of machine learning based on the obtained results and collected information. We summarized the studies and extracted the main characteristics such as type of surface, problem types, timeline, type of network, techniques, and datasets. Among the most relevant results of our analysis, we found that the metallic surface is the most used, as it is the one found in 62.71% of the studies, and the most prevalent problem type is classification, accounting for 49.15% of the total. Furthermore, we observe that transfer learning was employed in 83.05% of the studies, while data augmentation was utilized in 59.32%. Our findings also provide insights into the cameras most frequently employed, along with the strategies adopted to address illumination challenges present in certain articles and the approach to creating datasets for real-world applications. The main results presented in this review allow for a quick and efficient search of information for researchers and professionals interested in improving the results of their defect detection projects. Finally, we analyzed the trends that could open new fields of study for future research in the area of surface defect detection.