RESUMEN
Gaze understandinga suggested precursor for understanding others' intentionsrequires recovery of gaze direction from the observed person's head and eye position. This challenging computation is naturally acquired at infancy without explicit external guidance, but can it be learned later if vision is extremely poor throughout early childhood? We addressed this question by studying gaze following in Ethiopian patients with early bilateral congenital cataracts diagnosed and treated by us only at late childhood. This sight restoration provided a unique opportunity to directly address basic issues on the roles of "nature" and "nurture" in development, as it caused a selective perturbation to the natural process, eliminating some gaze-direction cues while leaving others still available. Following surgery, the patients' visual acuity typically improved substantially, allowing discrimination of pupil position in the eye. Yet, the patients failed to show eye gaze-following effects and fixated less than controls on the eyestwo spontaneous behaviors typically seen in controls. Our model for unsupervised learning of gaze direction explains how head-based gaze following can develop under severe image blur, resembling preoperative conditions. It also suggests why, despite acquiring sufficient resolution to extract eye position, automatic eye gaze following is not established after surgery due to lack of detailed early visual experience. We suggest that visual skills acquired in infancy in an unsupervised manner will be difficult or impossible to acquire when internal guidance is no longer available, even when sufficient image resolution for the task is restored. This creates fundamental barriers to spontaneous vision recovery following prolonged deprivation in early age.
Asunto(s)
Fijación Ocular , Visión Ocular , Atención , Ceguera , Niño , Humanos , Agudeza VisualRESUMEN
Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.
Asunto(s)
Interferón beta/metabolismo , SARS-CoV-2/inmunología , Proteínas Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Chlorocebus aethiops , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Humanos , Interferón beta/genética , Interferón beta/farmacología , SARS-CoV-2/efectos de los fármacos , Factor de Transcripción STAT1/metabolismo , Células Vero , Proteínas Virales/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009800.].
RESUMEN
The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has profound activity in chronic lymphocytic leukemia (CLL) but limited curative potential by itself. Residual signaling pathways that maintain survival of CLL cells might be targeted to improve ibrutinib's therapeutic activity, but the nature of these pathways is unclear. Ongoing activation of IFN receptors in patients on ibrutinib was suggested by the presence of type I and II IFN in blood together with the cycling behavior of IFN-stimulated gene (ISG) products when IFN signaling was blocked intermittently with the JAK inhibitor ruxolitinib. IFN signaling in CLL cells from human patients was not prevented by ibrutinib in vitro or in vivo, but ISG expression was significantly attenuated in vitro. ISGs such as CXCL10 that require concomitant activation of NF-κB were decreased when this pathway was inhibited by ibrutinib. Other ISGs, exemplified by LAG3, were decreased as a result of inhibited protein translation. Effects of IFN on survival remained intact as type I and II IFN-protected CLL cells from ibrutinib in vitro, which could be prevented by ruxolitinib and IFNR blocking Abs. These observations suggest that IFNs may help CLL cells persist and specific targeting of IFN signaling might deepen clinical responses of patients on ibrutinib.
Asunto(s)
Adenina/análogos & derivados , Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Piperidinas/farmacología , Adenina/farmacología , Adenina/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Resistencia a Antineoplásicos/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/inmunología , Nitrilos , Piperidinas/uso terapéutico , Cultivo Primario de Células , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas , Receptores de Interferón/antagonistas & inhibidores , Receptores de Interferón/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Células Tumorales CultivadasRESUMEN
The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteínas Virales de Fusión/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Activación de Linfocitos , Fusión de Membrana , Ratones , Ratones Endogámicos C57BL , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Visual object recognition is performed effortlessly by humans notwithstanding the fact that it requires a series of complex computations, which are, as yet, not well understood. Here, we tested a novel account of the representations used for visual recognition and their neural correlates using fMRI. The rationale is based on previous research showing that a set of representations, termed "minimal recognizable configurations" (MIRCs), which are computationally derived and have unique psychophysical characteristics, serve as the building blocks of object recognition. We contrasted the BOLD responses elicited by MIRC images, derived from different categories (faces, objects, and places), sub-MIRCs, which are visually similar to MIRCs, but, instead, result in poor recognition and scrambled, unrecognizable images. Stimuli were presented in blocks, and participants indicated yes/no recognition for each image. We confirmed that MIRCs elicited higher recognition performance compared to sub-MIRCs for all three categories. Whereas fMRI activation in early visual cortex for both MIRCs and sub-MIRCs of each category did not differ from that elicited by scrambled images, high-level visual regions exhibited overall greater activation for MIRCs compared to sub-MIRCs or scrambled images. Moreover, MIRCs and sub-MIRCs from each category elicited enhanced activation in corresponding category-selective regions including fusiform face area and occipital face area (faces), lateral occipital cortex (objects), and parahippocampal place area and transverse occipital sulcus (places). These findings reveal the psychological and neural relevance of MIRCs and enable us to make progress in developing a more complete account of object recognition.
Asunto(s)
Reconocimiento Visual de Modelos/fisiología , Reconocimiento en Psicología/fisiología , Corteza Visual/fisiología , Adulto , Encéfalo/fisiología , Mapeo Encefálico , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Estimulación Luminosa , Adulto JovenRESUMEN
Discovering the visual features and representations used by the brain to recognize objects is a central problem in the study of vision. Recently, neural network models of visual object recognition, including biological and deep network models, have shown remarkable progress and have begun to rival human performance in some challenging tasks. These models are trained on image examples and learn to extract features and representations and to use them for categorization. It remains unclear, however, whether the representations and learning processes discovered by current models are similar to those used by the human visual system. Here we show, by introducing and using minimal recognizable images, that the human visual system uses features and processes that are not used by current models and that are critical for recognition. We found by psychophysical studies that at the level of minimal recognizable images a minute change in the image can have a drastic effect on recognition, thus identifying features that are critical for the task. Simulations then showed that current models cannot explain this sensitivity to precise feature configurations and, more generally, do not learn to recognize minimal images at a human level. The role of the features shown here is revealed uniquely at the minimal level, where the contribution of each feature is essential. A full understanding of the learning and use of such features will extend our understanding of visual recognition and its cortical mechanisms and will enhance the capacity of computational models to learn from visual experience and to deal with recognition and detailed image interpretation.
Asunto(s)
Redes Neurales de la Computación , Reconocimiento Visual de Modelos/fisiología , Visión Ocular/fisiología , Percepción Visual/fisiología , Encéfalo/fisiología , Humanos , Modelos Neurológicos , Red Nerviosa/fisiología , Estimulación Luminosa , Psicofísica/métodos , Corteza Visual/fisiología , Vías Visuales/fisiologíaRESUMEN
We analysed gene expression microarray data from whole blood samples from 228 multiple sclerosis (MS) patients either untreated or treated with one of three alternative commonly used interferon beta (IFNß) disease modifying drugs: Avonex (×1 weekly), Betaseron (every second day) or Rebif (×3 weekly). Patient injections were not timed to coordinate sample collections, thus providing a global transcriptomic profile for each population of patients studied. Three hundred and fifty one genes were significantly differentially expressed by at least one of the IFNß drugs. Despite the different drug sources with distinct injection and dosage protocols, a striking similarity was found in the identity and functional classes of the differentially expressed genes induced. Using the 25 most-upregulated genes, we defined a robust IFNß gene expression signature that quantifies the IFN activation state per blood sample collected irrespective of the type of IFNß therapy. This 25-gene signature also defined basal IFN activation states among untreated MS patients, which differed among individuals but remained relatively constant per patient with time. The maximum drug-induced IFN-activation state was similar for all three drugs despite a 1.7-2.0-fold diminished average effect for Avonex. This and a more erratic effect of Avonex per patient across longitudinal measurements is likely a result of its reduced injection frequency. In summary, we have defined a robust blood-derived type I IFN gene signature from MS patients. This signature could potentially serve to generically quantify the systemic Type I IFN activation status for any other clinical manifestation, inclusive of other autoimmune diseases.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Interferón beta-1a/farmacología , Interferon beta-1b/farmacología , Esclerosis Múltiple/sangre , Transcriptoma/efectos de los fármacos , Adyuvantes Inmunológicos/uso terapéutico , Humanos , Interferón beta-1a/uso terapéutico , Interferon beta-1b/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patologíaRESUMEN
Body image disturbances are a prominent feature of eating disorders (EDs). Our aim was to test and evaluate a computerized assessment of body image (CABI), to compare the body image disturbances in different ED types, and to assess the factors affecting body image. The body image of 22 individuals undergoing inpatient treatment with restricting anorexia nervosa (AN-R), 22 with binge/purge AN (AN-B/P), 20 with bulimia nervosa (BN), and 41 healthy controls was assessed using the Contour Drawing Rating Scale (CDRS), the CABI, which simulated the participants' self-image in different levels of weight changes, and the Eating Disorder Inventory-2-Body Dissatisfaction (EDI-2-BD) scale. Severity of depression and anxiety was also assessed. Significant differences were found among the three scales assessing body image, although most of their dimensions differentiated between patients with EDs and controls. Our findings support the use of the CABI in the comparison of body image disturbances in patients with EDs vs. CONTROLS: Moreover, the use of different assessment tools allows for a better understanding of the differences in body image disturbances in different ED types.
Asunto(s)
Anorexia Nerviosa/psicología , Imagen Corporal , Bulimia Nerviosa/psicología , Computadores , Autoimagen , Adolescente , Adulto , Ansiedad/complicaciones , Ansiedad/psicología , Estudios de Casos y Controles , Depresión/complicaciones , Depresión/psicología , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Israel , Índice de Severidad de la Enfermedad , Encuestas y Cuestionarios , Adulto JovenRESUMEN
IFNß is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNSα8) that is built on the backbone of a low affinity IFNα but modified to exhibit higher receptor affinity than even for IFNß. Here, YNSα8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via "PASylation." PAS-YNSα8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG35-55-induced experimental autoimmune encephalomyelitis compared with IFNß, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b(+)/CD45(hi) myeloid lineage cells detectable in the CNS, as well as a decrease in IBA(+) cells in spinal cord sections determined by immunohistochemistry for PAS-YNSα8-treated animals. Importantly, PAS-YNSα8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4(+) cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Interferón Tipo I/agonistas , Interferón Tipo I/farmacología , Péptidos/química , Animales , Separación Celular , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Citometría de Flujo , Humanos , Interferón beta/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/tratamiento farmacológico , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Resonancia por Plasmón de Superficie , Resultado del TratamientoRESUMEN
Early in development, infants learn to solve visual problems that are highly challenging for current computational methods. We present a model that deals with two fundamental problems in which the gap between computational difficulty and infant learning is particularly striking: learning to recognize hands and learning to recognize gaze direction. The model is shown a stream of natural videos and learns without any supervision to detect human hands by appearance and by context, as well as direction of gaze, in complex natural scenes. The algorithm is guided by an empirically motivated innate mechanism--the detection of "mover" events in dynamic images, which are the events of a moving image region causing a stationary region to move or change after contact. Mover events provide an internal teaching signal, which is shown to be more effective than alternative cues and sufficient for the efficient acquisition of hand and gaze representations. The implications go beyond the specific tasks, by showing how domain-specific "proto concepts" can guide the system to acquire meaningful concepts, which are significant to the observer but statistically inconspicuous in the sensory input.
Asunto(s)
Percepción Visual , Mano , Humanos , Análisis y Desempeño de TareasRESUMEN
Introduction: Chronic lymphocytic leukemia (CLL) is characterized by an aberrant cytokine network that can support tumor growth by triggering janus kinase (JAK)/STAT pathways. Targeting cytokine-signaling should then be a rational therapeutic strategy but the JAK inhibitor ruxolitinib failed to control and seemingly accelerated the disease in clinical trials. Methods: The effect of ruxolitinib on primary human CLL cells was studied in vitro and in vivo. Results: Ruxolitinib increased phosphorylation of IRAK4, an important toll-like receptor (TLR)- signaling intermediate, in circulating CLL cells in vitro. It also enhanced p38 and NFKB1 phosphorylation while lowering STAT3 phosphorylation in CLL cells activated with TLR-7/8 agonists and IL-2. Among the cytokines made by activated CLL cells, high levels of IL-10 contributed strongly to STAT3 phosphorylation and inhibited TLR7 activity. Ruxolitinib limited TLR-mediated IL10 transcription and markedly reduced IL-10 production in vitro. It also decreased blood levels of IL-10 while increasing TNFα along with phospho-p38 expression and gene sets associated with TLR-activation in CLL cells in vivo. The bruton's tyrosine kinase inhibitor ibrutinib decreased IL-10 production in vitro but, in contrast to ruxolitinib, blocked initial IL10 transcription induced by TLR-signaling in vitro, decreased TNFα production, and deactivates CLL cells in vivo. Discussion: These findings suggest the possible benefits of inhibiting growth factors with JAK inhibitors in CLL are outweighed by negative effects on potential tumor suppressors such as IL-10 that allow unrestrained activation of NFκB by drivers such as TLRs. Specific inhibition of growth-promoting cytokines with blocking antibodies or infusing suppressive cytokines like IL-10 might be better strategies to manipulate cytokines in CLL.
RESUMEN
Delivering medication to the lungs via nebulization of pharmaceuticals is a noninvasive and efficient therapy route, particularly for respiratory diseases. The recent worldwide severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic urges the development of such therapies as an effective alternative to vaccines. The main difficulties in using inhalation therapy are the development of effective medicine and methods to stabilize the biological molecules and transfer them to the lungs efficiently following nebulization. We have developed a high-affinity angiotensin-converting enzyme 2 (ACE2) receptor-binding domain (RBD-62) that can be used as a medication to inhibit infection with SARS-CoV-2 and its variants. In this study, we established a nebulization protocol for drug delivery by inhalation using two commercial vibrating mesh (VM) nebulizers (Aerogen Solo and PARI eFlow) that generate similar mist size distribution in a size range that allows efficient deposition in the small respiratory airway. In a series of experiments, we show the high activity of RBD-62, interferon-α2 (IFN-α2), and other proteins following nebulization. The addition of gelatin significantly stabilizes the proteins and enhances the fractions of active proteins after nebulization, minimizing the medication dosage. Furthermore, hamster inhalation experiments verified the feasibility of the protocol in pulmonary drug delivery. In short, the gelatin-modified RBD-62 formulation in coordination with VM nebulizer can be used as a therapy to cure SARS-CoV-2.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Gelatina , Aerosoles/química , Humanos , Pulmón , SARS-CoV-2RESUMEN
SARS-CoV-2 variants of interest and concern will continue to emerge for the duration of the COVID-19 pandemic. To map mutations in the receptor-binding domain (RBD) of the spike protein that affect binding to angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, we applied in vitro evolution to affinity-mature the RBD. Multiple rounds of random mutagenic libraries of the RBD were sorted against decreasing concentrations of ACE2, resulting in the selection of higher affinity RBD binders. We found that mutations present in more transmissible viruses (S477N, E484K and N501Y) were preferentially selected in our high-throughput screen. Evolved RBD mutants include prominently the amino acid substitutions found in the RBDs of B.1.620, B.1.1.7 (Alpha), B1.351 (Beta) and P.1 (Gamma) variants. Moreover, the incidence of RBD mutations in the population as presented in the GISAID database (April 2021) is positively correlated with increased binding affinity to ACE2. Further in vitro evolution increased binding by 1,000-fold and identified mutations that may be more infectious if they evolve in the circulating viral population, for example, Q498R is epistatic to N501Y. We show that our high-affinity variant RBD-62 can be used as a drug to inhibit infection with SARS-CoV-2 and variants Alpha, Beta and Gamma in vitro. In a model of SARS-CoV-2 challenge in hamster, RBD-62 significantly reduced clinical disease when administered before or after infection. A 2.9 Å cryo-electron microscopy structure of the high-affinity complex of RBD-62 and ACE2, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/administración & dosificación , COVID-19/virología , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Animales , Antivirales/química , COVID-19/genética , COVID-19/metabolismo , Cricetinae , Diseño de Fármacos , Evolución Molecular , Femenino , Humanos , Masculino , Mesocricetus , Simulación de Dinámica Molecular , Mutación , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Receptores Virales/genética , Receptores Virales/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del Virus/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Rapid developments in the fields of learning and object recognition have been obtained by successfully developing and using methods for learning from a large number of labeled image examples. However, such current methods cannot explain infants' learning of new concepts based on their visual experience, in particular, the ability to learn complex concepts without external guidance, as well as the natural order in which related concepts are acquired. A remarkable example of early visual learning is the category of 'containers' and the notion of 'containment'. Surprisingly, this is one of the earliest spatial relations to be learned, starting already around 3â¯month of age, and preceding other common relations (e.g., 'support', 'in-between'). In this work we present a model, which explains infants' capacity of learning 'containment' and related concepts by 'just looking', together with their empirical development trajectory. Learning occurs in the model fast and without external guidance, relying only on perceptual processes that are present in the first months of life. Instead of labeled training examples, the system provides its own internal supervision to guide the learning process. We show how the detection of so-called 'paradoxical occlusion' provides natural internal supervision, which guides the system to gradually acquire a range of useful containment-related concepts. Similar mechanisms of using implicit internal supervision can have broad application in other cognitive domains as well as artificial intelligent systems, because they alleviate the need for supplying extensive external supervision, and because they can guide the learning process to extract concepts that are meaningful to the observer, even if they are not by themselves obvious, or salient in the input.
Asunto(s)
Desarrollo Infantil/fisiología , Aprendizaje/fisiología , Modelos Teóricos , Percepción Espacial/fisiología , Percepción Visual/fisiología , Humanos , LactanteRESUMEN
LIM kinase 1 (LIMK1) is a serine protein kinase that regulates the actin cytoskeleton by phosphorylation and inactivation of actin depolymerizing factor cofilin. LIMK1 activity is regulated by the Rho-GTPases via their serine/threonine kinase effectors Rho-kinase and p21-activated kinases 1 and 4 that phosphorylate LIMK1 on threonine 508 in its activation loop. The purpose of this study was to elucidate the pathway leading to the stability of LIMK1, a protein with a half-life of approximately 20 h. Because the half-life of kinase-dead LIMK1 is only 4 h, it is suggestive that trans- or auto-phosphorylation is responsible for the stabilization of LIMK1. Using known Hsp90 inhibitors, we have shown that the half-life of LIMK1 in cells depends on the presence of active Hsp90. Furthermore, endogenous LIMK1 coimmunoprecipitated with endogenous Hsp90 and this interaction promoted LIMK1 homodimer formation as seen by cross-linking experiments. Hsp90 binds LIMK1 via a recognition sequence within the LIMK1 kinase domain, homologous to that of ErbB-2. Mutation of a proline residue within this sequence to glutamic acid reduces its interaction with Hsp90, inhibits homodimer formation, and reduces its half-life to 4 h. These findings implicate Hsp90 in the stabilization of LIMK1 by promoting homodimer formation and transphosphorylation.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Quinasas/metabolismo , Sustitución de Aminoácidos , Animales , Dominio Catalítico , Dimerización , Activación Enzimática , Estabilidad de Enzimas , Semivida , Quinasas Lim , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genéticaRESUMEN
Type I interferons are multi-potent cytokines that serve as first line of defense against viruses and other pathogens, posses immunomudolatory functions and elicit a growth inhibitory response. In recent years it has been shown that interferons are also detrimental, for example in lupus, AIDS, tuberculosis and cognitive decline, highlighted the need to develop interferon antagonists. We have previously developed the antagonist IFN-1ant, with much reduced binding to the IFNAR1 receptor and enhanced binding to IFNAR2. Here, we further tune the IFN-1ant by producing three additional antagonists based on IFN-1ant but with altered activity profiles. We show that in all three cases the antiproliferative activity of interferons is blocked and the induction of gene transcription of immunomudolatory and antiproliferative associated genes are substantially decreased. Conversely, each of the new antagonists elicits a different degree of antiviral response, STAT phosphorylation and related gene induction. Two of the new antagonists promote decreased activity in relation to the original IFN-1ant, while one of them promotes increased activity. As we do not know the exact causes of the detrimental effects of IFNs, the four antagonists that were produced and analyzed provide the opportunity to investigate the extent of antagonistic and agonistic activity optimal for a given condition.
Asunto(s)
Interferón Tipo I/genética , Proteínas Mutantes/genética , Mutación , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Virus de la Encefalomiocarditis/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/metabolismo , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Receptor de Interferón alfa y beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacosRESUMEN
The neuregulin (NRG)/epidermal growth factor (EGF) family of growth factors consists of several ligands that specifically activate four erbB receptor-tyrosine kinases, namely erbB-1 (EGF-R), erbB-2 (neu), erbB-3, and erbB-4. We have previously shown that islet morphogenesis is impaired and beta-cell differentiation delayed in mice lacking functional EGF-R [EGF-R (-/-)]. The present study aims to clarify which erbB ligands are important for islet development. Pancreatic expression of EGF, TGF-alpha, heparin-binding EGF, betacellulin (BTC), and NRG-4 was detected as early as embryonic d 13 (E13). Effects of these ligands were studied in E12.5 pancreatic explant cultures grown for 5 d ex vivo. None of the growth factors affected the ratio of endocrine to exocrine cells. However, significant effects within the endocrine cell populations were induced by EGF, BTC, and NRG-4. beta-Cell development was augmented by BTC, whereas the development of somatostatin-expressing delta-cells was stimulated by NRG-4. Both ligands decreased the numbers of glucagon-containing alpha-cells. The effect of BTC was abolished in the EGF-R (-/-) mice. A soluble erbB-4 binding fusion protein totally inhibited the effects of NRG-4 but not of BTC. Neutralization of endogenous NRG-4 activity in the model system effectively inhibited delta-cell development, indicating that this erbB4-ligand is an essential factor for delineation of the somatostatin-producing delta-cells. Our results suggest that ligands of the EGF-R/erbB-1 and erbB-4 receptors regulate the lineage determination of islet cells during pancreatic development. BTC, acting through EGF-R/erbB-1, is important for the differentiation of beta-cells. This could be applied in the targeted differentiation of stem cells into insulin-producing cells.
Asunto(s)
Receptores ErbB/fisiología , Islotes Pancreáticos/embriología , Transducción de Señal , Animales , Anticuerpos/farmacología , Betacelulina , Diferenciación Celular , Factor de Crecimiento Epidérmico/análisis , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/análisis , Receptores ErbB/deficiencia , Receptores ErbB/genética , Expresión Génica , Edad Gestacional , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Islotes Pancreáticos/citología , Ratones , Ratones Noqueados , Neurregulinas/análisis , Neurregulinas/genética , Neurregulinas/inmunología , Neurregulinas/farmacología , Técnicas de Cultivo de Órganos , ARN Mensajero/análisis , Receptor ErbB-4 , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador alfa/análisis , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These "HyBNAR" (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNß, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.