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Purpose: The effects of FLASH-level dose rates delivered at the spread-out Bragg peak (SOBP) on normal tissue damage in mice were investigated. Materials and Methods: Fifty nontumor-bearing mice received abdominal irradiation, 30 at FLASH dose rates (100 Gy/s) and 20 at conventional dose rates (0.1 Gy/s). Total dose values ranged from 10 to 19 Gy, delivered in a single spot by a synchrocyclotron proton therapy system. Centered on the abdomen, the collimated field delivered was an 11-mm diameter circle with a water-equivalent depth of 2.4 cm from entrance to distal 80% dose. A ridge filter was used to provide dose uniformity over the full 2.4-cm range. The spatial distribution was identical for both the FLASH and conventional deliveries. Results: Overall survival and individual mouse weights were tracked for 21 days after the exposure date, and LD50 values were compared for the FLASH and conventional dose rate groups. Mice exposed to FLASH dose rates had a higher LD50 value as compared with mice exposed to conventional dose rates, with a dose-dependent improvement in survivability of 10% to 20%. The FLASH cohort also showed greater or equal percent population survival for each day of the study. Conclusion: These results are preliminary confirmation of the potential for the combination of the advantages of the Bragg peak with the normal tissue sparing benefits of FLASH treatments. This experiment also confirms that pulsed synchrocyclotrons can be used for the purpose of FLASH research and treatment.
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Neoadjuvant immunotherapy can induce immune responses within the tumor microenvironment. Gene expression can be used to assess responses with limited amounts of conventionally-fixed patient-derived samples. We aim to assess the cross-platform concordance of immune-related gene expression data. We performed comparisons across three panels in two platforms: Nanostring nCounter® PanCancer Immune Profiling Panel (nS), HTG EdgeSeq Oncology Biomarker Panel (HTG OBP) and Precision Immuno-Oncology Panel (HTG PIP). All tissue samples of 14 neoadjuvant GM-CSF treated, 14 neoadjuvant Provenge treated, and 12 untreated prostate cancer patients were radical prostatectomy (RP) tissues, while 6 prostatitis patients and 6 non-prostatitis subjects were biopsies. For all 52 patients, more than 90% of the common genes were significantly correlated (p < 0.05) and more than 76% of the common genes were highly correlated (r > 0.5) between any two panels. Co-inertia analysis also demonstrated high overall dataset structure similarity (correlation>0.84). Although both dimensionality reduction visualization analysis and unsupervised hierarchical cluster analysis for highly correlated common genes (r > 0.9) suggested a high-level of consistency across the panels, there were subsets of genes that were differentially expressed across the panels. In addition, while the effect size of the differential testing for neoadjuvant treated vs. untreated localized prostate cancer patients across the panels were significantly correlated, some genes were only differentially expressed in the HTG panels. Finally, the HTG PIP panel had the best classification performance among the 3 panels. These differences detected may be a result of the different panels or platforms due to their technical setting and focus. Thus, researchers should be aware of those potential differences when deciding which platform and panel to use.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/fisiología , Terapia Neoadyuvante/métodos , Próstata/metabolismo , Neoplasias de la Próstata/terapia , Biología Computacional , Conjuntos de Datos como Asunto , Perfilación de la Expresión Génica , Humanos , Inmunidad Celular/genética , Masculino , Nanoestructuras , Próstata/patología , Prostatectomía , TranscriptomaRESUMEN
Taking advantage of Bragg peak and small spot size, pencil beam scanning proton therapy can deliver a highly conformal dose distribution to target while sparing normal tissues. However, such dose distributions can be highly sensitive to the proton range uncertainty which can reach 5% or higher in lung tissue. One proposed method for reducing range uncertainty is to measure the water equivalent path length (WEPL) by proton radiography. In this study, we followed a newly proposed proton beam radiography technique based on energy resolved dose functions (ERDF) to construct a Monte Carlo model for a single detector energy-resolved proton radiography system (SDPRS). This SDPRS model was constructed in the Monte Carlo software package TOPAS (TOol for PArticle Simulation) and it includes the Mevion HYPERSCAN™ pencil beam scanning treatment head and a 2D dose detector positioned downstream as the imager. A calibration phantom containing a number of tissue equivalent materials was simulated to evaluate the accuracy in WEPL measurement by SDPRS. The mean deviation of the obtained relative stopping power (RSP) from the reference values was 0.31%. Proton radiographs of an anthropomorphic head phantom were also generated to demonstrate the clinical relevance of the technique. Effects of different energy layer spacing and measurement noise were also studied.
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Cabeza/diagnóstico por imagen , Método de Montecarlo , Fantasmas de Imagen , Protones , Radiografía/métodos , Calibración , HumanosRESUMEN
OBJECTIVE:: To investigate whether the Mevion S250i with HYPERSCAN clinical proton system could be used for pre-clinical research with millimetric beams. METHODS:: The nozzle of the proton beam line, consisting of an energy modulation system (EMS) and an adaptive aperture (AA), was modelled with the TOPAS Monte Carlo Simulation Toolkit. With the EMS, the 230 MeV beam nominal range can be decreased in multiples of 2.1 mm. Monte Carlo dose calculations were performed in a mouse lung tumour CT image. The AA allows fields as small as 5 × 1 mm2 to be used for irradiation. The best plans to give 2 Gy to the tumour were derived from a set of discrete energies allowed by the EMS, different field sizes and beam directions. The final proton plans were compared to a precision photon irradiation plan. Treatment times were also assessed. RESULTS:: Seven different proton beam plans were investigated, with a good coverage to the tumour (D95 > 1.95 Gy, D5 < 2.3 Gy) and with potentially less damage to the organs at risk than the photon plan. For very small fields and low energies, the number of protons arriving to the target drops to 1-3%, nevertheless the treatment times would be below 5 s. CONCLUSION:: The proton plans made in this study, collimated by an AA, could be used for animal irradiation. ADVANCES IN KNOWLEDGE:: This is one of the first study to demonstrate the feasibility of pre-clinical research with a clinical proton beam with an adaptive aperture used to create small fields.
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Neoplasias Pulmonares/radioterapia , Terapia de Protones/métodos , Dosificación Radioterapéutica/veterinaria , Planificación de la Radioterapia Asistida por Computador/métodos , Animales , Simulación por Computador , Estudios de Factibilidad , Ratones , Método de Montecarlo , Terapia de Protones/instrumentación , Terapia de Protones/veterinaria , Planificación de la Radioterapia Asistida por Computador/veterinaria , Tomografía Computarizada por Rayos X/métodosRESUMEN
OBJECTIVE: To compare the biomechanical strength and histologic features of 3-0 Glycomer™ 631 barbed suture (V-LOC™ 90 Absorbable Wound Closure Device, Covidien, Mansfield, MA) to non-barbed 3-0 Glycomer™ 631 suture (Biosyn™, Covidien) for intradermal skin wound closure in the dog. STUDY DESIGN: Randomized, factorial, in vivo. ANIMALS: Eighteen purpose-bred, mature male, and female hound dogs. METHODS: Eighteen adult hound dogs were randomly assigned to 1 of 3 groups designated by postoperative day of assessment. Six skin incisions were made along the dorsum in the thoracolumbar region of each dog with an equal number (n=3) randomly assigned to closure with barbed or non-barbed suture. Six dogs were euthanatized on postoperative days 3, 10, and 14, respectively. Two additional incisions were made on each dog after euthanasia for baseline data (Day 0). The skin incision specimens were harvested for biomechanical testing and histologic evaluation. RESULTS: Non-barbed closure had significantly higher maximum load at failure (P<.001) and stiffness (P<.001) than barbed closure regardless of day. The average tissue reaction score was significantly higher for barbed closure (P=.008), regardless of day. Suturing time for barbed closures was significantly shorter. There was no significant difference in frequency of complications between closures. CONCLUSION: Barbed Glycomer™ 631 closures had a significantly lower maximum load at failure and stiffness, and higher average tissue reaction scores, but showed no difference in short term outcome for intradermal closure of dorsally located skin incisions in dogs.
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Perros/cirugía , Piel/patología , Técnicas de Sutura/veterinaria , Suturas/veterinaria , Cicatrización de Heridas , Animales , Fenómenos Biomecánicos , Procedimientos Quirúrgicos Dermatologicos/veterinaria , Femenino , Masculino , Ensayo de MaterialesRESUMEN
Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.
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Cadherinas/metabolismo , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Gastrulación , Secuencia de Aminoácidos , Animales , Cadherinas/química , Movimiento Celular , Pollos , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Humanos , Mesodermo/citología , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Transforming growth factor-beta (TGFß) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFß signaling is propagated through one of three TGFß ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFß signaling is regulated partly by the combinatorial expression patterns of TGFß receptors and ligands, a comprehensive gene expression analysis has not been published. RESULTS: Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFß ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFß signaling. CONCLUSIONS: TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis.
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Proteínas Aviares/biosíntesis , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Proteínas Aviares/genética , Embrión de Pollo , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Survivin, a member of the inhibitor of apoptosis protein (IAP) family proteins, has essential roles in cell division and inhibition of apoptosis. Several clinical studies in cancer patients have shown that the elevated levels of survivin correlate with aggressiveness of the disease and resistance to radiation and chemotherapeutic treatments. Survivin is an integral component of chromosomal passenger complex (CPC) where it binds to borealin and INCENP through its dimerization interface. Thus, disruption of functional survivin along its dimer interface with a small molecule is hypothesized to inhibit the proliferation of cancer cells and sensitize them to therapeutic agents and radiation. Recently, a small molecule (Abbott8) was reported to bind at the dimerization interface of survivin. Further development of this compound was accomplished by computational modeling of the molecular interactions along the dimerization interface, which has led to the design of promising survivin dimerization modulators. Two of the most potent survivin modulators, LLP3 and LLP9 at concentrations between 50 and 100nM, caused delay in mitotic progression and major mitotic defects in proliferating human umbilical vein endothelial cells (HUVEC) and prostate cancer cells (PC3).
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Clorofenoles/química , Clorofenoles/farmacología , Diseño de Fármacos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Mitosis/efectos de los fármacos , Piridonas/química , Piridonas/farmacología , Antineoplásicos/farmacología , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dimerización , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Modelos Moleculares , Survivin , Factores de TiempoRESUMEN
Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual's response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after therapeutic radiation.
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Bioensayo/métodos , MicroARNs/sangre , Radiometría/métodos , Suero/metabolismo , Animales , Biomarcadores/sangre , Exposición a Riesgos Ambientales/efectos adversos , Exposición a Riesgos Ambientales/análisis , Humanos , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Dosis de Radiación , Radiación Ionizante , Liberación de Radiactividad PeligrosaRESUMEN
Cell-matrix adhesion strongly influences developmental signaling. Resulting impacts on cell migration and tissue morphogenesis are well characterized. However, the in vivo impact of adhesion on fate induction remains ambiguous. Here, we employ the invertebrate chordate Ciona intestinalis to delineate an essential in vivo role for matrix adhesion in heart progenitor induction. In Ciona pre-cardiac founder cells, invasion of the underlying epidermis promotes localized induction of the heart progenitor lineage. We found that these epidermal invasions are associated with matrix adhesion along the pre-cardiac cell/epidermal boundary. Through targeted manipulations of RAP GTPase activity, we were able to manipulate pre-cardiac cell-matrix adhesion. Targeted disruption of pre-cardiac cell-matrix adhesion blocked heart progenitor induction. Conversely, increased matrix adhesion generated expanded induction. We were also able to selectively restore cell-matrix adhesion and heart progenitor induction through targeted expression of Ci-Integrin ß2. These results indicate that matrix adhesion functions as a necessary and sufficient extrinsic cue for regional heart progenitor induction. Furthermore, time-lapse imaging suggests that cytokinesis acts as an intrinsic temporal regulator of heart progenitor adhesion and induction. Our findings highlight a potentially conserved role for matrix adhesion in early steps of vertebrate heart progenitor specification.
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Polaridad Celular/fisiología , Uniones Célula-Matriz/fisiología , Ciona intestinalis/embriología , Inducción Embrionaria , Corazón/embriología , Células Madre/fisiología , Animales , Animales Modificados Genéticamente , Adhesión Celular/genética , Adhesión Celular/fisiología , Polaridad Celular/genética , Uniones Célula-Matriz/genética , Uniones Célula-Matriz/metabolismo , Cordados/embriología , Cordados/genética , Cordados/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Embrión no Mamífero , Inducción Embrionaria/genética , Inducción Embrionaria/fisiología , Invertebrados/embriología , Invertebrados/genética , Invertebrados/metabolismo , Modelos Biológicos , Células Madre/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/fisiologíaRESUMEN
RATIONALE: The aim of this study was to investigate the mass spectral fragmentation of a small set of stimulants in a high-resolution time-of-flight mass spectrometer equipped with a soft ionization source using vacuum ultraviolet (VUV) photons emitted from different plasma gases. It was postulated that the use of a plasma gas such as Xe, which emits photons at a lower energy than Kr or Ar, would lead to softer ionization of the test compounds, and thus to less fragmentation. METHODS: A set of nine stimulants: cocaine, codeine, nicotine, methadone, phenmetrazine, pentylenetetrazole, niketamide, fencamfamine, and caffeine, was analyzed by gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) in positive ion mode with this soft ionization source, using either Xe, Kr, or Ar as plasma gases. Working solutions of the test compounds at 0.1 to 100 ng/µL were used to establish instrument sensitivity and linearity. RESULTS: All test compounds, except methadone and pentylenetetrazole, exhibited strong molecular ions and no fragmentation with Xe-microplasma photoionization (MPPI). Methadone exhibited significant fragmentation not only with Xe, but also with Kr and Ar, and pentylenetetrazole could not be ionized with Xe, probably because its ionization energy is above 8.44 eV. The Kr- and Ar-MPPI mass spectra of the test compounds showed that the relative intensity of the molecular ion decreased as the photon energy increased. CONCLUSIONS: When coupled to a TOF mass spectrometer this soft ionization source has demonstrated signal-to-noise (S/N) ratios from 7 to 730 at 100 pg per injection (depending on the compound), and a dynamic range of three orders of magnitude (100 pg to 100 ng) for some of the test compounds.
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Estimulantes del Sistema Nervioso Central/análisis , Cromatografía de Gases y Espectrometría de Masas/instrumentación , Cafeína/análisis , Cocaína/análisis , Codeína/análisis , Inhibidores de Captación de Dopamina/análisis , Diseño de Equipo , Antagonistas del GABA/análisis , Estimulantes Ganglionares/análisis , Iones/química , Metadona/análisis , Narcóticos/análisis , Nicotina/análisis , Niquetamida/análisis , Norbornanos/análisis , Pentilenotetrazol/análisis , Fenmetrazina/análisis , Sensibilidad y EspecificidadRESUMEN
Mobile health apps are proliferating, but they fail to deliver on a key patient and caregiver requirement - the ability to collaborate using key phone features while leveraging existing web services. Typically, mobile health apps are for single use, proprietary, and deliver closed-world solutions. By making use of web services, both open and proprietary, mobile health apps can be created to support the caregiver network in the community. The full value of telehealth will only be achieved when the spectrum of trusted health care services (preventive, promotion, curative, and rehabilitative) is delivered to the collaborating network of caregivers.
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Teléfono Celular , Computadoras de Mano , Conducta Cooperativa , Registros Electrónicos de Salud , Almacenamiento y Recuperación de la Información/métodos , Medicina de Precisión/métodos , Programas Informáticos , Telemedicina/métodosRESUMEN
Survivin is a critical regulator of mitosis, and an inhibitor of apoptosis which is overexpressed in almost all cancers. In the current study, cell cycle profiles of normal proliferating human umbilical vein endothelial cells, prostate cancer, and lung cancer cell lines expressing varying levels of survivin and its splice variants were compared using a novel functional complementation assay. Defects in chromosome segregation and cytokinesis that were observed after depletion of endogenous survivin were not complemented by any of the survivin splice variants: survivin-2B, survivin-3B, survivin-ΔEx3, or survivin-2A when expressed exogenously at a level comparable to endogenous full-length survivin. Survivin variants were not detectable at the endogenous protein level. Cancer cells with higher levels of full-length survivin and survivin-2B expression, exhibited reduced caspase-3 activation following doxorubicin treatment and radiation. Whereas earlier studies focused on function and expression levels of survivin specific to cancer cells, the current study brings forward the essential role of survivin in normal dividing cells. Full-length survivin was found to be associated with Aurora-B kinase in the chromosomal passenger complex and depletion of survivin mimics mitotic phenotypes observed after Aurora-B kinase inhibition, in cancer as well as normal proliferating cells. Thus, our study establishes survivin as a marker of proliferation, rather than a cancer specific marker. Therefore, systemic therapeutic interventions targeting survivin will affect cancer as well as normal proliferating cells.
RESUMEN
Cells must make appropriate fate decisions within a complex and dynamic environment. In vitro studies indicate that the cytoskeleton acts as an integrative platform for this environmental input. External signals regulate cytoskeletal dynamics and the cytoskeleton reciprocally modulates signal transduction. However, in vivo studies linking cytoskeleton/signalling interactions to embryonic cell fate specification remain limited. Here we show that the cytoskeleton modulates heart progenitor cell fate. Our studies focus on differential induction of heart fate in the basal chordate Ciona intestinalis. We have found that differential induction does not simply reflect differential exposure to the inductive signal. Instead, pre-cardiac cells employ polarized, invasive protrusions to localize their response to an ungraded signal. Through targeted manipulation of the cytoskeletal regulator CDC42, we are able to depolarize protrusive activity and generate uniform heart progenitor fate specification. Furthermore, we are able to restore differential induction by repolarizing protrusive activity. These findings illustrate how bi-directional interactions between intercellular signalling and the cytoskeleton can influence embryonic development. In particular, these studies highlight the potential for dynamic cytoskeletal changes to refine cell fate specification in response to crude signal gradients.
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Polaridad Celular/fisiología , Citoesqueleto/metabolismo , Corazón/embriología , Animales , Animales Modificados Genéticamente , Linaje de la Célula/fisiología , Ciona intestinalis/citología , Ciona intestinalis/embriología , Inducción Embrionaria , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Modelos Biológicos , Transducción de Señal , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Ilyanassa obsoleta is a marine gastropod that is a long-standing and very useful model for studies of embryonic development. It is especially important as a model for the spiralian development program, a distinctive mode of early development shared by a large group of animal phyla, but poorly understood. Ilyanassa adults are readily obtainable and easy to keep in the laboratory, and they produce large numbers of embryos throughout most of the year. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. In this article, we present an overview of aspects of its biology and use as a model organism.
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Biología Evolutiva/métodos , Modelos Animales , Caracoles/embriología , Caracoles/fisiología , Animales , Técnicas Genéticas , Genómica , Modelos Anatómicos , Caracoles/genética , Factores de TiempoRESUMEN
The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa adults are readily obtainable and easy to keep in the laboratory. Although the normal spawning season for Ilyanassa is during early summer, they can produce high-quality embryos nearly year-round in the laboratory. Snails collected in the late fall, winter, or spring can be induced to deposit zygotes before the natural spawning season by warming them to room temperature, and snails collected before the natural spawning season can be made to postpone zygote deposition until needed (up to at least 6 mo) by maintaining them in tanks in a cold room at 4 degrees C-8 degrees C. This protocol describes how to induce embryo production in Ilyanassa snails, collect the embryos, and rear them to the stage required for study.
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Biología Evolutiva/métodos , Caracoles/embriología , Caracoles/fisiología , Animales , Femenino , Modelos Anatómicos , Estaciones del Año , Temperatura , Factores de Tiempo , Cigoto/fisiologíaRESUMEN
The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa adults are readily obtainable and easy to keep in the laboratory, and they can produce high-quality embryos nearly year-round in the laboratory. After hatching from capsules, larval Ilyanassa can be maintained in culture, feeding on single-celled algae. The larvae will become competent to undergo metamorphosis after approximately 3 wk in culture. Metamorphosis can be induced artificially by treating with either the neurotransmitter serotonin or the nitric oxide synthase inhibitor 7-nitroindazole. Both of these reagents have been shown to induce metamorphosis in >75% of larvae within 48 h. This protocol describes the induction of metamorphosis in snail larvae.
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Biología Evolutiva/métodos , Técnicas Genéticas , Larva/genética , Metamorfosis Biológica/genética , Caracoles/embriología , Caracoles/fisiología , Animales , Diseño de Equipo , Larva/crecimiento & desarrollo , Modelos BiológicosRESUMEN
The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques, as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Intracellular microinjection is an important tool, especially for lineage tracing and perturbations of specific genes by knockdown approaches and synthetic mRNA injections. Two methods for the introduction of lineage tracers into particular cells are routine in Ilyanassa. Iontophoresis of charged molecules, such as fluorophore-dextran conjugates can be accomplished using a simply built current generator. Injection of an oil-based solution containing the fluorescent probe 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) is also straightforward. However, injection of oil-based solutions and iontophoresis have not been useful for delivering water-soluble reagents to perturb gene function, and pressure injection of aqueous solutions has been more challenging. This protocol describes a recently optimized procedure for the pressure injection of aqueous solutions into Ilyanassa embryos and zygotes with high rates of survival and normal development. The key parameters seem to be the injection needles, injection media, and the stage of injected embryos.
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Biología Evolutiva/métodos , Técnicas Genéticas , Larva/genética , Microinyecciones/métodos , Caracoles/embriología , Caracoles/fisiología , Animales , Carbocianinas/química , Dextranos/química , Colorantes Fluorescentes/química , Larva/crecimiento & desarrollo , ARN Mensajero/metabolismo , Agua/químicaRESUMEN
The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques, as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa embryos are particularly well suited for RNA and protein localization studies because of the relatively large cells and favorable properties for imaging. This protocol describes how to fix and store Ilyanassa embryos and larvae for use in whole-mount in situ hybridization and immunohistochemical studies.
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Biología Evolutiva/métodos , Embrión no Mamífero/embriología , Técnicas Genéticas , Larva/genética , Caracoles/embriología , Caracoles/fisiología , Animales , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Larva/crecimiento & desarrollo , Metamorfosis Biológica , ARN/metabolismoRESUMEN
The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa is host to several species of parasitic trematode worms, so care must be taken to avoid contamination of Ilyanassa genomic DNA with that of the parasites. The easiest way to avoid this contamination is to isolate DNA from veliger larvae, which are not parasitized. This also avoids other problems that can be encountered when isolating DNA from adult mollusc tissues, such as the presence of large amounts of polysaccharides. This protocol describes the isolation of genomic DNA from Ilyanassa larvae.