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Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.
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Aprendizaje Profundo , Microscopía Confocal/métodos , Microscopía Confocal/normas , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Línea Celular Tumoral , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Humanos , Discos Imaginales/citología , Ratones , Mioblastos/citología , Especificidad de Órganos , Análisis de la Célula Individual , Fijación del TejidoRESUMEN
Fibroblast growth factors (FGF) are essential signaling proteins that regulate diverse cellular functions in developmental and metabolic processes. In Drosophila, the FGF homolog, branchless (bnl) is expressed in a dynamic and spatiotemporally restricted pattern to induce branching morphogenesis of the trachea, which expresses the Bnl-receptor, breathless (btl). Here we have developed a new strategy to determine bnl- expressing cells and study their interactions with the btl-expressing cells in the range of tissue patterning during Drosophila development. To enable targeted gene expression specifically in the bnl expressing cells, a new LexA based bnl enhancer trap line was generated using CRISPR/Cas9 based genome editing. Analyses of the spatiotemporal expression of the reporter in various embryonic stages, larval or adult tissues and in metabolic hypoxia, confirmed its target specificity and versatility. With this tool, new bnl expressing cells, their unique organization and functional interactions with the btl-expressing cells were uncovered in a larval tracheoblast niche in the leg imaginal discs, in larval photoreceptors of the developing retina, and in the embryonic central nervous system. The targeted expression system also facilitated live imaging of simultaneously labeled Bnl sources and tracheal cells, which revealed a unique morphogenetic movement of the embryonic bnl- source. Migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branch. The genetic tool and the comprehensive profile of expression, organization, and activity of various types of bnl-expressing cells described in this study provided us with an important foundation for future research investigating the mechanisms underlying Bnl signaling in tissue morphogenesis.
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Movimiento Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Morfogénesis/genética , Tráquea/metabolismo , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Hipoxia , Hibridación in Situ , Larva/genética , Larva/metabolismo , Microscopía Confocal , Técnicas de Cultivo de Órganos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de Tiempo/métodos , Tráquea/citología , Tráquea/embriologíaRESUMEN
Development creates a vast array of forms and patterns with elegant economy, using a small vocabulary of pattern-generating proteins such as BMPs, FGFs and Hh in similar ways in many different contexts. Despite much theoretical and experimental work, the signaling mechanisms that disperse these morphogen signaling proteins remain controversial. Here, we review the conceptual background and evidence that establishes a fundamental and essential role for cytonemes as specialized filopodia that transport signaling proteins between signaling cells. This evidence suggests that cytoneme-mediated signaling is a dispersal mechanism that delivers signaling proteins directly at sites of cell-cell contact.
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Comunicación Celular/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Morfogénesis/fisiología , Seudópodos/fisiología , Transducción de Señal/fisiología , Animales , Extensiones de la Superficie Celular/fisiología , Drosophila , Discos Imaginales/citología , Modelos Biológicos , Transporte de Proteínas/fisiologíaRESUMEN
Recent findings in several organ systems show that cytoneme-mediated signaling transports signaling proteins along cellular extensions and targets cell-to-cell exchanges to synaptic contacts. This mechanism of paracrine signaling may be a general one that is used by many (or all) cell types in many (or all) organs. We briefly review these findings in this perspective. We also describe the properties of several signaling systems that have previously been interpreted to support a passive diffusion mechanism of signaling protein dispersion, but can now be understood in the context of the cytoneme mechanism. Also watch the Video Abstract.
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Comunicación Celular , Comunicación Paracrina , Transducción de Señal , Animales , Difusión , Matriz Extracelular/metabolismo , Humanos , Modelos BiológicosRESUMEN
Dinoflagellates are microscopic, eukaryotic, and primarily marine plankton. Temporary cyst formation is a well-known physiological response of dinoflagellate cells to environmental stresses. However, the molecular underpinnings of cold-induced cyst physiology have never been described. Cultures of the photosynthetic dinoflagellate Lingulodinium polyedrum readily form temporary cysts when placed at low (8°C±1°C) temperature and excyst to form normal motile cells following a return to normal temperature (18°C±1°C). The normal circadian bioluminescence rhythm and the expected changes in Luciferin Binding Protein abundance were arrested in L. polyedrum cysts. Furthermore, after excystment, the bioluminescence rhythm initiates at a time corresponding to zeitgeber 12, independent of the time when the cells encysted. Phosphoprotein staining after two-dimensional polyacrylamide gel electrophoresis, as well as column-based phosphoprotein enrichment followed by liquid chromatography tandem mass spectrometry, showed cyst proteins are hypophosphorylated when compared with those from motile cells, with the most marked decreases found for predicted Casein Kinase2 target sites. In contrast to the phosphoproteome, the cyst proteome is not markedly different from motile cells, as assessed by two-dimensional polyacrylamide gel electrophoresis. In addition to changes in the phosphoproteome, RNA sequencing revealed that cysts show a significant decrease in the levels of 132 RNAs. Of the 42 RNAs that were identified by sequence analysis, 21 correspond to plastid-encoded gene products and 11 to nuclear-encoded cell wall/plasma membrane components. Our data are consistent with a model in which the highly reduced metabolism in cysts is achieved primarily by alterations in the phosphoproteome. The stalling of the circadian rhythm suggests temporary cysts may provide an interesting model to address the circadian system of dinoflagellates.
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Ritmo Circadiano , Frío , Dinoflagelados/fisiología , Luminiscencia , Fotosíntesis , Proteínas Protozoarias/metabolismo , Relojes Biológicos , Quinasa de la Caseína II/metabolismo , Movimiento Celular , Dinoflagelados/ultraestructura , Regulación hacia Abajo/genética , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Plastidios/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Análisis de Secuencia de ARNRESUMEN
Dinoflagellates are an important component of the marine biota, but a large genome with high-copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.
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Dinoflagelados/genética , Genes Protozoarios/fisiología , ARN Protozoario/genética , Secuencia de Bases , Dinoflagelados/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Protozoario/metabolismo , Transcripción Genética/fisiologíaRESUMEN
BACKGROUND: Almost all cells display circadian rhythms, approximately 24-hour period changes in their biochemistry, physiology or behavior. These rhythms are orchestrated by an endogenous circadian clock whose mechanism is based on transcription-translation feedback loops (TTFL) where the translated products of clock genes act to inhibit their own transcription. RESULTS: We have used RNA-Seq to measure the abundance of all transcripts in an RNA-Seq-derived de novo gene catalog in two different experiments. One compared midday and midnight in a light-dark cycle (ZT6 and ZT18) and under constant light (CT6 and CT18). The second compared four different times (ZT2, ZT6, ZT14 and ZT18) under a light dark cycle. We show here that despite an elaborate repertoire of biological rhythms, the unicellular dinoflagellate Lingulodinium had no detectable daily variation in the abundance of any transcript in an RNA-Seq-derived de novo gene catalog. We also examined the timing of the bioluminescence and photosynthesis rhythms in the presence of the transcription inhibitors actinomycin D and cordycepin. We found that the timing of the two rhythms was unchanged even when transcription rates had decreased to roughly 5% the levels of untreated cells. CONCLUSIONS: The lack of detectable daily variation in transcript levels indicates that the endogenous circadian timer of Lingulodinium does not require rhythmic RNA. If the circadian timer is considered as a limit cycle oscillator, then cellular time in this organism must be defined by variations in state variables that do not include the amount of a clock gene transcript.
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Relojes Circadianos , Ritmo Circadiano , Dinoflagelados/genética , Dactinomicina/química , Desoxiadenosinas/química , Luz , Fotoperiodo , Inhibidores de la Síntesis de la Proteína/química , ARN/genética , ARN/aislamiento & purificación , Análisis de Secuencia de ARNRESUMEN
Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.
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Pregnenolona , Células Th2 , Pregnenolona/metabolismo , Pregnenolona/farmacología , Células Th2/metabolismo , Simulación de Dinámica Molecular , Esteroides , Proteínas Portadoras/metabolismoRESUMEN
OBJECTIVES: From the treatment of damaged teeth to replacing missing teeth, dental biomaterials cover the scientific interest of many fields. Dental biomaterials are one of the implants whose effective life depends vastly on their material and manufacturing techniques. The purpose of this review is to summarize the important aspects for metallic dental implants from biomedical, mechanical and materials science perspectives. The review article will focus on five major aspects as mentioned below. Tooth anatomy: Maximizing the implant performance depends on proper understanding of human tooth anatomy and the failure behavior of the implants. Major parts from tooth anatomy including saliva characteristics are explored in this section. Wear mechanisms: The prominent wear mechanisms having a high impact on dental wear are abrasive, adhesive, fatigue and corrosion wear. To imitate the physiological working condition of dental implants, reports on the broad range of mastication force and various composition of artificial saliva have been included in this section, which can affect the tribo-corrosion behavior of dental implants. Dental implants classifications: The review paper includes a dedicated discussion on major dental implants types and their details for better understanding their applicability and characteristics. Implant materials: As of today, the most established dental implant materials are SS316L, cobalt chrome alloy and titanium. Detailed discussion on their material properties, microstructures, phase transformations and chemical compositions have been discussed here. Manufacturing techniques: In terms of different production methods, the lost wax casting method as traditional manufacturing is considered. Selective Laser Melting (SLM) and Directed Energy Deposition (DED) as additive manufacturing techniques (AM) have been discussed. For AM, the relationships between process-property-performance details have been explored briefly. The effectiveness of different manufacturing techniques was compared based on porosity distribution, mechanical and biomechanical properties. SUMMARY: Despite having substantial research available on dental implants, there is a lack of systematic reviews to present a holistic viewpoint combining state-of-the-art from biomedical, mechanical, materials science and manufacturing perspectives. This review article attempts to combine a wide variety of analyzing approaches from those interdisciplinary fields to deliver deeper insights to researchers both in academia and industry to develop next-generation dental implants.
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Dinoflagellate clocks are unique as they show no resemblance to any known model eukaryotic or prokaryotic clock architecture. Dinoflagellates are unicellular, photosynthetic, primarily marine eukaryotes are known for their unique biology and rhythmic physiology. Their physiological rhythms are driven by an internal oscillator whose molecular underpinnings are yet unknown. One of the primary reasons that slowed the progression of their molecular studies is their extremely large and repetitive genomes. Dinoflagellates are primary contributors to the global carbon cycle and oxygen levels, therefore, comprehending their internal clock architecture and its interaction with their physiology becomes a subject of utmost importance. The advent of high throughput Omics technology provided the momentum to understand the molecular architecture and functioning of the dinoflagellate clocks. We use these extensive databases to perform meta-analysis to reveal the status of clock components in dinoflagellates. In this article, we will delve deep into the various "Omics" studies that catered to various breakthroughs in the field of circadian biology in these organisms that were not possible earlier. The overall inference from these omics studies points toward an uncommon eukaryotic clock model, which can provide promising leads to understand the evolution of molecular clocks.
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How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and promotes target-specific cytoneme contacts and contact-dependent FGF release. FGF-source and ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through cell-adhesion-molecule (CAM)-like FGF-FGFR binding. Contact-mediated FGF-FGFR interactions induce bidirectional responses in ASP and source cells that, in turn, polarize FGF-sending and FGF-receiving cytonemes toward each other to reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion.
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Proteínas de Drosophila , Discos Imaginales , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Discos Imaginales/metabolismo , Alas de Animales/metabolismoRESUMEN
Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Niche-specific AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Músculos/metabolismoRESUMEN
Bacterial FtsE gene codes for the ATP-binding protein, FtsE, which in complex with the transmembrane protein, FtsX, participates in diverse cellular processes. Therefore, regulated expression of FtsE and FtsX might be critical to the human pathogen, Mycobacterium tuberculosis, under stress conditions. Although ftsX gene of M. tuberculosis (MtftsX) is known to be transcribed from a promoter inside the upstream gene, ftsE, the transcriptional status of ftsE gene of M. tuberculosis (MtftsE) remains unknown. Therefore, the authors initiated transcriptional analyses of MtftsE, using total RNA from M. tuberculosis cells that were grown under stress conditions, which the pathogen is exposed to, in granuloma in tuberculosis patients. Primer extension experiments showed the presence of putative transcripts, T1, T2, T3, and T4. T1 originated from the intergenic region between the upstream gene, MRA_3135, and MtftsE. T2 and T3 were found initiated from within MRA_3135. T4 was transcribed from a region upstream of MRA_3135. RT-PCR confirmed co-transcription of MRA_3135 and MtftsE. The cloned putative promoter regions for T1, T2, and T3 elicited transcriptional activity in Mycobacterium smegmatis transformants. T1, T2, and T3, but no new transcript, were present in the M. tuberculosis cells that were grown under the stress conditions, which the pathogen is exposed to in granuloma in tuberculosis patients. It showed lack of modulation of MtftsE transcripts under the stress conditions tested, indicating that ftsE may not have a stress response-specific function in M. tuberculosis.
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Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Transcripción Genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ADN Intergénico , Humanos , Datos de Secuencia Molecular , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Regiones Promotoras Genéticas , Tuberculosis/microbiologíaRESUMEN
Echinococcus granulosus causes a zoonotic infection called Cystic Echinococcosis (CE). Surgeons meet with hydatid cysts of the liver and lungs with reasonable frequency. However hydatid cyst may also appear in other parts of the body. A 30 years old lady presented with a smooth slow-growing subcutaneous nodule on the anteromedial side of the right thigh with no detectable primary site in the liver or lung. The case was subsequently diagnosed as hydatid cyst of muscle and radical surgery was done under coverage of anihelminthic drug. The common practice in this type of case is to do fine needle aspiration cytology (FNAC) taking the lesion to be a soft tissue neoplasm. The aim of this case presentation is to be aware of that in case of a diffuse non-tender swelling with history of gradual increase in size, hydatid cyst should be considered in the differential diagnosis.
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Equinococosis/diagnóstico , Echinococcus granulosus/aislamiento & purificación , Enfermedades Musculares/diagnóstico , Adulto , Animales , Diagnóstico Diferencial , Equinococosis/cirugía , Femenino , Humanos , Enfermedades Musculares/parasitología , MusloRESUMEN
Pregnenolone (P5) promotes prostate cancer cell growth, and de novo synthesis of intratumoural P5 is a potential cause of development of castration resistance. Immune cells can also synthesize P5 de novo. Despite its biological importance, little is known about P5's mode of actions, which appears to be context dependent and pleiotropic. A comprehensive proteome-wide spectrum of P5-binding proteins that are involved in its trafficking and functionality remains unknown. Here, we describe an approach that integrates chemical biology for probe synthesis with chemoproteomics to map P5-protein interactions in live prostate cancer cells and murine CD8+ T cells. We subsequently identified P5-binding proteins potentially involved in P5-trafficking and in P5's non-genomic action that may drive the promotion of castrate-resistance prostate cancer and regulate CD8+ T cell function. We envisage that this methodology could be employed for other steroids to map their interactomes directly in a broad range of living cells, tissues, and organisms.
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This work investigated the linear thermal expansion properties of a multi-material specimen fabricated with Invar M93 and A36 steel. A sequence of tests was performed to investigate the viability of additively manufactured Invar M93 for lowering the coefficient of thermal expansion (CTE) in multi-material part tooling. Invar beads were additively manufactured on a steel base plate using a fiber laser system, and samples were taken from the steel, Invar, and the interface between the two materials. The CTE of the samples was measured between 40 °C and 150 °C using a thermomechanical analyzer, and the elemental composition was studied with energy dispersive X-ray spectroscopy. The CTE of samples taken from the steel and the interface remained comparable to that of A36 steel; however, deviations between the thermal expansion values were prevalent due to element diffusion in and around the heat-affected zone. The CTEs measured from the Invar bead were lower than those from the other sections with the largest and smallest thermal expansion values being 10.40 µm/m-K and 2.09 µm/m-K. In each of the sections, the largest CTE was measured from samples taken from the end of the weld beads. An additional test was performed to measure the aggregate expansion of multi-material tools. Invar beads were welded on an A36 steel plate. The invar was machined, and the sample was heated in an oven from 40 °C and 160 °C. Strain gauges were placed on the surface of the part and were used to analyze how the combined thermal expansions of the invar and steel would affect the thermal expansion on the surface of a tool. There were small deviations between the expansion values measured by gauges placed in different orientations, and the elongation of the sample was greatest along the dimension containing a larger percentage of steel. On average, the expansion of the machined Invar surface was 42% less than the expansion of the steel surface. The results of this work demonstrate that additively manufactured Invar can be utilized to decrease the CTE for multi-material part tooling.
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Gold (Au) and copper (Cu)-based nanostructures are of great interest due to their applicability in various areas including catalysis, sensing and optoelectronics. Nanostructures synthesized by the galvanic displacement method often lead to non-uniform density and poor size distribution. Here, density and size-controlled synthesis of Au and Cu-based nanostructures was made possible by galvanic displacement with limited exposure to hydrofluoric (HF) acid and the use of surfactants like L-cysteine (L-Cys) and cetyltrimethylammonium bromide (CTAB). An approach involving cyclic exposure to HF acid regulated the nanostructure density. Further, the use of surfactants generated monodisperse nanoparticles in the initial stages of the deposition with increased density. The characterization of Au and Cu-based nanostructures was performed by scanning electron microscopy, atomic force microscopy, UV-Visible spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and X-ray diffraction. The surface enhanced Raman spectroscopic measurements demonstrated an increase in the Raman intensity by two to three orders of magnitude for analyte molecules like Rhodamine 6G dye and paraoxon.
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Wire-based metal additive manufacturing utilizes the ability of additive manufacturing to fabricate complex geometries with high deposition rates (above 7 kg/h), thus finding applications in the fabrication of large-scale components, such as stamping dies. Traditionally, the workhorse materials for stamping dies have been martensitic steels. However, the complex thermal gyrations induced during additive manufacturing can cause the evolution of an inhomogeneous microstructure, which leads to a significant scatter in the mechanical properties, especially the toughness. Therefore, to understand these phenomena, arc-based additive AISI 410 samples were fabricated using robotic gas metal arc welding (GMAW) and were subjected to a detailed characterization campaign. The results show significant scatter in the tensile properties as well as Charpy V-notch impact toughness data, which was then correlated to the microstructural heterogeneity and delta (δ) ferrite formation. Post-processing (austenitizing and tempering) treatments were developed and an ~70% reduction in the scatter of tensile data and a four-times improvement in the toughness were obtained. The changes in mechanical properties were rationalized based on the microstructure evolution during additive manufacturing. Based on these, an outline to tailor the composition of "printable" steels for tooling with isotropic and uniform mechanical properties is presented and discussed.
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Furin is an evolutionarily conserved proprotein convertase (PC) family enzyme with a broad range of substrates that are essential for developmental, homeostatic, and disease pathways. Classical genetic approaches and in vitro biochemical or cell biological assays identified that precursor forms of most growth factor family proteins are processed by Furin. To quantitatively assess the potential role of Furin in cleaving and modulating intercellular dispersion of a Drosophila signaling protein, we developed a simple assay by combining genetics, ex vivo organ culture, pharmacological treatment, and imaging analyses. The protocol herein describes how to ex vivo culture Drosophila wing imaginal discs expressing a fluorescently tagged Drosophila Fibroblast Growth Factor (FGF, Branchless/Bnl) over a long period of time in the presence of Furin inhibitors and monitor the cleavage and intercellular dispersion of the truncated Bnl parts using microscopy. Although the assay described here is for assessing the effect of Furin inhibition on Bnl cleavage in the Drosophila larval wing imaginal disc, the principle and methodology can easily be adopted for any other signals, tissue systems, or organisms. This strategy and protocol provide an assay for examining Furin activity on a specific substrate by directly visualizing the spatiotemporal distribution of its truncated parts in an ex vivo-cultured organ.
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How morphogenetic signals are prepared for intercellular dispersal and signaling is fundamental to the understanding of tissue morphogenesis. We discovered an intracellular mechanism that prepares Drosophila melanogaster FGF Branchless (Bnl) for cytoneme-mediated intercellular dispersal during the development of the larval Air-Sac-Primordium (ASP). Wing-disc cells express Bnl as a proprotein that is cleaved by Furin1 in the Golgi. Truncated Bnl sorts asymmetrically to the basal surface, where it is received by cytonemes that extend from the recipient ASP cells. Uncleavable mutant Bnl has signaling activity but is mistargeted to the apical side, reducing its bioavailability. Since Bnl signaling levels feedback control cytoneme production in the ASP, the reduced availability of mutant Bnl on the source basal surface decreases ASP cytoneme numbers, leading to a reduced range of signal/signaling gradient and impaired ASP growth. Thus, enzymatic cleavage ensures polarized intracellular sorting and availability of Bnl to its signaling site, thereby determining its tissue-specific intercellular dispersal and signaling range.