Project description:Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.
Project description:The morphological analysis of the end of boron nitride nanotubes (BNNTs) using high-resolution transmission electron microscopy (HR-TEM) can provide valuable insight into the growth mechanism in high temperature pressure (HTP) laser ablation where the best quality of BNNT materials can be obtained so far. Two growth modes of BNNT coexisting during the synthesis process have been proposed based on HR-TEM observation and length analysis. One is the root growth mode, in which boron nitride (BN) species formed via the surface interaction between surrounding N2 molecules and boron nanodroplets incorporate into the tubular structure. Another mode called open-end growth mode means the prolongation of tube growth from the exposed BN edge surrounding the surface of boron nanodroplets which is constructed by the heterogeneous nucleation of absorbed BN radicals from the gas plume. The statistical data, the proportions of end structures and the length of BNNTs, could be fitted to two growth modes, and the open-end growth mode is found to be especially effective in producing longer nanotubes with a higher growth rate. The scientific understanding of the growth mechanism is believed to provide the control for optimized production of BNNTs.
Project description:Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.
Project description:We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.
Project description:Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic "toolkits," such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis. LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species. Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.
Project description:Reversible protein phosphorylation represents a key mechanism by which signals are transduced in eukaryotic cells. Dysregulated phosphorylation is also a hallmark of carcinogenesis and represents key drug targets in the precision medicine space. Thus, methods that preserve phosphoprotein integrity in the context of clinical tissue analyses are crucially important in cancer research. Here we investigated the impact of UV laser microdissection (UV LMD) and IR laser capture microdissection (IR LCM) on phosphoprotein abundance of key cancer signaling protein targets assessed by reverse-phase protein microarray (RPPA). Tumor epithelial cells from consecutive thin sections obtained from four high-grade serous ovarian cancers were harvested using either UV LMD or IR LCM methods. Phosphoprotein abundances for ten phosphoproteins that represent important drug targets were assessed by RPPA and revealed no significant differences in phosphoprotein integrity from those obtained using higher-energy UV versus the lower-energy IR laser methods.
Project description:Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus(XT) instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.
Project description:The goal of the study was to characterize the whole genome transcriptome profiles of pure sample population of human ameloblastoma epithelial cells for examination of molecular pathways.
Project description:To improve the ability of an external cavity laser (ECL) biosensor to more easily distinguish true signals caused by biomolecular binding from a variety of sources of background noise, two photonic crystal (PC) resonant reflectors were incorporated into a single flow cell, with one of the PCs performing the detection function and the other one serving as a reference sensor. The ECL-based sensor system simultaneously emits at two distinct wavelengths corresponding to two different longitudinal cavity modes selected by the sensing and reference PC reflectors. The surface of the sensing PC filter was functionalized by a biomolecule recognition layer and exhibited narrowband reflection with the peak reflection wavelength at 856 nm. The reference PC was untreated and had the peak reflection wavelength at 859 nm. The PCs were bond to the upper and lower surfaces of a thin chamber frame, forming a flow cell. Utilizing the reference external cavity mode, the dual-mode ECL sensor system eliminated common-mode noise sources, including thermal drift, refractive index variations of the analyte solution, and nonspecific biomolecule binding.
Project description:An artificial synapse, such as a memristive electronic synapse, has caught world-wide attention due to its potential in neuromorphic computing, which may tremendously reduce computer volume and energy consumption. The introduction of layered two-dimensional materials has been reported to enhance the performance of the memristive electronic synapse. However, it is still a challenge to fabricate large-area layered two-dimensional films by scalable methods, which has greatly limited the industrial application potential of two-dimensional materials. In this work, a scalable pulsed laser deposition (PLD) method has been utilized to fabricate large-area layered SnSe films, which are used as the functional layers of the memristive electronic synapse with dual modes. Both long-term memristive behaviour with gradually changed resistance (Mode 1) and short-term memristive behavior with abruptly reduced resistance (Mode 2) have been achieved in this SnSe-based memristive electronic synapse. The switching between Mode 1 and Mode 2 can be realized by a series of voltage sweeping and programmed pulses. The formation and recovery of Sn vacancies were believed to induce the short-term memristive behaviour, and the joint action of Ag filament formation/rupture and Schottky barrier modulation can be the origin of long-term memristive behaviour. DFT calculations were performed to further illustrate how Ag atoms and Sn vacancies diffuse through the SnSe layer and form filaments. The successful emulation of synaptic functions by the layered chalcogenide memristor fabricated by the PLD method suggests the application potential in future neuromorphic computers.