Laser Microdissection Technique Research Articles

Tissue-specific chemical expression and quantitative analysis of bioactive components of Moutan Cortex by laser-microdissection combined with UPLC-Q-Orbitrap-MS technique

Moutan Cortex, is the root bark of Paeonia suffruticosa Andrews, which is classified into three specifications according to whether or not it is peeled and cored: Liandanpi, Guadanpi and whole root. In this study, the cork layer, cortex, phloem and xylem of P. suffruticosa fresh root were precisely separated by laser microdissection technique. UPLC-Q-Orbitrap-MS and UPLC-QQQ-MS techniques were used to analyse the differences in the chemical composition of different tissue parts of P. suffruticosa fresh root and Liandanpi, and to determine the optimal processing method of P. suffruticosa root. As a result, a total of 90 compounds were characterised, among which the cork layer had more types and higher contents of chemical constituents, and the xylem had fewer types and lower contents of chemical constituents. The proportion of xylem is larger, while the type and content of active ingredients is smaller. Therefore, the processing method of removing the wood core and retaining the cork bark can be used in the processing of Moutan Cortex. In this study, laser microdissection and ultra performance liquid chromatography-mass spectrometry were used to provide a theoretical basis for optimising the processing method of Moutan Cortex to enhance its pharmacological effects.

Developing a simple and rapid method for cell-specific transcriptome analysis through laser microdissection: insights from citrus rind with broader implications

BackgroundWith the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed.ResultsWe omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach.ConclusionsWe developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues.

Neoplastic cell enrichment of tumor tissues using coring and laser microdissection for proteomic and genomic analyses of pancreatic ductal adenocarcinoma

BackgroundThe identification of differentially expressed tumor-associated proteins and genomic alterations driving neoplasia is critical in the development of clinical assays to detect cancers and forms the foundation for understanding cancer biology. One of the challenges in the analysis of pancreatic ductal adenocarcinoma (PDAC) is the low neoplastic cellularity and heterogeneous composition of bulk tumors. To enrich neoplastic cells from bulk tumor tissue, coring, and laser microdissection (LMD) sampling techniques have been employed. In this study, we assessed the protein and KRAS mutation changes associated with samples obtained by these enrichment techniques and evaluated the fraction of neoplastic cells in PDAC for proteomic and genomic analyses.MethodsThree fresh frozen PDAC tumors and their tumor-matched normal adjacent tissues (NATs) were obtained from three sampling techniques using bulk, coring, and LMD; and analyzed by TMT-based quantitative proteomics. The protein profiles and characterizations of differentially expressed proteins in three sampling groups were determined. These three PDACs and samples of five additional PDACs obtained by the same three sampling techniques were also subjected to genomic analysis to characterize KRAS mutations.ResultsThe neoplastic cellularity of eight PDACs ranged from less than 10% to over 80% based on morphological review. Distinctive proteomic patterns and abundances of certain tumor-associated proteins were revealed when comparing the tumors and NATs by different sampling techniques. Coring and bulk tissues had comparable proteome profiles, while LMD samples had the most distinct proteome composition compared to bulk tissues. Further genomic analysis of bulk, cored, or LMD samples demonstrated that KRAS mutations were significantly enriched in LMD samples while coring was less effective in enriching for KRAS mutations when bulk tissues contained a relatively low neoplastic cellularity.ConclusionsIn addition to bulk tissues, samples from LMD and coring techniques can be used for proteogenomic studies. The greatest enrichment of neoplastic cellularity is obtained with the LMD technique.

The histological and microbiological characteristics of bacterial microcolonies in paediatric tonsillar hyperplasia

IntroductionPaediatric tonsillar hyperplasia (TH) is associated with a spectrum of presentations ranging from recurrent tonsillitis (RT) to sleep-disordered breathing (SDB). The underlying pathogenesis of tonsillar hyperplasia remains poorly understood. Previous studies have implicated bacterial microcolonies as targets of host inflammatory cells and as a potential driver of the chronic inflammation seen in TH. The role of atopy in tonsillar hyperplasia is also largely unknown. In this study, we aimed to determine the allergic responses and microbial factors that may influence TH in children. Materials and methodsPaired tonsils and a serum sample were collected from 21 children undergoing tonsillectomy for RT or SDB in the Auckland region. The disposition of immunoglobulin isotypes (IgG, A, M and E) and local inflammatory cells on histological sections of tonsil tissue were determined using immunohistochemistry techniques. Aeroallergen specific IgE (sIgE) and Staphylococcal enterotoxin C specific IgE (SEC-specific IgE) were measured in serum and tonsil tissue using the ImmunoCAP® system. Finally, tonsil bacterial microcolonies were then excised from histological slides using laser microdissection techniques, before undergoing bacterial and fungal amplicon sequencing. ResultsThere were no significant differences in any of the measured variables between children with RT and SDB symptoms. IgE staining was not associated with increased levels of mast cells, leukocytes or plasma cells. However, sIgE positivity was more frequently found in local tissue than in serum (p = 0.025). A significant association was observed between tissue sIgE levels and tissue SEC-specific IgE levels (r2 = 0.95, p = 0.0001). The most abundant bacterial and fungal genera identified in the microcolonies were Fusobacterium, Sphingomonas, Porphyromonas, Prevotella and Malassezia. DiscussionThese results suggest that there is a local IgE response in children with TH. Local IgE production is unrelated to systemic atopy and may play a key role in the pathogenesis of TH. This is the first study to determine the microbial composition of microcolonies in tonsil tissue. These findings enhance current understanding of the microbiology of tonsils in children with TH and have important implications for antibiotic strategies.

Abstract 1535: Identification of novel epithelial-mesenchymal transition inducers and their clinical significance in colorectal cancer

Abstract Purpose. Cancer tissues contain a substantial amount of stromal tissues, and transcriptome derived from bulk samples originates from a mixture of the cancer epithelium and stroma. Gene signature of epithelial-mesenchymal transition (EMT) cells is similar to that of stromal cells; therefore, gene signature that we considered to be related EMT might be only contamination of stroma. Here, we identify novel EMT inducers by analysis of independent gene expression profiles in the colorectal cancer epithelium and stroma. Materials and Methods. Gene expression data of pure epithelial tissue was obtained using laser microdissection technique from 13 colorectal cancer tissues. Next, the ratio of each gene expression in the stroma to in the epithelium and correlation of each gene expression with a target gene derived from The Cancer Genome Atlas (TCGA) is plotted in two dimensions, and correlation coefficients are calculated. We regard genes with high correlation coefficients but highly expressed in the epithelium as candidates of EMT inducers. We manipulate the expression of some candidate genes in colorectal cancer cell lines to investigate the alterations of EMT markers and migration ability in vitro. Results. We extract 91 new putative EMT-related genes in silico. They are highly expressed in similar cell lines to known EMT markers in a public database. Furthermore, some candidates have reported having a relation to EMT, and that indicates the validity of this procedure. The knockdown of three candidate genes upregulates epithelial gene expression and downregulates mesenchymal gene expression. The knockdown of one of the three genes reduces cell migration. In contrast, overexpression of the gene downregulates epithelial gene expression and leads to cell migration. Conclusion. We identify novel EMT inducers successfully using purified gene expression data. They could be new prognostic markers and therapeutic targets of colorectal cancer. Citation Format: Satoshi Ishikawa, Naohiro Nishida, Shiki Fujino, Takayuki Ogino, Hidekazu Takahashi, Norikatsu Miyoshi, Mamoru Uemura, Chu Matsuda, Tsunekazu Mizusima, Hirofumi Yamamoto, Koshi Mimori, Masaki Mori, Yuichiro Doki, Hidedoshi Eguchi. Identification of novel epithelial-mesenchymal transition inducers and their clinical significance in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1535.

Regenerative activity of Hericium erinaceus on axonal injury model using in vitro laser microdissection technique

ABSTRACTObjective: Peripheral nerve injury (PNI) is an important global health problem. Nerve Growth Factor (NGF) plays crucial role in the survival, growth, and maintenance of various neurons in the mammalian nervous system, human included. Hericium erinaceus (HE), an edible and medicinal mushroom, has been extensively studied for its neuroprotective properties. In this study, the neuroprotective and neurotogenic effects of HE and NGF were compared on mouse PNI model by using a laser microdissection technique.Methods: Neuronal cultures were prepared from dorsal root ganglia (DRG) of 6–8 week aged mice, pretreated them with phosphate-buffered saline (PBS), NGF, HE, or the combination of NGF and HE. To model axonal injury in vitro, axons were cut (axotomy) with a microscope-controlled laser beam. Axotomized neurons were imaged under the microscope. Axotomized neurons’ survival ratios were calculated using the propidium iodide (PI), which is a red-fluorescent nuclear dye. Their axon lengths were measured using the AxioVision 4.8 software.Results: Although both HE and NGF have neuroprotective and regenerative effects on axotomized peripheral sensory neurons, HE exhibits a higher neuroprotective activity compared to the NGF. The combination of HE and NGF maximizes axonal regeneration ability of axotomized neurons.Conclusion: HE has capabilities of preventing the death of neurons and regenerating their axons in the experimental axonal injury model. Our findings provide experimental evidence that HE may serve as a neuroprotective and regenerative candidate for treating peripheral nerve injuries. Present study warrants further investigation of HE as a potential natural compound to remedy PNI.

Thymoquinone protects DRG neurons from axotomy-induced cell death

ABSTRACTObjective: Peripheral nerve injury (PNI) is a significant health problem that is linked to sensory, motor, and autonomic deficits. This pathological condition leads to a reduced quality of life in most affected individuals. Schwann cells (SCs) play a crucial role in the repair of PNI. Effective agents that promote SC activation may facilitate and accelerate peripheral nerve repair. Thymoquinone (TQ), a bioactive component of Nigella sativa seeds, has an antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective properties. In the present study, the neuroprotective efficacy of TQ was investigated by using a laser microdissection technique in a mouse PNI model.Methods: Single cells were isolated from dorsal root ganglions (DRGs) of 6–8-week-old mice, maintained in defined culture conditions and treated with or without TQ at different concentrations. Axons were cut (axotomy) using a controllable laser microbeam to model axonal injury in vitro. Under fluorescence microscopy, cell viability was evaluated using the fluorescent dyes. The behavior of the cells was continuously monitored with time-lapse video microscopy.Results: TQ significantly increased neuronal survival by promoting the survival and proliferation of SCs and fibroblasts, as well as the migration of SCs. Furthermore, TQ improved the ability to extend neurites of axotomized neurons. The regenerative effect of TQ was dose-dependent suggesting a target specificity. Our studies warrant further preclinical and clinical investigations of TQ as a potential regenerative agent to treat peripheral nerve injuries.Conclusion: TQ exhibits a regenerative potential for the treatment of damaged peripheral nerves.

NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood samples because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1st-gen NanoVelcro Chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2nd-gen NanoVelcro Chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3rd-gen Thermoresponsive NanoVelcro Chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4th-gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC assays, and the clinical applications of each generation of devices.

Adaptation of Laser Microdissection Technique to Nanostring RNA Analysis in the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model.

The mouse model characterized by spontaneous lung metastasis from JygMC (A) cells closely resembles the human triple negative breast cancer (TNBC) subtype. The primary tumors morphologically present both epithelial and spindle-like cells, but metastases in lung parenchyma display only adenocarcinoma properties. In the study of molecular signatures, laser capture microdissection (LCM) on frozen tissue sections was used to separate the following regions of interest: the epithelial-mesenchymal transition (EMT), mesenchymal-epithelial transition (MET), carcinoma, lung metastases, normal mammary gland and normal lung parenchyma. NanoString was selected for the study of molecular signatures in LCM targets as a reliable downstream gene expression platform allowing analysis of tissue lysates without RNA extraction and amplification. This chapter provides detailed protocols for the collection of tissue, LCM sample preparation and dissection, production of lysates, extraction, and quality control of RNA for NanoString analysis, as well as the methodology of Nanostring gene expression profiling experiment.

LC-MS/MS imaging with thermal film-based laser microdissection.

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 μm; for imaging purposes in this study, the diameter was fixed at 40 μm, allowing acquisition of LC-MS/MS images at a 40-μm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.

Experimental evidence for hybridization of closely related lineages in Plasmodium relictum

Over 50 avian Plasmodium species have been described. However, PCR-based information shows much broader diversity of genetic lineages in these parasites. This discrepancy indicates insufficient knowledge about taxonomic diversity and boundaries of a single species in avian Plasmodium species. In recent taxonomy, most of genetically closely related lineages that share the same morphology and development patterns are attributed to the same biological species, but there is no information if these lineages are able to cross. This information is crucial to understand if these lineages form single or multiple evolutionary units.Due to presence of sexual process and sporogonic development of Plasmodium parasites in mosquitoes, self and cross-fertilization can occur and be identified during the oocyst stage. We initiated in vivo hybridization experiments of two widespread Plasmodium relictum lineages (pSGS1 and pGRW11) in experimentally infected Culex pipiens pipiens form molestus mosquitoes. To study putative hybrid oocysts, we used a laser microdissection technique together with PCR-based analyses of mitochondrial and nuclear genes. We demonstrate that both pSGS1 and pGRW11 lineages develop in infected mosquitoes in parallel, but also form hybrid oocysts of these two lineages. Our results are in accord to a recent global phylogeographic study of P. relictum that suggested that cross-fertilization between pSGS1 and pGRW11 might occur. This information helps to understand population structure, gene flow and the evolutionary process of haemosporidian parasites.

Primary Pancreatic Secretinoma: Further Evidence Supporting Secretin as a Diarrheogenic Hormone.

To document the existence of primary pancreatic secretinoma in patients with watery diarrhea syndrome (WDS) and achlorhydria and establish secretin as a diarrheogenic hormone. Vasoactive intestinal peptide (VIP) has been widely accepted as the main mediator of WDS. However, in 1968, Zollinger et al reported 2 female patients with pancreatic neuroendocrine tumors, WDS, and achlorhydria. During surgery on the first, a 24-year-old patient, they noticed distended duodenum filled with fluid and a dilated gallbladder containing dilute bile with high bicarbonate concentration. After excision of the tumor, WDS ceased and gastric acid secretion returned. The second, a 47-year-old, patient's metastatic tumor extract given intravenously in dogs, produced significantly increased pancreatic and biliary fluid rich in bicarbonate. They suggested a secretin-like hormone of islet cell origin explains WDS and achlorhydria. These observations, however, predated radioimmunoassay, immunohistochemical staining, and other molecular studies. The first patient's tumor tissue was investigated for secretin and VIP. Using both immunohistochemistry and laser microdissection and pressure catapulting technique for RNA isolation and subsequent reverse transcription polymerase chain reaction, the expression levels of secretin, and VIP were measured. Immunoreactive secretin and its mRNA were predominantly found in the tumor tissue whereas VIP and its mRNA were scarce. The findings strongly support that the WDS and achlorhydria in this patient may have been caused by secretin as originally proposed in 1968 and that secretin may act as a diarrheogenic hormone.

Molecular bases for phyllomorph development in a one-leaf plant, Monophyllaea glabra.

The aboveground part of one-leaf plants (Gesneriaceae) consists of a unique shoot-like unit called a phyllomorph, which is composed of a growing lamina and one petiolode (a stem- and petiole-like organ). The phyllomorph has three meristems: a basal meristem (BM; involved in lamina growth), a petiolode meristem (PM; required for midrib and petiolode growth), and a groove meristem (GM; contributes to inflorescence and new phyllomorph development). Although the GM has a tunica-corpus structure like a conventional shoot apical meristem (SAM), the vegetative GM of a one-leaf plant does not form any organ primordia and has been considered a defective or suppressed SAM. In this study, we evaluated the nature of the three meristems to reconsider their roles in phyllomorph development in the one-leaf species Monophyllaea glabra. Mitotic activities of meristem cells were monitored by incorporation of a thymidine analog (EdU) into DNA. Orthologs of SHOOTMERISTEMLESS (STM) and PHANTASTICA, ROUGH SHEATH2, ASYMMETRIC LEAVES1 (PHAN/RS2/AS1) were isolated from M. glabra, and their expression patterns were investigated by quantitative reverse transcription polymerase chain reaction in combination with a laser microdissection technique. Mitotic activities were detected in all three phyllomorph meristems. Strong expression of the STM and PHAN/RS2/AS1 orthologs was detected in the vegetative GM and BM, respectively. The vegetative GM is an active meristem that expresses the STM ortholog. We postulate that the GM is required for growth of a phyllomorph by providing undifferentiated cells and/or growth regulators to the BM and/or PM.

Involvement of the different lung compartments in the pathogenesis of pH1N1 influenza virus infection in ferrets.

Severe cases after pH1N1 infection are consequence of interstitial pneumonia triggered by alveolar viral replication and an exacerbated host immune response, characterized by the up-regulation of pro-inflammatory cytokines and the influx of inflammatory leukocytes to the lungs. Different lung cell populations have been suggested as culprits in the unregulated innate immune responses observed in these cases. This study aims to clarify this question by studying the different induction of innate immune molecules by the distinct lung anatomic compartments (vascular, alveolar and bronchiolar) of ferrets intratracheally infected with a human pH1N1 viral isolate, by means of laser microdissection techniques. The obtained results were then analysed in relation to viral quantification in the different anatomic areas and the histopathological lesions observed. More severe lung lesions were observed at 24 h post infection (hpi) correlating with viral antigen detection in bronchiolar and alveolar epithelial cells. However, high levels of viral RNA were detected in all anatomic compartments throughout infection. Bronchiolar areas were the first source of IFN-α and most pro-inflammatory cytokines, through the activation of RIG-I. In contrast, vascular areas contributed with the highest induction of CCL2 and other pro-inflammatory cytokines, through the activation of TLR3.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0395-0) contains supplementary material, which is available to authorized users.

Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies.

Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM.

Laser Microdissection of Grapevine Leaves Reveals Site-Specific Regulation of Transcriptional Response toPlasmopara viticola

Grapevine is one of the most important fruit crops in the world, and it is highly susceptible to downy mildew caused by the biotrophic oomycete Plasmopara viticola. Gene expression profiling has been used extensively to investigate the regulation processes of grapevine-P. viticola interaction, but all studies to date have involved the use of whole leaves. However, only a small fraction of host cells is in contact with the pathogen, so highly localized transcriptional changes of infected cells may be masked by the large portion of non-infected cells when analyzing the whole leaf. In order to understand the transcriptional regulation of the plant reaction at the sites of pathogen infection, we optimized a laser microdissection protocol and analyzed the transcriptional changes in stomata cells and surrounding areas of grapevine leaves at early stages of P. viticola infection. The results indicate that the expression levels of seven P. viticola-responsive genes were greater in microdissected cells than in whole leaves, highlighting the site-specific transcriptional regulation of the host response. The gene modulation was restricted to the stomata cells and to the surrounding areas of infected tissues, indicating that the host response is mainly located at the infection sites and that short-distance signals are implicated. In addition, due to the high sensitivity of the laser microdissection technique, significant modulations of three genes that were completely masked in the whole tissue analysis were detected. The protocol validated in this study could greatly increase the sensitivity of further transcriptomic studies of the grapevine-P. viticola interaction.

Analysis of steroidogenic pathway key transcripts in interrenal cells isolated by laser microdissection (LMD) in stressed rainbow trout

An assessment of the key transcripts expression of the steroidogenesis-related genes in rainbow trout subjected to either acute or chronic stress was performed in both interrenal cells and whole head kidney tissue. The analysis of interrenal cells was possible thanks to the use, for the first time in this specific type of cells, of the technique of laser microdissection (LMD) which allows to isolate specific cells and process them independently of other surrounding cells in the tissue. The results indicated that both acute and chronic stressors induced a significant up-regulation of the steroidogenesis-related genes with a higher but expected degree in the isolated cells. In addition, under acute stress a delay between cortisol levels and transcript expression was found. Under chronic stress a clear relation between plasma cortisol levels, mRNA transcription and interrenal tissue area was observed, since all parameters were concomitantly increased at day 5 after stress. Moreover results indicated that the LMD technique allowed ascertaining with more precision and accuracy whether and when the steroidogenesis-related genes were significantly expressed, disregarding the noise produced by other cells present in the head kidney. Results also showed a typical physiological response in plasma parameters and a positive relationship between plasma cortisol data and transcript abundance in isolated cells. The present results may help to better understand the mechanisms behind the interrenal response to stress challenges in fish.

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis.

Brain function in normal aging and neurological diseases has long been a subject of interest. With current technology, it is possible to go beyond descriptive analyses to characterize brain cell populations at the molecular level. However, the brain comprises over 100 billion highly specialized cells, and it is a challenge to discriminate different cell groups for analyses. Isolating intact neurons is not feasible with traditional methods, such as tissue homogenization techniques. The advent of laser microdissection techniques promises to overcome previous limitations in the isolation of specific cells. Here, we provide a detailed protocol for isolating and analyzing neurons from postmortem human brain tissue samples. We describe a workflow for successfully freezing, sectioning and staining tissue for laser microdissection. This protocol was validated by mass spectrometric analysis. Isolated neurons can also be employed for western blotting or PCR. This protocol will enable further examinations of brain cell-specific molecular pathways and aid in elucidating distinct brain functions.

68. Development of Thioaptamers and X-aptamers Targeting Human Ovarian Cancer Cells

Aptamers are oligonucleotide or peptide molecules that bind to a specific target with high affinity and specificity. A diverse range of modifications have been reported for either enhancing aptamers’ nuclease resistance or expanding their chemical functionalities. We created a combinatorial DNA aptamer library that had been modified with thio substitution of the phosphate backbone (thioaptamer, TA) at selected 5’dA positions for cell and tissue based selections. Based on morphological assessment of the tissue, we used an image-directed laser microdissection (LMD) technique to dissect Regions of Interest (ROIs) bound with thioaptamers, and further identified the targeted proteins. Using a Next Generation Sequencing (NGS) approach and alignments by our recently developed Aptaligner program, we are able to select tissue specific TAs in a more representative way from the TA libraries. We have successfully identified TAs that bind specifically to tumor endothelial cells and characterized the lead candidate TA, Endo28, as an Annexin A2-specific TA that allows for selective delivery to the tumor vasculature. Based on the primary sequences of several of the previously selected TAs and observed variations, we developed an X-aptamer (XA) library by conjugating drug-like ligands (X) to the DNA bases in a random fashion. We were able to select an XA with high affinity to CD44 hyaluronic acid binding domain (HABD) from a large combinatorial TA library modified with a drug (N-acetyl-2,3-dehydro-2- deoxyneuraminic acid, ADDA). The selected CD44 X-aptamers (amine form and ADDA form) show enhanced binding affinities with ADDA to CD44 over-expressed on human ovarian cancer IGROV cells. Using a systems biology approach that combines the use of 15 mm bead-based XA libraries with flow cytometry sorting and mass spectrometry, we collected beads binding to proteins labeled with different fluorescence dyes and identified their associated XA sequences by PCR and NGS. The differentially expressed proteins can be identified by an XA beads pulldown experiment to discover the potential biomarkers for ultimately TA or XA targeted nanoparticle delivery of drugs to tumors.

Increased microRNA-34b and -34c predominantly expressed in stromal tissues is associated with poor prognosis in human colon cancer.

The microRNA-34 family (miR-34a, -34b and -34c) have been reported to be tumor suppressor microRNAs (miRNAs) that are regulated by the TP53 and DNA hypermethylation. However, the expression, regulation, and prognostic value of the miR-34 family have not been systematically studied in colon cancer. To elucidate the roles of miR-34 family in colon carcinogenesis, miR-34a/b/c were measured in tumors and adjacent noncancerous tissues from 159 American and 113 Chinese colon cancer patients using quantitative RT-PCR, and we examined associations between miR-34a/b/c expression with TNM staging, cancer-specific mortality, TP53 mutation status and Affymetrix microarray data. All miR-34 family members were significantly increased in colon tumors, counter to the proposed tumor suppressor role for these miRNAs. Increased miR-34b/c were observed in more advanced tumors in two independent cohorts and increased expression of miR-34b/c was associated with poor cancer-specific mortality. While the expression of miR-34 family was not associated with TP53 mutation status, TP53 transcriptional activity was associated with miR-34a/b/c expression that is consistent with the proposed regulation of miR-34a/b/c by TP53. To examine where the miR-34 family is expressed, the expression of miR-34 family was compared between epitheliums and stromal tissues using laser microdissection technique. The expression of miR-34b/c was increased significantly in stromal tissues, especially in cancer stroma, compared with epithelial tissue. In conclusion, increased miR-34b/c predominantly expressed in stromal tissues is associated with poor prognosis in colon cancer. MiR-34 may contribute to cancer-stromal interaction associated with colon cancer progression.