RNA Isolation from Nematode-Induced Feeding Sites in Arabidopsis Roots Using Laser Capture Microdissection.

Nematodes are diverse multicellular organisms that are most abundantly found in the soil. Most nematodes are free-living and feed on a range of organisms. Based on their feeding habits, soil nematodes can be classified into four groups: bacterial, omnivorous, fungal, and plant-feeding. Plant-parasitic nematodes (PPNs) are a serious threat to global food security, causing substantial losses to the agricultural sector. Root-knot and cyst nematodes are the most important of PPNs, significantly limiting the yield of commercial crops such as sugar beet, mustard, and cauliflower. The life cycle of these nematodes consists of four molting stages (J1-J4) that precede adulthood. Nonetheless, only second-stage juveniles (J2), which hatch from eggs, are infective worms that can parasitize the host's roots. The freshly hatched juveniles (J2) of beet cyst nematode, Heterodera schachtii, establish a permanent feeding site inside the roots of the host plant. A cocktail of proteinaceous secretions is injected into a selected cell which later develops into a syncytium via local cell wall dissolution of several hundred neighboring cells. The formation of syncytium is accompanied by massive transcriptional, metabolic, and proteomic changes inside the host tissues. It creates a metabolic sink in which solutes are translocated to feed the nematodes throughout their life cycle. Deciphering the molecular signaling cascades during syncytium establishment is thus essential in studying the plant-nematode interactions and ensuring sustainability in agricultural practices. However, isolating RNA, protein, and metabolites from syncytial cells remains challenging. Extensive use of laser capture microdissection (LCM) in animal and human tissues has shown this approach to be a powerful technique for isolating a single cell from complex tissues. Here, we describe a simplified protocol for Arabidopsis-Heterodera schachtii infection assays, which is routinely applied in several plant-nematode laboratories. Next, we provide a detailed protocol for isolating high-quality RNA from syncytial cells induced by Heterodera schachtii in the roots of Arabidopsis thaliana plants.

Laser-Assisted Microdissection of Plant Embryos for Transcriptional Profiling.

Transcriptomic studies have proven powerful and effective as a tool to study the molecular underpinnings of plant development. Still, it remains challenging to disentangle cell- or tissue-specific transcriptomes in complex structures like the plant seed. In particular, the embryo of flowering plants is embedded in the endosperm, a nurturing tissue, which, in turn, is enclosed by the maternal seed coat. Here, we describe laser-assisted microdissection (LAM) to isolate highly pure embryo tissue from whole seeds. This technique is applicable to virtually any plant seed, and we illustrate the use of LAM to isolate embryos from species of the Boechera and Solanum genera. LAM is a tool that will greatly help to increase the repertoires of tissue-specific transcriptomes, including those of embryos and parts thereof, in nonmodel plants.

Laser Capture Microdissection-Liquid Vortex Capture Mass Spectrometry Metabolic Profiling of Single Onion Epidermis and Microalgae Cells.

Laser capture microdissection is a valuable technique in individually isolating single cells whether in tissue networks or deposited from a cell suspension. New developments have enabled coupling of laser capture microdissection with mass spectrometry via liquid vortex capture sampling probe. This enables online metabolic profiling of sectioned cells. Here, we describe the protocol used to deposit, isolate, and individually chemically characterize single Allium cepa and Chlamydomonas reinhardtii cells by laser capture microdissection-liquid vortex capture mass spectrometry.

High levels of PIWI-interacting RNAs are present in the small RNA landscape of prostate epithelium from vitamin D clinical trial specimens.

BACKGROUND: Vitamin D, a hormone that acts through the nuclear vitamin D receptor (VDR), upregulates antitumorigenic microRNA in prostate epithelium. This may contribute to the lower levels of aggressive prostate cancer (PCa) observed in patients with high serum vitamin D. The small noncoding RNA (ncRNA) landscape includes many other RNA species that remain uncharacterized in prostate epithelium and their potential regulation by vitamin D is unknown. METHODS: Laser capture microdissection (LCM) followed by small-RNA sequencing was used to identify ncRNAs in the prostate epithelium of tissues from a vitamin D-supplementation trial. VDR chromatin immunoprecipitation-sequencing was performed to identify vitamin D genomic targets in primary prostate epithelial cells. RESULTS: Isolation of epithelium by LCM increased sample homogeneity and captured more diversity in ncRNA species compared with publicly available small-RNA sequencing data from benign whole prostate. An abundance of PIWI-interacting RNAs (piRNAs) was detected in normal prostate epithelium. The obligate binding partners of piRNAs, PIWI-like (PIWIL) proteins, were also detected in prostate epithelium. High prostatic vitamin D levels were associated with increased expression of piRNAs. VDR binding sites were located near several ncRNA biogenesis genes and genes regulating translation and differentiation. CONCLUSIONS: Benign prostate epithelium expresses both piRNA and PIWIL proteins, suggesting that these small ncRNA may serve an unknown function in the prostate. Vitamin D may increase the expression of prostatic piRNAs. VDR binding sites in primary prostate epithelial cells are consistent with its reported antitumorigenic functions and a role in ncRNA biogenesis.

Connectivity Analyses of Bioenergetic Changes in Schizophrenia: Identification of Novel Treatments.

We utilized a cell-level approach to examine glycolytic pathways in the DLPFC of subjects with schizophrenia (n = 16) and control (n = 16) and found decreased mRNA expression of glycolytic enzymes in pyramidal neurons, but not astrocytes. To replicate these novel bioenergetic findings, we probed independent datasets for bioenergetic targets and found similar abnormalities. Next, we used a novel strategy to build a schizophrenia bioenergetic profile by a tailored application of the Library of Integrated Network-Based Cellular Signatures data portal (iLINCS) and investigated connected cellular pathways, kinases, and transcription factors using Enrichr. Finally, with the goal of identifying drugs capable of "reversing" the bioenergetic schizophrenia signature, we performed a connectivity analysis with iLINCS and identified peroxisome proliferator-activated receptor (PPAR) agonists as promising therapeutic targets. We administered a PPAR agonist to the GluN1 knockdown model of schizophrenia and found it improved long-term memory. Taken together, our findings suggest that tailored bioinformatics approaches, coupled with the LINCS library of transcriptional signatures of chemical and genetic perturbagens, may be employed to identify novel treatment strategies for schizophrenia and related diseases.

Increased expression of BDNF mRNA in the frontal cortex of autistic patients.

Autistic spectrum disorders (ASDs) are neurodevelopmental disorders for which genetic components have been well defined. However, specific gene deregulations related to synapse function in the autistic brain have not been as extensively described. Based on a candidate genes approach, we present in this study the expression data of 4 transcripts of interest (BDNF, CAMK2a, NR-CAM and RIMS1) located at the synapse in two regions of interest in the context of the ASDs; the lobule VI of cerebellum and the Brodmann area 46. We have also genotyped in our cohort the coding single nucleotide polymorphism rs6265, located in the BDNF gene. After correction for age and sex, whereas no change was observed in the lobule VI between controls and autistic patients, we found a significant increase of BDNF expression level in the BA46 from autistic patients. No significant interaction between the rs6265 genotype and autism was observed for the BDNF expression. However, "A" allele carriers are more likely to have increased BDNF levels. Finally, we found a significant positive correlation between BDNF and RIMS1 expression levels. Our data suggest that these two molecules which are involved in cell signalling at the synapse, might have coordinated expressions and, that BDNF regulation in the brain has to be investigated further in the context of ASDs.

Neuron-specific deficits of bioenergetic processes in the dorsolateral prefrontal cortex in schizophrenia.

Schizophrenia is a devastating illness that affects over 2 million people in the United States and costs society billions of dollars annually. New insights into the pathophysiology of schizophrenia are needed to provide the conceptual framework to facilitate development of new treatment strategies. We examined bioenergetic pathways in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and control subjects using western blot analysis, quantitative real-time polymerase chain reaction, and enzyme/substrate assays. Laser-capture microdissection-quantitative polymerase chain reaction was used to examine these pathways at the cellular level. We found decreases in hexokinase (HXK) and phosphofructokinase (PFK) activity in the DLPFC, as well as decreased PFK1 mRNA expression. In pyramidal neurons, we found an increase in monocarboxylate transporter 1 mRNA expression, and decreases in HXK1, PFK1, glucose transporter 1 (GLUT1), and GLUT3 mRNA expression. These results suggest abnormal bioenergetic function, as well as a neuron-specific defect in glucose utilization, in the DLPFC in schizophrenia.

Oligomeric amyloid ß preferentially targets neuronal and not glial mitochondrial-encoded mRNAs.

INTRODUCTION: Our laboratories have demonstrated that accumulation of oligomeric amyloid ß (OAß) in neurons is an essential step leading to OAß-mediated mitochondrial dysfunction. METHODS: Alzheimer's disease (AD) and matching control hippocampal neurons, astrocytes, and microglia were isolated by laser-captured microdissection from the same subjects, followed by whole-transcriptome sequencing. Complementary in vitro work was performed in OAß-treated differentiated SH-SY5Y, followed by the use of a novel CoQ10 analogue for protection. This compound is believed to be effective both in suppressing reactive oxygen species and also functioning in mitochondrial electron transport. RESULTS: We report decreases in the same mitochondrial-encoded mRNAs in Alzheimer's disease laser-captured CA1 neurons and in OAß-treated SH-SY5Y cells, but not in laser-captured microglia and astrocytes. Pretreatment with a novel CoQ10 analogue, protects neuronal mitochondria from OAß-induced mitochondrial changes. DISCUSSION: Similarity of expression changes in neurons from Alzheimer's disease brain and neuronal cells treated with OAß, and the effect of a CoQ10 analogue on the latter, suggests a pretreatment option to prevent OAß toxicity, long before the damage is apparent.

Laser microdissection hyphenated with high performance gel permeation chromatography-charged aerosol detector and ultra performance liquid chromatography-triple quadrupole mass spectrometry for histochemical analysis of polysaccharides in herbal medicine: Ginseng, a case study.

This study establishes a new combinatorial approach for histochemical analysis of polysaccharides in herbal medicines using laser microdissection followed by high performance gel permeation chromatography coupled with charged aerosol detector and ultra-performance liquid chromatography hyphenated with triple quadrupole mass spectrometry. Ginseng was employed as a study model. Tissue-specific qualitative and quantitative characterization of ginseng polysaccharides was performed by determining their molar masses and monosaccharide compositions in three macro-dissected parts (rhizome, main and branched roots) and five micro-dissected tissues (cork, cortex, xylem, phloem and resin canal). The results showed that ginseng "flesh" (xylem, phloem and resin canal) contained more polysaccharides with larger molecular weights and higher ratios of glucose residue, whereas ginseng "skin" (cork and cortex) had fewer polysaccharides with smaller molecular weights and higher ratios of non-glucose constituents (e.g. galacturonic acid, galactose, arabinose and rhamnose). These findings suggested that the polysaccharides of the "flesh" were predominantly starch-like glucans, while those of the "skin" were of a higher proportion of acidic pectins. The revealed histologic distribution and accumulation pattern of ginseng polysaccharides contributes to the scientific understanding of ginseng regarding the biosynthesis and transportation of polysaccharides, medicinal quality evaluation as well as empirical clinical application.

Hypothalamic leptin action is mediated by histone deacetylase 5.

Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and greater diet-induced obesity when fed high-fat diet. Pharmacological and genetic inhibition of HDAC5 activity in the mediobasal hypothalamus increases food intake and modulates pathways implicated in leptin signalling. We show HDAC5 directly regulates STAT3 localization and transcriptional activity via reciprocal STAT3 deacetylation at Lys685 and phosphorylation at Tyr705. In vivo, leptin sensitivity is substantially impaired in HDAC5 loss-of-function mice. Hypothalamic HDAC5 overexpression improves leptin action and partially protects against HFD-induced leptin resistance and obesity. Overall, our data suggest that hypothalamic HDAC5 activity is a regulator of leptin signalling that adapts food intake and body weight to our dietary environment.

Laser dissection sampling modes for direct mass spectral analysis.

RATIONALE: Laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. METHODS: The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis of single cells and tissue. RESULTS: Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (~4-15 µm) even when agglomerated together. Turbid Allium Cepa cells (~150 µm) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. CONCLUSIONS: Laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.

5-HT2A and 5-HT2C receptors as hypothalamic targets of developmental programming in male rats.

Although obesity is a global epidemic, the physiological mechanisms involved are not well understood. Recent advances reveal that susceptibility to obesity can be programmed by maternal and neonatal nutrition. Specifically, a maternal low-protein diet during pregnancy causes decreased intrauterine growth, rapid postnatal catch-up growth and an increased risk for diet-induced obesity. Given that the synthesis of the neurotransmitter 5-hydroxytryptamine (5-HT) is nutritionally regulated and 5-HT is a trophic factor, we hypothesised that maternal diet influences fetal 5-HT exposure, which then influences development of the central appetite network and the subsequent efficacy of 5-HT to control energy balance in later life. Consistent with our hypothesis, pregnant rats fed a low-protein diet exhibited elevated serum levels of 5-HT, which was also evident in the placenta and fetal brains at embryonic day 16.5. This increase was associated with reduced levels of 5-HT2CR, the primary 5-HT receptor influencing appetite, in the fetal, neonatal and adult hypothalamus. As expected, a reduction of 5-HT2CR was associated with impaired sensitivity to 5-HT-mediated appetite suppression in adulthood. 5-HT primarily achieves effects on appetite by 5-HT2CR stimulation of pro-opiomelanocortin (POMC) peptides within the arcuate nucleus of the hypothalamus (ARC). We show that 5-HT2ARs are also anatomically positioned to influence the activity of ARC POMC neurons and that mRNA encoding 5-HT2AR is increased in the hypothalamus ofin uterogrowth-restricted offspring that underwent rapid postnatal catch-up growth. Furthermore, these animals at 3 months of age are more sensitive to appetite suppression induced by 5-HT2AR agonists. These findings not only reveal a 5-HT-mediated mechanism underlying the programming of susceptibility to obesity, but also provide a promising means to correct it, by treatment with a 5-HT2AR agonist.

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood encephalopathy.

Transcriptional analysis of phloem-associated cells of potato.

BACKGROUND: Numerous signal molecules, including proteins and mRNAs, are transported through the architecture of plants via the vascular system. As the connection between leaves and other organs, the petiole and stem are especially important in their transport function, which is carried out by the phloem and xylem, especially by the sieve elements in the phloem system. The phloem is an important conduit for transporting photosynthate and signal molecules like metabolites, proteins, small RNAs, and full-length mRNAs. Phloem sap has been used as an unadulterated source to profile phloem proteins and RNAs, but unfortunately, pure phloem sap cannot be obtained in most plant species. RESULTS: Here we make use of laser capture microdissection (LCM) and RNA-seq for an in-depth transcriptional profile of phloem-associated cells of both petioles and stems of potato. To expedite our analysis, we have taken advantage of the potato genome that has recently been fully sequenced and annotated. Out of the 27 k transcripts assembled that we identified, approximately 15 k were present in phloem-associated cells of petiole and stem with greater than ten reads. Among these genes, roughly 10 k are affected by photoperiod. Several RNAs from this day length-regulated group are also abundant in phloem cells of petioles and encode for proteins involved in signaling or transcriptional control. Approximately 22 % of the transcripts in phloem cells contained at least one binding motif for Pumilio, Nova, or polypyrimidine tract-binding proteins in their downstream sequences. Highlighting the predominance of binding processes identified in the gene ontology analysis of active genes from phloem cells, 78 % of the 464 RNA-binding proteins present in the potato genome were detected in our phloem transcriptome. CONCLUSIONS: As a reasonable alternative when phloem sap collection is not possible, LCM can be used to isolate RNA from specific cell types, and along with RNA-seq, provides practical access to expression profiles of phloem tissue. The combination of these techniques provides a useful approach to the study of phloem and a comprehensive picture of the mechanisms associated with long-distance signaling. The data presented here provide valuable insights into potentially novel phloem-mobile mRNAs and phloem-associated RNA-binding proteins.

Localization of ginsenosides in the rhizome and root of Panax ginseng by laser microdissection and liquid chromatography-quadrupole/time of flight-mass spectrometry.

The root and rhizome of Panax ginseng C.A. Mey, known as ginseng, is a commonly used medicinal plant. Ginsenosides are the major active components responsible for the tonic effects of this herb. Here, the combination of laser microdissection and ultra-high performance liquid chromatography quadrupole/time of flight-mass spectrometry (UHPLC-QTOF-MS) was applied to investigate the localization of ginsenosides in root and rhizome of P. ginseng. Five kinds of tissue cells were separated from the rhizome, main root and branch root of ginseng. Fifty-nine ginsenosides were identified and the results showed that the cork contained more kinds of ginsenosides than did the cortex, phloem, xylem and resin canals. It is interesting that the phloem, xylem and resin canals from branch root contained a greater number of ginsenosides than did from main root. This study provides solid evidence on the accumulation of ginsenosides in cork, cortex, phloem and xylem.

Histochemical evaluation of alkaloids in rhizome of Coptis chinensis using laser microdissection and liquid chromatography/mass spectrometry.

Traditional macroscopic and microscopic identification methods of medicinal materials are economical and practical, but usually experience-based due to few chemical supports. Here histochemical evaluation on bioactive components of Coptidis Rhizoma (CR) in anatomic sections using laser microdissection and liquid chromatography-mass spectrometry (LMD-LC-MS) was developed to correlate the inner quality and outer features of materials from different growing areas. Results of a total 33 peaks representing potential different alkaloids were detected and 8 common peaks were identified as the major alkaloids, namely magnoflorine, thalifendine, columbamine, epiberberine, jatrorrhizine, coptisine, palmatine, and berberine. Six major alkaloids were quantified in the top and middle sections of raw materials and in their tissues and cells at the same time. Histochemical analyses showed consistent results with direct determination in raw materials and explained the reason why top sections of all samples contained higher contents of alkaloids by giving out attributions of each alkaloid in different anatomic sections. Besides, results manifested the distribution and accumulation rules of alkaloids in diverse tissues and cells of CR. This study demonstrates an effective and scientific way to correlate bioactive components and morphological features of medicinal materials, which is beneficial to future research, agriculture and application.

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury.

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, ß2, ß3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, ß2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei.

Application of laser microdissection ICP-MS for high resolution elemental mapping in mouse brain tissue: a comparative study with laser ablation ICP-MS.

Mapping of elements in biological tissue by laser induced mass spectrometry is a fast growing analytical methodology in life sciences. This method provides a multitude of useful information of metal, nonmetal, metalloid and isotopic distribution at major, minor and trace concentration ranges, usually with a lateral resolution of 12-160 µm. Selected applications in medical research require an improved lateral resolution of laser induced mass spectrometric technique at the low micrometre scale and below. The present work demonstrates the applicability of a recently developed analytical methodology - laser microdissection associated to inductively coupled plasma mass spectrometry (LMD ICP-MS) - to obtain elemental images of different solid biological samples at high lateral resolution. LMD ICP-MS images of mouse brain tissue samples stained with uranium and native are shown, and a direct comparison of LMD and laser ablation (LA) ICP-MS imaging methodologies, in terms of elemental quantification, is performed.

Cytochrome P450-generated metabolites derived from ω-3 fatty acids attenuate neovascularization.

Ocular neovascularization, including age-related macular degeneration (AMD), is a primary cause of blindness in individuals of industrialized countries. With a projected increase in the prevalence of these blinding neovascular diseases, there is an urgent need for new pharmacological interventions for their treatment or prevention. Increasing evidence has implicated eicosanoid-like metabolites of long-chain polyunsaturated fatty acids (LCPUFAs) in the regulation of neovascular disease. In particular, metabolites generated by the cytochrome P450 (CYP)-epoxygenase pathway have been shown to be potent modulators of angiogenesis, making this pathway a reasonable previously unidentified target for intervention in neovascular ocular disease. Here we show that dietary supplementation with ω-3 LCPUFAs promotes regression of choroidal neovessels in a well-characterized mouse model of neovascular AMD. Leukocyte recruitment and adhesion molecule expression in choroidal neovascular lesions were down-regulated in mice fed ω-3 LCPUFAs. The serum of these mice showed increased levels of anti-inflammatory eicosanoids derived from eicosapentaenoic acid and docosahexaenoic acid. 17,18-epoxyeicosatetraenoic acid and 19,20-epoxydocosapentaenoic acid, the major CYP-generated metabolites of these primary ω-3 LCPUFAs, were identified as key lipid mediators of disease resolution. We conclude that CYP-derived bioactive lipid metabolites from ω-3 LCPUFAs are potent inhibitors of intraocular neovascular disease and show promising therapeutic potential for resolution of neovascular AMD.

Cell biological analyses of anther morphogenesis and pollen viability in Arabidopsis and rice.

Major advances have been made in recent years in our understanding of anther development through a combination of genetic studies, cell biological technologies, biochemical analysis, microarray and high-throughput sequencing-based approaches. In this chapter, we summarize the widely used protocols for pollen viability staining; the investigation of anther morphogenesis by light microscopy of semi-thin sections; TUNEL assay for programmed tapetum cell death; and laser microdissection procedures to obtain specialized cells or cell layers for carrying out transcriptomics.