Combining Laser Microdissection Research Articles

Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus

The physiological role of amyloid precursor protein (APP) has been extensively investigated in the rodent hippocampus. Evidence suggests that APP plays a role in synaptic plasticity, dendritic and spine morphogenesis, neuroprotection and—at the behavioral level—hippocampus-dependent forms of learning and memory. Intriguingly, however, studies focusing on the role of APP in synaptic plasticity have reported diverging results and considerable differences in effect size between the dentate gyrus (DG) and area CA1 of the mouse hippocampus. We speculated that regional differences in APP expression could underlie these discrepancies and studied the expression of APP in both regions using immunostaining, in situ hybridization (ISH), and laser microdissection (LMD) in combination with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. In sum, our results show that APP is approximately 1.7-fold higher expressed in pyramidal cells of Ammon’s horn than in granule cells of the DG. This regional difference in APP expression may explain why loss-of-function approaches using APP-deficient mice revealed a role for APP in Hebbian plasticity in area CA1, whereas this could not be shown in the DG of the same APP mutants.

Renal Amyloidosis Associated With 5 Novel Variants in the Fibrinogen A Alpha Chain Protein

IntroductionFibrinogen A alpha chain amyloidosis is an autosomal dominant disease associated with mutations in the fibrinogen A alpha chain (FGA) gene, and it is the most common cause of hereditary renal amyloidosis in the UK. Patients typically present with kidney impairment and progress to end-stage renal disease over a median time of 4.6 years.MethodsSix patients presented with proteinuria, hypertension, and/or lower limb edema and underwent detailed clinical and laboratory investigations.ResultsA novel FGA gene mutation was identified in each case: 2 frameshift mutations F521Sfs*27 and G519Efs*30 and 4 single base substitutions G555F, E526K, E524K, R554H. In 5 subjects, extensive amyloid deposits were found solely within the glomeruli, which stained specifically with antibodies to fibrinogen A alpha chain, and in one of these cases, we found coexistent fibrinogen A alpha chain amyloidosis and anti-glomerular basement membrane antibody disease. One patient was diagnosed with light-chain amyloidosis after a bone marrow examination revealed a small clonal plasma cell population, and laser microdissection of the amyloid deposits followed by liquid chromatography and tandem mass spectrometry identified kappa light chain as the fibril protein.DiscussionWe report 6 novel mutations in the FGA gene: 5 were associated with renal fibrinogen A alpha chain amyloidosis and 1 was found to be incidental to light-chain amyloid deposits discovered in a patient with a plasma cell dyscrasia. Clinical awareness and suspicion of hereditary amyloidosis corroborated by genetic analysis and adequate typing using combined immunohistochemistry and laser microdissection and mass spectrometry is valuable to avoid misdiagnosis, especially when a family history of amyloidosis is absent.

Continued improvement in overall survival in elderly multiple myeloma patients after 2008; a population based study from the Danish Multiple Myeloma Registry

Graduate School of Health Sciences, Kumamoto University; Department of Morphological and Physiological Sciences, Kumamoto University; Faculty of Life Sciences, Department of Neurology, Kumamoto University; Diagnostic Unit for Amyloidosis, Kumamoto University Hospital; Faculty of Life Sciences, Department of Hematology, Kumamoto University; Department of Surgical Pathology, Kumamoto General Hospital; Division of Informative Clinical Sciences, Kumamoto University Introduction: AL amyloidosis is a disease featuring deposition of Ig light chain (LC). Diagnosis is determined if the presence of LC in amyloid deposits is detected with anti-LC chain antibodies. Poor prognosis cases, like detected amyloid deposits in heart, require the prompt diagnoses. However, in several cases, the existence of LC is not detected within amyloid deposits with immunohistochemistry (IHC) using conventional anti-LC antibodies although monoclonal lambda light chain in serum or urine was proved. In such cases, we found deposition of particular light chain constant region (immunoglobulin light chain constant region 2: IGLC2) at amyloid tissues. Materials and Methods: Amyloid tissues were either stained with conventional anti-human lambda LC rabbit antiserum (DAKO PO-0130) or rabbit anti-IGLC2 antibodies (made by SIGMA ALDLICH). Deposited peptide within amyloid lesion was examined using Congo Red staining followed by combination of laser micro-dissection and mass-spectrometry combining (LC-MS/MS). Plasma cells were purified with CD138-immunomagnetic beads from bone marrow aspirated obtained from two cases. Recombination of LC DNA in purified plasma cells was analyzed using PCR and direct nucleic acid sequencing. Results and Discussion: LC-MS/MS analysis revealed the presence of IGLC2 in all cases without showing positive signal by conventional antibody. Recombination of IGLC2 was detected in plasma cells. Deposition of IGLC2 was confirmed by IHC using anti-IGLC2 antibodies. The present data imply that “IGLC2-related amyloidosis” might be overlooked by conventional IHC unless utilizing mass-spectrometry. Utilization of antibodies for IGLC2 should contribute to improve quality of diagnosis for AL-amyloidosis. We consider that investigation of IGLC2 is important for not only making diagnosis but also analyzing pathogenesis of AL-amyloidosis.

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer

More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins-SLC3A2, STMN1, and TAGLN2-in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant overexpression in individual bladder cancer tissues and urine specimens, and thus represents a potential biomarker for noninvasive screening for bladder cancer. Our findings highlight the value of bladder tissue proteome in providing valuable information for future validation studies of potential biomarkers in urothelial carcinoma.

αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell-related features.

Objectives:To characterize phenotypes of T cells that accumulated in multiple sclerosis (MS) lesions, to compare the lesional T-cell receptor (TCR) repertoire of T-cell subsets to peripheral blood, and to identify paired α and β chains from single CD8+ T cells from an index patient who we followed for 18 years.Methods:We combined immunohistochemistry, laser microdissection, and single-cell multiplex PCR to characterize T-cell subtypes and identify paired TCRα and TCRβ chains from individual brain-infiltrating T cells in frozen brain sections. The lesional and peripheral TCR repertoires were analyzed by pyrosequencing.Results:We found that a TCR Vβ1+ T-cell population that was strikingly expanded in active brain lesions at clinical onset comprises several subclones expressing distinct yet closely related Vα7.2+ α chains, including a canonical Vα7.2-Jα33 chain of mucosal-associated invariant T (MAIT) cells. Three other α chains bear striking similarities in their antigen-recognizing, hypervariable complementarity determining region 3. Longitudinal repertoire studies revealed that the TCR chains that were massively expanded in brain at onset persisted for several years in blood or CSF but subsequently disappeared except for the canonical Vα7.2+ MAIT cell and a few other TCR sequences that were still detectable in blood after 18 years.Conclusions:Our observation that a massively expanded TCR Vβ1-Jβ2.3 chain paired with distinct yet closely related canonical or atypical MAIT cell–related α chains strongly points to an antigen-driven process in early active MS brain lesions.

Transcriptional characteristics and differences in Arabidopsis stigmatic papilla cells pre- and post-pollination.

Pollination is an important early step in sexual plant reproduction. In Arabidopsis thaliana, sequential pollination events, from pollen adhesion onto the stigma surface to pollen tube germination and elongation, occur on the stigmatic papilla cells. Following successful completion of these events, the pollen tube penetrates the stigma and finally fertilizes a female gametophyte. The pollination events are thought to be initiated and regulated by interactions between papilla cells and pollen. Here, we report the characterization of gene expression profiles of unpollinated (UP), compatible pollinated (CP) and incompatible pollinated (IP) papilla cells in A. thaliana. Based on cell type-specific transcriptome analysis from a combination of laser microdissection and RNA sequencing, 15,475, 17,360 and 16,918 genes were identified as expressed in UP, CP and IP papilla cells, respectively, and, of these, 14,392 genes were present in all three data sets. Differentially expressed gene (DEG) analyses identified 147 and 71 genes up-regulated in CP and IP papilla cells, respectively, and 115 and 46 genes down-regulated. Gene Ontology and metabolic pathway analyses revealed that papilla cells play an active role as the female reproductive component in pollination, particularly in information exchange, signal transduction, internal physiological changes and external morphological modification. This study provides fundamental information on the molecular mechanisms involved in pollination in papilla cells, furthering our understanding of the reproductive role of papilla cells.

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.

Deposition of Lambda Chain Constant Region within AL-Amyloid Lesion

[Introduction]Diagnosis of AL amyloidosis is dependent on the proof of light chains in amyloid lesions. However, immunostaining does not always successfully prove the presence of light chains in lesions in AL amylidosis patients. Here we report that the constant region of immunoglobulin lambda light chain (IGLC2) is seen in amyloid lesions where no positive signals are found with regular immunostaining.[Materials and Methods]Amyloid samples were stained with anti-human lambda light chain antibody (DAKO PO-0130) and analyzed with mass-spectrometry combining laser micro-dissection. Bone marrow samples were obtained from patients with amyloidosis, who gave written informed consent, and were subjected to plasma cell purification using CD138-immunomagnetic beads. Expression of immunoglobulin light chain mRNA was examined with RT-PCR. Anti-human IGLL5 antibody, capable of detecting immunoglobulin light chain constant region 2 (IGLC2) in paraffin embedded samples, was utilized.[Results and Discussion]We performed immunostaining for immunoglobulin light chains with 18 samples and found that six and eight cases were positive for kappa and lambda light chains, respectively, whereas light chains were not detected in remaining four cases (immunostaining-negative amyloidosis; INA). However, interestingly, mass spectrometry analysis revealed the presence of IGLC2 in all of the INA cases. RT-PCR analysis revealed the presence of IGLC2 mRNA in plasma cells from such INA cases. Surprisingly, amyloid lesions in all of the INA cases were positively stained with anti-IGLL5 antibody, whereas no staining was found in other samples positively stained with DAKO PO-0130. These observations suggest that the deposition of IGLC2 may cause AL amyloidosis, which otherwise could not be diagnosed with regular immunostaining. Although high dose chemotherapy produced hematological remission, half of such cases died within one year, suggesting irreversible and life-threatening amyloid fibril depositions in critical organs in IGLC2-related cases. We further examined additional twelve cases with AL amyloidosis to determine the incidence of IGLC2-related amyloidosis by immunostaining. With regular immunostaining, kappa and lambda chain were found in three and five cases, respectively. Interestingly, the remaining four cases were negative with regular immunostaining but positive with anti-IGLL5 antibody. Taken these observations together, eight IGLC2-related amyloidosis cases and thirteen lambda type amyloidosis were identified. Thus, the incidence of IGLC2-related amyloidosis should be approximately 38% (8/21) among lambda type AL amyloidosis.We conclude that diagnosis of IGLC2-related AL amyloidosis was possible only with the use of anti-IGLL5 antibody, but not with regular immunostaining. Given the relatively high incidence and often poor prognosis of IGLC2-related amyloidosis, it is important that this clinical entity is recognized to potentially improve outcomes of treatments. Analysis of mechanisms regulating amyloid formation with IGLC2 peptides is currently underway. DisclosuresNo relevant conflicts of interest to declare.

G.P.64: Proteomic analysis of rimmed vacuoles in sporadic inclusion body myositis (sIBM)

Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy in patients >50 years and characterized by both degenerative and autoimmune features. Rimmed vacuoles (RV) are a typical histological finding in sIBM. The aim of our study was to identify proteins that accumulate in RV by using a proteomic approach. Skeletal muscle samples of 18 sIBM patients were included in this study. RV samples and intraindividual control samples (CS; from muscle fibers without vacuolar changes) were collected from 10 μm H&E stained muscle sections by laser microdissection and analyzed by a highly sensitive mass spectrometry approach. A total of 3875 different proteins were identified and 215 of these showed a statistically significant accumulation in RV samples compared to CS. Proteins of the extracellular matrix and of the basal lamina, sarcolemmal proteins and intermediate filaments were the most abundant over-represented proteins in RV. In addition, we found an accumulation of proteins that play a role in protein degradation and of several chaperones including BiP and calreticulin, two proteins that are involved in unfolded protein response and SR stress, and components of a chaperone complex that was not described in the context of sIBM so far. Our proteomic approach also revealed an over-representation of proteins that play a role in inflammatory pathways, e.g. interferon-induced proteins and proteins involved in T-cell activation. In conclusion, our proteomic data provide essential new insights into the composition of rimmed vacuoles in sIBM. The application of a combined laser microdissection and mass spectrometry approach enabled the identification of more than 200 proteins that accumulate in RV areas. The results not only confirm previous findings but expand our knowledge about proteins and pathways that seem to be relevant in pathogenesis of sIBM.

Placenta‐specific miRNA (miR‐512‐3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells

The microRNAs (miRNAs) derived from the chromosome 19 miRNA cluster (C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19MC miRNAs are not fully understood. The purpose of this study was to elucidate which cells express C19MC miRNAs in chorionic villi and identify their miRNA targets. A combination of laser microdissection (LMD) and real-time polymerase chain reaction (PCR) to examine the localization of five C19MC miRNAs (i.e. miR-512-3p, miR-518b, miR-520a, miR-524 and miR-1323) in the human placenta was performed. Furthermore, to identify miR-512-3p-target genes, we analyzed gene expression profiles of the trophoblast cell line BeWo using a DNA microarray. Predicted target genes were validated by real-time PCR, western blotting, and 3'-untranslated region reporter assay. By LMD and subsequent PCR analysis, five C19MC miRNAs examined in this study were predominantly expressed in villous trophoblast cells; little expression, if any, was observed in villous stroma cells or fetal endothelial cells. Microarray data showed that 334 genes were downregulated in BeWo cells treated with Pre-miR-512-3p (mature miR-512-3p mimic). We found six candidate target genes of miR-512-3p using DNA microarray data and target prediction software. Furthermore, we revealed that protein phosphatase 3, regulatory subunit B, alpha (PPP3R1), one of the six genes, was a miR-512-3p target using an in vitro experimental validation system. These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.

P.15.6 Desminopathy or myotilinopathy? An integrated proteomics approach for diagnosis

Protein aggregation in skeletal muscle fibers is a hallmark of myofibrillar myopathies and relevant in pathogenesis. Our previous proteomic studies deciphered details of aggregate composition and revealed specific proteomic profiles in different MFM subtypes. The aim of this study was to validate if these profiles are helpful in differential diagnosis in a patient with new mutations in two different MFM genes. The index patient presented with adult-onset weakness affecting limb and respiratory muscles. Two novel and heterozygous nucleotide exchanges in exon 1 of DES and exon 2 of MYOT, both predicted missense mutations, were identified and MFM was proven on the muscle biopsy. Protein aggregates from abnormal fibers and control samples from normally looking muscle fibers were collected by laser microdissection and analyzed by a combination of mass spectrometry (LC-MS/MS) and spectral index calculation. Our proteomic approach detected 113 proteins that were over-represented in intramyoplasmic aggregates of the index patient with a ratio >1.8 compared to control sample. The proteomic profile was consistent with desminopathy: desmin, filamin C, XIRP2, N-Rap and α B-crystallin showed the highest spectral indices of over-represented proteins. Accumulation of the desmin binding partner desmuslin also pointed to a pathogenic desmin mutation. The myotilinopathy markers plectin and obscurin were not over-represented in aggregates of the index patient and spectral index and ratio of myotilin also argued against a MFM-causing MYOT mutation. The results of our study indicate that the index patient harbors a disease-causing desmin mutation and demonstrate that our combined laser microdissection and mass spectrometric approach is a helpful new tool in differential diagnostics of MFM patients. Subtype-specific proteomic profiles can contribute to evaluate the pathogenicity of new mutations in MFM genes.

Abstract 1514: Proteomics of human dense breast stroma.

Abstract Dense breast contains glandular and connective tissue. Less dense breasts are mainly comprised of fat tissue. Dense breast tissue itself is a risk factor for breast cancer with the risk being 4-6 fold higher in women with extremely dense breasts compared to fatty breasts. Typically, breast cancer is more likely to develop in the denser areas of the breast. Furthermore, on mammogram, a tumor is more difficult to detect in dense tissue than in fatty tissue because the tumor looks similar to the tissue that surrounds it. Recognition of the important role of the stroma during the progression of breast cancer leads to the possibility of new targets for the prevention or treatment of the primary breast cancer. In addition, clinically useful biomarkers for the early detection of risk for breast cancer in dense breasts could lead to a significant reduction of mortality. In this investigation we describe a detailed analysis of normal and dense breast stroma using laser microdissection in combination with mass spectrometry and immunohistochemistry of normal and dense breast stroma. Using this approach several potential markers for dense breast were identified and compared to stromal markers associated with breast cancer. Citation Format: Jay W. Fox, Leann Mikesh, Lavakumar R. Aramadhaka, Christopher A. Moskulak. Proteomics of human dense breast stroma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2013-1514

The Arabidopsis leaf provascular cell transcriptome is enriched in genes with roles in vein patterning

Several classes of genes have been associated, by mutant phenotypes or cell biology, with the formation of vein patterns during early leaf development, including genes for certain transcription factors, auxin transport and response factors, endomembrane traffic components and other signaling pathway components. The majority of these are expressed with spatial and temporal specificity that includes expression in the precursors of vascular cells - provascular (PV) and procambial (PC) cells - suggesting that other PV/PC-specific genes might have roles in vein patterning. We inventoried the PV/PC transcriptome of Arabidopsis leaves using a combination of laser microdissection and microarray expression profiling, and determined the phenotypes of knock-outs of previously uncharacterized PV/PC-specific genes. As examples, we observed vein pattern defects in knock-out lines of KEG and a CCCH zinc finger protein. This strategy of gene discovery, based on the identification of a gene set co-expressed in the same cells during the targeted developmental event, appears to be an efficient means of identifying genes functionally relevant to the event. In the case of vein patterning, this strategy would have identified many or most of the genes previously obtained by labor-intensive screening for pattern-defective mutants.

Evaluation of the Use of Induced Pluripotent Stem Cells (iPSCs) for the Regeneration of Tracheal Cartilage

The treatment of laryngotracheal stenosis remains a challenge as treatment often requires multistaged procedures, and successful decannulation sometimes fails after a series of operations. Induced pluripotent stem cells (iPSCs) were generated in 2006. These cells are capable of unlimited symmetrical self-renewal, thus providing an unlimited cell source for tissue-engineering applications. We have previously reported tracheal wall regeneration using a three-dimensional (3D) scaffold containing iPSCs. However, the efficiency of differentiation into cartilage was low. In addition, it could not be proven that the cartilage tissues were in fact derived from the implanted iPSCs. The purpose of this study was to evaluate and improve the use of iPSCs for the regeneration of tracheal cartilage. iPSCs were cultured in vitro in a 3D scaffold in chondrocyte differentiation medium. After cultivation, differentiation into chondrocytes was examined. The ratio of undifferentiated cells was analyzed by flow cytometry. The 3D scaffolds were implanted into tracheal defects, as an injury site, in 24 nude rats. Differentiation into chondrocytes in vitro was confirmed histologically, phenotypically, and genetically. Flow cytometric analysis demonstrated that the population of undifferentiated cells was decreased. Cartilage tissue was observed in the regenerated tracheal wall in 6 of 11 rats implanted with induced iPSCs, but in none of 13 rats implanted with the control and noninduced iPSCs. The expression of cartilage-specific protein was also demonstrated in vivo in 3D scaffolds containing iPSCs. The presence of the GFP gene derived from iPSCs was confirmed in samples of cartilage tissue by the combination of laser microdissection (LMD) and polymerase chain reaction (PCR) techniques. Our study demonstrated that iPSCs have the potential to differentiate into chondrogenic cells in vitro. Cartilage tissue was regenerated in vivo. Our results suggest that iPSCs could be a new cell source for the regeneration of tracheal cartilage.

A Combined Laser Microdissection and Mass Spectrometry Approach Reveals New Disease Relevant Proteins Accumulating in Aggregates of Filaminopathy Patients

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.

A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis.

BackgroundAbout 80% of today’s land plants are able to establish an arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi to improve their access to nutrients and water in the soil. On the molecular level, the development of AM symbioses is only partly understood, due to the asynchronous development of the microsymbionts in the host roots. Although many genes specifically activated during fungal colonization have been identified, genome-wide information on the exact place and time point of their activation remains limited.ResultsIn this study, we relied on a combination of laser-microdissection and the use of Medicago GeneChips to perform a genome-wide analysis of transcription patterns in defined cell-types of Medicago truncatula roots mycorrhized with Glomus intraradices. To cover major stages of AM development, we harvested cells at 5-6 and at 21 days post inoculation (dpi). Early developmental stages of the AM symbiosis were analysed by monitoring gene expression in appressorial and non-appressorial areas from roots harbouring infection units at 5-6 dpi. Here, the use of laser-microdissection for the first time enabled the targeted harvest of those sites, where fungal hyphae first penetrate the root. Circumventing contamination with developing arbuscules, we were able to specifically detect gene expression related to early infection events. To cover the late stages of AM formation, we studied arbusculated cells, cortical cells colonized by intraradical hyphae, and epidermal cells from mature mycorrhizal roots at 21 dpi. Taken together, the cell-specific expression patterns of 18014 genes were revealed, including 1392 genes whose transcription was influenced by mycorrhizal colonization at different stages, namely the pre-contact phase, the infection of roots via fungal appressoria, the subsequent colonization of the cortex by fungal hyphae, and finally the formation of arbuscules. Our cellular expression patterns identified distinct groups of AM-activated genes governing the sequential reprogramming of host roots towards an accommodation of microsymbionts, including 42 AM-activated transcription factor genes.ConclusionsOur genome-wide analysis provides novel information on the cell-specific activity of AM-activated genes during both early and late stages of AM development, together revealing the road map of fine-tuned adjustments of transcript accumulation within root tissues during AM fungal colonization.

Fluorescence laser microdissection reveals a distinct pattern of gene activation in the mouse hippocampal region

A histoanatomical context is imperative in an analysis of gene expression in a cell in a tissue to elucidate physiological function of the cell. In this study, we made technical advances in fluorescence laser microdissection (LMD) in combination with the absolute quantification of small amounts of mRNAs from a region of interest (ROI) in fluorescence-labeled tissue sections. We demonstrate that our fluorescence LMD-RTqPCR method has three orders of dynamic range, with the lower limit of ROI-size corresponding to a single cell. The absolute quantification of the expression levels of the immediate early genes in an ROI equivalent to a few hundred neurons in the hippocampus revealed that mice transferred from their home cage to a novel environment have distinct activation profiles in the hippocampal regions (CA1, CA3, and DG) and that the gene expression pattern in CA1, but not in the other regions, follows a power law distribution.

Sample Preparation for Laser-Microdissection of Soybean Shoot Apical Meristem

The shoot apical meristem houses stem cells responsible for the continuous formation of aerial plant organs including leaves and stems throughout the life of plants. Laser-microdissection in combination with high-throughput technology such as next generation sequencing permits an in-depth analysis of molecular events associated with specific cell type of interest. Sample preparation is the most critical step in ensuring good quality RNA to be extracted from samples following laser-microdissection. Here, we optimized the sample preparation for a major legume crop, soybean. We used Farmer’s solution as a fixative and paraffin as the embedding medium for soybean shoot apical meristem tissue without the use of any specialized equipment. Shorter time for tissue fixation (two days) was found to be critical for the preservation of RNA in soybean shoot apical meristem. We further demonstrated the utility of this method for different tissues derived from soybean and rice. The method outlined here shall facilitate studies on crop plants involving laser-microdissection.

Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development.

BackgroundDuring sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of sexual spores. Fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression. However, little is known about the spatial distribution of gene expression in fungal development. Here, we used laser microdissection (LM) and RNA-seq to determine gene expression patterns in young fruiting bodies (protoperithecia) and non-reproductive mycelia of the ascomycete Sordaria macrospora.ResultsQuantitative analysis showed major differences in the gene expression patterns between protoperithecia and total mycelium. Among the genes strongly up-regulated in protoperithecia were the pheromone precursor genes ppg1 and ppg2. The up-regulation was confirmed by fluorescence microscopy of egfp expression under the control of ppg1 regulatory sequences. RNA-seq analysis of protoperithecia from the sterile mutant pro1 showed that many genes that are differentially regulated in these structures are under the genetic control of transcription factor PRO1.ConclusionsWe have generated transcriptional profiles of young fungal sexual structures using a combination of LM and RNA-seq. This allowed a high spatial resolution and sensitivity, and yielded a detailed picture of gene expression during development. Our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor PRO1 is involved in the regulation of many genes expressed specifically in sexual structures. The LM/RNA-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated.

Tissue-specific metabolite profiling of alkaloids in Sinomenii Caulis using laser microdissection and liquid chromatography–quadrupole/time of flight-mass spectrometry

Secondary metabolites accumulated in different tissues and cells of herbs are usually bioactive components of herbal medicines. Thus, tissue- and cell-specific phytochemical profiling should be useful for indicating relationship between herbal tissues and chemicals, and evaluating the quality of a medicinal herb. Here, a method that combining laser microdissection and ultra-performance liquid chromatography–quadrupole/time-of-flight mass spectrometry (LMD with UPLC–Q/TOF-MS) was established to achieve simultaneous localization and determination of bioactive components in herbal medicines. Sinomenii Caulis, sourced from the stems of Sinomenium acutum (Thunb.) Rehd. et Wils., was set as an illustrative case, and its phytochemicals were profiled by the present method through analyses of different microdissected tissues and cells, involving epidermis, cortex, stone cells, pericycle, vascular bundles and pith. Results revealed that different tissues and cells contained varied alkaloids, among which six alkaloids, i.e. 6-Me-ether-12-O-β-d-glucopyranoside-laudanosoline (peak 4), sinomenine (peak 6), N-norsinoacutine (peak 7), magnoflorine (peak 11), laurifoline (peak 16) and menisperine (peak 17) were detected in all microdissected parts, and sinomenine and magnoflorine were the two most abundant components. By further quantitative determination, alkaloids were generally demonstrated to distribute in the outer part of the cortex, phloem and xylem. According to the relationship between alkaloids and tissues revealed in our study, Sinomenii Caulis of larger diameter has proportionately more bioactive components, and is therefore of higher quality for medicinal use. The method of LMD with UPLC–Q/TOF-MS developed in this study was initially applied to the research of medicinal herbs, and proved to be high sensitive, low cost, convenient and practical.