Tissue Spatial Distribution Research Articles

Unraveling Spatial Domain Characterization in Spatially Resolved Transcriptomics with Robust Graph Contrastive Clustering.

Spatial transcriptomics can quantify gene expression and its spatial distribution in tissues, thus revealing molecular mechanisms of cellular interactions underlying tissue heterogeneity, tissue regeneration, and spatially localized disease mechanisms. However, existing spatial clustering methods often fail to exploit the full potential of spatial information, resulting in inaccurate identification of spatial domains. In this paper, we develop a deep graph contrastive clustering framework, stDGCC, that accurately uncovers underlying spatial domains via explicitly modeling spatial information and gene expression profiles from spatial transcriptomics data. The stDGCC framework proposes a spatially informed graph node embedding model to preserve the topological information of spots and to learn the informative and discriminative characterization of spatial transcriptomics data through self-supervised contrastive learning. By simultaneously optimizing the contrastive learning loss, reconstruction loss, and Kullback-Leibler (KL) divergence loss, stDGCC achieves joint optimization of feature learning and topology structure preservation in an end-to-end manner. We validate the effectiveness of stDGCC on various spatial transcriptomics datasets acquired from different platforms, each with varying spatial resolutions. Our extensive experiments demonstrate the superiority of stDGCC over various state-of-the-art clustering methods in accurately identifying cellular-level biological structures. Code and data are available from https://github.com/TimE9527/stDGCC and https://figshare.com/projects/stDGCC/186525. Supplementary data are available at Bioinformatics online.

Trans-eyelid distribution of epinastine to the conjunctiva following eyelid application in rabbits

PurposeTo reveal the penetration of epinastine, an anti-allergic ophthalmic agent, into the eyelid and its distribution to the conjunctiva after administration of a cream formulation on rabbit eyelid skin.Study designExperimental study.MethodsRabbits were treated with 0.5% epinastine cream on hair-shaved eyelids, followed by preparation of eyelid tissue slices to determine spatial tissue distribution of epinastine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification using laser-microdissected tissues and desorption electrospray ionization mass spectrometry imaging (DESI-MSI). In addition, following either eyelid application of 0.5% epinastine cream or ocular instillation of 0.1% epinastine eye drops, concentration-time profiles of epinastine in the palpebral conjunctiva and bulbar conjunctiva were determined using LC-MS/MS.ResultsLaser microdissection coupled with LC-MS/MS analysis detected high concentrations of epinastine around the outermost layer of the eyelid at 0.5 h post-administration that gradually diffused deeper into the eyelid and was distributed in the conjunctival layer at 8 and 24 h post-administration. Similar time-dependent drug distribution was observed in high-spatial-resolution images obtained using DESI-MSI. Epinastine concentrations in the conjunctival tissues peaked at 4–8 h after administration of 0.5% epinastine cream and then decreased slowly over 72 h post-administration. In contrast, epinastine concentrations peaked quickly and decreased sharply after epinastine eye drop administration.ConclusionAfter the application of epinastine cream to the eyelid skin, epinastine gradually permeated the eyelid. The compound was retained in the conjunctiva for 8–24 h post-administration, indicating that epinastine cream is a promising long-acting formulation for treating allergic conjunctivitis.

P099 Neutrophil extracellular traps enhance profibrotic activity of intestinal fibroblasts in Crohn's disease through TLR2/NF-kB pathway

Abstract Background Neutrophil extracellular traps (NETs) consist of DNA filaments and cytoplasmic protein granules, extruded by neutrophils after PAD4-dependent activation. NETs release occurs early during inflammation in various immune-related diseases, further aggravating tissue injury and contributing to fibrosis. Our aim was to investigate the potential profibrotic effect of NETs in Crohn’s disease (CD). Methods NETs and activated fibroblasts were labelled by multiplex immunofluorescence staining on unaffected, inflamed and fibrotic ileum derived from patients with stricturing CD undergoing ileocaecal resection. Intestinal fibroblasts isolated from unaffected CD ileum were co-cultured with NETs for 24 hours to analyse their transcriptome and quantify collagen released with SIRCOL®. Fibroblast migratory activity was investigated with a scratch test. Immunofluorescence intensity of collagen and fibroblast activation protein (FAP) was quantified using the Operetta® Analysis System. Transfection of fibroblasts with NF-kB-luciferase reporter plasmid was performed to evaluate the TLR2/NF-kB pathway, and a specific TLR2 inhibitor (TL2-C29) tested. Finally, mice with selective deletion of PAD4 in neutrophils (PAD4fl/flMRP8Cre+) were subjected to chronic Dextran Sulphate Sodium (DSS) colitis to assess the role of PAD4-mediated NETosis in intestinal fibrosis development. Results We found spatial tissue distribution of clusters of NETs adjacent to FAP+ fibroblasts in ileal ulcerations in patients with active CD. Transcriptomics demonstrated upregulation of various profibrotic processes in fibroblasts stimulated with NETs, including TGFβ-receptor signalling pathway, positive regulation of collagen metabolic process, and TLR-signalling pathways. This correlated with increased proliferation rate (p<0.0001), slower wound healing capability (p<0.0001) and higher collagen release in the medium (p<0.01) in the NET-treated group, as well as higher expression of collagens and FAP. Among TLR genes, TLR2 was found upregulated after stimulation with NETs (p=0.02). Transfection data showed significant upregulation (p<0.05) of NF-kB in the NETs-treated group, whereas its expression and soluble collagen release decreased with the addition of the TLR2 antagonist TL2-C29. In line, a significantly lower amount of collagen deposition was observed in the colon of PAD4fl/flMRP8Cre+ mice subjected to chronic DSS colitis, as well as modulation of MMP2/TIMP2 balance and reduced fibroblast activation. Conclusion NETs may represent an early trigger of intestinal fibroblast activation via the TLR2/NF-kB axis. As NETs are early players during inflammation, blocking PAD4 may reduce inflammation and thus fibrogenesis in CD.

Characterization of ginsenosides from Panax japonicus var. major (Zhu-Zi-Shen) based on ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry and desorption electrospray ionization-mass spectrometry imaging

BackgroundPanax japonicus var. major (PJM) belongs to the well-known ginseng species used in west China for hundreds of years, which has the effects of lung tonifying and yin nourishing, and exerts the analgesic, antitussive, and hemostatic activities. Compared with the other Panax species, the chemical composition and the spatial tissue distribution of the bioactive ginsenosides in PJM have seldom been investigated.MethodsUltra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) and desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) were integrated for the systematic characterization and spatial tissue distribution studies of ginsenosides in the rhizome of PJM. Considering the great difficulty in exposing the minor saponins, apart from the conventional Auto MS/MS (M1), two different precursor ions list-including data-dependent acquisition (PIL-DDA) approaches, involving the direct input of an in-house library containing 579 known ginsenosides (M2) and the inclusion of the target precursors screened from the MS1 data by mass defect filtering (M3), were developed. The in situ spatial distribution of various ginsenosides in PJM was profiled based on DESI-MSI with a mass range of m/z 100–1500 in the negative ion mode, with the imaging data processed by the High Definition Imaging (HDI) software.ResultsUnder the optimized condition, 272 ginsenosides were identified or tentatively characterized, and 138 thereof were possibly not ever reported from the Panax genus. They were composed by 75 oleanolic acid type, 22 protopanaxadiol type, 52 protopanaxatriol type, 16 octillol type, 19 malonylated, 35 C-17 side-chain varied, and 53 others. In addition, the DESI-MSI experiment unveiled the differentiated distribution of saponins, but the main location in the cork layer and phloem of the rhizome. The abundance of the oleanolic acid ginsenosides was high in the rhizome slice of PJM, which was consistent with the results obtained by UHPLC/QTOF-MS.ConclusionComprehensive characterization of the ginsenosides in the rhizome of PJM was achieved, with a large amount of unknown structures unveiled primarily. We, for the first time, reported the spatial tissue distribution of different subtypes of ginsenosides in the rhizome slice of PJM. These results can benefit the quality control and further development of PJM and the other ginseng species.

Bubble-based microrobots enable digital assembly of heterogeneous microtissue modules

The specific spatial distribution of tissue generates a heterogeneous micromechanical environment that provides ideal conditions for diverse functions such as regeneration and angiogenesis. However, to manufacture microscale multicellular heterogeneous tissue modules in vitro and then assemble them into specific functional units is still a challenging task. In this study, a novel method for the digital assembly of heterogeneous microtissue modules is proposed. This technique utilizes the flexibility of digital micromirror device-based optical projection lithography and the manipulability of bubble-based microrobots in a liquid environment. The results indicate that multicellular microstructures can be fabricated by increasing the inlets of the microfluidic chip. Upon altering the exposure time, the Young’s modulus of the entire module and different regions of each module can be fine-tuned to mimic normal tissue. The surface morphology, mechanical properties, and internal structure of the constructed bionic peritoneum were similar to those of the real peritoneum. Overall, this work demonstrates the potential of this system to produce and control the posture of modules and simulate peritoneal metastasis using reconfigurable manipulation.

Rapid Automated Annotation and Analysis of N-Glycan Mass Spectrometry Imaging Data Sets Using NGlycDB in METASPACE.

Imaging N-glycan spatial distribution in tissues using mass spectrometry imaging (MSI) is emerging as a promising tool in biological and clinical applications. However, there is currently no high-throughput tool for visualization and molecular annotation of N-glycans in MSI data, which significantly slows down data processing and hampers the applicability of this approach. Here, we present how METASPACE, an open-source cloud engine for molecular annotation of MSI data, can be used to automatically annotate, visualize, analyze, and interpret high-resolution mass spectrometry-based spatial N-glycomics data. METASPACE is an emerging tool in spatial metabolomics, but the lack of compatible glycan databases has limited its application for comprehensive N-glycan annotations from MSI data sets. We created NGlycDB, a public database of N-glycans, by adapting available glycan databases. We demonstrate the applicability of NGlycDB in METASPACE by analyzing MALDI-MSI data from formalin-fixed paraffin-embedded (FFPE) human kidney and mouse lung tissue sections. We added NGlycDB to METASPACE for public use, thus, facilitating applications of MSI in glycobiology.

Noninvasive evaluation of tumor immune microenvironment in patients with clear cell renal cell carcinoma using metabolic parameter from preoperative 2-[18F]FDG PET/CT.

Nowadays, it is necessary to explore effective biomarkers associated with tumor immune microenvironment (TIME) noninvasively. Here, we investigated whether the metabolic parameter from preoperative 2-[18F]FDG PET/CT could provide information related to TIME in patients with clear cell renal cell carcinoma (ccRCC). Ninety patients with newly diagnosed ccRCC who underwent 2-[18F]FDG PET/CT prior to surgery were retrospectively reviewed. The immunological features included tumor-infiltrating lymphocytes (TILs) density, programmed death-ligand 1 (PD-L1) expression, and tumor immune microenvironment types (TIMTs). TIMTs were classified as TIMT I (positive PD-L1 and high TILs), TIMT II (negative PD-L1 and low TILs), TIMT III (positive PD-L1 and low TILs), and TIMT IV (negative PD-L1 and high TILs). The relationship between maximum standardized uptake value (SUVmax) in the primary lesion from 2-[18F]FDG PET/CT and immunological features was analyzed. Cox proportional hazards analyses were performed to identify the prognostic factors for disease-free survival (DFS) after nephrectomy. Tumors with high TILs infiltration showed remarkable correlation with elevated SUVmax and aggressive clinicopathological characteristics, such as high World Health Organization/International Society of Urological Pathology (WHO/ISUP) grade. PD-L1 expression on tumor cells was positively associated with WHO/ISUP grade and negatively correlated with body mass index (BMI). However, no correlation was observed between SUVmax and PD-L1 expression, regardless of its spatial tissue distribution. SUVmax of TIMT I and IV was higher than that of TIMT II, but there was remarkable difference merely between TIMT II and IV. In multivariate analysis, SUVmax (P = 0.022, HR 3.120, 95% CI 1.175-8.284) and WHO/ISUP grade (P = 0.046, HR 2.613, 95% CI 1.017-6.710) were the significant prognostic factors for DFS. Six cases (16.2%) with normal SUVmax showed disease progression, while 25 cases (71.4%) with elevated SUVmax experienced disease progression. Conversely, the immunological features held no prognostic value. Our findings demonstrated that 2-[18F]FDG PET/CT could provide metabolic information of TIME for ccRCC patients and develop image-guided therapeutic strategies accordingly. Patients with elevated preoperative SUVmax should be seriously considered, and perioperative immunotherapy might be beneficial for them.

K-means clustering of coherent Raman spectra from extracellular vesicles visualized by label-free multiphoton imaging.

Extracellular vesicles (EVs) have emerged as potential biomarkers in cancer research and for clinical diagnosis. Little is known, however, about their spatial distributions in tissue and the different subpopulations that may exist. Here we report the use of label-free nonlinear optical imaging techniques to provide spatially resolved chemical information of EVs within untreated tissues. A multimodal nonlinear optical imaging system incorporating multiphoton autofluorescence and hyperspectral coherent anti-Stokes Raman scattering (CARS) imaging was built to visualize the spatial tissue distribution and probe the spectra of EVs. K-means clustering is performed on the CARS spectra from EVs in rat mammary tissues and human breast tumor tissue to reveal both the spatial distribution of EV clusters and their different chemical signatures. Correlations are identified between EV clusters and metabolic information.

The density and spatial tissue distribution of CD8+ and CD163+ immune cells predict response and outcome in melanoma patients receiving MAPK inhibitors

BackgroundClinical response to MAPK inhibitors in metastatic melanoma patients is heterogeneous for reasons still needing to be elucidated. As the patient immune activity contributes to treatment clinical benefit, the pre-existing level of immunity at tumor site may provide biomarkers of disease outcome to therapy. Here we investigated whether assessing the density and spatial tissue distribution of key immune cells in the tumor microenvironment could identify patients predisposed to respond to MAPK inhibitors.MethodsPretreatment tumor biopsies from a total of 213 patients (158 for the training set and 55 for the validation set) treated with BRAF or BRAF/MEK inhibitors within the Italian Melanoma Intergroup were stained with selected immune markers (CD8, CD163, β-catenin, PD-L1, PD-L2). Results, obtained by blinded immunohistochemical scoring and digital image analysis, were correlated with clinical response and outcome by multivariate logistic models on response to treatment and clinical outcome, adjusted for American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment received.ResultsPatients with high intratumoral, but not peritumoral, CD8+ T cells and concomitantly low CD163+ myeloid cells displayed higher probability of response (OR 9.91, 95% CI 2.23–44.0, p = 0.003) and longer overall survival (HR 0.34, 95% CI 0.16–0.72, p = 0.005) compared to those with intratumoral low CD8+ T cells and high CD163+ myeloid cells. The latter phenotype was instead associated with a shorter progression free survival (p = 0.010). In contrast, PD-L1 and PD-L2 did not correlate with clinical outcome while tumor β-catenin overexpression showed association with lower probability of response (OR 0.48, 95% CI 0.21–1.06, p = 0.068).ConclusionsAnalysis of the spatially constrained distribution of CD8+ and CD163+ cells, representative of the opposite circuits of antitumor vs protumor immunity, respectively, may assist in identifying melanoma patients with improved response and better outcome upon treatment with MAPK inhibitors. These data underline the role of endogenous immune microenvironment in predisposing metastatic melanoma patients to benefit from therapies targeting driver-oncogenic pathways.

Quantitative seasonal variation of the falcarinol‐type polyacetylene (3S)‐16,17‐didehydrofalcarinol and its spatial tissue distribution in Tridax procumbens

(3S)-16,17-Didehydrofalcarinol (1) has been isolated from Tridax procumbens and has proved to have notorious bioactivity against Leishmania mexicana. In this study, hexane fractions obtained from the methanol extract of each plant part (roots, stems, leaves, flowers, and fruits) of T. procumbens collected monthly during a year were analysed in order to determine the quantity of 1 associated with biotic variables. The aim of this study was to find the season of the year in which the bioactive metabolite 1 is at the highest concentration and to correlate it with temperature, length of day light, and rainfall. Hexane fractions were obtained by liquid-liquid extraction and an accurate quantitation of 1 was performed using gas chromatography with a flame ionisation detector (GC-FID) employing pelargonic acid vanillyl amide (2) as internal standard. Partial validation was based on linearity and precision. Our results indicated that the total content of 1 has significant variation (P≤0.05) during the different collecting months. The total content of the metabolite reached its highest level in the roots of the plant during June in the rainfall season (0.0358±0.001mg/g), and its lowest values in February and March during the drought season (0.0015±0.000 and 0.0008±0.000mg/g, respectively). Our study provided evidence that the content of 1 in roots is strongly influenced by the variables of the harvesting season, also indicating that the biosynthesis of the active metabolite is enhanced during the warm and rainy months.

Spatial Distribution of Sclerenchyma in Leaf Blades of Some Fescues (Festuca L., Gramineae Juss.)

Leaves’ anatomical characteristics are considered to be important diagnostic features for identifying grasses. Cross sections from the middle part of leaf blades have traditionally been used for such purposes. Leaf anatomy has been of key interest for identifying the representatives of the Festuca L. genus because of great morphological similarity between the species. Particular attention is paid to the arrangement of sclerenchyma in the middle part of the leaf blade. The lack of knowledge regarding the spatial distribution of tissues throughout the leaf blades of grasses does not allow one to evaluate the diagnostic significance of data obtained from a single, two-dimensional cross section. The aim of the work is to make 3D models of sclerenchyma from serial sections of leaf blades of two narrow-leaved fescues: Festuca valesiaca Gaud. and Festuca beckeri (Hack.) Trautv. Cross-sectional areas of sclerenchyma increase just above the blade/sheath junction and then gradually decrease up to the tips of the leaf blades in both species. Subepidermal sclerenchyma of the fescues appears as an uneven layer. The layer of F. beckeri is more or less solid, the layer of F. valesiaca is dissected by four long splits. Changes in the shape of sclerenchyma sections along the leaf blades are more pronounced in F. valesiaca than in F. beckeri. Variability of the sclerenchyma arrangements along the leaf blades should be considered when using 2D sections of the leaf for diagnostics of fescues.

Algorithms of Three-Dimensional Reconstructionfor Building a Model of Spatial Distributionof Tissue Properties (on the Exampleof Articular Cartilage)

The paper demonstrates relationship between morphological characteristics of articular cartilage and its functional properties in osteoarthritis. An automated analysis of the sections of the rat articular cartilage has revealed these mutual dependences. An algorithm for assessing the functional properties of cartilage by certain characteristics of its tissue in a natural full-color reconstruction is proposed.

Characterization and spatiotemporal expression analysis of nine bone morphogenetic protein family genes during intermuscular bone development in blunt snout bream

Intermuscular bones (IBs) only exist in the myosepta of lower teleosts and its molecular mechanism remains to be clarified. Bone morphogenetic proteins (BMPs) have been demonstrated to be involved in various physiological processes, including bone and cartilage formation. In this study, we firstly obtained and characterized nine bmp genes for Megalobrama amblycephala, which belongs to Cyprinidae and have a certain amount of IBs. Sequence alignment and phylogenetic analysis both documented that the mature proteins of M. amblycephala bmp genes were highly conserved with other corresponding homologs, respectively, indicating that the function of each bmp gene has been conserved throughout evolution. As a step to characterize potential involvement of bmp genes in IB formation and development, spatiotemporal expressions of nine bmp genes (bmp2a, bmp2b, bmp3, bmp4, bmp5, bmp7b, bmp8a, bmp14 and bmp16) were investigated during the key development stages of IBs. During the ossification process from stage I (the IBs haven't emerged) to stage IV (all of the IBs ossified in the tail with the mature morphology), the expression profiles revealed that bmp16 was the most abundant transcript while bmp4 had the lowest abundance. The mRNA levels of bmp3, bmp4, bmp5 and bmp8a increased significantly at stage II, suggesting their roles in stimulating IB formation. The expression of bmp7b reached the highest level at stage III (the rapid period of IB development), suggesting potential involvement of bmp7b in promoting osteoblast differentiation. With the exception of bmp7b and bmp16, most bmp genes appeared a significant increase at IB maturation phase (stage IV), which means that they may play important roles in maintenance of IB morphogenesis. Spatial tissue distribution of bmp genes showed that most bmp genes were observed at the highest level in developing IBs at one year old fish. Spatiotemporal expression patterns suggest the potential key roles of these bmp genes in IBs formation and maintenance in fish, being as possible promoters or inhibitors.

MALDI-MS Imaging of Urushiols in Poison Ivy Stem.

Urushiols are the allergenic components of Toxicodendron radicans (poison ivy) as well as other Toxicodendron species. They are alk-(en)-yl catechol derivatives with a 15- or 17-carbon side chain having different degrees of unsaturation. Although several methods have been developed for analysis of urushiols in plant tissues, the in situ localization of the different urushiol congeners has not been reported. Here, we report on the first analysis of urushiols in poison ivy stems by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Our results show that the urushiol congeners with 15-carbon side chains are mainly localized to the resin ducts, while those with 17-carbon side chains are widely distributed in cortex and vascular tissues. The presence of these urushiols in stem extracts of poison ivy seedlings was confirmed by GC-MS. These novel findings provide new insights into the spatial tissue distribution of urushiols that might be biosynthetically or functionally relevant.

Modeling Therapy Response and Spatial Tissue Distribution of Erlotinib in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. The aim of this study was to investigate the emerging technology of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery and spatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small-molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term-treated mice to measure drug tissue distribution. Histologic and statistical analyses were performed to correlate morphology, drug distribution, and survival. We found that erlotinib levels were significantly lower in PDAC compared with healthy tissue (P = 0.0078). Survival of long-term-treated mice did not correlate with overall levels of erlotinib or with overall histologic tumor grade but did correlate both with the percentage of atypical glands in the cancer (P = 0.021, rs = 0.59) and the level of erlotinib in those atypical glands (P = 0.019, rs = 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution of compounds in a preclinical setting and support drug imaging-based translational approaches. Mol Cancer Ther; 15(5); 1145-52. ©2016 AACR.

Analysis of small molecule antibody-drug conjugate catabolites in rat liver and tumor tissue by liquid extraction surface analysis micro-capillary liquid chromatography/tandem mass spectrometry.

Antibody-drug conjugates (ADCs) are some of the most promising antibody-related therapeutics. The fate of the cytotoxic moiety of ADCs in vivo after proteolytic degradation of the antibody needs to be well understood in order to mitigate toxicity risks and design proper first in patient studies. The feasibility of liquid extraction surface analysis micro-capillary liquid chromatography/tandem mass spectrometry (LESA-μLC/MS/MS) was tested for direct surface sampling of two possible ADC catabolites composed of synthetically modified maytansinoid (DM1) and 4-[N-maleimidomethyl]cyclohexane-1-carbonyl (MCC) from rat liver and tumor tissue. Moreover, the iMatrixSpray was incorporated to prepare calibration standards (Cs) and quality control (QC) samples by spraying analyte solution at different concentrations directly on blank tissue. Lys-MCC-DM1 sprayed on blank liver tissue was homogeneously distributed (12.3% variability). The assay was selective (inference ≤20%) and linear from 50.0 to 1000 ng/mL without any carry-over. Inter-run accuracy and precision were ≤2.3% and ≤25.9% meeting acceptance. Lys-MCC-DM1 was the only catabolite detected in liver and tumor tissue and was most likely responsible for the total radioactivity signal in liver tissue 72 h post-dose measured by quantitative whole body autoradiography (QWBA). Both analytical assays (LESA-μLC/MS/MS and QWBA) are complementary to each other and provide useful quantitative and qualitative information in spatial tissue distribution of ADCs and their related catabolites. Copyright © 2016 John Wiley & Sons, Ltd.

In situ drug and metabolite analysis [corrected] in biological and clinical research by MALDI MS imaging.

In recent years the analysis in mass spectrometry (MS) [corrected] imaging has been expanded to detect a wide variety of low molecular weight compounds (LMWC), including exogenous and endogenous compounds. The high sensitivity and selectivity of MS imaging combined with visualization of molecular spatial distribution in tissues, makes it a valuable [corrected] platform in targeted drug and untargeted metabolomic analysis [corrected] in biological and clinical research. Here, we review the current and potential applications of MALDI MS imaging in these areas. The aim of advancing MALDI MS imaging in the field of LMWC is to support clinical applications by understanding drug and drug-metabolite distribution, investigating toxicity and discovering [corrected] new biomarkers.

Comparative anatomy of leaflets of <i>Zamia acuminata</i> and <i>Z. pseudomonticola</i> (Zamiaceae) in Costa Rica

The genus Zamia is morphologically and ecologically the most diverse of the order Cycadales. Throughout its history this genus has been restricted to the New World and is presently almost entirely restricted to the Neotropics. Unusual anatomical traits of the leaflets, such as the sunken stomata and thick cuticle, are common in this and related genera. The objective of this research was to study and compare the leaflet anatomy of Zamia acuminata and Z. pseudomonticola and establish possible phylogenetic relationships between the anatomical traits and the near relatives of these species. The leaf material was obtained from living plants and then processed for electron microscopy study. We found that both species are very similar to each other and to Z. fairchildiana, and that they share several unusual traits with other species of the genus, such as the parenchyma morphology, the spatial distribution of tissues between the veins and the stomata morphology. The main differences between these species were seen in their fiber clusters and in the abundance of trichome basal cells on the epidermis. The anatomical similarities between the three species could be the result of their close phylogenetic relationship and the divergences between them could be the result of recent speciation during the Pleistocene, resulting from geological changes in Southern Costa Rica.

Micromechanics of the human vertebral body for forward flexion

To provide mechanistic insight into the etiology of osteoporotic wedge fractures, we investigated the spatial distribution of tissue at the highest risk of initial failure within the human vertebral body for both forward flexion and uniform compression loading conditions. Micro-CT-based linear elastic finite element analysis was used to virtually load 22 human T9 vertebral bodies in either 5° of forward flexion or uniform compression; we also ran analyses replacing the simulated compliant disc (E=8MPa) with stiff polymethylmethacrylate (PMMA, E=2500MPa). As expected, we found that, compared to uniform compression, forward flexion increased the overall endplate axial load on the anterior half of the vertebra and shifted the spatial distribution of high-risk tissue within the vertebra towards the anterior aspect of the vertebral body. However, despite that shift, the high-risk tissue remained primarily within the central regions of the trabecular bone and endplates, and forward flexion only slightly altered the ratio of cortical-to-trabecular load sharing at the mid-vertebral level (mean±SD for n=22: 41.3±7.4% compression; 44.1±8.2% forward flexion). When the compliant disc was replaced with PMMA, the anterior shift of high-risk tissue was much more severe. We conclude that, for a compliant disc, a moderate degree of forward flexion does not appreciably alter the spatial distribution of stress within the vertebral body.

Mapping Retinoids in Live P19 Cells with Autofluorescence Phasorflim Imaging

Recently a label-free FLIM imaging method has been proposed to distinguish metabolic states of germ cells (Stringari et al, 2011). This method, based on the phasor analysis of autofluorescence lifetime (AF-phasorFLIM), avoids some problems of state-of-art approaches due to multi-exponential fitting. The capability of phasorFLIM analysis to separate the different molecular species in a pixel lies on the rule of phasor addition. PhasorFLIM identifies molecules by their position in the phasor plot because every molecular species has a specific phasor that can be calibrated in reference samples.We applied phasorFLIM to detect retinoids autofluorescence in live P19 embryonal carcinoma cells that undergo differentiation when treated with retinoic acid (RA).RA and retinol are essential for cell-cell signaling during vertebrate organogenesis. RA is involved in stem cell differentiation and it is a morphogen. It is a diffusible signal that generates patterns in embryo because it induces different cellular responses depending on its local concentration. The presence of RA in tissues has been inferred through gene activation, and the RA average tissular concentration measured by HPLC. However, the spatial distribution in tissues and cells of the active molecule has never been observed or quantified directly.We built a database of phasorFLIM standards of the major molecules that contribute to autofluorescence. The database allows assigning the contribution of fluorescent molecules to P19 autofluorescence.P19 cells were treated with retinol to follow in time the effect of the molecule on cellular metabolism. PhasorFLIM analysis was performed using SimFCS and showed an increase of retinoids fluorescence after treatment. In cells treated with retinol, we observed retinol in plasma and nuclear membrane and an increase of RA in the nucleus.AF-phasorFLIM allowed determining, distinguishing and localizing the contribution of retinoids to autofluorescence in live P19 cells.