scholarly journals Single-Cell Mapping and 3D Tissue Reconstruction using Cryosection-derived Images and Tissue Mapper software

2021 ◽  
Author(s):  
Shanina Robbins ◽  
Rajanikanth Vadigepalli ◽  
James Schwaber
Keyword(s):  
3D Visualization ◽  
Molecular Data ◽  
Single Neurons ◽  
Cell Mapping ◽  
Face Images ◽  
Tissue Reconstruction ◽  
Block Face ◽  
The Individual ◽  
Anatomical Information ◽  
Laser Capture

Abstract The present protocol describes the process of using the MBF Bioscience Tissue Mapper software to create a 3D reconstructed stack from contouring the block face images of serial cryosections and mark the individual immunolabeled cells onto the contoured images. The stack of aligned, contoured and annotated images can be visualized as a 3D reconstruction that depicts the tissue shape as it was embedded and cryosectioned, relative positions of various tissue features, and locations of individually marked neurons within the tissue, yielding a 3D anatomical map of the distribution of cells of interest with a tissue. This protocol can be used in conjunction with other protocols for sampling spatially-tracked single neurons using laser capture micro-dissection and assessed for molecular profiles using transcriptomics approaches. Such integration extends the present protocol to enable 3D visualization of molecular data in conjunction with the anatomical information for spatial transcriptomic analysis.

scholarly journals Cryosectioning, block face imaging and Nissl staining fluorescently labeled pig heart

2021 ◽  
Author(s):  
Shaina Robbins ◽  
Rajanikanth Vadigepalli ◽  
James Schwaber
Keyword(s):  
Molecular Data ◽  
Heart Tissue ◽  
Single Neurons ◽  
Nissl Staining ◽  
Microscopic Imaging ◽  
Tissue Sections ◽  
Face Images ◽  
Fresh Frozen ◽  
Block Face ◽  
Laser Capture

Abstract The present protocol describes the process of cryosectioning OCT-embedded fresh frozen pig heart tissue, along with the acquisition of block face images, followed by Nissl staining of the tissue sections and microscopic imaging of the sections for localizing the fluorescently-labeled neurons in the tissue. This tissue section preparation workflow was optimized for use in laser capture microdissection of single neurons from the pig heart tissue, which are used in downstream transcriptomic analysis and mapping of the neuronal location and molecular data to a 3D reconstructed contour stack of block face images.

PATH-39. INTEGRATED MOLECULAR-MORPHOLOGICAL MENINGIOMA CLASSIFICATION: A MULTICENTER RETROSPECTIVE ANALYSIS, RETRO- AND PROSPECTIVELY VALIDATED

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi123-vi124
Author(s):  
Sybren Maas ◽  
Damian Stichel ◽  
Thomas Hielscher ◽  
Philipp Sievers ◽  
Anna Berghoff ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Copy Number ◽  
Retrospective Cohort ◽  
Clinical Decision Making ◽  
Clinical Decision ◽  
Molecular Data ◽  
Copy Number Variations ◽  
Who Grade ◽  
Risk Of Progression ◽  
The Individual

Abstract PURPOSE Meningiomas are the most frequent primary intracranial tumors. Patient outcome varies widely from cases with benign to highly aggressive, ultimately fatal courses. Reliable identification of risk of progression for the individual patient is of pivotal importance in clinical management. However, only biomarkers for highly aggressive tumors are established at present (CDKN2A/B and TERT), while no molecularly-based stratification exists for the broad spectrum of low- and intermediate-risk meningioma patients. PATIENTS AND METHODS DNA methylation data and copy-number information were generated for 3,031 meningiomas of 2,868 individual patients, with mutation data for 858 samples. DNA methylation subgroups, copy-number variations (CNV), mutations and WHO grading were comparatively analyzed. Prediction power for outcome of these parameters was assessed in an initial retrospective cohort of 514 patients, and validated on a retrospective cohort of 184, and on a prospective cohort of 287 multi-center cases, respectively. RESULTS Both CNV and methylation family- (MF)-based subgrouping independently resulted in an increase in prediction accuracy of risk of recurrence compared to the WHO classification (c-indexes WHO 2016, CNV, and MF 0.699, 0.706 and 0.721, respectively). Merging all independently powerful risk stratification approaches into an integrated molecular-morphological score resulted in a further, substantial increase in accuracy (c-index 0.744). This integrated score consistently provided superior accuracy in all three cohorts, significantly outperforming WHO grading (c-index difference p=0.005). Besides the overall stratification advantage, the integrated score separates more precisely for risk of progression at the diagnostically challenging interface of WHO grade 1 and grade 2 tumors (HR 4.56 [2.97;7.00], 4.34 [2.48;7.57] and 3.34 [1.28; 8.72] for discovery, retrospective, and prospective validation cohort, respectively). CONCLUSIONS Merging these layers of histological and molecular data into an integrated, three-tiered score significantly improves the precision in meningioma stratification. Implementation into diagnostic routine informs clinical decision-making for meningioma patients on the basis of robust outcome prediction.

Evolution of mutation rate and virulence among human retroviruses

1994 ◽  
Vol 346 (1317) ◽  
pp. 333-343 ◽  
Keyword(s):  
Immune System ◽  
Mutation Rate ◽  
Hiv Infections ◽  
Molecular Data ◽  
Mutation Rates ◽  
Rapid Progression ◽  
Human T Cell ◽  
Large Numbers ◽  
The Individual ◽  
Multicellular Organisms

High mutation rates are generally considered to be detrimental to the fitness of multicellular organisms because mutations untune finely tuned biological machinery. However, high mutation rates may be favoured by a need to evade an immune system that has been strongly stimulated to recognize those variants that reproduced earlier during the infection, hiv infections conform to this situation because they are characterized by large numbers of viruses that are continually breaking latency and large numbers that are actively replicating throughout a long period of infection. To be transmitted, HIVS are thus generally exposed to an immune system that has been activated to destroy them in response to prior viral replication in the individual. Increases in sexual contact should contribute to this predicament by favouring evolution toward relatively high rates of replication early during infection. Because rapid replication and high mutation rate probably contribute to rapid progression of infections to aids, the interplay of sexual activity, replication rate, and mutation rate helps explain why HIV-1 has only recently caused a lethal pandemic, even though molecular data suggest that it may have been present in humans for more than a century. This interplay also offers an explanation for geographic differences in progression to cancer found among infections due to the other major group of human retroviruses, human T-cell lymphotropic viruses (HTLV). Finally, it suggests ways in which we can use natural selection as a tool to control the aids pandemic and prevent similar pandemics from arising in the future.

Single-Neuron Responses to Rapidly Presented Temporal Sequences in the Primary Auditory Cortex of the Awake Macaque Monkey

2007 ◽  
Vol 97 (2) ◽  
pp. 1726-1737 ◽  
Author(s):  
M. L. Phan ◽  
G. H. Recanzone
Keyword(s):  
Auditory Cortex ◽  
Cortical Neurons ◽  
Primary Auditory Cortex ◽  
Macaque Monkey ◽  
Auditory Task ◽  
Single Neurons ◽  
Animal Vocalizations ◽  
Early Processing ◽  
The Individual ◽  
Temporal Rate

One fundamental process of the auditory system is to process rapidly occurring acoustic stimuli, which are fundamental components of complex stimuli such as animal vocalizations and human speech. Although the auditory cortex is known to subserve the perception of acoustic temporal events, relatively little is currently understood about how single neurons respond to such stimuli. We recorded the responses of single neurons in the primary auditory cortex of alert monkeys performing an auditory task. The stimuli consisted of four tone pips with equal duration and interpip interval, with the first and last pip of the sequence being near the characteristic frequency of the neuron under study. We manipulated the rate of presentation, the frequency of the middle two tone pips, and the order by which they were presented. Our results indicate that single cortical neurons are ineffective at responding to the individual tone pips of the sequence for pip durations of <12 ms, but did begin to respond synchronously to each pip of the sequence at 18-ms durations. In addition, roughly 40% of the neurons tested were able to discriminate the order that the two middle tone pips were presented in at durations of ≥24 ms. These data place the primate primary auditory cortex at an early processing stage of temporal rate discrimination.

scholarly journals Phylogenetic information of freshwater copepod (Diaptomus sicilis) with special reference to 18S rRNA

2016 ◽  
Vol 4 (1) ◽  
pp. 25 ◽  
Author(s):  
Gomathi Jeyam Mookkaiah ◽  
Ramanibai Ravichandran
Keyword(s):  
Tamil Nadu ◽  
18S Rrna ◽  
Molecular Data ◽  
Small Subunit ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Calanoid Copepods ◽  
Universal Primer ◽  
Pcr Products ◽  
The Individual

<p>In the present investigation to isolate freshwater calanoid copepods (<em>Diaptomus sicilis</em>) was characterized and identify the organisms by 18S rRNA sequencing. Plankton samples containing <em>D. sicilis</em> were collected during January 2014 (Post-monsoon) from Madippakkam Lake (12°57'41"N80°11'27"E) Chennai, Tamil Nadu. Immediately after sampling, specimens for genetic analyses were fixed in 95% ethyl alcohol. The total DNA was extracted from the individual copepod <em>D. sicilis</em> using Qiagen Blood tissue kit. The nuclear small subunit 18S rRNA gene was amplified using the Universal primer LCO —1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’). PCR products were loaded onto a 1% TAE agarose gel. Sequences were carried out an automated sequencer. The nucleotide sequence of 1282 base pair region of 18S rRNA was determined for D. sicilis. The similarity of sequences of <em>D. sicilis</em> was retrieved by BLASTn pro­gram and maximum identity and E-value was 76% and 0.00, respectively. The PCR products of <em>D. sicilis</em> individuals showed 80% similarity with the partial nuclear small subunit 18S rRNA gene region of other calanoid copepods. Based on molecular data the freshwater Calanoid copepods showed different algorithms and similar types of topologies useful for designing molecular analyses using phylogeny tree construction.Present molecular stud­ies on the relationship of D. sicilis with other freshwater calanoid copepods indicate that this species is close to <em>D. castor</em> followed by <em>D. keniraensis</em><em>.</em></p>

scholarly journals Unravelling the Complexity of Plant Defense Induced by a Simultaneous and Sequential Mite and Aphid Infestation

2019 ◽  
Vol 20 (4) ◽  
pp. 806 ◽  
Author(s):  
Małgorzata Kiełkiewicz ◽  
Anna Barczak-Brzyżek ◽  
Barbara Karpińska ◽  
Marcin Filipecki
Keyword(s):  
Spider Mite ◽  
Green Peach Aphid ◽  
Marker Gene ◽  
Molecular Data ◽  
Aphid Infestation ◽  
Aphid Feeding ◽  
Two Spotted Spider Mite ◽  
The Individual ◽  
Ja Signaling ◽  
Arabidopsis Defense

In natural and agricultural conditions, plants are attacked by a community of herbivores, including aphids and mites. The green peach aphid and the two-spotted spider mite, both economically important pests, may share the same plant. Therefore, an important question arises as to how plants integrate signals induced by dual herbivore attack into the optimal defensive response. We showed that regardless of which attacker was first, 24 h of infestation allowed for efficient priming of the Arabidopsis defense, which decreased the reproductive performance of one of the subsequent herbivores. The expression analysis of several defense-related genes demonstrated that the individual impact of mite and aphid feeding spread systematically, engaging the salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Interestingly, aphids feeding on the systemic leaf of the plant simultaneously attacked by mites, efficiently reduced the magnitude of the SA and JA activation, whereas mites feeding remotely increased the aphid-induced SA marker gene expression, while the JA-dependent response was completely abolished. We also indicated that the weaker performance of mites and aphids in double infestation essays might be attributed to aliphatic glucosinolates. Our report is the first to provide molecular data on signaling cross-talk when representatives of two distinct taxonomical classes within the phylum Arthropoda co-infest the same plant.

scholarly journals Deep fusion of contextual and object-based representations for delineation of multiple nuclear phenotypes

2019 ◽  
Vol 35 (22) ◽  
pp. 4860-4861
Author(s):  
Mina Khoshdeli ◽  
Garrett Winkelmaier ◽  
Bahram Parvin
Keyword(s):  
Contextual Information ◽  
Automated Analysis ◽  
Molecular Data ◽  
Supplementary Information ◽  
Genome Wide ◽  
Object Based ◽  
Code Base ◽  
Individual Signature ◽  
Biological Heterogeneity ◽  
The Individual

Abstract Motivation Nuclear delineation and phenotypic profiling are important steps in the automated analysis of histology sections. However, these are challenging problems due to (i) technical variations (e.g. fixation, staining) that originate as a result of sample preparation; (ii) biological heterogeneity (e.g. vesicular versus high chromatin phenotypes, nuclear atypia) and (iii) overlapping nuclei. This Application-Note couples contextual information about the cellular organization with the individual signature of nuclei to improve performance. As a result, routine delineation of nuclei in H&E stained histology sections is enabled for either computer-aided pathology or integration with genome-wide molecular data. Results The method has been evaluated on two independent datasets. One dataset originates from our lab and includes H&E stained sections of brain and breast samples. The second dataset is publicly available through IEEE with a focus on gland-based tissue architecture. We report an approximate AJI of 0.592 and an F1-score 0.93 on both datasets. Availability and implementation The code-base, modified dataset and results are publicly available. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Correlative microscopy and block-face imaging (CoMBI) method for both paraffin-embedded and frozen specimens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nobukazu Ishii ◽  
Yuki Tajika ◽  
Tohru Murakami ◽  
Josephine Galipon ◽  
Hiroyoshi Shirahata ◽  
...  
Keyword(s):  
Single Neuron ◽  
Bile Canaliculus ◽  
Face Image ◽  
Correlative Microscopy ◽  
Preparation Methods ◽  
Microscopic Images ◽  
3 Dimensional ◽  
Face Images ◽  
Block Face ◽  
New System

AbstractCorrelative microscopy and block-face imaging (CoMBI), a method that we previously developed, is characterized by the ability to correlate between serial block-face images as 3-dimensional (3D) datasets and sections as 2-dimensional (2D) microscopic images. CoMBI has been performed for the morphological analyses of various biological specimens, and its use is expanding. However, the conventional CoMBI system utilizes a cryostat, which limits its compatibility to only frozen blocks and the resolution of the block-face image. We developed a new CoMBI system that can be applied to not only frozen blocks but also paraffin blocks, and it has an improved magnification for block-face imaging. The new system, called CoMBI-S, comprises sliding-type sectioning devices and imaging devices, and it conducts block slicing and block-face imaging automatically. Sections can also be collected and processed for microscopy as required. We also developed sample preparation methods for improving the qualities of the block-face images and 3D rendered volumes. We successfully obtained correlative 3D datasets and 2D microscopic images of zebrafish, mice, and fruit flies, which were paraffin-embedded or frozen. In addition, the 3D datasets at the highest magnification could depict a single neuron and bile canaliculus.

scholarly journals Quantifying the individual impact of artificial barriers in freshwaters: A standardized and absolute genetic index of fragmentation

2019 ◽  
Author(s):  
Jérôme G. Prunier ◽  
Camille Poesy ◽  
Vincent Dubut ◽  
Charlotte Veyssière ◽  
Géraldine Loot ◽  
...  
Keyword(s):  
Genetic Differentiation ◽  
Molecular Data ◽  
Freshwater Biodiversity ◽  
Effective Population ◽  
Barrier Effects ◽  
Population Sizes ◽  
The Individual ◽  
Independent Assessment ◽  
Objective Tool ◽  
Genetic Index

AbstractFragmentation by artificial barriers is an important threat to freshwater biodiversity. Mitigating the negative aftermaths of fragmentation is of crucial importance, and it is now essential for environmental managers to benefit from a precise estimate of the individual impact of weirs and dams on river connectivity. Although the indirect monitoring of fragmentation using molecular data constitutes a promising approach, it is plagued with several constraints preventing a standardized and individual quantification of barrier effects. Indeed, observed levels of genetic differentiation depend on both the age of the obstacle and the effective size of the populations it separates, making difficult comparisons of the actual barrier effect of different obstacles. Here, we developed a standardized genetic index of fragmentation (FINDEX), allowing an absolute and independent assessment of the individual effects of obstacles on connectivity. The FINDEX is the standardized ratio (expressed as a percentage) between the observed genetic differentiation between pairs of populations located on either side of an obstacle and the genetic differentiation expected if this obstacle completely prevented gene flow. The expected genetic differentiation is calculated from simulations taking into account two nuisance parameters: the number of generations since barrier creation (the age of the obstacle) and the expected heterozygosity of the targeted populations, a proxy for effective population sizes. Using both simulated and published empirical datasets, we explored and discussed the validity and the limits of the FINDEX. We demonstrated that it allows quantifying genetic effects of fragmentation only from a few generations after barrier creation and provides valid comparisons among populations (or species) of different effective populations sizes and obstacles of different ages. The computation of the FINDEX requires a minimum amount of fieldwork and genotypic data, and solves some of the difficulties inherent to the study of artificial fragmentation in rivers and potentially in other ecosystems. This makes the FINDEX a promising and objective tool for managers aiming at at planning restoration programs and at evaluating the efficiency of these programs.

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