Microtome Sections Research Articles

Targeted 2D histology and ultrastructural bone analysis based on 3D microCT anatomical locations

Histological processing of mineralised tissue (e.g. bone) allows examining the anatomy of cells and tissues as well as the material properties of the tissue. However, resin-embedding offers limited control over the specimen position for cutting. Moreover, specific anatomical planes (coronal, sagittal) or defined landmarks are often missed with standard microtome sectioning. Here we describe a method to precisely locate a specific anatomical 2D plane or any anatomical feature of interest (e.g. bone lesions, newly formed bone, etc.) using 3D micro computed tomography (microCT), and to expose it using controlled-angle microtome cutting. The resulting sections and corresponding specimen's block surface offer correlative information of the same anatomical location, which can then be analysed using multiscale imaging. Moreover, this method can be combined with immunohistochemistry (IHC) to further identify any component of the bone microenvironment (cells, extracellular matrix, proteins, etc.) and guide subsequent in-depth analysis. Overall, this method allows to:•Cut your specimens in a consistent position and precise manner using microCT-based controlled-angle microtome sectioning.•Locate and expose a specific anatomical plane (coronal, sagittal plane) or any other anatomical landmarks of interest based on microCT.•Identify any cell or tissue markers based on IHC to guide further in-depth examination of those regions of interest.

4D-Printed Transformable Tube Array for High-Throughput 3D Cell Culture and Histology.

3D cell cultures are rapidly emerging as a promising tool to model various human physiologies and pathologies by closely recapitulating key characteristics and functions of in vivo microenvironment. While high-throughput 3D culture is readily available using multi-well plates, assessing the internal microstructure of 3D cell cultures still remains extremely slow because of the manual, laborious, and time-consuming histological procedures. Here, a 4D-printed transformable tube array (TTA) using a shape-memory polymer that enables massively parallel histological analysis of 3D cultures is presented. The interconnected TTA can be programmed to be expanded by 3.6 times of its printed dimension to match the size of a multi-well plate, with the ability to restore its original dimension for transferring all cultures to a histology cassette in order. Being compatible with microtome sectioning, the TTA allows for parallel histology processing for the entire samples cultured in a multi-well plate. The test result with human neural progenitor cell spheroids suggests a remarkable reduction in histology processing time by an order of magnitude. High-throughput analysis of 3D cultures enabled by this TTA has great potential to further accelerate innovations in various 3D culture applications such as high-throughput/content screening, drug discovery, disease modeling, and personalized medicine.

Rapid Processing and Drug Evaluation in Glioblastoma Patient-Derived Organoid Models with 4D Bioprinted Arrays

SummaryGlioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.

Comparative leaf and wood anatomical characteristics of Chrysophyllum (Sapotaceae) relate to taxonomy of the species in Thailand

Abstract. Prasawang S, Srinual A. 2020. Comparative leaf and wood anatomical characteristics of Chrysophyllum (Sapotaceae) relate to taxonomy of the species in Thailand. Biodiversitas 21: 1578-1587. Anatomical attributes are important tools for taxonomic studies of plants. The present study compared anatomical characteristics (i.e. lamina, petiole and wood) of two species of Chrysophyllum (namely Chrysophyllum cainito L. and C. roxburghii G.Don) belonging to family Sapotaceae in Thailand to develop taxonomic identification keys for two species of Chrysophyllum. Peeling and clearing methods were used for epidermal study, paraffin method for lamina and petiole transverse sections and standard microtome sectioning for anatomical analyses of wood. The findings of this study demonstrated the leaf and wood anatomical features presenting in C. cainito differs from those in the species of C. roxburghii including 1) shape and outline of epidermal cell wall 2) presence or absence of T-shaped trichome 3) presence or absence of inclusions 4) shape of vascular bundle and accessory bundle in midrib 5) shape of petiole and vascular bundle 6) grouping type of vessel 7) type of axial parenchyma 8) presence or absence of inclusions in rays and 9) thickness of fiber walls. Therefore, these characteristics can be used valuably as additional data in case of taxonomic studies of Thai Chrysophyllum.

Morphometric analysis of structural arrangement of the testial arteries in postresectional arterial pulmonary hypertension

Introduction. Removal of the right or left lung can lead to postresection pulmonary arterial hypertension, venous stasis in the organs of the great circle of blood circulation and structural and functional changes in them. Morphological changes in endothelial cells of arteries of testes in postresection pulmonary arterial hypertension are insufficiently studied.Objective of the research: to study features of structural reorganization of testicular arteries at postresection pulmonary arterial hypertension.Materials of the research and their discussion. Morphological methods were used to study the structural rearrangement of the testicular arteries of 40 rats, which were divided into 3 groups. The 1st group included 11 intact animals, the 2nd - 19 rats with arterial hypertension in the small circulation and compensated pulmonary heart, the 3rd - 10 experimental animals with pulmonary arterial hypertension and decompensated pulmonary heart. Postresection pulmonary arterial hypertension and pulmonary heart were modeled by performing right pulmonectomy in rats. One month after the start of the experiment, rats were euthanized by bloodletting under conditions of thiopental anesthesia. A separate weighing of the chambers of the heart, their planimetry. Pieces were cut from the testicles, which were fixed in 10% formalin solution, passed through ethyl alcohols of increasing concentration and placed in paraffin. Microtome sections after dewaxing were stained with different dyes. In the left and right testes, the outer and inner diameters of small-caliber arteries, media thickness, Kernogan index, endothelial cell height, diameter of their nuclei, nuclear-cytoplasmic ratios in endotheliocytes, and relative volume of damaged cells were measured. Quantitative indicators were processed statistically.Results of the research and their discussion. Morphometrically, it was found that with decompensation of the postresection pulmonary heart, the lumen of the small-caliber arteries of the left testicle decreased by 12.6 %. the Kernogan index - by 11.7 %, and the right - by 9.5 and 9.9 %, respectively. The relative volume of damaged endothelial cells of the studied vessels in right ventricular failure increased 11.6 times, and the right - 9.3 times.Histological examination of the testicles micropreparations of experimental animals after right-sided pulmonectomy showed severe vascular disorders (plethora of mostly venous vessels of the microhemocirculatory tract, stasis, thrombosis, diapedetic perivasal hemorrhage), stromal and perivasal edema, noncystic edema, dystrophy, dystrophy.Conclusions. It is established that in the conditions of the simulated pathology the wall thickens markedly, the lumen of the studied arteries narrows, endotheliocytes are damaged, structural cellular homeostasis is disturbed, which is complicated by their dysfunction, deterioration of blood supply, hypoxia, dystrophic, necrobiotic lesions. Structural changes in the arteries predominated in the left testis and in pulmonary heart decompensation.

Floral anatomy of Tristerix longebracteatus (Loranthaceae)

Introduction: Most of the New World members of the Loranthaceae comprise a clade that corresponds to the tribe Psittacantheae. Previous studies on floral anatomy and development in this tribe have concentrated on the highly diversified subtribe Psittacanthinae, while the smaller subtribe Ligarineae has received less attention. A detailed anatomical description of Tristerix longebracteatus helps to fill this information gap. Objetive: The present research analyzes the anatomy of Tristerix longebracteatus flowers, detailing the structure of androecium and gynoecium, including megasporogenesis and microsporogenesis. Methodology: Anatomical serial sections of flowers at different stages of development were prepared, following processing with fixation techniques, incorporation in paraffin, microtome sectioning and staining with Astra-blue and basic fuchsin. Results: The large-sized flowers of Tristerix longebracteatus present a complex pattern of vascularization with 18-20 vascular bundles at the base of the inferior ovary. A group of three vascular bundles irrigate the 4-5 petals and associated stamens, and ten bundles continue through the gynoecium. The androecium is composed of four or five anthers with simultaneous microsporogenesis. The gynoecium as a single ovarian cavity with a central mamelon in which the archesporial tissue is oriented towards the style. The base of the style forms a nectary similar to that found in the sister genus Ligaria. Conclusions: The gynoecium with a single ovarian cavity and central mamelon is a condition shared by Tristerix (subtribe Ligarinae) and all the genera of the subtribe Psittacanthinae, except Tripodanthus. The base of the style forms a nectary similar to that found in the sister genus Ligaria. This type of stylar nectary is of taxonomic value for grouping species of the subtribe Ligarinae and difers from the annular nectary of subtribe Psittacanthinae.

Roots Structure and Development of Austrobaileya scandens (Austrobaileyaceae) and Implications for Their Evolution in Angiosperms

Since the resolution of the ANA grade [Amborellales, Nymphaeales, Austrobaileyales] as sister to all other flowering plants, a few comparative studies of root structure have suggested that some of their anatomical traits could be of importance to understanding root evolutionary development and angiosperm phylogeny. However, there is still a paucity of information on root structure and apical meristems (RAMs) in these lineages and especially the sister to all other Austrobaileyales, Austrobaileya scandens. We used microtome sections and bright field, epifluorescence, laser confocal, and scanning electron microscopy to study adventitious root RAMs and tissues of A. scandens. Our results indicate that root structure is relatively simple in A. scandens. The epidermis has a thick cuticle and lacks root hairs. The stele is typically diarch, or some modification thereof, and surrounded by a cortex differentiated into a uniseriate endodermis, a middle region sometimes packed with starch, some oil cells, and colonized by arbuscular mycorrhizal fungi, and a multiseriate exodermis. Secondary growth produced many vessel elements in the secondary xylem and scattered sclerenchymatous fibers in secondary phloem. The absence of distinct patterning within the RAM and between the RAM and derivative differentiating tissues shows that the RAM is open and characterized by common initials. Roots structure and anatomy of A. scandens are thus essentially similar to some previously described in Amborella or Illicium in the ANA grade and many magnoliids, and suggest that the first woody flowering plants likely had an open RAM with common initials. Their functional and evolutionary significance in woody early-diverging and basal lineages of flowering plants and gymnosperms remains unclear, but they are clearly ancestral traits.

Synthesis, characterization and optimization of in vitro properties of NIR-fluorescent cyclic α-MSH peptides for melanoma imaging.

Melanoma are malignant tumors derived from melanocytes being responsible for the majority of skin cancer deaths with an increasing rate of incidence. The Melanocortin-1 receptor (MC1R) has been recognized as a molecular target for melanoma detection. Here, we report on the development and optimization of molecular probes which are based on novel conjugates of near-infrared (NIR) fluorescent indocyanine dyes and an MC1R-targeting peptide intended for optical fluorescence imaging enabling an early, specific, accurate and sensitive diagnosis of malignant melanomas. The introduction of anionic groups into the aromatic ring of the indolenine substructure of the conjugated dyes has shown to result in a strong fluorescence in aqueous solution and a concomitant increase of binding affinities of the peptide conjugates to the target receptor. The length and flexibility of the PEG chain introduced as a linker, as well as the nature of its attachment to the dye also affect the binding affinities, albeit to a lower extent. The conjugates have been successfully applied in the MC1R-specific staining of B16F10 melanoma cells, both in cell cultures and in microtome sections of solid tumors.

Multiple shoot regeneration, establishment of callus and cell suspension cultures in Salacia macrosperma Wight

Salacia macrosperma Wight. - a potent medicinal plant facing the verge of rare and endemic status to the Western Ghat of southern India. Effective protocol has been established for induction of callus and multiple shoot regeneration using leaf and nodal explants on MS medium fortified with various plant growth regulators like 2,4-dichlorophenoxyacetic acid (2,4-D), benzyl amino purine (BAP), thidiazuron (TDZ), Indole acetic acid (IAA), Kinetin (Kn), Naphthalene acetic acid (NAA) and Indole butyric acid (IBA). Leaf explants produced more calli (98.33%) than nodal explants at 2.5 + 1.5 mg L-1 of 2, 4-D and BAP in combination. MS medium with 1.5 mg L-1 of 2, 4-D, 2.0 mg L-1 of BAP and 1.5 mg L-1 of TDZ along with 1% activated charcoal was apt for multiple shoot regenerations (93.33%) from nodal explants with embryogenic callus. Rhizogenesis was achieved in liquid MS medium supplemented with 1.0 mg L-1 of IAA. Friable callus when agitated in liquid MS medium at 100 rpm embryogenic callus followed by morphological changes were observed. Cytogenetic variations such as multinucleate, multinucleolate, cytodifferentiation, chromosomal bridges were noticed, besides normal dividing stages. Anatomical features were observed by microtome sections of the embryogenic callus revealed the presence of active centers, embryoids, and shoot bud formation. Further, by scanning electron micrograph (SEM) analysis of embryogenic callus different stages of morphogenic developmental features were recorded.

Nonlinear microscopy for detection of prostate cancer: analysis of sensitivity and specificity in radical prostatectomies.

Intraoperative evaluation of specimens during radical prostatectomy using frozen sections can be time and labor intensive. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can rapidly generate images that closely resemble H&E histology in freshly excised tissue, without requiring freezing or microtome sectioning. Specimens are stained with nuclear and cytoplasmic/stromal fluorophores, and nonlinear microscopy evaluation can begin within 3 minutes of grossing. Fluorescence signals can be displayed using an H&E color scale, facilitating pathologist interpretation. This study evaluates the accuracy of prostate cancer detection in blinded reading of nonlinear microscopy images compared to the gold-standard of formalin fixed, paraffin embedded H&E histology.A total of 122 freshly excised prostate specimens were obtained from 40 patients undergoing radical prostatectomy. The prostates were grossed, dissected into specimens of ~10×10 mm with 1–4 mm thickness, stained for 2 minutes for nuclear and cytoplasmic/stromal contrast, and then rinsed with saline for 30 seconds. Nonlinear microscopy images were acquired and multiple images were stitched together to generate large field of view, centimeter-scale digital images suitable for reading. Specimens were then processed for standard paraffin H&E. The study protocol consisted of training, pre-testing, and blinded reading phases. After a washout period, pathologists read corresponding paraffin H&E slides.Three pathologists achieved a 95% or greater sensitivity with 100% specificity for detecting cancer on nonlinear microscopy compared to paraffin H&E. Pooled sensitivity and specificity was 97.3% (93.7%−99.1%; 95% confidence interval) and 100.0% (97%−100%), respectively. Interobserver agreement for nonlinear microscopy reading had a Fleiss κ=0.95. The high cancer detection accuracy and rapid specimen preparation suggest that nonlinear microscopy may be useful for intraoperative evaluation in radical prostatectomy.

Production of high-quality extremely-thin histological sections by ultrasonically assisted cutting

Modern-day histology of biological tissues requires precision cutting of a wide variety of tissue samples for histological analyses. Lots of common problems can be identified at the conventional microtome sectioning including creation of curling sections and sections stick to the blade, which made high-quality sections hard to obtain. This paper deals with the development of next generation of microtomes employing introduction of a controlled ultrasonic vibration to process biological tissues. Based on a combination of advanced experimental and numerical studies of a novel cutting system with multi-body dynamics, this study investigated effects of cutting parameters and characteristics of ultrasonic excitation with the aim to design and manufacture an ultrasonically assisted cutting device (UACD) for microtomy. The cutting mechanism was detailed to show the advantages of the ultrasonically assisted cutting in the creation of high quality, thin sections. The novel prototype was designed and developed to conduct conventional cutting (CC) and ultrasonically assisted cutting (UAC) of biological tissues embedded in wax. Cutting forces, blade wear, blade damage and section quality for these cutting processes were assessed. It was found that the efficiency and quality of cutting were dependent on the level of cutting forces, which were lower in UAC compared with CC. The quality of cut samples with a thickness of 4 μm was better in UAC than CC. The developed ultrasonically assisted cutting device also enables successfully sectioning of the thin biological samples with high precision, reduced blade wear and less blade damage. This will increase the blade life making both environmental and economic impacts.

High Resolution Nanoscale Probing of Bacteriophages in an Inhalable Dry Powder Formulation for Pulmonary Infections.

Use of powder phage formulations for the treatment of multiple-drug-resistant pulmonary infections is gaining attention. To achieve therapeutic benefits, it is critical for phages to remain stable in the formulation. Assessment of phage stability relies on plaque assay (bioactivity), which requires powder samples to be reconstituted in liquid. The purpose of this study was to develop an innovative approach using photothermal-induced resonance-enhanced atomic force microscopy infrared spectroscopy (AFM-IR) to assess the presence of phages and investigate their protein conformation in the solid state. Staphylococcal phage S83 was spray-dried with lactose and sodium stearate using spray-drying. The phage powder recrystallized at 60% relative humidity (RH), so it was stored and handled below this RH. For the AFM-IR measurements, spray-dried Staphylococcal phage Sa83 powder was embedded in resin, followed by microtome sectioning. AFM-IR spectra collected from different regions within the microtomed sections revealed the presence of phage proteins with amide I and amide II bands at 1640 and 1550 cm-1, respectively. The phages were confirmed to be stable, as the plaque assay showed negligible titer reduction after spray-drying. Our results thus demonstrated the utility of AFM-IR for characterization of nanosized phages present in extremely low quantity in spray-dried particles. These biologically active phages were shown to retain their physical and chemical integrity in the spray-dried particles.

Histopathological factors affecting the extraction of high quality genomic DNA from tissue sections for next-generation sequencing.

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR: 4.30, 95% CI: 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR: 0.23, 95% CI: 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR: 4.43, 95% CI: 1.87-10.49; and P=0.0003, OR: 5.51, 95% CI: 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of <3 years allows the extraction of genomic DNA suitable for NGS.

Phylogenetic relationships of Coreanomecon (Papaveraceae: Chelidonioideae) inferred from seed morphology and nrITS sequence data

The phylogenetic position of the Coreanomecon, a monotypic genus endemic to Korea, has been controversial for a long time. Coreanomecon has variously been placed in its own genus or combined with Hylomecon or Chelidonium. The main purpose of this study was to examine the phylogenetic position of Coreanomecon in relation to genera of the subfamily Chelidonioideae using seed morphology and molecular data. The seed morphology of 10 genera of Chelidonioideae was examined using microtome sections and scanning electron microscopy. The shape and size of exotestal cells varied between genera. The exotestal cells were tangentially elongated in Chelidonium majus, Stylophorum diphyllum and Hylomecon vernalis, whereas the cells were sub‐orbicular and broad in Coreanomecon hylomeconoides. The endotesta was represented by thick palisade‐like cells that contain small rectangular crystals near their outer walls. The mesotesta was totally collapsed in Co. hylomeconoides but wholly or partly persistent in Ch. majus and H. vernalis. The seed surface of Co. hylomeconoides was well ornamented with a unique echinate seed surface, whereas other genera showed plain and reticulate seed surfaces. A phylogenetic analysis of the genera including Coreanomecon was conducted with nuclear ribosomal internal transcribed spacer (nrITS) sequences using genetic distance, maximum parsimony, maximum likelihood and Bayesian inference methods. The results confirm that Co. hylomeconoides is separated from both Hylomecon species and is a sister group to Chelidonium majus and Stylophorum diphyllum with robust bootstrap support. In addition to differences in the absence of cauline leaves and rhizomes, the presence of a hairy stem and leaves, and 12‐pericolpate pollen, seed characters and molecular data strongly support the recognition of Coreanomecon as an independent genus, distinct from Hylomecon and Chelidonium.

Characterization of mechano-microenvironment of acral melanoma and its potential impact on tumor cell invasion.

e21062 Background: Acral melanoma is the main subtype in Asia, and tends to be insensitive to conventional and novel therapies compared with cutaneous melanomas. It has been proved that biomechanical alterations inside the tumor microenvironment significantly affect tumor progression in breast, pancreatic and other cancers(Hepatology. 2016 July ; 64(1): 261–275; Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXXI) . Hence, we hypothesized that the biomechanical microenvironment plays critical roles in proliferation and invasion in acral melanoma. In this study, the main purpose is to observe whether melanoma cells have the same ability called “durotaxis” as other tumors, which to respond to a gradient of extracellular stiffness and migrate in a directed fashion. Methods: In this study, the stiffness was compared between the acral melanoma tissue and the normal tissue by using the atomic force microscope (AFM) with fresh samples and the freezing microtome sectioning specimen (J. Matthew Barnes et al., Nature Cell Biology 2018.) . A stiffness gradient polyacrylamide hydrogel system was constructed to observe how the acral melanoma cells response. Using automated tracking (Curr Protoc Cell Biol. ; 76: 12.12.1–12.12.16.) of positional data for large sample size of single migrating cells to systematically analyze polarized melanoma cell migration in response to stiffness gradient. Results: The results showed that 1) all the acral melanoma cell lines tested displayed strong anti-durotactic migratory response, which was in accordance with the AFM measurement that the melanoma microenvironment gradually softened; 2) Acral melanoma cells tend to move toward softer areas on gradient gels, which is contrary to the migration behavior of most other tumor cells. Conclusions: In this study, we presented the different migratory pattern of acral melanoma cells. It may illustrate the invasion mechanism for aral melanoma cells. It could also herald that extracellular matrix may be a potential therapeutic target in acral melanomas.

NanoSIMS reveals unusual enrichment of acetate and propionate by an anammox consortium dominated by Jettenia asiatica

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite into N2 in a chemolithoautotrophic way, meaning that they utilize CO2/HCO3 solely as their carbon sources. Such autotrophic behavior limits their competitiveness with heterotrophic microorganisms in both natural environments and engineered systems. Recently, environmental metagenomic results have indicated the capability of anammox bacteria to metabolize short-chain fatty acids, further confirmed by limited experimental evidence based on highly enriched cultures. However, clear evidence is difficult to get because of the limits of traditional methodologies which rely on the availability of a pure anammox culture. In this study, we identified and quantified the uptake of acetate and propionate, on a single-cell level, by an anammox consortium that was dominated by Candidatus Jettenia asiatica (relative abundance of 96%). The consortium, growing in granular form with an average relative abundance of anammox bacteria of 96.0%, was firstly incubated in a13C-labelled acetate or propionate medium; then microtome sections were scanned by a nanometer-scale secondary ion mass spectrometer (NanoSIMS). The NanoSIMS scannings revealed that the consortium enriched acetate and propionate at a >10 times higher efficiency than bicarbonate incorporation. Our results also suggest that acetate or propionate was likely not assimilated by J. asiatica directly, but firstly oxidized to CO2, which then served as carbon sources for the follow-up autotrophy in J. asiatica cells. Furthermore, more [15N]ammonium was enriched by the propionate-fed consortium than the acetate-fed consortium despite that exactly the same amount of 13C atoms were supplied. Our study strongly indicates an alternative lifestyle, namely organotrophy, in addition to chemolithoautotrophy of anammox bacteria, making it more versatile than often expected. It suggests that the niche of anammox bacteria in both natural and engineered ecosystems can be much broader than usual assumed. Recognising this is important for their role in wastewater treatment and the global nitrogen turn-over rates.

Abstract TP333: Motor Recovery After Transient Middle Cerebral Artery Occlusion May be Impacted by the Disruption of Nucleus Basalis-Cortical Cholinergic Pathways

Stroke survivors often have chronic motor deficits that significantly impact their quality of life. The most common vessel affected by ischemic events is the middle cerebral artery. These strokes commonly affect the basal ganglia and internal capsule in humans and in rodent models of stroke. Our lab studies the neurotransmitter acetylcholine for its potential to drive cortical plasticity that may support recovery after cortical spinal tract damage. Cholinergic cells in the nucleus basalis (NB), a major cholinergic nucleus with axonal projections throughout the cortex, can drive the beneficial motor map reorganization induced by vagus nerve stimulation after stroke in rats (Kilgard et al., 2016). Our lab has previously shown that selective injury to NB cholinergic cells reduces motor recovery (Becker et al., Soc. for Neuroscience abstract. 800.09, 2014.). In the current experiments, we mapped NB projection pathways in transgenic mice expressing fluorescent proteins in cholinergic neurons. We crossed mice expressing Cre driven by the choline acetyltransferase (ChAT) promoter with mice expressing channelrhodopsin and EYFP with a floxed stop cassette driven by the CAG promoter, both obtained from Jackson Laboratories. Fixed brains were imaged with high-throughput fluorescence microscopy using either whole slide imaging of freezing microtome sections (Nanozoomer, Hamamatsu) or serial two-photon tomography of whole brains (TissueCyte 1000, TissueVision). Cholinergic cell bodies in the NB and their proximal processes were visually tracked from the basal forebrain to the cortex. We found that some NB cholinergic projections to motor cortex were immediately adjacent to the globus pallidus interna, which is typically injured by transient middle cerebral artery occlusion. If the axons of NB cholinergic cells that are projecting to the cortex are damaged by stroke, this pathway may impact the ability for stroke patients to recover motor functions by disrupting central motor learning pathways. Understanding neuromodulatory circuits involved in cortical plasticity and motor learning after stroke is essential for developing new therapeutic methods.

Changes in Anatomical Features and Protein Pattern of Sunflower Partially Resistant and Susceptible Lines During Infection By Virulence Factors of Sclerotinia Sclerotiorum

Helianthus annuus L. as an oil seed crop is widely grown throughout the world. One of the most destructive diseases of sunflower is stem rot caused by Sclerotinia sclerotiorum. Oxalic acid is the major virulence factor of this necrotrophic pathogen. It is important to further investigate plant responses to this non-specific toxin. The basal stems of sunflower partially resistant and susceptible lines were treated with 40 mM OA (pH 3.7) of fungus culture filtrate and samples were collected at 24, 48 and 72 hours post treatment. SDS-PAGE protein pattern and total soluble protein were determined in both lines after treatment. In SDS-PAGE protein pattern new protein bands appeared in both lines after treatment. These observations suggest induction of stress-related proteins upon culture filtrate treatment. The identities of these new proteins need to be more clarify in future investigations. The changes in xylem morphology and degree of lignification of both lines was studied by light microscopy and microtome sectioning techniques after treatment with S. sclerotiorum culture filtrate. Anatomical investigations revealed changes in xylem diameter and xylem lignification of treated lines at various time points. More lignin deposition in xylem vessels of partially resistant line has been observed after treatment. In addition, the size of xylem vessels in partially resistant line has been sharply decreased upon pathogen filtrate treatment. The results of this study will help us gain a more complete understanding of resistance mechanisms to this cosmopolitan and devastating pathogen.

Histological comparison between laser microtome sections and ground specimens of implant-containing tissues

Evaluation of bone regeneration and peri-implant bone apposition can only be accomplished using laboratory techniques that allow assessment of decalcified hard tissue. It is known that 5–15μm thick sections can be prepared with the cutting-grinding technique, but their production causes a high material loss (≥0.5mm) between two sections and requires years of training and experience. With the development of the laser microtome it has become possible to cut decalcified bone without high sample material loss. Many scientific publications deal with the application possibilities of the individual methods So far, there is no comparison work between the cutting-grinding technique and laser microtomy. For this reason, new tissue sections were prepared by laser microtome and analyzed histologically from samples that had been previously been prepared by the cutting-grinding technique. Using both methods, it could be demonstrated that the different implants were completely surrounded by a connective tissue layer. In sections (50–100μm) produced by the routine cutting-grinding technique, magnifications up to 20× revealed no detailed histological information because cell structures could not be clearly identified. By contrast, laser microtome sections (10μm) revealed these information as e.g. osteocytes are already clearly visible at 10× magnification. Furthermore, the interface between implant and the surrounding bone could be clearly demonstrated due to visible demarcation between a capsule and connective tissue. At the histological level, laser microtome sections were clearly superior at thicknesses ≥30μm compared to sections produced by the cutting-grinding technique. In addition, laser microtomy has the advantages of time saving and markedly reduced sample loss, especially in cases of the production of serial sections.

Fruit Structure of Calycanthaceae (Laurales): Histology and Development

Premise of research. Fruit structure, in contrast to flower structure, has not been the subject of detailed comparative studies in many angiosperm lineages. This study focuses on fruit morphology, anatomy, and histology of the family Calycanthaceae (Laurales), including all genera: Calycanthus (including Sinocalycanthus), Chimonanthus, and Idiospermum. The results are compared with the fruit structures of all other families of Laurales.Methodology. We use standard methods of histological research, microtome sectioning, light microscopy, and scanning electron microscopy. Histology is studied in young, midstage, and mature fruits.Pivotal results. The fruiting carpels in Calycanthaceae are covered by the floral cup (receptacle) but not fused with it. In Calycanthus and Chimonanthus, the fruitlets are drupes of the Laurus type with the stone consisting of only the endocarp (one cell layer). In Idiospermum, the fruiting carpel is technically a berry. Development shows that fruitlets of Calycanthoideae have a thin mesocarp and a lignified palisade endocarp, whereas in Idiospermoideae there is a multicell-layered mesocarp with histological subzones and a parenchymatous endocarp.Conclusions. The sclerenchymatous palisade endocarp, and the mode of its development, is similar in drupes of the Laurus type of Calycanthoideae, Hernandiaceae, and Lauraceae. In contrast, the structure of the parenchymatous pericarp, which may be partly obliterated in fruiting carpels in berries of Idiospermoideae, is similar to that in some Atherospermataceae and Monimiaceae with a berry fruit type.