Anatomical Characteristics of African Cherry (Prunus Africana) Medicinal Plant for its Accurate Taxonomic Identification

Background: The genus Prunus (Family Rosaceae) comprises over 400 plant species and exhibits vast biodiversity worldwide. Due to its wide distribution, its taxonomic classification is important. Anatomical characters are conserved and stable and thus can be used as an important tool in plant taxonomic characterization. Thus, this study aimed at examining and documenting P. africana leaf, stem, and seed anatomy using micrographs and photographs for possible use in identification, quality control, and phylogenetic studies of the species.Methods: P. africana leaves, stems, and seeds were fixed, dehydrated in ascending ethanol series (50–100 %), embedded in Technovit resin, and sectioned using a microtome for mounting histological slides for anatomical observation under a microscope and subsequent description.Results: The anatomical sections of a young stem revealed a cortex consisting of isodiametric parenchyma cells, druse crystals, primary vascular bundles, and pith. The mature stem bark consisted majorly of rhytidome with periderm densely arranged in multiple layers, a cluster of stone cells, and sclerenchyma. The sections of the leaf were hypostomatic with stomata size ranging between 18.90– (22.34)–26.90 × 15.41– (18.40)–21.22 μm. The leaf sections showed the presence of characteristic druse crystals, vascular bundles, and mesophyll layers. The pericarp showed the presence of epicarp, mesocarp, and endocarp with a thickness of approximately 350–400, 300–350, and 30–50 μm, respectively and a seed testa with a thickness of approximately 50–60 μm. Conclusion: The characteristic morphological and anatomical features observed in P. africana leaves, stems, and seeds in this study could provide useful data in taxonomical identification of this species.

Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs

A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.

Visualization of Purkinje Neurons of Hypothyroid Brain under Golgi Cox Stain

Background: Golgi staining was invented hundreds of years ago but it is still a reliable method to study the anatomy of neurons.Objective: This experiment was conducted to study the structure of Purkinje neurons of cerebellum of neonate rats born from hypothyroid dams under golgi cox stain and compare it to the control group.Material and Methods: For this purpose 10 female wistar rats were divided equally into 2 groups control (A) and hypothyroid (B) groups and allowed to conceive. For inducing hypothyroidism in dams, Propylthiouracyl (PTU) was administered in a dose of 15mg/kg/day orally mixed with chow daily a week before mating and throughout the period of gestation and weaning uptil 22nd day after delivery. On the 23rd day, 10 neonatal rats from each group were sacrificed and blood samples were immediately collected for evaluating serum levels of T3, T4 and TSH. The freshly extracted brains were placed in Golgi Cox solution in complete darkness for 18 to 21 days and the solution waschanged every alternate day. The brains were then removed from this stain, processed, infiltrated with parafin, cut into 80ìm thick sections by a microtome and mounted on gelatin coated slides, which were now incubated in 20% ammonium hydroxide for ten minutes.They were then washed in distilled water for 2 minutes and passed through ascending ethanol series 70%, 95%, 100% (five minutes each) and two times xylene (10 minutes each). The sections were now coverslipped, visualized and photographed under a research microscope.Results: Serum enzyme analysis revealed that group PP pups had significantly increased serum levels of TSH and control CC pups showed normal levels of this hormone. T3 and T4 levels were not significantly altered. The number of dendrites seen in purkinje neurons were less in group PP as compared to CC. Normal Purkinje cell count in both the groups were not significantly variable, but the apoptotic cells were significantly more in PP group.Conclusion; Hypothyroidism decreases the number of dendrites ofpurkinje neurons in cerebellum, causing impaired neuronalconnectivity.

Identification of Active Compounds on Muntingia calabura L.Leaves using Different Polarity Solvents

Plant of Muntingia calabura L are often known as “kersen”, "seri or "cherry". Leaves of Muntingia calabura L. contains many benefits but its properties are still little known to the public. It contains secondary metabolites which have many uses. This study was aimed to determine the content of secondary metabolites in this leaf. Leaves extracts were obtained by maceration extraction for 3 times 24 hours using polar, semi-polar and non-polar solvents to determine the solubility of secondary metabolite compounds in each solvent. The solvents used were ethanol, ethyl acetate and n-hexane. The leaveswere dissolved a lot in polar solvents, marked by the formation of a dark green color in ethanol-series extracts, the color fades more in semi-polar and non-polar solvents. The three leaves extracts were tested for secondary metabolite contents by phytochemical screening tests. Phytochemical screening was an initial selection stage to detect classes of chemical compounds contained in plant. Phytochemical screening were included alkaloid, terpenoid, steroid, tannin, flavonoidand saponin tests. Based on the results of phytochemical screening tests, the leaf was contained several secondary metabolite compounds, namely flavonoids, saponins, steroids, terpenoids, alkaloids, phenols and tannins.

The difference of saline and sterile water for tetracycline hydrochloride solvents in cementum demineralization

Background: The root cementum demineralization is an important step in regenerative periodontal therapy to smear layer removal on the root surface. Smear layer on the root surface becomes a barrier of the new attachment between periodontal tissues with the root surface. The use of tetracycline capsules as root surface demineralizing agent cannot be applied directly on the root surface and solvents such as saline or sterile water are needed. Purpose: The aim of this study was to determine differences between sterile water and saline solvent for tetracycline HCl (tetra HCl) as a cementum demineralization. Method: In this study the specimens were divided into three groups: a control, tetra HCl dissolved in saline, and tetra HCl dissolved in sterile water. Application using burnishing method for 3 minutes. Samples were dehydrated with ethanol series of 30% to 100%. Results of the root demineralization observed by scanning electron microscopy (SEM). Statistical analysis was performed using the Kruskal-Wallis followed by a Mann-Whitney nonparametric test. Result: Upon statistical analysis showed that the sterile water as a solvent of tetra HCl is more effective in smear layer removal and collagen structure exposure in the cementum. Conclusion: Tetra HCl dissolved in sterile water was found to be the best root cementum demineralization agent.

Characterization of Magnesium Alloy Degradation in Whole Blood and Platelet Rich Plasma

Magnesium (Mg) is an attractive biomaterial due to its desirable biodegradable and mechanical properties. In this study, we compared the degradation behavior of Mg and a new Mg alloy incubated in both whole blood and platelet rich plasma (PRP) for two hours under standard cell culture conditions. To avoid settling of red blood cells, tubes with whole blood were under constant rotation during the incubation. Post-incubation solutions were collected, centrifuged, and analyzed for pH and Mg ion concentration. Mg and Mg alloy samples were fixed with a 3% glutaraldehyde solution, dehydrated using an ethanol series, critical point dried, sputter coated, and imaged with a field emission scanning electron microscope. Analysis of the post-incubation solutions showed PRP had greater concentrations of Mg ions and higher pH values when compared with whole blood. This indicated that the Mg and Mg alloy degraded faster when incubated in PRP than in whole blood. When comparing the surface of the materials after incubation with whole blood and PRP, the surfaces of Mg and Mg alloy that was incubated in PRP had larger cracks and grain boundaries than the samples incubated in whole blood. Additionally, more particulate microstructures were observed on the samples incubated in PRP as opposed to whole blood. Further studies are still needed to elucidate the differences in degradation of Mg alloys in whole blood and PRP.

Thrips atactus (Thysanoptera, Thripidae): first records of the male and host plant, intercepted in quarantine at Japan

Thrips atactus Bhatti was described from a single female taken without host data at Sibpore, West Bengal (Bhatti, 1967, 1980), and this has remained the only known specimen apart from some adults recorded by Palmer (1992) from Nepal. Thus no biological information is available about this species. In Japanese plant quarantine, this species has been intercepted several times on Eryngium foetidum [Umbelliferae] from Laos and Thailand, also on roses from Nepal. Moreover, a male adult was intercepted recently together with a female adult. The male adult is here described for the first time, and the host plant recorded. The specimens were intercepted on imported plants by plant quarantine inspectors at Narita airport and Chubu airport, Japan. Thereafter, all specimens were mounted into Canada balsam after dehydration through an ethanol series, and slide mounted for microscope study. Abbreviations as follows are used: CB=Chubu airport, CPS=campaniform sensorium, NR=Narita airport.

366 SEXING OF GOAT BLASTOCYSTS PRODUCED IN VITRO BY FISH USING CHROMOSOME X AND Y OVINE SPECIFIC PROBES

The aim of this study was to test a FISH approach using ovine-painted probes specific for the chromosomes X and Y, on goat interphase and metaphase nuclei of blastocyst cells. Oocytes of prepubertal goats were recovered at a slaughterhouse and selected by morphological criteria. Oocytes were matured in TCM-199 supplemented with hormones, serum and cysteine at 38.5°C for 27 h. IVM-oocytes were fertilized in vitro and the presumptive zygotes were cultured for 10 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. The blastocyst nuclei were spread using a modified Tarkowski method (1966). Briefly, individual embryos were immersed into hypotonic solution for 5 min, followed by fixative solution of methanol/acetic acid (Carnoy’s solution) until the embryos acquired a transparent appearance. Next, the embryos were transferred to a Superfrost plus Slide (Menzel Gläser, Braunschweig, Germany) in a small droplet mixture of distilled water and Carnoy The zona pellucida and the blastomere cytoplasm dissolved gradually and Carnoy solution was added dropwise to the slide before the nuclei dried out. The morphology and total number of nuclei in each embryo were analyzed under phase contrast microscope and stored at -18°C. Embryos with appropriate fixation were then subjected to hybridization with ovine-painted probes specific to chromosomes X (Fluorocrom green-FITC) and Y (Fluorocrom orange-TAMRA) (Chrombios-Molecular Cytogenetics GmbH, Raubling, Germany) according to the manufacturers protocol and adjusted for the caprine species. Briefly, slides were then incubated at 60°C for 1 h. The chromosomal DNA was then denatured by immersing slides in 70% formamide/30% 2 × SSC at 70°C for 1.5 min, and immediately dehydrated in an ethanol series (70%, 90%, and 100%), 4 min duration per solution and air dried. In parallel, X- and Y-probes were added to the hybridization solution (50% deionized formamide, 10% dextran sulfate, 2 × SSC) and denatured at 75°C for 10 min. Aliquots (0.5 to 1.5 μL) of this solution were placed on each slide and sealed with a coverslip and glue prior to incubation at 37°C (Hybrite; Vysis Inc, Dowers Grove, IL, USA). After 22 to 24 h the coverslip was removed and the slides were washed three times. The first and third washes were performed with 2 × SSC at room temperature while the second wash was in a 0.4 × SSC/0.1% Tween at 73°C for 3 min. Nuclear DNA was counterstained with diamino-phenyl-indole solution (DAPI) and examined with a fluorescence microscope (Olympus BX61, Olympus America Inc., Melville, NY, USA) equipped with appropriate filters. From a total of 69 blastocysts, 11 355 blastomeres were analyzed and 7,825 were correctly hybridized (68.9%). The results of the embryo sexing were: 24 embryos XX, 11 XY, 22 polyploid embryos (of which 13 presented more than 80% of cells XX), 3 haploid embryos (X0), 2 tetraploid embryos, and 5 no result. In summary, goat blastocysts were successfully sexed using FISH with painted ovine X- and Y-specific probes. Grant sponsor Spanish Ministery of Science and Innovation.Code: AGL2007-60227-CO2-01.

Ultrastructure and molecular cytogenetics of human metaphase II oocytes with granular vacuoles

Studies demonstrated poor fertilization rates and compromised early embryo development in cases of oocytes with dark and granular ooplasm, or with central granular ooplasm and presence of a refractile body. Many other structural abnormalities of human mature oocytes do exist, but remain to be evaluated. In the present study we characterized granular vacuoles (GVa) of mature metaphase II human oocytes. Surplus abnormal MII oocytes (n=6) with GVa were used under informed consent, after controlled superovulation during IVF treatment cycles. In all cases, patients had normal karyotypes. For electron microscopy oocytes were fixed with karnovsky fixative for 2h at 4°C, post-fixed in 1-2% OsO4 in buffer containing 0,8% K3Fe3+(CN)6, dehydrated in ethanol series, equilibrated with propylene oxide and embedded in Epon. Semithin sections were stained with aqueous azur II and methylene blue (1:1). Ultrathin sections were stained with 3% aquous uranyl acetate (20 min) and Reynols lead citrate (10 min). Sections were observed with a transmission electron microscope JEOL 100CXII operated at 60kV.

Plasma etching and ashing: a technique for demonstrating internal structures of helminths using scanning electron microscopy

Plasma etching and ashing for demonstrating the three-dimensional ultrastructure of the internal organs of helminths is described. Adult worms of the cestode Caryophyllaeides fennica were dehydrated through an ethanol series, critical point dried (Polaron E3000) and sputter coated with 60% gold-palladium (Polaron E5100) and glued to a standard scanning electron microscope (SEM) stub positioned as required for ashing. After initial SEM viewing of worm surfaces for orientation, stubs were placed individually in the reactor chamber of a PT7150 plasma etching and ashing machine. Worms were exposed to a radio frequency (RF) potential in a low pressure (0.2 mbar) oxygen atmosphere at room temperature. The oxidation process was controlled by varying the times of exposure to the RF potential between 2 to 30 min, depending on the depth of surface tissue to be removed to expose target organs or tissues. After each exposure the oxidized layer was blown from the surface with compressed air, the specimen sputter-coated, and viewed by SEM. The procedure was repeated as necessary, to progressively expose successive layers. Fine details of organs, cells within, and cell contents were revealed. Ashing has the advantage of providing three dimensional images of the arrangement of organs that are impossible to visualize by any other procedure, for example facilitating testes counts in cestodes. Both freshly-fixed and long-term stored helminths can be ashed. Ashing times to obtain the desired results were determined by trial so that some duplicate material was needed.