Dry Tissue Samples Research Articles

Effect of in vitro Incubation Time and Drying on Genomic DNA Extracted from Bovine Ear Tissue Samples

Background: Use of ear punch samples as a competent source of nucleic acids from bovines, gained momentum around two decades ago, as their collection is quite easy and can be transported with minimal quantities of preservatives at ambient conditions. However, the auricular cartilage makes them more resistant to lysis, resulting in poor genomic DNA yields. The objective of the current study was to investigate whether drying can enhance the gDNA yields from ear tissue samples as a few past experiments gave an unanticipated good gDNA yields from completely dried tissue samples. Methods: Upon receipt, ear tissue samples of the cattle and buffaloes were kept at room temperature for different time intervals (in vitro incubation) and then dried at various temperatures prior to gDNA extraction, with commercially available gDNA extraction kits from two manufacturers (MN and QIAGEN). The extracted gDNA samples were assessed with spectrophotometry, flourometry and agarose gel electrophoresis for concentration, purity and integrity. Result: gDNA yields were gradually increasing in parallel to in vitro incubation time till 250 days and after that there was a decline. Further, gDNA yields were significantly higher for ear tissue samples that were not dried prior to extraction, which did not comply with the past experimental observations. Fluorometry-based quantifications revealed that drying of ear tissue samples at greater than 50°C temperatures reduced double-stranded DNA, however up to 50°C the gDNA yield increased, indicating the tissue samples can be safely transported up to 50°C in the TSUs.

Yield performance, mineral profile, and nitrate content in a selection of seventeen microgreen species.

Originally regarded as garnish greens, microgreens are increasingly valued for their nutritional profile, including their mineral content. A study was conducted under controlled environmental conditions utilizing a selection of seventeen microgreen species belonging to seven different botanical families to investigate the genetic variation of macro- and micro-minerals and nitrate (NO3 -) content. Plants were grown in a soilless system using a natural fiber mat as the substrate. After germination, microgreens were fertigated with a modified half-strength Hoagland solution prepared using deionized water and without adding microelements. At harvest (10 to 19 days after sowing, based on the species), yield components were measured and dry tissue samples were analyzed for the concentration of total nitrogen (N), NO3 -, P, K, Ca, Mg, S, Na, Fe, Zn, Mn, Cu, and B. Genotypic variations were observed for all of the examined parameters. Nitrogen and K were the principal macronutrients accounting for 38.4% and 33.8% of the total macro-minerals concentration, respectively, followed in order by Ca, P, S, and Mg. Except for sunflower (Helianthus annuus L.), all the tested species accumulated high (1,000-2,500 mg kg-1 FW) or very high (>2,500 mg kg-1 FW) NO3 - levels. Eight of the studied species had a K concentration above 300 mg 100 g-1 FW and could be considered as a good dietary source of K. On the other hand, scallion (Allium fistulosum L.), red cabbage (Brassica oleracea L. var. capitata), amaranth (Amaranthus tricolor L.), and Genovese basil (Ocinum basilicum L.) microgreens were a good source of Ca. Among micro-minerals, the most abundant was Fe followed by Zn, Mn, B, and Cu. Sunflower, scallion, and shiso (Perilla frutescens (L.) Britton) were a good source of Cu. Moreover, sunflower was a good source of Zn, whereas none of the other species examined could be considered a good source of Fe and Zn, suggesting that supplementary fertilization may be required to biofortify microgreens with essential microminerals. In conclusion, the tested microgreens can be a good source of minerals showing a high potential to address different dietary needs; however, their yield potential and mineral profile are largely determined by the genotype.

Preliminary Data Regarding Total Chlorophylls, Carotenoids and Flavonoids Content in Flavoparmelia Caperata (L.) Hale Lichens Species

Flavoparmelia caperata L. Hale (common greenshield lichen) is an Ascomycete, foliose lichen usually growing on tree bark in most mesophytic-forested habitats in Romania. Lichen samples were collected from three stations in Romania (Craiova, Timișoara, Hoia-Baciu Forest, Cluj) and extracted in 96% ethanol. Both dry lichen tissue powder and extracts were analyzed by UV-Vis spectrophotometry for determining photosynthetic pigment concentrations (chlorophylls a and b and total carotenoids). Dry tissue samples had chlorophyll a concentrations of 95.12-109.59 mg/kg DW, chlorophyll b concentrations of 88.53-110.98 mg/kg and total carotenoids 88.89-102.85 mg/kg. Alcoholic extracts of fresh lichen tissue showed an extreme variability, with 715.97-10331.50 mg/kg chlorophyll a, 527.77-8124.20 mg/kg chlorophyll b, 1125.72-8714.90 mg/kg carotenoids pigments, with the highest values in samples from Craiova. A lower variability was observed in flavonoid contents, with 461.78-966.02 mg/kg DW in dry powder and 815.56-2734.135 mg/kg in ethanolic extracts. Extracts of lichens from Craiova had a decreased content of total flavonoids compared with the other two lichens ethanol extracts.

Association of wheat chaff derived silica fiber and esophageal cancer in north China

BackgroundDespite decades of research and intervention programs, the epidemic of esophageal squamous cell carcinoma (ESCC) in the Taihang Mountain area of north China has not seen convincing explanation by any risk factor yet and the incidence has not seen a substantial decrease. Based on recently disclosed association of aridity and wheat consumption with esophageal cancer, we revisited the hypothesis of biogenic silica in esophageal cancer development. MethodsFrom the archives of the Pathology Department of Heping Hospital, Changzhi Medical College, we selected three pairs of formalin-fixed samples, tumor tissues and distant normal tissues, of three patients operated for ESCC who had no history of workplace exposure to silica dust. Two pairs of dried tissue samples were used for phytolith (silica body) analysis and another pair for microanalysis with Transmission Electron Microscope (TEM). ResultsOne of the phytoliths in ESCC tumor tissue was similar to the prickle hair on the surface of wheat bract. In the mineral particles detected in the tumor tissue the predominant elements were Si, Ca, and P, whereas Si signals were not obvious in the distant normal tissue. ConclusionsThe preliminary findings on the detection of phytoliths and the higher than normal Si concentration in ESCC tumor tissue warrants further testing the role of biogenic silica in esophageal cancer.

Tissue distribution, excretion and effects on genotoxicity of tritium following oral administration to rats

Abstract Tritium is an important nuclide that must be monitored for radiation safety management. In this study, HTO was orally administered to rats at the level of 37 kBq (1 μCi) or 370 kBq (10 μCi) to examine tissue distribution and excretion levels. After sacrifice, wet and dry tissue samples were weighed and analyzed for tissue free-water tritium (TFWT) and organically bound tritium (OBT). The mean tissue concentrations of TFWT (OBT) were 30.9 (17.8) and 4.4 (8.1) Bq/g on days 7 and 13 at the 37 kBq level and 30.8 (64.6) Bq/g on day 17 at the 370 kBq level. To assess the cytogenetic damage due to tritium exposure, a cytokinesis-blocked micronucleus (MN) assay was performed in blood samples from rats exposed to HTO for 14 and 21 days after oral administration. There was no significant difference in the MN frequencies between the control and exposed rats.

Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit.Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at −20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%.Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier.

DETECTION OF STRAWBERRY VIRUSES IN EGYPT

As part of a USAID-MERC funded project, 'Disease-indexing and mass propagation of superior strawberry cultivars', an effort was made to evaluate the virus status of strawberries in Egypt. Diagnostic reverse transcription-polymerase chain reaction (RT-PCR) tests for Strawberry mottle virus, Strawberry crinkle virus, Strawberry vein banding virus (SVBV), Strawberry mild yellow edge virus (SMYEV), Strawberry chlorotic fleck virus, Strawberry necrotic shock virus, Strawberry latent ringspot virus, Apple mosaic virus, Fragaria chiloensis latent virus, Strawberry pallidosis associated virus (SPaV) and Beet pseudo yellows virus (BPYV) were developed and/or evaluated at the USDA-ARS laboratory in Corvallis, Oregon. Positive controls for the testing consisted of dried leaf samples of strawberries infected with each of the above viruses, shipped to Egypt and Israel under import permits. Collection of strawberry test samples was done in production fields (cultivars unknown) and from nuclear stock plants grown in Egypt ('Tamar' and 'Yael' originating from Israel). RNA extraction was carried out using Qiagen kits or a previously described protocol. Reverse transcription and polymerase chain reactions were carried as per manufacturer's recommendations. Amplicons were visualized after separation on an agarose gel and staining with ethidium bromide. The RT-PCR detection of viruses from RNA extracted from positive controls that were vacuum dried was successful for several but not all of the viruses used in this study. Thus, extracted RNA that can be shipped under ethanol may be a better positive control that dried tissue samples. Material carried back to the USA from Egypt as extracted RNA under ethanol was not impacted by storage at room temperature during transport. Initial testing of strawberry material from fields in Egypt showed that SMYEV, SVBV, BPYV and SPaV were detected in field samples. The plants tested from the nuclear stocks tested negative for each of the viruses tested.

Laboratory Study of Arsenic Accumulation and Toxicity in Eichhornia crassipes and Salvinia auriculata

The evaluation of the toxicity effects of arsenic (As) and the tolerance potential of Eichhornia crassipes and Salvinia auriculata regarding to this element, is an important step in the selection of plants for the purpose of phytoremediation. Both species were collected at clean water bodies, disinfected, acclimatized and submitted to treatments with As as sodium arsenate at the concentrations: 0, 0.5, 2.5 and 5.0 mg.L–1. After seven days of exposure to As, the plants were washed in a solution of HCl 0.1N. Shoot and root tissues were separated, dried and weighed. The As content analyses were carried out by acid digestion of dried tissue samples followed by measurement of total concentration of arsenic by Inductively-Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). The results indicate that S. auriculata accumulated more As, presenting marginal necroses in leaves, while, E. crassipes didn’t present any visible morphological alteration. The As absorption for both species increased substantially with the external concentration and S. auriculata was more sensitive. This effect was found either for roots and shoots in both species, highlight, the higher accumulation in roots. In the treatment of 5.0 mg.L–1 S. auriculata accumulated in roots an average of 146.66 μg As g–1 dry weight and E. crassipes accumulated 56.29 μg g–1 As dry weight. Probably, the highest As accumulation in this concentration in both species, was caused by the highest proportion of arsenic/phosphate in the solution, since the arsenate absorption competes with phosphate. It was observed, along the experiment, that the “parent” plants of S. auriculata, although with leaves damaged, produced “daughter” plants healthy. This probably tolerance mechanism can be related to some process that blocks the translocation of arsenic to “daughter” plants or can be a process of acclimatization to the pollution.

Extracellular Matrix Changes in Urethral Stricture Disease

Glycosaminoglycans (GAGs) and collagen are major components of the extracellular matrix and they have key roles in fibrotic diseases. Little is known about the molecular environment in urethral stricture and the majority of the studies available focused on collagen analysis. However, to our knowledge there are no data on GAG composition in urethral stricture disease. Bulbar urethral strictured segments were obtained from 10 patients 18 to 61 years old (mean age 41.8) who underwent end-to-end anastomotic urethroplasty. GAGs in dry tissue samples were extracted by papain digestion and cetylpyridinium chloride/ethanol precipitation. The concentration of total GAGs was assessed by hexuronic acid assay and expressed in microg. hexuronic acid per mg. dry tissue, while the proportion of sulfated GAGs was determined by agarose gel electrophoresis. The concentration of hyaluronic acid was determined by ion exchange chromatography and total tissue collagen was estimated as its hydroxyproline content. The control group consisted of 10 bulbar urethras obtained from fresh normal cadavers 22 to 53 years old (mean age 32.8). Mean total GAG concentration plus or minus standard deviation in the stricture group was 1.09 +/- 0.13, which was significantly lower than in controls (p <0.05). While the predominant GAG in normal bulbar urethras was hyaluronic acid, dermatan sulfate predominated in strictured urethras (mean 44.1% +/- 8.4 and 45.6% +/- 7.7%, respectively). Hyaluronic acid decreased 49.9% and dermatan sulfate increased 68.3%. There were no significant changes in the concentration of heparan sulfate or chondroitin sulfate in normal and strictured bulbar urethras. Mean total collagen significantly increased 32.3% (p <0.05). Composition changes in GAGs in strictured urethras could contribute to the noncompliant nature of urethral scar tissue and cause functional changes. These results may be useful for defining new targets for therapy for urethral stricture disease.

Determination of Silicone in Breast Tissue by Graphite Furnace Continuum Source Atomic Absorption Spectrometry

A method has been developed for the determination of silicone in breast tissue samples using a graphite furnace continuum source atomic absorption spectrometer. Silicone was measured as Si present in a heptane extract of the dried tissue samples. Thirty tissue samples were examined from 16 women having silicone gel-filled breast implants. Concentrations of Si in the dried tissue samples ranged from below the method detection limit of 3 ppm, to 6%.

Immunohistochemical antigen detection in dried tissue samples

Investigations were carried out on dried tissue samples for the identification of ABH antigens and human hemoglobin using the indirect immunoperoxidase technique (PAP). Samples from various organs were stored at room temperature over a period of 1 year and periodically examined immunohistochemically. By means of a rehydration medium, blood group and species identification were successfully demonstrated in the complete experimental series.

Multi-Elemental X-Ray Analysis of Rat Liver by Proton Excitation of Thick Samples in Air

Abstract Rat liver samples were irradiated in air by 4 MeV protons for elemental analysis by X-ray emission spectroscopy. The standard instrumental procedure was modified in our study, resulting in optimum detection limits being attained. The analytical system was accurately calibrated with standard biological reference materials, compressed into 2 mm thick disks. Ten macro and trace elements were quantitatively determined in the tissue specimens : eight essential elements (S, CI, K, Ca, Mn, Fe, Cu, and Zn) and two with undefined biological roles (Br and Kb). Detection limits in the dry tissue samples were estimated to be 0.1 ppm for Mn, Fe, Cu and Zn. The method was employed to examine the effects of a low level of phytate supplementation on the trace element status of the rat liver, since phytate feeding potentiated tumor yields when carcinogens were administered. The results are discussed in terms of current hypotheses.

The determination of selenium in biological material by thermal neutron activation analysis and atomic absorption spectrometry

Neutron activation analysis and atomic absorption spectrometry (graphite furnace) methods for the analysis of selenium in human tissue are described. The sensitivity (10–30 ng/sample), accuracy and precision are of the same order for both techniques and the choice can only be made on grounds of urgency or convenience. AAS should be chosen for the analysis of wet tissue or the urgent analysis of small numbers of dry tissue. NAA should be chosen for the analysis of large numbers of dry tissue samples where time is not important. The selenium concentration of human liver is shown to be in the region of 1 to 2 ppm (dry weight). Selenium may be lost from tissue during freeze drying if the samples are not maintained at −35°C.

Comparison of the elemental composition of normal and diseased human tissues by PIXE analysis

The proton beam from a 4 MV Van de Graaff accelerator has been used in conjunction with a Si(Li) detector system to perform elemental analysis of biological samples by proton-induced X-ray emision (PIXE). Dried tissue samples from a number of different organs were analyzed; including kidney, lung, liver, colon, breast, larynx, and aorta. For each organ, two specimens were taken of the same cellular type: normal tissue and diseased tissue. Therefore, a direct comparison of the effect of the disease state on the elemental composition could easily be obtained. For the aorta sets the disease state studied compared tissue from atherosclerotic lesions to normal aorta tissue while for the other organs, a sample of cancerous tissue was compared to a healthy tissue. The present results show a marked decrease in the Cd concentration as well as for the Se and Pb, when present, for the malignant kidney tissue compared to the normal one. It is found that the Zn/Cu ratio also decreases for all malignant kidney cases. Preliminary results indicate the same trend for this ratio for the colon and liver but an increase for the breast, larynx, and lung sets. Except for Br and Cu, the aorta sets show a general increase in all elements, particularly for Ca and Sr, when the lesion tissue is compared to the normal aorta tissue.