Unwashed Specimens Research Articles

Element Cycling at Thermally Active Coal-Waste Dumps: A Case Study of Calamagrostis epigejos and Solidago canadensis

Coal-waste dumps in the Upper Silesian Coal Basin are usually colonized by tall grass Calamagrostis epigejos and Solidago canadensis, which influence the direction of vegetation formation and the soil chemistry. The aim of this study is to analyze and determine the content of major elements (Fe, Ca, P, Mg, Al, Na, K, S) and trace elements (Mo, Cu, Pb, Zn, Ni, Co, Mn, Sr, Cd, Cr) in aboveground and underground parts of the plants and the soil at the thermally active coal-waste dump. Analysis of the heavy metal concentrations reveals that they are higher in plant materials than in soil materials within the root zone of the plants. Environmental indicator analysis (geoaccumulation index, enrichment factor, translocation factor) shows that the studied species exhibit varying degrees of pollution, with cadmium and zinc showing the highest accumulation rates. The content of elements in the analyzed species, both in washed and unwashed specimens, does not show significant differences, which is confirmed by the enrichment factor. Statistical analysis shows a positive correlation between the amount of microelements in plants (roots, aerial part) and soil samples in both thermally active and inactive zones. These findings broaden the scientific inquiry and hold practical significance for the reclamation of post-industrial areas.

360 Waste not, test more: Innovations in tissue processing to expand the testing of clinical specimens

OBJECTIVES/GOALS: Clinical tissue specimens are primarily destined for formalin fixed, paraffin embedded processing to create a basis for diagnosis by microscopic examination. Innovations in specimen processing are required to expand its availability for inclusion as the substrate in assays that can contribute to the further development of Precision Medicine. METHODS/STUDY POPULATION: Transurethral resection of bladder tumors were selected for testing based on availability and tissue composition. A wash step was used to generate daughter aliquots composed of dislodged cells and a solution with prior contact to the parent tissue. This wash step served two purposes: 1) reduce the amount of contaminating material from spreading to other cases, a problem known to be associated with this type of specimen; and 2) create aliquots from which additional informative data could be generated. These daughter aliquots were then examined to determine their value as a source for exosome profiling, metabolomic studies, molecular characterization and organoid development. The parent tissue was not compromised, was able to undergo conventional processing and yielded results equivalent to unwashed specimens. RESULTS/ANTICIPATED RESULTS: Exosomes secreted by the tumor cells were identified to be present in the daughter aliquots by a combination of their isolation using CD31 and detection of miR-21 expression. These exosomes were confirmed to be not related to fragmented cells from testing for beta-tubulin. A global/discovery-based approach using mass spectrometry provided insights into early characterization of metabolomic profiles present in these tumor cells. Ample amounts of high quality DNA (226 ng/ul concentrations; 11.3 ug total) were recovered from the dislodged, excess cells in the wash for molecular studies. Finally, from viable cells recovered in one of the daughter wash aliquots, the ability to grow organoids was proven to be possible and reproducible. DISCUSSION/SIGNIFICANCE: Based on these results, the value of the clinical specimen can be markedly expanded for utilization in research and possible clinical use without detracting from the parent tissue. This non-destructive, easy to adopt wash procedure can potentially lead to an influx of data that may ultimately prove useful in improving patient care.

Impact of Washing Processes on RNA Quantity and Quality in Patient-Derived Colorectal Cancer Tissues.

Background: Colorectal cancer (CRC) is a common and lethal cancer worldwide. Extraction of high-quality RNA from CRC samples plays a key role in scientific research and translational medicine. Specimen collection and washing methods that do not compromise RNA quality or quantity are needed to ensure high quality specimens for gene expression analysis and other RNA-based downstream applications. We investigated the effect of tissue specimen collection and different preparation processes on the quality and quantity of RNA extracted from surgical CRC tissues. Materials and Methods: After surgical resection, tissues were harvested and prepared with various washing processes in a room adjacent to the operating room. One hundred fourteen tissues from 36 CRC patients were separately washed in either cold phosphate-buffered saline reagent (n = 34) or Dulbecco's modified Eagle's medium (DMEM; n = 34) for 2-3 minutes until the stool was removed, and unwashed specimens served as controls (n = 34). Six tissue specimens were washed and immersed in DMEM for up to 1 hour at 4°C. Before RNA extraction, all specimens were kept in the stabilizing reagent for 3 months at -80°C. RNA was extracted, and the concentration per milligram of tissue was measured. RNA quality was assessed using the RNA integrity number (RIN) value. Results: Different washing processes did not result in significant differences in RNA quantity or RIN values. In the six tissues that were washed and immersed in DMEM for 1 hour, RIN values significantly decreased. The quality of the extracted RNA from most specimens was excellent with the average RIN greater than 7. Conclusions: RNA is stable in specimens washed in different processes for short periods, but RIN values may decrease with prolonged wash times.

The influence of the washing cycles on the functionality of the electric-embroidered element

In modern production, embroidered elements perform not only traditional functions of decorativeness, informativeness, but are also used in such areas as medical diagnostics and rehabilitation (e.g. embroidered sensors), implant production, smart home interior elements, smart clothing or electronic textile production. The aim of the work is to investigate and evaluate the influence of different washing cycles performed during operation on the functionality of an electric-embroidered element. It was found that after the first washing, the electrical resistance of the embroidered elements decreased compared to the unwashed specimens. After seven washings, the electrical resistance increased. In all cases, the obtained strong linear dependence between the electrical resistance and the operating cycles indicates that, with a larger number of washing cycles, the electrical conductivity of the embroidered elements decreases. The results showed that the conductivity of the closed embroidered chain of embroidered 14 mm contour width elements was obtained from ∼ 34% to ∼ 61% higher than the conductivity of 6 mm contour width elements.

Ca, Cu, and Li in washed and unwashed specimens of needles, bark, and branches of the blue spruce (Picea pungens) in the city of Ankara.

The use of certain types of plants to determine the accumulation of HMs (heavy metals) has yielded quite consistent results in the research fields. Many researches have focused on particular types of HMs due to their common presence in the air (Pb, Cd, Ni, Co, Cr to name a few). However, it is equally as important to shed light on other types of HMs and the scale of their existence in our atmosphere, hence this paper. Blue spruce (Picea pungens) tree organs were used in an experiment to calculate the recent concentration of HMs. The research concentrates on Ca, Cu, and Li elements in the washed and unwashed needles, branches, and barks, and these organs were evaluated depending on the organ age. The study results showed that the concentration of the elements subjected to the research changed depending on the organ, washing status and organ age, and that the lowest concentrations of Ca and Cu elements were obtained in the barks in general. In relation to the organ age, it was found that there was an increase in the concentration of Ca with age, and that the concentration of Li element was inversely proportional to age.

Preparation of Semen Specimens for Cryopreservation: Morning or Afternoon Specimen Collection, Washed Versus Unwashed. Which is better?

Objective To determine differences in semen parameters between morning or afternoon collection, response to cryopreservative addition and washed versus unwashed specimens. Design Retrospective analysis of sperm bank database between 2014 and 2018. Materials and Methods Of 1179 specimens from 22 donors, 720 were collected before noon, 458 after noon. 252 ejaculates were cryopreserved as neat, unwashed semen, while 932 specimens were gradient prepared prior to cryopreservation. A standard semen analysis was performed 30 minutes after ejaculation. Raw semen was diluted 1:1 with TEST-yolk buffer (Irvine Scientific), placed in vials, and frozen in liquid nitrogen vapor prior to storage in the liquid phase. For gradient preparation, raw semen was layered onto an 80% layer followed by centrifugation at 320g for 20 minutes. The pellet was washed, resuspended in HTF, followed by addition of the cryopreservative and frozen as above. Following a minimum of 48 hours, the test vials were thawed, and the post thaw count and % motility were determined. Data was analyzed using a 2-tailed, unpaired t-test with significance set at p ≤ 0.05. Results When all donor data was compared, there was no statistical difference between morning and afternoon semen collection for volume (p = 0.006), count (p = 0.49), motile count (p = 0.59) and % motility (p = 0.95). When AM to PM collections were compared for each donor, no statistical difference was seen for all parameters in only two donors. Volume was significantly higher in 5 donors; count was higher in AM for 2 donors and in PM for 2 donors; % motility higher for 1 donor in AM and in PM for 1 other donor. The gradient prepared specimens showed significantly (P = 0.0001) higher post-thaw motility and cryosurvival rate than unwashed specimens. Conclusions There appears to be no difference in semen specimens collected in the morning versus in the afternoon, although there was a trend for higher semen volume in the afternoon specimens. There were differences in individuals between morning and afternoon collections which should be considered when evaluating sperm donors as well as clients, particularly for use in ART procedures. Gradient preparation prior to freezing results in better motility, motile count and cryosurvival before freezing and after thaw, suggesting that this method is more favorable for donor sperm cryopreservation. Disclosures Nothing to disclose. Funding None.

Spectral Signatures of Immature Lucilia sericata (Meigen) (Diptera: Calliphoridae).

Hyperspectral remote sensing is an innovative technology with applications in many sciences and is a non-destructive method that may offer more precise aging within development stages. Hyperspectral reflectance measurements from the anterior, midsection, and posterior of Lucilia sericata (Meigen) larvae and pupae were conducted daily from samples of the developing insects beginning at second instar. Only midsection measurements were conducted on second instar larvae due to their size, to ensure that the measurement was not of reflective surroundings. Once measured, all insects were washed with deionized water, blotted with filter paper, and re-measured. Daily age prediction during the post-feeding stage was not impacted by the unwashed insect measurements and was best predicted based on posterior measurements. The second and third instar larvae, which move about their food source, had different contributing coefficients to the functional regression model for the hyperspectral measurements of the washed compared with unwashed specimens. Although washing did not affect the daily prediction within these stages, it is still encouraged in order to decrease the effect of food source on spectral reflectance. Days within the intra-puparial period were best predicted based on anterior measurements and were not well distinguished from one another in the first few days based on midsection and posterior measurements.

Salt weathering of limestones: susceptibility of petrographical features (SEM study)

Salt weathering is one of the main processes affecting rock materials applied in the built environment. Scanning electron microscopy (SEM) studies can contribute to the understanding of the susceptibility of petrographic features of rocks to salt weathering.SEM studies were performed in limestones (grainstones with the commercial designations of Semi-rijo and Moca Creme and a travertine) submitted to salt weathering tests (EN 12370 using sodium sulphate solutions and cubic specimens) consisting of 15 cycles of immersion/drying followed by water washing after the 15th cycle. Fragments from small cubes of the grainstones subjected to salt weathering cycles without the final water cleaning were also studied. SEM observation before and after the tests allows the discussion of the petrographical characteristics of these rocks that contribute to erosive decay under salt weathering.In the case of the grainstones specimens the texture can be described in general as allochemical particles cemented by sparry calcite (Figure 1 a) and it is visible from the preparation of the polished surfaces the interface between the sparry cement and the allochemical components. After the salt weathering tests, SEM studies show (Figure 1 b) extensive fissuring in the sparry cement and separation between grains and sparry cement. Observations of disaggregation products showed an important amount of apparently intact grains. These results are similar to those observed by with the sparry cement being affect by salt crystallisation and lesser impact on the grains. Studies in the unwashed specimens showed the presence of sodium sulphate in the interface between allochemical grains and the sparry cement (Figure 1 c,d). Travertine specimens show heterogeneous patterns of erosion attributed to the heterogeneous texture of this rock, with more terrigenous (especially clay-rich) portions that favour a higher erosive susceptibility and the presence of clays has been frequently connected with salt weathering susceptibility in limestones. In Figure 1e can be observed the heterogeneity of a polished surface, with the more irregular portions showing a chemical spectra (Figure 1 f) indicative of the terrigenous component and the presence of sodium and sulphate (resulting from the solutions used in these weathering tests).The authors acknowledge the support of the Fundação para a Ciência e Tecnologia (POCTI/CTA/44940/2002; PEst-OE/CTE/UI0697/2011 and Pest-OE/CTE/UI0098/2011, funding by the European Union and national budget of the Portuguese Republic). Acknowledgments to Eng. Teresa Luís, Eng. Sónia Pereira and Enterprise Mármores Galrão for the rock blocks.

Increase in the fracture toughness and bond energy of clay by a root exudate

SummaryRoot exudates help drive the formation of the rhizosphere by binding soil particles, but the underlying physical mechanisms have not been quantified. This was addressed by measuring the impact of a major component of root exudates, polygalacturonic acid (PGA), on the interparticle bond energy and fracture toughness of clay. Pure kaolinite was mixed with 0, 1.2, 2.4, 4.9 or 12.2 g PGA kg−1to form test specimens. Half of the specimens were washed repeatedly to remove unbound PGA and evaluate the persistence of the effects, similar to weathering in natural soils. Fracture toughness,KIC, increased exponentially with added PGA, with washing increasing this trend. In unwashed specimensKICranged from 54.3 ± 2.5 kPa m−1/2for 0 g PGA kg−1to 86.9 ± 4.7 kPa m−1/2for 12.2 g PGA kg−1. Washing increasedKICto 61.3 ± 1.2 kPa m−1/2for 0 g PGA kg−1and 132.1 ± 4.9 kPa m−1/2for 12.2 g PGA kg−1. The apparent bond energy, γ, of the fracture surface increased from 5.9 ± 0.6 J m−2for 0 g kg−1to 12.0 ± 1.1 J m−2for 12.2 g kg−1PGA in the unwashed specimens. The washed specimens had γ of 13.0 ± 1.9 J m−2for 0 g kg−1and 21.3 ± 2.6 J m−2for 12.2 g PGA kg−1. Thus PGA, a major component of root exudates, has a large impact on the fracture toughness and bond energy of clay, and is likely to be a major determinant in the formation of the rhizosphere. This quantification of the thermodynamics of fracture will be useful for modelling rhizosphere formation and stability.

Pre-washed frozen donor sperm is associated with lower pregnancy rates compared to semen processed immediately prior to donor insemination.

Objective: Pre-washed frozen sperm has recently been promoted by some commercial sperm banks. The obvious benefit is reduced need for laboratory resources for physicians wishing to perform donor intrauterine insemination (IUI-D). Although many physicians would consider this a major advantage, comparison to outcome using traditional semen cryopreservation (unwashed) and post-thaw processing is very limited. The purpose of this study is to compare sperm motility parameters and cycle outcome in women undergoing intrauterine insemination (IUI) with either prewashed donor sperm or unwashed donor sperm requiring post thaw preparation. Design: Retrospective analysis of women undergoing LH timed IUI-D and FSH stimulated IUI-D with pre-washed or unwashed frozen donor sperm from January 1, 1999 through January 31, 2001. Materials/Methods: Patients undergoing donor insemination using sperm from a commercial bank (Cryobiology–Ohio) ordered pre-washed or unwashed frozen based primarily on availability. Pre-washed specimens were centrifuged over a density gradient and re-suspended in a solution of test-yolk buffer (TYB) to achieve a desired density of motile sperm prior to cryopreservation. Unwashed semen samples were diluted in TYB and centrifuged and resuspended as necessary (no density gradient) to achieve a desired density of motile sperm prior to freeze. Pre-washed specimens were thawed immediately prior to insemination requiring no further preparation. Unwashed specimens were thawed on the day of insemination, and processed via centrifugation over a density gradient just prior to IUI. All specimens were analyzed by computer assisted semen analyzer (CASA–IVOS/Hamilton Thorne) post thaw. Unwashed specimens were additionally analyzed following processing and prior to IUI. Results: Please refer to table. Results. ∗Type of cycle:Timed IUIFSH IUIType of vial:Unwashed vialsPre-washed vialsUnwashed vialsPre-washed vialsTotal number cycles:15178125Average age:32.8534.1432.7536.20Mean total progressive (M):7.868.7910.2310.14Mean progressive motility:37.97%33.11%44.63%36.06%Positive hCG:401.141Percent positive hCG:26.49%∗p < 0.5T (Timed IUI: Unwashed vs. Prewashed; Fisher’s Exact).14.10%33.33%20.00%∗ p < 0.5T (Timed IUI: Unwashed vs. Prewashed; Fisher’s Exact). Open table in a new tab Conclusions: Pre-washed semen had relatively lower percent progressive sperm motility than sperm processed post-thaw. The pregnancy rates following insemination with pre-washed sperm was markedly lower than the rates following insemination with sperm processed post thaw. Although the use of pre-washed sperm inarguably allows IUI-D to be performed by physicians with limited access to reproductive laboratories, this may come at the cost of lower fecundity. Further refinement of sperm pre-washing techniques may ultimately lead to improved outcome. At the present time, physicians with limited access to reproductive laboratories may wish to consider other alternatives to IUI with pre-washed donor sperm.

Comparison of Semen Characteristics and Pregnancy Rates Using Washed Versus Unwashed Donor Sperm for Intrauterine Insemination (IUI)

Objective: Many commercial sperm banks offer washed frozen donor semen specimens that are convenient and ready to use, albeit more costly. If the staff and facilities are available, less expensive unwashed specimens can be purchased, thawed and washed prior to use. Our objective was to compare semen characteristics and pregnancy rates associated with washed and unwashed specimens used for donor insemination (DI) in a group of women with proven fertility.

The silver reaction of nucleolar proteins in the main structural compartments of ring-shaped nucleoli in smear preparations

The present study was undertaken to provide more information on the conditions which result in preferential silver staining of the main nucleolar structural compartments using silver stainable proteins as their markers at the light microscopic level. For this study the mostly used method in cytology and pathology in which the nucleolar silver-positive structures are "developed" with the colloidal developer (Howell and Black, 1980; Ploton et al., 1986) was selected as silver reaction. Ring-shaped nucleoli of mature human lymphocytes represent a convenient model for such a study because they consist of one large fibrillar center, adjacent nucleolar regions with dense fibrillar components and the nucleolar peripheral shell with dense granular components. All these nucleolar compartments are known to possess characteristic silver stainable proteins. The results demonstrated that proteins of the fibrillar center and possibly adjacent nucleolar regions reacted preferentially with silver after a relatively long fixation with formaldehyde or methanol in unwashed specimens before the silver reaction. In contrast, the preferential staining of proteins in the nucleolar peripheral shell with silver was achieved after the fixation with acidified methanol or ethanol as well as after short fixation with formaldehyde vapors. In addition, the commonly used fixation before the silver reaction are not necessary and may be omitted for the visualization of all silver stainable proteins present in the fibrillar center as well as in the adjacent nucleolar regions and the nucleolar peripheral shell. In addition, similar results were achieved for the simultaneous visualization of proteins in the fibrillar center and nucleolar peripheral shell after fixation with ethanol.

The effect of sunlight on the colour of Ghanaian cotton printed fabrics

Abstract In a 3·2·2 factorial experiment in three replicates, Ghanaian cotton prints (Real Wax, Real Java, Batik) were studied. Specimens were hand washed in soap solution, rinsed in clean water and dried in shade or sunlight twice a week for 3 weeks. Unwashed specimens were similarly sun‐dried or aired. All specimens were exposed to a total of 42 h in the sun or shade. Colour loss was measured by means of a grey scale. The specimens lost significantly more colour (P &lt; 0·01) when dried in the sun (3·96) than when dried in the shade (4·09). Irrespective of the drying method, washed fabrics lost significantly more colour (P&lt;0·01) (3·96) than unwashed ones (4·09). There was no significant interaction effect (P &gt; 0·01) between method of drying and fabric type. However, there was a significant interaction (P&lt;0·01) between fabric type and washing or airing. The grey scale mean for washing was 4·03 for Wax, 3·81 for Batik and 3·75 for Java. For airing, Batik, Wax and Java had means of 4·22, 4·19 and 4·17 respectively. Thus, Real Wax was the most stable when washed, whereas Batik was the most stable to airing. It is suggested that drying or airing of these fabrics be performed in the shade to preserve their colour.

Evaluating glass, polystyrene, and polypropylene containers for semen collection and sperm washing.

The effects of glass, polystyrene, and polypropylene containers on semen parameters as measured by Cell Soft computer-assisted semen analysis and hypoosmotic swelling (HOS) tests were assessed in unwashed specimens and again after sperm washing and swim-up procedures. A superiority in unwashed specimens was observed in glass versus polystyrene concerning velocity, motility percentage, and HOS testing (p less than 0.01). Also, superiority of glass versus polystyrene was demonstrated for linearity and ALH (p less than 0.05). Glass was only superior to polypropylene in categories of motility and HOS testing (p less than 0.05 and p less than 0.01, respectively). There was no category in which plastic was statistically superior to glass in the unwashed specimens. Concerning washed and swim-up specimens, there were no semen parameters in which there was any superiority demonstrated of either glass or plastic. Unless some future proof that the differences in semen parameters demonstrated in this study have no clinical significance, these data suggest that the collection of semen samples for sperm analysis or therapeutic use should be performed in glass containers.

Mercury Contamination of Vegetation due to the Application of Sewage Sludge as a Fertilizer

Abstract The mercury content of vegetation grown on sludged and control plots is given. Both washed and unwashed specimens are tested. Material analysed is, leaves and seeds of wild barley and quack grass; tomato roots, plants and fruit; bean roots, plants and pods; carrot roots and tops and lettuce leaf. No significant increase in mercury levels for washed plant material grown on sludged plots was noted except in the case of tomato fruit which showed levels up to 50 times higher compared to specimens from unsludged plots. Surface contamination of vegetation by mercury containing dusts and soils can cause elevated levels in unwashed or poorly washed material.

A DEMINERALIZATION PROCEDURE FOR ENZYMATIC HISTOCHEMICAL USE: A QUANTITATIVE SUCCINIC DEHYDROGENASE ASSAY.

Thinnest practical slices of bones or teeth are suspended at 4 C in a 10% solution of EDTA in 0.1 M tris buffer brought to pH 6.95 with KOH pellets. The solution is stirred moderately and continuously with a magnetic stirrer until specimens are demineralized. Unwashed specimens, taken directly from the demineralizing fluid, are frozen on a block of CO2 ice, mounted on a tissue carrier and sectioned at 6 μ in a cryostat. Histochemical stains conducted on sections stored in a slide holder enclosed in a plastic bag in a refrigerator for as long as 2 wk were successful. Quantitative studies on the preservation of succinic dehydrogenase showed nearly all of it present in specimens demineralized for 2 days, and approximately 50% remaining in specimens demineralized for 7 days. Qualitative studies with other dehydrogenases indicate that several may be similarly affected.