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Principles of forensic science and crime scene investigation
Published in Jason Payne-James, Richard Jones, Simpson's Forensic Medicine, 2019
Jason Payne-James, Richard Jones
There are many different methods of enhancing footwear marks, some of which are used in the enhancement of fingerprints. Often photographing under controlled-lighting conditions, or the addition of specialist light sources, can improve the detail within a mark. When a mark warrants a more intensive examination (e.g., in a serious assault) the enhancement may be carried out using chemicals. For soil deposits, potassium or ammonium thiocyanate can be used, which reacts with metallic ions in the soil. Marks in blood may be enhanced using Amido Black solution, which reacts with the proteins in the blood. There are many other methods of chemically enhancing marks.
Method of Fractionating Subcellular Particles to Determine Intracellular Localization of Radiotracers
Published in Lelio G. Colombetti, Principles of Radiopharmacology, 2019
H. Orii, K. Samezime, K. Komine
A Hitachi 65p-7 ultracentrifuge and a Hitachi RPZ48T zonal rotor were used. The sucrose gradient was obtained by use of a microtube pump (Tokyo Rika Co., MP4001). A linear gradient of 25 to 35% w/w in 0.01 M tris buffer was pumped in the rotor running at 3000 rpm, followed by 170 mℓ of 50% sucrose supplemented with cesium chloride to raise the density to 1.36 g/mℓ. On top of the homogenate in the center side, 10 to 20 mℓ of liver homogenate and 50 mℓ of 0.25M sucrose were carefully added. Edge unloading of samples resulted in increased pressure compared to center unloading. Cross leaking was checked with amido black solution. The critical pressure was found to be 0.8 kg/cm2. The absorbance of the effluent from the edge was monitored by a Uvicord®II (LKB Co.) at 280 nm and recorded. By use of an Ultrorack®(LKB), 10 mℓ fractions were collected. All experiments were performed at 5°C.
Isolation, Fractionation, and Analysis of Nonhistone Chromosomal Proteins
Published in Lubomir S. Hnilica, Chromosomal Nonhistone Proteins, 2018
Leokadia Klyszejko-Stefanowicz, Lubomir S. Hnilica
Analytical gel electrofocusing can be carried out in tubes, in horizontal flat beds, or vertical gel slabs.316 In any case, a gel is chosen with a pore size which offers low resistance to the passage of proteins and thus acts mainly as an anticonvectant medium. One can usually apply the sample at the desired gel surface in the same manner as for PAGE, i.e., after addition of a dense medium, by layering under the electrolyte, or in the case of sample volumes exceeding 200 μℓ by overlayering the electrolyte.316 The resolved proteins can be visualized as white precipitation bands by immersing the gels in trichloroacetic acid (5%). The gels, washed repeatedly in this solution to remove the ampholytes, can be then stained, e.g., in amido-black or Coomassie® blue and destained by washing in acetic acid (7%). The pH gradient in gels is determined by cutting 2-mm sections from an unstained gel run simultaneously with stained gels, soaking each section in 2 mℓ water for several hours and measuring the pH of the extracts. The pH gradient is reproducible for gels run simultaneously.332
In Vitro Efficacy of Chlorhexidine and a riboflavin/UVA Combination on Fungal Agents of Keratitis
Published in Current Eye Research, 2020
Zeynep Kunt, Meltem Yağmur, Hazal Kandemir, Inan Harbiyeli, Elif Erdem, Ayşe Kalkancı, G.Sybren De Hoog, Macit Ilkit
We performed the riboflavin/UVA treatment combination in U-bottomed, 96-well microplates using the “black plate” method, as described by Bilgihan et al.19 Following this method, we painted the backside of wells harboring microorganisms black to prevent reflection of UVA light and interaction effects between wells. To mimic the condition of cells without fungi, we added 200 μL of amido black 10B stain (Merck) to wells without microorganisms. Then we added 100 μL of phosphate-buffered saline solution and 10 μL of fungal suspensions to each well prior to riboflavin/UVA combination. For Groups B–D, we added 5 μL of antifungal drugs seven times at 30-min intervals, at 37°C. Then we added 33 μL of 0.1% riboflavin (MedioCross TE; Peschke Meditrade, Zug, Switzerland) and performed the riboflavin/UVA combination procedure under 9 mW/cm2 illumination for 10 min at 365-nm UVA (Peschke Meditrade, Zurich, Switzerland). In addition, for all strains we tested control groups in each plate to assess the effect of UVA alone and with 0.1% riboflavin alone.
Placental glucocorticoid receptors are not affected by maternal depression or SSRI treatment
Published in Upsala Journal of Medical Sciences, 2020
Åsa Edvinsson, Angela Hoyer, Malin Hansson, Theodora Kunovac Kallak, Inger Sundström-Poromaa, Alkistis Skalkidou, Susanne Lager
Placental villous tissue samples were homogenized in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; cat# 89900, Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail (cat# 87785, Thermo Fisher Scientific). Protein concentrations of homogenates were determined using the Bradford assay (B6919, Sigma-Aldrich, St. Louis, MO). Western blot was performed on 4–12% Bis-Tris pre-cast gels (NP0323, Thermo Fisher Scientific, Waltham, MA) and transferred onto PVDF membranes (IPFL00010, Merck Millipore, Burlington, MA). After transfer, membranes were stained for total protein with Amido Black Staining Solution (A8181, Sigma-Aldrich) and/or Ponceau S Staining Solution (P7170, Sigma-Aldrich). Total protein stains have been previously proposed as a good reference for Western blotting of placental proteins (21). Thereafter, membranes were blocked for 1 h at room temperature in Odyssey Blocking buffer (927–40000, LI-COR Biosciences, Lincoln, NE). After blocking, the membranes were probed with beta-actin (final concentration 0.2 µg/mL; SC-47778, Santa Cruz Biotechnology, Dallas, TX; 1 h at room temperature) or glucocorticoid receptor (NR3C1; final concentration 81 ng/mL; ab109022, Abcam, Cambridge, UK; overnight at +4 °C). Immunolabeling was visualized with fluorescently labelled secondary antibodies (926–68070 or 926–32211, LI-COR Biosciences) in an Odyssey Sa scanner (LI-COR Biosciences). Images were analysed with ImageJ (version 1.52a). Placental levels of the glucocorticoid receptor (GR) were adjusted for total protein staining intensity (Ponceau S staining).
Candida biome of severe early childhood caries (S-ECC) and its cariogenic virulence traits
Published in Journal of Oral Microbiology, 2020
Kausar Sadia Fakhruddin, Lakshman Perera Samaranayake, Hiroshi Egusa, Hien Chi Ngo, Chamila Panduwawala, Thenmozhi Venkatachalam, Allagappan Kumarappan, Siripen Pesee
Proteinase enzyme activity of Candida spp. was performed in terms of bovine serum albumin (BSA) degradation as per the technique of Ruma-Haynes et al. with some modifications [37]. The BSA test medium consisted of 20 g of dextrose, 1 g K2HPO4, 0.5 g MgSO4, 0.2 g yeast extract, 15 g of agar, 2 g of bovine serum albumin. Briefly, an 18 h yeast cell suspension adjusted to 108 cells was prepared, and the 10 µl suspension was inoculated onto the BSA plate and incubated at 37°C for five days. The plates were flooded with 1.25% Amido black stain (MB165-Amido black 10B) in 90% methanol and 10% acetic acid and allowed to stand for 10 minutes for the staining. After de-staining using 15% acetic acid for 20 minutes, plates were washed twice with PBS and allowed to dry at room temperature. Proteinase activity (Prz) was determined as the ratio of the colony diameter to that of clear zone proteolysis expressed in mm.