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Behind the scenes of the forensic lab
Published in Rachel E. Lovell, Jennifer Langhinrichsen-Rohling, Sexual Assault Kits and Reforming the Response to Rape, 2023
An added complexity when looking at the cost and number of samples to test is that one sample does not necessarily yield one DNA extract. SAK samples are first presumed to have sperm present and go through what is called a differential extraction process.1 In this process, the DNA is separated into two fractions: the sperm fraction and the epithelial fraction. The epithelial fraction contains skin cells that are non-specific to male or female DNA, but the sperm fraction specifically targets DNA from spermatozoa (Butler, 2009). This means that if one sample is selected for testing in a SAK, and is presumed to have sperm present, it will yield two DNA extracts. The number of samples dictated at the beginning of testing oftentimes does not take this into account, which can have downstream effects for the cost determination. Due to the costs, one study recommends tiered testing with the first rounds of testing to only include the three most probative samples (Wang et al., 2020). This study also found that there are no significant effects in applying this methodology to SAK testing (Wang et al., 2020).
Clinical Detection of Exposure to Chemical Warfare Agents *
Published in Brian J. Lukey, James A. Romano, Salem Harry, Chemical Warfare Agents, 2019
Benedict R. Capacio, J. Richard Smith, Robert C. diTargiani, M. Ross Pennington, Richard K. Gordon, Julian R. Haigh, John R. Barr, Brian J. Lukey, Daniel Noort
Radioactive assays for AChE activity are very sensitive but require special handling and disposal. Although used in a research setting, radioactive assays have not been developed for general clinical laboratory or field use. These methods are based on the measurement of the production of [14C]carbon dioxide or choline or [3H]acetate from the hydrolysis of appropriately 14C- or 3H-labeled acetylcholine, respectively. The radioactive species are separated by Reinecke salt precipitation, differential extraction into organic solvent (Johnson and Russell, 1975), and ion-exchange chromatography or ion-exchange disks (Gordon et al., 1982). The advantage of radioactive methods is that they overcome the potentially high background in some biological samples from SH groups that may react with the Ellman reagent. On the other hand, there are notable disadvantages, including disposal of the radioactive waste, and the assays are not readily adaptable for kinetic analysis, since aliquots of the radioactive mixture must be removed for scintillation counting at specific time intervals.
Pitfalls and Practical Solutions
Published in Joseph Chamberlain, The Analysis of Drugs in Biological Fluids, 2018
Some of the procedures mentioned above are applicable on the normal laboratory analytical scale. One of the features of ultramicro analysis is the need to keep the addition of any chemicals or solvents to a minimum and many workers have endeavored to devise microsystems of analysis. These procedures also have the intrinsic merit of being relatively rapid. For example Bonato and Lanchote devised a procedure for differential extraction of drugs and metabolites from apolar compounds (thought to be detrimental to reverse-phase columns) using partitioning between hexane and the mobile phase before analysis and claimed longer column life as a result.1425 Salting out solvent extraction for preconcentration of benzaikonium chloride prior to high-performance liquid chromatography has also been claimed to increase concentration tenfold.1426 Van der Vlis et al. also claimed on-line trace enrichment of doxorubicin on iron(III)-loaded 8-hydroxyquinoline-bonded silica.1427
mRNA expression of the P5 ATPase ATP13A4 is increased in Broca’s area from subjects with schizophrenia
Published in The World Journal of Biological Psychiatry, 2020
Andrew S. Gibbons, Laura M. Bell, Madhara Udawela, Brian Dean
The significant differences in ATP13A4 expression in BA 8 between subjects with schizophrenia and controls in reference gene normalised data but not in the raw data could, therefore, be due to differential extraction of mRNA in the two diagnostic groups in that CNS region, which has been corrected upon normalisation. Alternatively, levels of ATP13A4 mRNA may not differ with diagnosis in BA 8 but this variation was introduced as an artefact of reference gene normalisation. We previously showed that expressing levels of expression of a gene of interest as a ratio of the geometric mean of two reference genes can (1) give results similar to that using three reference genes and (2) help identify if one reference gene may be overly influencing comparisons of data between subjects with schizophrenia and controls (Dean et al. 2016). Hence, when ATP13A4 levels were normalised to geometric means of different pair combinations of our reference genes, ATP13A4 levels remained significantly different between control and schizophrenia groups in data normalised to GAPDH and TFB1M (U = 247, P < 0.01), GAPDH and SKP1 (U = 249, P < 0.01), and TFB1M and SKP1 (U = 249, P < 0.01). Therefore, no single reference gene could account for the variation in normalised ATP13A4 levels between the control and schizophrenia groups and for the increase in ATP13A4 to be explained as an artefact of normalisation, the expression of all three unrelated reference genes from study would need to be preferentially decreased in BA 8 but not BA 44 in subjects with schizophrenia.
Antibacterial effects of 18 medicinal plants used by the Khyang tribe in Bangladesh
Published in Pharmaceutical Biology, 2018
Md Shahadat Hossan, Hassan Jindal, Sarah Maisha, Chandramathi Samudi Raju, Shamala Devi Sekaran, Veeranoot Nissapatorn, Fatima Kaharudin, Lim Su Yi, Teng Jin Khoo, Mohammed Rahmatullah, Christophe Wiart
The plant powders (20–60 g) were mixed at room temperature sequentially with organic solvents of increasing polarity starting with hexane (Friendemann Schmidt, Parkwood, Australia), ethyl acetate (Friendemann Schmidt, Parkwood, Australia) and 95% (v/v) ethanol (AR grade, John Kollin Corporation, Midlothian, UK) for differential extraction of non-polar, mid-polar and polar extracts, respectively (Harborne 1998). Each extraction was performed in triplicate by maceration of plant powder-to-solvent ratio of 1:5 (w/v) for three days at room temperature. The respective liquid extracts were subsequently filtered through qualitative filter papers No. 1 (Whatman International Ltd., Maidstone, UK) using aspirator pump (EW-35031-00, 18 L/min, 9.5 L Bath, 115 VAC), and the filtrates were concentrated to dryness under reduced pressure at 40 °C using rotary evaporator (Buchi Labortechnik AG, Flawil, Switzerland). The dry extracts obtained were weighed with an analytical balance (Sartorius AG, Göttingen, Germany) and stored in tightly closed glass scintillation vials (Kimble, Rockwood, TN) at −20 °C until further use. For stock solutions, each crude extract was dissolved in 100% dimethyl sulphoxide (DMSO) (R&M Chemicals, Chelmsford, UK) to a concentration of 100 µg/µL. A yield for each extract was calculated.