Complete Knockout of Endogenous Mdr1 (Abcb1) in MDCK Cells by CRISPR-Cas9.

Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.

Gunshot residue preservation in seawater.

Little is known about the persistence of gunshot residue (GSR) in soft tissue and bones during decomposition in marine environments. For a better understanding, qualitative and quantitative data were obtained on GSR retention on soft tissue and bony gunshot wounds (GSWs). A quantity of 36 fleshed and 36 defleshed bovine ribs were shot at contact range with 0.22 calibre hollow point ammunition using a Stirling 0.22 calibre long rifle. Bone specimens in triplicate were placed in three environments: submerged, intertidal and in supralittoral zone. Sets of triplicates were recovered on day 3, 10, 24 and 38, and analysed with scanning electron microscopy with energy dispersive X-ray spectrometry (SEM-EDX), and inductive coupled plasma mass spectrometry (ICP-MS). The SEM-EDX recorded GSR-indicative particles surrounding the bullet entrance on all bone types (fleshed and defleshed) in all environments throughout the study. GSR-unique particles were only detected on the supralittoral bones. The ICP-MS analysis showed faster GSR loss on submerged than intertidal and supralittoral defleshed specimens. Fleshed specimens showed a faster GSR loss on intertidal than submerged and supralittoral specimens. In conclusion, the GSR disappeared faster from submerged and intertidal than non-submerged specimens. The difference of detection of GSR between analysed specimens (defleshed versus fleshed) disappeared upon defleshing. This study highlights the potential of finding evidence of GSR in a submerged body and the potential of microscopic and analytical methods for examining suspected GSW in highly decomposed bodies in marine habitats.

Development of a skull/brain model for military wound ballistics studies.

Reports on penetrating ballistic head injuries in the literature are dominated by case studies of suicides; the penetrating ammunition usually being .22 rimfire or shotgun. The dominating cause of injuries in modern warfare is fragmentation and hence, this is the primary threat that military helmets protect the brain from. When helmets are perforated, this is usually by bullets. In combat, 20% of penetrating injuries occur to the head and its wounding accounts for 50% of combat deaths. A number of head simulants are described in the academic literature, in ballistic test methods for helmets (including measurement of behind helmet blunt trauma, BHBT) and in the 'open' and 'closed' government literature of several nations. The majority of these models are not anatomically correct and are not assessed with high-velocity rifle ammunition. In this article, an anatomically correct 'skull' (manufactured from polyurethane) and 'brain' (manufactured from 10%, by mass, gelatine) model for use in military wound ballistic studies is described. Filling the cranium completely with gelatine resulted in a similar 'skull' fracture pattern as an anatomically correct 'brain' combined with a representation of cerebrospinal fluid. In particular, posterior cranial fossa and occipital fractures and brain ejection were observed. This pattern of injury compared favourably to reported case studies of actual incidents in the literature.