Mineral absorption by submerged bone in marine environments as a potential PMSI indicator

Human remains enter marine environments in a number of ways ranging from homicides, suicides, accidental drownings, shipwrecks, to burials at sea. Once the remains are discovered, a legal and forensic investigation begins. A key component to this investigation is the postmortem submergence interval (PMSI). Determining this range on skeletonized remains is a complicated process in which there is no accurate test; although barnacle growth data was previously used to determine PMSI, there are still limitations with that method. Therefore, a more reliable component of bone needs to be used as a potential PMSI indicator, such as its elemental composition. Diagenesis starts affecting bones immediately and continues for thousands of years. Although diagenesis is a slow process, an exchange of elements between bone and the marine environment continually occurs. The purpose of the present study is to determine whether an increase in marine elements is found within the composition of bone after being submerged in a marine environment for up to 20 months. The present study will also determine whether bones submerged in different aquatic environments have significantly different elemental concentrations. For the time trials, pig femora were submerged in lobster cages off the coast of the University of Massachusetts Boston for 2-20 months. For the salinity trials, pig femora were submerged in a freshwater pond (Holliston, MA), the Inner Boston Harbor, and an ocean inlet near Woods Hole, MA for 18 months. All bone samples were dried, milled, homogenized, and analyzed by ED-XRF under He purge. The initially produced mass percentages of the identified elements were corrected with certified values of standard reference materials (NIST 1486, 1646a, and 2702). A Pearson’s correlation test determined that the concentrations for K, Fe, Zn, Sr, Si, S, Cr, Mn, Cl, Br, Ta, and W were significantly correlated to the amount of time submerged in the water. An ANCOVA analysis was applied to the significant elements noted above. After adjusting for the amount of time submerged, the concentrations of K, Fe, Sr, Si, S, Cl, Br, and Ta were determined to be significantly different between the control samples (never submerged) and the submerged samples (submerged for 2-20 months). K was the only element that had greater concentrations in the control samples than the submerged samples, most likely because of the decrease in mass percent as other environmental elements were incorporated into the bone. S and W were significantly related to the number of months submerged, with S being positively influenced and W being negatively. A multivariable linear regression was run in order to identify a means of predicting the amount of time submerged from the elemental concentrations of an unknown bone from a marine environment. The regression produced an equation that used the concentrations for K, Sr, Si, S, Cr, Cl, and Br to predict the PMSI in months. For the salinity trials, a one-way ANOVA was performed on all the elemental concentrations from the different salinity environments. Post hoc tests determined significant differences in elemental concentrations for K, Fe, Si, S, Al, Ti, Cr, Ni, Mn, Cl and Br among the different submergence locations; elemental concentrations of S, Fe, Mn, Cl, K, and Br were either significantly different between the fresh, brackish, and saltwaters or the freshwater and some form of marine water (brackish and salt). The trends in the other elemental concentrations were less obvious due to the impact of pollution within the surrounding environments. The linear regression equation created in the present study accounted for the majority of the variance in the outcome (R2 = 80.2%); however, this equation should not currently be applied in forensic investigations. The study needs to be repeated a number of times with other bone samples from the same and different submergence locations, in order to determine the accuracy and usefulness of the equation. Although not verified, this regression equation may be useful in analyzing samples from brackish and saltwater environments, because the majority of the variables within the equation (K, Sr, S, Cl, Br) were consistent among the fresh, brackish, and saltwater samples. Time constraints, small sample sizes, and variance among samples were the major limitations of the present study. Even with limitations, significant results were produced by the ED-XRF analysis. Future research should expand upon the methodologies of XRF analyses of bones, especially those from marine environments. Because of their relevance to forensic investigations and PMSI, future research should include longer experimental periods, more salinity locations, more information on the surrounding water components, and more comparisons among instrumentation

Structured inquiry-based learning: Drosophila GAL4 enhancer trap characterization in an undergraduate laboratory course.

We have developed and tested two linked but separable structured inquiry exercises using a set of Drosophila melanogaster GAL4 enhancer trap strains for an upper-level undergraduate laboratory methods course at Bucknell University. In the first, students learn to perform inverse PCR to identify the genomic location of the GAL4 insertion, using FlyBase to identify flanking sequences and the primary literature to synthesize current knowledge regarding the nearest gene. In the second, we cross each GAL4 strain to a UAS-CD8-GFP reporter strain, and students perform whole mount CNS dissection, immunohistochemistry, confocal imaging, and analysis of developmental expression patterns. We have found these exercises to be very effective in teaching the uses and limitations of PCR and antibody-based techniques as well as critical reading of the primary literature and scientific writing. Students appreciate the opportunity to apply what they learn by generating novel data of use to the wider research community

Counteraction of Tetherin Antiviral Activity by Two Closely Related SIVs Differing by the Presence of a Vpu Gene

In different primate lentiviruses, three proteins (Vpu, Env and Nef) have been shown to have anti-tetherin activities. SIVden is a primate lentivirus harbored by a Cercopithecus denti (C. denti) whose genome code for a Vpu gene. We have compared the activity of HIV-1 Vpu and of SIVden Vpu on tetherin proteins from humans, from C. denti and from Cercopithecus neglectus (C. neglectus), a monkey species that is naturally infected by SIVdeb, a virus closely related to SIVden but which does not encode a Vpu protein. Here, we demonstrate that SIVden Vpu, is active against C. denti tetherin, but not against human tetherin. Interestingly, C. neglectus tetherin was more sensitive to SIVden Vpu than to HIV-1 Vpu. We also identify residues in the tetherin transmembrane domains that are responsible for the species-specific Vpu effect. Simultaneous mutation (P40L and T45I) of human tetherin conferred sensitivity to SIVden Vpu, while abolishing its sensitivity to HIV-1 Vpu. We next analyzed the anti-tetherin activity of the Nef proteins from HIV-1, SIVden and SIVdeb. All three Nef proteins were unable to rescue virus release in the presence of human or C. denti tetherin. Conversely, SIVdeb Nef enhanced virus release in the presence of C. neglectus tetherin, suggesting that SIVdeb relies on Nef in its natural host. Finally, while HIV-1 Vpu not only removed human tetherin from the cell surface but also directed it for degradation, SIVden Vpu only induced the redistribution of both C. denti and C. neglectus tetherins, resulting in a predominantly perinuclear localization

Effect of tetherin from different hosts on virus release.

<p>A. Virus particle release was tested in 293T cells transfected with HIV-1ΔVpuΔNef NL4.3 proviral DNA (500 ng) in the presence of human, <i>C. denti</i> and <i>C. neglectus</i> tetherin. Virus production expressed as a percentage of maximal particle release in the absence of tetherin is shown for increasing amounts of plasmid DNA encoding for the three tetherin constructs (100, 330 and 1000 ng). Differences in the amount of plasmid DNA in each transfection were compensated by the addition of control vector (pcDNA3). After 42 h, the amount of virus released into cell culture supernatant was measured by HIV-1 p24 ELISA. This figure is representative of three independent experiments. B. Expression of Vpu constructs. Western blot analysis was performed to determine the expression levels of the three plasmids encoding HA-tagged tetherins. 293T cells were cotransfected with 1000 ng of each plasmid. Tetherin migrated as several species in SDS-PAGE analyses, presumably as a result of heterogeneus glycosylation. Forty-two hours after transfection cell lysates were collected and analyzed by Western blot. The expression level of HIV-1 Gag protein was monitored with an anti-p24 antibody. Actin was used as a loading control.</p

Virion release from cells tranfected with vectors expressing tetherin mutants.

<p>HIV-1 Vpu and SIVden Vpu were tested for their ability to rescue p24 release for HIV-1ΔVpuΔNef in 293T cells expressing human tetherin delta 25–26 (A) or <i>C. denti</i> tetherin insertion 28–29 (B). Twenty-four h after transfection the p24 content of the supernatant from each sample was quantified using a p24 ELISA. and expressed as the percent of p24 of the control without tetherin. Results are the mean ± SD for 5 independent experiments. The indicated p values correspond to the comparison with the «no vpu» sample.</p

Alignment of tetherin amino acid sequences.

<p>A. Alignment of the cytoplasmic tail and transmembrane domains of tetherins from 16 primates. The accession numbers of the sequences used in this figure are: Human NP 004326; <i>Gorilla gorilla</i> ADI58594; <i>P. troglodytes</i> ADI58593; <i>P. paniscus</i> ADI58595; <i>C. nictitans</i> ACX46125; <i>C. mona</i> ACX46126; <i>C. cephus</i> ACX46508; <i>P. nemaeus</i> ADI58604; <i>C. aethiops</i> ADI58600; <i>E. patas</i> ADI58599; <i>P. pygmaeus</i> ADI58596; <i>M. mulatta</i> ADI58602; <i>N. leucogenys</i> ADI58597; <i>H. agilis</i> ADI58598; <i>C. guereza kikuyuensis</i> ADI58603; <i>M. talapoin</i> ADI58601. B. Differences in the TM domain of human, <i>C. denti</i> and <i>C. neglectus</i> tetherins are highlighted in the blue box. Red numbers and letters indicate the amino acids we mutated in this study. In both panels, amino acid identity is indicated by dots and sequence gaps are indicated by dashes.</p

SIV Vpu does not reduce total tetherin levels but leads to depletion of tetherin from the cell surface.

<p>(A) 293T cells were cotransfected with HIV-1ΔVpuΔNef proviral construct (500 ng) and a plasmid encoding HA-tagged tetherins (human (100 ng), <i>C. denti</i> (1000 ng) and <i>C. neglectus</i> (1000 ng) and with or without HIV-1 Vpu plasmid (250 ng) or SIVden Vpu plasmid (250 ng). The effect of the two Vpu constructs on tetherin protein levels was monitored by Western blotting, using an anti-HA antibody. Tetherin migrated as several species in SDS-PAGE analyses, presumably as a result of heterogeneus glycosylation. The depicted gel is representative of three independent experiments. (B) Subcellular localization of tetherin. 293T cells were cotransfected with 1000 ng of DNA encoding the indicated tetherin proteins (HA-tagged), with a proviral construct (1000 ng) and with a HIV-1 Vpu or SIV Vpu plasmid (500 ng). 24 h after transfection the cells were fixed, permeabilized, and stained with rat anti-HA (green) monoclonal antibody (clone 3F10, Roche Applied Science). Nuclei are stained with DAPI (blue). Cells were examined by confocal microscopy. Images are representative of three independent experiments. Scale bars represent 10 µm.</p

Structured Inquiry-Based Learning: Drosophila GAL4 Enhancer Trap Characterization in an Undergraduate Laboratory Course

We have developed and tested two linked but separable structured inquiry exercises using a set of Drosophila melanogaster GAL4 enhancer trap strains for an upper-level undergraduate laboratory methods course at Bucknell University. In the first, students learn to perform inverse PCR to identify the genomic location of the GAL4 insertion, using FlyBase to identify flanking sequences and the primary literature to synthesize current knowledge regarding the nearest gene. In the second, we cross each GAL4 strain to a UAS-CD8-GFP reporter strain, and students perform whole mount CNS dissection, immunohistochemistry, confocal imaging, and analysis of developmental expression patterns. We have found these exercises to be very effective in teaching the uses and limitations of PCR and antibody-based techniques as well as critical reading of the primary literature and scientific writing. Students appreciate the opportunity to apply what they learn by generating novel data of use to the wider research community

Structured Inquiry-Based Learning: <i>Drosophila</i> GAL4 Enhancer Trap Characterization in an Undergraduate Laboratory Course

<div><p>We have developed and tested two linked but separable structured inquiry exercises using a set of <i>Drosophila melanogaster</i> GAL4 enhancer trap strains for an upper-level undergraduate laboratory methods course at Bucknell University. In the first, students learn to perform inverse PCR to identify the genomic location of the GAL4 insertion, using FlyBase to identify flanking sequences and the primary literature to synthesize current knowledge regarding the nearest gene. In the second, we cross each GAL4 strain to a UAS-CD8-GFP reporter strain, and students perform whole mount CNS dissection, immunohistochemistry, confocal imaging, and analysis of developmental expression patterns. We have found these exercises to be very effective in teaching the uses and limitations of PCR and antibody-based techniques as well as critical reading of the primary literature and scientific writing. Students appreciate the opportunity to apply what they learn by generating novel data of use to the wider research community.</p></div