Non‐invasive measurement of retinal permeability in a diabetic rat model

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this recordObjective The gold standard for measuring blood‐retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non‐specific tissue binding and toxicity. This study describes a non‐toxic, high throughput and cost effective alternative technique that minimizes animal usage. Methods Sodium fluorescein fundus angiography was performed in non‐ and diabetic Brown Norway rats on days 0, 7, 14, 21 and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post study eyes were fixed, dissected and stained (isolectin B4) to measure required parameters for permeability quantification including: Total vessel length per retinal volume, radius and thickness. Results In the non‐diabetic cohort retinal permeability remained constant over the 28‐day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from day 14 to 28 (p<0.01, p<0.001, p<0.0001). Conclusions This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non‐invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non‐toxic, rapid, sensitive and cost‐effective alternative to the Evans blue assay.Medical Research Council (MRC)National Eye Research CentreMasonic Charitable Foundatio

Robotic milking technologies and renegotiating situated ethical relationships on UK dairy farms

Robotic or automatic milking systems (AMS) are novel technologies that take over the labor of dairy farming and reduce the need for human-animal interactions. Because robotic milking involves the replacement of 'conventional' twice-a-day milking managed by people with a system that supposedly allows cows the freedom to be milked automatically whenever they choose, some claim robotic milking has health and welfare benefits for cows, increases productivity, and has lifestyle advantages for dairy farmers. This paper examines how established ethical relations on dairy farms are unsettled by the intervention of a radically different technology such as AMS. The renegotiation of ethical relationships is thus an important dimension of how the actors involved are re-assembled around a new technology. The paper draws on in-depth research on UK dairy farms comparing those using conventional milking technologies with those using AMS. We explore the situated ethical relations that are negotiated in practice, focusing on the contingent and complex nature of human-animal-technology interactions. We show that ethical relations are situated and emergent, and that as the identities, roles, and subjectivities of humans and animals are unsettled through the intervention of a new technology, the ethical relations also shift. © 2013 Springer Science+Business Media Dordrecht

Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution

<p>Abstract</p> <p>Background</p> <p>Vaccination of macaques with live attenuated simian immunodeficiency virus (SIV) provides significant protection against the wild-type virus. The use of a live attenuated human immunodeficiency virus (HIV) as AIDS vaccine in humans is however considered unsafe because of the risk that the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. We earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (dox). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient dox administration. Since the effectiveness and safety of such a conditionally live virus vaccine should be tested in macaques, we constructed a similar dox-dependent SIV variant. The Tat-TAR transcription control mechanism in this virus was inactivated through mutation and functionally replaced by the dox-inducible Tet-On regulatory system. This SIV-rtTA variant replicated in a dox-dependent manner in T cell lines, but not as efficiently as the parental SIVmac239 strain. Since macaque studies will likely require an efficiently replicating variant, we set out to optimize SIV-rtTA through in vitro viral evolution.</p> <p>Results</p> <p>Upon long-term culturing of SIV-rtTA, additional nucleotide substitutions were observed in TAR that affect the structure of this RNA element but that do not restore Tat binding. We demonstrate that the bulge and loop mutations that we had introduced in the TAR element of SIV-rtTA to inactivate the Tat-TAR mechanism, shifted the equilibrium between two alternative conformations of TAR. The additional TAR mutations observed in the evolved variants partially or completely restored this equilibrium, which suggests that the balance between the two TAR conformations is important for efficient viral replication. Moreover, SIV-rtTA acquired mutations in the U3 promoter region. We demonstrate that these TAR and U3 changes improve viral replication in T-cell lines and macaque peripheral blood mononuclear cells (PBMC) but do not affect dox-control.</p> <p>Conclusion</p> <p>The dox-dependent SIV-rtTA variant was optimized by viral evolution, yielding variants that can be used to test the conditionally live virus vaccine approach and as a tool in SIV biology studies and vaccine research.</p

Absence of Evidence not equal Evidence of Absence: Statistical Analysis of Inclusions in Multiferroic Thin Films

Assertions that a new material may offer particularly advantageous properties should always be subjected to careful critical evaluation, especially when those properties can be affected by the presence of inclusions at trace level. This is particularly important for claims relating to new multiferroic compounds, which can easily be confounded by unobserved second phase magnetic inclusions. We demonstrate an original methodology for the detection, localization and quantification of second phase inclusions in thin Aurivillius type films. Additionally, we develop a dedicated statistical model and demonstrate its application to the analysis of Bi6Ti2.8Fe1.52Mn0.68O18 (B6TFMO) thin films, that makes it possible to put a high, defined confidence level (e.g. 99.5%) to the statement of ‘new single phase multiferroic materials’. While our methodology has been specifically developed for magnetic inclusions, it can easily be adapted to any other material system that can be affected by low level inclusions