Development and preclinical evaluation of a novel AAV vector-based gene therapy to treat retinitis pigmentosa type 45

Loss-of-function mutations in the cyclic nucleotide-gated channel beta 1 subunit (Cngb1) gene are known to cause Retinitis pigmentosa type 45 (RP45), an incurable retinal disorder characterized by primary functional loss and degeneration of rod photoreceptors, followed by a non-cell autonomous cone death, often resulting in legal blindness. Here, a novel recombinant adeno-associated virus vector for gene supplementation therapy of Cngb1-linked RP was developed and tested for its efficacy in two preclinical Cngb1 knockout (KO) animal models of RP45. The full-length human CNGB1 coding sequence was packaged in recombinant AAV5 under the control of a short human rhodopsin promoter, as CNGB1 is expressed natively in rod photoreceptors. Due to the limited cargo capacity of the AAV, an optimized shortened rhodopsin promoter was designed (hRHO194) in order to produce the entire expression cassette in cis and to ensure rod-specific expression of hCNGB1. The resulting viral vector (rAAV5.hCNGB1) was produced by triple-transfection in HEK293 cells as well as in HeLa cells as a producer cell line. For the preclinical validation of the therapy, the efficacy of rAAV5.hCNGB1 was assessed in a Cngb1 KO mouse model as well as in a Cngb1 KO dog model. The animals were treated with rAAV5.CNGB1 delivered via subretinal injection to assess transgene expression as well as biological activity. The effect of the treatment was read out with multiple outcome measures. Proper availability, integrity, and localization of the introduced gene product was assessed by immunohistochemistry. Retinal function was examined via ERG measurements and morphological effects on photoreceptor degeneration were monitored in vivo by OCT imaging. Behavioral tests were implemented to validate visual function. Additionally, critical aspects including studies on long-term efficacy, biologically active dose range, time window for a therapeutic intervention, as well as on potential toxicities were addressed, to provide further insights on the feasibility and safety of the drug administration. The study demonstrated efficient, specific, and long-term hCNGB1 expression in murine and canine rod photoreceptors driven by the short hRHO194 promoter. By treatment with rAAV5.hCNGB1, a substantial preservation of rod and cone photoreceptors was achieved in both Cngb1 KO mice and dogs. Furthermore, the efficacy of rAAV5.hCNGB1 was dose-dependent manifesting itself as an increasing expression level with ascending dose, resulting in enhanced biological efficacy in both animal models. In addition, a toxic side effect was identified for the vector produced in HeLa cells emerging in a dose-dependent manner, which was not observed for the vector produced in HEK293 cells. It was further shown, that the viral vector-introduced human CNGB1 subunit was capable of forming functional chimeric rod-specific CNG channels together with the endogenous murine or canine CNGA1 subunits leading to a recovery of rod photoreceptor function as well as markedly improved vision-guided behavior in both animal models. Furthermore, retinal stress in the Cngb1 KO mouse, manifesting as Müller cell gliosis, was reduced by rAAV5.hCNGB1, even beyond the treated region of the retina. Finally, it was shown that a degeneration-induced inflammation was diminished by the treatment in the Cngb1 KO dog model, shown as reduced microglia activation, while in one treated dog an increased immune reaction was observed, manifesting as increase activation of microglia within the retina. These results demonstrated the efficacy of the rAAV5.hCNGB1 gene supplementation therapy in small and large animals and thus showed the transferability of this approach to human application. Thereby, the work covered a major part of the preclinical phase and proved that rAAV5.hCNGB1 is a suitable candidate for a clinical application. The results of this study provided novel insights into the transferability of a gene therapy from mouse to man and will serve as a basis for a design of preclinical toxicology studies and early-phase clinical application of rAAV5.hCNGB1

Sublimity of the New Mother in Gothic Film: The Babadook and Goodnight Mommy

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Ta(l)king Women out of the Picture: How Extreme Republican Discourse Framed Women Outside Women's Issues: 2010-2012.

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Characterization of perillyl alcohol as a cell migration-inhibiting compound

Inhibiting cancer cell migration could greatly improve the prognosis of cancer patients. Prior to commercialization of the Plopper laboratory designed a 96-well plate, we optimized our in vitro migration and cell viability assays to meet the standards set by the National Cancer Institute for use in a large-scale drug screen for anti-migration compounds. Using these assays, we measured the cytotoxicity and migration-inhibiting effect of dimethyl sulfuoxide, a drug solvent, in three cancer and one non-cancer cell lines. Next, we investigated plant-derived compounds called monoterpenes, known chemopreventive/chemotherapeutic agents, for non-cytotoxic, migration-inhibiting properties in MCF-10A normal and MDA-MB 435 cancerous breast cells. Perillyl alcohol at 0.5 mM inhibited normal breast cell migration while it failed to affect cancer cell migration. Finally, we examined the loss of prenylation of RhoA, a small GTPase protein involved in the formation of focal adhesions and stress fibers, and corresponding morphological changes following exposure to perillyl alcohol

Corporate Psychological Responsibility: CPR Is Needed

Corporate Psychological Responsibility (CPR) is the responsibility of an organization to focus on psychological factors that impact employees to create a competitive advantage for all stakeholders (Sweet, 2020). This manuscript seeks to introduce a framework and model for CPR. A literature review of components that impact employees psychologically in motivational theory and in positive organizational behavior was conducted to identify elements that should be managed to become a psychologically responsible organization. Five pillars of CPR are introduced: health and safety, psychological capital, positive relationships, valued rewards, and fair organizational policies/practices. Organizations that take CPR seriously can create a competitive advantage

Successfully releasing jackdaws, Corvus monedula: spatial dispersion and the fusion of social groups

In der vorliegenden Studie untersuchten wir eine Auswilderung von in einer Voliere gehaltenen Dohlen (Corvus monedula), die in einer erfolgreichen Etablierung einer Wildkolonie mündete. Die Auswilderung erfolgte in zwei Stufen in den Jahren 2007 und 2009. Im ersten Projektjahr lag der Fokus unserer Untersuchung auf der räumlichen Ausbreitung der ausgewilderten Individuen, die zunächst nur allmählich, dann jedoch fast sprunghaft erfolgte. Mit der räumlichen Ausbreitung der Individuen auf andere als die in unmittelbarer Nähe zur Voliere gelegenen Bereiche war auch eine veränderte funktionelle Nutzung des Raumes verbunden, in der vor allem das Zentrum der Aktivitäten der Vögel verlagert wurde. Das zweite Projektjahr war der Untersuchung gewidmet, wie sich eine neuerlich ausgewilderte Dohlengruppe mit der bereits bestehenden Wildkolonie zu einer sozialen Gruppe zusammenschließen würde. In diesem Prozess benutzten die beiden Kolonien unterschiedliche Strategien der Annäherung. Während Individuen der Wildkolonie mit den hinzugekommenen Individuen häufiger in aggressiver Weise Kontakt aufnahmen, zeigten umgekehrt die neu ausgewilderten Individuen gegenüber Individuen der Wildkolonie vor allem soziopositives Verhalten. Obwohl die beiden Kolonien nach nur rund zwei Wochen räumlich als eine Kolonie betrachtet werden durften, zeigen unsere Ergebnisse, dass der soziale Zusammenschluss erst nach etwa zwei Monaten erreicht war. Unsere Studie zeigt auch Kontextfaktoren bzw. Faktoren der Biologie von Dohlen auf, die für eine erfolgreiche Auswilderung von besonderer Bedeutung sind: das visuelle Kennenlernen der neuen Umgebung, die Dynamik von Dohlenkolonien als offene Gruppen, in denen Abwanderungen und Neuzugänge ein häufiges Phänomen sind und für Auswilderungszwecke genutzt werden können und die Wichtigkeit einer etablierten Dominanzhierarchie in der dominante Individuen Führungsrollen übernehmen und Artgenossen diesen folgen können.The focus of the present study was a release project on jackdaws (Corvus monedula) which was carried out in two steps in 2007 and 2009. In 2007 we focused on the spatial dispersion of individuals which started gradually but then turned into a stepwise increase. A change in the functional use of space was associated with the birds’ spatial dispersion into areas other than those immediately surrounding the aviary in which especially the centre of the birds’ activities was relocated. 2009 focused on the analysis of the process how a newly released group of jackdaws and the already existing wild colony of birds would unite into a single social group. In this process both colonies used different strategies: in contrast to the individuals of the wild colony that mainly approached the newly released individuals in an aggressive manner, the latter initiated more sociopositive interactions towards the former. Although after two weeks the two colonies could be considered as one when referring to their spatial cohesiveness, our results show that their social cohesiveness was achieved only after about two months. Furthermore, our study indicates which factors of the context and the biology of jackdaws, respectively, may be especially important for a successful release in these birds: visual acquaintance with the new environment, social dynamics of jackdaw colonies that represent free entry groups in which emigration and immigration are frequent phenomena and can be used for releasing purposes, and the importance of an established dominance hierarchy due to which dominant individuals can take the lead while subdominant conspecifics may follow them

Is volumetric pricing for drinking water an effective revenue strategy in rural Mali?

Africa lags behind the world on operational and financial progress to maintain safe drinking water services. In rural Mali, we explore the implications of monthly flat fee contributions and volumetric (pay-as-you-fetch) payments for water use and revenue generation. By assessing 4413 months of data across 177 handpumps, we find that once payment modalities switch from volumetric payments to monthly flat fees, a waterpoint registers a more than three-times higher monthly revenue. While flat fees cover a higher share of the operational costs of providing reliable water services, a subsidy gap persists. Flat fees appear to stimulate daily water use which more than doubles compared to volumetric payments. We estimate that a 1 °C increase in average monthly temperature is associated with 180 more litres of water used every day per handpump, emphasising the importance of climate-resilient water supplies. Based on these insights, we discuss the role of professional service delivery models to support reliable drinking water services for rural communities

Jody Cardinal, Deirdre E. Egan-Ryan, and Julia Lisella (eds.), Modernist Women Writers and American Social Engagement

The “new modernist studies”, coined by Mao and Walkowitz in their titular article in the May 2008 issue of PMLA, has been a boon for scholars who study women writers. As modernism has “softened its definitional gaze and relinquished its gatekeeping function” (451), as stated by Paul Saint-Amour in 2018, its scholarship has blossomed in variation and kind. An interest in gender, i.e. women writers, a long-policed, tacit demarcation in literary modernism, has increased and continues to prosper ..