AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.     

Does the Establishment of Sustainable Use Reserves Affect Fire Management in the Humid Tropics?

Tropical forests are experiencing a growing fire problem driven by climatic change, agricultural expansion and forest degradation. Protected areas are an important feature of forest protection strategies, and sustainable use reserves (SURs) may be reducing fire prevalence since they promote sustainable livelihoods and resource management. However, the use of fire in swidden agriculture, and other forms of land management, may be undermining the effectiveness of SURs in meeting their conservation and sustainable development goals. We analyse MODIS derived hot pixels, TRMM rainfall data, Terra-Class land cover data, socio-ecological data from the Brazilian agro-census and the spatial extent of rivers and roads to evaluate whether the designation of SURs reduces fire occurrence in the Brazilian Amazon. Specifically, we ask (1) a. Is SUR location (i.e., de facto) or (1) b. designation (i.e. de jure) the driving factor affecting performance in terms of the spatial density of fires?, and (2), Does SUR creation affect fire management (i.e., the timing of fires in relation to previous rainfall)? We demonstrate that pre-protection baselines are crucial for understanding reserve performance. We show that reserve creation had no discernible impact on fire density, and that fires were less prevalent in SURs due to their characteristics of sparser human settlement and remoteness, rather than their status de jure. In addition, the timing of fires in relation to rainfall, indicative of local fire management and adherence to environmental law, did not improve following SUR creation. These results challenge the notion that SURs promote environmentally sensitive fire-management, and suggest that SURs in Amazonia will require special attention if they are to curtail future accidental wildfires, particularly as plans to expand the road infrastructure throughout the region are realised. Greater investment to support improved fire management by farmers living in reserves, in addition to other fire users, will be necessary to help ameliorate these threats.     

Kinetic Characterization of 100 Glycoside Hydrolase Mutants Enables the Discovery of Structural Features Correlated with Kinetic Constants

The use of computational modeling algorithms to guide the design of novel enzyme catalysts is a rapidly growing field. Force-field based methods have now been used to engineer both enzyme specificity and activity. However, the proportion of designed mutants with the intended function is often less than ten percent. One potential reason for this is that current force-field based approaches are trained on indirect measures of function rather than direct correlation to experimentally-determined functional effects of mutations. We hypothesize that this is partially due to the lack of data sets for which a large panel of enzyme variants has been produced, purified, and kinetically characterized. Here we report the k_cat and K_M values of 100 purified mutants of a glycoside hydrolase enzyme. We demonstrate the utility of this data set by using machine learning to train a new algorithm that enables prediction of each kinetic parameter based on readily-modeled structural features. The generated dataset and analyses carried out in this study not only provide insight into how this enzyme functions, they also provide a clear path forward for the improvement of computational enzyme redesign algorithms.     

Bioecological Drivers of Rabies Virus Circulation in a Neotropical Bat Community

Introduction

In addition to the commonly accepted importance of the vampire bat in the maintenance and transmission of the rabies virus (RABV) in South America, RABV infection of other species is widely evidenced, challenging their role in the viral cycle.

Methodology / Principles findings

To identify the bioecological drivers of RABV circulation in neotropical bat communities, we conducted a molecular and serological survey on almost 1,000 bats from 30 species, and a 4-year longitudinal survey in two colonies of vampire bats in French Guiana. RABV was molecularly detected in a common vampire and in a frugivorous bat. The sequences corresponded to haematophagous bat-related strains and were close to viruses circulating in the Brazilian Amazon region. Species’ seroprevalence ranged from 0 to 20%, and the risk of seropositivity was higher in bats with a haematophagous diet, living in monospecific colonies and in dense forests. The longitudinal survey showed substantial temporal fluctuations, with individual waves of seroconversions and waning immunity. The high prevalences observed in bat communities, in most habitats and in species that do not share the same microhabitats and bioecological patterns, the temporal variations, and a rather short period of detectable antibodies as observed in recaptured vampires suggest (i) frequent exposure of animals, (ii) an ability of the infected host to control and eliminate the virus, (iii) more relaxed modes of exposure between bats than the commonly assumed infection via direct contact with saliva of infected animals, all of which should be further investigated.

Conclusions / significance

We hypothesize that RABV circulation in French Guiana is mainly maintained in the pristine forest habitats that may provide sufficient food resources to allow vampire bats, the main prevalent species, to survive and RABV to be propagated. However, on the forest edge and in disturbed areas, human activities may induce more insidious effects such as defaunation. One of the ecological consequences is the disappearance of resources for tertiary or secondary consumers. Populations of vampires may then shift to alternative resources such as cattle, domestic animals and humans. Therefore, a good forest status, allowing both a dilution effect in highly rich bat communities and the maintenance of large populations of medium-sized and large mammals used as prey by vampires, should prevent their migration to anthropized areas.     

Impact of New Diagnostic Approaches for Invasive Candidiasis on Antifungal Stewardship

Background

Invasive candidiasis (IC) including candidemia and deep-seated candidiasis is associated with up to 50 % overall mortality and up to $80,000 in attributable cost (2015 US$). Rapid diagnostic tests (RDTs) for Candida have been developed. However, whether RDTs along with real-time decision support translate to better attainment of stewardship goals—improve clinical outcomes, minimize unintended consequences of antifungal use, and reduce healthcare costs—is unknown. The purpose of this systematic review was to provide an up-to-date review of recently published studies that have assessed how RDTs for IC impact attainment of these goals.

Methods

Three electronic bibliographic databases were searched using a pre-defined search strategy evaluating the impact of RDTs for IC on attainment of antifungal stewardship goals. Quality assessments were performed by two reviewers using established study methodology metrics.

Results and Conclusions

Eight studies were identified of which five had sufficient information to be included in the review. Despite the limitations of the various studies and the different methodologies employed, the studies all produced similar conclusions. Compared to conventional methods and baseline stewardship activities, the integration of RDTs for IC and real-time decision support, mainly through antifungal stewardship, was associated with decreased mortality, more optimal use of antifungals, and reduced healthcare costs. However, larger clinical studies are needed to confirm these trends.

     

Environmental DNA from Residual Saliva for Efficient Noninvasive Genetic Monitoring of Brown Bears (Ursus arctos)

Noninvasive genetic sampling is an important tool in wildlife ecology and management, typically relying on hair snaring or scat sampling techniques, but hair snaring is labor and cost intensive, and scats yield relatively low quality DNA. New approaches utilizing environmental DNA (eDNA) may provide supplementary, cost-effective tools for noninvasive genetic sampling. We tested whether eDNA from residual saliva on partially-consumed Pacific salmon (Oncorhynchus spp.) carcasses might yield suitable DNA quality for noninvasive monitoring of brown bears (Ursus arctos). We compared the efficiency of monitoring brown bear populations using both fecal DNA and salivary eDNA collected from partially-consumed salmon carcasses in Southeast Alaska. We swabbed a range of tissue types from 156 partially-consumed salmon carcasses from a midseason run of lakeshore-spawning sockeye (O. nerka) and a late season run of stream-spawning chum (O. keta) salmon in 2014. We also swabbed a total of 272 scats from the same locations. Saliva swabs collected from the braincases of salmon had the best amplification rate, followed by swabs taken from individual bite holes. Saliva collected from salmon carcasses identified unique individuals more quickly and required much less labor to locate than scat samples. Salmon carcass swabbing is a promising method to aid in efficient and affordable monitoring of bear populations, and suggests that the swabbing of food remains or consumed baits from other animals may be an additional cost-effective and valuable tool in the study of the ecology and population biology of many elusive and/or wide-ranging species.     

Genome Wide Association Study Identifies New Loci Associated with Undesired Coat Color Phenotypes in Saanen Goats

This paper reports a quantitative genetics and genomic analysis of undesirable coat color patterns in goats. Two undesirable coat colors have routinely been recorded for the past 15 years in French Saanen goats. One fifth of Saanen females have been phenotyped “pink” (8.0%) or “pink neck” (11.5%) and consequently have not been included in the breeding program as elite animals. Heritability of the binary “pink” and “pink neck” phenotype, estimated from 103,443 females was 0.26 for “pink” and 0.21 for “pink neck”. Genome wide association studies (using haplotypes or single SNPs) were implemented using a daughter design of 810 Saanen goats sired by 9 Artificial Insemination bucks genotyped with the goatSNP50 chip. A highly significant signal (-log10pvalue = 10.2) was associated with the “pink neck” phenotype on chromosome 11, suggesting the presence of a major gene. Highly significant signals for the “pink” phenotype were found on chromosomes 5 and 13 (-log10p values of 7.2 and, 7.7 respectively). The most significant SNP on chromosome 13 was in the ASIP gene region, well known for its association with coat color phenotypes. Nine significant signals were also found for both traits. The highest signal for each trait was detected by both single SNP and haplotype approaches, whereas the smaller signals were not consistently detected by the two methods. Altogether these results demonstrated a strong genetic control of the “pink” and “pink neck” phenotypes in French Saanen goats suggesting that SNP information could be used to identify and remove undesired colored animals from the breeding program.     

Using simulations to evaluate Mantel‐based methods for assessing landscape resistance to gene flow

Mantel‐based tests have been the primary analytical methods for understanding how landscape features influence observed spatial genetic structure. Simulation studies examining Mantel‐based approaches have highlighted major challenges associated with the use of such tests and fueled debate on when the Mantel test is appropriate for landscape genetics studies. We aim to provide some clarity in this debate using spatially explicit, individual‐based, genetic simulations to examine the effects of the following on the performance of Mantel‐based methods: (1) landscape configuration, (2) spatial genetic nonequilibrium, (3) nonlinear relationships between genetic and cost distances, and (4) correlation among cost distances derived from competing resistance models. Under most conditions, Mantel‐based methods performed poorly. Causal modeling identified the true model only 22% of the time. Using relative support and simple Mantel r values boosted performance to approximately 50%. Across all methods, performance increased when landscapes were more fragmented, spatial genetic equilibrium was reached, and the relationship between cost distance and genetic distance was linearized. Performance depended on cost distance correlations among resistance models rather than cell‐wise resistance correlations. Given these results, we suggest that the use of Mantel tests with linearized relationships is appropriate for discriminating among resistance models that have cost distance correlations <0.85 with each other for causal modeling, or <0.95 for relative support or simple Mantel r. Because most alternative parameterizations of resistance for the same landscape variable will result in highly correlated cost distances, the use of Mantel test‐based methods to fine‐tune resistance values will often not be effective.     

Actin exposure upon tissue injury is a targetable wound site-specific protein marker

BackgroundIdentification of wound-specific markers would represent an important step toward damaged tissue detection and targeted delivery of biologically important materials to injured sites. Such delivery could minimize the amount of therapeutic materials that must be administered and limit potential collateral damage on nearby normal tissues. Yet, biological markers that are specific for injured tissue sites remain elusive.MethodsIn this study, we have developed an immunohistological approach for identification of protein epitopes specifically exposed in wounded tissue sites.ResultsUsing ex-vivo tissue samples in combination with fluorescently-labeled antibodies we show that actin, an intracellular cytoskeletal protein, is specifically exposed upon injury. The targetability of actin in injured sites has been demonstrated in vivo through the specific delivery of anti-actin conjugated particles to the wounded tissue in a lethal rat model of grade IV liver injury.ConclusionsThese results illustrate that identification of injury-specific protein markers and their targetability for specific delivery is feasible.General significanceIdentification of wound-specific targets has important medical applications as it could enable specific delivery of various products, such as expression vectors, therapeutic drugs, hemostatic materials, tissue healing, or scar prevention agents, to internal sites of penetrating or surgical wounds regardless of origin, geometry or location.