A library of infectious hepatitis C viruses with engineered mutations in the E2 gene reveals growth-adaptive mutations that modulate interactions with scavenger receptor class B type I

While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties. IMPORTANCE Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability

Enrichment of clinical trials in MCI due to AD using markers of amyloid and neurodegeneration

Objective: To investigate the effect of enriching mild cognitive impairment (MCI) clinical trials using combined markers of amyloid pathology and neurodegeneration. Methods: We evaluate an implementation of the recent National Institute for Aging–Alzheimer's Association (NIA-AA) diagnostic criteria for MCI due to Alzheimer disease (AD) as inclusion criteria in clinical trials and assess the effect of enrichment with amyloid (A+), neurodegeneration (N+), and their combination (A+N+) on the rate of clinical progression, required sample sizes, and estimates of trial time and cost. Results: Enrichment based on an individual marker (A+ or N+) substantially improves all assessed trial characteristics. Combined enrichment (A+N+) further improves these results with a reduction in required sample sizes by 45% to 60%, depending on the endpoint. Conclusions: Operationalizing the NIA-AA diagnostic criteria for clinical trial screening has the potential to substantially improve the statistical power of trials in MCI due to AD by identifying a more rapidly progressing patient population

Memory concerns in the early Alzheimer’s disease prodrome: Regional association with tau deposition

Introduction: Relationship between self- and informant memory concerns and tau aggregation was assessed in adults at risk for Alzheimer's disease (AD). Methods: Regional mean standardized uptake value ratios were extracted from [18F]flortaucipir positron emission tomography (PET) scans of 82 at-risk adults in the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. Associations between self- and informant ECog memory scores and tau aggregation were analyzed on both regional and voxelwise bases. Analyses were completed both on the whole sample and restricted to amyloid-positive individuals only. Results: Memory concerns were associated with tau aggregation. Self-perception was more associated with frontal tau. In contrast, informant scores were more associated with parietal tau. This source-by-region interaction was more prominent in amyloid-positive participants and observed in both regional and voxelwise analyses. Discussion: Quantitative assessment of perceived memory functioning may be useful for screening older adults at risk for Alzheimer's disease. Individuals and their informants may provide complementary information relating to the anatomical distribution of tau

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers

An acetylcholine alpha7 positive allosteric modulator rescues a schizophrenia-associated brain endophenotype in the 15q13.3 microdeletion, encompassing CHRNA7

The 15q13.3 microdeletion copy number variation is strongly associated with schizophrenia and epilepsy. The CHRNA7 gene, encoding nicotinic acetylcholine alpha 7 receptors (nAChA7Rs), is hypothesized to be one of the main genes in this deletion causing the neuropsychiatric phenotype. Here we used a recently developed 15q13.3 microdeletion mouse model to explore whether an established schizophrenia-associated connectivity phenotype is replicated in a murine model, and whether positive modulation of nAChA7 receptor might pharmacologically normalize the connectivity patterns. Resting-state fMRI data were acquired from male mice carrying a hemizygous 15q13.3 microdeletion (N=9) and from wild-type mice (N=9). To study the connectivity profile of 15q13.3 mice and test the effect of nAChA7 positive allosteric modulation, the 15q13.3 mice underwent two imaging sessions, one week apart, receiving a single intraperitoneal injection of either 15 mg/kg Lu AF58801 or saline. The control group comprised wild-type mice treated with saline. We performed seed-based functional connectivity analysis to delineate aberrant connectivity patterns associated with the deletion (15q13.3 mice (saline treatment) versus wild-type mice (saline treatment)) and their modulation by Lu AF58801 (15q13.3 mice (Lu AF58801 treatment) versus 15q13.3 mice (saline treatment)). Compared to wild-type mice, 15q13.3 mice evidenced a predominant hyperconnectivity pattern. The main effect of Lu AF58801 was a normalization of elevated functional connectivity between prefrontal and frontal, hippocampal, striatal, thalamic and auditory regions. The strongest effects were observed in brain regions expressing nAChA7Rs, namely hippocampus, cerebral cortex and thalamus. These effects may underlie the antiepileptic, pro-cognitive and auditory gating deficit-reversal effects of nAChA7R stimulation

Topographic staging of tau positron emission tomography images

Introduction: It has been proposed that the signal distribution on tau positron emission tomography (PET) images could be used to define pathologic stages similar to those seen in neuropathology. Methods: Three topographic staging schemes for tau PET, two sampling the temporal and occipital subregions only and one sampling cortical gray matter across the major brain lobes, were evaluated on flortaucipir F 18 PET images in a test-retest scenario and from Alzheimer's Disease Neuroimaging Initiative 2. Results: All three schemes estimated stages that were significantly associated with amyloid status and when dichotomized to tau positive or negative were 90% to 94% concordant in the populations identified. However, the schemes with fewer regions and simpler decision rules yielded more robust performance in terms of fewer unclassified scans and increased test-retest reproducibility of assigned stage. Discussion: Tau PET staging schemes could be useful tools to concisely index the regional involvement of tau pathology in living subjects. Simpler schemes may be more robust

Tracer-specific PET and SPECT templates for automatic co-registration of functional rat brain images

Objectives: Template based spatial co-registration of PET and SPECT data is an important first step in its semi- automatic processing, facilitating VOI- and voxel-based analysis. Although this procedure is standard in human, using corresponding MRI images, these systems are often not accessible for preclinical research. Alternatively, manual co-registration of images to a MRI template is often performed. However, this is operator dependent and can introduce bias. Therefore, we constructed several tracer-specific PET and SPECT rat brain templates for automatic co-registration, spatially aligned with a widely used MRI-based template in Paxinos stereotactic space [1]. Methods: PET (18F-FDG, 11C-PK11195, and 11C-MeDAS) and SPECT (99mTc-HMPAO) brain scans were acquired from healthy male Sprague-Dawley and Wistar rats. Symmetrical left-right templates were constructed by averaging the scans. Within-modality registration was performed by minimizing the sum of squared difference and template to MRI registration by normalized mutual information maximization algorithm. For validation purposes, PET scans were acquired from a rat model of multiple sclerosis (MS) where focal demyelination was induced by injection of lysolecithin (or control saline) in right corpus callosum and striatum. Parametric SUV images were created for automatic co-registration. The validity of the templates was assessed by estimation of registration accuracy errors, inter-subject variability, right-to-left asymmetry indices, and voxel-based analysis of the MS model [2]. Results: The obtained mean registration errors were 0.097-1.277mm for PET, and 0.059-0.477mm for SPECT. These values are below spatial resolution of the cameras (1.4mm and 0.8mm, respectively) and in agreement with human literature [3]. Results from voxel-based analyses (Figure 1) correspond with those previously reported using VOI-based analysis [4], and correlate with the regions where lesion was induced. Conclusion: The constructed tracer-specific templates allow accurate registration of functional rat brain data, using automatic normalization algorithms available in standard packages (e.g., SPM, FSL), supporting either VOI- or voxel-based analysis. The templates will be made freely available for the research community

A Standardized Method for the Construction of Tracer Specific PET and SPECT Rat Brain Templates:Validation and Implementation of a Toolbox

High-resolution anatomical image data in preclinical brain PET and SPECT studies is often not available, and inter-modality spatial normalization to an MRI brain template is frequently performed. However, this procedure can be challenging for tracers where substantial anatomical structures present limited tracer uptake. Therefore, we constructed and validated strain- and tracer-specific rat brain templates in Paxinos space to allow intra-modal registration. PET [18F]FDG, [11C]flumazenil, [11C]MeDAS, [11C]PK11195 and [11C]raclopride, and SPECT [99mTc]HMPAO brain scans were acquired from healthy male rats. Tracer-specific templates were constructed by averaging the scans, and by spatial normalization to a widely used MRI-based template. The added value of tracer-specific templates was evaluated by quantification of the residual error between original and realigned voxels after random misalignments of the data set. Additionally, the impact of strain differences, disease uptake patterns (focal and diffuse lesion), and the effect of image and template size on the registration errors were explored. Mean registration errors were 0.70 ± 0.32 mm for [18F]FDG (n = 25), 0.23 ± 0.10mm for [11C]flumazenil (n = 13), 0.88 ± 0.20 mm for [11C]MeDAS (n = 15), 0.64 ± 0.28 mm for [11C]PK11195 (n = 19), 0.34 ± 0.15 mm for [11C]raclopride (n = 6), and 0.40 ± 0.13 mm for [99mTc]HMPAO (n = 15). These values were smallest with tracer-specific templates, when compared to the use of [18F]FDG as reference template (p<0.001). Additionally, registration errors were smallest with strain-specific templates (p<0.05), and when images and templates had the same size (p ≤ 0.001). Moreover, highest registration errors were found for the focal lesion group (p<0.005) and the diffuse lesion group (p = n.s.). In the voxel-based analysis, the reported coordinates of the focal lesion model are consistent with the stereotaxic injection procedure. The use of PET/SPECT strain- and tracer-specific templates allows accurate registration of functional rat brain data, independent of disease specific uptake patterns and with registration error below spatial resolution of the cameras. The templates and the SAMIT package will be freely available for the research community [corrected]