Long-term vitamin A supplementation in a preclinical mouse model for RhoD190N-associated retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Anterior N2 enhancement is not a general electrophysiological index of concealed information.

Event-related potentials (ERPs) have been used with the concealed information test (CIT) to detect concealed recognition of specific stimuli (i.e., "probes"). While most research has focused on the P300 component, which is larger for infrequent probes than for frequent control stimuli (i.e., "irrelevants"), some studies have investigated an earlier ERP component, the anterior N2, with mixed results. Although some studies have reported a larger anterior N2 for probes than irrelevants (N2 enhancement), other studies, including our own, have not found such an effect. The present study aimed to replicate and extend our previous findings using the same CIT paradigm and measurement parameters. Results of Bayesian analyses show strong evidence against the hypothesis of anterior N2 enhancement by probes, replicating our previous work. Bayesian analyses also show strong evidence against the hypothesis of N2 enhancement for the three components revealed by a temporal principal component analysis (PCA) conducted to disentangle potentially overlapping ERP effects. In conclusion, whereas the CIT has shown promise in detecting recognition of specific information, anterior N2 enhancement cannot be used as an electrophysiological measure of concealed information across CIT paradigms.

CRISPR genome surgery in a novel humanized model for autosomal dominant retinitis pigmentosa.

Mutations in rhodopsin (RHO) are the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 20% to 30% of all cases worldwide. However, the high degree of genetic heterogeneity makes development of effective therapies cumbersome. To provide a universal solution to RHO-related adRP, we devised a CRISPR-based, mutation-independent gene ablation and replacement (AR) compound therapy carried by a dual AAV2/8 system. Moreover, we developed a novel hRHOC110R/hRHOWT humanized mouse model to assess the AR treatment in vivo. Results show that this humanized RHO mouse model exhibits progressive rod-cone degeneration that phenocopies hRHOC110R/hRHOWT patients. In vivo transduction of AR AAV8 dual vectors remarkably ablates endogenous RHO expression and overexpresses exogenous WT hRHO. Furthermore, the administration of AR during adulthood significantly hampers photoreceptor degeneration both histologically and functionally for at least 6 months compared with sole gene replacement or surgical trauma control. This study demonstrates the effectiveness of AR treatment of adRP in the human genomic context while revealing the feasibility of its application for other autosomal dominant disorders.

Effect of the Brief Instructional Video Intervention on the Quality of Cardiopulmonary Resuscitation.

Background: Chest compressions are the basis of cardiopulmonary resuscitation (CPR), and high-quality chest compressions can improve survival rate in patients with out-of-hospital cardiac arrest. Although many efforts have been made to improve the quality of CPR in inexperienced adults, the results are still not high, especially during emergencies. The primary purpose of this study is to investigate whether a brief instructional chest compression-only CPR video could improve chest compression quality in inexperienced adults. Methods: One hundred adults with no CPR experience (age: 20.28 ± 2.28 years; women: 50, men: 50) participated in this study. Participants completed body composition and handgrip strength measurements, and performed two CPR quality tests on the Laerdal® Little Anne QCPR Manikin, namely without video-CPR (WV-CPR) and video-CPR (V-CPR). The WV-CPR quality test was performed first. After 2 minutes of continuous chest compression, the participants rested for 10 seconds and repeated 3 cycles (phase 1, phase 2, and phase 3). After resting for more than 72 hours, V-CPR quality test was conducted. During the V-CPR with video intervention, the participants also continued to compress the chest for 2 minutes, and then rested for 10 seconds, repeating 3 cycles. Results: In phase 1, compared with WV-CPR, the V-CPR has a significant increase (p < 0.001) in chest compression fraction (CCF) (56.31 ± 33.22% vs. 41.82 ± 32.30%) and percent of correct compression rate (PCCR) (96.17 ± 8.45% vs. 26.31 ± 37.55%). In addition, the V-CPR has significantly lower (p < 0.001) chest compression rate (CCR) (110.85 ± 2.40 cpm vs. 128.86 ± 24.52 cpm) and rating of perceived exertion (RPE) (11.89 ± 2.25 vs. 12.87 ± 2.25). For phases 2 through 3, V-CPR and WV-CPR achieved significant differences in CCF, CCD, CCR, PCCR, and RPE (p < 0.01). There were significant differences (p < 0.05) in CCF, CCD, chest compression rebound rate, and RPE among the different administration stages of both WV-CPR and V-CPR. Conclusions: The results of this study revealed that a brief instructional chest compression-only CPR video could improve chest compression quality for inexperienced adults by reducing fatigue and CCR, and increasing CCF and PCCR.

Magnetic-Field Universality of the Kondo Effect Revealed by Thermocurrent Spectroscopy.

Probing the universal low-temperature magnetic-field scaling of Kondo-correlated quantum dots via electrical conductance has proved to be experimentally challenging. Here, we show how to probe this in nonlinear thermocurrent spectroscopy applied to a molecular quantum dot in the Kondo regime. Our results demonstrate that the bias-dependent thermocurrent is a sensitive probe of universal Kondo physics, directly measures the splitting of the Kondo resonance in a magnetic field, and opens up possibilities for investigating nanosystems far from thermal and electrical equilibrium.

Controlling the Entropy of a Single-Molecule Junction.

Single molecules are nanoscale thermodynamic systems with few degrees of freedom. Thus, the knowledge of their entropy can reveal the presence of microscopic electron transfer dynamics that are difficult to observe otherwise. Here, we apply thermocurrent spectroscopy to directly measure the entropy of a single free radical molecule in a magnetic field. Our results allow us to uncover the presence of a singlet to triplet transition in one of the redox states of the molecule, not detected by conventional charge transport measurements. This highlights the power of thermoelectric measurements which can be used to determine the difference in configurational entropy between the redox states of a nanoscale system involved in conductance without any prior assumptions about its structure or microscopic dynamics.

Substitution Pattern Controlled Quantum Interference in [2.2]Paracyclophane-Based Single-Molecule Junctions.

Quantum interference (QI) of electron waves passing through a single-molecule junction provides a powerful means to influence its electrical properties. Here, we investigate the correlation between substitution pattern, conductance, and mechanosensitivity in [2.2]paracyclophane (PCP)-based molecular wires in a mechanically controlled break junction experiment. The effect of the meta versus para connectivity in both the central PCP core and the phenyl ring connecting the terminal anchoring group is studied. We find that the meta-phenyl-anchored PCP yields such low conductance levels that molecular features cannot be resolved; in the case of para-phenyl-coupled anchoring, however, large variations in conductance values for modulations of the electrode separation occur for the pseudo-para-coupled PCP core, while this mechanosensitivity is absent for the pseudo-meta-PCP core. The experimental findings are interpreted in terms of QI effects between molecular frontier orbitals by theoretical calculations based on density functional theory and the Landauer formalism.

Hypoxic drive caused type 3 neovascularization in a preclinical model of exudative age-related macular degeneration.

Hypoxia associated with the high metabolic demand of rods has been implicated in the pathology of age-related macular degeneration (AMD), the most common cause of adult blindness in the developed world. The majority of AMD-associated severe vision loss cases are due to exudative AMD, characterized by neovascularization. To further investigate the causes and histopathology of exudative AMD, we conditionally induced hypoxia in a novel preclinical AMD model (Pde6gcreERT2/+;Vhl-/-) by targeting Vhl and used multimodal imaging and immunohistochemistry to track the development of hypoxia-induced neovascularization. In addition to developing a preclinical model that phenocopies exudative AMD, our studies revealed that the photoreceptor hypoxic response initiates and drives type 3 neovascularization, mainly in the outer retina. Activation of the VHL-HIF1a-VEGF-EPO pathway in the adult retina led to long-term neovascularization, retinal hemorrhages and compromised retinal layers. Our novel preclinical model would accelerate the testing of therapies that use metabolomic approaches to ameliorate AMD.

Mouse Models of Achromatopsia in Addressing Temporal "Point of No Return" in Gene-Therapy.

Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form "functional" CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.

Synthesis and Transport Studies of a Cofacial Porphyrin Cyclophane.

Porphyrin cyclophane 1, consisting of two rigidly fixed but still movable cofacial porphyrins and exposing acetate-masked thiols in opposed directions of the macrocycle, is designed, synthesized, and characterized. The functional cyclophane 1, as pioneer of mechanosensitive 3D materials, forms stable single-molecule junctions in a mechanically controlled break-junction setup. Its reliable integration in a single-molecule junction is a fundamental prerequisite to explore the potential of these structures as mechanically triggered functional units and devices.

Mechanisms of neurodegeneration in a preclinical autosomal dominant retinitis pigmentosa knock-in model with a RhoD190N mutation.

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.

Genetic rescue models refute nonautonomous rod cell death in retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease, in which the death of mutant rod photoreceptors leads secondarily to the non-cell autonomous death of cone photoreceptors. Gene therapy is a promising treatment strategy. Unfortunately, current methods of gene delivery treat only a fraction of diseased cells, yielding retinas that are a mosaic of treated and untreated rods, as well as cones. In this study, we created two RP mouse models to test whether dying, untreated rods negatively impact treated, rescued rods. In one model, treated and untreated rods were segregated. In the second model, treated and untreated rods were diffusely intermixed, and their ratio was controlled to achieve low-, medium-, or high-efficiency rescue. Analysis of these mosaic retinas demonstrated that rescued rods (and cones) survive, even when they are greatly outnumbered by dying photoreceptors. On the other hand, the rescued photoreceptors did exhibit long-term defects in their outer segments (OSs), which were less severe when more photoreceptors were treated. In summary, our study suggests that even low-efficiency gene therapy may achieve stable survival of rescued photoreceptors in RP patients, albeit with OS dysgenesis.

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials.

Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 KO mouse model. In humans, CAPN5 loss-of-function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.

The effect of mental countermeasures on neuroimaging-based concealed information tests.

During the last decade and a half, functional magnetic resonance imaging (fMRI) has been used to determine whether it is possible to detect concealed knowledge by examining brain activation patterns, with mixed results. Concealed information tests rely on the logic that a familiar item (probe) elicits a stronger response than unfamiliar, but otherwise comparable items (irrelevants). Previous work has shown that physical countermeasures can artificially modulate neural responses in concealed information tests, decreasing the accuracy of these methods. However, the question remains as to whether purely mental countermeasures, which are much more difficult to detect than physical ones, can also be effective. An fMRI study was conducted to address this question by assessing the effect of attentional countermeasures on the accuracy of the classification between knowledge and no-knowledge cases using both univariate and multivariate analyses. Results replicate previous work and show reliable group activation differences between the probe and the irrelevants in fronto-parietal networks. Critically, classification accuracy was generally reduced by the mental countermeasures, but only significantly so with region of interest analyses (both univariate and multivariate). For whole-brain analyses, classification accuracy was relatively low, but it was not significantly reduced by the countermeasures. These results indicate that mental countermeasure need to be addressed before these paradigms can be used in applied settings and that methods to defeat countermeasures, or at least to detect their use, need to be developed. HIGHLIGHTS: FMRI-based concealed information tests are vulnerable to mental countermeasures Measures based on regions of interest are affected by mental countermeasures Whole-brain analyses may be more robust than region of interest ones Methods to detect mental countermeasure use are needed for forensic applications.

Genetic Rescue Reverses Microglial Activation in Preclinical Models of Retinitis Pigmentosa.

Microglia cells (MGCs) play a key role in scavenging pathogens and phagocytosing cellular debris in the central nervous system and retina. Their activation, however, contributes to the progression of multiple degenerative diseases. Given the potential damage created by MGCs, it is important to better understand their mechanism of activation. Here, we explored the role of MGCs in the context of retinitis pigmentosa (RP) by using four independent preclinical mouse models. For therapeutic modeling, tamoxifen-inducible CreER was introduced to explore changes in MGCs when RP progression halted. The phenotypes of the MGCs were observed using live optical coherence tomography, live autofluorescence, and immunohistochemistry. We found that, regardless of genetic background, MGCs were activated in neurodegenerative conditions and migrated beyond the layers where they are typically found to the inner and outer segments, where degeneration was ongoing. Genetic rescue not only halted degeneration but also deactivated MGCs, regardless of whether the intervention occurred at the early, middle, or late stage of the disease. These findings suggest that halting long-term disease progression may be more successful by downregulating MGC activity while co-administering the therapeutic intervention.

Large Conductance Variations in a Mechanosensitive Single-Molecule Junction.

An appealing feature of molecular electronics is the possibility of inducing changes in the orbital structure through external stimuli. This can provide functionality on the single-molecule level that can be employed for sensing or switching purposes if the associated conductance changes are sizable upon application of the stimuli. Here, we show that the room-temperature conductance of a spring-like molecule can be mechanically controlled up to an order of magnitude by compressing or elongating it. Quantum-chemistry calculations indicate that the large conductance variations are the result of destructive quantum interference effects between the frontier orbitals that can be lifted by applying either compressive or tensile strain to the molecule. When periodically modulating the electrode separation, a conductance modulation at double the driving frequency is observed, providing a direct proof for the presence of quantum interference. Furthermore, oscillations in the conductance occur when the stress built up in the molecule is high enough to allow the anchoring groups to move along the surface in a stick-slip-like fashion. The mechanical control of quantum interference effects results in the largest-gauge factor reported for single-molecule devices up to now, which may open the door for applications in, e.g., a nanoscale mechanosensitive sensing device that is functional at room temperature.

Reprogramming towards anabolism impedes degeneration in a preclinical model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an incurable neurodegenerative condition featuring photoreceptor death that leads to blindness. Currently, there is no approved therapeutic for photoreceptor degenerative conditions like RP and atrophic age-related macular degeneration (AMD). Although there are promising results in human gene therapy, RP is a genetically diverse disorder, such that gene-specific therapies would be practical in a small fraction of patients with RP. Here, we explore a non-gene-specific strategy that entails reprogramming photoreceptors towards anabolism by upregulating the mechanistic target of rapamycin (mTOR) pathway. We conditionally ablated the tuberous sclerosis complex 1 (Tsc1) gene, an mTOR inhibitor, in the rods of the Pde6bH620Q/H620Q preclinical RP mouse model and observed, functionally and morphologically, an improvement in the survival of rods and cones at early and late disease stages. These results elucidate the ability of reprogramming the metabolome to slow photoreceptor degeneration. This strategy may also be applicable to a wider range of neurodegenerative diseases, as enhancement of nutrient uptake is not gene-specific and is implicated in multiple pathologies. Enhancing anabolism promoted neuronal survival and function and could potentially benefit a number of photoreceptor and other degenerative conditions.

Catenin delta-1 (CTNND1) phosphorylation controls the mesenchymal to epithelial transition in astrocytic tumors.

Inactivating mutations of the TSC1/TSC2 complex (TSC1/2) cause tuberous sclerosis (TSC), a hereditary syndrome with neurological symptoms and benign hamartoma tumours in the brain. Since TSC effectors are largely unknown in the human brain, TSC patient cortical tubers were used to uncover hyperphosphorylation unique to TSC primary astrocytes, the cell type affected in the brain. We found abnormal hyperphosphorylation of catenin delta-1 S268, which was reversible by mTOR-specific inhibitors. In contrast, in three metastatic astrocytoma cell lines, S268 was under phosphorylated, suggesting S268 phosphorylation controls metastasis. TSC astrocytes appeared epithelial (i.e. tightly adherent, less motile, and epithelial (E)-cadherin positive), whereas wild-type astrocytes were mesenchymal (i.e. E-cadherin negative and highly motile). Despite their epithelial phenotype, TSC astrocytes outgrew contact inhibition, and monolayers sporadically generated tuberous foci, a phenotype blocked by the mTOR inhibitor, Torin1. Also, mTOR-regulated phosphokinase C epsilon (PKCe) activity induced phosphorylation of catenin delta-1 S268, which in turn mediated cell-cell adhesion in astrocytes. The mTOR-dependent, epithelial phenotype of TSC astrocytes suggests TSC1/2 and mTOR tune the phosphorylation level of catenin delta-1 by controlling PKCe activity, thereby regulating the mesenchymal-epithelial-transition (MET). Thus, some forms of TSC could be treated with PKCe inhibitors, while metastasis of astrocytomas might be blocked by PKCe stimulators.

Is anterior N2 enhancement a reliable electrophysiological index of concealed information?

Concealed information tests (CITs) are used to determine whether an individual possesses information about an item of interest. Event-related potential (ERP) measures in CITs have focused almost exclusively on the P3b component, showing that this component is larger when lying about the item of interest (probe) than telling the truth about control items (irrelevants). Recent studies have begun to examine other ERP components, such as the anterior N2, with mixed results. A seminal CIT study found that visual probes elicit a larger anterior N2 than irrelevants (Gamer and Berti, 2010) and suggested that this component indexes cognitive control processes engaged when lying about probes. However, this study did not control for potential intrinsic differences among the stimuli: the same probe and irrelevants were used for all participants, and there was no control condition composed of uninformed participants. Here, first we show that the N2 effect found in the study by Gamer and Berti (2010) was in large part due to stimulus differences, as the effect observed in a concealed information condition was comparable to that found in two matched control conditions without any concealed information (Experiments 1 and 2). Next, we addressed the issue of the generality of the N2 findings by counterbalancing a new set of stimuli across participants and by using a control condition with uninformed participants (Experiment 3). Results show that the probe did not elicit a larger anterior N2 than the irrelevants under these controlled conditions. These findings suggest that caution should be taken in using the N2 as an index of concealed information in CITs. Furthermore, they are a reminder that results of CIT studies (not only with ERPs) performed without stimulus counterbalancing and suitable control conditions may be confounded by differential intrinsic properties of the stimuli employed.

Validation of genome-wide association study (GWAS)-identified disease risk alleles with patient-specific stem cell lines.

While the past decade has seen great progress in mapping loci for common diseases, studying how these risk alleles lead to pathology remains a challenge. Age-related macular degeneration (AMD) affects 9 million older Americans, and is characterized by the loss of the retinal pigment epithelium (RPE). Although the closely linked genome-wide association studies ARMS2/HTRA1 genes, located at the chromosome 10q26 locus, are strongly associated with the risk of AMD, their downstream targets are unknown. Low population frequencies of risk alleles in tissue banks make it impractical to study their function in cells derived from autopsied tissue. Moreover, autopsy eyes from end-stage AMD patients, where age-related RPE atrophy and fibrosis are already present, cannot be used to determine how abnormal ARMS2/HTRA1 expression can initiate RPE pathology. Instead, induced pluripotent stem (iPS) cell-derived RPE from patients provides us with earlier stage AMD patient-specific cells and allows us to analyze the underlying mechanisms at this critical time point. An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines identified superoxide dismutase 2 (SOD2)-mediated antioxidative defense in the genetic allele's susceptibility of AMD. The AMD-associated risk haplotype (T-in/del-A) impairs the ability of the RPE to defend against aging-related oxidative stress. SOD2 defense is impaired in RPE homozygous for the risk haplotype (T-in/del-A; T-in/del-A), while the effect was less pronounced in RPE homozygous for the protective haplotype (G-Wt-G; G-Wt-G). ARMS2/HTRA1 risk alleles decrease SOD2 defense, making RPE more susceptible to oxidative damage and thereby contributing to AMD pathogenesis.