Sequencing by avidity enables high accuracy with low reagent consumption.

We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer-nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.

Utilization of the retinal organoid model to evaluate the feasibility of genetic strategies to ameliorate retinal disease(s).

Organoid models have quickly become a popular research tool to evaluate novel therapeutics on 3-D recapitulated tissue. This has enabled researchers to use physiologically relevant human tissue in vitro to augment the standard use of immortalized cells and animal models. Organoids can also provide a model when an engineered animal cannot recreate a specific disease phenotype. In particular, the retinal research field has taken advantage of this burgeoning technology to provide insight into inherited retinal disease(s) mechanisms and therapeutic intervention to ameliorate their effects. In this review we will discuss the use of both wild-type and patient-specific retinal organoids to further gene therapy research that could potentially prevent retinal disease(s) progression. Furthermore, we will discuss the pitfalls of current retinal organoid technology and present potential solutions that could overcome these hurdles in the near future.

Human subthalamic nucleus neurons differentially encode speech and limb movement.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN), which consistently improves limb motor functions, shows mixed effects on speech functions in Parkinson's disease (PD). One possible explanation for this discrepancy is that STN neurons may differentially encode speech and limb movement. However, this hypothesis has not yet been tested. We examined how STN is modulated by limb movement and speech by recording 69 single- and multi-unit neuronal clusters in 12 intraoperative PD patients. Our findings indicated: (1) diverse patterns of modulation in neuronal firing rates in STN for speech and limb movement; (2) a higher number of STN neurons were modulated by speech vs. limb movement; (3) an overall increase in neuronal firing rates for speech vs. limb movement; and (4) participants with longer disease duration had higher firing rates. These data provide new insights into the role of STN neurons in speech and limb movement.

Nicotinamide Promotes Formation of Retinal Organoids From Human Pluripotent Stem Cells via Enhanced Neural Cell Fate Commitment.

Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide (NAM) treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggests that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines.

Aberrant Neural Response to Social Exclusion Without Significantly Greater Distress in Youth With Bipolar Disorder: Preliminary Findings.

Background: Little is known about the effects of social exclusion on youth with bipolar disorder (BD). Understanding these effects and the functional neural correlates of social exclusion in youth with BD may establish differences from healthy youth and help identify areas of intervention. Methods: We investigated brain function in 19 youth with BD and 14 age and gender matched healthy control (HC) participants while performing Cyberball, an fMRI social exclusion task. Whole brain activation, region-of-interest, and functional connectivity were compared between groups and examined with behavioral measures. Results: Compared with the HC group, youth with BD exhibited greater activation in the left fusiform gyrus (FFG) during social exclusion. Functional connectivity between the left FFG and the posterior cingulate/precuneus was significantly greater in the HC compared with the BD group. For the HC group only, age and subjective distress during Cyberball significantly predicted mean FFG activation. No significant differences in distress during social exclusion were found between groups. Conclusion: Although preliminary due to small sample size, these data suggest that youth with BD process social exclusion in a manner that focuses on basic visual information while healthy youth make use of past experiences to interpret current social encounters. This difference may account for the social cognitive issues experienced by youth with BD, which can lead to more severe anxiety and mood symptoms.

Primary cilia biogenesis and associated retinal ciliopathies.

The primary cilium is a ubiquitous microtubule-based organelle that senses external environment and modulates diverse signaling pathways in different cell types and tissues. The cilium originates from the mother centriole through a complex set of cellular events requiring hundreds of distinct components. Aberrant ciliogenesis or ciliary transport leads to a broad spectrum of clinical entities with overlapping yet highly variable phenotypes, collectively called ciliopathies, which include sensory defects and syndromic disorders with multi-organ pathologies. For efficient light detection, photoreceptors in the retina elaborate a modified cilium known as the outer segment, which is packed with membranous discs enriched for components of the phototransduction machinery. Retinopathy phenotype involves dysfunction and/or degeneration of the light sensing photoreceptors and is highly penetrant in ciliopathies. This review will discuss primary cilia biogenesis and ciliopathies, with a focus on the retina, and the role of CP110-CEP290-CC2D2A network. We will also explore how recent technologies can advance our understanding of cilia biology and discuss new paradigms for developing potential therapies of retinal ciliopathies.

HIPRO: A High-Efficiency, Hypoxia-Induced Protocol for Generation of Photoreceptors in Retinal Organoids from Mouse Pluripotent Stem Cells.

Mouse pluripotent stem cells can be efficiently differentiated into retinal organoids with polarized, laminated neural retina harboring all retinal cell types by the Hypoxia-Induced Generation of Photoreceptor in Retinal Organoids (HIPRO) protocol. In our recent publication, we modified the HIPRO protocol on the basis of comparative transcriptome analyses to facilitate photoreceptor biogenesis and maturation. Here, we provide a detailed protocol for efficient generation of retinal organoids from mouse pluripotent stem cells. For complete details on the use and execution of this protocol, please refer to (Chen et al., 2016, DiStefano et al., 2018, Brooks et al., 2019).

Accelerated Development of Rod Photoreceptors in Retinal Organoids Derived from Human Pluripotent Stem Cells by Supplementation with 9-cis Retinal.

Human pluripotent stem cells (PSCs) can be differentiated into retinal organoids with proper neural layer organization, yet the protocols are technically challenging and time consuming. We have modified a widely used differentiation protocol by switching all-trans retinoic acid with 9-cis retinal to accelerate photoreceptor differentiation and improve morphogenesis. In this report, we provide a detailed and improved protocol to generate retinal organoids from human pluripotent stem cells. For complete details on the use and execution of this protocol, please refer to Kaya et al. (2019).

Manufacturing of Dexamethasone-Poly(d,l-Lactide-co-Glycolide) Implants Using Hot-Melt Extrusion: Within- and Between-Batch Product Performance Comparisons.

Purpose: Reliable drug therapy with injectable intravitreal implants requires implants of consistent quality. The purpose of this study was to prepare dexamethasone-poly(d,l-lactide-co-glycolide) (PLGA) biodegradable implants and assess implant quality within and between batches for different polymer compositions. Methods: Implants containing 20% w/w dexamethasone with 3 theoretical rates of release (fast, intermediate, and slow) were manufactured with decreasing proportion of acid-terminated PLGA (50:50) and increasing proportion of ester-terminated PLGA (50:50) in a batch process using hot-melt extrusion. The implants were manufactured without and with in-process modification of extrusion/conveyor speed in the late phase of each batch. Implant samples collected at early, middle, and late phases of each batch were analyzed for diameter, drug loading, mechanical properties (strength and toughness), and drug release. Results: With a fixed process, unlike a modified process with an increase in extrusion speed and reduction of conveyor speed in the late phase, all implant formulations tended to decrease in diameter and mechanical properties in the late phase. Drug release profiles for the intermediate and slow release compositions were similar with or without process modification, unlike the fast release composition. Addition of ester-terminated PLGA resulted in a slower drug release. When all formulations are grouped together, the implant diameter exhibited a moderate correlation with mechanical properties, but no correlation was observed with drug release. Conclusions: Within a hot-melt extrusion batch process, the dexamethasone-PLGA implant diameter and hence toughness and strength tend to decline in the latter phase. In-process adjustment of extrusion and conveyor speeds can improve batch consistency and, potentially, implant integrity or performance during or after injection. Process changes did not affect drug release for 2 of the 3 implant compositions.

A simple and efficient method for generating human retinal organoids.

Purpose: Retinal organoids (ROs) derived from human pluripotent stem cells largely recapitulate key features of in vivo retinal development, thus permitting the study of retinogenesis, disease modeling, and therapeutic development. However, the complexities of current protocols limit the use of this in vitro system in applications requiring large-scale production of organoids. Currently, widely used methods require the isolation of presumed optic vesicle-like structures from adherent cultures by dissection, a labor-intensive and time-consuming step that involves extensive practice and training. Method: We report a simple and efficient method for generating ROs by scraping the entire adherent culture and growing the resulting cell aggregates in a free-floating condition. Results: Within 1 to 7 days following the procedure, emerging morphologically well-defined optic vesicles can be identified and harvested with ease. The transition from two-dimensional (2D) to 3D culture condition favored the formation of ROs from areas devoid of typical optic vesicle-like structures, thus increasing the RO yield. Moreover, ROs generated by this approach were more often associated with the pigment epithelium. Conclusions: This improved, robust, and efficient protocol should facilitate large-scale differentiation of pluripotent stem cells into retinal organoids in support of human disease modeling and therapy development.

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal.

PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.

Improved Retinal Organoid Differentiation by Modulating Signaling Pathways Revealed by Comparative Transcriptome Analyses with Development In Vivo.

Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.

Effect of deep brain stimulation on vocal motor control mechanisms in Parkinson's disease.

INTRODUCTION: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective treatment for limb motor symptoms in Parkinson's disease (PD); however, its effect on vocal motor function has yielded conflicted and highly variable results. The present study investigated the effects of STN-DBS on the mechanisms of vocal production and motor control. METHODS: A total of 10 PD subjects with bilateral STN-DBS implantation were tested with DBS ON and OFF while they performed steady vowel vocalizations and received randomized upward or downward pitch-shift stimuli (±100 cents) in their voice auditory feedback. RESULTS: Data showed that the magnitude of vocal compensation responses to pitch-shift stimuli was significantly attenuated during DBS ON vs. OFF (p = 0.012). This effect was direction-specific and was only observed when subjects raised their voice fundamental frequency (F0) in the opposite direction to downward stimuli (p = 0.019). In addition, we found that voice F0 perturbation (i.e. jitter) was significantly reduced during DBS ON vs. OFF (p = 0.022), and this DBS-induced modulation was positively correlated with the attenuation of vocal compensation responses to downward pitch-shift stimuli (r = +0.57, p = 0.028). CONCLUSIONS: These findings provide the first data supporting the role of STN in vocal F0 motor control in response to altered auditory feedback. The DBS-induced attenuation of vocal compensation responses may result from increased inhibitory effects of the subcortical hyperdirect (fronto-subthalamic) pathways on the vocal motor cortex, which can help stabilize voice F0 and ameliorate vocal motor symptoms by impeding PD subjects' abnormal (i.e. overshooting) vocal responses to alterations in the auditory feedback.

Brain Distribution and Metabolism of Flupirtine, a Nonopioid Analgesic Drug with Antiseizure Effects, in Neonatal Rats.

Flupirtine, a nonopioid analgesic drug, is effective in treating neonatal seizures. However, its brain delivery and pharmacokinetics are unknown in neonatal mammals. The purpose of this study was to determine the pharmacokinetics of flupirtine and the formation of its active metabolite D-13223 in various tissues such as brain in neonate animals. On postnatal day 7, rat pups received 25 mg/kg of flupirtine intraperitoneally. Liver; heart; kidney; lung; spleen; retina; serum; and brain regions hippocampus, cortex, and the remaining brain (devoid of cerebellum) were harvested up to 24-h postdosing. An LC-MS/MS assay was developed to quantify flupirtine and D-13223. Flupirtine was delivered to all tissues assessed, with the highest area under the concentration vs. time curve (AUC0⁻24h) in liver (488 µg·h/g tissue) and the lowest in spleen (82 µg·h/g tissue). Flupirtine reached the brain, including the hippocampus and cortex, within 1 h of dosing and persisted at 24 h. Flupirtine AUC in various brain regions was approximately 195 µg·h/g tissue. The half-life of flupirtine in various tissues ranged from 3.1 to 5.2 h. D-13223 was formed in vivo and detected in all tissues assessed, with the concentrations being the highest in the liver. Incubation of isolated neonatal rat liver, heart, kidney, lung, spleen, whole eye, serum, or whole brain with flupirtine for 3 h at 37 °C formed D-13223 in all tissues, except serum. D-13223 formation was the highest in isolated liver tissue. Tissue partition coefficients based on isolated tissue uptake correlated well with in vivo tissue:serum drug exposure ratios. Thus, flupirtine reaches the target brain tissues from the systemic route in neonatal rats, and brain tissue forms the active metabolite D-13223.

Neurofunctional Correlates of Response to Quetiapine in Adolescents with Bipolar Depression.

OBJECTIVES: Prior studies have shown that youth with bipolar disorder demonstrate neurofunctional changes in key prefrontal and subcortical brain regions implicated in emotional regulation following treatment with pharmacological agents. We recently reported a large response rate (>60%) to quetiapine (QUET) for treating depressive symptoms in adolescents with bipolar depression. This study investigates the neurofunctional effects of QUET using functional magnetic resonance imaging (fMRI). METHODS: Thirty-three unmedicated subjects, 10-17 years of age, with a current depressive episode (Children's Depression Rating Scale-Revised [CDRS-R] > 40) associated with bipolar I or II disorder were recruited in a two-site randomized, placebo (PBO)-controlled trial of QUET monotherapy for treatment of bipolar depression in adolescents. Twenty-three of these participants (nine male) underwent an MRI scan at baseline, then were randomized to QUET or PBO, followed for 8 weeks, and at the end of their study participation underwent another MRI scan. During the fMRI scan, subjects viewed negative and neutral pictures and rated the valence of each picture. RESULTS: Sixteen subjects had usable data at both time points: 10 subjects randomized to QUET, and 6 randomized to PBO. For QUET subjects, lower baseline activation in the left dorsolateral prefrontal cortex (p < 0.005) and higher baseline activation in the left ventrolateral prefrontal cortex (p = 0.0024) predicted greater improvement in CDRS-R scores from baseline to follow-up. When QUET and PBO groups were combined (n = 16), region-of-interest activation did not significantly predict change in CDRS-R. CONCLUSIONS: Baseline activation patterns in dorsal and ventral portions of the prefrontal cortex that are critical for the regulation of emotion-predicted response, but only within the QUET group. Thus, specific medications may be more effective in the context of specific prefrontal activation patterns in youth with bipolar depression. Larger studies of these youth would help to clarify the effects of QUET on brain activation.

Retbindin Is Capable of Protecting Photoreceptors from Flavin-Sensitized Light-Mediated Cell Death In Vitro.

Retbindin (Rtbdn) is a novel protein of unknown function found exclusively in the retina. Recently, our group has suggested, from in silico analysis of the peptide sequence and in vitro binding data, that Rtbdn could function to bind riboflavin (RF) and its derivatives flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), collectively known as flavins. Here we confirm that Rtbdn is capable of flavin binding and that this characteristic can protect photoreceptors from flavin-sensitized light damage.

DNA nanoparticles are safe and nontoxic in non-human primate eyes.

INTRODUCTION: DNA nanoparticles (NPs) comprising polylysine conjugated to polyethylene glycol efficiently target murine photoreceptors and the retinal pigment epithelium (RPE) and lead to long-term phenotypic improvement in models of retinal degeneration. Advancing this technology requires testing in a large animal model, particularly with regard to safety. So, herein we evaluate NPs in non-human primates (baboon). METHODS AND RESULTS: NPs with plasmids carrying GFP and a ubiquitous, RPE-specific, or photoreceptor-specific promoter were delivered by either subretinal or intravitreal injection. We detected GFP message and protein in the retina/RPE from eyes dosed with NPs carrying ubiquitously expressed and RPE-specific vectors, and GFP message in eyes injected with NPs carrying photoreceptor-specific vectors. Importantly, we observed NP DNA in the retina/RPE following intravitreal injection, indicating the inner limiting membrane does not prevent NP diffusion into the outer retina. We did not observe any adverse events in any baboon, and there were no NP-associated changes in retinal function. Furthermore, no systemic or local inflammatory reaction to the vectors/injections was observed, and no NP DNA was found outside the eye. CONCLUSION: Taken together with the well-established rodent safety and efficacy data, these findings suggest that DNA NPs may be a safe and potentially clinically viable nonviral ocular therapy platform for retinal diseases.

A human prefrontal-subthalamic circuit for cognitive control.

The subthalamic nucleus is a key site controlling motor function in humans. Deep brain stimulation of the subthalamic nucleus can improve movements in patients with Parkinson's disease; however, for unclear reasons, it can also have cognitive effects. Here, we show that the human subthalamic nucleus is monosynaptically connected with cognitive brain areas such as the prefrontal cortex. Single neurons and field potentials in the subthalamic nucleus are modulated during cognitive processing and are coherent with 4-Hz oscillations in medial prefrontal cortex. These data predict that low-frequency deep brain stimulation may alleviate cognitive deficits in Parkinson's disease patients. In line with this idea, we found that novel 4-Hz deep brain stimulation of the subthalamic nucleus improved cognitive performance. These data support a role for the human hyperdirect pathway in cognitive control, which could have relevance for brain-stimulation therapies aimed at cognitive symptoms of human brain disease.awx300media15660002226001.

Ablation of the riboflavin-binding protein retbindin reduces flavin levels and leads to progressive and dose-dependent degeneration of rods and cones.

The interface between the neural retina and the retinal pigment epithelium (RPE) is critical for several processes, including visual pigment regeneration and retinal attachment to the RPE. One of its most important functions is the exchange of metabolites between the photoreceptors and RPE because photoreceptor cells have very high energy demands, largely satisfied by oxidative metabolism. The riboflavin (RF) cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), are two key cofactors involved in oxidative metabolism. We have previously shown that retbindin is a photoreceptor-specific RF-binding protein exclusively expressed in the rods and present in the interphotoreceptor matrix at the interface between the RPE and photoreceptor outer segments. Here, we show that retbindin ablation in mice causes a retinal phenotype characterized by time- and dose-dependent declines in rod and cone photoreceptor functions as early as 120 days of age. Whereas minor retinal ultrastructural defects were observed at all ages examined, a significant decline occurred in photoreceptor nuclei at 240 days of age (∼36.8% rods and ∼19.9% cones). Interestingly, significant reductions in FAD and FMN levels were observed before the onset of degeneration (∼46.1% FAD and ∼45% FMN). These findings suggest that the reduced levels of these flavins result in the disruption of intracellular mechanisms, leading to photoreceptor cell death. Altogether, our results suggest that retbindin is a key player in the acquisition and retention of flavins in the neural retina, warranting future investigation into retbindin's role in photoreceptor cell death in models of retinal degenerative disorders.

Anomalous prefrontal-limbic activation and connectivity in youth at high-risk for bipolar disorder.

OBJECTIVE: Abnormal prefrontal-limbic brain activation in response to facial expressions has been reported in pediatric bipolar disorder (BD). However, it is less clear whether these abnormalities exist prior to onset of mania, thus representing a biomarker predicting development of BD. METHODS: We examined brain activation in 50 youth at high risk for BD (HR-BD), compared with 29 age- and gender-matched healthy control (HC) subjects. HR-BD was defined as having a parent with BD, as well as current mood or attentiondeficit/ hyperactivity disorder (ADHD) symptoms, or a history of at least one depressive episode. FMRI data were collected during an implicit emotion perception task using facial expression stimuli. Activation to fearful faces versus calm faces was compared between HR-BD and HC groups, including analyses of functional connectivity, and comparison of allele subgroups of the serotonin transporter (5-HTTLPR) gene. RESULTS: While viewing fearful versus calm faces, HR-BD youth had significantly greater activation than HC youth in the right amygdala, ventrolateral prefrontal cortex (VLPFC), superior frontal cortex, cerebellum, and lingual gyrus. HR-BD youth, relative to HC youth, had greater functional connectivity between the right amygdala and the VLPFC as well as visual cortical regions Within the HR-BD group, youth with the s-allele had a trend for greater activation in the right amygdala and subgenual cingulate cortex CONCLUSIONS: Similar to youth with BD, youth at high risk for BD have greater activation than healthy controls in the amygdala and ventrolateral prefrontal cortex in response to fearful faces, as well greater functional connectivity between these regions. HR-BD youth with the s-allele of the 5-HTTLPR gene may be at greatest risk for developing BD.